Ministry of Agriculture, Govt. of India Plot No.

86, Sector -18, Institutional Area, Gurgaon - 122 015 Website: www.novodboard.com

6th R&D Report on Tree Borne Oilseeds 2010-11

EEDITOREDITOR-IN- CHIEF EDITOR IN- CHIEF

Shri V.K. Gaur, Executive Director

EDITORIAL TEAM EDITORIAL TEAM

Dr. Virendra Kumar, Asstt. Director (Oilseeds) Shri Ashutosh Pandey, Technical Assistant

NATIONAL OILSEEDS AND VEGETABLE OILS DEVELOPMENT BOARD

Ministry of Agriculture, Govt. of India Plot No. 86, Sector-18, Institutional Area, Gurgaon-122 015 (Haryana)

Sl.No.

6th R&D Report on TBOs 2010-11

Contents
Introduction 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Researchable issues Survey and collection of CPTs of Jatropha Recommended Genotypes in Progeny trial, Zonal trial & National trial Progeny trials Zonal Trial National Trial I of Jatropha National trial-II of Jatropha National trial-III of Jatropha National trial-IV Agri- Silvicultural Trial in Jatropha Standardization of package of practices Infestation of Insects and pests Disease infestation Development of Data base of TBOs Hybridization in Jatropha Tissue culture DNA finger printing and Molecular characterization of Jatropha Value addition of TBOs Survey & collection of CPTs of Karanja Progeny, Zonal & National Trial Recommended genotypes of Karanja Molecular analysis Agri-silvicultural trial in Karanja Survey & collection of CPTs of Wild Apricot Progeny, zonal and national trail Research on other TBOs Chemical evaluation of TBOs Abbreviations

Page No.

1 2 3 4 6 6 8 9 10 10 10 16 21 22 25 30 51 53 84 84 86 87 90 92 94 95 101 105

Introduction
India is the biggest importer of edible oils in the world. Approximately, 16.6 million tones of edible oils consumed each year in India. Consumption of edible oils is increasing at the rate of 5 percent per annum . Demand of processed food like pastry, chocolate and ice-cream are also increasing worldwide. The edible oil is an important constituent of these food products. More than 50% of the total consumption of edible

oils in India is imported from other countries. Indonesia, Melasia, Brazil and Argentina are the major exporter of palm oil and soya oil to India. This has pressed hard the country to import of this amount of edible oils resulting in outflow of Indian currency. Therefore, we need to increase our edible and non-edible oilseeds production and productivity to meet our domestic requirements for ever increasing population . There

are several tree borne oilseeds like Simarouba, Neem, Jojoba, Karanja, Mahua, Wild apricot, Jatropha, Cheura, Kokum, Coconut, Oilpalm & Tung. They can be grown and established in marginal and semi-marginal land under varied agro-climatic conditions. Tree-Borne Oilseeds (TBOs) may play an important role to supplement the vegetable oil production in the country. Hence, National Oilseeds & Vegetable Oils Development (NOVOD) Board is promoting various tree borne oilseeds under various R&D schemes in all the states of the country.

NOVOD Board has constituted

National Research Network on Tree Borne

Oilseeds (TBOs) was constituted by involving State Agricultural Universities, Institutions of ICAR, CSIR, ICFRE, CFTRI and IIT during 2004-05. In order to strengthen the R&D programme on other important research aspects, some new projects as well as centres were included during 2008-09 and 2009-10. Since, there is no authentic data on state wise acreage under cultivation of various TBOs, availability of statewise land of different category underTBO cultivation, therefore, a project has been given to ICFRE, Dehradun to complete the task within two years. Five more projects on TBOs including

development of synthetic variety of jatropha have been approved during 2010-11. There are a total of 80 R&D centres in the above R&D network.

In addition to previous researchable issues viz; identification of elite planting material of TBOs, seed resources assessment, collection & storage; developing suitable package of practices; tree improvement to develop high yielding varieties with better quality of the reliable seed source etc. The new objectives namely Germplasm

collection, evaluation and development of varieties of mahua to increase oil content, yield and oil quality; Development of agri-hortisilvicultural models for wasteland in Chhatisgarh through mahua plantation; Standarization of mass clonal propogation protocols for karanja; Development of synthetic variety of Jatropha have been incorporated in the R&D network programme during 2010-11

The participating institutes have collected germplasm about 1433 CPTs/superior genotypes of TBOs i.e. Jatropha (1077), Karanja (228), Wild apricot (26), Simarouba (33), Mahua (50) and Kokum (19) having high yield and oil contents. Out of which a total of 964 CPTs of Jatropha (584), Karanja (170) & Wild apricot (210) preserved in gene bank at NBPGR, New Delhi. were cryo-

Seed samples of various TBOs are

regularly receiving in the Oil Analysis Laboratory for determination of oil contents and other quality parameters. A total of 723 seed samples have been received from the participating R&D institutions during 2010-11 respectively. Out of which a total of 1187 seed samples have been analyzed at NOVOD Board, Oil Analysis Lab during 2010-11 respectively. Reports are being sent to the respective institutions regularly. A standard package of practices regarding in propagation technique, nutrient management, water requirement, plant protection measures and post harvest management alongwith varietal improvement at location specific has also been developed.

Besides, the DNA fingerprinting & molecular characterization of 101

genotypes

of various TBOs i.e. Jatropha (17), Karanja (41), Wild apricot (26) and Simarouba (17) has been carried out. The efforts have also been made for development of control measures for insect-pest and diseases, value addition for preparation of quality grade defatted cakes/meal from identified TBOs, design and development of cost effective and efficient post harvest technology etc.

6th R&D Report on Tree Borne Oilseeds

A National Research Network on Tree Borne Oilseeds (TBOs) was constituted by involving State Agricultural Universities, Institutions of ICAR, CSIR, ICFRE, CFTRI and IIT during 200405. In order to strengthen the R&D programme on other important research aspects, some new projects as well as centres were included during 2008-09 and 2009-10. Since, there is no authentic data on state wise acreage under cultivation of various TBOs, availability of statewise land of different category underTBO cultivation, therefore, a project has been given to ICFRE, Dehradun to complete the task within two years. Five more projects on TBOs including development of synthetic variety of jatropha have been approved during 2010-11. There are a total of 80 R&D centres in the above R&D network. In addition to previous researchable issues viz; identification of elite planting material of TBOs, seed resources assessment, collection & storage; developing suitable package of practices; tree improvement to develop high yielding varieties with better quality of the reliable seed source etc. following four objectives have been incorporated in the R&D network programme during 2010-11 Researchable issues : i) ii) iii) iv) Germplasm collection, evaluation and development of varieties of mahua to increase oil content, yield and oil quality. Development of agri-hortisilvicultural models for wasteland in Chhatisgarh through mahua plantation. Standarization of mass clonal propogation protocols for karanja. Development of synthetic variety of jatropha

JATROPHA (Jatropha curcas)

The following salient achievements have been made during 2010-11 The R&D network programme on Jatropha is being implemented by involving 54 research institutes/universities of ICAR, CSIR, ICFRE, IIT etc in 27 states/UTs i.e. Andaman & Nicobar Island (1), Andhra Pradesh (3), Arunachal Pradesh (1), Assam (1), Bihar (1), Chhattisgarh (1), Delhi (3), Gujarat (3), Haryana (5), H.P. (1), J&K, (1), Jharkhand (1), Karnataka (3), Kerala (2), Maharashtra (3), M.P. (3), Meghalaya (1), Manipur 1), Nagaland (1), Orissa (2), Punjab (1), Rajasthan (10, Tamil Nadu (1), Tripura (1), Uttar Pradesh (6), Uttrakhand (3), and West Bengal (3) .

A. Survey and collection of superior planting material (seeds / cuttings from different agro-climatic zones) A total of 1077 candidate plus trees (CPTs) have been identified by various research institutions in respect of Jatropha totalling to 4368 CPTs since inception of the project. Sl. No. Name of Research Institutions No. of CPTs/Seed Source identified. Upto 2009- 2010-11 10 29 123 120 420 36 14 153 51 94 2 5 3 250 6 24 35 88 19 66 31 9 26 160 687 1077 Total

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

CSFER, Allahabad ICAR R.C.NEH, Nagaland ICAR R.C.NEH, Tripura SDAU, Gujarat GBPUA&T, Pantnagar NDUA&T,Faizabad BAU, Ranchi OUAT, Bhubaneshwar SVBPUAT, Meerut UBKV, Coochbehar ANGRAU, Palem UAS, GKVK, Bangalore CARI, Portblair KAU, Thrissur AAU, Anand
CCS,HAU, Hisar (Dr.Dhawan)

Total IGAU, Raipur

152 540 50 204 96 8 256 24 35 88 19 66 31 9 26 160 1764

Morphological variation in seed characteristics of different CPTs selected during 2010-11 of Jatropha Curcas in Chhattisgarh Seeds collected from various CPTs were studied for morphological characters and oil percent. Five samples were taken from each seed CPTs and seed parameters (viz, seed length, seed width, seed thickness) were measured with the help of digital vernier caliper. Seed length was maximum (18.50 mm) for Moran (Distt- Surguja), while it was minimum (16.41 mm) for Dhundra (Distt- Surguja). Aaturgaon (Distt- Kanker), Moran (Distt- Surguja) and Wadrafnagar (Distt- Surguja) had highest (11.34 mm) seed width, while minimum seed width was observed for Sonhat (Distt- Korea). Seed thickness was maximum (9.29 mm) for Wadrafnagar (Distt- Surguja), while it was minimum (8.15 mm) for Semra (Distt- Surguja). Seed weight was taken with the help of digital balance. 100 seed weight was maximum (79.36 gm) for Keshkal (Distt- Bastar) and minimum (46.63 gm) for Dhundra (Distt- Surguja). Oil per cent was estimated with the help of Socs plus solvent extractor. Oil per cent was highest (41 %) for Songara (Distt- Surguja), while it 2

was minimum (28 %) for Keshkal (Distt- Bastar), Sukhia nalah (Distt- Surguja), Parsapara (Distt- Surguja),Biharpur (Distt- Surguja),Jarhi (Distt- Surguja) and Rewati (Distt- Surguja). ANGRAU, Hyderabad At, RARS, Palem, 19 germplasm accessions of Jatropha which were planted during August 2007 are in the third year of evaluation. The wide variability was observed for important characteristics viz Plant height (98-242 cm), No. of branches (14.5-39.2), No. of fruits/cluster (6-23), No of fruit clusters/plant (3-14), weight of seed/ plant (130565g), No of seed/ capsule(2-3), 100 seed weight(38-56 g) and Oil content (24.6-33.8 %). Highest no of branches, No of fruit clusters/ plant, number of fruits/ cluster (Average 23.0) and seed yield (565 g/pl) were recorded in Kollapari (Local collection) C. Recommended genotypes in Progeny, Zonal & National trials of Jatropha The institute-wise recommended new genotypes based on germination percentage and growth parameters are given as here under. Progeny Trial Sl. No. Name of Recommended Genotypes/ remarks Research Institutes 1 FCRI(TNAU) TNMC 5 & TNMC 7 2 CRIDA, AccNo-3, AccNo-21, AccNo-12 Hyderabad 3 MPKV, Rahuri MPC-UD-42, MPC-UD-55, MPC-UD-38, MPC-UD16 4 TFRI, Jabalpur Khaerwani-1, Timrikhurd, Panna, Timrikala, Jogimuhar Daultpur - 25 (Seed yield / ha = 180.76 kg) and Kaladungi Rd 5 GBPUA&T, - 15(Seed yield / ha = 176.82 kg) Pantnagar CSFER, Allahabad Zonal Trial Sl. No. 1 2 3 4 Name of Research Institutes CRIDA, Hyderabad MPKV, Rahuri PDKV, Akola GBPUA&T, Pantnagar 6 CGKP-8

Recommended Genotypes/Remarks

TNMC-5, TNMC-6 Ludhiana, SKNJ-2, MPC-RS-K, MPC-CH-B JNKKV-JABALPUR-SAGAR, Pant Sel - 31(Seed yield / ha = 130.47 kg) and Jajhar Kotali (Seed yield / ha = 106.57 kg)

National Trial Sl. No. Name of Research Recommended Genotypes/ remarks Institutes

1 2

CRIDA, Hyderabad MPKV, Rahuri

PDKV, Akola

GBPUA&T, Pantnagar

National Trial-III LBJJ-23, TNCJC-19 National Trial I : Indore, TFRI-I, TNMC-2 National Trial II : MPJ-55, JA-9 National Trial III : CALD-14, NDJC-1 National Trial I PDKV-AKOLA-MALKAPUR National Trial II : NBJ-I National Trial III : Ranchi National Trial I
IGAU Raipur (Seed yield / ha = 292.04 kg), Pant Jatropha Selection -2 (Seed yield / ha = 199.46 kg), Sagar (SFRI. Jabalpur) (Seed yield / ha = 199.17 kg)

National Trial II
NBJ 1 ( Seed yield / ha = 106.23 kg ) and Pant J - 03103 (Seed yield / ha = 83.89 kg)

5 6 7

CSFER, Allahabad ICARRCNEH, Meghalaya CARI, Port Blair

Akola(PKVJ-MKU-1, PJ Sel-2

PJS-2 PantJCP4, CARI AN JC 2, JIP-12 and FCRI JC

PROGENY TRIAL The salient findings reported by some of the participating institutes are summarized here under : IGAU, Raipur: Plants were planted in July 2009 at Mudhipar (Bhatapara). Plant height varied from 53.88 cm (Teka) to 71.25 cm (Laberakhurd). Collar diameter ranged from 28.73 mm (Chicdo) to 50.61 mm (Laberakhurd). IMMT, Bhubaneshwar: Analysis of the field observations revealed that accession Baramunda performed better in terms of all the yield attributing characters followed by accession Mancheswar. In terms of seed oil content the low rankers were TNMC 3, PKVJ SJ 1, Pendra Road, Ambikapur, Kalyanpur, PKVJ MKU 1 and RJ 117. Rest of the accessions yielded more than 30% of seed oil content (w/w) with the maximum (36.78%) in Baramunda followed by (35.33%) in Mancheswar. ICARRCNEH, Tripura : In the progeny trial, 18 seed sources were planted in 2006 and growth characteristics estimated upto 2010 indicated that jatropha planted from seed samples of Mendipathar (East Garo hills) showed the maximum diameter of 11.74 cm

with 3.00 primary branches ,11.00 secondary branches and 63.00 tertiary branch . The lowest basal diameter (8.57 cm) was noted in Jatropha planted from Khowai (West Tripura) With 3 nos of primary branches 10 nos of secondary branches and 57.00 nos of tertiary branches . Jatropha from south Tripura (Udaipur), North Tripura (Dharmanagar) and Rajasthan showed the maximum no. of primary brances (4 nos) and Bilash pur showed the maximum no. of secondary branches ( 18 nos). Male/female ratio is a matter of great concern in jatropha plantation. Male /Female ratio underwent a variation from 14.78 to 31.24 with fruit yield from 398.33 to 2378.00 g/plant. Seed yield was variable from 174.44 to 786.00 g/plant with the shelling percent from 25.44 to 59.75 .1st flowering was recorded in the month of April, 2010. After 4th year of plantation, Jatropha from Dimapara ( South Garo Hills) showed the maximum fruit yield (2378.00 kg/tree) with 33.05 % recovery and Jatropha from Madhya Pradesh showed the lowest production of fruit yield (0.398 kg/tree) with 43.79 % recovery. Out of the progeny material from Tripura, Jatropha from Mohanpur ( West Tripura) showed the maximum production of fruit ( 1.296 kg/ tree) with 38.08 % recovery. RRSCCSHAU, Bawal: Progeny Trial-I, II and III were established in the field during 2005, 2006 and 2008 consisting of 20, 15 and 17 accessions, respectively. In Progeny Trial-I, IC 520601 showed maximum oil content (29.35%) and seed yield (879.4g/plant). Performance of BS-11 for oil content and seed yield (773.4g/plant) was better over other accessions included in the Progeny Trail-II. In Progeny Trail-III, IC 552898 and 552902 were better in respect of oil content and seed yield, respectively.
UBKV, Nadia : For Progeny trial, 20 genotypes of different accessions were planted with the spacing of 3mX3m. It was noticed that after one year growth UBKV-J/13 had attained the maximum height of 250.53 cm and UBKV-J/27 had attained the minimum height of 201.15 cm. The collar diameter was found to be maximum in UBKV-J/10 and minimum in UBKV-J/14 i.e.64.75 mm and 49.54 mm respectively. Maximum number of branches was found to be in UBKV-J/17 and minimum in UBKV-J/ 26 i.e.17.05 and 7.82 respectively.

NRCAF, Jhansi : The field data on progenies of the candidate plus trees (CPT) at the age of five years revealed that the seed yield varied from 30.12 g/plant in NRCJ39 to maximum of 128g/plant in NRCJ42. This was followed by 76.16g in NRCJ32 and 35.3g in NRCJ35 and NRCJ48. Hundred seed weight varied from a minimum of 48.04g (NRCJ32) to the maximum of 57.26g (NRCJ42). Hundred fruit weight was 188.6 g. Average fruit length and width was 26.51mm and 19.78mm, respectively. Average number of primary branches/plant was 6.8 and it ranged from a minimum of 5.3 in NRCJ36 to a maximum of 9.33 in NRCJ56. Other progenies that showed superiority for high primary branches/plant were NRCJ48 (8), NRCJ33 (7.6), NRCJ37 (7.6), and NRCJ38 (7.5). Average number of secondary branches/plant was 33.63 and it ranged from a minimum of 27 in NRCJ45 to a maximum of 43.33 in NRCJ38. Other accessions having high number of secondary branches were NRCJ56 (39.9), NRCJ32 (37) and NRCJ31 (36.75).

The data of progeny trial II (2004) consisting of nine candidate plus trees collected from Rajasthan and Uttar Pradesh revealed that average seed yield was 201.4 g/plant. The seed yield varied from a minimum of 89 g/plant in NRCJ60 to the maximum of 317g/plant in NRCJ64. This was followed by 304g in NRCJ65 and 226g in NRCJ63 and NRCJ70. The data of progeny trial III (2005) consisting of 12 CPT collected from Uttar Pradesh, Madhya Pradesh and Rajasthan revealed that the seed yield varied from minimum of 32.5 g/plant in NRCJ50 to maximum of 470g/plant in NRCJ92. This was followed by NRCJ41 (310g) and NRCJ40 (290g). Average seed yield was 160.38g/plant. Average 100 seed weight was 53.83g and 100 seed weight varied from a minimum of 48.8g (NRCJ50) to the maximum of 58.08g (NRCJ41). Fruit length varied from a minimum of 23.86mm (NRCJ50) to the maximum of 26.08mm (NRCJ40 and 66).
Zonal Trial :

RRSCCSHAU, Bawal: Two Zonal Trials comprising of 28 accessions were transplanted in the field during 2005 and 2006. Among both the trials BNK-21 from Bawal performed better in respect of oil content (30.05%) followed by SKNJ-11 from Gujrat (28.08%). In general, the yield was poor during this year. In Zonal Trial-I maximum seed yield (139.8g/plant) was recorded in accession MPCUDK collected from Rajasthan whereas accession SKNJ-20 from Gujrat showed minimum seed yield i.e. 93.8g/plant. The performance of Zonal Trial-II was better as compared to Zonal Trail-I. Maximum seed yield (810.5g/plant)as recorded in MPAU-3 followed by PP-2 (654.6g/plant). IMMT, Bhubaneshwar: The progenies of Baramunda and Mancheswar consistently performed better in terms of seed yield and oil content. In the sixth year the seed yield and oil content of Baramunda was 0.578 kg/ plant and 35.33 % and that of Mancheswar was 0.371 kg/ plant and 34.33 %, respectively. National Trial-I (2005-06) The main observations recorded by the participating institutes/centres are given as hereunder: CRIDA, Hyderabad
S. No Name of Genotype S.No Average seed yield (kg/ha)* 181.46 562.91 340.71 185.17 359.22 Oil content in seed (%) 30.41 31.04 32.50 30.81 27.87 9 10 11 12 13 Name of Genotype Average seed yield (kg/ha)* 444.40 327.75 138.88 229.61 196.28 Oil content in seed (%) 24.50 26.85 29.11 32.74 31.61

1 2 3 4 5

Bilaspur TNMC-4 Pendra Road TNMC-22 Indore

TNMC-16 PJ-Sel-2 AMBICAPUR RAK-29 TNMC-21

6 7 8

PJ-Sel-1 TNMC-20 PKVJ MKU-1

172.21 466.62 318.49

37.64 37.60 31.74

14 15 16

PKVJ DHW-1 TNMC-6 TFRI-1

444.40 462.92 214.79

33.53 25.30 29.05

* No Fruiting * * Seed yield was affected by severe attack of root borer.

CCS HAU, Bawal : A total of 25 accessions/genotypes were tested for yield and oil content. Bilaspur accession performed better in respect of oil content (36.76%) followed by PKVJDHW 1 (35.38%). It was observed that total no. of branches was highest in TNMC-4(18.4) followed by TNMC 3 (17) and Bilaspur(16). The highest plant height was observed in Sagar (299cm) followed by TNMC 22(292cm) and Set 2 Pantnagar (280cm). The seed yield (g/plant) was highest in Sergia (Raipur) followed by Bilaspur (319.0) and RJ 117 (298.7). Orissa accession yielded maximum seed (553.3g/plant) among 9 accessions. BAU, Ranchi : The total no. of branches was observed highest in RJ-117(22.7) followed by PKVJ-MKW1(18.9) and Pant J Sel 1(13.0). The seed yield (Kg/ha) was highest in PKVJMKW1(176) followed by RJ-117(58.32), Pant J Sel-1(38.53) and Kalyanpur (30.20). The Collar diameter was observed highest in RJ-117(8.6 cm) followed by PKVJ-MKW1(7.9 cm). The highest plant height was observed in RJ-117(8.6 cm) followed by PKVJ-MKW1(7.9 cm). CRIDA, Hyderabad : It was observed that total no. of branches were highest in TNMC-6(210.9) followed by PKVJ DHW-1(177.6 ). Similarly, the Collar diameter was highest in TNMC6(13.5cm) followed by PKVJ DHW-1(12.3cm). The estimated yield (kg.) /ha. was highest in Indore and PJ-Sel-1. TNAU Coimbatore: The first National Trial of Jatropha was established in 2006 at Forest College and Research Institute(TNAU), Mettupalayam. Sixteen genotypes provided by the various research centres were raised under NOVOD R&D network. The trial gave yields nominally till 2008 and was maintained under rainfed condition. After that, due to the incidence of mealy bugs, the plants of the trial got damaged and are no more existing. CSAUA&T, Kanpur : It is observed that the all genotypes survived better at initial period except Sarguja (68%). The height, collar diameter and branches/plant also showed progressive development of Jatropha at beginning period but, at succeeding period the plants do not show the progressive development in any character. It is also observed that plant height stunted and some plants died due to accumulation of bi-valent and monovalent ions in vicinity of the root zone of Jatropha plant. During the report year of 2010, the number of plants died in all genotypes due to the poor development of plant roots. 7

The rest plants of the experimental site under all genotypes showed slow growth of any genotypes under salt affected condition due to the biotic and abiotic factor. D. National Trial- II (2007) of Jatropha

A second national trial with superior genotypes was established during June-July, 2007 by 14 research institutions contributing 19 genotypes and following standard package of practices. The salient findings reported by some of the participating institutes are summarized hereunder : FCRI (TNAU), Mettupallayam: For seed yield per plant, entry Jabalpur showed significantly higher value (0.45 kg/plant) followed by Akola (0.41 Kg/plant). Entry JC 24 also showed higher seed yield followed by TNMC 7. For oil content, entry Jabalpur showed significantly higher value (26.5%), followed by Akola (25.6%). The genotype Jabalpur (6.0) showed higher average number of branches followed by Akola (5.6). For seed yield, entry Jabalpur (1135 Kg) showed significantly higher value, followed by Akola (1023.4 Kg). The better performance of the entry Jabalpur for the traits collar diameter, fruiting bunches, no: of branches, seed yield, oil content etc followed by Akola. TNMC 7 also showed good results in almost all the characters. CSAUA&T, Kanpur: The Jatropha plant survival was observed about 95 %. However plant height, collar diameter and branches/plant showed progressive development up to II year but between the period of II year and III year the plant height of all genotypes restricted due to the presence of sodium salt in the vicinity of roots resulting poor growth and development of plants.

