2 páginas.-- 1 tabla.-- 12 referencias.Research NoteThe article is a joint publication of the COST Action FA1003 "East-West Collaboration for Grapevine Diversity Exploration and Mobilization of Adaptive Traits for Breeding".Peer reviewe

Cantos, Manuel

García Fernández, José Luis

Maghranze, D.

Ocete Rubio, Rafael

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Vitis 54 (Special Issue), 257–258 (2015)long were chosen and disinfected by the following steps: (a) immersion in 70 % ethanol for 1 min; (b) immersion in 12 % sodium hypochlorite (10 % active chlorine) with some drops of Tween-20, for 12 min; and (c) rinsing with sterilized water (three times, 5 min. each time).After disinfection, uninodal explants (0.3-0.5 cm long) with one bud, were placed separately in sterile test tubes (25 x 150 mm) with 8 mL of culture medium (SARMIENTO et al. 1992), modified with 2.5 % sucrose, 0.072 mg∙L-1 of BAP, 0.024 mg∙L-1 of NAA and 0.6 % agar, pH 5.7. Each tube was covered with a plastic cap, sealed with parafilm and placed in a growth chamber at 23 ± 2 °C, 30 µmol∙m-2 s-1 and 16 h photoperiod. Explants from rootstock varieties 'Ramsey', '110-Richter' (110R), '161-49 Couderc' (161-49), '41B’ and 'CH' (from a saline semi-arid zone in Arica-Chile); from Vitis vinifera varieties 'Superior Seedless' (SS), 'Mal-vasia' and 'Pedro Ximénéz' (PX), and from the Andalusian wild grapevines 'Serag', '14/Córdoba/3' (CO3); '14/Rute/1' (CO9); '14/Montoro/4' (CO8); '14/Montoro/3' (CO7) and '23/Guarromán/2' (J3) (OCETE et al. 2007), were obtained from the in vitro germplasm bank of IRNAS-CSIC. Similar uninodal explants (Table) from these accessions were cul-tured in the same micropropagation conditions described above. After 60 d of in vitro culture the number of surviv-ing plants, plant size (length, bud number per shoot and axillary shoot number) and root development (percentage of rooting and root number per plant) were determined.For acclimatization, 10 rooted plants from each Geor-gian wild grapevine obtained in the micropropagation proc-ess were adapted to ex vitro conditions according to CANTOS et al. (1998).Statistical analysis was carried out using IBM SPSS Sta-tistics v. 22. Data were analysed using ANOVA. Tukey test was applied for identification of important contrasts. Differ-ences in percentages cases were compared using z test.Results and Discussion: The average survival of all tested accessions was 78.5 % (Table). All the Georgian ac-cessions, except G6 with a survival of 61 %, were above this value particularly G10 (100 %) and G17 (97.4 %). The three Georgian wild grapevines with higher survival abil-ity were statistically higher than J3, 41B, PX and G6. The average stem length was 2.1 cm. This value is smaller (p ≤ 0.05) than the length recorded for the Georgian accessions G10 (2.9 cm) and G17 (2.8 cm). These values are much lower than for 110R (4.5 cm) and CH (7.1 cm). The aver-age number of buds per shoot for investigated accessions was 3.5 and the shoot number was 0.5, but the maximum values of these traits were detected in the plants belong-ing to CH (11.4 and 1.9 respectively), much higher than in other accessions (p ≤ 0.05). The response of Georgian wild grapevines G10 and G17 was rather high, the average number of buds per shoot was 4.5 and the shoot number was 0.5 for the first sample and 5.4 and 1.0 for the second one which was significantly different in comparison with the same characteristics for the plants belonging to CH.Explants of all accessions developed root system in the same micropropagation medium without any modification of phytoregulators. This behavior of grapevine explants with this medium is well known (TRONCOSO et al., 1990; TRONCOSO et al., 1999). The average rooting percentage in our experiment was 70.2 %, less than that reported by TRON-COSO et al. (1999) (89.7 %). Georgian wild grapevines G10, G17 and G21 presented very high percentages of rooted plants, 87.5, 94.7 and 