OBTAINING PLANTS USING IN VITRO MICROGRAFTING METHOD IN SOME GRAPEVINE VARIETIES (VITIS VINIFERA L)

S. G. Tangolar1, K. Erik2, S. Tangolar2 University of ukurova, Faculty of Agriculture, Department of Plant Protection and Research Institute, 01330 Adana, Turkey1, University of ukurova, Faculty of Agriculture, Department of Horticulture, 01330 Adana, Turkey2

ABSTRACT

The aim of this study was to investigate the possibilities of application of in vitro micrografting method in vines. In the study, shoot tips with apical meristem and 1-2 leafprimordia of Early Cardinal and Yalova incisi were grafted on the cut surface of the hypocotyls of Dogridge, Salt Creek, 1613 C and 41 B American rootstocks under aseptic conditions. From all the grafting combinations tested grafted plants were produced at different rates of success. The best results were obtained from Early Cardinal/41 B and Early Cardinal/Salt Creek combinations with 80 % and 71.4 % grafting success rates, respectively. Considering the other characteristics Early Cardinal combinations produce better results than Yalova incisi.

Introduction

Turkey is located between 36 and 42 north latitudes. Because of its geographic position, Turkeys climate is very suitable for grape growing. In total agricultural production, viticulture has an important share. The vineyard area is about 535 000 hectares with annual production of 3.6 million tons; 1 500 000 tons of it are table grapes and 345 000 tons are raisin either seeded or seedless (1). One of the most important problems existing in Turkeys vineyards is the contamination with viruses and virus-like organism in the considerable part of national grape production area (2,3). Grapevine Fanleaf Virus (GFLV) and Grapevine Leafroll Virus (GLRV) are the most significant diseases. For this reason, for the
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new vineyard establishment, the use of virus-free material is required. Thermotherapy, meristem culture and micrografting techniques were used effectively for producing virus-free plants from contaminated plants (4). Micrografting is especially the most effective technique to obtain grafted plants free from virus and virus-like diseases for the regions in which vineyards have contaminated with Phylloxera and nematodes like Turkey (5, 6, 7). Micrografting were applied with the microscopy under in vitro conditions using sterilized media. This grafting technique in particular, provides the production of many grafted and rooted virus-free plants in a short time. Furthermore, this method provides earlier diagnosis of virus diseases (8) as well as rootstock-scion incompatibilities (9) in comparison with the
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grafting practiced under in vivo conditions. The success rate in micrografting can be affected by different factors such as the age of materials, grafting technique, sterilization conditions, the concentration and the application period of chemical used (7, 10). Baydar and Celik (11) in their research using Kober 5 BB rootstock found the micrografting success rates as 40.9 and 68.2% for Hafizali and Emir varieties respectively. Similarly Cantos et al. (12) succeed to transplant their micrografted plants to the soil with the success rate of 60% by using 110 R, 161-49 C and 41 B M.G. rootstocks with Palomino Fino, Pedro Ximenez and Zaiema varieties. Ben Abdallah et al. (7) reached an accomplishment rate up to 63% with the use of rootstocks germinated in rockwool. Ben Abdallah et al. (13) have also obtained success rates between 15-60% in another study in which the apical meristems of Asli, Beldi and Muscat de Rafraf cultivars were grafted on the rootstocks germinated on the solid medium under in vitro conditions. Cupidi and Barba (14) have examined the applied procedures of the best culture media with shoot tip isolation and the acclimatization of the in vitro germinated rootstocks in peach and grape rootstocks. As a result of their study, the success rates of grafts were found as 70% in peaches and 60% in grapes. In vitro micrografting techniques have being practiced to obtain grafted plants in citrus (15, 16, 17, 18,19); avocados (20); cherries (21,22); peaches (21,23) and apricots (24) as well as in grapes. In this study, by using shoot tips of some grape varieties and American rootstocks, possibilities of production of micrografted plants were searched
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Materials and Methods

In this study, Dogridge, Salt Creek, 1613 C and 41 B American rootstocks, and Early Cardinal and Yalova Incisi grape varieties, grown in the experimental vineyard of Department of Horticulture, Faculty of Agriculture, University of Cukurova, were used, as plant materials. Seeds taken from ripen berries were washed with tap water and then dried and stored in cloth bag under room temperature conditions for a short time. The seeds were then stratified in the wet sand which were autoclaved for 30 min then they were stored at 4 C for three months before sowing (25). In order to obtain seedlings, the stratified seeds were washed with tap water and then sterilized in 30 % sodium hypo chloride containing 0.1% Tween 20 for 45 min. After sterilization the seeds were rinsed three times with distilled water. Excision of the embryos from the seeds was prepared under sterile conditions. These embryos were then cultured on MS medium (26) containing 0.5 mg/1 GAs in petri dishes. Preparation of the scions Shoot tips taken from the scion varieties were sterilized 20 min in 20 % sodium hypo chloride containing 0.1 % Tween 20. The sterilized shoot tips were shortened to 3-4 mm length and placed on MS medium containing 1 mg/1 IBA in test tubes. The shoot tips were transferred to the fresh medium at 3 or 4 weeks intervals for proliferation. Grafting The rootstock seedlings were decapitated just below cotyledons and the roots were shortened to 3-4 cm in length (Figure Ib). Apical meristems 0.1-0.2 mm in length containing 2-3 leafprimordia were cut
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from the shoot tips and then placed on cambial zone in the cut surface of the rootstock seedlings (7,10). The grafted plants were placed on MS medium supplemented with 1 mg/1 IBA. These procedures were carried out under sterile conditions with the aid of a dissecting microscope. Culture conditions The rootstock seedlings, the scions and the grafted plants were kept at 25 1 C with a 16 hour daily photoperiod and 3000-4000 lux light intensity. For the determination of the differences between the rootstocks and the scions, the rate of successful grafts, the rate of shooting, shoot length, number of leaves, number and length of roots were examined.