NRCAF, Jhansi : A large variation was recorded in the seed yield which differed
significantly among the genotypes. Average seed yield was 167.23kg/ha. Maximum seed yield of 388.5 kg/ha was recorded in NRCJ2. It was followed by JA9 (336.07 kg/ha), TNMC8 (252.19 kg/ha), PDKV Nov3 (227.75 kg/ha) and Bawal Sel (208.86kg/ha). Minimum seed yield was recorded in PantJ03103 (61.10kg/ha). The seed yield/plant was also found in these genotypes at the same rank. Seven genotypes had seed yield higher than the average seed yield. Hundred seed weight was found maximum in SKNJ2-1 (62.89g). This was followed by NBJ9 (61.26g) and PantJ03103 (59.34g). Minimum 100 seed weight was recorded in JA9 (50.06g). Average hundred seed weight was 56.80g. Number of primary branches/plant ranged from a minimum of 3.8 (Orissa) to a maximum of 8 (TNMC33). Other promising genotypes were SKNJ2-1(6.07), TNMC8 (6), NBJ1 (5.8) and HAUJ39 (5.75). Out of 17 genotypes, ten genotypes were recorded for primary branches/plant above the population mean. Average number of secondary branches/plant was 42.88 and it ranged from a minimum of 33.83 (Bawal Sel) to a maximum of 49.14 (SKNJ2-1). Other accessions having high number of secondary branches were TNMC8 (49), TNMC19 (47) and PDKVNov3 (46.79). Out of 17 genotypes, seven genotypes were recorded for secondary branches/plant above the population mean. CCS HAU, Hisar: The highest mean seed yield of 6.15q/ha was recorded in genotype Orissa followed by JA-9 ( 6.03 q/ ha) and NBJ-1 (4.56 q/ha). The highest average no of branches was recorded in genotype NBJ-1 (68.7) which is followed by JA-9 (68.3),

PDKV-NOV-3 and MPJ-55(65.3). The no of fruiting branches was highest in NBJ-1 (36.0) followed by JA-9 (35.3), Orissa (33.3) and PDKV-NOV-3 (30.3). E. National Trial- III (2008) of Jatropha

A second national trial with superior genotypes was established during June-July, 2008 by 14 research institutions contributing 22 genotypes. The main observations recorded by the participating institutes/centres are given as hereunder: NRCAF, Jhansi : A large variation was recorded in the seed yield among the genotypes. Average seed yield was 183.46kg/ha. Maximum seed yield of 219 kg/ha was recorded in MNJ001. It was followed by NRCJ2 (218 kg/ha), TFRI07 (215.5 kg/ha) NRCJ89 (211 kg/ha) and TNCJC19 (206.5kg/ha). Minimum seed yield was recorded in PantJCP1 (109.5kg/ha). Ten genotypes had seed yield higher than the average yield. Number of primary branches/plant ranged from a minimum of 2.88 (MNJ006) to a maximum of 4.73 (TFRI07). Out of 18 accessions, nine accessions were recorded for primary branches/plant above the population mean. Other promising accessions were PDKV Nov19 (4.3), NRCJ-89 (4.1) JJ-2 and CRJ-29 (4). Average number of secondary branches/plant was 12.08 and it ranged from a minimum of 6.7 (MNJ006) to a maximum of 20.6 (PantJCP1). Other genotype having high number of secondary branches were TNCJC20 (16.12), CRJ-29 (14.8) and NRCJ-18 (14.3). Out of 18 genotype, eight genotype were recorded for secondary branches/plant above the population mean. FCRI(TNAU), Mettupallayam: Among the 14 genotypes viz TNCJC-19, TNCJC-20, JJ-2, Pant JCP-1, Pant JCP-2, NRCJ-7, NRCJ-18, NRCJ-89, PDKV Nov.19, TFRI-07, LBJJ-23, TR-4, CALD-14 and NDJC-1, the TNCJC-20 showed significant higher values followed by LBJJ-23. The observations for most of the genotypes were not recorded owing to the absence of plants due to mealy bug attack. Inspite of all these measures, the mealy bug attack could not be controlled effectively. IGAU, Raipur: Plants were planted in August 2008 at Mudhipar (Bhatapara). Plant height varied from 52.72 cm (TFRI-07) to 71.36 cm (PDKV-NOV-1). Collar diameter ranged from 22.57 mm (Pant JCP-1) to 28.98 mm (CRJ-29). ICARRCNEH, Tripura: A National trial with genotypes LBJJ-23 (Ranchi). NDJC-1 (Faizabad), TFRI -07 (Jabalpur), JIP-12-520621 (Jammu), JJ2 (Jabalpur), Panth JCP-1 (Uttarakhand), JCP-2 (Uttarakhand) and PDKVNOV -19 (Akola ) was laid out. Jatropha planted from seed samples of PDKVNOV-19 (Akola) showed the maximum basal diameter of 7.75 cm with 1 primary branches , 5.66 no of secondary branch and 3.00 no of tertiary branch. The lowest basal diameter (5.73 cm ) was noted in Jatropha planted from Pant-JCP-1 with 1 no of primary branches,6.50 no of secondary branch and 4.00 no of tertiary branch.

F.

National Trial- IV (2009)

ANGRAU, Hyderabad This trial was initiated during 2009-10 with the seed Jatropha CPTs received from various R&D institutions. The highest plant height was recorded in MPCUD 55 (141cm) jatropha accession. However, it was statistically on par with PJA-1, PANT JCP-3, TJGS37, PANT JCP-4, HAU J -37 and AJA-1. The highest collar diameter was recorded in PJA-1 (8.1cm) jatropha accession. However it was statistically on par with TJGS-37, HAU J -37 and AJA-1. The highest number of branches was recorded in RJ-124 (10) jatropha accession. However it was statistically on par with AJA-1. UBKV, Nadia : After attaining an age of one year it was seen that JCP-4 had reached the maximum height of 257.26 cm and TNAU had the minimum height of 176.48 cm. The collar diameter was also found to be highest in JCP-4 i.e.81.84 mm and lowest in NAUJ16 (58.85mm).It was observed that PJA-1 had highest no. of branches (6.55) and JCP-3 had minimum no. of branches (4.04). UAS, GKVK, Bangalore For this trial, seeds of 10 genotypes viz., TFRI 1/09 from Jabalpur, MPC UD 55 from Udaipur, Ludhiana 1/09 from Ludhiana, PJA- 1 from Hyderabad, Pant JCP-3 & Pant JCP-4 from Pantnagar, RJ -124 from Rahori, JIP -12 from Jammu and one entry without label from Coimbatore have been received during 2009. However, seeds of only seven entries viz TFRI 1/09, PJA-1, Ludhiana 1/09, Pant JCP-3, Pant JCP-4, JIP-12 and Coimbatore have germinated and produced seedlings for planting and hence included in this trial. G. Agri-silvicultural Trials (Inter-cropping) with Jatropha

UBKV, Nadia : For Agro forestry trial also 20 genotypes of different accessions were planted
with the spacing of 4mX3m. Mung var.PDM-139 and Kalai var. P.U-30 were intercropped in 1st year and Mustard var.B-9 and Jhumuka were intercropped in 2nd year. Here it was observed that after 1 year of planting UBKV-J/1 had attained the maximum height of 294.93 cm and UBKVJ/19 with minimum height of 135.40 cm. Collar diameter was highest in UBKV-J/1 and minimum in UBKV-J/24 with 79.65 mm and 50.64 mm respectively. Number of branches was found to be highest in UBKV-J/ 1and lowest in UBKV-J/9 i.e. 8.00 and 2.29 respectively.

Standardization of package of practices Spacing Trial GBPUA&T, Pantnagar: The experiment for plant density /ha in jatropha with soybean and wheat as intercrop has been studied. The maximum jatropha seed yield of 4.2 q/ha was obtained at planting density 4444 plants/ha i.e. 1.5mx1.5m. At 3.0mx3.0m spacing (1111plants/ha) jatropha seed yield obtained were 1.35 q/ha and wheat yield of 25.16 q/ha.The study suggest that the wider spacing reduce the number of jatropha plants, thus reducing the jatropha seed yield also. The broad based agronomic trials combining 10

pruning, plant density and intercrops are required to reach the recommendation for farmers. This data is at the second year cropping of jatropha and the agronomic specification beyond third year also need to be studied as the increasing foliage will affect the intercrops. It was also observed that more plant height was recorded in agroforestry than sole crop
of jatropha. Maximum plant height of jatropha both in agroforestry and sole crop was recorded with 20 g N/plant. Branches/plant were more in sole crop of Jatropha than Jatropha with crops. Like height, branching in jatropha was also recorded maximum at 20 g N/plant both in agroforestry and sole crop of Jatropha. No seed yield was recorded because late flowering resulted in poor fertilization as a result of which kernals remained shriveled. The grain yield (4.5 q/ha) of moongbean intercropped with jatropha decreased by 47 per cent over control (8.5 q/ha). The main reason for reduced yield of moongbean intercropped with jatropha was loss of about 40 per cent space to jatropha plants. Similarly, during the winter season, mustard yield (2.5 q/ha) was reduced by 59.0 per cent due to loss of space over control (6.1 q/ha) reported at CCS HAU, Hissar.

Pruning GBPUA&T, Pantnagar :Under the intercropping system the maximum jatropha seed yield (q/ha) obtained was 3.31 and the corresponding wheat yield (q/ha) of 26.53 were obtained when no pruning was done in jatropha while 50 cm pruning in jatropha gave 31.20 q/ha wheat yield and 2.94 q/ha yield of jatropha seed. The treatment of 100 cm pruning of jatropha from the ground level yielded 29.91 q/ha wheat yield and 3.25 q/ha jatropha seed. This treatment appears appropriate under intercropping system for both wheat and jatropha yield. PAU, Ludhiana : This centre reported that Pruning in Jatropha is necessary to increase the number of branches and maintaining the shape of plants. The main branches were pruned to 2/3rd and 1/2 levels annually, 2/3rd and 1/2 branches pruned once initially and a control (unpruned). The analysis of the data reveals that minimum height (3.51 m) was observed in T1 treatment and the maximum (3.83 m) was observed in T5 treatments. Collar girth was found to be maximum (57.9 cm) in T5 treatment and the minimum (42.4 cm) was observed in T1 treatment. The yield of unpruned plant was significantly higher (555 g/plant, 0.617 mg/ha) than the pruned plants. The yield of plants pruned 2/3rd annually was minimum (223 g/plant or 0.248 mg/ha). ICARRCNEH, Tripura : It is observed that there was an increment of the pruning on the secondary branches of Jatropha .Jatropha from Dimapara (south Garo Hills) showed the maximum no of secondary branches (18-19) thus indicating the maximum fruit/seed yield after 4 years of planting. Standardization of spacing and pruning intensity of Jatropha The experiment on pruning treatments revealed that the height of plants was maximum (2.94 m) under 3 x 2 m spacing with 1/2 pruning of branches and minimum (2.14 m) under 3 x 4 m spacing with 2/3rd pruning of branches. The collar girth was

11

maximum (31.9 cm) in 3 x 4 m spacing with 1/2 pruning of branches and minimum (24.1 cm) in 3 x 2 m spacing with 2/3rd pruning of branches. The seed yield/plant was highest (478 g/plant) in 3 x 3 m spacing having 2/3rd branches pruned, whereas the yield was minimum (258 g/plant) in 3 x 2 m spacing with 2/3rd branches pruned. The yield on per ha basis was higher in 3 x 3 m (0.461-0.531 Mg/ha) and 3 x 2 m (0.430-0.550 Mg/ha) spacings than 3 x 4 m spacings (PAU, Ludhiana). IGAU, Raipur: In kharif 2010 soybean var. JS-335 was taken as intercrop between Jatropha & Karanja. Soybean yield varied from (24.13-48.26 Q/ha). It was found maximum in Control followed by J6 K6 (30.00) > J5 K5, J4 K4 (26.93). CSAUA&T, Kanpur : Among the tested summer groundnut varieties, Dh 86 and ICGV 93468 gave significant higher pod yield by 1.81 tha-1 and 1.75 tha-1, respectively in the companion cropping system of Jatropha + summer groundnut. Cultivar JL 24 used as check variety gave yield by 0.68 tha-1 in the parallel cropping system. The order of varietals performance was Dh 86 (1.81 tha-1) & ICGV 93468 (1.75 tha-1)> NRCG 2063 (0.70 tha1) )> NRCG 2063(0.69 tha-1 )> NRCG 12082 (0.69 tha-1)> TG 26 (0.68 tha-1)> JL 24 (0.0.68 tha-1) >BG 95(0.68 tha-1) and NRCG 7150 (0.66 tha-1) under agroforestry system of Jatropha + summer groundnut . FCRI(TNAU), Mettupallayam Effect of different spacing and intercrops on the yield of 5 years old Jatropha curcas (Kg ha-1)
Tree Spacing
M1 (3m x 3 m) M2 (4 mx 2 m) M3 (3m x 4 m) M4 (4m x 3 m) Mean

Blackgr am
950 1600 710 750 1003

Greengra m
940 1650 730 775 1023

Cowpe a
990 1760 800 880 1108

Groundn ut
945 1840 780 890 1114

Gingell y
880 1550 690 790 978

Sunflowe r
870 1520 690 780 965

Pure Tree s
1100 1900 850 910 1190

Mean

954 1689 750 825

Effect of different spacing of Jatropha and intercrops on gross income from 5 year old Jatropha curcas (Rs.ha-1)
Tree Spacing
M1 (3m x 3 m) M3 (4 mx 2 m) M2 (3m x 4 m) M4 (4m x 3 m) Mean

Blackg ram 4750 8000 3550 3750 5013

Greengra m 4700 8250 3650 3875 5119

Cowpe a 4950 8800 4000 4400 5538

Groundn ut 4725 9200 3900 4450 5569

Gingell y 4400 7750 3450 3950 4888

Sunflowe r 4350 7600 3450 3900 4825

Pure Tree s 1100 1900 850 910 1190

Mean
4645 8266 3666 4054

12

Soil pH The results revealed that after 5 years of intercropping with Jatropha, a slight decrease in soil pH was observed in all tree-crop combinations and trees alone over the initial pH. Among the effect of intercrops on soil pH , the highest pH was recorded with gingelly (7.73) and lowest with greengram (7.68).However, there was no significant difference among the treatments. Soil EC The result showed that there was an increase in soluble salt content after five years of intercropping with Jatropha due to intercrops and spacing when compare to initial value. Among the different spacing, 4mx2m spacing of Jatropha recorded the highest EC followed by 3mx3m.Whereas 3mx4m spacing registered the lowest. However the differences among the treatments were meager. Among the intercrops, highest EC was recorded with cowpea (0.056 dSm-1) followed by green gram (0.054 dSm-1) and the least with gingelly 0.049 dSm-1) Soil Available Nitrogen The initial available N content of the experiment field was found to be 245 kg/ ha. The data revealed that there was a built up of soil available nitrogen after five years of intercropping over initial value . Among the spacing the highest available N was recorded in 3mx4m (274 kg/ ha) followed by 4mx3m and lowest was in 4mx2m (256 kg/ ha). Among the intercrops, the highest available N was found in cowpea (271 kg/ ha) followed by groundnut and green gram and lowest was with gingelly (263 kg/ ha) and sunflower (261 kg/ ha).It was observed that Jatropha intercropped with pulse crops registered significantly higher nitrogen content compare to other agricultural crops. This might be due to N fixing capacity of pulses and groundnut. Soil Available Phosphorus The initial soil phosphorus content of the soil was 15 kg/ ha. The available phosphorus status of soil was increased slightly after five years of intercropping with Jatropha when compared to initial value. Though there was no significant difference in phosphorus content between the spacing and intercrops, the highest value was recorded in 4mx2m spacing followed by 3mx3m. Among the intercrops, the cowpea-Jatropha combination registered highest value (21.1 kg/ ha) followed by groundnut. However the difference among the intercrops was leas. Soil Available Potassium The initial soil available potassium was 215 kg/ ha. .After five years of experiment, increased soil K content was observed. Among the spacing, 4mx2m recorded highest available K (245 kg/ ha) followed by 3mx3m (243 kg/ ha) and lowest in 4mx3m (237 kg/ ha). Among the intercrops, the groundnut recorded highest available with a value of 246 kg/ ha followed by cowpea (244 kg/ ha) and the lowest was in gingelly (234 kg/ ha).

13

Standardization of propagation techniques through seeds& cuttings in Jatropha Germination of Jatropha seeds after different pre treatments: BAU, Ranchi : An experiment was conducted on germination of Jatropha seeds after different pretreatments viz. water ( 24 hrs), 10 ppm IBA, 20ppm IBA, 30ppm IBA, 10ppm GA3, 20 PPM GA3, 30 PPM GA3, Conc H2SO4 ( 30 Sec), Conc H2SO4 ( 60 Sec). Results indicated that 30ppm IBA gave best result with 70% germination. IMMT, Bhubaneshwar: To achieve maximize rate of germination, pre-treatment experiments with cold water, hot water, 1 N dilute H2SO4 and cowdung slurry were carried out. Soaking in cowdung slurry for 12 hr was found to be the most effective to obtain highest percentage of germination (96 %). Propagation through cuttings Semi hard wood & hard wood cuttings of 15-25 cm length and 2-2.5 cm diameter resulted good response, whereas apical cuttings are poor in success. Soaking of cuttings in 100ppm IBA & 300ppm NAA (10 min.) showed the good results in sprouting and best season for planting of cuttings was mid March to mid April. The cuttings may be dip in 0.1% Carbandazime solution for better result. 100% budding was obtained in the cuttings pre treated with Seridex solution (3000ppm) for 24 hours. IMMT, Bhubaneshwar : Highest root production (10-12 no.) and survival rate (96 %) was noticed with 7.1 8.5 mm diameter class of cuttings. Shoot cuttings of lengths between 7.1 and 8.5 cm was found to be most effective as propagating materials. The month of August was observed to be the best time for preparing cuttings. Pre-treatment of cuttings with IBA @ 200 ppm gave good results of rooting in summer months. SKUA&T,Jammu The maximum i.e. 79.86% sprouting was found in cuttings of diameter size 1.5 to 2.0 cm when treated with 100 ppm IBA and planted during mid-April. However, the average no. of roots per cutting were found maximum ( 19.53) when 1.0-1.5 cm diameter size cutting were treated with 200 ppm IBA and planted during mid of April. Specific results : Effect of Pruning GBPUA&T, Pantnagar: One pruning in first two years during 15- Feb-15 March increases in No.of branches.The 25%, 50% & 75% branches were pruned in rows having 50 plants along with one row of control in February. The data for plant height, collar diameter and number of branches suggests a superiority of 50% pruning. The average number of branches in un-pruned row were 28 while 50% pruned row were 47. The data suggests that pruning is necessary for better growth.

14

IMMT, Bhubaneshwar: One soft pruning every year during dormant stage for first five years is necessary. 25 % percent of the existing branches should be pruned. Second fortnight of November is the best time of pruning. Seed treatment ICAR, RC NEH, Manipur: The results showed that among the various pre-treatments tried, untreated seeds resulted in the maximum germination percentage over all the periods of observation which was followed by seeds treated with 20ppm GA3. The lowest germination (18.18%) was recorded at 60 days after sowing with the seeds soaked in water. This could be attributed to the release of phenolic compounds while soaking the seeds with water or other substances which in turn could have interfered with the germination. IMMT, Bhubneshwar: Maximum Germination Percentage (GP) can be obtained by soaking the seeds in cow-dung slurry (1 kg dung in 5 lit. of well water). Jatropha seeds lose viability with the age. This is confirmed by Tetrazolium Biochemical method (0.1% 2,3, 5-Triphenyltetrazolium chloride-Dehydrogenase-Triphenylformazone). With the help of GP, unknown seeds can easily be determined of their age i.e. > 90 % of GP means that the seeds are 1-year old; if the GP is around 60% then, the seeds are of 2-years old. Contrary to this, if the seeds register around 40 % of GP, the seeds must be 3 years old and less than 30 % of GP means, they are 4 or more than 4 years old seeds. Planting geometry in relation to soil type: GBPUA&T, Pantnagar: A minimum of 3x3 m. is required for tractor operation but plant population reduces to around 1200/ha. However, an spacing 6x1 m. has to be tried in which all food crops (Wheat, Mustard, Soybean etc.) can be harvested by combine & also keeping population around 1700/ha. This will gives almost same income from base crop and additional income from Jatropha. Effect of saline & sodic soils on Jatropha curcas CSAUA&T, Kanpur: On the basis of experiments conducted at Regional Research Station, Daleepnagar, Kanpur on sodic land having pH value more than 9.5, ESP >80 and EC >1.30 dSm-1, it is recommended that these lands are not suitable, technically feasible and economically viable for the plantation of Jatropha, as in this case the root development and growth of Jatropha plants gets restricted which leads to the dying of the plant due to harmful ecology. Effect of number and stages of irrigation IMMT, Bhubaneshwar : Number and stages of irrigation: Experiments were conducted on irrigation at IW/CPE ratio and irrigation at critical stages of growth during

15

2010-11. It was observed that irrigation at IW/CPE 1.25 during pre- monsoon period enhances yield (416.16 kg/ ha) up to 43.82 % above control (182.37 kg/ha). It was also found that pre-monsoon irrigation at leaf emergence, flowering, fruiting and seed filling stages @ 70mm/ basin yielded more fruits in comparison to the rain-fed crops. Effect of various fertilizer doses in different soil types GBPUA&T, Pantnagar: The fertilizer scheduling is under study although in practice 1Kg. Vermi compost, 100g SSP was applied per plant. Vermi compost was pretreated with phosphate solubilizing bacteria @ litre per ton of Vermi compost. The fertilizer is applied after pruning in month of Feb.-March. IMMT, Bhubaneshwar: The significant variations were observed in all the treatments of fertilizer application. Plant height (234.47 cm) and number of branches (34.87) were maximum in T1 (N60). Increased number of female flowers was observed in T12 (N50K60) in both the flowering periods. Number of fruits was found highest (197.86) in T3 (K75) in the first flush but it was maximum (393.53) in T1 in the second flush. Similarly the total seed yield (1st + 2nd flush) was found maximum (591.44 kg/ha) in T1 (N60) followed by T5 (N60K75) (502.21 kg/ha). The oil content of seeds showed no significant variation among the treatments, although maximum content (36.74 %) was obtained in T14 (N50P100K60) followed by T1 (N60). Analysis of the results of fertilizer trial experiments indicate that nitrogen seems to have desirable effects on the yield attributing characters and oil content. Although plants receiving N50P100K60 had maximum oil content, the overall yield attributes exhibited only marginal increase over the control plants. On the other hand, plants undergoing N60 treatment showed an increase of about 66.82% in seed yield with comparable oil content. BAU, Ranchi : In fertilizer trial , among 14 treatments tried , maximum height was observed in plants of treatment N15P15K15 followed by 50% FYM and maximum collar diameter was observed in 50% FYM treatment followed by 50% FYM+ TSP. Infestation of Insects and Pests GBPUA&T, Pantnagar: Mite : It affects during young leaf stage. The leaves get shrunken. No damage observed as the leaves recovered with growth. It is controlled by spray of a herbal Drug calnova @ ml per liter. IMMT, Bhubaneshwar Thrips: Thrips attack during cloudy days of monsoon. It sucks the tender leaf sap and spreads viral diseases. It is controlled by spraying Cypermethrin @ 0.1% at 10 days interval up to one month during first appearance of thrips.

16

Scales: Scales attack on Jatropha during March after the plants put forth new growth. They remain adhered at the growing points particularly at the inflorescences and suck the cell sap. The inflorescences dry up gradually and yield reduces drastically. It is controlled successfully by spraying Cypermethrin @ 1.5 ml / l at 15 days interval eliminates the problem. TNAU, Mettupalayam Mealy bug - the common name of insect in Pseudococcidae, a family of scale insects found in moist, warm climates. These insects look like small balls of cotton. They suck the fluids from leaves and stems. In Jatropha, mealy bugs damaged all parts of the plant, especially tender new growing leaves. The infested plants were covered with a white mass of wool like small mealy bugs. Few days after infestation, the plant surface was covered with sticky colourless drops, better known as honeydew. Frequently, sooty moulds (black mould) formed on honeydew secretion on the plant surfaces. The growth of the infested plants thus reduced and shows sicky appearance with distorted stem and growing point deformation and started to shrivel. Leaves were reduced in size, discoloured, wilted and easily drop prematurely. Honey dew secretion invites lot of Ants. These insects also help to the spread of mealy bug from one plant to another which resulted in epidemic status of the insect. To control the mealy bug incidence, the following measures were taken. Before spraying insecticides, the plants were watered to decrease the risk of chemical burning. Different systemic and non systemic insecticides were sprayed at regular interval. Bio formulations were also used to control the mealy bugs. The spray of Imidacloprid (2 ml/Litre of water), Dimethoate (2 ml/Litre of water), Profenofos (2 ml/Litre of water), Veticilium lecanii+ Neem oil +Profenofos (6ml+3ml+2ml/ Litre of water) and Kerosene (1ml/Litre of water) has been found useful for control of mealy bugs.

17

BHU, Varanasi : The following insects were recorded.

Jatropha leaf-cum fruit webber, Pempelia morosalis (Saalm Uller)

Pupa

Adult

Larvae

Nezara viridula

Crysocoris perpureus

Golden flea beetle

Ferrisia virgata (Ckll.)

Leptoglossus natus (Dallus)

18

Occurrence and abundance of arthropod pest species recorded on Jatropha curcas L. during 2010-11
Name of the species and order 1. Hemiptera Scutellera nobilis (Fabricius) Chrysocoris purpureus (Westw.) Ferrisia virgata (Ckll.) Nezara viridula (Linnaeus) Leptoglossus zonatus (Dallus) 2. Lepidoptera Pempelia morosalis (Saalm Uller) Plutella Spp. Achaea janata (Linnaeus) Stomphastis thraustica 3. Coleoptera Myllocerus maculosus (Guer.) Aphthona Spp. 4. Orthoptera Oedaleus senegalensis Krauss Atractomorpha crenulata(Fabricius) 5. Isoptera Odentotermes Spp. 6. Arachnida Brevipalpus phoenicis (Geijskes) Tetranychus urticae Koch Polyphagotarsonamus latus (Banks) Amblyseius lorgoensis (Mum Sum Mean S.D. SEm Diversity index Species richness

177 21 15 17 9 229 303 24 16 10 3 22 30 130

11.80 1.40 1.00 1.33 0.60 15.27 20.20 1.60 1.07 0.67 0.20 1.47 2.00 8.67

4.9289 1.5406 1.2110 1.2037 0.6110 5.6270 4.8607 1.4514 1.1813 0.8692 0.40 1.4079 1.7889 4.9755

1.2726 0.3978 0.3127 0.3108 0.1578 1.4529 1.2550 0.3748 0.3050 0.2244 0.1024 0.3636 0.4619 1.2847

0.5648

0.4424

0.6153

0.5022

2 1

23
22

1.53
1.47 1.07

1.3597
1.40797 1.1813

0.3511
0.3636 0.3050

16 4

0.2933

0.27

0.442

0.1142

19

Among all the insect recorded, 05, 04, 02, 02, 01 species were recorded from insect orders, Hemiptera, Lepidoptera, Coleoptera, Orthoptera and Isoptera, respectively. Whereas from class arachnida, 03 phytophagous and 01 predaceous mite was recorded from Jatropha. Among all the insects recorded, scutellerid bug appeared to be most damaging and the field experiments were carried out to evaluate damage potential of this insect pest. and efficacy of some IPM components against scutellerid bug infesting Jatropha during 2010. The experiment on the damage potential of Scutellera nobilis showed an increase in damage with increasing population. Two bugs/plants did not cause significant damage, while 20 bugs per plant resulted in more significant damage than previous one. Two foliar sprays given at monthly interval revealed that lambda-cyhalothrin 5 EC @ 25 gm a.i./ha and imidacloprid 17.8 SL 100 ml/ha were most effective treatments followed by carbosulfan 25 EC @ 250 g a.i./ha and monocrotophos 36 SL @ 500 g a.i./ha (Table 2). Next effective treatments were spinosad, endosulfan, Beauveria bassiana, Metarhizium anisopliae resulted in moderately reduction in pest population. All Insecticides showed maximum reduction of bug population during 3rd days after treatments in both sprays, whereas in case of botanical and bioagents maximum reduction was noticed 7th days after treatments in both sprays. Chlorpyriphos 50 EC @ 250 gm a.i./ha and neem oil (2%) were found least effective and exhibited minimum reduction in bug population as compared to other IPM components.

20

J.

