Study on virus-free propagation of in-vitro leaf organ regeneration for grapes of Sweet Scarlet and Shine Muscat

【Objective】Grapes are highly susceptible to various virus infection. Heat treatment combined with apical meristem culture is currently a commonly used method for virus elimination and is effective against most grape viruses, but it is less effective in eliminating Grapevine Rupestris Stem Pitting associated Virus (GRSPaV). Organ regeneration refers to the in vitro culture of plant stems, leaves, or other organs under sterile conditions to regenerate new tissues and organs. This process accelerates the rate of cell division and development, thereby in creasing the probability of obtaining virus-free tissues and cells, from which adventitious buds can be induced to regenerate virus-free plants. This experiment aims to eliminate GRSPaV and other viruses through organ regeneration, thereby cultivate primary virus- free planets and provide support for the theoretical and practical development of grape virus elimination techniques.【Methods】The in vitro plantlets of Sweet Scarlet grape infected with GRSPaV and Shine Muscat grape infected with Grapevine Virus E (GVE), grapevine fabavirus (GFabV), and GRSPaV were studied. The subculture medium for Sweet Scarlet grape and Shine Muscat grape was B5+0.5 mg·L-1 IAA+25 g·L-1 sucrose+ 6 g·L-1 agar, and B5+0.3 mg·L-1 IBA+0.5 mg·L-1 IAA+25 g·L-1 glucose+6 g ·L-1 agar, respectively. The appropriate amount of cytokinin and auxin in B5 basal medium were screened by supplied (4.0, 5.0 mg·L-1 ) BA+(0.1, 0.2, 0.3 mg·L-1 ) IAA, and 2.0 mg·L-1 TDZ+(0.1, 0.2, 0.3 mg·L-1 ) IAA or (0.1, 0.2, 0.3 mg·L-1 ) NAA. The addition of TDZ to B5 basal medium were tested at concentrations of 0.5, 1.0, 2.0, 2.5, and 3.0 mg · L- 1 levels. The sodium nitroprusside (SNP) was supplied to MS or B5 basal medium at concentrations of 0, 4, 6, 8, 10, and 12 mg·L-1 to evaluate its efficiency for the regeneration of adventitious buds from the plantlets in vitro. Based on the optimal medium obtained from the above experiments, Sweet Scarlet grape was directly induced to regenerate adventitious buds from leaves in vitro. Subsequently, the adventitious buds were excised and cultured in the subculture medium. A total of sixty-three plantlets from the seven clones were obtained and subjected for the virus detection by RT- PCR analysis. For Shine Muscat grape plantlets, the in vitro cultured leaves were firstly induced to generate callus. The callus was then subcultured onto B5+1.0 mg·L-1 BA+ 0.1 mg·L-1 NAA+30 g·L-1 glucose medium, with subculture interval lasting 20-30 d. During each subculture interval, six clones of callus were randomly selected for the virus detection. The virus-free callus was then transferred to B5+2.0 mg ·L-1 TDZ+0.2 mg ·L-1 NAA+15 g ·L-1 glucose medium to induce the regeneration of adventitious buds. After virus detection, the buds were cut and subcultured to develop into plants.【Results】The combination of TDZ and IAA was suitable for in vitro regeneration of adventitious buds from Sweet Scarlet grape leaves and for inducing callus from Shine Muscat grape leaves. In vitro leaves of Sweet Scarlet grape treated with TDZ at 1.0 mg·L-1 showed the highest adventitious bud rate (18.06%) and the number of buds per leaf disk (0.96), significantly higher than those under other TDZ concentrations. The optimal medium for adventitious bud regeneration from the leaves of Sweet Scarlet grape plantlets was B5+1.0 mg·L-1 TDZ+0.1 mg·L-1 IAA+15 g·L-1 glucose+10 mg·L-1 SNP. Within the 0.5-1.0 cm leaf disk, the average number of the adventitious buds reached 1.82, representing an 114.12% increase compared to the treatment without SNP. For Shine Muscat grape, the in vitro cultured leaf discs failed to induce adventitious bud in medium with different cytokinin and auxin factors or different concentrations of TDZ. However, the adventitious bud regeneration was successfully induced on medium supplemented with SNP, and the optimal medium was B5+1.0 mg · L-1 TDZ+ 0.1 mg ·L-1 IAA+ 15 g ·L-1 glucose+8 mg ·L-1 SNP, yielding an adventitious bud rate of 17.03% and an average of 1.80 buds per plantlet. The adventitious buds regenerated from the leaves of Sweet Scarlet grape were in vitro cultured into plants. Five out of seven tested clones were free of GRSPaV, achieving a virus removing rate of 71.4%. Callus was induced from the leaves of Shine Muscat grape plantlets carrying GVE, GFabV, and GRSPaV. The first-generation callus retained all viruses presented in the original plants. Only GRSPaV was detected in the second-generation, while none of the viruses were found in the third- generation. Virus testing for the callus of the fourth generation and the subsequent confirmed complete virus- free status. Adventitious buds were then regenerated from the virus- free callus, successfully yielding the primary virus- free plants.【Conclusion】The addition of SNP in the medium significantly promoted the adventitious bud regeneration from the leaves of Sweet Scarlet grape and Shine Muscat grape plantlets in vitro. Virus- free plants could be directly obtained via the adventitious bud regeneration either from the leaves in vitro in Sweet Scarlet grape or from the callus of the leaves in vitro in Shine Muscat grape.