Bud break and multiple shoots were induced in apical and axillary meristems derived from one month old seedlings of Sapindus mukorossi. Forest trees in general have proved to be difficult to mass propagate by tissue culture. Some success however has been achieved in a few woody tree species.
Bud break and multiple shoots were induced in apical and axillary meristems derived from one month old seedlings of Sapindus mukorossi. Forest trees in general have proved to be difficult to mass propagate by tissue culture. Some success however has been achieved in a few woody tree species.
Bud break and multiple shoots were induced in apical and axillary meristems derived from one month old seedlings of Sapindus mukorossi. Forest trees in general have proved to be difficult to mass propagate by tissue culture. Some success however has been achieved in a few woody tree species.
Micropropagation of Sapindus mukorossi Gaertn N S Philomi na* & J V S Rao Department of Botany, S. V. University, Tirupati 517 502, India Received 13 April 1999; revised 16 December 1999 Bud break and multiple shoots were induced in apical and axillary meristems derived from one month old seedlings of S. mukorossi on Murashige and Skoog (MS) medium supplemented with benzylamino purine (BAP) 0.4 J.1M or 0.8 J.1M alone. A combination of BAP and gibberellic acid (GA 3 ) 0.4 J.1M and 2.8 J.1M produced elongated multiple shoots from both types of explants. Excised shoots were rooted on MS medium respectively with indole-3-butyric acid (IBA) 3.4 J.1M or 2.4 J.!M. The regenerated plantlets were successfully acclimatized and transferred to soil. Forest trees in general have proved to be difficult to mass propagate by tissue culture. Some success however has been achieved in a few woody tree species. Importance of plant tissue culture for mass propagation of forest trees I ike Eucalyptus 1 , Sandal wood 2 and Rose wood 3 has already been demonstrated. Micropropagation by the method of organogenesis and by multiple shoot production of axillary meristems of seedling explants have been reported in Leucaena 4 , Albizia 5 and Acacia 6 . Shoot tip cultures were established from germinated seedlings in Redsanders 7 and Teak 8 So far there are very few reports of Sapindaceae like Sapindus trifoliatus 9 established by tissue culture. Sapindus mukorossi Gaertn. or Sapindus detergens Roxb, soapnut is a perennial tree belonging to the family Sapindaceae, indigenous to northern India. Oil from the seed kernel of soapnut is of interest to the soap industry. The oil is quite useful industrially because of its most valuable phytochemicals like saponins or trigl yceri des 10 The exhausted cake is used as a filler and fertilizer and the shells for making lignin based adhesives or boards 11 Vegetative propagation of soapnut did not yield satisfactory results and propagation through seed is unreliabl e because the per cent survival of the seedlings proved to be meagre due to heavy incidence of mortality at seedling stage in the natural . habi-at 12
Micropropagation of soapnut tree is at a stage of infancy in forest tree species which has great importance in the soap industry and social forestry programmes. In this communication for the first time an in vitro micropropagation method for Sapindus *Present address: Dr. N.S. Philomina, Oo. Smt. Y. Elizabethamma, H.S.G. II P.A. Head Post Office, Cuddapah 516 001, Indi a mukorossi tree using apical and axillary meri stem explants has been presented. Materials and Methods Seeds of soapnut were obtained from Biotechnology Research Centre for Tree Improvement (BIOTRIM), Andhra Pradesh Forest Department Nursery, Tirupati and soaked in cone. H 2 S0 4 for 90 min and washed thoroughly with running tap water. The seeds were surface sterilized with 0.1 % HgCI 2 for 15 min and rinsed several times with sterile distilled water. Agar water medium (0.8%) without growth regulators was used for seed germination. Seeds inoculated on this medium were incubated at 24 2 C in the dark or light at a photon flux density of 15 11Em 2 S of white fluorescent tubes for thirty days after which seedlings were used for apical and axillary meri stems di ssection. The apical and axillary meristems (2-4 mm) were collected from one month old aseptic seedlings and cultured on Murashige and Skoog 13 (MS) basal