Diseases Infestation:

GBPUA&T, Pantnagar ( Rizoctonia, Fusarium & sclerotium complex) : It affects during wet months of July Aug. Root zone rots first then color zone rots killing the whole plant. The disease is predisposed by water stagnation. In upland no diseases appeared after 3 Month of plant age. It is controlled by dipping of seedlings & cutting in a solution of Bavistin+ viatavax @ 1g/lt. controls the disease. The soil is to be treated with trichoderma @ 2Kg/ acre and pusdomonas @ 1Kg / acre before plantation. MPKV, Rahuri: Fusarium wilt. The infestation spreads during summer season and wilting and drying of plant occurs during rainy season. It is controlled by application of insecticide Phorate @100 g/plant, drenching of Bavistin @ 0.2 % or Blitox 0.25 %, treatments of soil drenching with Copper Oxychloride (Blitox) @ 0.25% and Carbendazim (Bavistin) @ 0.20%. Studies on etiology, epidemiology and management of diseases UAS, Dharwad : Survey conducted in different districts of Karnataka indicated that different diseases were observed in different localities and important diseases observed were leaf blight, root rot, powdery mildew, stem canker, anthracnose. Root rot was observed for the first time in Karnataka. Pathogens were isolated by tissue isolation method and pathogenicity was proved. Seed rot caused by Sclerotium rolfsii was observed in Dharwad. Experiments have been successfully completed on following aspects. Optimum temperature for the growth of Rhizoctonia sp. causing root rot of Jatropha was identified. (30 C) Best medium (V-8 juice broth ) and Incubation period required for the optimum growth of Rhizoctonia sp. was standardized (12days). Best medium (PDB) and Incubation period required for the optimum growth Sclerotium rolfsii was identified Strategies were developed to manage sclerotium wilt of Jatropha. Botanicals were evaluated against Colletotrichum gloeosporioides causing anthracnose of Jatropha. This pathogen on fruits can reduce the per cent seed germination. Biochemical changes due to infection by foliar pathogen was studied. Colletotrichum gloeosporioides has resulted in 20.43% reduction in reducing sugar, 27.54% increase in total phenol and 61.1% in OD phenols. Studies are conducted to know the host range for Sclerotium rolfsii and Rhizoctonia sp. Experiment conducted for management of root rot of Jatropha indicated that disease can be effectively managed by soil drenching with carbendazim @ 0.1%

21

Other techniques are standardized by some research institutes PAU, Ludhiana Standardization of irrigation and nutrient requirements for Jatropha The irrigation had significant effect on the plant height of Jatropha. The minimum height was observed in unirrigated plants (3.54 m) which increased significantly to 3.79 m at I3 level (7-8 irrigations in a year) of irrigation. At this irrigation level, irrigation is applied at an interval of about 15-20 days in summer and 2.5-3 months in winters. Amongst the fertilizer levels, the minimum height (3.45 m) was observed in unfertilized plants and maximum (3.91 m) was observed in F5 level. Irrigation and nutrients had significant effect on the collar girth of branches. Minimum girth (25.9 cm) was observed in unirrigated plants, which increased significantly with the application of irrigation to plants. Among the nutrient levels, the minimum girth (24.9 cm) was observed in F1 (unfertilized) treatment and maximum (29.5 cm) was observed in F5 treatment. The interaction of irrigation and nutrient was found to be significant. The seed yield of Jatropha can be enhanced significantly by application of 7-8 irrigations (I3) in a year from that of unirrigated plants. The irrigation application at CPE of 120 mm produced highest seed yield after 4 year growth of plants. Flowering stage is critical for irrigation application. Better flowering and subsequent higher seed production is observed under irrigated conditions. Amongst the different fertilizer levels, the minimum (302/g plant) seed yield was observed in unfertilized plants and maximum (369/g plant) at F5 (1st-2nd-3rd-4th-5th years: N60P20-N60P20-N75P30-N75P30-N90P40 g/plant) level of fertilizers. The interaction effects between irrigation and fertilizer were found to be non-significant. Development of a database on Tree-borne Oilseeds in India A project on development of a database on TBOs is being implemented by Indian Council of Forestry Research and Education (ICFRE), Dehradun with the following objectives: 1. Estimation of state-wise acreage under cultivation of various TBOs covering individual farmers, Government Organizations, research institutions, etc. 2. Estimation of category wise land under TBO cultivation and its effect on land use change State-wise. 3. Design of a Relational Database Management System (RDBMS) for data storage and retrieval. Snow-ball sampling for survey in farmers field is being followed with information obtained from district agriculture offices and agricultural boards apart from boards like bio-fuel boards, etc. For organizations, questionnaires have been canvassed directly. For natural distribution, State Forest Departments and Agriculture Departments 22

have been approached for information and survey is also being carried out in hotspots. Sampling is being resorted to for estimating the acreage in plantations and avenue planting, especially in cities. A sampling approach has been developed for assessment of line plantations and acreage which is being tested and assessed. Historical data of landuse prior to TBO cultivation is being collected. Analysis will reveal the land-use change and land use patterns of TBO cultivation. A RDBMS is being designed. The first version has been designed and is under testing. Modifications are being carried out to achieve a consistency with the data being recorded. Homogenizing units and factors is also being undertaken. Data from Himachal Pradesh, Karnataka, Madhya Pradesh, Tamil Nadu, Jharkhand, West Bengal and some specific North-eastern states has been collected and is in the process of collation. Data from other areas is being collected. ICFRE has prepared a format for survey and separate Questionnaire for each TBO under NOVOD Board sponsored project. A sample secondary information collation form has also been developed containing information of statewise name, natural distribution, flowering & collection time, oil per cent, insects, pests & disease management, major uses etc. The sample snap shots of the first version of the database has been developed and is under testing. The following information have been identified for returning in a query based DBMS Front end: 1. Type of planting stock used (with estimated percentage of each) 2. Source of planting stock (with estimated percentage of each) 3. Type of plantation (with estimated percentage) 4. Area planted 5. Estimated expenditure per unit area, species-wise 6. Funding agencies (if any) and estimated subsidies 7. Land use change with estimated percentage in each category 8. Availability of marketing channels with utilization (excluding market dynamics) 9. Estimated periodic expenses in maintenance (species-wise) 10. Information on storage aspects of seeds (duration, shelf life, method, etc.) 11. Main identified problems in TBO cultivation and marketing as perceived by planters 12. Irrigation regimes 13. Survival percentage species wise and state wise 14. Characteristics of soil on which TBO plantation has been done and the success information 15. Silvicultural operations carried out along with benefits/losses of such operations 16. Major fertilizers used species wise 17. Major insect pests and their management 18. Major diseases of TBOs and their management 19. Estimated mandays invested in raising TBO plantations and maintaining them 20. Estimated quantity of seeds collected species wise and state wise 21. Information on industry consuming material from the identified 14 TBOs The database has been designed with the desired output and contents of the database comprise of Species, Distribution, Insect, Pests, Utilization, Estimated oil content, State / UT and Status of research. The salient features of front end (with mapping on back-end) are adding data to the database by authorized person; to select species by check boxes for

23

information; to select the information desired to be returned by the database and to select the state / UT on which information is sought, include one option for selecting total. The information may require a username/password for accessing. Following information may be desirable from the use point of view: Geographical distribution Core areas Utilization Oil content Average size of seeds, their number per kilogram, etc., as available from FTSL, FRI, Dehradun Acreage under the crop Mixed with any crop in agro-horti-fodder system Important insect pests and their effect Important diseases and their management Current status of research Land-use prior to planting TBO and after planting the TBO Total land-use change in sleeted state / UT or country Hotspots of the selected TBO state-wise and total

24

Hybridization in Jatropha curcas

The hybridization programme is being implemented in T.N. (TNAU), Haryana (CCS, HAU), Maharashtra (MPKV), Uttrakhand (GBPUA&T), U.P (NRCAF) and Gujarat (SDAU). The progress made by the institutions concerned are summarized as follows : Tamil Nadu Agricultural University, Mettupalayam: The inter specific hybridization between different species of Jatropha viz., J.curcas, J.villosa, J.multifida, J.podagarica, J.integerrima, J.maheswari, J.glandulifera and J.gossypifolia have been attempted to transfer desirable traits to the cultivated species of Jatropha in order to enhance the existing genetic variability. The crossed seeds are being raised to study the compatibility for desirable traits and for further selection. Back crossings were carried out on available clones with the hybrids (F1) as female parents and J. curcas as male parent and generated BC1F1 and subsequently BC2F1, BC3 F1 and BC4F1 populations. Table.3 Back crossing before pruning
Sl No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Crosses 3-66-19-5 x TNMC 7 3-97-5-4 x TNMC 7 3-97-5-6 x TNMC 7 3-66-19-4 x TNMC 7 3-95-7-5 x TNMC 7 3-66-17-2 x TNMC 7 3-82-7-5 x TNMC 7 3-75-4-5 x TNMC 7 3-66-19-6 x TNMC 7 3-66-25-1 x TNMC 7 3-82-7-5 x TNMC 7 3-66-59-4 x TNMC 7 3-97-5-4 x TNMC 7 3-66-35-1 x TNMC 7 3-66-36-4 x TNMC 7 3-66-8-c13-3 x TNMC 7 3-75-4-3 x TNMC 7 3-66-52-3 x TNMC 7 3-66-25-2 x TNMC 7 3-83-4-6 x TNMC 7 3-66-C18-4 x TNMC 7 3-56-5-NT 5 x TNMC 7 3-66-2-C12-1 x TNMC 7 3-66-2-C 12-3 x TNMC7 3-7-10-4 x TNMC 7 3-75-9-5 x TNMC 7 No of flowers crossed 4 9 10 19 9 16 7 10 9 8 7 5 9 18 10 17 19 13 8 4 4 9 7 5 9 10 No of fruits obtained 2 5 6 13 6 12 5 6 3 5 4 3 6 13 8 12 13 6 4 2 2 5 4 3 6 8 Percentag e of fruit set (%) 50 55 60 68 66 75 71 60 33 62 57 60 66 72 80 70 68 46 50 50 50 55 57 60 67 80 No of seedlings obtained 5 12 12 24 12 28 9 9 6 12 9 6 11 30 17 28 27 12 9 4 4 12 9 9 12 19
Percentage of germination (%)

83.3 80 66.7 61.5 66.7 77.8 60 50 66.7 80 75 66.7 61.1 76.9 70.8 77.8 69.2 66.7 75 66.7 66.7 80 75 100 66.7 79.2

25

Sl No 27 28

Crosses 3-66-29-1 x TNMC 7 3-56-13-1 x TNMC 7

No of flowers crossed 10 7

No of fruits obtained 6 5

Percentag e of fruit set (%) 60 71

No of seedlings obtained 12 9

Percentage of germination (%)

66.7 60

CCS HAU, Hisar: Genetic improvement through hybridization To develop high yielding and cold resistant genotypes in Jatropha curcas, inter and intraspecific hybridization were attempted. The cross compatibility study among different Jatropha species was conducted. Five species of Jatropha viz., J. curcas, J. gossypifolia, J. multifida, Jatropha integerrima and J. podagrica were crossed in all possible combinations. The preliminary results of this study showed that four species viz. J. curcas, J. gossypifolia, J. multifida and J. podagrica were found cross incompatible to each other. However, the crosses were successful when J. multifida was used as male () and J. curcas as female () parent. A strong reciprocal compatibility was also found between J. curcas and J. integerrima and successful hybrids between J. curcas and J. integerrima were developed. The seeds so obtained from this cross were sown in earthen pots, filled with potting media, the successful germinated hybrid plants were maintained and transplanted in the field for further breeding programme. These hybrids showed vigorous growth and earliness in comparison to both the parents in nursery as well as in field after transplantation. The hybrid plants were critically observed for reproductive parameters and a lot of variability was observed in morphological characters and inflorescence pigmentation. The back crosses were also attempted by pollinating J. curcas (selected plants) with the pollen of hybrid plants (F1). However, the reciprocal back crosses were not successful due to incompatibility of hybrid female flowers. Moreover, the pollen infertility in hybrid plants was observed and there was poor fruit setting even in open pollinated flowers. A wide variability was recorded in seed morphological characters of F1, BC1 and J. curcas (female parent).

The seeds so obtained from different population were raised in nursery. The F2 plants showed very slow growth as compared to BC1 and J. curcas plants in nursery. BC1 plants also showed intra-population variability in growth and reproductive parameters. During current flowering season back crosses were attempted to produce BC3 population. Self and open pollinated seeds were also collected for subsequent study. Increase of femaleness in Jatropha inflorescence To increase the female flowers in J. curcas inflorescence, the growth hormone GA3 and Etheral (generally used for fruit ripening) were used at different concentration levels (25 ppm to 250 ppm). The preliminary results were not encouraging, as different treatments reduced the period of panicle and floral bud development followed by high 26

flower drop. In the coming flowering season, the experiment will continue at lower concentration levels of sex changing growth hormones.

Growth variability in hybrids, BC1 and parent( J.curcas)

Fig. 4: Morphological variability hybrids, BC1 and parent( J.curcas)

Hybrids Population of Inter-Specific crosses between J.curcas and J.Integerrima The hybrids between Jatropha curcas and J. integrrima were developed during year 200708 . The seeds were harvested and raised in polyhouse of Forestry department during first fortnight of Feb. 2008 and transplanted in research field at the end of September 2008 . Majority of the hybrids (F1) started flowering at the age of one year . The significant variability was observed among different hybrids with respect to morphological and reproductive parameters . The bud initiation started at the end of June and continued up to September . The average number of buds exhibited a wide variability ranging from 42.13 to 82.40. Morphological and Reproductive studies of Jatropha curcas Hybrids
Sr.no. Date of buds intiation 29/6/10 30/6/10 30/6/10 30/6/10 30/6/10 30/6/10 12/7/2010 12/7/2010 12/7/2010 12/7/2010 AV.No.of inflorosence 15 5 12 10 11 20 12 31 29 12 AV.No.of buds AV.No.of female flower 11.50 9.80 4.83 6.20 5.82 4.85 5.08 4.48 6.59 6.00 AV.No of male flower 110.63 72.60 61.00 65.90 50.91 40.10 42.08 38.00 52.48 52.00 Female male Ratio 1:10 1:7 1:12 1:10 1:8 1:8 1:8 1:8 1:8 1:9 % Fruit Set/ infloroscence 17.66 11.89 7.34 8.60 10.26 10.79 10.78 10.64 11.15 10.34

H5 H9 H13 H24 H 15 H 26 H 37 H18 H 29 H 34

65.13 82.40 65.83 72.10 56.73 44.95 47.17 42.13 59.07 58.00

27

Sr.no.

Date of buds intiation 12/7/2010 12/7/2010 18/7/10 19/7/10 19/7/10 26/7/10 26/7/10 26/7/10 30/7/10 2/8/2010 13/8/10 18/8/10 27/8/10 27/8/10 27/8/10 27/8/10 27/8/10 27/8/10 27/8/10 27/8/10 27/8/10 1/9/2010 1/9/2010 1/9/2010 3/9/2010 3/9/2010 13/9/10 13/9/10 13/9/10 13/9/10 Mean Range CD

AV.No.of inflorosence 8 23 11 6 16 4 11 8 11 6 4 2 3 2 10 2 5 3 6 5 5 1 5 3 1 2 2 2 1 1 8.15 1--31 6.39

AV.No.of buds

AV.No.of female flower 6.13 6.74 6.55 8.67 7.81 5.75 7.36 7.13 7.18 5.50 4.75 8.00 12.33 7.00 8.00 6.00 8.00 7.00 8.83 8.40 5.60 8.00 6.80 10.67 13.00 4.50 7.00 5.5 5.00 10.00 7.25 4.48--13.00

AV.No of male flower 50.50 45.04 49.45 60.00 52.81 52.00 52.73 49.00 48.45 36.83 41.50 64.00 50.00 41.50 57.80 43.50 47.00 72.67 54.33 43.40 42.20 45.00 53.80 63.67 67.00 46.50 56.00 71.00 52.00 61.00 53.91 36.83-110.63

Female male Ratio 1:8 1:7 1:7 1:7 1:7 1:9 1:7 1:7 1:7 1:7 1:9 1:8 1:5 1:6 1:7 1:7 1:6 1:10 1:6 1:5 1:7 1:6 1:8 1:6 1:5 1:10 1:8 1:13 1:10 1:6 1:5-1:13

H 10 H 21 H 32 H23 H 14 H 27 H 36 H 17 H 28 H 39 H 30 H 31 H 12 H 33 H 11 H7 H 16 H6 H 20 H 19 H1 H4 H 22 H8 H 35 H 25 H2 H 38 H 40 H3

56.63 51.78 56.00 68.67 60.63 57.75 60.09 55.38 55.64 42.33 46.25 72.00 62.33 48.50 65.80 49.50 55.00 79.67 63.17 51.80 47.80 53.00 60.60 74.33 80.00 51.00 63.00 76.5 51.00 71.00 59.08 42.13-82.40

% Fruit Set/ infloroscence 10.82 13.01 11.69 12.62 12.89 9.96 12.25 12.87 12.91 12.99 10.27 11.11 19.79 14.43 12.16 12.12 14.55 8.79 13.98 16.22 11.72 15.09 11.22 14.35 16.25 8.82 11.11 7.19 9.80 14.08 12.11 2.64-19.79

It was interesting to note that in hybrids number of female flowers increased as compare to parents. In hybrids female and male ratio was observed from 1:5 to 1:13. However, parent fruit set per inflorescence was very poor which ranged from 2.64-19.79. Further, the fruit size was also found small in hybrids comparison to J. curcas.

28

G.B.Pant University of Agriculture & Technology, Pantnagar, Uttrakhand Hybridization Programme (A) Inter varietals hybridization There was positive and significant correlation exist between seed yield / plant and number of cluster and Fruits / plant , while the number of cluster and fruit / plant had significant and positive association with plant height and number of branches. Thus among the present day genotypes the tall plants with more number of branches are desirable for good yield. MPKV, Rahuri Inter varietal Hybridization Programme in Jatropha: In inter varietal hybridization programme, four crosses were studies for eleven characters. Among all the crosses the cross RJ-127xNOVOD was found promising for seed yield plant.1 and oil content. In this cross B1 generation recorded the highest seed yield per plant (425 g) followed by B2 (380g) and F1 (345g). Similarly in cross RJ127xRJ-89 F1 (361g) generation recorded highest seed yield followed by B1 (326g) and B2 (281.7g) generations.

29

Tissue Culture
Development of an efficient protocol for in vitro multiplication (Tissue culture) in Jatropha The application of advanced tissue culture techniques like protoplast fusion, gene transfer, inducts of somaclonal variations, in vitro mutagenesis, cell culture and subsequent plant regenerations may be opened a new awareness in jatropha improvement. In vitro technique is a highly sought technique to create novel variability in especially cross pollinated tree crop like jatropha. Somaclonal varieties that occur in successful in vitro cultivars may give desirable variants with the defects rectified. Hence tissue culture technique may be useful and effective for getting superior quality vegetable oil from jatropha in future. Keeping in view of above, the efforts are being made in Punjab (PAU), Haryana (RRS CCS HAU, Karnal), Orissa (IMMT), Rajasthan (MPUA&T), Delhi (DU), Gujarat (SDAU) and T.N. (TNAU). Punjab Agricultural University, Ludhiana: Establishment of hedge orchard of elite genotypes A hedge orchard of elite genotypes (BNK-1, BNK-2, BNK-5, BNK- 19, BNK-20, and MPAU-4) was established to provide the explant material for the present investigations on micropropagation. For this purpose, vegetative propagules of elite genotypes were procured from Regional Research Station (CCS HAU) Bawal.

Standardization of sterilization procedure

Maximum culture establishment was obtained on treatment combination with bavistin ( 0.2%) for 45 minutes + HgCl2 (0.1%) for 4 minutes for shoot apices and nodal explants collected from glass house and field grown plants. Higher concentration of HgCl2 resulted in yellowing and killing of explants. Standardization of media for shoot regeneration and multiplication Explants (nodal segments and apical shoots) were collected and inoculated on Murashige and Skoog Medium (1962) supplemented with different concentrations of BAP, IAA and GA3. Following hormone combinations were tried for shoot regeneration: (i) MS (ii) MS+BAP(1.0mg/l) (iii) MS+BAP(2.0mg/l) (iv) MS+BAP(2.0mg/l) +IAA(2.0mg/l) (v) MS+BAP(2.0mg/l) +IAA(2.0mg/l) + GA3(3.0mg/l)+ Glutamine (50.0mg/l) (vi) MS+BAP(1.0mg/l) + IAA(0.25.0mg/l)

30

Differential response was observed for shoot regeneration on various media compositions. Among the various combinations tried, MS + BAP (1.0 mg/l) was found to be best combination for shoot regeneration for apical buds and nodal segments (Plate 2). In vitro regenerated shoots were further subcultured on different media for shoot multiplication. Maximum shoot multiplication (Plate 3A & B) was observed on MS+BAP(1.0mg/l) + GA3(7.0mg/l)+ Glutamine (50.0mg/l).

Plate 2 Shot regeneration on MS + BAP(1.0 mg/l)

(A)

(B)

Plate 3 Multiple shoot formation of different genotypes on MS + BAP (1.0mg/l) + GA3 (7.0mg/l) + Glutamine (50mg/l) Rooting of in vitro regenerated shoots 31

In vitro regenerated shoots were cultured on half-strength MS media supplemented with IBA (2.0 and 3.0 mg/l) to induce rooting. Root induction was obtained on MS + IBA (2.0mg/l) and completed within three to four weeks (Plate 4). The plantlets with well developed roots and shoots were transferred to polythene bags containing mixture of sand and soil and kept under glasshouse conditions (Plate 5).

Plate 4 Rooting of shoots

Plate 5 Plantlets shifted to glasshouse

Response of different genotypes to in vitro culturing An experiment was conducted to study the response of different genotypes for shoot regeneration under in vitro conditions by using genotypes namely BNK-1, BNK-2, MPAU-4, BNK-5, BNK-20, BNK-19. Explants collected from field grown plants of these genotypes were cultured individually on media MS + BAP (1.0mg/l).Maximum shoot regeneration in nodal segments (82.6%) and apical shoots (85.1%) was recorded in genotype BNK-5. Regenerated shoots were sub-cultured on MS+BAP (1.0mg/l) + GA3 (7.0mg/l) + Glutamine (50.0mg/l) for shoot multiplication. BNK-5 attained the higher shoot multiplication as compared to other genotypes.

Effect of season on culture establishment To study the effect of season on culture establishment, explants (apical shoots and nodal segments) collected from glasshouse and field grown conditions were cultured on MS + BAP (1.0mg/l) during different seasons (spring, summer, monsoon, autumn and winter). Maximum culture establishment was observed during spring, summer and monsoon season (70.0 to 80.0%). Culture establishment was reduced during autumn (55.5%) and was poor during winter season.

32

Effect of different carbon sources on culture establishment In an effort to reduce the cost of in vitro regenerated plants, a trial was conducted to find the cheap substitutes of sucrose for shoot regeneration. For this purpose, four carbon sources were tried individually viz., normal table sugar, branded table sugar, branded sugar cubes and sucrose, all used at the same concentration (3 %). All carbon sources were tried at the same media i.e. MS + BAP (1.0mg/l). Differential response was observed among various carbon sources for shoot regeneration. Highest shoot regeneration was observed on media with sucrose as carbon source. Number of days taken for shoot formation was also less on media supplemented with sucrose as compared to other carbon sources tried. Very less callus formation was observed on standard sucrose while it was moderate in sugar cubes and branded sugar and high on normal table sugar. Comparison of agriculture grade chemicals with laboratory grade chemicals In an another experiment to reduce the cost of in vitro regenerated plants, an effort was made to explore the possibility of using agricultural grade chemicals instead of laboratory grade chemicals. For this purpose, different agricultural grade chemical fertilizers viz., zinc sulphate, boron, manganese sulphate, potassoum nitrate, ferrous sulphate and magnesium sulphate were procured from the local market. MS media were prepared using these agricultural grade chemical fertilizers instead of laboratory grade chemicals. No culture establishment was observed with the addition of these fertilizers due to drying of explants probably due to toxic effects of these chemicals to the tender tissues of explants. CCS HAU Regional Research Station, Karnal: Surface Sterilization: Explants were obtained from Apical/Axillary buds collected from Elite plants established in the field. Buds were surface sterilized with Teepol washing followed by Bavistin. This was followed by treatment with 0.1% Mercuric Chloride for 2.30 min and rinsed 4 times with sterile double distilled water. Yellowing problem Initiation of cultures from the nodal explants did not pose a major problem. During initiation, the explants did not show any leaching or browning of tissues, but later on cultures showed symptoms of yellowing. The best way to overcome the Yellowing problem encountered after 1 month of culturing is by frequent sub-culturing, reducing the concentration of MS media, reducing sucrose conc. and increase in calcium chloride level as compared to adding 10gm/l fructose addition. Also various additives like Glutamine (Glu) 50mg/l, Adenine Sulphate (AS) 25mg/l, Citric acid 25mg/l, ascorbic acid 25mg/l, Silver nitrate (SN)1- 15 mg/l and Arginine (Ar) 30mg/l were added in different combinations. As per previous various reports, lowering the concentration of BAP

33

(~1mg/l or less than) is best for proliferation in Jatropha curcus, to prevent yellowing & defoliation of leaves. When explants were inoculated in MS media+ Glu+ AS containing: BAP 0.5mg/l, 1.0mg/l & 2.0mg/l), it was observed that media having BAP 0.5mg/l, & 1.0mg/l showed good proliferation without any callus formation & yellowing of leaves as compared to other media tried. Shoot regeneration through nodal segments & apical buds A total of 20 Combinations attempted so far during 2010-2011. Nodal segments and apical buds were collected during different months of the year, reared from coppice shoots & cultured on MS media supplemented with different concentrations of BAP, IAA, IBA, TDZ along with the GA3. Differential response of explants apical/axillary was reported for shoot regeneration on different media compositions. Among the various combination tried, the best response of apical/axillary explants was observed when cultured in media (MS+ BAP 1mgl/l +IAA 0.25mg/l). Multiple shoot induction For multiplication of shoots, in vitro regenerated shoots were cultured on MS media with lower concentration of cytokinins, auxins & addition of additives to prevent from defoliation of leaves. Explants transferred to Media ( MS+ BAP 0.25mg/l+ IAA 0.1mg/l + Glu+ AS) after 15-20 days showed multiple shoots. Again following media combinations of MS & MS were tried (ten repeats of each media) to reduce callus formation & get multiple shoots induction: (i) Media MS +BAP 1.0mg/l + IAA 0.25mg/l is better than MS +BAP 1.0 mg/l + IAA 0.5 mg/l. (ii) Media MS + BAP 1.0 mg/l + IAA 0.1 mg/l is better than MS + BAP 2.0 mg/l + IAA 0.2 mg/l (iii) Multiple shoots also observed in MS Media (BAP 1.5mg/l + IBA 0.5mg/l+ AS + Ar). We Also tried MS media containing BAP 1.5 mg/l with IAA 0.1 mg/l & 0.2 mg/l for comparison (Experiment in progress). (iv) Media MS+ BAP 2mg/l + TDZ 0.2mg/l+ AS+ Glu, MS+ TDZ 0.2mg/l + AS & MS+ BAP 2mg/l + IAA 0.2mg/l+ AS showed good proliferation & gave 2 to 3 multiple shoots from explants occasionally with slight callus formation. (v) Response shown to different level of Silver nitrate (5, 10 & 15 mg/lt) supplement with MS media +BA 1.0mg/lt, as result 5mg/lt of SN was best & helped in better proliferation.

