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

Abstract

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

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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Construction of High Efficient Genetic Transformation System for Diploid Potatoes

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