medium containing 2% sucrose and lower concentrations of BAP or KIN ranging from 0.4, 0.8, 1.7 and 2.6 1-l.M, higher concentrations ranging from 4.4,8.8 and 13.2 1-l.M, combination of BAP and KIN 0.4 or 0.4 1-l.M, 13.2 and 13.2 11M and combination of BAP or KIN with auxin naphthalene acetic acid (NAA) 0.4 and 0.4 11M to 13.2 and 13.2 11M were used for inducing muliple shoots. For muliplication and elongation of established shoots different combinations of GA, (0.5 and 2.8 11M) alone, . . combination of BAP and GA 3 (0.4 and 0.5 1-l.M, 0.8 and 0.5 1-l.M, 0.4 and 2.8 1-l.M, 0.8 and 2.8 11M) were tried. For root induction, 2-3 em long shoots were transferred to MS medium with IBA or NAA (0.4, 2.4, 3.4 and 4.9 1-l.M) . Media were sterilized at 15 622 INDIAN J EXP BIOL, JUNE 2000 Ib/sq inch for 20 min. Twenty explants were cultured at 25 2 C in the light (16hr photoperiod) . Rooted plantlets were acclimatized gradually in a green house. Results are mean of three culture cycles with 20 replicates per experiment. Results and Discussion Experiments on explant type, shoot tips and axillary meristems for multiple shoot formation were tested. In all the BAP concentrations tested, 0.4 and 0.8f.tM concentrations were more effective for inducing 6 to 8 multiple shoots within a month from axillary meristems. However on the same medium shoot tips were proliferated and 4 to 6 shoots were formed in 4-5 weeks. Addition of hi gher concentration of BAP (8.8 and 13.2 f.!M) to MS medium induced more callus formation in both the explants within a week from the cut surface, and along the surface from the apical to basal part of the explants. Initiation of callus was faster in all the higher concentrations of BAP. In order to test the passaging on shoot multiplication, the shoots obtained Fig. I-Formation of multi ple shoots regenerated from axill ary buds of S. mukorossi after 30 day of culture; Fig. 2 - Rooting of shoots ~ MS + 3.4 J.lM IBA + 2% sucrose after 15 days of culture; Fig. 3 -In vitro raised S. mukorossi plant 30 days after transplanting to soil. PHILOMINA& RAO: MICROPROPAGATION OF SAPINDUS MUKOROSSI GAERTN. 623 Table I-Effect of growth regul ators on in vitro response of apical and axillary buds derived form one month old aseptic seedlings of S. mukorossi. [Values are means SE from 20 replicates/treatments] Growth Number of shoots/culture Shoot length (em) regul ators Apical bud Axillary bud Apical bud Axillary bud (JlM) BAPor KIN 0.4 6.00.20 8.00.90 2.00.109 3.0 0.214 0.8 4.0 0. 14 6.0 0.20 2.0 0.118 3.5 0.248 1.7 1.0 0.0 2.6 + 4.4 + 8.8 + 13.2 + BAP 0.4+0.4 1.0 0.0 +KIN 13.2+1 3.2 1.0 0.0 BAP 0.4+0.4 + +NAA 13.2+13.2 + Culture response scored 30 days after inocul ati on +=callusing on the ex plants from apical and axillary meristems were separated and recultured on to the same shoot multiplication media (MS with 0.4 or 0.8 f1M BAP) and shoot multiplication was determined after second and third subcultures. Highest number of shoots (8-1 0) were recorded from a single explant within three weeks (Fig. I). No increase in shoot multiplication was observed by prolonging the culture period beyond sixth subculture. Shoots obtained by this method were divided into 5-8mm nodal explants with single axillary bud for further proliferation to increase the number of shoots. These buds proliferated into 5 to 8 multiple shoots in 4 weeks on MS medium with BAP 0.4 and 0.8f.l.M individually. Experiments conducted with BAP in combination of kinetin and auxin showed single shoots with callus formation. Of the two cytokinins BAP was most effective for inducing bud break and shoot proliferation in apical and axillary meristem (Table 1) . Simil ar results were reported in Madhuca I if . [' 14 at1 ow . Within 8 weeks of culture, the regenerated shoots elongated upto 2-3 em in height. Prolonged culture on the same medium did not increase the shoot length. For shoot elongation the shoots were separated and grown on MS medium with GA 3 (0.5, 2.8J.1M)in combination with BAP (0.4, 0.8 f.l.M) treatments. The shoots were elongated upto 5 to 7 em in 4 weeks in all the BAP and GA 3 treatments. Lower concentrations of BAP (0.4 or 0.8 JlM) were favorable for bud proliferation and application of GA 3 to these shoots 1.0 0.0 2.00.119 3. 