Abstract: 【Objective】 Persimmon (Diospyroskaki Thunb.) is generally propagated by grafting. The sweet persimmon 'Fuyu' is known for its excellent fruit quality. However, when grafted with the common rootstock D. lotus (Diospyros lotus L.), it has shown low survival rate, slow growth or incompatibility in the later stage. This is particularly evident in traditional producing areas in China where D. lotus is commonly used to be common rootstock. So that ‘Fuyu’ could not be produced on a large scale in our country.The germplasm resources of ‘Xiaoguo Tianshi’ persimmon (Diospyros kaki Thunb.) were collected in Dabieshan Mountain area of Hubei Province. And through field observations, in vitro stem segment wedge grafting and callus tissue approach grafting experiments, it has been found that ‘Xiaoguo Tianshi’ persimmon and ‘Fuyu’ persimmon varieties graft affinity. Following its widespread planting and popularity throughout China, it was noted that the ‘Xiaoguo Tianshi’ persimmon showed inadequate resistance to frost, which resulted in a greater mortality rate when exposed to unfavorable weather circumstances.Compared to molecular breeding, traditional breeding through seedlings is time-consuming. Introducing resistance traits via transgenic methods could enhance the frost resistance of ‘Xiaoguo Tianshi’ persimmon. To date, a leaf regeneration system and a stable genetic transformation system of ‘Xiaoguo Tianshi’ persimmon have not been reported. This study aims to establish a leaf regeneration system of ‘Xiaoguo Tianshi’ persimmon and optimize its stable genetic transformation system, thereby facilitating the development of frost-resistant rootstock germplasm through molecular breeding technology.【Methods】Utilizing leaf discs from ‘Xiaoguo Tianshi’ persimmon as explants, our initial investigations focused on the influence of varying concentrations of indoleacetic acid (IAA) and cytokinins (zeatin [ZT] and thidiazuron [TDZ]) on the induction of callus formation and the regeneration of adventitious buds. Subsequently, the sensitivity of ‘Xiaoguo Tianshi’ persimmon leaf discs to various antibiotic concentration combinations was assessed. Moreover, leveraging an Agrobacterium tumefaciens-mediated stable genetic transformation system facilitated through the aforementioned explants, we further examined the impact of overexpressing the cold resistance gene DlCBF1 from D. lotus on the frost resistance of regenerated plants derived from ‘Xiaoguo Tianshi’ persimmon leaf discs.【Results】The highest callus formation rate (64.71%) in ‘Xiaoguo Tianshi’ persimmon was achieved when leaf explants were cultured on Murashige and Skoog medium containing half the normal nitrogen concentration (1/2N) supplemented with 2.0 mg/L zeatin (ZT) and 2.0 mg/L thidiazuron (TDZ). When these callus tissues were transferred to a germination medium of MS (1/2N) + ZT 2.0 mg/L + IAA 0.1 mg/L, the rate of adventitious bud regeneration and the average number of buds generated were 31.11% and 2.37±0.11, respectively. During the optimization of stable genetic transformation conditions for ‘Xiaoguo Tianshi’ persimmon, it was found that a concentration of 400 mg/L cefotaxime could inhibit the growth of Agrobacterium tumefaciens, this led to a low rate of callus induction (9.84%). Kanamycin significantly affected the regeneration of ‘Xiaoguo Tianshi’ persimmon leaf explants, with a concentration of 20 mg/L reducing the induction rate of callus formation to just 11.57%. Concentration of 10 mg/L kanamycin was found to be optimal for the stable genetic transformation of ‘Xiaoguo Tianshi’ persimmon leaf explants, resulting in a callus induction rate of 62.28%. Following stable genetic transformation of ‘Xiaoguo Tianshi’ persimmon to overexpress the DlCBF1 gene from D. lotus, a total of 73 regenerated plants were obtained. DNA-PCR identification revealed 5 transgenic plants. Quantitative fluorescence analysis showed that the expression levels of the DlCBF1 gene in transgenic plants #1 and #47 were higher, being 13 and 11 times that of the wild-type plants, respectively. In frost resistance experiment, after treatment at -4 °C for 8 hours, the transgenic lines showed significantly reduced damage compared to wild-type plants, which exhibited browned leaves and signs of dehydration. The chlorophyll fluorescence signal in wild-type plants was weaker, indicating reduced photosynthetic efficiency after low temperature treatment. After cold treatment, electrical conductivity increased in wild-type plants, indicating that the leaf tissue membrane had been damaged.Staining with DAB and NBT after cold treatment demonstrated that wild-type plants accumulated more H2O2 and O2- in their leaf tissues than transgenic lines.These findings show that, in comparison to plants of the wild type, the transgenic lines had increased cold tolerance.【Conclusion】This study, building upon the optimization of a leaf explant regeneration and stable genetic transformation system for ‘Xiaoguo Tianshi’ persimmon, has successfully achieved stable overexpression of D. lotus cold resistance gene DlCBF1 within ‘Xiaoguo Tianshi’ persimmon of leaf tissue. This resulted in 6.85% of the regenerated plants being transgenic positive. Further experimental results have convincingly demonstrated that overexpression of the DlCBF1 gene significantly enhances the frost resistance of the regenerated ‘Xiaoguo Tianshi’ persimmon plants. These findings provide a scientific basis for the cultivation of new rootstock germplasm with enhanced cold resistance.
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