89.7 % respectively. G1 explants re-Research NoteMicropropagation and in vitro germplasm conservation of Georgian wild grapevinesD. MAGHRADZE1), R. OCETE2), J. L. GARCÍA3) and M. CANTOS3)1) Institute of Horticulture, Viticulture and Oenology. Georgian Agricultural University, Tbilisi, Georgia2) Applied Entomology Laboratory, Faculty of Biology, Sevilla University, Spain3) Institute of Natural Resources and Agrobiology of Sevilla, (IRNAS, CSIC), Sevilla, SpainK e y  w o r d s :  tissue culture; South Caucasus; plant de-velopment.Introduction: Wild grapevine Vitis vinifera ssp sylves-tris Gmel., considered the wild ancestor of the cultivated grapevine Vitis vinifera ssp sativa D.C., is a typical repre-sentative of the Georgian flora (MAGHRADZE et al. 2012). Fifty populations of wild grapevine have been described in this zone of the south of Caucasus. Population size var-ies from 1 to 20 plants, with an average of 3.8 plants per site (CHKHARTISHVILI et al. 2005, MAGHRADZE et al. 2006). It grows mainly in river gorges, the common habitat of wild grapevine. The lack of human selection has resulted in a highly conserved genetic diversity, so it can play an impor-tant role as plant genetic resource for further improvement of grapevine cultivars.In vitro vegetative propagation of grapevine plants (micropropagation) has been used successfully by differ-ent authors (CANTOS et al. 1993, NICHOLSON et al. 2012). These authors reported the development of a high number of plants starting with a few initial explants, in a relatively small space. In vitro growth is often strong due to rejuvena-tion, disease free, optimal nutrition balance and independ-ence of the seasonal period. This technique is usually used for a suitable material conservation.The objective of this research was testing the response of five Georgian wild grapevine accessions to micropropa-gation and comparing the resulting plants with other com-mercial and wild grapevines.Material and Methods: Cuttings from five wild grape-vine plants from two Georgian populations, Bagichala 10 (G10) and Tedotsminda 01 (G1), 06 (G6), 17 (G17) and 21 (G21) located in Aragvi river basin at Dusheti district and in Liakhvi river basin at Gori district correspondently, Kartli Province, East Georgia were taken, base-dipped in a solution with 2.5 % sucrose and 0.6 % of the fungicide benomyl and placed in a growth chamber with 23 ± 2 °C, 111 µmol∙m-2∙s-1 light intensity and 16 h photoperiod. From these cuttings, sprouting shoots with 3-4 buds, 1.5 cm Correspondence to: Dr. M. CANTOS BARRAGÁN, Dept. Biotecnología Vegetal, Spanish National Research Council (IRNAS-CSIC). Seville, Av. Reina Mercedes, 10, 41012 Sevilla, Spain. E-mail: cantos@irnase.csic.es 258 D. MAGHRADZE et al.duced this percentage to 82.9 and the lowest rooting was observed for G6, only 56.1 % with the trend described for the aerial part. The average number of roots was 2.99 for the plants of all 19 accessions. Plants of the five Georgian wild grapevines presented a lower number of roots, in a range between 2.6 (G17) and 1.6 (G10). These values were particularly low (p ≤ 0.05) when compared to plants of Su-perior Seedless, 5.8.Bud number per shoot was significantly correlated (p < 0.0001) with stem length (r = 0.96, p < 0.0001) for all accessions, including Georgian wild grapes. Other re-lationships detected were between shoot number and stem length (r = 0.88, p < 0.0001) and between shoot number and bud number per shoot (r = 0.92, p < 0.0001). There was also a significant correlation between survival and rooting (r = 0.91, p < 0.0001). On the other hand root number did not correlate with survival or rooting. These results dem-onstrated that the rooting was a more important parameter than root number for successful explant culture.The Georgian plants showed in all cases a 100 % ad-aptation from in vitro to ex vitro conditions, indicating the suitable quality of the root system obtained by