1 0.119 1.0 0.0 + 1.90.115 + + 2.00.115 + + + + + + 1.0 0.0 2.1 0.122 2.70.169 1.0 0.0 2.3 0.123 2.9 0.217 + + + + + + Table 2-Effect of auxins on in vitro rooting of regenerated shoots of S. mukorossi after 30 days of inculbation Auxins Control IBA NAA IBA + NAA Concentrations ( J..LM) 0.0 0.4 2.4 3.4 4.9 0.5 2.6 5.3 7.9 0.4 (each) 2.4 (each) * 20 repli cates/treat ment repeated thri ce +=callusing at the basal end Mean percentage of rooting ( SE) * 0.0 10.5 0.275 18.9 0.260 68.0 0.478 59.0 0.366 + + + + + + increased their length. For elongated multiple shoot formation with a combination of BAP (0.4JlM) and GA 3 (2.8 JlM) was optimum. In the present study combination of GA 3 with a cytokinin was effective in inducing shoot elongation. Different concentrations of GA 3 alone failed to increase the shoot elongation however when GA 3 was applied in combination with BAP effectiveness of gibberellic acid was improved in causing shoot elongation. Application of GA 3 to in vitro regenerated shoots increased their length in Azadirachta indica 15
The regenerated shoots were transferred to MS medium with IDA and NAA of different concentrations for rooting. Among these concentrations the regenerated shoots were rooted in IDA (3.4 and 4.9JlM) 624 INDIAN 1 EXP BIOL, JUNE 2000 m 15 days of culture (Fig. 2). IBA and NAA (0.4 and 2 4 ~ induced callus at the base of the shoots with poor rooting after 30 days of incubation (Table 2). A combination of IBA with NAA inhibited roots formation and showed only callus at the basal cut ends. Regenerated plantlets were transferred to plastic containers fill ed with vermiculite. During first week the potted pl antlets were covered with polythene bags to provide high humidity. Transpl antation success was 60% (Fig. 3). Plantlets were subsequently transferred to larger pots and gradually acclimatized to outdoor conditions. In the present study multiplication by multiple shoot methods from shoot tip or axill ary meri stems was developed for successful in vitro propagation of Sapindus mukorossi an economi cally important tree. References I Gupta P K, Meht a UJ & Mascarenhas A F, A tissue culture method for clonal propagation of mature trees of Eulayptus torelliana and Eucalyptus camaldulensis, Plant Cell Rep, 2 ( 1983) 296. 2 Rao PS & Bapat VA, Vegetative propagati on of sandalwood plants through tissue culture, Can J Bot, 56 ( 1978) 1153 .. 3 Lakshmi Sita G & Raghava Swamy BY, Regeneration of plantlets from leaf di sc cultures of rose wood: control of leaf abscission and shoot tip necrosis, Plant Sci, 88 ( 1993) 107. 4 Dhawan V & Bhojwani SS, In vitro vegetat ive propagat ion of Leucaena leucocephala Landewit, Plalll Cell Rep, 4 ( 1985) 3 15. 5 Upadhyaya S & Chandra N, Shoot and Plantl et formation in organ and callus cultures of Albizia lebbeck Benth, Ann Bot , 52 ( 1983) 421. 6 Mittal A, Agarwal R & Gupta SC, In vitro development of Pl ant lets from axillary buds of Acacia auriculariformis, Plant Cell Tiss Org Cult , 19 (1990) 65. 7 Lakshmi Sita G, Sreenatha KS & Suj ata S, Plantlet production from shoot tip cultures of red sandal wood (Pterocarpus santalinus L), Curr Sci, 7 ( 1992) 532. 8 Sunitibala Devi Y, Mukherjee BB & Gupta S, Rapid cloning of elite Teak (Tectono grandis L) by in vitro mulipl e shoot production, Indian J Exp Bioi, 32 ( 1994) 668. 9 Desai HV, Bhatt P N & Mehta A R, Pl ant regeneration of Sapindus trifoliatus L (soapnut) through somati c embryogenesis, Plant Cell Rep, 3 ( 1986)-190. 10 Dev I & Guha S R D, Glyceride compos iti on of Sapindus mukorossi (soapnut) oil , Indian J For, 2( 1979) 261. II Karnik M G, Sharma 0 P & Dev I, Studi es on the chemi cal composition and possible utiliti es of soapnuts (Sapindus mukorossi) Indian For, 8 ( 1971) 462. 12 Troup R S & Sapindaceae, The silviculture of Indian forest trees, Vol. I (Claredon Press, Oxford U.K (1 92 1) 232. 13 Murashi ge T & Skoog F, A revised medi um for rapid growth and bi oassays wi th tobacco ti ssue cultures , Physiol Plant, 15 (1962) 473. 14 Rout C R & Das P, Mi cropropagat ion of Madhuca longifolia, Plant Cell Rep, 12 ( 1993) 5 13. 15 Ramesh K & Padhya M A, In vitro propagation of neem (Azadirachta indica A. Juss), Indian J Exp Bioi, 28 ( 1990) 932.
Rachmawati Et Al. 2020. Adventitious Shoots Derived From Leaf Explants in in Vitro Mass Propagation of Indonesian Selected Anthurium Clones (#674085) - 1145272