34

Rooting of excised shoots: The in vitro raised shoots from apical/axillary bud failed to root on hormone free media. Best response was obtained in media containing MS+ IBA 1mg/l.
Somatic embryogenesis from leaf disc& Cotyledonary leaves: For development of callus and somatic embryogenesis fresh & young leaf explants were taken from adult plants & Cotyledonary leaves from freshly germinated seed. Both the explants were kept under tap water for washing, followed by 0.1% Mercuric Chloride for 2 mins. After drying, these were dissected into pieces of 1cm size & cultured them into callus induction media. Cotyledonary leaves initiated good callus in MS+ 2, 4-D 1mg/lt+BAP 4mg/lt. leaf explants showed different response such as: (i) Media1:- MS+ No response was observed in leaf parts. (ii) Media2:- MS +BAP-5mg/l -slight response was in leaf parts after a long time. (iii) Media3:- MS+ BAP 2mg/l + TDZ; 0.2mg/l- There was better response in leaf parts, but browning took place. (iv) Media4:- MS+ BAP-2mg/l+ 2, 4-D-1mg/l & BAP-4mg/l+ 2, 4-D-1mg/l - There was better response to formed, good amount of form callus (white granular structure).

Further Callus via leaf explants put into media containing BAP 2mg/l + TDZ 0.2mg/l+ AS+ Glu for induction of regeneration shoots, and it formed multiple shoots from callus.

Institute of Minerals & Materials Technology, Bhubaneshwar: Standardization of media to control contamination The main problem faced during the in vitro culture of Jatropha explants is contaminations. The contaminants could be of endophytic or epiphytic origin. Inducible endophytes are most frequently encountered during in vitro culture, since in nature, they are epiphytes of the parent plant .The use of anti-microbial agents (anti-bacterial as well as anti-fungal) to control contamination is the preferred method in plant tissue culture. However their indiscriminate use may lead to phytotoxicity problems. Hence, proper concentration and combination is a prerequisite for successful regeneration of plants under in vitro. We have formulated a media comprising of an antibiotic (rifampicin) and an anti-fungus (Bavistin) chemical which reduced the infection rate to 10 20 % as against the non antibiotic and anti-fungus media which showed 80-90 % contamination. Out of various combination and concentration tested, MS + 1.5 mg/l Zt + 35 mg/l Rifampicin + 1.5 g/l Bavistin yielded best response. Both rifampicin and bavistin was filter sterilized and added to the medium.

35

Plant regeneration from Shoot tip and axillary bud culture: In order to get juvenile shoots, stem cuttings from Baramunda clone were collected and rooted under a green-house chamber. Shoots produced from such cuttings were severed from which shoot tip and axillary bud explants were collected and cultured on to MS basal medium supplemented antibiotic and antifungus solution with different concentrations and combinations of BA, Kn, Zt and IAA. Multiple shoot induction was achieved in the media consisted of MS+ Zt 1.5 mg/l + IAA 0.25 mg/l + 35 mg/l Rifampicin + 1.5 g/l Bavistin, wherein 5 6 shootlets could be obtained from a single explant. Plant regeneration from hypocotyl explants Multiple shoots were induced on hypocotyl segments reared from in vitro germinated seedlings. MS bsal medium supplemented with BA and kin either alone or in combination with IAA were tried. A combination of 7.05 M kin and 1.425 M IAA resulted in the highest shoot number production. In such combination shoot bud ranging from 10 22 numbers (average 12.1) was produced per explant. The regenerated microshoots were transferred to root induction medium containing half-strength MS salts supplemented with 9.8 M IBA. Rooted plantlets could be acclimatized under a green house condition and the plantlets showed 70 % survival. Plant regeneration from leaf explants via direct organogenesis For induction of callus on leaves tissue, leaves from in vitro grown cultures were obtained and the leaf segments (1 cm dia) were cultured on to various media composition consisting of MS basal medium supplemented with 2,4-D either alone or in combination with Zt or BA or Kn. Shoot bud formation was obtained when they were cultured on to MS + 1.0 mg/l BA + 1.0 mg/l 2,4-D. About 10-12 shoot buds were emerged from each segments without any intervening callus. Then, they were transferred to shoot elongation media containing half-strength MS and 0.5 mg/l BA + 0.5 mg/l 2,4-D. The elongated shoots were rooted on the medium contained MS + 1.5 mg/l IBA. Induction of somatic embryogenesis Leaf discs (1 cm diameter) obtained from in vitro plants were cultured on to MS basal media supplemented with different concentration and combinations of 2,4-D, BA and kn. Profuse callusing was achieved on MS basal medium supplemented with 2,4-D 2.0 mg/l + BA 0.5 mg/l. After two subcultures on the same medium, the proliferated calli were transferred to somatic embryogenesis induction media containing MS + 0.5 mg/l BA + 0.25 mg/l 2,4-D + 0.2 mg/l GA3. The experiment is still continuing.

36

University of Delhi, Delhi: Establishment of aseptic cultures of zygotic embryos excised from seeds of Jatropha curcas: In order to obtain aseptic seedlings, zygotic embryos excised from 48 hour soaked seeds (after 30 mins of sterilization with 50 mg/l Cefotaxime and 2% bavistin) of the elite clone of J. curcas were inoculated on MS medium supplemented with various concentrations (0, 10, 25, 50, 100 & 200 mg/l) of cefotaxime, antibiotic. The percentage of aseptic seedlings observed was dose dependant. At 0, 10, 25 and 50 mg/l Cefotaxime, the percentage of aseptic seedlings observed were 5, 17, 28, 52 and 100, respectively (Table 1). The nature of the aseptic seedlings was green and healthy at these concentrations. The optimum percentage (100%) of aseptic seedlings was achieved on 100 mg/l cefotaxime augmented medium. At higher concentration of cefotaxime i.e. 200 mg/l, though the maximum percentage of aseptic seedlings was achieved but the conditions of the seedlings were abnormal. Table 1: Percentage of aseptic seedlings of zygotic embryos excised from mature seeds on MS medium (1% sucrose) augmented with different concentrations of Cefotaxime, antibiotic after two weeks of inoculation.

Sl. No. 1 2 3 4 5 6
*

Cefotaxime (mg/l) 0 10 25 50 100 200

Mean of 12 explants.

% Aseptic seedlings

Nature of seedlings

5 17 28 52 100 100

Green and normal Green and normal Green and healthy Green and healthy Green and healthy Green and stout

Optimization of media for zygotic embryo germination: Zygotic embryos excised from 48 hour soaked seeds (after 30 mins of sterilization with 50 mg/l Cefotaxime and 2% bavistin) of the elite clone of J. curcas were inoculated on various culture media viz. MS, 2MS, MS, B5, Knops and WPM supplemented with 1% sucrose and 100 mg/l of Cefotaxime. The percentage seed germination response observed in the different culture media was variable and optimum germination was achieved on Knops medium with a germination response of 70% followed by MS medium with a germination response of 60% after two weeks of inoculation (Table 2). The nature of the seedlings observed at both this culture medium

37

was normal with normal radicle and roots. The percentage germination response recorded on double strength MS, half strength B5 and WPM medium were 45, 45, 55 and 50%, respectively. The nature of the seedlings observed on this culture medium was abnormal either with their radicals stunted or without roots. Table 2: Germination response of zygotic embryos excised from 48 hours soaked mature seeds of Jatropha curcas on different culture media after two weeks of inoculation.
Sl. No.

1 2 3 4 5 6
*

Culture % Seed % medium germination Aseptic (augmented seedlings with 100 mg/l Cefotaxime) MS 60 83 2MS MS B5 Knops WPM
Mean of 12 explants.

Nature of seedlings

45 45 55 70 50

92 92 83 92 75

Seedling with normal radicle and roots Seedling with stunted radicle and improper roots Seedlings with stunted radicle having single root Seedlings with stunted radicle without roots Seedlings with normal radicle and roots Seedlings with stunted radicle and roots

Establishment of aseptic cultures of shoot tips excised from mature garden raised plants: Shoot tip explants were excised from two-year-old garden raised elite plants of J. curcas and after proper sterilization and treatment with 100 mg/l cefotaxime and 2% bavistin for 15 minutes, were inoculated on MS + 10 mg/l BA + 5 mg/l Glutathione + 10 M AgNO3 medium supplemented with various concentrations (0, 10, 25, 50, 100 & 200 mg/l) of Cefotaxime (Table 3). The percentage of aseptic cultures observed was dose dependant. At control, i.e. on MS medium without cefotaxime, aseptic cultures of shoot tip explants could not be raised, the percentage of aseptic cultures being 0%. Cefotaxime at 300 or 500 mg/l, respectively augmented to MS + 10 mg/l BA + 5 mg/l Glutathione + 10 M AgNO3 medium, displayed the maximum (100%) percentage of aseptic cultures after 3-weeks of culture. However, the aseptic explants gradually turned brown after three weeks of culture. Cefotaxime at 200 mg/l induced 94% aseptic cultures that were green and healthy. At 50 and 100 mg/l Cefotaxime, the percentage of aseptic cultures recorded was 70 and 78%, respectively. The nature of the shoot tip explants was green and healthy at these concentrations.

38

Table 3: Depicts the optimum dose of cefotaxime antibiotic supplemented to MS + 10 mg/l BA + 5 mg/l Glutathione + 10M AgNO3 medium for raising aseptic cultures of shoot-tip explants of Jatropha curcas. Sl. Cefotaxime No. (mg/l) 1 0 2 3 4 5 6
*

*Percentage of Aseptic Nature of explants cultures ( % ) 0 Infected at the basal portion 70 78 94 100 100 Explants green Explants green Explants green and proliferating Explants turns brown Explants turns dark brown

50 100 200 300 500

Mean of 12 explants.

Establishment of aseptic cultures of nodal explants excised from garden raised plants: Nodal explants were excised from 2-year-old garden raised elite plants of J. curcas and after proper sterilization and treatment with 100 mg/l Cefotaxime and 2% bavistin for 15 minutes were inoculated on MS + 10mg/l BA + 5 mg/l Glutathione + 10M AgNO3 medium supplemented with various concentrations (0, 50, 100, 200, 300 & 500 mg/l) of Cefotaxime (Table 4). The percentage of aseptic cultures observed was dose dependant. At control i.e. on MS medium without cefotaxime, the percentage of aseptic cultures was 0% (Table 4; Fig.3A). At 50, 100 and 200 mg/l Cefotaxime, the percentages of aseptic cultures recorded were 50, 60 and 65, respectively the nature of the shoot tip explants being green and healthy at these concentrations (Table 4). Cefotaxime at 300 mg/l augmented to MS + 10 mg/l BA + 5 mg/l Glutathione + 10 M AgNO3 medium induced the optimum response with 95% aseptic cultures that were green and proliferating after 3-weeks of culture. Cefotaxime at 500 mg/l augmented to MS + 10 mg/l BA + 5 mg/l Glutathione + 10 M AgNO3 medium, displayed the maximum (100%) percentage of aseptic cultures after 3-weeks of culture. However at this concentration of cefotaxime, the aseptic explants gradually turned dark brown after 3-weeks of culture. Table 4: Depicts the doses of the Cefotaxime antibiotic augmented to MS + 10 mg/l BA + 5 mg/l Glutathione + 10M AgNO3 medium for raising aseptic cultures of nodal explants of Jatropha curcas. Sl. No. 1 2 Cefotaxime (mg/l) *Percentage of Nature of explants Aseptic cultures (%) 0 Infected at the basal portion 50 Explants green

0 50

39

3 4 5 6
*

100 200 300 500

60 65 95 100

Explants green Explants green Explants green and healt hy Explants turns dark brown

Mean of 12 explants.

Establishment of callus cultures of embryonal axes dissected from fresh green seeds: Embryonal axis explants excised from green seeds of one-year-old garden raised plants of Jatropha curcas were cultured on MS + 2mg/l BA + 2 mg/l TDZ + 100 mg/l Cefotaxime medium containing different concentrations (0, 1, 2.5, 5, and 10mg/l) of IBA. Table 5: Effect of IBA augmented to MS + 2mg/l BA + 2mg/l TDZ + 100 mg/l Cefotaxime medium zygotic embryo explants of Jatropha curcas. MS + 2mg/l BA + 2 mg/l TDZ + 100 mg/l Cefotaxime+ IBA mg/l 0 1 2.5
*

Explants inducing callus (%)

Colour, nature and amount of callus**

10 25 55

Brownish green (+) Brownish green (+) Brownish (++) green, compact

5 10
*

100 100

Green, compact (++++) Green, compact (++++)

**

Mean of 12 explants. Relative amounts of callus: Nil (); scanty (+); moderate (++); good (+++); better (++++).

Of all the different combinations tried, MS + 2mg/l BA + 2mg/l TDZ + 100 mg/l Cefotaxime medium augmented with 5 mg/l IBA induced optimum callus in 100% of the cultures within two weeks of culture (Table 5). The calli developed were green, compact (++++) and morphogenic. Calli developed on MS + 2mg/l BA + 2 mg/l TDZ + 100 mg/l Cefotaxime medium augmented with 1 and 2.5mg/l IBA, respectively were brownish green with the amount of calli being scanty and moderate, respectively. The calli developed on MS + 2mg/l BA + 2 mg/l TDZ + 100 mg/l Cefotaxime medium augmented with 10mg/l IBA were green and compact (++++) .

40

Morphogenic response of calli explants raised from zygotic embryos on MS + 2mg/l BA + 2mg/l Kn + 2 mg/l 2iP + 1mg/l IAA + 1 mg/l IBA + 100 mg/l Cefotaxime medium: Induction of shoot buds: Experiments were carried out with two week old calli raised through embryonal axis explants (raised on MS + 2mg/l BA + 2mg/l TDZ + 2mg/l IBA + 100 mg/l Cefotaxime) on MS medium supplemented with different combinations of growth regulators such as BA, 2iP, Kn and TDZ and varying the concentrations of one of the cytokinins (BA/ 2iP/ Kn/ TDZ) tried. Table 6: Effect of TDZ, augmented to MS + 2mg/l BA + 2mg/l Kn + 2 mg/l 2iP + 100 mg/l + 1mg/l IAA + 1 mg/l IBA+ 100 mg/l Cefotaxime medium, on morphogenic response of callus derived from zygotic embryo explants .
MS + 2mg/l TDZ + 2mg/l Kn + 2 mg/l 2iP + 1mg/l IAA + 1 mg/l IBA 100 mg/l Cefotaxime + TDZ (mg/l) 0 0 0.00 0.00 0.00 0.00 0.00 0.00 1.50 0.30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.10 0.28 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.50 0.21 0.00 0.00 Green, granular Green compact Green, compact Green, slightly compact Green, slightly compact Callus developing shoots and shoot buds (%) Average No. of shoots Average shoot length (cm) Average No. of shoot buds per culture Colour and nature of the callus

40

7.5

Mean of 12 explants S.E.

Experiments carried out by varying the concentrations (0, 2, 3, 5 and 7.5 mg/l) of TDZ augmented to MS + 2mg/l BA + 2mg/l Kn + 2 mg/l 2iP + 100 mg/l + 1mg/l IAA + 1 mg/l IBA + 100 mg/l Cefotaxime medium revealed positive results. The optimum response for shoot bud initiation from the aforesaid calli explants was achieved on MS + 2mg/l BA + 2mg/l Kn + 2 mg/l 2iP + 1mg/l IAA + 1 mg/l IBA + 41

100 mg/l Cefotaxime medium supplemented with 5mg/l TDZ wherein, an average of 1.50 0.30 shoots with an average shoot length of 2.10 0.28 cm and 2.50 0.21 shoot buds were induced in 40% of the explants after the third sub-culture on the same fresh medium in an interval of two-weeks (Table 6). Conclusion : i. Optimum percentage (100%) of aseptic cultures of zygotic embryos (excised from 48 hours soaked mature seeds) of Jatropha curcas was achieved on MS medium augmented with 100 mg/l cefotaxime (antibiotic). ii. Amongst the different culture media (MS, 2MS, 1/2MS, B5, Knops and WPM) employed for germination of zygotic embryos (excised from 48 hours soaked mature seeds of Jatropha curcas), Knops medium proved optimum with a percentage germination of 70%. iii. Cefotaxime at 200 and 300 mg/l, respectively proved optimum for raising aseptic cultures of shoot tip and nodal explants on MS + 10 mg/l BA + 5 mg/l Glutathione + 10 M AgNO3 medium from garden raised elite plants of Jatropha curcas. iv. The optimum amount of callus induction from zygotic embryonal axis excised from green seeds of Jatropha curcas was achieved on MS + 2mg/l BA+ 2 mg/l TDZ+ 100 mg/l Cefotaxime + 5 mg/l IBA. v. Optimum induction of shoots from the aforesaid callus derived from zygotic embryonal axis (excised from green seeds of Jatropha curcas) was achieved on MS + 2 mg/l BA + 2 mg/l Kn + 2 mg/l 2iP + 1 mg/l IAA+ 1 mg/l IBA + 100 mg/l Cefotaxime + 5mg/l TDZ. The induction of shoot bud occurred after second subculture of 15 days each.

Maharana Pratap University of Agriculture & Technology, Udaipur Effect of Ascorbic acid (10 ml and 5.0 ml of 0.01% con.) on recovery of shoot necrosis and multiplications of in vitro shoots of Jatropha: Results: After 1 week Bud breaks 5-6 days in all cultures. Shooting started 10-15 days in cultures poured with 5.0 ml ascorbic acid, where as 10 ml ascorbic acid treatment showed delayed shooting. After 3rd week More shooting in 5.0 ml poured ascorbic acid cultures compared to 10 ml treated cultures. In control, more callus at the base of microshoots and almost all shoots were drying at the tip. Therefore, less shoot bud induction.

42

After 9th week Culture treated with 5.0 ml ascorbic acid showed total 73 shoots. Cultures poured with 10 ml ascorbic acids, showed total 25 shoots. Control exhibit 17 shoots. From 10 ml ascorbic acid treatment, albino type shoots were raised but not considered in the final data. Albino type shoots were found because of high amount of ascorbic acid (10 ml), that somewhat interfere in normal development of shoots. However the shoot tip necrosis was reduced considerably in 10 and 5 ml treatments (Table 1, Fig.1 and 2).It is recommended from our experiment that Ascorbic acid (5.0 ml of 0.01% concentration in culture bottles (5.3 x 12.5 cm, containing 40 ml media) can be used to prevent shoot necrosis at some extend during culture of shoot tips form Jatropha.

Fig.1 Effect of Ascorbic acid on shoot induction (5.0 ml of 0.01% con.)

Table:1 Effect of Ascorbic acid on shoot necrosis in in vitro shoots of Jatropha:

43

Media

No of shoots culture

1.0 mg /l BAP + 0.25 10+10+10+10+10+10 mg/l IAA(Control) 1.0 mg /l BAP + 0.25 10+10+10+10+10+10 mg/l IAA (poured with 5.0 ml Ascorbic Acid (0.01%) 1.0 mg /l BAP + 0.25 10+10+10+10+10+10 mg/l IAA (poured with 10 ml Ascorbic Acid (0.01%) * Only shoot tip containing shoots were counted

No of shoots after 9 weeks*/types of shoot 3+2+2+3+6+1/ 50%necrotic 12+16+11+12+10+12/10 % necrotic

5+3+4+4+3+6/60-70% albino,20-30% necrotic

1. Effect of combinations of PGRs, PVP, Adenine sulphate and CuSO4 on shoot

induction from leaf explants of Jatropha . Results: 1. Shoot induction were observed in the media (L1 to L12) within 4-5 weeks. 2. Bud induction was found maximum in L10 media (44 %) followed by L5 (40 %), L9 (38%) and L6 (20%). 3. Average lengths of shoots also found maximum on L10 media ( 2.0 cm). 4. Shoot multiplied in 7-8 weeks on shoot multiplication media (L11). 5. Explants cultured on L12 media gave some shoots in 4-5 weeks time, but media L13 and L14 did not have any effect on shoot induction. Media L13 and 14 induced mass callus induction at all margins of explants (Table 2 and Fig.3) 6. From above experiments we concluded that use of Adenine Sulphate at concentration of 40 mg/l (L10 media) could be used to achieve a higher % of shoot induction compared to other media tried Table: 2 Response in different treatments
SN Code Treatment No of explants induced shoots out of 40 explants 2 2 4 Response

1 2 3 4

L1 L2 L3 L4

BAP 1.0 mg/l + Kn. 0.5 mg/l TDZ 1.0 mg/l + BAP 0.5 mg/l BAP 1.0 mg/l + IBA 0.5 mg/l BAP 1.0 mg/ l+ TDZ 0.5 mg/l

Shoot induction starts in 4-5 weeks, Good growth, less shoots Shoot induction starts in 4-5 weeks, Less growth, more shoots All the explants showed callus formation, no shoot induction Explants become enlarge without any bud induction, approximately 10% explants

44

SN

Code Treatment

No of explants induced shoots out of 40 explants 16

Response

L5

BAP 0.5 mg/l +TDZ 0.5 mg/l +KIN 0.5 mg/l BAP 1.0 mg/l +TDZ 0.5 mg/l+ KIN 0.5 mg/l BAP 1.0 mg/l+ Kin 0.5 mg/l+ PVP 10 mg/l BAP 1.0 mg/l+ Kin 0.5 mg/l+ PVP 40 mg/l

exhibit callus formation Shoot induction starts in 4-5 weeks, 40 % explants showed bud induction Shoot induction starts in 4-5 weeks,20% explants showed bud indcution Shoot induction starts in 4- weeks, Less callus induction with 8.3 % bud induction. Length of shoot after 8-weeks average 0.5 cm Shoot induction starts in 4- weeks, Less callus induction compared to above experiment with 17.5 % bud induction. Length of shoots after 8-weeks recorded an average 1.0 cm Shoot induction starts in 4- weeks, Low amount of callus induction compared to PVP experiment with 38.0 % bud induction. Length of shoots after 8-weeks recorded an average 1.5 cm Shoot induction starts in 4- weeks, Very low amount of callus induction compared to above experiment with 44.4 % bud induction. Length of shoots after 8-weeks recorded an average 2.0 cm Shoot multiplication and elongation achieved in 7-8 weeks (20%) Shoot induction starts in 4- weeks in 2-3 cultures out of 40 explants. Less callus induction. Length of shoot after 8-weeks average 0.5 cm No shoots were obtained. Watery mass of callus induced from the surface of explants No shoot induction

L6

8 3

L7

L8

L9

BAP 1.0 mg/l+ Kin 0.5 mg/l+ Adenine Sulphate 10 mg/l

15

10

L10

BAP 1.0 mg/l+ Kin 0.5 mg/l+ Adenine Sulphate 40 mg/l

18

11

L11 L12

BAP1.0 mg/l +TDZ0.5 mg/l +KIN 0.5 mg/l BAP 1.0 mg/l+ Kin 0.5 mg/l+ Cu SO4 2.0 mg/l

12

13 14

L13 L14

BAP 1.0 mg/l+ Kin 0.5 mg/l+ Cu SO4 4.0 mg/l BAP 1.0 mg/l+ Kin 0.5 mg/l+ Cu SO4 6.0 mg/l

3. Shoot regeneration from seedling raised explants: Results: Response of cotyledon leaf explant on various media after 2 week of culture Media -1 Media -2 Moderate callus formation Moderate callus formation

45

Media -3 Media-4 Media-5 Media-6

Callus formation is less compared to above experiment. Bud induction found in one explants. Moderate callus induction with two explants showed bud induction Shoot induction in three explants Shoot induction in one explant and rest cultures have intensive callus formation than above treatments

Response of cotyledon leaf explants on various media after 5 week (Table 3 and Fig. 4) Media-1 Callus at cut ends, 8 small shoot induced Media-2 Callus at cut ends with same frequency as above, but 48 shoot buds induction noticed on culture cotyledon leaf explants Media-3 Callus formation is in-between above two treatments. Maximum shoot induction was recorded in this treatment (72 shoots). Media-5 Total 10 shoots rasied from explants Media-6 Total 2 shoots Response of hypocotyl on various media after 2 week Media-1 Less callus no shoot bud induction Media-2 More callus than above and no bud induction Media-3 Callus formation in-between of above two treatments and shoot bud induction in one explant Media-4 Callus formation is maximum and no shoot induction Media-5 Less callus than treatment No 4 and shoot induction in one explant Media-6 Callus formation is same as above

Response of hypocotyl on various media after 5 week (Table 4 and Fig. 5) Media-1 More callus, no shoot bud Media-2 Same as in week 2nd Media-3 Less callus with 16 shoot buds Media-4 Callus formation is maximum no shoot bud Media-5 Total 24 buds induced. Media-6 No bud induction but callus formed in all explants Table: 3 Shoot bud induction from seedling raised cotyledon leaf explant on different media. Media 1 2 3 4 5 6 Media 0.5 mg /l BAP + 0.25 mg/l IAA 1.0 mg /l BAP + 0.25 mg/l IAA 1.5 mg /l BAP + 0.25 mg/l IAA 0.5 mg/l BAP+0.25 mg/l IBA 1.0 mg/l BAP+0.25 mg/l IBA 1.5 mg/l BAP+0.25 mg/l IBA No of explants 40 40 40 40 40 40 No of shoots after 5 weeks/callus amount 8/less callus 48/less callus 72/less callus 8/more callus 48/more callus 16/less callus

46

Table: 4 Shoot bud induction from seedling raised hypocotyl explants on different media. Media code 1 2 3 4 5 6 Media 0.5 mg /l BAP + 0.25 mg/l IAA 1.0 mg /l BAP + 0.25 mg/l IAA 1.5 mg /l BAP + 0.25 mg/l IAA 0.5 mg/l BAP+0.25 mg/l IBA 1.0 mg/l BAP+0.25 mg/l IBA 1.5 mg/l BAP+0.25 mg/l IBA No of explants 40 40 40 40 40 40 No of shoots after 5 weeks/callus amount -/more callus -/more callus 16/less callus -/more callus 24/more callus -/more callus

4. Effect of CaCl2 and KH2PO4 on micropropagation of Jatropha Results In vitro shoot cultures of Jatropha curcas suffered from severe necrosis. The symptoms of necrosis started randomly in 6 to 7 weeks aged shoot cultures. The browning of the margin and tip of leaves were the first symptoms followed by leaf drop and death of shoots (Fig.6). The severity of necrosis increased (10 to 42 %) with culture duration. Forty two percent of shoots died due to lethal necrosis in 9- weeks under culture (Table 5). Initiation of necrosis varied amongst shoots in culture. Some cultures showed necrosis from old/lower leaf browning / blackening and gradually extended to apical meristem. Whereas some of symptoms started from tip of the shoots and extended down to the lower end . The ability of CaCl2 and K2H2PO4 to reduce shoot necrosis can be elucidated in the next experiments (Tables 6 and 7). In the first experiment, the influence of CaCl2 was examined. We found that CaCl2 at 60-80 mg/l significantly reduced shoot necrosis from about 42% (control) to 20-23 % without negative side effects on shoot growth. However, concentration of CaCl2 100 mg/l was detrimental as it reduced shoot fresh weight (Table 6). However, amendment of the basal medium with CaCl2 was effective to control necrosis, although death of 20-23.3 % of regenerated shoot still continued to occur (Fig. 6). In the second experiment, addition of various concentrations of K2H2PO4 to shoot growth medium fortified with 80 mg/l of CaCl2 were evaluated for mortality rate of in vitro grown shoots due to lethal necrosis. Compared to the control, 40-60 mg/l of K2H2PO4 reduced necrosis incidence to 11.6 -14 % without inhibiting growth (Table 7), but higher concentrations were harmful. We conclude from above experiment that addition of CaCl2 and K2 H2 PO4 is imperative for prevention of shoot necrosis in in vitro shoots of Jatropha.

