| Genetic transformation is crucial for the functional study of genes and the improvement of traits in Vitis species.However,establishing an efficient and stable transformation system remains a challenge for most grapevines.The aim of this study was to develop a stable grape genetic transformation system using immature zygotic embryos of ’Chardonnay’ as a research material.To achieve this,the in vitro regeneration efficiency of immature zygotic embryos of ’Chardonnay’ was optimized by examining the effects of antibiotic concentration,pretreatment methods,Agrobacterium concentration(OD600),acetosyringone concentration,infection duration,and infection methods in the genetic transformation system.The most suitable Agrobacterium infection method for ’Chardonnay’ immature zygotic embryos was determined by observing GFP(green fluorescent protein)expression and Western blot analysis,providing technical support for grapevine variety improvement,gene function analysis,and editing.The main results are as follows:1.An efficient germination and somatic embryo regeneration system was established for immature zygotic embryos of ’Chardonnay’.Using 80-day post-anthesis immature zygotic embryos of’Chardonnay’ as explants,dormancy was effectively broken by immersion in a 2.5 g·L-1 GA3 solution,followed by incubation in a 55℃ water bath for 15 minutes and then resting at room temperature for 24 hours.The germination efficiency of the embryos reached 73.8%± 5.75 by inoculating surfacesterilized embryos with an epidermal incision onto filter paper culture dishes containing 3 mL of sterile water.Germinated cotyledonary embryos were then inoculated onto various concentrations of 6-BA and NAA combination media,with M2(containing 3.0 mg·L-1 NAA+0.4 mg·L-1 6-BA+2.0 g·L-1 PVP),M5(containing 4.0 mg·L-1 NAA+0.4 mg·L-1 6-BA+3.0 g·L-1 PVP),and M8(containing 5.0 mg·L-1 NAA+0.4 mg·L-1 6-BA+1.0 g·L-1 PVP)media promoting callus induction and somatic embryo formation,resulting in embryo formation rates of 4.17%,14.58%,and 8.33%,respectively.Rapid germination of somatic embryos was confirmed on X6 media containing 30 g·L-1 sucrose.2.A callus tissue proliferation and expansion culture system was established using ’Chardonnay’flower buds as explants.Unopened small ’Chardonnay’ flower buds were used as explants,and after removal of the petioles,were placed on 16 embryogenic callus induction media in an orthogonal design.MU-2(containing 0.02 μM ABA),MU-7(containing 0.4μM Uni+0.04 μM ABA),MU-9(containing 0.7 μM Uni),MU-10(containing 0.7 μM Uni+0.02 μM ABA),and MU-16(containing 1.0 μM Uni+0.06 μM ABA)demonstrated the best callus induction effects at day 28,all reaching over 90%.The addition of 1.0 g·L-1 AC to the callus proliferation and expansion system significantly reduced browning in solid culture,and cell viability was higher in liquid culture at 30 g/100 mL inoculum,peaking at day 9.3.A stable genetic transformation system for ’Chardonnay’ immature zygotic embryos has been established.Through direct germination of immature zygotic embryos,factors such as antibiotic concentration,pre-treatment methods,Agrobacterium concentration(OD600),acetosyringone concentration,infection duration,and infection methods were further investigated.The results indicated that inoculating dormant embryos on MEL3 medium for 5 days for pre-culture,selecting embryos with cracked beaks and exposed whites,and placing them in Agrobacterium suspension(OD600=0.6)containing 200 μM acetosyringone for conventional infection for 20 minutes resulted in a transformation efficiency of 12.8%.Integration and expression of the GFP gene in the plants were further confirmed by fluorescence microscopy and Western blot analysis.This transformation method is simple and rapid,requiring approximately four months to obtain transgenic positive plants.Importantly,the method is convenient and not influenced by seasonal or human factors,providing robust technical support for grape gene function research. |
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