Iantcheva A, Vlahova M, Trinh T H., Brown S C., Slater A, Elliott M C., Atanassov A
Institute of Genetic Engineering, 2232, Kostinbrod, Bulgaria
Plant Sci. 2001 Mar;160(4):621-627. doi: 10.1016/s0168-9452(00)00432-5.
To avoid polyploidy in regenerants the source of explant material should be monosomatic. Therefore, the leaf and petiole tissue of five diploid Medicago species (Medicago ciliaris, Medicago murex, Medicago orbicularis, Medicago polymorpha and Medicago truncatula cv. Jemalong, and the ecotype R108-1) was assessed for polysomaty by flow cytometry. For the species studied the frequency of 2C nuclei was about 90% in leaves compared with that in petioles. Embryos were readily formed from tissue of leaves in liquid media containing 1 mg l(-1) or 4 mg l(-1) dichlorophenoxyacetic acid (2,4-D). For embryo development two procedures were tested - prolonged use of induction medium and treatment with polyethylene glycol Mw 6000 (PEG). The highly regenerable genotypes M. truncatula cv. Jemalong and R108-1 showed efficient conversion of embryos after maturation in liquid medium. The regenerated plants were diploid and with normal phenotype.
为避免再生植株出现多倍体，外植体材料的来源应为单倍体。因此，通过流式细胞术评估了5种二倍体苜蓿属植物（睫毛苜蓿、硬刺苜蓿、圆叶苜蓿、多形苜蓿和蒺藜苜蓿品种Jemalong以及生态型R108-1）的叶片和叶柄组织的多体性。在所研究的物种中，叶片中2C核的频率约为90%，而叶柄中的频率与之相比。在含有1 mg l(-1)或4 mg l(-1)二氯苯氧乙酸（2,4-D）的液体培养基中，叶片组织很容易形成胚。对于胚的发育，测试了两种方法——延长诱导培养基的使用时间和用聚乙二醇Mw 6000（PEG）处理。高度可再生的基因型蒺藜苜蓿品种Jemalong和R108-1在液体培养基中成熟后显示出胚的高效转化。再生植株为二倍体，且具有正常表型。