47

Table: 5 Incidence of shoot necrosis in in vitro shoot cultures of Jatropha curcas. Shoots were grown on shoot growth medium (MS basal medium,0.5 mg/l BAP + 0.5 mg/l Kin + 0.25 mg/l IAA + 0.25 mg/l GA3). Data were recorded upon 6-to-9 weeks of culture Duration (week) 6 7 8 9 Necrotic shoots (%) 10.00.5c 26.30.8b 38.30.3a 42.01.0a Fresh weight (g) 0.50.07c 1.00.06b 1.40.15ab 1.50.08a Shoot length (cm) 1.30.03c 1.70.10b 2.00.03a 2.00.08a

Means se in column followed by the same letters are not significant at P 0.05 by TukeyKramer HSD test.

Table 6: Effect of CaCl2 added to MS basal medium on growth and shoot necrosis of Jatropha curcas shoot cultures . Shoot were cultured on shoot growth medium (MS basal medium, 0.5 mg/l BAP + 0.5 mg/l Kin+ 0.25 mg/l IAA + 0.25 mg/l GA3). Data were recorded by 9 weeks of culture. CaCl2 (mg/l) 0 20 40 60 80 100 Necrotic shoots (%) 42.00.5a 40.30.6a 31.31.2b 23.30.8c 20.00.5c 35.00.5b Fresh weight (g) 1.60.05c 1.50.06c 1.90.08b 2.50.03a 2.30.12a 1.60.03bc Shoot length (cm) 2.50.03c 2.50.03c 2.60.32bc 3.30.05a 3.20.03ab 2.70.03abc

Means se in column followed by the same letters are not significant at P 0.05 by TukeyKramer HSD test.

Table 7 Effect of K2H2PO4 on shoot necrosis in shoot cultures of Jatropha curcas (L) grown on a medium fortified with CaCl2. Cultures growth on a medium of MS basal medium, 0.5 mg/l BAP + 0.5 mg/l Kin+ 0.25 mg/l IAA + 0.25 mg/l GA3+ 80 mg/l CaCl2. Data were recorded by 9 weeks of culture. K2H2PO4 (mg/l) 0 Necrotic shoots (%) 21.60.5c Fresh weight (g) 2.50.08c Shoot length (cm) 2.50.03c

48

20 40 60 80 100

16.30.8d 11.60.4e 14.00.5de 28.30.2b 39.61.5a

2.50.05c 2.90.02b 3.50.01a 1.90.05d 1.30.08e

2.60.05c 3.00.02b 3.30.04a 1.60.04d 1.50.02d

Means se in column followed by the same letters are not significant at P 0.05 by TukeyKramer HSD test.

Control

CaCl2 (80 mg/l)

CaCl2 (80 mg/l+K2H2PO4(40 mg/l)

Fig.6 Effect of CaCl2 and K2H2PO4 on shoot necrosis in

Conclusions: 1. The use of Adenine Sulphate at concentration of 40 mg/l (L10 media) could be used to achieve quick and higher frequency of shoot induction from mature leaf explants of Jatropha compared to other media tried. MS basal media supplemented with 1.5 mg /l BAP + 0.25 mg/l IAA found to be very effctive for indcution of healty shoots from cotyledon leaf explants compared to hypocotyl explants. Mature leaf explant was used for micropropagation of Jatropha at large scale.We noticed from our experiments that in vitro shoot cultures of Jatropha 49

2.

3.

curcas suffered severe necrosis. Forty two percent of shoots died due to lethal necrosis in 9- weeks under culture. Addition of various concentrations of K2H2PO4 to shoot growth medium fortified with 80 mg/l of CaCl2 were evaluated for mortality rate of in vitro grown shoots due to lethal necrosis. Compared to the control, 40-60 mg/l of K2H2PO4 reduced necrosis incidence to 11.6 -14 % without inhibiting growth, but higher concentrations were harmful. We conclude from above experiment that addition of CaCl2 and K2H2PO4 is imperative for prevention of shoot necrosis in in vitro shoots of Jatropha. SDAU, S.K. Nagar: a. Experiments for shoot initiation, shoot multiplication, shoot elongation and root induction in Jatropha curcas (cv. SKJN 1) were conducted and the most economical media were selected, that gave a much earlier response, than others. This was done to lessen the incubation time needed for the desired response, in order to develop a commercially viable protocol for Jatropha tissue culture. b. Stable rooting of in vitro raised Jatropha plantlets is under progress. Various rooting media are being tried to over come the browning of roots and stabilize the regenerated plantlets, which result in drying of the regenerated shoots. Conclusion: (i) The in vitro regenerated multiple shoots of Jatropha curcas were excised and introduced into shoot elongation media (MS + BAP + GA3 and MS + KIN +GA3). The shoots not only elongated but also became stout as the girth of the shoots increased. The green colour of the leaves deepened and leaf enlargement was also observed. These regenerated shoots were subjected to rooting in the MS medium supplemented with various concentrations of IBA and NAA. However on rooting the plantlets lost stability and the leaves dried out. This reduces the survival percentage (20%) and thus, offers a big hindrance towards development of a commercially viable protocol for Jatropha curcas. Standardization of rooting media is under progress.

(ii) (iii) (iv)

(v)

TNAU, Coimbatore Micropropagation and Tissue culture techniques in Jatropha have been initiated using nodal and shoot tips as explants. Among the explants, nodal segments proved its superiority in terms of morphogenesis and organogenesis. Four different basal media were tried where in the MS media supplemented with 3 mg/litre each of BAP and Kinetin proved effective in shoot bud proliferation and secondary multiplication through subculturing. The microshoots were dissected out and rhizogenesis was observed in MS + 2.5 mg/ litre IBA. Further studies are on the way.

50

DNA Fingerprinting and molecular characterization of Jatropha germplasm collected from diverse agro-climatic zone of India
The NBPGR, New Delhi has cryostored 166 accessions of Jatropha curcas, and 47 accessions of Prunus armeniaca. Thus, a total of 892 accessions of Jatropha, 305acessions of karanja and 296 accessions of wild apricot has been cryostored under the project, so far. These accessions are collected from the different states of the country. In order to select a set of representative diversity of Jatropha for DNA profiling, selection was based on the basis of available data, namely, passport information, oil content and the information on the collection site etc. About 101 accessions of various TBOs viz. Jatropha (17) from Tripura, Maharashtra & U.P., Karanja (41) from A.P., Simarouba (17) from Maharashtra and Wild apricot (26) from J&K totalling to 101 accessions were selected on the basis of above available information. So far, a total of 358 accessions of Jatropha (245), Karanja (67), wild apricot 26) and Simarouba (20) has been selected for DNA finger printing. The seeds of these accessions have been sown to obtain leaf material for the DNA extractions and fingerprinting. The available elite Jatropha accessions in NBPGR were used for DNA extraction and screening of the primers for DNA profiling. Seventy-five AFLP primers were screened in addition to 100 SRAP and 50 ISSR primers in order to identify the markers polymorphic enough to distinguish distinct genotype of the Jatropha and Pongamia. A set of 50 RAPD, 20 ISSR and 25 AFLP primers were identified as suitable for genetic diversity analysis and DNA fingerprinting of the Jatropha genotypes. Cryopreservation of germplasm: A total of 793 accessions of Jatropha germplasm were received from some of the participating institutes of R&D network programme. Out of these, 584 samples were found acceptable for storage based on high viability and sufficient sample size. These 584 samples have been cryostored successfully. IC numbers have been allocated to these and numbers communicated to the respective collaborators. In addition, 184 accessions of Pongamia germplasm were received from 13 Network collaborators out of these 170 accessions were found acceptable for storage based on high viability and sufficient sample size. These have been cryostored successfully. IC numbers have been allotted to the genotypes provided with complete passport data and numbers communicated to the respective collaborators. About 295 accessions of Wild apricot germplasm were received from Srinagar & Palampur. Out of these 210 samples were found acceptable for storage based on high viability and sufficient sample size. These have been cryo stored successfully. IC numbers have been allocated to these. 51

Only 6 accessions of Diploknema butyracea were received from Nainital Network collaborator. These accessions did not qualify the cryobanking standards of high viability as it was below 30%. Therefore, they have not been cryostored. In addition, 20 accessions of Simarouba glauca were received from Akola Network collaborator. Only 2 accessions have been cryostored. Other accessions did not qualify the cryobanking standards of high viability as it was below 50%. Therefore, they have not yet been cryostored. So far, a total of 726 germplasms of Jatropha, 329 germplasms of karanja and 249 genotypes of wild apricot has been cryostored and IC No have been allotted by NBPGR.

52

Value Addition of Tree Borne Oilseeds

Research & Development projects on value addition of Tree Borne Oilseeds are under implementation with CFTRI, Mysore; IIT, Delhi; IIT, Kharagpur; OTRI, Anantapur and SVBPUA&T, Meerut with the objective to develop appropriate technologies for the preparation of quality grade defatted cakes/meal from the identified Tree Borne Oilseed such as jatropha, karanja, mahua, neem and simarouba for better utilization.

IIT, New Delhi

Salient achievements Growth of biocontrol agents on cakes - From the results it is clear that the PDB/PDA may be replaced by cake amended with dextrose for the growth of Paecilomyces . Effect of aqueous extracts and active components of cakes on mortality of a) Termites- Got encouraging results from water extracts but active components gave better results against termites . b) Nematodes- On the contrary, aqueous extracts have been found superior over active components of cakes in controlling nematodes .
Preparation of P. lilacinus, B. bassiana and M. anisopliae filtrates and using against termites and nematodes- All fungal filtrates were ineffective against termites but in case of nematodes, P.lilacinus filtrate proved lethal . SEM of Paecilomyces- Morphological changes in the sporulation and mycelia formation, when non edible oil cakes were used as growth medium, also observed with the help of Scanning Electron Microscopy .

Optimization of growth for better spore production of P.lilacinus on cakes- karanja cake with C/N 40 at pH 7 at 2800C supported maximum growth of P.lilacinus . Nursery study (No. of repeats-two) on effect of cakes on seed germination of vegetable crops- There was negative effect of all cakes on the germination of chilli but Jatropha enhanced germination in brinjal . Preparation of microbial biofertilizer formulation for potato- Use of cakes and microbial consortium (biofertilizers) based formulations have been found effective in supplementing the required nutrients to the potato . Effect of different cakes alone as well as in combination with chemical fertilizers on selected vegetables such as chilli and brinjal- Jatropha has been found to have better fertilizer effect as compared to other cakes on selected vegetable crops when used alone.

53

However, both Jatropha and Neem cakes have been found to reduce the dose of chemical fertilizers by 50% when used with chemical fertilizers at various proportions. Studies on Use of Cakes as Biopesticides (A) Termite Control Experiments on karanjin and phorbol esters against termites Karanjin and phorbol esters showed good results. Different dilutions 100 % (1 mg /ml), 50 % (0.5 mg /ml), 10% (0.1 mg /ml), 5% (0.05 mg /ml) and 1% (0.01 mg /ml) were prepared using acetone as solvent in case of karanjin and diethyl ether in case of phorbol esters. After getting 100 % mortality in 6 hours with 1 mg/ml karanjin and in 12 hours with 1 mg/ml of phorbol esters, the synergistic effect of karanjin with phorbol esters was also studied. Karanjin and crude phorbol esters were combined in different ratios and tested on termites. Table 1 : Toxic effect of the combinations of karanjin and phorbol ester on O.obesus Treatments Combinations Karanjin + Phorbol esters (1:1) Karanjin + Phorbol esters (1:3) Karanjin + Phorbol esters (3:1) Control Chemical Control 1 hour 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 100.00 0.0 % Mortality of termite workers (mean SE, % ) 3 hour 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 100.00 0.0 6 hour 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 100.00 0.0 12 hour 24 hour 48 hour 72 hour 36.67 56.67 80 8.2 93.3 9.43 4.71 4.7 20 0.0 66.67 4.7 36.67 4.71 0.00 0.0 100.00 0.0 76.67 9.43 73.33 4.7 10.00 0.0 100.00 0.0 86.67 9.43 86.67 4.71 10.00 0.0 100.00 0.0

16.67 4.71 0.00 0.0 100.00 0.0

Experiments on saponins and sapogenol against termites Mahua cake was dissolved in water (1:6 dilutions) and stirred at 50 rpm for about 12 hours. This gave approximately 50 % removal of saponins from the cake. The residual water left after the treatment had therefore some (approx. 50 %) saponins in it. Therefore, experiments were performed to test its efficacy. The water obtained after the treatment was designated as 100 % and there after, its different dilutions were made in water. The efficacy of all the concentrations was tested for about a month. The results showed that 75 % concentration was the most effective, but the efficacy decreased down after 15 days and even more drastically after a month.

54

The results with saponins extracted from mahua have already been shown and it is expected that the toxicity of the saponins is due to the non sugar part, ie., sapogenol. So, the study was performed accordingly by dissolving sapogenol in appropriate solvents. Different solvents were tried which showed minimal toxicity to the termite workers but also dissolved the sapogenol. It was found out that tetrahydrofuran (Thf) was the suitable solvent. The results (fig.1) showed that sapogenol is very effective in killing the termite workers as 0.5 g/ml sapogenol designated as 100 % concentration gave 100 % mortality in 2 hours and even concentration as low as 0.12 % was able to give 100 % mortality in 36 hours.

Plot of % mortality of termite workers with different sapogenol concentrations

120 100 80 60 40 20 0 1 2 4 8 16 24 28 36 48 72 Time H) 0.12% 0.25% 0.50% 1% 5% 10% 50% % Mortality

Fig 1: Mortality of the termite workers with sapogenol different time intervals. Karanjin in combination with other cake active component has been found more effective and hence a formulation having synergistic effect is being prepared and filed for patent. Beside cakes, fungal filterates of P.lilacinus and B.bassiana were also tried against termites but they were not found effective therefore no further work was proceeded.

55

(B) Nematode Control Efficacy of aqueous extracts and crude bioactive components (phorbol esters, karanjin and azadirachtins) against M.incognita Aqueous extracts of cakes were prepared by soaking the powders for 24 h in sterilized distilled water (SDW) in a ratio of 1:6 at 280C. After straining through muslin cloth the extracts were centrifuged at 2400 rpm for 10 min and supernatants were stored at 40C. This aqueous extracts were suitably diluted with sterilized distilled water to obtain concentrations of S (100%), S/2 (50%), S/4 (25%) and S/10 (10%). Crude bioactive constituents (1000ppm) were obtained through solvent extraction methods and all these extracts were tested against freshly hatched J2 larvae and it was observed that 100% jatropha extract was most effective. It was also observed that the crude bioactive constituents were not that effective as aqueous extracts of cakes against nematodes indicating the role of water solubles present in them. Optimization of growth for better spore production of P.lilacinus on cakes The growth of this fungus were observed on different substrates such as jatropha, neem and karanja cake and factors affecting its sporulation and mycelia production such as pH, C/N ratio, carbon concentrations were optimized. It was observed that karanja cake favoured the most as far as growth of fungus (colony diameter and spore count) is concerned Further, growth of fungus on karanja cake (C/N-8.5) was improved by increasing C/N ratio (10, 20, 40 and 50) at varying pH through sucrose addition. C/N 40 and 50 at pH 7 were best and at par with each other in producing maximum colony diameter and spore numbers of P .lilacinus. Studies on the efficacy of fungal filtrate grown on above mentioned optimized media against M. incognita showed that more the C/N ratio (40>20>10) more efficient the filtrate was in killing nematode larvae. Formulation of fungal filtrate is in progress.

SEM of Paecilomyces
Morphological changes in the sporulation and mycelia formation, when non edible oil cakes were used as growth medium, also observed with the help of Scanning Electron Microscopy (SEM). Since these oil cakes contain high amount of Nitrogen, creating the stress condition for the fungal growth and hence led to sporulation. Also, under stress the morphology of the spores change. The shape of the spore is found to be more oval when grown with cakes and round when grown in Potato Dextrose medium (PDB).

56

Studies on Use of Cakes (along with bioinoculants) as Biofertilizers Experiments on Potato Potatoes were planted in the field in the plots in randomized plot design. Total 8 treatments were applied and 4 replicates for each treatment have been taken. Vermicompost has been applied @ 4.5Kg per plot. The formulations were coated around the potato while sowing. The well sprouted seeds weighing 40 to 50 grams were taken and tubers were planted 20 cm apart. The following treatments have been applied: a) T1- Control b) T2- Telcom powder based formulation c) T3- Charcoal based formulation d) T4- Karanja cake based formulation e) T5- Chemical (with standard N, P, K ) f) T6 -50%Chemical +T2 g) T7- 50%Chemical +T3 h) T8 -50%Chemical +T4 The germination rate of potato was calculated after 4 weeks. The germination rate highest (81.73 %) was found in treatment with 50% chemical and Karanja cake based formulation. After the 3 months the wet fresh and dry weight analysis of biomass was done. The maximum plant height (42.4. 1.37 cm), no of leaves/plant (46.51.29), fresh weight (891.56.75) and dry weight (98.733.5) were observed in combined application of Karanja cake based formulation+50 % chemical (N, P, K) (table 2). The plant nutrient content N (41.72.86mg/g), P (11.55.23mg/g), K (33.31.56mg/g) (table 6). Chlorophyll Ch a (2.967.014 mg/g), Ch b (.234.004 mg/g) and total chlorophyll (3.469.026mg/g) were also found maximum in Karanja cake based formulations + 50% chemical required (fig 2)

Mortality (%)

100 50 0 50% J 25% J 100% J 10% J 12 h 48 h 24 h 12 h 48 h

Mortality (%)

100 50 0 100% N 50% N 25% N 10% N 12 h 48 h 24 12 h h 48 h

Treatment
Fig 2: Mortality of nematodes by Jatropha extract (aq.)

Treatment
Fig 3: Mortality of nematodes by Neem extract (aq.)

57

Table 2: Effect of various treatments on growth and biomass yield of potato

Treatment Plant height 27.10.86 32.301.73 No. of Leaves/plant 23.351.70 30.001.82 Fresh Weight (g/Plant) 250.2514.59 658.7510.56 Dry Weight (g/Plant) 23.566.05 44.791.86 Yield (t/ha) 5.35.45 8.33.78

T 1 (Control) T2 (Telcom powder based formulation) T3 (Charcoal based formulation) T4 (Karanja cake based (formulation) T5 (50%Chemical +T2) T6 (50%Chemical +T3) T7 (50%Chemical +T4) T8 Chemical (with standard N, P, K )

34.752.31

30.751.89

695.665.85

52.811.87

8.82.85

35.97.97

33.51.29

738.510.37

64.622.85

8.13.54

33.951.12 36.171.67 40.351.65 42.4. 1.37

37.75.95 42.51.73 45.501.29 46.51.29

759.759.10 778.628.57 876.758.65 891.56.75

67.632.06 76.342.61 85.214.08 98.733.5

9.33.46 9.90.73 11.58.25 10.45.82

Total sugars9.42.19 mg/g, total fibre 21.491.37 mg/g and starch content 12.11.90 g/kg was found higher in the treatment with application of 100% chemical (table 3). Table 3: Effect of various treatments on fibre & starch content of potato tubers Treatment Fibre crude (mg/Kg) 17.251.15 T 1Control T2Telcom powder based formulation 17.31.55 T3Charcoal based formulation T4 Karanja cake based formulation T5 50%Chemical +T2 T650%Chemical +T3 T7 50%Chemical +T4 T8 Chemical (with standard N, P, K ) 22.883.1 21.491.37 22..972.04 23.561.08 24.651.22 24.621.1 Starch (g/Kg) 8.47.15 9.34.62 9.18.47 9.62.43 9.82.39 10.25.25 11.35.19 12.11.90

However to verify the above results further experiments are in progress.

58

Experiments on Brinjal and Chilli Table 4 : Treatment details

Treatment No. T1 T2 T3 T4 T5 T6 T7 T8 T9

Treatment details
Control (No cake & No fertilizers) Chemical alone (NPK) Neem cake alone (N) Jatropha cake alone (J) Mahua cake alone (M) Karanja cake alone (Ka) Nitrogen 100% N + P+K 100% J + P+K 100% M + P+K

Treatment No. T10 T11 T12 T13 T14 T15 T16 T17 T18

Treatment details
100% Ka + P+K 75% N (25 % Urea) + P+K 75% J (25 % Urea) + P+K 75% M (25 % Urea) + P+K 75% Ka (25 % Urea) + P+K 50 % N (50% Urea) + P+K 50 % J (50% Urea) + P+K 50 % M (50% Urea) + P+K 50 % Ka (50% Urea) + P+K

It was observed that Jatropha 50% + P+K was most effective for brinjal and N 50%+ P+K for chilli (fig 14-15) in producing best growth in terms of physical parameters like plant height, fresh biomass and yield as well as macronutrient contents (NPK) of fruits (table 4 & 5). However, not much difference was observed in phosphorous content of fruits.
100 90 80 70 60 50 40 30 20 10 0 Plant height 1200 1000 Fresh biomass (g) 800 600 400 200 T1 T3 T5 T7 T9 T11T13T15T17 Treatments 0 120 Plant height (cm), No. of fruits/plant 100 80 60 40 20 Plant height No. of fruits/plant 350 300 200 150 100 50 0 T1 T3 T5 T7 T9 T11 T13 T15 T17 Treatments Fig 15: Growth contributing parameters of chilli influenced by the treatments. Fresh biomass (g) 250

Plant height (cm), Yield (t/ha)

Fig 14: Growth contributing parameters of brinjals influenced by the treatments

59

Table 5: Effect of various treatments on NPK content of fruits of brinjal and chilli Treatments
Brinjal T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 2.67 2.97 2.81 2.88 2.69 2.74 3.28 3.33 3.07 3.17 3.78 3.95 3.43 3.65 4.23 4.3 4.03 4.17

N%
Chilli 1.7 2.06 1.98 1.85 1.73 1.79 2.43 2.31 2.16 2.28 2.75 2.67 2.58 2.63 3.1 2.95 2.8 2.87 Brinjal 0.52 0.58 0.56 0.56 0.53 0.54 0.59 0.6 0.58 0.58 0.63 0.65 0.62 0.63 0.74 0.77 0.69 0.71

P%
Chilli 0.29 0.31 0.30 0.30 0.30 0.31 0.32 0.32 0.31 0.32 0.34 0.33 0.33 0.33 0.35 0.35 0.34 0.34

K%
Brinjal 2.13 2.89 2.65 2.73 2.21 2.54 3.14 3.17 3.06 3.11 3.21 3.27 3.18 3.18 3.34 3.40 3.27 3.32 Chilli 1.5 1.76 1.65 1.62 1.59 1.61 1.98 1.93 1.83 1.87 2.07 2.06 2.01 2.04 2.12 2.1 2.09 2.09

Repeat experiments for brinjal in winter season are in progress. Further, in order to minimize chemical fertilizers, bioinoculants such as Azotobacter and mycorrhizae have been used in brinjal and this experiment is also in progress. Conclusions: 1. Termite control- Although the bioactive constituents of cakes were effective, their effects got enhanced when the cakes were used in combination. Therefore, formulation with the combination of these constituents of different cakes is being developed and will be filed for patent. 2. Nematode control - The crude bioactive constituents of cakes were not that effective as aqueous extracts indicating the effects of combinations of many chemicals present in them. Order of efficacy of aqueous extracts of cakes against nematodes (M. incognita) was; Jatropha> Neem> Karanja 3. Karanja cake has been proved as a suitable low cost substrate for the growth of biocontrol fungus P.lilacinus and hence a formulation of the combination of Karanj cake and P. lilacinus is being developed and will be filed for patent.

60

4. Use of cakes and microbial consortium (Biofertilizers) based formulations have been found effective in supplementing the required nutrients to the potato. 5. Jatropha and neem cakes have been found to reduce the dose of chemical fertilizers by 50%. However a number of repeats are needed and being done.

