小果甜柿遗传转化体系建立及其抗冻性遗传改良的初步研究

Establishment of genetic transformation system and genetic improvement of frost resistance of Xiaoguo Tianshi persimmon

【目的】建立小果甜柿(Diospyros kaki Thunb.‘Xiaoguo Tianshi’)叶片再生和优化其遗传转化体系,以期通过分子育种技术获得抗冻性强的砧木新种质。【方法】以小果甜柿叶盘为外植体,首先通过不同生长素(IAA)质量浓度和细胞分裂素(ZT,TDZ)配比探讨对其愈伤诱导和不定芽再生的影响;进而探讨其叶片对不同抗生素质量浓度组合的敏感性;此外,利用根癌农杆菌介导的叶盘稳定遗传转化体系,进一步探讨超表达君迁子(D.lotus L.)抗寒基因DlCBF1对小果甜柿叶盘再生植株抗冻性的影响。【结果】叶圆片在MS(1/2N)+ZT 2.0 mg·L^(-1)+TDZ 2.0 mg·L^(-1)培养基中愈伤形成率最高(64.71%);将愈伤组织接种到MS(1/2N)+ZT 2.0 mg·L^(-1)+IAA 0.1mg·L^(-1)生芽培养基中,不定芽再生率和不定芽数分别为31.11%和2.37个。头孢霉素质量浓度为400 mg·L^(-1)时可抑制农杆菌生长,且叶片愈伤形成率低(9.84%);卡那霉素对叶片再生影响大,在质量浓度为20 mg·L^(-1)时其愈伤诱导率为11.57%。小果甜柿叶片稳定超表达君迁子DlCBF1基因,获得再生植株73株,其中阳性植株5株,荧光定量结果分析,阳性植株#1和#47中DlCBF1基因表达量分别为野生型的13倍和11倍。经过-4℃低温处理8 h后,超表达株系与野生型株系相比,叶片受伤害程度更小,光合作用效率变化不明显,电导率低,H_(2)O_(2)和O_(2)^(-)在组织中的积累少,阳性株系表现出比野生型株系更强的抗冻性。【结论】在优化小果甜柿叶片外植体再生和稳定遗传转化体系的基础上,通过在小果甜柿叶片中超表达君迁子DlCBF1基因,获得6.85%转基因阳性再生植株,稳定超表达DlCBF1基因可显著增强小果甜柿再生植株的抗冻性。研究结果为培育抗冻性强的柿砧木新种质提供了科学依据。

【Objective】Persimmon(Diospyros kaki Thunb.)is generally propagated by grafting.The sweet persimmon Fuyu is known for its excellent fruit quality.However,when grafted on the common rootstock Diospyros lotus L.,it has shown low survival rate,slow growth or incompatibility in later stage.This is particularly evident in traditional producing areas in China where D.lotus is commonly used as rootstock,so that Fuyu could not be produced on a large scale in our country.Xiaoguo Tianshi persimmon(D.kaki Thunb.)was collected from Dabie Mountain area of Hubei province.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 presented graft affinity.Following its widespread planting and popularity throughout China,it is noted that the Xiaoguo Tianshi persimmon has showed inadequate resistance to frost,which results in a greater mortality rate when exposed to unfavorable weather circumstances.Compared to molecular breeding,traditional cross breeding 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 reliable stable genetic transformation system of Xiaoguo Tianshi persimmon have not been reported.This study aims to establish a reliable leaf regeneration system for Xiaoguo Tianshi persimmon and optimize its stable genetic transformation system,thereby facilitating genetic improvement 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)or 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,by 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 the Murashige and Skoog medium containing half of the normal nitrogen concentration(1/2N)supplemented with 2.0 mg·L^(-1)zeatin(ZT)and 2.0 mg·L^(-1)thidiazuron(TDZ).When these callus tissues were transferred to a germination medium of MS(1/2N)+ZT 2.0 mg·L^(-1)+IAA 0.1 mg·L^(-1),the rate of adventitious bud regeneration and the average number of buds generated were 31.11%and 2.37,respectively.During the optimization of stable genetic transformation conditions for Xiaoguo Tianshi persimmon,it was found that a concentration of 400 mg·L^(-1)cefotaxime could inhibit the growth of Agrobacterium tumefaciens,meanwhile 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^(-1)reducing the induction rate of callus formation to just 11.57%.Concentration of 10 mg·L^(-1)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 cloned from D.lotus,a total of 73 regenerated plants were obtained.DNA-PCR identification revealed 5 positive transgenic plants.Quantitative fluorescence PCR analysis showed that the expression levels of the Dl-CBF1 gene in transgenic plants#1 and#47 were higher,being 13 and 11 times than those of the wildtype plants,respectively.In frost resistance determination experiment,after treatment at-4℃for 8 hours,the transgenic lines showed significantly weak damage compared to wild-type plants,which exhibited browned leaves and signs of dehydration.Weak chlorophyll fluorescence signal was observed in wild-type plants,thus indicating reduced photosynthetic efficiency in wild plants after low temperature treatment.After cold treatment,electrical conductivity increased in wild-type plants,implying that the leaf tissue membrane had been damaged.Staining with DAB and NBT after cold treatment showed that wild-type plants accumulated more H_(2)O_(2)and O_(2)^(-)in their leaf tissues than transgenic lines.These results exhibited that,in comparison to plants of the wild type,cold tolerance was enhanced in the transgenic lines.【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 leaf tissue of Xiaoguo Tianshi persimmon.There was 6.85%transgenic positive Xiaoguo Tianshi persimmon.Further experimental results had 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 genetic improvement of novel persimmon rootstock germplasm with enhanced cold resistance.