Results and Discussion

Germination of the embryos isolated from the seeds Embryo germination occurred in one week after the sowing and the embryo became complete plantlets in three weeks (Figu. la). The best seedling growth was observed from the embryos of Dogridge which was followed by 1613 C, 41 B and Salt Creek rootstocks, respectively. As shown in Table 1, the highest germination rate was 98.4% for 1613 C. The germination rate was 91.7, 88.2 and 75.4% for 41 B, Salt Creek and Dogridge, respectively. The results obtained from grafting combinations The highest grafting success (Figure Ic, Id) rate was obtained from Early Cardinal cultivar grafted on 41 B, which was followed by the combination of Early Cardinal/Salt Creek with the rate of 71.4%. The rate was 64.3, 58.3 and 57.1%
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for Early CardinaV1613 C, Early Cardinal/Dogridge and Yalova Incisi/41 B combinations, respectively. The lowest success was 45.1% with Yalova Incisi/1613 C combination (Table 2). The shoot development of grafting combinations ranged between 50% (for Early Cardinal/41 B and for Yalova tncisi/Salt Creek) and 22.2% (for Early Cardinal/ 1613 C) as can be seen from table 2. The shoot length was 5 cm for Early Cardinal grafted on Salt Creek; 4.6 cm for Yalova Incisi on Dogridge and 3.9 cm for Yalova Incisi on 41 B (Table 3). The number of leaves varied between 6.0 (Dogridge) and 9.5 (Salt Creek) for Early Cardinal and 4.8 (1613 C) and 7.0 (41 B) for Yalova incisi (Table 3). According to the characteristics investigated, 41 B and Salt Creek rootstocks for Early Cardinal, 41 B and Dogridge for Yalova incisi were found to be the leading rootstocks. The number and the length of the rootstock roots are presented in Table 4. High number of roots was obtained from Early Cardinal/1613 C and Yalova Incisi/Salt Creek combinations with 4.8 and 4.5, respectively. Yalova Incisi/1613 C combination exhibited better results in terms of root length (5.8 cm), which was followed by Yalova Incisi/Salt Creek combination (5.0 cm). The same values (4.2 cm) were obtained from both Early Cardinal/Dogridge and Early Cardinal/Salt Creek combinations. The shortest root length was determined from Early Cardinal/1613 C and Early Cardinal/41 B combinations, with the values of 1.9 and 1.8 cm, respectively (Table 4). In this study, success rates of grafting were investigated for different variety/rootstock combinations. The highest grafting success was obtained from Early Cardi52

Fig. 1. a. Germination of isolated from the seeds of rootstocks, b. Preparation of rootstock for grafting, c. Graft union point, d. Some examples of successful micrografts

TABLE 1 Germination of embryos isolated from the rootstock seeds Rootstock Dogridge Salt Creek 1613 C 41 B Number of cultured embryos 65 68 64 60 Number of germinated embryos 49 60 63 55 Germination rate (%) 75.4 88.2 98.4 91.7

nal/Salt Creek combination (71.4%). The results obtained in this study are in good agreement with the studies available in the literature. In some studies, the grafting success was reported to be 40.9 % with Hafizali/5 BB and 68.2 % with Emir/5 BB (11); 63% (7); 52-65% (22); 30-50% (15).
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The placement method of the scion apex on the rootstock is one of the most effective factors on successful grafting. Baydar (10) tested that the placement of the scion apex at the top cut, opposite T placement and cleft placement and reported that the most successful method was the top placement of the scion apex. Ben AbdalBiotechnol. & Biotechnol. Eq. 17/2003/2

TABLE 2 The effects of the rootstocks and the varieties on the success of in vitro micrografting Variety Rootstock Number of grafting 12 7 28 5 33 17 51 14 Number of succeed grafting 7 5 18 4 18 8 23 8 Rate of successful graft (%) 58.3 71.4 64.3 80.0 54.5 47.1 45.1 57.1 Number of shot graft 2 2 4 2 6 4 6 3 Shooting rate (%) 28.6 40.0 22.2 50.0 33.3 50.0 26.1 37.5

Dogridge Salt Creek 1613 C 41 B Dogridge Salt Creek 1613 C 41 B

Yalova incisi

Early Cardinal

TABLE 3 The effects of the rootstoeks and the varieties on the shoot characteristics

Variety Early Cardinal

Yalova incisi

Rootstock Dogridge Salt Creek 1613 C 41 B Dogridge Salt Creek 1613 C 41 B

Shoot length (cm) 3.8 4.6 3.1 5.0 3.9 2.4 2.8 3.2

Leaf number 6.0 9.5 6.8 8.5 6.5 5.8 4.8 7.0
TABLE 4

The root characteristics of grafting combinations (for the roots longer than 0,5 cm)

Scion Early Cardinal

Yalova incisi

Rootstock Dogridge Salt Creek 1613 C 41 B Dogridge Salt Creek 1613 C 41 B

Root number 2.5 4.0 4.8 1.5 3.5 4.5 1.6 2.7
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Root length (cm) 4.2 4.2 1.9 1.8 3.6 5.0 5.8 3.8

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lah et. al. (7) carried out a study by using following micro grafting types; Apex placed on the top cut surface ofhypocotyls in contact with cambial region Apex inserted inside the lateral notch on its horizontal surface, in contact with the cambial region Apex placed on the outward contical of the lateral notch surface. According to their report, the second method mentioned above produced the best result with the rate of 59% which was followed by the third one (57%). The results obtained in this study also show that the placement method of the scion apex can be used for successful micrografting. REFERENCES
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