Central Food Technological Research Institute (CFTRI), Mysore. Value addition to tree-borne oilseeds - detoxification of seed meal of Jatropha, Karanja and Simarouba Simarouba: The major challenge in the utilization of simarouba oil cake (by-product of oil extraction) was the presence of toxic components (saponins and alkaloids) at high concentration. Acidic ethanol wash and two step boiling with acetic acid have been found to be an effective treatment for removal of saponins, alkaloids and phenolics simultaneously from simarouba meal. It was found that the treatments can be applied to commercial simarouba oil cake containing 10-12% fat with the same detoxification method applied for defatted simarouba meal. Saponin one of the important phytochemicals from simarouba has been shown to inhibit pathogenic organisms like B.cereus, E-coli and S.aureus at considerably lower concentrations compared to saponins obtained from soybean and quillaja. Saponins, a by-product obtained after detoxification of simarouba oil cake, recovered simultaneously during detoxification can be a component in therapeutic formulations. Karanja: The additional step of solvent wash used had removed saponins more than 70% in the meal and minimized the bitterness. The detoxified meal was evaluated by feeding rats and improvement up to 85% in food consumption and body growth up to 80% were found in experimental rats compared to control animals. In the present study, for further improvement in detoxification process an aqueous system has been followed and achieved removal of saponins more than 75%. Saponins of karanja seeds were studied for their biological activity, studies were carried out to estimate metal chelating ability and anti-oxidant activity of saponins. The crude extracts of karanja and soy flour are compared for theier biological activities. Saponins obtained by butanol fractionation exhibited highest proton radical scavenging activity followed by methonolic extract of saponins. Karanjin, a furano flavonoid of karanja seed was assessed for its antioxidant properties.

61

Detoxification of simarouba oil cake Saponins and alkaloids have been identified as major toxic constituents of simarouba meal. Based on the type of toxic constituents different treatments were tried for detoxification of simarouba meal containing <1% fat prepared in the laboratory. Treatment with alkali, 2% acetic acid washing, boiling with 2% acetic acid in two steps, autoclaving with 2% acetic acid, boiling with water, 2% ethanolic acetic acid wash and percolation of 2% acetic acid after packing the meal in a column were the simple methods tried in the laboratory. Boiling with 2% acetic acid and washing with 2% ethanolic acetic acid was the most efficient treatments for removal of toxins (alkaloids and saponins) simultaneously. Further, work was carried out by boiling with 2% acetic acid since washing with 2% ethanolic acetic acid involves organic solvent involving solvent which is not advisable in industry. Simarouba meal detoxified by boiling with 2% acetic acid in two steps resulted in maximum level of reduction of saponins (95%), alkaloids (89%) and phenolics (91%). Simarouba kernel is usually used for extraction of oil. The simarouba oil cake obtained after expeller pressing contains usually 10-12% fat. In order to test the efficacy of detoxification by boiling with 2% acetic acid, simarouba oil cake containing 10-12% fat was (500g batch) treated by boiling with 2% acetic acid in two steps and evaluated for detoxification by estimation of saponins and alkaloids. The study showed that the treatment resulted in removal of 95% saponins, 89% alkaloids and 91% phenolics. The protein content of treated simarouba oil cake resulted in (60.2) compared to untreated simarouba oil cake (41.1) (Table 1). A close comparison of the saponin and alkaloid content of defatted simarouba meal (<1% fat) detoxified by similar methods 2% acetic acid wash showed that the extent of removal was similar. This shows that the detoxification procedure by treatment using 2% acetic acid is applicable to commercially available simarouba oilcake also (Fig. 1). Scale up studies on detoxification of simarouba oil cake is in progress. Table 1: Protein and toxic components of simarouba oil cake* detoxified by 2% acetic acid treatment in two steps
Sample Protein % 41.1 Saponins
Content % Reduction %

Alkaloids
Content % Reduction %

Phenolics
Content % Reduction %

Untreated simarouba oil cake Detoxified simarouba oil cake

3.4 60.2 0.17

95.00

0.91 0.10

89.01

0.91 0.08

91.20

*with 10-12% fat Evaluation of antimicrobial activity of saponins from simarouba glauca meal

62

The n-butanol purified saponin extract of simarouba meal was screened for antimicrobial activity against three pathogenic microbes viz; Escherichia coli, Staphylococcus aureus and Bacillus cereus. Saponins isolated from soybean and commercial quillaja bark saponin was taken for comparison. The three microorganisms were grown in Brain Heart infusion agar at 37 oC. Each bacterial strain was transferred from stored slants at 4-5 oC to 10ml of broth and cultivated overnight. A pre-culture was prepared by transforming 1ml of this culture to 9 ml Brain Heart infusion broth and cultivated for 48 hours. The cells were washed in saline and diluted to obtain approximately 106spores/ml and used for inoculation. Saponins from simarouba was dissolved in propylene glycol and added to sterilized nutrient agar media at different concentration (ppm) in conical flask and the bacteria to be tested was inoculated at suitable concentration (103cfu/ml) under aseptic conditions and the media was poured into disposable petriplates and incubated at 37oC for 20-24 hours. The colonies developed were counted and the % inhibition of bacteria at different concentration (ppm) of saponins was calculated. Comparison of the % inhibition of simarouba saponin, soybean saponin and quillaja saponin indicates that the percentage inhibition of S.aureus by quillaja bark saponin was only 45.67% at 125 ppm concentration. The inhibition increased up to 64.18% at 937.5 ppm concentration. Soybean saponin inhibited S.aureus by 84.18% at 937.5 ppm concentration. The extent of inhibition of S. aureus by simarouba saponin was much higher. At a concentration of 275 ppm simarouba saponin completely inhibited the growth of S.aureus while for soy it was only 67%. In the case of B.cereus the complete inhibition by simarouba saponin occurred at 375 ppm concentration and completely inhibited the growth of E-coli at 500 ppm. Study is in progress to arrive at the minimum inhibitory concentration (MIC) with respect to simarouba saponins for S.aureus, B.cereus and E.coli. The growth inhibition study will also be carried out using simarouba saponins against fungal and yeast cells. Karanja: Detoxification of karanja meal Although the karanja seed proteins are of good quality, the main problem in the nutritional exploitation is the presence of the anti-nutritional factors. The anti-nutritional factors present are tannins, phytates, trypsin inhibitors and saponins. However, raw cake is not normally used as feed since the presence of these anti-nutritional factors resulted in poor intake. Therefore, detoxification of the seed meal is necessary prior to their use as animal feed. It was reported earlier that 2% HCl wash followed by ethanol wash, was found effective in reducing the saponin content. The saponin content was reduced by more than 70% in the above detoxification procedure. This meal was fed to weanling rats for nutritional quality evaluation. The food intake by rats fed with detoxified karanja meal showed more than 50% response and caused no mortality up to 10% incorporation of seed proteins in the diet. The poor food intake and retardation in body weight was found

63

due to bitterness of the meal rendered by the presence of saponins making it nonpalatable. Hence, there was a requirement to improve the detoxification process for better palatability and make the meal more promising. Improved methods for detoxification of defatted karanja seed meal Present modified method of detoxification carried out was effective in removal of anti-nutrients from defatted karanja seed meal. The study also addresses their effect on protein contents. These methods involved washing of seed meal in 2% HCl, followed by soaking in salt solution (1-3% NaCl solution). Later extracts were filtered, residues were dried and saponin content was estimated. A set of experiments were carried out in three different concentrations of salt solution for detoxification of the meal. In the 1st set of experiment, salt solution washing was done at acidic pH. In the 2nd set it was done at neutral pH and in the 3rd set, process was carried out at alkaline pH. The results were tabulated in Table 3a-3c. The detoxification of karanja meal was achieved by soaking defatted meal in 2% HCl (1:5) for 2 hours and carried out the filtration. The residue was adjusted to pH 8 using 2N NaOH. The residue was further washed with 3% sodium chloride for 3 hours, filtered and washed with water adjusted to neutral pH and residue was dried at 50 C. Saponins from karanja seed meal: Estimation of saponins in crude extract and butanol fraction: The amount of saponins present in defatted seed meal of karanja was extracted using two different solvent systems (i) Methanolic crude extract and (ii) butanol fractionation and was quantified after evaporating the solvents under vacuum. Standard quillaja saponins was taken as reference saponins. Linear curve was obtained from 50-250g saponins concentration which is shown below in table 4. Similarly, sample is plotted on standard graph by drawing different concentrations of karanja saponins from defatted meal in crude form. Percentage of saponin is determined by back calculation. Table 5 shows the saponin content in karanja crude extracts. The crude extract obtained from meal showed the presence of saponins equivalents at 4.1-4.5% concentration, this is in par with saponins present in soybean reflecting its similarities among leguminous seeds. In another instance saponins were extracted and isolated by butanol fractionation. Similarly, isolated saponins from defatted karanja were taken again in different concentration to determine the saponins content. The results are described in table 4 showed 4.35-4.78% saponins. Comparative studies between table 4 and table 5 shows that isolated karanja saponins from defatted meal have more in amounts of saponins when compared to crude saponins in crude extract of karanja. This depicts that the method of extraction of saponins by butanol fractionation has made the compound freely available.

64

Polyphenols from karanja seed meal: Estimation of phenolics in crude extracts: The yield of polyphenols content of the defatted karanja extractives is presented. Methanol is found to have extracted the maximum anti - oxidant or bioactive component. Polyphenols are considered as one of the principle anti-oxidant component in plant and food system, they comprise of a wide range of components including phenols, tannins, etc. Total polyphenols content of the karanja seed are expressed as gallic acid equivalents. The polyphenols content in karanja seed was 110 115 mg/100g of the sample. As mentioned earlier polyphenols is consisted of a wide range of compounds and extraction of these depends on the extraction efficiency of different solvents hence a varied activity. Difference existed in the polyphenols content of defatted karanja seed and also among the extracts of different solvents. Standard phenolic estimation was done using gallic acid at 1mg/ml concentration. Standard calibration was done. The standard phenol concentration range was 30mg/ml. Similarly, crude of karanja was taken and result was determined using standard curve. The amount of phenol present in 5 microliter of crude karanja sample was 110mg/100g. Whereas, the amount of phenol present in 10 microliter of crude karanja sample was 115 mg/100g. Thus, the average was about 112.5mg/100g. Table 6 shows the total phenolic content in crude extract of karanja seed meal. In comparison to defatted soya flour was reported about 148 mg equivalent phenol g-1. Hence, we can infer that phenolic content was high in soya flour than compared to karanja flour. Total antioxidant activity The total antioxidant activity of the defatted karanja seed meal extracts in different solvents was analysed by phosphomolybdenum method. The method is based on the reduction of molybdenum to molybdenum by the anti - oxidant compounds and subsequent formation of green molybdenum complexes with the maximum absorption 695nm. A higher antioxidant activity of crude extract than isolated saponin could be attributed to the higher concentration of phenolic acid and their derivatives. The difference in activity between methanol and butanol fraction was found to be non significant. In case of defatted seed meal from methanol extract exhibited higher activity. This method is quantified as the activity expressed as the number of equivalence of ascorbic acid or -tocopherol depending on the composition of sample product analysed. The antioxidant mechanisms of vitamin C (ascorbic acid) are based on hydrogen atom and electron donation to lipid radicals, quenching of singlet oxygen and removal of molecular oxygen. Also, its antioxidant activity has been related with tocopherol and flavonoids regeneration. When values were compared to soya meal total antioxidant assay, it was reported about 55mg/mL and 66.9 mg/mL in both the crude extracts of saponins from karanja and soybean seed meals respectively. Hence, soya has relatively high total antioxidant assay when compared to karanja seed meal.

65

Indian Institute of Technology, Kharagpur. (Biotechnology-based value-addition of leaves, oilseeds, and cakes of Neem & Jatropha) Creation of a phytochemical libraryLibrary of purified fractions rich in quercetin, quercitin glucoside and chlorogenic acid from neem leaf, and quercitin glucoside, quercetin and ferulic acid from jatropha leaf have been made. A hydrogel formulation A hydrogel formulation with arabinogalactan (from gum Arabic) and Jatropha phenolics is ongoing. In silico study In silico probing of anti-microbial activity of hydrogel ingredients against 2RJT and 3HJT surface proteins (Streptococcus mutans) are in progress. This targets inhibition of biofilm formation during dental decay and skin infection. A. Harvesting of tissue for extraction of phenolics and flavonoid Plant material were collected, air dried crushed with liquid nitrogen using a mortar and pestle into fine powder and used immediately or kept in 20C for further use. B. Extraction methods: The basic protocol followed for the extraction of terpenoids and phenolics:

66

Hydrogel preparation LC-MS analysed phenolics rich fractions were used at 100 ug/kg levels in 1% gum Arabic gel. In vitro testing against Streptococcus mutans shows its efficacy in inhibiting growth. c) In silico evaluation of anti-microbial activity Docking experiments of quercetin/quercetin monoglucoside against 2RJT and 3HJT of S mutans show plausible interaction with the microbe and the gel. Chemical structure of the compounds was converted to PDB files using Dundee package. Protein crystal structure was used for docking, by patchdock/autodock softwares. Significant Results A. Library of phenolics rich fractions 1. Neem Phenolics /Flavonoids Mass 248.3 464.3 Identification Quercetin Quercetin glycoside 354.3 Chlorogenic acid 2. Jatropha Phenolics /Flavonoids Mass 464.4 338.7 194.2 Identification Quercetin glucoside Quercetin Ferulic acid

Oil Technological Research Institute, Anantpur, Andhra Pradesh Standardization of Quality Parameters of Oil and Bio-diesel of Jatropha and Karanja for commercial use. Esterification: A known amount of karanja / jatropha oil was taken in the abovementioned setup. Required amount of sulphuric acid and methanol were added to the oil and stirred continuously maintaining a steady temperature of 640C. Intermittently samples were collected at regular intervals (30min) and acid value was determined. After the confirmation of complete reduction of acid valve to less than 1.0, the heating was stopped and the products were cooled. The unreacted methanol was separated by separating funnel. The remaining product was analyzed for acid value and it was found that the acid value varied from 1.0 to 0.5. This oil sample was used for transesterified to obtain methyl esters.

67

Transesterification: In the same setup, known amount of esterified karanja / jatropha oil was charged. Required amount of catalyst KOH / NaOH was dissolved in methanol and the rest amount of methanol along with the catalyst solution was added to the oil sample. After proper closing of the flask it was put on the water bath. The system was maintained airtight to prevent the loss of alcohol. The reaction mixer was maintained at temperature just above the boiling point of the alcohol i.e. around 700C to speed up the reaction rate. Excess alcohol was used to ensure total conversion of the oil in to its methyl esters. The formation of methyl ester was monitored by measuring the viscosity of the reaction mixer. Reduction in viscosity confirms the formation of methyl esters. This procedure was followed for all the samples collected at regular interval of time to check the formation of methyl esters. After the confirmation of complete formation of methyl esters, the heating was stopped and the products were cooled and transferred to a separating funnel. Where the ester layer containing mainly methyl ester and methanol and glycerol layer containing mainly glycerol and methanol were separated. The pH level of both layers were measured and neutralized separately. For neutralization a known amount of sulfuric acid in methanol was added to both the layers separately to neutralize the sodium methoxide present in them. The traces of methanol present in ester layer were recovered in a distillation column under controlled vacuum. Distilled methanol was weighed and stored in sample bottle. Similar procedure was adopted to recover the traces of methanol present in glycerol layer. The methyl ester was washed and dried under vacuum to remove traces of moisture. A sample of esters was analyzed for acid value by using standard AOCS procedures for standardization. The sample of glycerol layer was analyzed for glycerol content by using AOCS procedure. The glycerol content was found from 80 to 85 %.

Fig: 1. Basic scheme for biodiesel production

68

(a) Fig: 2. Settling of (a) karanja and (b) jatropha bio diesels PHYSICAL PROPERTIES.

(b)

The physical properties of bio-diesel are compared to the petro-diesel in Table-1 Table-1: Physical properties of bio-diesel and petro-diesel S. No. 1. 2. 3. 4. 5 6 7 8. 9. 10. 11. Physical properties. Fuel composition. Petro-diesel Hydrocarbons Bio-diesel Fatty acids methyl esters. 1.9 to 6. 130 C 0.05 Max 47 min of

Kinematic Viscosity @ 2.0 to 5. 40 C in cst Flash point C 35 C Min Sulphur, % mass 0.25 max Cetane number 45Min Pour point 3 C for winter 15 C for summer Oxygen % Almost negligible. Carbon % Sulfur Lubricity Material Compatibility 87 (low sulphur diesel < 1.2 (low sulphur diesel) Low Degrades Natural rubber.

Up to 11% free oxygen. 77 None High No effect.

Table -2: Comparison of properties of pongam and jatropha bio-diesels

69

S.No..

Characteristics

Pongam Bio- Jatropha Bio-diesel diesel 0.89. 5.12 166C Nil Clear. 0 .25% 8.1 85% < 5.0 0.882 4.86 163C Nil Clear. 0.10 7.8 93% 1.5

1. 2. 3. 4. 5. 6. 7. 8. 9.

Specific gravity. Kinematic Viscosity at Cst, 40 C Flash point (Penskey- Martens closed cup apparatus) Water content Appearance Carbon residue % by mass (Rams bottom apparatus) PH (PH meter) Methyl ester% Acid value.

The viscosity of liquid is an important characteristic as it determine the ease of flow through pipeline, injector nozzle and orifices and formation of fuel in the cylinder. The viscosity of karanja oil was found to be higher compare to the Karanja bio-diesel. Flash point measures the tendency of the sample to form a flammability mixture with air. The flash point of Karanja bio-diesel was found to be higher in comparison to the HSD. The acid value can be used to guide in quality control of fuels. In the case of karanja oil it is found to be less than 5. Similarly higher the carbon residue value, the greater the expected carbon deposits in the combustion chamber. Therefore, carbon residue is intended to provide some indication of the relative coke forming properties. It was observed that the carbon residue of Karanja oil was higher than the karanja bio-diesel. Thus Bio-diesel has the potential to change the entire scenario in the field of energy. It is not only pollution free and renewable but it has the good impact on the economy of India.

Sardar Vallabh Bhai Patel University of Agriculture & Technology, Meerut Development of value added products from leaves and oil cakes of Neem, Jatropha, Mahua & Karanja by using them as substrate for mass multiplication of Trichoderma spp.

Effect of Different moisture level in Neem cakes on Growth and development of Trichoderma harzianum. Growth and development of Trichoderma were monitored after each 15 days. All flasks were taken out (Autoclaved/Non-autoclaved) from incubator at Room temperature and 1 gm cake was taken from all of them and used for colony count, using water agar media. Similer practices were repeated after 45 days, 70 days and 105 days. Following observations have been made :

70

S Moistur N e level (%)

CFUs of Trichoderm after different internals 15 days 45 days 70 days 105 days BOD Room BOD Room BOD Room BOD Room Temp. Temp. Temp. Temp.
Autoclave NonAutoclave NonAutoclave NonAutoclave Nond autoclave d autoclave d autoclave d autoclave d d d d

1 2 3 4 5 6

10 15 20 25 30 40

Avg. 17.66 18.33 41.00 48.00 27.33 28.33

Avg. 18.33 20.00 38.33 39.00 37.66 15.33

Avg. 39.33 35.00 60.33 52.00 27.66 42.00

Avg. 36.00 41.33 63.00 57.33 33.66 31.66

Avg. 21.66 37.66 81.33 40.66 35.33 34.00

Avg. 27.00 36.66 53.66 64.33 46.33 33.66

Avg. 10.00 18.33 32.66 31.66 23.00 15.66

Avg. 17.33 28.33 33.00 30.66 30.00 23.00

Results:- This experiment were conducted during the period April August 2010. It was observed that all cakes looks brown black after one month but in Non-autoclaved cakes containing 20% and 25% moisture they becomes powdery white. T. harzianum CFUs were recovered at 15 days, 45 days, 70 days and 105 days also from the Neem cake stored at 270C in the BOD and the Neem cakes stored at room temperature also. Experiment N0. 2 Effect of different moisture level in Jatropha cakes on growth and development of Trichoderma harzianum. Growth and development of Trichoderma on Jatropha cakes were monitored after each 15 days interval. For monitoring the growth after 15 days all flasks were taken out (Autoclaved/Non-Autoclaved) from incubator and 1gm of Jatropha cake was taken from each flask which was used for monitoring growth and development (CFUs) using water agar media. Similar practices were repeated after 25 days, 35 days and afterwards. Following observations have been made:

71

SN Moisture level (%)

1 2 3 4 5 6

10 15 20 25 30 40

CFUs of Trichoderm after different internals 15 days 25 days 35 days BOD Room BOD Room BOD Room Temp. Temp. Temp. Autoclaved Non- Autoclaved NonAutoclaved Nonautoclaved autoclaved autoclaved Avg. Avg. Avg. Avg. Avg. Avg. 19.00 17.66 33.00 28.66 34.66 21.33 28.66 28.66 29.00 24.33 45.00 19.66 20.00 27.66 63.33 45.33 46.33 41.66 45.33 49.66 67.00 42.33 68.66 66.00 62.00 83.00 33.33 39.66 47.33 51.66 79.00 29.00 50.66 34.66 42.00 45.66

Results:- This experiment were conducted during the period June July 2010. Abundant number of CFUs of T. harzianum were recovered from Jatopha cake upto 15, 25 and 35 days of incubation. Recovery of CFUs of T. harzianum after 35 days and on wards will be monitored in due course of time for which experiment is already in progress. Experiment N0. 3 Effect of different moisture level in Mahua cakes on growth and development of Trichoderma harzianum. Growth and development of Trichoderma were monitored after each 15 days interval. For monitoring the growth after 15 days all flasks were taken out (Autoclaved/NonAutoclaved) from incubator and 1gm weight of Mahua cake was taken from each flasks it was used for monitoring CFUs using water agar media. Similar practices were repeated after 15 days, 25 days and afterwards. Results have been given as hereunder : SN Moisture level (%) CFUs of Trichoderma after different internals 15 days 25 days BOD Room Temp. BOD Room Temp. Autoclaved Non-autoclaved Autoclaved Non-autoclaved Avg. Avg. Avg. Avg. 2.33 1.00 0.66 1.00 3.33 5.66 5.00 2.33 14.33 6.33 7.00 5.33 7.33 9.33 6.33 10.33

1 2 3 4

10 15 20 25

72

5 6

30 40

3.33 4.33

5.00 3.33

1.00 6.33

4.00 3.00

Results:- This experiment were conducted during the period June - July 2010. Abundant number CFUs of T. harzianum were recovered from Mahua cake at 15, and 25 days of incubation. Recovery of CFUs of T. harzianum after 25 days and on wards will be monitored in due course of time for which experiment is already in progress. Conclusion: 1. Initial trends showed that Neem cake was better than Jatropha and Mahua cake for mass multiplication of Trichoderma spp. as it supported longer survival i.e. upto 105 days. 2. Its was also noticed that sterilized cakes as substrate were better than non-sterilized cakes. 3. Autoclaved Neem cake with 20% moisture supported highest CFU upto 70 days. 4. Autoclaved Jatropha cake with 40% moisture resulted in highest CFU in 25 days and 35 days. 5. Non-Autoclaved Neem cake at all moisture level supported the survival of Trichoderma upto 45 days also. 6. Autoclaved Jatropha cake with 20, 25 and 30% moisture resulted good No. of CFU at all moisture level. 7. It was also observed that Trichoderma mass cultured on Neem, Jatropha, Mahua & Karanja cakes, when applied to soil containing pots and planted with tomato, there was recovery of Trichoderma CFUs upto 105 days. Experiments to determine, whether Trichoderma can survive any more longer is under progress. 8. Based on present investigation it can be further concluded that T. harzianum mass cultured on ckaes of Neem, Jatropha, Mahua and Karanja is able to survive upto 105 days in the rhizosphere of tomato and unplanted soil as well. Dr.Y.S.Parmar University of Horticulture and Forestry, Nauni, Solan, H.P. Development of post harvest equipments and refinement of technology for extraction and value addition of Wild apricot oil for commercial adoption Development of method for removal of bitterness component (HCN) from wild apricot kernels Wild apricot seeds are known to contain inherent bitterness component, the cyanogenic glucoside, amygdalin which is hydrolyzed by inherent enzyme glucosidase in the presence of moisture to HCN, glucose and benzaldehyde. The presence of HCN causes the seeds to be bitter. HCN being water soluble, only a small quantity of this component may be found in the mechanically pressed oil while press cake left after oil extraction contains substantial quantity of HCN and thus renders it unfit for use as animal feed. The press cake on the other hand is a rich source of proteins and can be used as an excellent animal feed if the toxic component causing bitterness is removed. Therefore this component must be removed from the seeds.

73

For removal of HCN, various trials were conducted on wild apricot kernels and oil press cake. To ascertain the nature of the compound which causes bitterness, the press cake was extracted sequentially with different solvents. Samples were evaluated qualitatively as bitter or non bitter. The bitter compound was found to be soluble in ethanol and water. Further, different experiments consisting of leaching, soaking and steaming of wild apricot kernels and press cake were conducted. Only the treatment consisting of crushing of kernels, soaking in excess of water followed by boiling or steam distillation for a specified period resulted in elimination of bitterness while leaching with water also reduced it substantially. However, even the presence of very small amounts of the bitter compound renders the material unacceptable, thus making its complete removal absolutely necessary. Further, the large excess of water and long period of heating necessary for removal of bitterness limit the use of the method on commercial scale. Therefore further work is being conducted to develop a commercially viable method of debittering of wild apricot kernels. Among different methods of removal of HCN, the immersion of apricot kernels in 10 % sodium thiosulphate solution for 30 minutes was found to be effective for complete detoxification of kernels, while 20 minute treatment resulted in oil with no HCN. Immersion of wild apricot kernels in 25% salt solution for 50-60 minutes was also found effective for complete removal of HCN from the kernels, while the oil was free of HCN after 30 minutes treatment. However, blanching of wild apricot kernels in boiling water for 10 min. followed by oil extraction was optimized as the oil was found completely free of HCN in a comparatively shorter time, i.e. 10 minutes. The initial level of inherent bittering component, hydrocyanic acid (HCN) was found to be 154.8, 81.9 and 39.6 mg/ 100g respectively in kernels, press cake and oil .

74

Fig 3. Traditional method of oil

extraction

Fig 4. Table oil expeller (Y2K)

Fig 5. Filter press for oil

Fig 6. Packaging of wild apricot oil in bottles and PE pouches

75

Demonstration and providing Service to the farmers on Wild apricot oil extraction: Technology optimized for extraction of oil at commercial scale has been transferred to six oil processing units established in different parts of the state and outside state for providing demonstration and/or training to the farmers of the wild apricot growing regions. The four oil processing units established in HP are: i) ii) iii) iv) Wild apricot oil extraction unit at KVK Shimla at Karalash (Rohru), Distt. Shimla. Wild apricot oil extraction unit at KVK Kinnaur at Reckong Peo, Distt. Kinnaur Wild apricot oil extraction unit at Horticultural Research Station, Seobag, Distt. Kullu Wild apricot oil extraction unit at Deptt. of Food Science and Technology, UHF, Nauni, Solan.

