二倍体栽培马铃薯高效遗传转化体系的建立

doi:10.3864/j.issn.0578-1752.2018.17.002

摘要/Abstract

摘要： 【背景】马铃薯是最重要的块茎类粮食作物。栽培马铃薯以四倍体为主，但四倍体遗传和薯块繁殖增加了马铃薯改良的难度。因此越来越多的科学家呼吁在二倍体水平进行马铃薯的再驯化，将马铃薯驯化成种子繁殖作物。自然界中存在4个二倍体马铃薯原始栽培种，这些二倍体栽培种中蕴含着丰富的遗传变异，但是它们的遗传转化体系尚未成熟。【目的】建立高效的二倍体栽培马铃薯遗传转化体系，为二倍体马铃薯优良等位基因和分子育种提供工具。【方法】以二倍体栽培种马铃薯（Solanum phureja）CIP 703541为试验材料，利用绿色荧光蛋白基因（green fluorescent protein，GFP）作为报告基因，对遗传转化体系进行摸索，主要包括：预处理培养时间、诱导再生的激素比例、抗生素浓度以及转化效率。另外，利用同样的激素组合对17份不同基因型的二倍体马铃薯原始栽培种进行了测试，比较各个材料在不同激素浓度条件下的再生效率，筛选出再生效率较高的激素组合进行大量遗传转化；对再生苗进行生根筛选、荧光观察和PCR鉴定，统计阳性植株的概率。用流式细胞仪检测阳性植株的倍性，以分析再生植株发生染色体加倍的频率。【结果】预处理培养时间为2 d的外植体浸染效率最高。二倍体材料CIP 703541最适合再生出芽的激素配方为2.0 mg·L^-1玉米素（zeatin，ZT）﹕0.01 mg·L^-1奈乙酸（naphthaleneacetic acid，NAA）。再生阶段抗生素卡那霉素（kanamycin，Kan）的最适筛选浓度为100 mg·L^-1，生根阶段的最适筛选浓度为50 mg·L^-1。通过对不同基因型进行筛选，获得3份再生能力较强的二倍体马铃薯材料（CIP 703308、CIP 703312和CIP 703541）。它们响应的激素配方各不相同，在添加了100 mg·L^-1卡那霉素的再生培养基中，3份材料的再生效率分别为45%、40%和52%。通过大规模遗传转化证明，3份材料的转化率分别为2.8%、4.2%和5.3%。经流式细胞仪检测，所获得的马铃薯阳性植株中四倍体的比例较高，CIP 703308、CIP 703312和CIP 703541的阳性转化植株中二倍体的比例分别为5.26%、10.53%和38.46%。【结论】建立了高效的二倍体马铃薯遗传转化体系，获得了3份再生能力较强的二倍体材料。另外，发现二倍体马铃薯在经过遗传转化之后再生植株中染色体加倍的比例较高。

关键词: 二倍体马铃薯, 遗传转化, 染色体加倍

Abstract: 【Background】Potato is the most important tuber crop. Cultivated potatoes are mainly tetraploid, of which the improvement is hampered by tetrasomic genetics and clonal propagation. Thus, more and more scientists are appealing to re-domesticate potato into an inbred line-based crop propagated by seeds at the diploid level. There are four diploid landraces in the nature, which contains abundant genetic variations. But the genetic transformation of diploid landraces is immature. 【Objective】This study will construct the high efficient genetic transformation system of diploid potato, which is necessary for exploiting the beneficial genes and molecular breeding in diploid potato. 【Method】In this study, we used GFP (green fluorescent protein) as a reporter gene, and explored the genetic transformation in diploid clones Solanum phureja CIP 703541. Different conditions were tested, including pre-culture time, ratio of plant hormones during regeneration, concentration of antibiotics, transformation efficiency.. In addition, we tested the screening system in another 17 diploid landraces. The regeneration efficiencies of different genotypes using various hormone concentrations were compared, and the genotypes with high regeneration efficiency were selected for large-scale transformation. Rooting screening and PCR amplification were performed for the regeneration seedlings, to obtain the positive transformants. The ploidy of regenerated seedlings were detected by flow cytometry to calculate the frequency of chromosome doubling. 【Result】The optimal pre-culture time was 2 days (d). The optimal ratio of hormones for regeneration of CIP 703541 was 2.0 mg·L^-1Zeatin (ZT) and 0.1 mg·L^-1Naphthaleneacetic acid (NAA). The optimal concentrations of kanamycin for regeneration and rooting were 100 mg·L^-1and 50 mg·L^-1, respectively. Three diploid clones (CIP 703308, CIP 703312 and CIP 703541) with high regeneration capacity were obtained, and the optimal ratio of hormones varied among these genotypes， the regeneration rates were 45%, 40% and 52%, respectively, on the regeneration medium with 100 mg·L^-1kanamycin, and their transformation rates were 2.8%, 4.2% and 5.3%, respectively. The frequency of chromosome doubling is high during the genetic transformation of diploid potatoes. The ratio diploid transformants in CIP 703308, CIP 703312 and CIP 703541 were 5.26%, 10.53% and 38.46%, respectively. 【Conclusion】We constructed the high efficient genetic transformation system in diploid potato, and obtained three diploid clones with high regeneration capacity. In addition, the ratio of regenerated seedlings with chromosome doubling is high in the positive transformants of diploid potatoes.

Key words: diploid potato, genetic transformation, chromosome doubling

叶明旺，张春芝，黄三文. 二倍体栽培马铃薯高效遗传转化体系的建立[J]. 中国农业科学, 2018, 51(17): 3249-3257.

YE MingWang, ZHANG ChunZhi, HUANG SanWen. Construction of High Efficient Genetic Transformation System for Diploid Potatoes[J]. Scientia Agricultura Sinica, 2018, 51(17): 3249-3257.

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