The effects of two retardants (CCC and paclobutrazol) and the new compound XE 1019, applied before grapevine anthesis, were studied in order to increase the number of fertilised embryos and growing plantlets derived from in ovulo culture of seedless cultivars CG 102.011, Emperatriz and Malvinas. No significant differences were detected between treatments with CCC (400 and 800 mg . l-1, applied 2, 3 and 4 weeks before bloom), and the control in the cv. CG 102.011. The number of growing plantlets at 10 weeks after anthesis and at maturity was significantly higher than that from 8 weeks. In applications closer to bloom, CCC treatments increased the number of ovules per berry in the cultivars assayed. From all cultivars, only CG 102.011 showed a significant increase in plantlet production after CCC treatment when clusters were harvested the 10th week after bloom. It is believed that CCC would act through inhibition of endogenous gibberellin synthesis as the cause of ovule abortion. The idea is based on the fact that gibberellic acid can induce seedlessness in some seeded cultivars

Agüero, C.

Riquelme, C.

Tizio, R.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 34 (2), 73-76 (1995) Embryo rescue from seedless grapevines (Vitis vinifera L.) treated with growth retardants by C. AGOERO, C. RIQUELME and R. TIZro Facultad de Ciencias Agrarias, Universidad Nac. de Cuyo, Mendoza, Argentina Sum mar y: The effects of two retardants (CCC and paclobutrazol) and the new compound XE 1019, applied before grapevine anthesis, were studied in order to increase the number of fertilised embryos and growing plantlets derived from in ovulo culture of seedless cultivars CG 102.011, Emperatriz and Malvinas. No significant differences were detected between treatments with CCC (400 and 800 mg-J-1, applied 2, 3 and 4 weeks before bloom), and the control in the cv. CG 102.011. The number of growing plantlets at 10 weeks after anthesis and at maturity was significantly higher than that from 8 weeks. In applications closer to bloom, CCC treatments increased the number of ovules per berry in the cultivars assayed. From all cultivars, only CG 102.0ll showed a signifi-cant increase in plantlet production after CCC treatment when clusters were harvested the lOth week after bloom. It is believed that CCC would act through inhibition of endogenous gibberellin synthesis as the cause of ovule abortion. The idea is based on the fact that gibberellic acid can induce seedlessness in some seeded cultivars. K e y w o r d s : in ovulo culture, embryo rescue, ovule abortion, seedlessness. Introduction The development of new culti vars of good quality, high yield and large berry size is the main purpose of grape breeding programs in order to satisfy the increasing inter-est in seedless grapes (EMERSHAD and RAMMING 1984; Bou-QUET and 0AVIS 1989). Conventional hybridization to obtain seedless prog-enies using seeded cultivars as female parents is of limited use due to a low proportion of seedless progeny, not higher than 10-15% (LOOMIS and WEINBERGER 1979; SPIEGEL-ROY et al. 1990; SrNGH and BRAR 1992), pernaps due to the fact that seedlessness appears to be controlled by single or few recessive genes (STOUT 1936; CAIN et al. 1983). The proportion of seedless progeny increases in con-nection with the level of seedlessness in the parentage of the seeded female parent. This fact allows to postulate that seedless crosses would be more efficient (BARLASS et al. 1988). In seedless table grapes fertilization takes place but embryo and/or endospermic development stops soon after anthesis and the seed aborts in different stages of growth which mainly depends on the cultivar in question (STOUT 1936; NITSCH et al. 1960; WINKLER et al. 1974; BOUQUET and 0AVIS 1989). The technique of embryo rescue from in ovulo culture has been adapted in grapevine. Results show that a mean of 85 % of the progeny from seedless x seedless crosses can be seedless (CAIN et al. 1983; EMERSHAD and RAMMING 1984; SPIEGEL-ROY et al. 1985; GRAY et al. 1987). The development of growing embryos into plantlets is the basis to obtain new and promising genotypes of table grapes from crosses between seedless cultivars (EMERSHAD and RAMMING 