Role of melatonin in the induction of somatic embryogenesis from seeds of Vitis vinifera‘Chardonnay’

Abstract:【Objective】The role of melatonin in the induction of somatic embryogenesis and induced cir-culation establishment from wine grape seeds was investigated with the aim to establish a somatic em-bryo induction system based on seed induction, and to provide a theoretical basis for shortening the cy-cle of grapevine somatic embryo induction.【Methods】Seeds from European wine grape varieties Char-donnay, Marselan and Merlot were used as explants, which were collected at 60 d, 70 d and 80 d after flowering, respectively. The seeds were sterilized with 70% ethanol and 1.5% NaClO, and then cut and placed in four ways, namely, transversely cut and flatwise, transversely cut and anti-put, longitudinally cut and flatwise, and longitudinally cut and anti-put, to investigate the effect of different cutting and placing patterns on the germination of primary cotyledon embryos. In addition, five different concentra- tions (0.2, 0.4, 0.6, 0.8, 1.0 mg·L^-1) of melatonin were explored on their primary cotyledon embryos ger-mination, and the germinated primary cotyledon embryos were further cut to 0.5 cm sections to investi-gate the cyclic induction of their secondary somatic embryos by five different concentrations (0.2, 0.4, 0.6, 0.8, 1.0 mg · L^{- 1}) of melatonin.【Results】Seeds of the three wine grape varieties and different seed maturity levels differed significantly in the induction rate of primary cotyledon embryos on the medium supplemented with different concentrations of melatonin. Among them, the cotyledon embryo germina-tion rates were all proportional to seed maturity on MC, MEL0, MEL1, MEL2, MEL3, MEL4 and MEL5 medium, with induction rates ranging from 0.9 to 16.56% at 60 d after flowering, 1.34% to 17.3% at 70 d, and up to 18.33% at 80 d. Among the three test varieties, Chardonnay had the highest germination rate of 18.33% at 80 d after flowering induced by 0.6 mg· L^-1 melatonin, while the control 2,4-D induced germination with the worst effect of 2.98%. The difference in germination rate between varieties may be related to genotype. The seeds of Chardonnay were inoculated into the medium in dif- ferent ways, and the seed coat started to discolor at 3 d. The germination of cotyledon embryos started at 10 d. The germination rate of primary cotyledon embryos reached the highest rate of 33.07% at 30 d when the explants were inoculated by the longitudinally cut and flatwise, and the germination rates de-creased in longitudinally cut and anti-put, transversely cut and flatwise, transversely cut and anti-put, with 10.98%, 5.71%, and 3.24%. The results showed that longitudinal cut and flatwise was the most suitable inoculation method, which helped to improve the germination rate of primary cotyledon embry-os. Melatonin-induced germination of primary cotyledon embryos was stronger than 2,4-D in all three wine grape varieties, among which seeds from Chardonnay could occurred the highest primary cotyle-don embryos induction rate of 18.33% in MS + 0.6 mg·L^-1 melatonin + 2.0 mg·L^-1 6-BA + 30 g·L^-1 su-crose + 3 g·L^-1 phytogel + 0.5 g·L^-1 activated charcoal for 30 d. The secondary somatic embryos were induced by using Chardonnay primary cotyledon embryo cuttings in a cyclic manner, and callus tissues were formed in different media at 14 d. At 60 d, secondary somatic embryos began to appear, and fur- ther induction resulted in the formation of spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos. The highest induction rate of 14.4% was induced at 90 d with MS + 0.6 mg·L-1melatonin + 2.0 mg·L^-1 6-BA + 30 g·L^-1 sucrose + 3 g·L^-1 phytogel + 0.5 g·L^-1 activated carbon, fol-lowed by 0.4 mg·L^-1 melatonin with the induction rate of 3.13%, 0.2 mg·L^-1 occurred the induction rateof 1.89%, while no secondary embryos were formed either on medium supplemented with 0.8, and 1.0 mg·L^-1 melatonin or on medium supplemented with 2,4-D. The plant materials induced by 2,4-D gradu-ally browned and died during the succession process. The induction of Chardonnay seeds with melato-nin resulted in the formation of primary cotyledon embryos, and after cyclic induction, primary embryo clusters were produced and secondary embryos were formed, and seedlings could be established after transplanting.【Conclusion】The role of melatonin in the induction of grapevine somatic embryos was investigated with wine grape seeds as explant material. The results showed seed maturity was positively correlated with the germination rate of primary cotyledon embryos. The longitudinally cut and flatwise was beneficial to the germination of primary cotyledon embryos. Low concentration of melatonin had the effect of shortening the induction time for germination of primary cotyledon embryos and secondary embryogenesis in Chardonnay seeds. The primary cotyledon embryos from Chardonnay could induce the production of primary embryo clusters and somatic embryos, which could help to increase the num-bers of embryo materials and provide experimental materials for grapevine genetic transformation.