摘要
【目的】建立‘嘎拉’苹果小孢子来源纯合基因型植株高效再生体系,探索一个简单有效的苹果纯合基因型株系叶片外植体诱导再生不定芽的方法。【方法】以‘嘎拉’苹果花药离体培养获得的纯合基因型株系离体叶片为外植体,进行再生培养,检测‘嘎拉’各纯合基因型株系的不定芽再生能力;通过优化植物生长调节剂、叶片预处理和培养方式,建立苹果纯合基因型植株高效再生体系。【结果】苹果纯合基因型株系中双单倍体株系DH2-10的再生率最高,四倍体纯系DH2-15次之,单倍体株系DH2-3再生率较低,三倍体纯系DH2-40在培养中难以再生。不同基因型适宜的激素质量浓度不同,相较于双单倍体株系,四倍体纯系DH2-15在较高细胞分裂素水平再生率较好。再生培养过程中,整叶带伤口的外植体预处理方式优于叶片切块接种的方式,接种苗龄40 d左右的叶片有更好的再生率。【结论】建立了苹果纯合基因型高频高效离体再生体系:双单倍体株系DH2-10在最适再生培养基MS+0.5 mg·L^(-1)IBA+5 mg·L^(-1)6-BA上培养,再生周期为23~35 d,再生系数为7.27,再生率达100%。四倍体纯合基因型株系DH2-15适宜在MS+0.5 mg·L^(-1)IBA+6 mg·L^(-1)6-BA培养基上进行再生培养,再生率为93.33%。建立的再生体系可用于纯合基因型株系的快速增殖和遗传转化。
【Objective】Apple is highly heterozygous due to its self-incompatibility, but the microsporesderived plantlets from the in vitro culture of‘Gala＇apple anther are highly homozygous and can be a better material for genetic transformation. This study aimed to establish a stable and highly efficient in vitro regeneration system from leaves of the homozygous microspores-derived‘Gala＇plantlets, so as to provide a good system for genetic transformation. These plantlets were regenerated on MS medium containing phytohormone（6-BA and IBA）, and the method was optimized by optimizing the combinationof plant growth regulators, the way of leaf wounding and dark culture【.Methods】The genotype of microspores-derived homozygous plantlets from anther culture of‘Gala＇apple differed among plantlets.The leaves of microspores-derived plantlets with cutting wounds were used as the explants and cultured on MS medium with different hormone combinations and concentrations（M1： MS＋1.0 mg· L^-1 IBA＋4 mg· L^-16-BA; M2： MS＋1.0 mg· L^-1 IBA＋5 mg· L^-16-BA; M3： MS＋1.0 mg· L^-1 IBA＋6 mg· L^-16-BA;M4：MS＋0.5 mg· L^-1 IBA＋4 mg· L^-16-BA; M5： MS＋0.5 mg· L^-1 IBA＋5 mg· L^-16-BA; M6： MS＋0.5 mg· L^-1 IBA＋6 mg· L^-16-BA）, and were incubated at 25 ℃/17℃（14/10 h）for 14 days in dark. The leaves of 40 d, 80 d and 120 d were used as the explants for regeneration culture to explore the effect of leaf age on regeneration. Both the entire leaves with cutting wounds and leaflet slices were used as the explants for regeneration culture to find the best explant form. And the explants were cultured under light or in darkness for 14 days. The progresses of regeneration were observed, and the callus rate and regeneration rate were calculated. The regenerated buds were subcultured on MS medium containing 0.1 mg · L^-1 IBA＋1 mg· L^-16-BA, and 1/2 MS medium＋3 mg· L^-1 IBA was used as the rooting medium for the regenerated plantlets.【Results】The anther culture obtained homozygous plants, which had been analyzed by flow cytometry（BD Accuri C6） and proved to be haploid（plantlets of DH2-3 and DH2-41）, diploids（plantlets of DH2-10, DH2-20, DH2-24 and DH2-35）, triploid（plantlet of DH2-40）or tetraploid（plantlet of DH2-15）. Shoot regeneration frequency was different among plantlets. The regeneration rate of the diploid plantlet DH2-10 was the highest, followed by tetraploids DH2-15 and haploid plantlets, and the tritraploid DH2-41 had the lowest regeneration frequency. The regeneration frequency of the haploid plantlets DH2-10 and DH2-20 was 31.1% and 5.0%, respectively. That of the diploid plantlets DH2-10, DH2-20, DH2-24, DH2-35 was 100%, 78.89%, 60% and 33.33%, respectively. The regeneration frequency of the tetraploid plantlets DH2-15 was 93.33%. The results showed that plant genotype greatly affected plant regeneration. DH2-10 was a good material for highly efficient plant regeneration. The period of regeneration of the diploid DH2-10 was only 23-35 d, shorter than the other plantlets, and its average bud number per explant was 7.27. Furthermore, hormones had impact on plant regeneration. MS medium containing 6-BA and IBA could be used for callus differentiation and for adventitious bud regeneration. When the concentration of 6-BA was 5 mg · L^-1, the induction of callus from leaf explants was good. The best hormone combination for different genotypes varied. The optimal auxin concentration suitable for the haploid plantlets was higher than those for the diploid plantlets and tetraploid plantlets. The regeneration of the tetraploid DH2-15 required a higher level of cytokinin than that of the diploid plantlets and haploid plantlets. In regeneration process, the explant of whole leaf with wounds had a higher regeneration rate than that of small leaf slices. A higher regeneration rate was obtained using the 40 day old leaves as the explants. Culture under dark increased the regeneration rate significantly.The regenerated plants were subcultured and rooted plants were obtained after root induction【.Conclusion】A high-frequency in vitro regeneration system for homozygous apple plantlets was established.The diploid plantlets of DH2-10 had a regeneration rate of 100% after 14 d dark culture in the optimized regeneration medium of MS＋0.5 mg· L^-1 IBA＋5 mg· L^-16-BA. The tetraploid homozygous plantlets DH2-15, had a regeneration rate of 93.33% after 14 d dark culture in the optimized regeneration medium of MS＋0.5 mg· L^-1 IBA＋6 mg· L^-16-BA. The regeneration system could be used for the rapid propagation and genetic transformation.
作者
李芙蓉
邓舒
张春芬
肖蓉
侯丽媛
石江鹏
董艳辉
聂园军
曹秋芬
LI Furong1, DENG Shu2, ZHANG Chunfen2, XIAO Rong2, HOU Liyuan3, SHI Jiangpeng1, DONG Yanhui3, NIE Yuanjun4, CAO Qiufen1,3(1College of Biological Engineering, Shanxi University, Taiyuan 030006, Shanxi, China; 2 Shanxi Academy of Agricultural Sciences, Pomology Institute, Taiyuan 030001, Shanxi, China; 3Biotechnology Research Center, Shanxi Academy of Agriculture Sciences, Taiyuan 030031, Shanxi, China; 4Agricultural Resource and Economic Research Institute, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, Shanxi, Chin)
出处
《果树学报》
CAS
CSCD
北大核心
2018年第5期620-630,共11页
Journal of Fruit Science
基金
国家自然科学基金面上项目(31372033)
山西省重点研发(国际合作)项目(201603D421001)
山西省回国留学人员科研资助项目(2016-131
2016-132)
山西省基础研究项目青年科技研究基金(201701D221168)
作者简介
李芙蓉，女，在读硕士研究生，研究方向为植物遗传育种。Tel：18103514897，E-mail：lifuronghello@163．com;通信作者Author for correspondence．Tel：13753480017，E-mail：qiufengcao@163．com
