Embryogenesis And VaCOLD1 Gene Transformation In Immature Zygotic Embryos Of ’Cabernet Sauvignon’

Grapes(Vitis L.)are extensively cultivated due to their great economic worth and long history of agriculture.Grapes,however,are subject to abiotic factors such as low temperature and drought during cultivation and production,which often result in substantial yield and quality losses.Utilizing transgenic technology to specifically enhance grapevines’ tolerance to stress is an effective solution to this issue.For the transgenic development of grape stress tolerance,the establishment of a quick and effective grapevine genetic transformation system is of significant theoretical and practical benefit.In this study,we investigated the best treatment to promote the germination of primary embryos and the effect of different hormone combinations on the mode of secondary somatic embryogenesis of ’Cabernet Sauvignon’ using immature conidia 80 d after flowering as test material,in order to break the short period of explant collection and the limited operation procedure,so as to increase the germination rate of primordial embryos and provide a reference for the establishment of a standard operating procedure.On this basis,Agrobacteriummediated genetic transformation of the CHILLING-TOLERANCE DIVERGENCE 1(VaCOLD1)gene in grapevine was performed using immature zygotic embryos of ’Cabernet Sauvignon’ as the recipient material,providing a theoretical foundation for optimizing the genetic transformation system of Vitis vinifera.This research offers a theoretical foundation for enhancing the genetic transformation mechanism in grapevine.The following are the main findings of the study:1.The germination efficiency of primary embryos was enhanced by employing immature zygotic embryos of ’Cabernet Sauvignon’ as explants.To induce germination of primary embryos in immature zygotic embryos,different concentrations(1.0,1.5,2.0,2.5,3.0 g·L-1)of gibberellin(GA3)treatment,different durations of ultrasonication(2,5,10,15,30 min),different methods of breaking the skin(cutting seed back,cutting beak+cutting seed back,and sandpaper abrasion)and soaking time(12,24,36,48 h)were used.The three treatments of 2.5 g·L-1 gibberellin,ultrasonication for 2 minutes,and cut beak+cut seed back considerably raised the induction rate of primordial embryos compared to the control group,which was 68.42%.Greening of cotyledons and thickening of embryonic axis were evident in germinated primordial embryos treated with ultrasound for 2 minutes,and the induction efficacy was much greater than that of other treatments throughout the following callus induction process.2.Establishing a somatic embryo cycle regeneration method using immature zygotic embryos derived from ’Cabernet Sauvignon’.The primary embryos obtained from germination following gibberellin,sonication,skin breaking,and seed soaking were cut into cotyledons,embryonic axes,and embryonic roots before being placed in MS medium supplemented with four different combinations of 2,4-D(0.5,1.0,1.5,and 2.0 mg·L-1)and 6-BA(1.0,2.0,3.0,and 4.0 mg·L-1)hormones to induce secondary embryogenesis,respectively.MS medium containing 0.5,1.0,and 1.5 m g·L-1 2,4-D and 1.0,2.0,and 3.0 m g·L-1 6-BA induced indirect production of somatic embryos with embryogenesis rates between 6.45%and 17.15%at 90 days.In MS medium enriched with 2.0 mg·L-1 2,4-D and 4.0 mg·L-1 6-BA,secondary somatic embryos were generated immediately upon cotyledon browning,and the 90-day embryogenesis rate reached 21.33 percent.94%of normal secondary embryos were able to germinate and grow plants at a rate of 100%.3.The genetic transformation system of Agrobacterium tumefaciens with immature zygotic embryos of ’Cabernet Sauvignon’ as the recipient material was established.The immature zygotic embryos of Cabernet Sauvignon’ at 80 days after flowering were used as explants,and coldtolerant membrane protein VaCOLD1 from Vitis amurensis Rupr.was used as the target gene for genetic transformation.The genetic transformation of’Cabernet Sauvignon’ was enhanced by modifying the types of phytohormones,antibiotic concentrations,and screening methods in the preculture medium.The result showed that MC medium(NN69+0.55 mg·L-1 2,4-D+0.5 mg·L-1 NOA+1.24 mg·L-1 4-CPPU+2.0 mg·L-1 6-BA+0.5 g·L-1 activated charcoal+3 g·L-1 phytogel+30 g·L-1 sucrose)was advantageous for the induction and screening of healing tissues,whereas MEL3 medium(MS+0.6 mg·L-1 0.6 mg·L-1 Melatonin+2.0 mg·L-1 6-BA+0.5 g·L-1 activated charcocal+3 g·L-1 phytogel+30 g·L-1 sucrose)was advantageous for the germination and screening of primary embryos.Using a dual-wavelength fluorescent protein excitation light source,42.9%of the converted healing tissues displayed evident GFP green fluorescence.After screening with various doses of kanamycin,it was determined that 50 mg·L-1 Kan screening was the most successful,since some plant leaves became reddish-brown and died after 30 days.The experiment yielded a total of 43 plants,eight of which were WB protein assay-positive for overexpression,representing a transformation rate of 18.6%.The feasibility of the genetic transformation method in this study was initially demonstrated.