1984; BOUQUET and DAVIS 1989) but the pro-portion of growing normal plantlets is still very low, 7-10 % (BARLASS et al. 1988). The action and interaction of the following parameters have been studied: 1) culture date, since embryos become arrested and/or abort during different stages of ovule de-velopment; 2) genotype; 3) composition of culture media (EMERSHAD and RAMMING 1984; EMERSHAD et al. 1989). Differences in endogenous gibberellins between seeded and seedless grapes have been described in connection with early seed abortion (COOMBE 1960; NITSCH et al. 1960; BHULLAR and DHILLON 1977). The aim of this work was to study the effects of two retardants (CCC: - 2-chloroethyl-trimethylammonium chlo-ride, and paclobutrazol: ~ (( 4-chlorophenyl)methyl)-a-( 1,1-dimethylethyl)-1H-1 ,2,4-triazole-1-ethanol and the new compound XE 1019 ( (E)-1-( 4-chlorophenyl)-4,4-dimethyl-2-(1,2,4 triazol-1-yl)pent-1-en-3-ol) applied before grape-vine anthesis in order to increase the number of fertilised embryos derived from in ovulo culture. The use of those retardants is based on their capacity to inhibit gibberellin biosynthesis as a possible factor of seed abortion in seed-less cultivars (NITSCH et al. 1960; BHULLAR and DHILLON 1977; HEDDEN and GRAEBE 1985). XE 1019 significantly increases seed germination of the stenospermic cultivar C35-33 applied 35 d before bloom (LEDBETTER and SHONNARD 1990). Materials and methods F i r s t y e a r ( 1 9 9 1 ) : The stenospermic cultivar CG 102.011 was ground under forrow irrigation in a vine-yard at San Rafael, province of Mendoza, Argentina. Vines were previously cane-pruned to 4 canes/vine. Clusters were immersed for 1 h in a 400 or 800 mgl1 CCC aqueous solution 2, 3 or 4 weeks before bloom. A 4th treatment was performed with the same concentrations of the retardant applied 3 times (2, 3 and 4 weeks before anthesis). 10 clusters were selected for each treatment, harvested 8 and 10 weeks after bloom and at berry matu-rity. Correspondence to: Dr. RICARDO Trzro, Catedra de Fisiologia Vegetal, Alte Brown 500, Chacras de Coria, 5505 Mendoza, Argentina 74 C. AoOERO, C. RIQUELME and R. Tiz1o Berries of each cluster and treatment, were surface sterilised with 20 % sodium hypochlorite (80 g/1 of active Cl) for 20 min. Fertilised ovules were excised and cul-tured in the solid medium of NITSCH and NITSCH (1969) supplemented with 10·6 M gibberellic acid (GA3) and 10·5 M indolacetic acid (IAA) (SPIEGEL-Rov et al. 1985; GRIBAUDO 1993). 15 ovules were cultured in each 360 ml flasks. All treatments were placed at 24±1 oc day (16 h; 25 f.1mol·cm·2·s-1) and 20±1 °C night. After 2 months the ovules were dissected for embryo rescue. The excised embryos were then cultured in the solid medium of MURASHIGE and SKOOG (MS, 1962) half diluted and supplemented with 1 f.!M benzylaminopurine (BAP). All treatments were placed on the same conditions of daylength, light intensity and temperatures as before, dur-ing 15 d. Then, the material was placed in the same envi-ronmental conditions (but light intensity 100 J.t.Mcm·2·s-1). S e c o n d y e a r ( 1 9 9 2 ) : The cultivars were selected taking in account their in vitro capacity to pro-duce growing embryos: high, medium and weak embryo-genic rate (BouQUET and DAVIS 1989) corresponding with cultivars CG 102.011, Emperatriz and Malvinas, resp., as determined in a previous work (AoOERO et al. in press). Two shoots of uniform length carrying cluster primordia were selected in 9 plants of cv. CG 102.011, and 12 plants of Emperatriz and Malvinas. Plants were treated with XE 1019 240 mgl· 1 and CCC 400 mgl1• Emperatriz and Malvinas were also treated with paclobutrazol 5 mg·l-1• Two applications were made, in November 8 and 18, with all regulators, which were sprinkled on foliage and clus-ters. Blooming started from November 15 for cvs Malvinas and CG 102.011, and from November 18 for Emperatriz. Clusters of all treatments were harvested 8 and 10 weeks after anthesis. Excised ovules were cultured as before, using 20 ovules per flask for Malvinas and 25 for CG 102.011 and Empera-triz. The flasks were placed at random into the growth chambers. Embryo rescue was made after 2 months of in vitro culture, and then cultured in MS medium half di-luted and deprived of growth regulators. Results and discussion First ye a r: The analysis of variance and Duncan's test, when the differences were significant, were employed to analyze the number of embryos (>0.7 mm in length) and the number of growing plantlets. There were no significant differences between treat-ments with CCC 400 mg1·1 and the control but 800 mgl1 gave some negative results. 