All the four units are equipped with one set each of mechanical decorticator, table oil expeller and oil filter press along with other accessories for extraction of oil from wild apricot stones. Besides, the technology has been successfully transferred and adopted by following two self help groups in District Kinnaur in Himachal Pradesh. i) World Wide Fund (WWF) for Nature-India, Rakcham (Sangla Valley) Distt Kinnaur HP has adopted the technology of apricot oil extraction and established oil extraction unit. ii) Sushree Khormoshu, Self Help Group, Spillow, Distt Kinnaur has adopted the technology of apricot oil extraction and established oil extraction unit. During the period under report 525 kg of wild apricot stones were decorticated to separate 167 kg kernels. The service for wild apricot extraction consisting of breaking of stones as well as oil extraction was provided to 10 farmers against nominal charges. Conclusion: Post-harvest equipments for decortication/breaking of wild apricot stones using modified mechanical decorticator with additional screens and Mechanical separator for separation of kernels have been developed. Improvement in mechanical decorticator consists of attaching additional screens (3 vertically arranged sieve trays of 12.46mm, 7.8mm and 6.15 mm dia) with the decorticator which can be used for grading of stones as well. The unbroken stones thus collected in a separate tray can be recycled for decortication, thus preventing mixing of small stones with kernels. Four size grades as large, medium, small and very small were found in wild apricot stones. Additional screens with decorticator can reduce the quantity of broken kernels to 2.7% against 11.3% without screens. Specific gravity separation method has been optimized and recommended for separation of the kernels from the crushed mass. But due to apprehension of absorption of 76

salt in the kernels and its presence in the extracted oil, efforts were made to develop a mechanical separator for separation of kernels.. The modified mechanical separator consisting of coarse rubber belt moving in opposite directions from flow of decorticated mass of mixed shells and kernels which are dropped from height to allow the spherical kernels to roll on belt whereas shells being coarse are carried by the moving belt to the other end of separator. Dipping of decorticated mass in water for overnight has been optimized to cause swelling of kernels which facilitate rolling on the belt thus resulting in separation up to 70% as against 47% separation obtained without dipping of stones. However, the wild apricot kernels are bitter in taste due to the presence of inherent amygdaline which upon hydrolysis release glucose, benzaldehyde and HCN which causes bitterness in the kernels. This bitterness component needs to be removed for the use of extracted oil in cosmetic preparations and utilization of press cake as animal feed. Out of different combinations, soaking of kernels in water followed by boiling or steam distillation for a specified period resulted in elimination of bitterness while leaching with water also reduced it substantially. However, on the basis of preliminary observations, blanching of wild apricot kernels in boiling water for 10 min. followed by oil extraction was optimized as the oil was found completely free of HCN in a comparatively shorter time, i.e. 10 minutes. Further, the technology optimized for oil extraction at commercial scale has been transferred to six oil processing units established in different parts of the state for providing demonstration and/or training to the farmers of the wild apricot growing regions. Four oil processing units have been established in HP under different research stations of Dr YS Parmar, University of Horticulture & Forestry well equipped with one set each of mechanical decorticator, table oil expeller and oil filter press along with other accessories for extraction of oil from wild apricot stones. Two Shelf Help Groups have also adopted this technology and installed oil processing units. The service has been provided to 10 farmers which included decortication, kernel separation and oil extraction. TNAU, Coimbatore Standardization of seed enhancement and storage technique, seed certification standards for Jatropha and Pungamia and standardization of seed handling techniques in Jatropha The evaluation of seed testing procedure and standards for biofuel crops viz., Jatropha, Pungam, Mahua, Neem and Simarouba expressed the following 1. Seed Testing Procedures
Seed testing methodology/standards Submitted sample size Working sample size Jatropha Pungam Mahua Neem Simarouba

Physical purity factors

1kg 1kg 1kg 1kg 1kg 1 kg 1 kg 1 kg 1 kg 600 to 700 g *(1880g to *(4070 g *(4170 to *(1786 g to *(570-625g) 2113 g) to 4498 g) 4433 g) 1961 g) *Actual weight of working sample Pure seed, inert matter and other crop seeds

77

Purity standards

Pure seed (%) Inert matter (%) Other crop seed 16 25 41 23

98% 2% Nil 23

Germination period (days to final count) Seedling evaluation

Normal seedlings include seedlings with good root and shoot growth, bald headed and twine seedlings. Abnormal seedlings includes diseased, deformed and albino seedlings. Germination (%) after 15-63% nine months (as depending 23% 10-11% bulk)** on sources Least germination at 27% after 2 12% after specific periods** months 4 months **To be conformed and improved with seed management techniques River Sand followed by vermiculite In sand method (sowing at 2 cm depth) 75.8 to 174.1 to 172.2 to 23.5 to 94.1 to 81.2 g 183.7 g 191.0 g 25.1 g 102.4 g Eight Preconditioning - 16h soaking Preparation - bisecting the seed Tetrazolium Staining Concentration Incubation period (400C) Depth of sowing at STL Orientation of sowing at STL Sand moisture holding capacity (dry weight basis of sand media) Moisture estimation Moisture content 2- 3 cm Radicle downwards 50-60% 2-3 cm Flat 2 cm Radicle downwards 50 % 1% 3h 1-2 cm Flat 70 % 2-3 cm Flat 50-60%

Media for germination Methodology Range on test weight (100 seed weight) Number of replications seed viability testing (Plate.1 for Jatropha)

Seed broken into pieces and kept at 130 0c for 3-4h Seed moisture content vary widely with fresh and dry seeds as ageing 9.1 - 9.7% 10-11% 8.5 10% proceeds. To be confirmed.

78

2. Seed Handling Techniques Jatropha Karanja Pod colour variation, observed both in fresh and dried pods revealed that germination percentage were maximum in yellowish brown pods followed by light brown seeds (90, 83 and 61, 65 respectively) and it accompanied with higher vigour characters and better storability. On colour grading, Yellowish brown seeds recorded the maximum germination percentage both as fresh and after three months of storage (82 and 58 % respectively). The seed germination was also accompanied with additive seedling vigour characteristics. Comparison of differential seeds per pod (single and double seeded pods) based on color and size expressed that single seeded pods recorded the maximum seed quality characters both initially and after storage. However seeds from double seeded pods recorded higher germination as fresh, but the seedling quality characters both initially and after storage were inferior. In both categories yellowish brown and bigger seeds recorded higher seed quality characters (Plate 2, 3 and 4). Specific gravity grading of pod and seed and selection of heavy pods / seeds improved the quality of seed both in fresh and as well as in the stored pods. In handling stored seeds, size grading and selection of medium size seeds recorded 8% higher germination than bulk seeds. Yellow color of fruit served as physiological maturity index and seeds at this stage recorded the highest germination percentage (88%). Size grading of seeds with 10mm width round perforated metal sieves recorded the highest recovery of 84% and germination percentage of 95%. Specific gravity grading and selection of heavy seeds improved the seed germination (78%) and seed storability (15% germination after one year).

Mahua

On size grading medium sized seeds recorded higher seed germination (53%) and was followed by the bigger sized seed (45%) irrespective of seed source. On size grading of kernels/ true seed, medium and bigger sized seeds (30%) recorded higher seed germination than bulk (25%) by 5% with Pollachi seed source.

79

Neem Size grading of seeds using 8mm round perforated metal sieves enhanced the seed germination by 10% compared to bulk.

Simarouba The studies on fresh fruit color variation (yellowish green, pink and dark pink) on seed quality characters expressed that light and dark pink fruit recorded 100% germination and higher seedling vigour. Specific gravity grading of seeds (fruit) expressed that selection of medium and heavy seeds improved the seed and seedling quality characters.

3. SEED ENHANCEMENT TECHNIQUES Jatropha Karanja Seed fortification with 1.0% Phosphobaterium with the soaking duration of 1h had better seed invigourative effect, which was 15 and 18% higher than water and control respectively with respect to germination percentage. In nursery also the performance was better. In organic fortification, seeds soaked in 0.50% Nutrigold (byproduct of bone meal) recorded the highest germination and seedling quality characteristics which were 22% ans 21% higher than control in respect of germination and dry weight of seedlings respectively. Seeds soaking with 100 ppm GA3 for 16 h improved the seed germination by 18% in fresh seeds and by 19% with stored seeds. Seed fortification with 0.5% K2SO4 or KH2PO4 improved the germination by 13 and 8 per cent respectively. Bio priming with 0.5% liquid biofertilizer viz., Azospirillum and Phosphobacterium improved the germination by 17 to 16 % respectively.

Mahua

Seed fortification with 2%Thiourea with the soaking duration of 24h had better seed invigourative effect, which was 33.8% and 46.1% higher than water and control respectively with respect to germination percentage (plate 5).

80

Simarouba Seed fortification with 0.5% MgSO4, or 0.5 per cent KH2PO4 or one per cent KCl for 24 h improved the seed germination by 88 to 86% which was 7% higher than the control.

4. Seed Storage Studies Jatropha Seed storage studies conducted with seed coating, seed pelleting, fungicidal treatment and grading revealed that management techniques could extend the germination to longer periods which has to be confirmed. % increase over control Bulk Based on initial germination and seed source storability of seeds very The seeds with 60 to 65% germination recorded 13 to 27 % germination after 9 months. The seeds with 80 to 90 % germination recorded 35 to 39 % germination after 9 months. Graded seed 10 mm round perforated 5 months 19 metal sieve (retained seeds) Polymer coated 2.5 ml /kg with 5 ml of 9 months 12 seed water Pelleted seed Pungam pelleting 9 months 22 Fungicidal Bavistin 4g /kg 7 months 33 treatment Karanja Storage studies with fresh seeds revealed that (Coimbatore seed source) germination reduced to 10%, after 9 months of storage without any management technique. Seed pelleting with neem leaf powder recorded higher germination both initially and after storage (23% after nine months). Seed pelleting with Azophos recorded higher germination under germination room and nursery condition. Even after 6 months of storage Asophos pelleting recorded 16% higher germination than control. Polycoating with red polymer @ 0.25ml to 0.5ml diluted with 5ml of water recorded 5 % higher germination even after 7 months of storage. Seedling vigour was also maintained at higher order Storage technique best treatment Storage period

81

Mahua

Storage studies with fresh seeds revealed that (Pollachi and Pattukkottai seed source) seed could maintain 27% per cent germination after two months of storage.

Neem Storage studies with fresh seeds of Arur and Pattukkottai seed sources revealed that Arur seeds can be stored upto 3 months with 18% germination while seeds of Pattukkottai stored upto 4 months with 15% germination.

Simaruba Seed storage studies with 5 different seed sources revealed that seed can be stored upto one year with 19% germination. Seed pelleting with adathoda leaf powder recorded higher germination both initially and after one year (60% after one year). Storage studies with fresh seeds revealed that (Mettupalayam seed source) seed treated with bavistin @ 2g /kg could maintain 63 per cent germination after 12 months of storage.

5. Studies on seed structure The studies on seed structure, structural components and their percentage contribution in seed structural formation in biofuel crops revealed the following Contribution of structural components in biofuel crops Components of fruit used Components of true as seed seed/ kernel True seed/ Endosper Fruit coat Seed coat Embryo kernel m 20 41 80 59 45 11 1 5 5 89 99 95 50 -

Seed Jatropha Pungam Mahua * Neem *

Simarouba* 70 30 7 93 * Seed with endocarp (nut) is used as common seed for sowing. 6. Studies on seed maturation Karanja

Studies on seed development and maturation in pungam expressed the seed color changed from yellowish white to light brown with advance in maturation and

82

were germinable from 16th weeks after anthesis and the maximum was recorded at 26th weeks after anthesis (Plate 11). Simarouba Simarouba seeds attained physiological maturity 63 days after anthesis with the maturation symptom of dark pink coloration of fruit (Plate 12).

83

KARANJA (Pongamia pinnata)

The National Network on Karanja is being implemented through 24 R&D institutes in 17 states viz; Andaman & Nicobar Island (1), Andhra Pradesh (3), Bihar (1), Chhattisgarh (1), Delhi (1), Gujarat (1), Haryana (1), Karnataka (2), Kerala (1), Maharashtra (3), M.P. (2), Orissa (1), Punjab (1), Rajasthan (14), Tamil Nadu (1), Uttar Pradesh (2) and West Bengal (1)

A.

Survey and collection of superior planting material (seeds / cuttings from different agro-climatic zones)

The number of CPTs identified by various research organizations in respect of Karanja are here as under
Sl. No. Name of Research Institutes No. of CPTs /Seed Source identified Upto 2009-10 2010-11

Total

1 2
3

NAU, Navsari TNAU, Mettupalayam CCS, HAU, Hisar (biotic and abiotic)

43 64 33 200 340

3 124 18 26 22 24 11 228

46 188 51

4. 5 6 7

CCS, HAU, Hisar (Dr. Hooda) OUAT, Bhubaneshwar CARI, Portblair KAU, Thrissur Total

226 22 24 11 568

B.

Progeny, Zonal & National trials of superior planting material

FCRI(TNAU), Mettupallayam: Karanja Progeny evaluation trial showed that significant highest tree height was recorded in TNMP12 (2.2 m). Collar diameter was high in TNMP 37(70.3mm), TNMP 44(69.3mm) and TNMP 45 (69.2mm). High Number of branches was recorded in TNMP 44(6.7 Nos), TNMP 37(6.2 Nos) and TNMP 1(5.4Nos). CCSHAU Hisar: The seed samples obtained from different locations of Uttar Pradesh showed a wide range of variation in terms of growth characters. Seedling height varied from 7.42 cm (UP-10) to 10.33 cm (UP-16) with a general mean value of 8.77 cm

84

whereas, minimum and maximum seedling diameter was recorded in UP-4 (2.81 mm) and UP-13 (3.61 mm), respectively. A considerable variation was also observed among the no. of leaves of seedling raised from seed samples of selected 20 CPTs from Uttar Pradesh which ranged between 6.17-11.50 in UP-15 and UP-17, respecticely. Germination percentage of the seedlings was observed 71.43-96.43% in UP-19 and UP16, respectively whereas germinative energy was minimum in UP-10 & UP-2 (28.57) and maximum in UP-14 (75%) with a general mean value of 51.07%. The 21CPTs of Karanj from Rajasthan were selected from different districts namely Udaipur, Rajsamand Chittorgarh, Bhilwada, Banswada, Pratapgarh etc based on seed bearing capacity as well as on the criteria used for the selection of plus trees and grown in screen house. Wide variations were observed in the growth characters of seedlings. Seedling height ranged between 7.75-12.50 cm with a mean value of 9.46 cm. Seedling diameter ranged from 2.61 to 3.82 mm. Similarly no. of leaves varied from 6.00-13.17, the highest being reported from RJ-20 followed by accession RJ-18 (10.83). Germination percentage of the seedlings also exhibited variations. In the present investigation, RJ-18 showed 100% germination among seed samples collected from various districts of Rajasthan whereas only 25% germination was observed in RJ-3. RJ15 showed the highest (75%) germinative energy followed by RJ-8 (71.43%). A total of 12 CPTs of karanj were selected from the different locations of Haryana namely Hisar, Rewari and Gurgaon districts. The growth characters of the seedlings were measured. Seedling height varied from 8.17 cm (H-2) to 12.08 cm (H-9, H-11) with a general mean value of 9.51 cm, whereas, maximum and minimum seedling diameter was recorded as 3.41 mm in H-4 and 2.13 mm in H-1. The no. of leaves also varied considerably among the seed samples of selected CPTs which ranged between 5.17-11.33. Wide variations for germination of seed were also observed and H-4, H-5, H10 and H-12 showed 100% germination whereas the lowest germination (75%) was recorded in H-9. In case of germinative energy maximum (89.29%) was found in H-7 and minimum (25%) was recorded in H-11 indicating ample scope in genetic improvement of this versatile plant through individual plant selection. JNKVV, Jabalpur: Growth parameters: Seeds of 22 provenances (collected 2005-06) were transplanted in the field at the age of 45 days. At the age of 5 years (Dec. 2010) all the provenances showed no significant difference in plant height, basal diameter and diameter at breast height. However provenance T13 (Nagod road, Satna) recorded higher plant height (372 cm) and dbh (40.07 mm), whereas T21 (KVK Seoni) recorded lowest plant height (231 cm), basal diameter (34.57 mm) and dbh (26.10 mm). Reproductive biology Out of 22 provenances, six provenances viz., T8 (Bandole-2), T7 (Cantt area), T12 (Lalpur Nagod Road) ,T14 ( 4th mile Mandla), T17 (Lalpur-2, Nagod road) and T 20

85

(Khajurahoo road) bear flower at the age of 5 years. In all the six provenances flower was started from first week of april and maximum well developed buds were observed during 2nd week of April to 1st week of May. Total period of flowering varied from 24 to 49 days. Initially in the month of May 2010 number of pods/tree in different progenies were counted from 20 to 220 number. After six month (i.e. Dec. 2010) number of pods /tree retained varies from 13 to 105. At present pods are green and in development stage. Multi-locational trial In multi-locational trial conducted during 2005-06, all the seven accession received from MPKVV(5) and TNAU (2) showed no significant difference in plant height, basal diameter and diameter at breast height. However accession T2 PKVK PRW recorded maximum plant height (308 cm), basal diameter (52.83 mm) and dbh (28.13 mm) whereas accession T5 (RAK-22) recorded lowest plant height (272 cm), basal diameter (44.53 mm) and dbh (207 mm). Recommended genotypes based on performance in progeny trial : Name of Research Recommended genotypes Institution CRIDA, Hyderabad Progeny Trial: Acc-21, Acc-23, Acc-4 & Acc-7 Zonal Trial: Acc-14, TNMP-23 & TNMP-9 National Trial: RAK-22, RAK=103, RAK-19 & TNMP-1 Progeny Trial: RAK-4, RAK-13, RAK-14, RAK-35 and RAK-28 National Trial: RAK-103, TNMP-21 Zonal trial : TNMP-9, TNMP-23, DWRP-1, RAK-6 TNMP-6, T18-Lalpur Satna and T6-GCF Jabalpur Zonal trial : T9 -TFRI-3 and T1-JNKVV-12 NAU Karanja-8, NAU Karanja-9
TNMP12,TNMP 37,TNMP44, TNPM 45

Sl. No

MPKV,Rahuri

3 4 5

TFRI, Jabalpur NAU, Navsari TNAU, Mettupalayam

6 7 8 9 10

NRCAF, Jhansi PDKV, Akola MPUA&T, Udaipur CARI, Port Blair UAS, Dharwad

NRCP-16,13 &24 National Trial: Maharastra RAK-22 Zonal trial : KZ-1 RPK-14 CARI ANK-1 and 2 Zonal trial : CPT-28 (Shimoga) & CPT-29 (Dharwad)

86

NRCAF, Jhansi Molecular analysis in P. pinnata The genetic diversity of 18 Pongamia germplasm was also assessed by Randomly Amplified Polymorphic DNA (RAPD) molecular marker. Initially 160 random primers obtained from Operon technologies Inc., USA were used in the study. After preliminary testing on a few samples, 40 primers that gave clear and reproducible banding patterns were selected for the final study. These 40 random primers produced 396 RAPD loci (average of 9.9 bands per primer) across the 18 accessions studied, out of which 341 loci (86.2%) were polymorphic (fig. 1). Primer informativeness was measured by utilizing four marker utility parameters viz. Polymorphic Information Content (PIC), Resolving power (Rp), Effective Multiplex Ratio (EMR) and Marker Index (MI). RAPD primers viz. OPC13, OPT2 and OPT5 having resolving power of 25, 22 and 21 were able to distinguish most of the Pongamia accessions under the study. The average Shannon index (H) of 2.1 indicated a relatively high genetic variation in the germplasm collection. Cluster analysis based on Jaccards similarity coefficient using UPGMA grouped all the 18 accessions into two major clusters (fig. 2). Distribution of the accessions among the clusters did not result in comprehensively distinct clustering patterns based on their geographical origin. The pair wise genetic similarity among 18 accessions ranged from 0.39 to 1.00 with an average of 0.66, suggesting that the current germplasm collection preserved the vast majority of the natural variation in Pongamia pinnata. Grouping of accessions using cluster analysis and principal coordinate analysis (PCoA) were similar and no discrepancy was observed. The result of the study showed that the analyzed accessions have wide range of diversity at morphological and molecular level. The investigations will also be very useful in choosing the precious accessions for further breeding programs. The observations demonstrate the usefulness, limitations and resolution power of molecular markers in comparison to classical morphological descriptors for analyzing the P. pinnata genome for which no prior genome sequence information is available.

87

Fig.1. RAPD profile of 18 genotypes of Karanj (Pongamia pinnata)

Fig 2. Dendrogram depicting the classification of 18 Pongamia pinnata accessions constructed through UPGMA method and based RAPD data. The scale at the bottom is Jaccard coefficient of genetic similarity.

88

NAU, Navsari Molecular characterization Total thirty genotypes selected for molecular and biochemical characterization. Large amount of variation could be observed. Total forty RAPD and Twenty ISSR primers were screened and used for molecular characterization. More number of primers are under investigation since larger the number of primer better the precision. Dendrogram of Karanj genotypes using RAPD and ISSR markers

Dendrogram of 30 genotypes in Karanja using ISSR marker

Dendrogram of 30 genotypes in Karanja using RAPD marker

89

RAPD and ISSR profile of 30 genotypes of Karanja

OPE-14

OPM-3

DE-3

DE-5

DE-15

DE-10

a) Biochemical characterization: Major stress related enzymes diversity and its correlation with growth performance is under observation. Development of harsh site tolerant clone: Cuttings of 30 genotypes has been established. In green house, mimic condition of harsh site will be created to evaluate the clones for harsh site tolerance.the selected clone will be subjected to national trial. Agri-silvicultural trial (Inter-cropping) : FCRI(TNAU), Mettupallayam: It was found that soybean was not performing well under Pongamia when trees attain 3 years. Hence, soybean was not included as intercrops during fourth and fifth intercropping.

90

During fourth and fifth intercropping also, cowpea and groundnut followed by blackgram were performed better as intercrops under Pongamia pinnata. Hence it is concluded that the agricultural crops viz.,, cowpea, groundnut and blackgram could be the suitable intercrops for karanja based agroforestry system. Soil pH The results showed that the pH under karanja based agroforestry system was decreased when compare to initial value (7.22). Among the treatments, the lowest pH was recorded with green gram and red gram (7.17).However among the treatments, the difference was insignificant. However, the differences among the treatments were meager. Soil EC Increased soil EC under karanja based agroforestry system after four years of intercropping was observed when compare to initial value. Among the treatments, highest EC was recorded with cowpea and red gram (0.079dSm-1) followed by green gram (0.078dSm-1) and the least with sunflower and grain amaranth (0.073 dSm-1) . Soil Available Nitrogen The initial available N content of the experiment field was found to be 225 Kg ha-1. An increase in soil available nitrogen was recorded in all treatments including karanja alone. Among the intercrops, the highest available N was found in redgram (238 Kg ha-1) followed by cowpea (236 Kg ha-1) and green gram (235 Kg ha-1) and black gram( 23 5 Kg ha-1 )and lowest was with grain amaranth and sunflower(230Kg ha-1). Soil Available Phosphorus The initial soil phosphorus content of the soil was 13 Kg ha-1. The available phosphorus was increased slightly from its initial value including karanja alone treatment. Among the intercrop, the groundnut registered highest P with a value of 17 Kg ha-1 followed by redgram (16 Kg ha-1). However the difference among the treatments was minimum. Soil Available Potassium The initial soil recorded 275 Kg ha-1 of soil available potassium. The soil K content was increased in all the treatments. Among the intercrops, groundnut recorded highest available K with a value of 295 Kg ha-1 followed by redgram (292 Kg ha-1 ) and the lowest in grainamaranth (282 Kg ha-1). Noticeable difference was observed between the treatments. Seed viability of Karanja NAU, Navsari For development of clonal propagation technique, the cuttings were subjected to various doses of Auxin. Among various auxin and the doses tested, 2000 ppm IBA gave maximum rooting and establishment (appx. 80-90 %). Good proliferation observed but problems occurs during rooting and further hardening.

91

Wild apricot (Prunus armeniaca)

The National Network programme on Wild Apricot is being implemented by involving 4 R&D centres and one each in Delhi, H.P., J&K and Uttrakhand. Survey and collection of superior planting material (seeds/cuttings) from different agro-climatic zones
Additional 26 CPTs of wild apricot has been identified by SKUA&T, Srinagar totalling to 152 CPTs identified by the institute so, far.

GBPUA&T, REC, Chaubattia: The genotype T-24 collected from field of Smt Tara Joshi, Village - Charree Dunagiri Block Dwarhat) Almora, had maximum oil content of 50.98%. Almora, Nainital, Uttarkashi, Chamoli, Tehri and Dehradun (Chakrata) are the potential area for wild apricot. HPKV, Palampur : Wild apricot seeds collected from 167 selected trees were evaluated and considerable genetic variation for oil content, stone and kernel characters were recorded. Oil content ranged from 50.05 - 57.97%, while range of stone length, breadth and thickness was from 14.64 - 26.48, 12.26 - 21.49 and 8.63 - 14.65 mm, respectively. Stone and kernel weight varied between 66.60 - 295.10 and 18.20 - 68.18 g. Kernel size (length, breadth and thickness) and weight are most desirable characters which affect oil percentage. On the basis of the correlation studies, it was found that kernel thickness (0.292) and kernel weight (0.236) was positively and highly significantly correlated with the oil content along with the stone thickness (0.211). However, kernel weight has positive and highly significant correlation with all the stone and kernel characters. SKUAT, Srinagar : Progenies from 126 identified CPTs of Kashmir, Ladakh and Chenab Valley during the period 2006 to 2009 were evaluated in the nursery from time to time. On nursery evaluation these seed sources showed great variation in germination, survival and morphological parameters. The maximum germination of 76.33 percent was recorded in seed of Zainatrag (Pulwama) CPT (WA)-88 and minimum germination of 5.33 percent was recorded in Ajas (Bandipora) CPT (WA)-65 whereas maximum survival of 66 percent was recorded in Mahawara (Budgam) CPT (WA)-76. On recording the morphological parameters it was found that height of the seedlings ranged between 46.33 and 189.2 cms and collar diameter between 3.17 and 12.28 cms. Growth performance of 26 CPTs [CPT(WA)-76 to CPT(WA)-101] in the field after one year of transplantation, recorded maximum average height of 107.97 cm in Ishgam (Budgam) CPT(WA) - 82 whereas maximum average collar diameter (8.72 mm) was recorded in Chowgam (Shopian) CPT(WA) -99. The minimum average height of 58.46 cm was recorded in D.K. Pora (Shopian) CPT(WA) 96 and minimum collar diameter (5.40 mm) was recorded in Khrew(Pulwama) CPT(WA)- 87.