micropropa-gation.Conclusion: In conclusion, we observed a good re-sponse of the considered Georgian wild grapevines to micropropagation and adaptation, surpassing other wild grapevines and cultivars with great importance in viticul-ture. In consequence, in vitro culture can be an appropriate conservation system for this Georgian material.The article is a joint publication of the COST Action FA1003 "East-West Collaboration for Grapevine Diversity Exploration and Mobilization of Adaptive Traits for Breeding".T a b l eComparison among the average values of parameters obtained in the micropropagation of Georgian wild grapevine accessions (in grey colour) and the other considered accessionsAccessionNumber of explantsSurvival (%)Stem length (cm)Buds number per shootsShoots numberRooting (%)Roots numberG10 8 100 e* 2.9 c 4.5 def 0.5 abcd 87.5 f 1.6 aG17 29 97.4 e 2.8 c 5.3 ef 1.00 cd 94.7 f 2.6 abRamsey 53 96.3 e 1.3 abc 2.3 abcd 0.1 a 94.3 ef 2.9 abG21 57 93.2 de 1.7 abc 3.6 bcdef 0.4 abc 89.7 def 2.3 abG1 33 91.4 bcde 1.8 abc 3.0 abcde 0.1 ab 82.9 bcdef 2.2 ab110R 56 90.3 bcde 4.5 d 5.9 f 1.0 cd 75.8 bcdef 2.7 abSerag 77 89.4 bcde 1.9 abc 4.5 def 0.7 bcd 77.4 bcdef 3.5 abCH 39 88.4 bcde 7.1 e 11.4 g 1.9 e 74.4 bcdef 2.6 abSS 122 83.6 bcde 0.3 a 0.8 a 0.1 a 82.8 cdef 5.7 cCO3 36 78.6 bcde 1.3 abc 3.1 abcde 0.5 abcd 69.0 abcdef 2.4 abCO9 44 77.8 bcde 0.9 ab 1.4 ab 0.2 ab 71.1 bcdef 3.7 abc161-49 24 77.8 abcde 1.5 abc 2.9 abcde 0.1 ab 59.3 abcdef 2.9 abMalvasía 67 74.7 bcde 1.4 abc 3.2 abcde 0.3 ab 51.9 ab 2.5 abCO8 42 68.8 abcd 1.3 abc 1.6 abc 0.1 ab 51.6 ab 2.9 abCO7 43 64.9 abcd 2.3 bc 3.4 bcdef 0.4 abc 64.9 abcd 3.6 abcJ3 42 61.8 a 1.1 ab 2.2 abcd 0.4 ab 56.4 ab 2.6 abG6 28 61 ab 1.4 abc 2.4 abcd 0.2 ab 56.1 abce 2.0 aPX 30 56.9 ab 2.2 bc 4.2 cdef 1.07 d 56.9 ab 4.4 bc41B 37 40 a 1.3 abc 1.5 ab 0.2 ab 36.7 a 3.7 abcAverage 78.5 2.1 3.5 0.5 70.2 2.9* In each column, means followed by the same letter are not statistically different (p = 0.05).CANTOS, M.; CUERVAS, J.; ZÁRATE, R.; TRONCOSO, A.; 1998: Embryo rescue and development of Juniperus oxycedrus subsp. oxycedrus and mac-rocarpa. Seed Sci. Tech. 26, 193-198. CHKHARTISHVILI, N.; MAGHRADZE, D.; GOGISHVILI, K.; TCHIPASHVILI, R.; 2005: CWR: Grapevine, small and minor fruits of Georgia (The Caucasus). 1st Int. Conf. Crop Wild Relative Conservation and Use. Agrigento, 14-17 September.MAGHRADZE, D.; RUSTIONI, L.; TUROK, J.; SCIENZA, A.; FAILLA, O.; 2012: Caucasus and Northern Black Sea Region Ampelography. Vitis (Special issue). MAGHRADZE, D.; MDINARADZE, I.; CHKHARTISHVILI, N.; GOGISHVILI, K.; CHIPASHVILI, R.; 2006: Inventory of wildly growing grapevines in Eastern Georgia. J. Vinodelie i Vinogradarsvo (Winemaking and Viticulture) 6, 39 (in Russian).NICHOLSON, K. L.; TARLYN, N.; ARMOUR, T.; 2012: Effect of phyllotactic position and cultural treatments toward successful direct shoot or-ganogenesis in dwarf 'Pixie' grapevine (Vitis vinifera L.). Plant Cell Tiss. Org. Cult. 111, 123-129.OCETE, R.; CANTOS, M.; LÓPEZ, M. A.; GALLARDO, A.; PÉREZ, M. A.; TRON-COSO, A.; LARA, M.; FAILLA, O.; FARRAGUT, F. J.;  LIÑÁN, J.; 2007: Caracterización y conservación del recurso fitogenético vid silvestre en Andalucía. FALCOR y Consejería de Medio Ambiente. Junta de Andalucía.SARMIENTO, R.; VILLEGAS, A.; MAZUELOS, C.; GARCÍA, J. L.; TRONCOSO, A.; 1992: Influence of the nitrogen source and concentration on N fractions and free amino acid levels of grape vine explants. Plant Soil 144, 255-258.TRONCOSO, A.; VILLEGAS, A.; MAZUELOS, C.; CANTOS, M.; 1990: Growth and mineral composition of grape-vine rootstock cultured in vitro with dif-ferent levels of ammonium nitrate. Plant Nutrit. Physiol. Applic. 41, 653-654. TRONCOSO, A.; MATTE, C.; CANTOS, M. LAVEE, S.; 1999: Evaluation of salt tolerance in vitro-grown grapevine rootstock varieties. Vitis 38, 55-60.

Micropropagation and in vitro germplasm conservation of Georgian wild grapevines

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