3 consecutive applications of CCC (400 and 800 mgl1) exerted a strong phytotoxic ef-fect on clusters inducing flower abortion. The number of growing plantlets obtained in week 8 was significantly lower than those from the week 10 and maturity (multifactorial analysis of variance). S e c o n d y e a r : Marked differences have been observed in berry size between treatments. Smallest ber-ries were produced by plants treated with CCC, XE 1019 and paclobutrazol. Parallel to that, great differences in the mean number of ovules per berry related to that of the controls were observed (Figure). 8week 'El-- tO week ... ...._ ezLl ,-----------lmporolrtz r---------- - f-- ml ----- - ------ ·-C01CI:I.011 --- -- --r:l ··-1 .JI 1::) -rtilr j::J .AI J r -- --ccc :g 1011 ccc :U1018 Figure: Mean number of ovules per berry of cultivars Malvinas, Emperatriz and CG 102.011, treated with CCC, XE 1019 and paclobutrazol, 8 and 10 weeks after bloom. Concerning embryo rescue and plantlet production, the behaviour of the cultivars was very different: CG 102.011 showed a marked effect of XE 1019 and CCC on ovule number in each berry (Figure). This behav-iour could be attributed to a minor competition for nutri-ents and hormones between shoots and clusters, a conse-quence of CCC application as has been suggested by SKENE (1969). When treated with CCC, the number of developed plantlets significantly increased when the ovules were ex-cised the lOth week, in contrast to data obtained the 8th week after bloom. These results would suggest that at the lOth week the embryos reached a suitable level of devel-opment as to continue their normal growth in vitro, as ob-served by SINGH and BRAR (1992) in other cultivars (Ta-ble). - XE 1019 did not exert the same effect as CCC did. It is possible that the concentration used was inadequate. With Emperatriz CCC, XE 1019 and paclobutrazol treatments also produced an increase in ovule number per berry. Nevertheless, treatments with those regulators did not increase the number of growing embryos and plantlets from the 8th week after anthesis . On the contrary, CCC and XE 1019 significantly reduced the number of growing embryos and plantlets when the clusters were harvested during the lOth week (Table). With Malvinas a slightly positive effect of CCC on ovule number per berry has also been observed. This be-haviour is desirable, considering the extremely low number of ovules normally developed. Results can not be statisti-cally analysed due to the inadequate number of repetitions which was a consequence of a very low number of berries per cluster (Table). Conclusions The effects of both growth regulators on cultivars CG 102.011 and Emperatriz were more marked when the material was harvested during the 1Oth week compared with those obtained from the 8th week after anthesis. This is probably due to the lower capacity of the younger em-bryos to survive under in vitro conditions. In Malvinas and Embryo rescue from seedless grapevines 75 Tab le Number of ovules, rescued embryos and plantlets from cultivars Malvinas, Emperatriz and CG 102.011, 8 and 10 weeks after anthesis 8th week lOth week Treatment Control ccc Paclo- Control ccc Paclo- XE 1019 butrazol butrazol Malvinas Ovule number 48 100 21 81 80 56 80 Embryo number 2 11 3 7 20 11 11 Plantlet number 1 1 2 3 5 3 2 Emperatriz Pr>F Pr>F Ovule number 275 275 275 150 150 150 150 Embryo number 9 7 2 0.336 34 a 14 b 25 ab 14 b 0.001 Plantlet number 5 3 0 0.079 19 a 6b 11a 5b 0 .022 CG 102.011 Ovule number 275 275 150 150 150 Embryo number 72 65 0.373 85 103 83 0.087 Plantlet number 44 37 0.446 40b 66 a 42 b 0.044 References: Emperatriz and CG 102.011, 11 blocks at random. When ANOVA detected significative differences between treatments, mean values were compared with Duncan. Means followed by