92

Estimation of oil content extracted from the kernel samples Candidate Plus Trees :

of identified

SKUAT, Srinagar : The estimation of the oil content extracted from the kernel samples of 126 CPTs identified from Kashmir, Ladakh and Chenab valley till 2009-10 reveled maximum percentage of oil content i.e. 56.38 % in the kernels of seeds collected from Changund (Kargil) provenance CPT (WA)-13 and minimum oil content of 25.52 % in the samples of Akchmal (Kargil) provenance CPT (WA)-15. Whereas, in case of CPTs identified from Kashmir and Chenab valley ( excluding Ladakh) till 2009-10 ,the maximum oil content was recorded in the kernel samples of Budgam district. Among the CPTs of this district the maximum oil content of 54.2 % was recorded in the kernels samples of Karewa CPT (WA)-77. Estimation of oil from kernels samples obtained from 26 CPTs of Wild apricot identified during the current year i.e.2010-11 of Kashmir Valley recorded maximum oil content of 52.7 % in the kernels samples of Dooru (Budgam) provenance CPT (WA)-148 and minimum oil content of 47.6 % was recorded in the kernels samples of Tengpora (Pulwama) provenance CPT (WA)-136. The average oil content in kernel samples of CPTs identified from Kashmir, Ladakh and Chenab valley was found to be 48.6%,

93

Progeny, Zonal & National trials of superior planting material SKUAT, Srinagar After completion of the nursery studies, the saplings of 17 seed sources were transplanted in the field during March 2006, at Shuhama to study their growth performance in the field. On recording the growth parameters after four years of transplantation it was found that the maximum average height of 517.34 cm was attained by the saplings of Onigam provenance and minimum 385.77 cm by Chitternar provenance whereas the maximum average diameter of 64.94 mm was recorded in Saspool (Kargil) provenance and minimum of 53.30 mm was recorded in Budgam provenance. National Trial : SKUAT, Srinagar Plants raised from wild apricot seeds received from three different states viz. J&K, HP & Uttrakhand were transplanted in the field for evaluation during 2007. Four years data with respect to the growth parameters in the field revealed that Maximum average height of 148.25cm was recorded by the Minji (J&K) source and Minimum average height of 95.56cm by the provenance from Gudam (Uttranchal). The Maximum average stem diameter of 20.27mm was recorded in Poyen (J&K) and minimum average collar diameter 13.12mm in Gudam (Uttranchal) provenance. CSK HPKV, Palampur : The studies were carried out to find out the effect of stratification and chemical treatments on seed germination and subsequent seedling growth of wild apricot. Seeds collected from mature, healthy and disease free fruits were divided into two groups (stratified and non stratified). Two groups of seeds (stratified and unstratified) were subjected to soaking in various chemical solutions of GA3, Thiourea and KNO3 at room temperature for 24 hours and sown in the field. Stratified seeds had exhibited significantly higher germination (80.13%), seedling survival (64.55%), seedling height (138.8 cm) and seedling biomass (287.0g). However, stem diameter, sturdiness quotient, leaf area and shoot/ root biomass ratio was not significantly affected by stratification treatment. Amongst the chemical treatments, KNO3 @ 0.3% had shown the significantly higher seed germination (91.24%), seedling survival (92.19%) and shoot to root biomass ratio (2.16). Whereas, mean seedling height (156.2cm), stem diameter (11.24mm), leaf area (19.20cm2) and total biomass of seedlings (348.7g) were recorded to be maximum under GA3 @ 500 ppm treatment. Seeds subjected to stratification followed by KNO3 @ 0.3% had significantly higher seed germination (94.00%), seedlings survival (93.63%), seedlings total biomass (367.9g) and shoot/ root biomass ratio (2.29). However, seedlings height (159.4cm), stem diameter (11.44mm) and leaf area (20.00cm2) was

94

found to be significantly higher in seedlings raised from seeds stratified first and then treated with GA3 @ 500 ppm. Recommendation: 1. The best commercial method of propagation is tongue grafting. 2. The cuttings cannot be used for the commercial propagation of wild apricot plants 3. Mound layering does increase the number of plants, however, this technique cannot be used commercially for plants multiplication, though in future this technique may be exploited when clonal rootstocks suitable for different agroclimatic conditions are evolved . The Green wood grafting may be practised if one misse the season for tongue grafting, however, the success rate will be approximately 70%.

Research on other Tree Borne Oilseeds

The R&D efforts are also being made on Mahua, Simarouba, Kokum, Neem, Jojoba and Tung by involving following R&D centres : S. No 1 Name of TBOs Simarouba Name of participating centre Dr PDKV, Akola Acharya N.G. Ranga Agricultural University(ANGRAU), Hyderabad CCS HAU, Hissar (Dr.Dhillon) Dr PDKV, Akola, IGAU, Raipur CCS HAU, Hissar (Dr.Dhillon), NDUA&T, Faizabad SVBPUA&T, Meerut Dr BSKKV, Dapoli

2 3 4

Mahua Neem Kokum

The number of CPTs identified by participating centres in respect of simarouba, mahua and kokum are hereunder:
S.No Name of TBOs Name of Institute No of CPTs identified (2010-11) Seed samples sent to NBPGR for cryopreservation

Simarouba

2 3

Mahua Kokum Total

Dr PDKV, Akola ANGRAU, Hyderabad CCS HAU, Hissar (Dr.Dhillon) CCS HAU, Hissar (Dr.Dhillon) Dr BSKKV, Dapoli

10 8 15 50 19 102

2 0 0 0 2 4

95

Simmarouba (Simarouba glauca)

PDKV, Akola :

Survey and collection of superior planting material (seeds / cuttings from different agro-climatic zones)
All the participating institutes have conducted survey in their agro-climatic zones and selected Candidate Plus Trees (CPTs)/potential seed sources. After selection, seeds/cuttings were collected from these sources. The seeds were characterized for their morphological features like seed length, diameter, test weight and oil content (%).The oil content of some of the genotypes is given as hereunder:
Genotype Kernel Oil (%) Genotype Kernel Oil (%) Genotype Kernel Oil (%)

PDKV SG-1 PDKV SG-2 PDKV SG-3 PDKV SG-4 PDKV SG-5 PDKV SG-6 PDKV SG-7

58.12 48.04 54.85 50.00 49.80 53.47 54.57

PDKV SG-8 PDKV SG-9 PDKV SG-10 PDKV SG-11 PDKV SG-12 PDKV SG-13 PDKV SG-14

53.71 54.49 47.67 48.13 46.90 55.99 54.13

PDKV SG-15 PDKV SG-16 PDKV SG-17 PDKV SG-18 PDKV SG-19 PDKV SG-20

53.33 49.61 58.23 52.54 53.57 58.59

Vegetative propagation of Simarouba: Grafting experiment of Simarouba is being undertaken in College nursery primacies .The cutting of female mother trees were grafted on one year old seedlings of Simarouba raised in the nursery in the month of June July 2010. It was found grafting of pencil size girth were successful. The survival of successful graft is satisfactory. Air laying experiment was undertaken on mother female trees with different root initiation growth hormone i.e. IBA, NAA & IAA with different concentrations i.e. 500ppm, 1000ppm, 1500ppm, 2000ppm & 2500ppm. The best result shown in IBA with concentration more than 1500 and 2500ppm. Cutting experiment was undertaken on Simarouba glauca with IBA, NAA & IAA with different concentrations i.e. 500ppm, 1000ppm, 1500ppm, 2000ppm & 2500ppm with above the chemicals but this experiment could not result in success. The highest survival & growth parameter were observed in seeds sown on bed compared to that sown on polythene bags and seedling transplanted from bed to polythene bags. CCSHAU, Hissar A total of 15 CPTs of Simmarouba glauca was identified and seed material was collected by conducting an intensive survey in Gujarat. The selections of CPTs was made on the basis of characters of economic interest In addition, four seed sample of CPTs

96

were obtained each from PDKV, Akola and ANGRAU, Hyderabad. The seed and kernel morphological characters were recorded. A wide variation in seed and kernel character (length, breadth and thickness) was recorded among the selected CPTs. Hundred seed and kernel weight varied from 74 148g and 25-41gm, respectively. At the time of chemical analysis of seeds, moisture content in the seed sample varied from10-11.6% . The protein content also showed considerable variability among different seed sample and it ranged from 18.5-21.4%, with general mean of 19.63%. The maximum protein was analyzed from the seed of SG-9 and SG-10 CPTs from Gujarat which were closely followed by SG-5 CPT. The oil content in the collected seed samples varied from 44-58%, having general mean of 50.09% indicating ample scope of genetic improvement of this versatile tree species. To raise the progenies of individual CPTs, seeds were sown in polythene bag filled with equal proportion of sand, soil and FYM. For each CPTs, 800 seed were sown in 400 polybags and these bags were kept in four block representing four replications. Regular irrigation was applied up to seed germination. After one month of seed sowing, germination percentage was recorded and it varied from 25-76. The plant growth characters of the progenies were recorded at an interval of 3 months in nursery. The significant variation was recorded in plant height and collar diameter after 3, 6, 9 and 12 months of age. The nine months old nursery raised progenies of individual CPTs were transplanted in the field at a spacing of 4x4 meter following Randomized Block Design in the month of March, 2010. The growth and reproductive parameter are under critical observations and will be recorded at an interval of one year. National trials- I(2009) The trial of simarouba was conducted in 3 replications both under irrigated and rainfed conditions with standard package of practices. A total of 12 genotypes were incorporated in NT-I from 3 states i.e. Maharashtra (Dr. PDKV), Hisar (CCS HAU) and A.P. (ANGRAU): Dr. PDKV, Akola The genotype Palam 1 showed maximum plant height (16.31 cm) followed by PDKV SG 1 (16.16 cm). Equal girth of 0.37 cm was recorded in Palam 1, Palam 2 and PDKV SG 4. However, the maximum canopy was recorded 21.76 cm & 21.41 cm in genotype Palam 4 & HAUP 09, respectively. ANGRAU, Hyderabad Data was collected from the transplanted seedlings in terms of plant height, collar diameter and number of branches, during year 2010-11. The highest plant height was recorded in Palem-3 (117cm). However, it was statistically on par with PDKV SG-025 and PDKV-SG-023. Highest collar diameter was recorded in Palem-3, PDKV SG-025 and HAU-P-13 (4.4cm) simarouba accession. However, it was statistically on par with Palem-4, Palem-2, Grafts and PDKV-SG-30. The highest number of branches was

97

recorded in Grafts (8) simarouba accession. However it was statistically on par with Palem-2, HAU-P-28, HAU-P-13, PDKV SG-025 and PDKV-SG- 27. Intercropping trial in Simarouba: In view of the potential offered by the Simarouba for intercropping owing to its wider spacing (5m x 5m) intercropping trial was initiated with popular crops grown in the Southern Telangana Zone of Andhra Pradesh. In total six crops were evaluated as intercrops during 2010-11. The intercrops were sown during 1st week of July, 2010. The inter crops and their varieties / hybrids evaluated were Sorghum (Palem-1), Pearl Millet (PHB-3), Redgram (PRG-158) and Castor (PCH-111). The highest grain yield of 23.80 q/ha was recoded in case of bajra crop and highest gross returns of Rs. 33,337 / ha was recorded in case of redgram crops due its higher market price. Details of the yield and gross returns in different crops tested are as following: S.No 1. 2. 3. 4. Yield Gross Income Yield (q/ha) (Rs/ha) (q/acre) Sorghum 3.1 7.75 7,750 (@Rs. 1000/q) Pearl Millet 10.1 23.80 21,431 (@Rs. 900/q) Redgram 4.0 11.11 33,337 (@Rs. 3000/q) Castor 3.2 8.0 28,000 (@Rs. 3500/q) Mahua (Madhuca indica) Crop

CCSHAU, Hisar Identification of Candidate Plus tree selection: Ripened fruits of Madhuca latifolia (Mahua) plus trees were collected from different parts of Haryana and Uttar Pradesh. In total 50 CPTs were identified. During survey, a lot of variability was observed in morphological characters and fruiting intensity of selected CPTs. Seeds were separated from fruits and seed samples were measured for seed traits. Seeds from individual plus tree were also shown in polythene bags having potting media to test the performance of progenies. The significant variation was found in seed morphological characters of selected plus trees. Seed length and breadth ranged from 25.73-36.91 and 12.66-21.03, respectively. The 100-seed weight varied between 113.17 and 229.85g. The maximum 100-seed weight of 229.85g was found in seeds samples from MPT-38 and minimum of 113.17g was recorded in MPT-14. The progenies of selected plus trees of mahua were raised in nursery in the polythene bags filled with germination media and aftercare was undertaken adopting customary management practices. The percent germination varied from 63.7 to 86.7, the highest being was recorded in seed sources MPT-28. The minimum germination of 63.7 per cent was observed in MPT-05 seed sample. At the age of three months, a wide variation in growth characters (shoot & root length, basal diameter, number of leaves) was found in the progenies of different candidate plus trees at nursery stage. The nursery raised progenies were transplanted in field at a spacing of 5x5m following RBD design during first fortnight of November, 2010.

98

Air-layering in mahua To standardize propagation techniques in different mahua through air-layering, one year old healthy branches with 1.5 to 2.0 cm girth were selected. Girdles of 3.0 to 4.0 cm length were made with a budding knife at 30 cm from the branch base. The layers were made during March-April 2006. The plant growth regulators used were IAA, IBA and NAA with different concentrations viz., 500, 1000, 1500, 2000 ppm and control (treated with distilled water). Under each treatment, 5 girdles were made. The cut surface was covered with moist sphagnum moss grass and polythene sleeves. Both the ends were tightened with thread. The observations on percent rooting, no. of roots/layer and root length in different air-layers was recorded after three months. The successful rooted airlayers were detached from the parent plant and were transplanted in pots filled with sand, soil and FYM in equal proportion. Different growth, regulators showed different responses. No rooting was observed in air-layers treated with 500 ppm NAA and 1000 ppm IAA . Maximum percent rooting was recorded with higher concentration (2000 ppm) of IBA and NAA. The average number of roots/layers was found in 1500 ppm IAA followed by 500 ppm IBA. Mean maximum root length (3.3 cm) was recorded in air-layers treated with 1000 ppm IBA and minimum (0.58 cm) in 1500 ppm NAA treatment.

Jojoba (Simmondsia chinensis)

CCSHAU, Hisar Germplasm collection: In jojoba (Simmondsia chinensis), there is no natural/planted population in Haryana. The seeds of different elite genotypes of jojoba were collected from Association of the Rajasthan Jojoba Plantation and Research Project (AJORP), Dhand and Fatehpur Farms, Rajasthan. The seeds of each genotype were sown in nursery. Seed characters, oil content, per cent germination and seedling characters were observed. Seed morphological traits exhibited significant variation among different genotypes. A wide variation in seed length, breadth and thickness was recorded. 100-seed weight ranged between 72.8 (Q102) to 95.8 g (Q-104) with a general mean of 85.9 g. Oil content varied from 43.6 per cent (C-64) to 51.9 per cent (Q-104), with general mean of 48.7 per cent. Maximum germination of 90.5 per cent was recorded in seed sample of local genotype followed by Q-104. The minimum percent germination of 46.2 was observed in C-64. Similar variation in seedling growth character was also recorded in progenies of different jojoba genotypes. During seed germination few albino plants were also observed but these plants survived for 30-35 days and after that they died. The albino plants showed stunted growth as compared to normal plants. Since albino seedlings do have chlorophyll pigment, they die as soon as the reserve food material stored in the endosperm is exhausted by the seedlings. The albinos are homozygous recessive lethals arising either from selfing or crossing between the two heterozygous individuals. Besides, mutations, whether induced or spontaneous, albino seedlings may be produced by mating of albino carriers under natural conditions. 99

Kokum (Garcinia indica)

Dr.Balasahab Sawant Konkan Krishi Vidyapeeth, Dapoli (Ratnagiri), Maharashtra

1. A primary survey & collection of various early bearing & high yielding CPTs of Kokum was done from various parts of Konkan region of Maharashtra. 2. It was observed that kokum is mostly distributed in Kudal, Walawal, Dabholi, Adiware, Pawas, Sangameshwar, Dapoli areas of Konkan as well as Goa part. 3. A total of 19 high yielding CPTs have been identified from Konkan region of Maharashtra, in which four belonged to early bearing type and remaining are mid late to late bearing individuals. 4. In Goa region, 2 CPTs are selected which show high yielding and bold type nature. 5. The overall fruit weight of kokum ranged from 17.72 to 71.82 g; the fresh rind weight varied from 6.73 to 34.73 g (Table No. 2). 6. Rind thickness also showed significant variation and it ranged from 0.08 to 3.75 mm. 7. Number of seeds per fruit, seed weight and T.S.S. also showed significant variation among 19 different genotypes. 8. The total seed weight per fruit ranged from 1.73 to 6.55 g. 9. Oil content in selected CPTs was recorded using SFE (Super Critical Fluid Extraction System) with CO2 as the supercritical liquid and it ranged from 31.34 to 52.73% from the selected CPT. 10. The seeds of the selected CPTs have been subjected to progeny testing and are at the nursery level. 11. The grafting of total 40 selected CPTs will be done in January, 2011 and the material will be maintained in the nursery. 12. The Eco-physiological studies of the early bearing Kokum CPTs is under progress.

100

Chemical Evaluation of Tree Borne Oilseeds

Chemical Evaluation of estimation of oil content and other quality parameters in the seeds of jatropha, karanja, wild apricot , cheura and other TBOs is under progress. The following significant achievement have been made: Oil Analysis Laboratory NOVOD BOARD Determination of oil content would be of great value for identification of Germplasm (CPTs) and various field trials such as Progeny, zonal and national trial of the genotypes and to thus create a data bank/ seed bank under the National Network Program of NOVOD Board for augmentation of total production and productivity of vegetable oils from quality seed material. The oil analysis laboratory are already equipped with vital analytical equipments such as electronic weighing balance, SOXTEC- 2045(2-unit), moisture meters, Gas Liquid Chromatography(GLC), Distillation unit etc. and other allied accessories such as chemicals and glass wares for determination of oil content and fatty acid composition. Seed samples are regularly receiving from the various participating R&D institutes under the National Network Program of TBOs such as Jatropha, Karanj, Wild Apricot, Cheura, Simarouba and Mahua. No. of samples have been received from the participation institutes during 2009-10 and 2010-11 are 662 and 723 respectively. Thus total No. of samples have been received during the 2009-11 is 1385 out of which Jatropha(1113), Karanj(160), Wild Apricot(80), Cheur(3), Simarouba(24) and Mahua(5). Oil Analysis Laboratory has started functioning in March, 2010. No. of samples have been analysed during 2009-10 and 2010-11 are 58 and 1187 respectively. Thus, total No. Accession have been analysed during 2009-11 is 1245 of which Jatropha(984), Karanj(149), Wild Apricot(80), Cheur(3), Simarouba(24) and Mahua(5). Report are being sent to the respective institutes regularly. Institute wise samples received and analysed is in Table-1. TBO wise samples have been analysed is in Table-2. Analysis work of seed samples are in progress in oil analysis laboratory. Table-1: Institute wise and TBO-wise seed samples received
S N Name of the Institute Jatroph a Sample received 200910 SVPUA&T, Meerut, UP CSK HPKV, Palampur, (HP) NAU, GUJARAT ICAR, TRIPURA CRIDA, HYDERABAD ICAR, MEGHALAYA DR.YSPUH&T, HP BAU, RANCHI SDAU, GUJARAT HSCS&T, HARYANA CSFER, UP PAU, PUNJAB Karanj Wild apricot 3 4 68 20 64 9 2 19 31 50 38 21 Sample Reciived Cheura Simarouba Mahua Total Reced. 3 4 68 20 64 9 2 19 31 50 38 21

1 2 3 4 5 6 7 8 9 10 11 12

101

13 14 15 16 17 18 19

20 21 22 23 24

25 26 27 28 29 30 31 32

33 34 35 36 37 38 39

40 41 42 43 44 45 46

GBPUA&T, UTTARAKHAND MPAU&T, RAJASTHAN SKUAS&T, J&K PDKV, MAHARASHTRA NAU, GUJARAT PDKV, MAHARASHTRA FRI, UTTRAKHAND Total sample received 2009-10 Sample received 201011 RRS, BAWAL, HARYANA TFRI, JABALPUR, MP NDAU&T, UP KAU, KERALA RARS, UAS, DHARWAD, KARNATAKA BHU, UP UVKV, COOCH BEHAR, WB SDAU, GUJARAT IIT, Khragpur, West Bengal CSFER, ALLHABAD, UP ICAR RESEARCH COMPLEX , MANIPUR AAU, Anand, Gujarat Department of Plant Pathology, UAS, DHARWAD CSKHPKV, PALAMPUR, HP CARI, Andaman & Nicober Islands RRS, BAWAL, HARYANA CSAU&T, UP CCS HAU, Dept. of Forestry, Hisar(Beniwal) MPKV, RAHURI, MAHARASHTRA TNAU, METTUPALLAYAM, TNAU TNAU, Coimbatore BCKV, WB DR.YSPUH&T, HP CARI, Andaman & Nicober Islands CCS HAU, Dept. of Forestry, Hisar(Beniwal) TNAU, Coimbatore University of Agricultural Sciences, Bangalore

28 114

10

41 114 25 24 8 72 49 662

25 24 8 72 541 49 57 37 3 24

43 5 4 2 3

43 5 4 2 3

1 7 20 1 37 12 10 1

1 7 20 1 37 12 10 2

14 2 64 5 27 1 20

14 3 84 5 47 2 5 15

20 2

1 20 3 1 15 2 55

1 20 3 1 15 2 55

102

S N

Name of the Institute Sample received 2010-11 Jatropha Karanj

Sample Received Wild aprico t Cheur a Simaroub a Mahu a Total Reced . 5 46 26 26 11 19 81 39 31 59 11 723 1385

47 48 49 50 51 52 53 54 55

State Forest Research Institute, MP University of Agricultural Sciences, Bangalore SKUAS&T, J&K ICAR RESEARCH COMPLEX , Nagaland CRIDA, HYDERABAD CSFER, UP MPKV, RAHURI, MAHARASHTRA TFRI, JABALPUR, MP KAU, KERALA Total sample received(2010-11) Total received(2009-11)

5 46

11
62 39 31 59 572 1113 11 103 160 43 80 3 24 5 5

Details of the institute wise samples received and analysed is at below given Table. Analysis work of seed samples are in progress in oil analysis laboratory. Table-2: Institute & TBO-wise seed samples analysed
S N Name of Institute Jatropha Karanj Wild aprico t Cheur a Simaroub a Mahu a Total analyse d 3 4 68 20 64 9 2 19 31 50 38 21 3 41 114 25 24 8 72 49 24 8 72 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

SVPUA&T, Meerut, UP CSK HPKV, Palampur, (HP) NAU, GUJARAT ICAR, TRIPURA CRIDA, HYDERABAD ICAR, MEGHALAYA DR.YSPUH&T, HP BAU, RANCHI SDAU, GUJARAT HSCS&T, HARYANA CSFER, UP PAU, PUNJAB GBPUA&T, UTTARAKHAND MPAU&T, RAJASTHAN SKUAS&T, J&K PDKV, MAHARASHTRA NAU, GUJARAT PDKV, MAHARASHTRA FRI, UTTRAKHAND

3 4 68 20 64 9 2 19 31 50 38 21 28 114 10 25

103

20 21 22 23

24 25 26 27 28 29 30 31

32 33 34 35 36 37

RRS, BAWAL, HARYANA TFRI, JABALPUR, MP NDAU&T, UP KAU, KERALA RARS, UAS, DHARWAD, KARNATAKA BHU, UP UVKV, COOCH BEHAR, WB SDAU, GUJARAT IIT, Khragpur, West Bengal CSFER, ALLHABAD, UP ICAR RESEARCH COMPLEX , MANIPUR AAU, Anand, Gujarat Department of Plant Pathology, UAS, DHARWAD CSKHPKV, PALAMPUR, HP CARI, Andaman & Nicober Islands RRS, BAWAL, HARYANA CSAU&T, UP CCS HAU, Dept. of Forestry, Hisar(Beniwal) MPKV, RAHURI, MAHARASHTRA TNAU, METTUPALLAYAM, TNAU TNAU, Coimbatore BCKV, WB DR.YSPUH&T, HP CARI, Andaman & Nicober Islands CCS HAU, Dept. of Forestry, Hisar(Beniwal) TNAU, Coimbatore University of Agricultural Sciences, Bangalore State Forest Research Institute, MP University of Agricultural Sciences, Bangalore SKUAS&T, J&K ICAR RESEARCH COMPLEX , Nagaland CRIDA, HYDERABAD Total samples analysed(2009-11)

43 5 4 2

43 5 4 2

3 1 7 20 1 37 12 10

3 1 7 20 1 37 12 10

1 0 2 64 5 27

1 14 1 20

2 14 3 84 5 47

20

38

39 40 41 42 43 44 45 46 47 48 49 50 51

5 1 20

3 1 15 2 55 5 46 26

15 1 20 3 1 15 2 55 5 46 26 11 81

11
62 984 19 149 80 3 24 5

1245

104

Abbreviations Used
2-4-D AFLP BAP CPTs IAA NAA RDBMS RAPD ISSR UPGMA MS SEM DNA BA GA3 WPM PGR PVP AS PSB PPM CPE TDZ : : : : : : : : : : : : : :
:

2-4. Dichlorodiphenyl Trichloro Eathene Amplified Fragment Length Polymorphism Bengyl Amino Purine Candidate Plus Trees Indole.3 Acetic Acid Nepthalene Acetic Acid Rationale Data Base Management System Randomly Amplified Polymorphic DNA Inter Simple Sequence Repeat Unweighted Pair Group Method with Arithmetic Mean Murashige and Skoog Medium Scanning Electron Microscopy Dioxyribo Nuclic Acid Benzyle Adenine Gibberellic Acid Woody Plant Medium Plant Growth Regulators Polyvinyl Pyrrilidene Adenine Sulphate Phosphate Solubilizing Bacteria Part Per Million Cumulative Pan Evaporation Thiodiazuron

: : : : : : : :

105

Published by NOVOD Board, Gurgaon & Printed by M/s Aryavart Mudranalaya, New Delhi-45 Ph. 011-25053794