摘要
外源基因删除系统在近年来成为了解决植物转基因生物安全性问题的一种极具潜力的方法。本文将以小叶杨叶片以及叶柄为外植体来建立小叶杨高效再生体系、农杆菌介导法转化小叶杨体系以及不定根发生体系,这些结果将为小叶杨的遗传改良提供基础,具有重要的实际应用价值和理论指导意义。为了开发新的小叶杨基因保存技术并提高其抗病虫害能力,本研究以杂种小叶杨(Populus simonii Carr.×P. deltoides cv.'Shanhaiguanensis')无性系的叶片作为外植体,研究了杂种无性系的的离体培养及叶片再生体系,探讨了不同外源激素组合对杂种无性系不定芽的诱导、分化以及生根的影响。主要结果如下：
1.最适于杂种小叶杨叶片分化的培养基是MS+0.1mg／L6-BA+0.08mg/LNAA+0.005mg/L TDZ,不定芽分化率和平均分化芽芽数分别达到最高的90%和6.8,芽长为2.14cm：最佳生根培养基为1/2MS+0.1mg/L IBA,生根率达90‰最后将这些再生植株成功驯化并移栽到温室。。
2.以小叶杨叶柄作为外植体,建立再生体系,详细分析了外源激素各个组合对叶柄分化和不定芽生根的影响。接种到MS+0.1mg/L6-BA+0.02mg/L NAA离体再生培养基上,不定芽的分化率、平均分化芽的芽数均达到最高的90%和6,芽长为2.25cm。
3.叶片的最佳放置方式是叶片形态学上端朝上；最有利于叶片、叶柄分化的最佳取材位置均是：形态学上部。
4.小叶杨叶片的诱导分化在Km浓度为20mg／L时被抑制,小叶杨叶柄不定芽的生根在Km浓度为20mg/L时被抑制；小叶杨叶柄的诱导分化在Km浓度为10mg／L时被抑制。
5.对Cef抑制农杆菌生长效果的实验表明：浓度为200mg/L的Cef能有效抑制住农杆菌的生长,同时该浓度条件下小叶杨叶片外植体分化和不定芽生根不受影响。因此,将200mg/L Cef作为抑制农杆菌生长的最适浓度。
In recently years,'GM gene deletion'system had becoming a prospectively popular tool for solving the biological safety of transgenic plants. The objective of this study was to establish a highly efficiency in vitro regeneration system and Agrobactirium-mediated transformation system and adventitious buds'rooting system using leaf and petiole as explants. The results will support the later study of Populus simonii's genetic breeding. In order to develop new technology for Populus simonii gene preservation and enhancing its resistance to disease and pests, leaves of hybrid clone off. simonii Carr.×P. deltoides cv.'Shanhaiguanensis'were used as explants to study culture and leaf regeneration system in vitro and to explore the effect of different prescriptions of hormone on induction, differentiation of adventitious bud and rooting. The main results that we had gained were as follows:
1. The optimum medium for adventitious buds regeneration of P. simonii Carr.×P. deltoides cv.'Shanhaiguanensis'was MS+0.1mg/L6-BA+0.08mg/L NAA+0.005mg/L TDZ, with the maximum adventitious bud frequency (90%), number of shoots (6.8), shoot length (2.14cm). The optimum medium for rooting of shoots was1/2MS+0.1mg/L IBA, with a rooting efficiency of90%. Thus the obtained plantlets were successfully hardened and transplanted to greenhouse.
2. We established a regeneration system using petiole as explant and analyzed the influence of differentiation and adventitious bud'rooting in different hormone combinations. The results showed that:the optimum medium for adventitious buds regeneration of the Populus simonii Carr.×P. deltoides cv.'Shanhaiguanensis'was MS+0.1mg/L6-BA+0.02mg/L NAA, with the maximum adventitious bud frequency (90%), number of shoots (6), shoot length (2.25cm).
3. Put foliages'face up using leaves as explant led to the best regeneration efficiency. The best leaves and petioles for adventitious buds regeneration all came from the upper stems.
4.20mg/L kanamycin could inhibit the bud regeneration of "P. simonii Carr.×P. deltoides cv.'Shanhaiguanensis'" when using leaf as explants,10mg/L kanamycin could inhibit the bud regeneration of "P. simonii Carr. XP. deltoides cv.'Shanhaiguanensis"' when using petiole as explants,20mg/L kanamycin could inhibit the root regeneration of P. simonii Carr. XP. deltoides cv.'Shanhaiguanensis'from the shoots.
5.The results of bacteriostasises effect of Cef research showed that:When the concentration of Cef reached to200mg/L, this concentration could restrain the growth of agrobacterium and had no obvious side-effects on adventitious bud regenerations and rooting of adventitious buds of the P. simonii Carr.×P. deltoides cv.'Shanhaiguanensis'. So, we had chosen the200mg/L Cef as the best antibacterial concentration.
引文
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