the same letter are not significantly different (P<0.05). CG 102.011, CCC increased ovule number per berry. This fact is technically important because it allows a greater number of embryo rescue per cluster in these cultivars. From all cultivars studied, only CG 102.011 showed a significant increase in plantlet production after CCC treat-ment, when the clusters were harvested 10 weeks after bloom. It is possible that CCC would act through inhibi-tion of gibberellin synthesis at folliage and cluster level as the probable cause of ovule abortion. Moreover, the action of endogenous gibberellins coming from growing shoot tips cannot be discarded. Combinations of retardant treat-ments with periodic elimination of growing shoot tips could be important to make clear the role of endogenous gibberellins on ovule abortion in seedless grape cultivars. This is related to the fact that applications of GA3 can in-duce seedlessness through ovule abortion in some treated cultivars of seeded grapevines (PRATI and SHAULIS 1961 ; CLORE 1965; PHARIS and KING 1985; FELLMAN et al. 1991). References AoOERO, C.; GREGORI, M. T.; PoNcE, M. T.; IANDOLINo, A.; TIZio, R.; 1995: Clasificaci6n de cu1tivares estenospermicos en funci6n del crecimiento "in vitro" de embriones. (In press) BARLASS, M.; RAMMING, D. W.; DAvis, H. P.; 1988: In ovulo embryo culture: a breeding technique to rescue seedless x seedless table grape crosses. Grapegrower Winemaker (292), 123-125. BHULLAR, J. S.; DHILLON, B. S.; 1977: Gibberellin-like activity in berries of seeded and seedless grapes. Indian. J. Hort. 31, 362-371. BouQUET, A.; DAvis, H. P.; 1989: Culture in vitro d'ovules et d'embryons de vigne (Vitis vinifera L.) appliquee a la selection de varietes de raisins de table sans pepins. Agronomie 9, 565-574. CAIN, D. W.; EMERSHAD, R. L.; TARAILO, R. E.; 1983: In ovulo embryo cul-ture and seedling development of seeded and seedless grapes (Vitis vinifera L.). Vitis 22, 9-14. CLORE, W. J. ; 1965: Responses of Delaware grapes to gibberellin. Proc. Amer. Soc. Hort. Sci. 87, 259-263. CooMBE, B. G.; 1960: Relationship of growth and development to changes in sugars, auxins and gibberellins in fruits of varieties with and with-out seeds of Vitis vinifera. Plant Physiol. 35, 241-250. EMERSHAD, R. L.; RAMMING, D. W.; 1984: In-ovu[o embryo culture of Vitis vinifera L. cv. Thompson Seedless. Amer. J. Bot. 71, 873-877. - -;- -; SERPE, M.; 1989: In ovulo embryo development and plant formation from stenospermic genotypes of Vitis vinifera. Amer. J. Bot. 76, 397-402. FELLMAN, C.; HoovER, E.; AscHER, P. D. ; LuBY, J.; 1991: Gibberellic acid-induced seedlessness in field-grown vines of Swenson Red grape. HortScience 26, 873-875. GRAY, D. J. ; FISHER, L. C.; MoRTENSEN, J.A.; 1987: Comparison of method-ologies for in-ovulo embryo rescue of seedless grapes. HortScience 22, 1334-1335. GRIBAUDO, 1.; ZANETTI, R.; BorrA, R.; V ALLANIA, R.; EYNARD, 1.; 1993: In ovulo embryo culture of stenospermic grapes. Vitis 32, 9-14. HEDDEN, P.; GRAEBE, J. E.; 1985: Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates of Cucurbita maxima endosperm and Malus pumilla embryos. J. Plant Growth Regul. 4, lll-122. LEDBETTER, C. A.; SHONNARD, C. B.; 1990: Improved seed development and germination of stenospermic grapes by plant growth regulators. J. Hort. Sci. 65, 269-274. 76 C. AGDERO, C. RIQUELME and R. TIZIO LooMis, N. H.; WEINBERGER, J. H.; 1979: Inheritance studies of seedlessness in grapes. J. Amer. Soc. Hort. Sci. 104, 181-184. MuRASHIGE, T.; SKooo, F. M.; 1962: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497. NITSCH, J. P.; PRATT, C.; NrrscH, C.; SHAULIS, N. J.; 1960: Natural growth substances in Concord and Concord Seedless grapes in relation to berry development. Amer. J. Bot. 47, 566-576. - -; NrrscH, C.; 1969: Haploid plants from pollen grains. Science 163, 85-87. PHARIS, R. P.; KiNG, R. 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Embryo rescue from seedless grapevines (Vitis vinifera L.) treated with growth retardants

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Embryo rescue from seedless grapevines (Vitis vinifera L.) treated with growth retardants

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