摘要
杨树作为三大速生树种之一,在湖北省林业产业中具有重要的地位。杨树无性系LH05-143、SN04-31和LN05-51作为F1代杂交无性系,在湖北省石首市杨树研究所试验林中生长表现良好,具有一定超亲优势,并已经进入品种鉴定阶段。
本论文以杨树无性系LH05-143和SN04-31叶柄为外植体,建立了2个无性系的再生体系,并以杨树无性系LN05-51为受体材料,开展了农杆菌介导下的抗虫基因Cry1C+9C转化初步研究,获得了主要研究结果如下：
1.以无性系LH05-143为研究对象,得出再生体系的最佳外植体为叶柄。叶柄的最佳消毒方法为：75%酒精消毒30s后灭菌水清洗2-3次,再用0.1%HgCl2消毒5min,LH05-143的污染率为10%,褐化率为5%。
2.在诱导愈伤组织的研究中,叶柄的放置方式对愈伤诱导率影响不显著,但诱导愈伤数存在显著差异,并最终确定杨树无性系再生材料叶柄的放置方式以横放最佳。
3.间接再生诱导中,LH05-143和SN04-31的最佳愈伤诱导培养基配方均为MS+6-BA 0.2mg/L+NAA 1.0mg/L,此时获得的愈伤组织紧实,颜色浅绿；在获得愈伤组织的分化试验中,发现LH05-143的最佳分化培养基配方为MS+6-BA 0.5mg/L+NAA0.1mg/L,而SN04-31则为MS+6-BA 1.0mg/L+NAA 0.1mg/L。
4.直接再生过程中,本文利用二次回归正交试验设计优化了LH05-143不定芽分化试验中激素6-BA(x1)和NAA(x2)的浓度及配比,获得了二次回归方程y=2.5699+0.4101x1-0.2694x2+0.0093x1x2-0.9183x12-0.2537x22
并通过方程得到了关于LH05-143不定芽分化的最佳激素浓度为6-BA1.75mg/L、NAA 0.30mg/L；对SN04-31的不定芽诱导采用了双因素试验,得到其最佳分化培养基配方为MS+6-BA 0.5mg/L+NAA 0.1mg/L。
5.所得不定芽的继代增殖试验中,LH05-143的最佳培养基配方为MS+蔗糖30g/L+琼脂6g/L+6-BA 0.1mg/L+NAA 0.05mg/L,SN04-31的最佳培养基配方为MS+蔗糖30g/L+琼脂7g/L+6-BA 0.1mg/L+NAA 0.02mg/L。
6.LH05-143的最佳生根培养基配方为1/2MS+IBA 0.5mg/L,SN04-31的最佳生根培养基配方为1/2MS+IBA 0.2mg/L。
7.进行了杨树无性系LN05-51叶片不定芽分化和无菌苗生根的卡那霉素敏感性试验,确定了叶片分化的临界浓度为20mg/L,生根的临界浓度为15 mg/L。
8.对转化过程中农杆菌的抑制试验发现最佳的抑菌措施是在筛选培养基中添加头孢霉素400mg/L,另在培养基表层覆盖一层灭菌滤纸,并需要及时更换培养基。
9.采用叶盘法,对无性系LN05-51进行了抗虫基因Cry1C+9C的转化,发现在共培培养基中添加乙酰丁香酮对抗性苗的获得不存在显著影响。
Poplar is one of three fast-growing trees, it has an important position in the forestry industry in Hubei Province. Clones of'LH05-143','SN04-31'andLN05-51 are F1 hybrid generations, which grew well at the Institute of Experimental Forest of Populus in 'Shishou'City, Hubei Province. It also has significant heterosis, and has been testing of cultivars identification.
In the experiment, the regeneration systems have established from petioles of 2 poplar clones(LH05-143 and SN04-31),and we also have study on transformation system of clone LN05-51,which transforms the insect-resistant gene Cry1C+9C by Agrobacterium. The main results that we have are summarized as follow:
1.In the experiment of regeneration, the petioles of poplar were the best explants. The optimal sterilizing methods of petioles were dipping to 75% Ethanol for 30sec, and then were washed by steriled water 2 or 3 times, and then treating with 0.1% HgCl2 for 5min, the rate of contamination was 10%, and the rate of browning was 5% also.
2.In the study of callus induction, the placement of the petioles had no significant effect on the rate of callus induction, but the number of induced callus were significantly different.and ultimately we choosed the style horizontally of the petioles of poplar clones as the best type in order to induce callus.
3.In the indirect induction of regeneration, the optimal callus indcuction medium of LH05-143 and SN04-31 was MS+6-BA 0.2mg/L+NAA 1.0mg/L, and the callus were tight with green color. In another experiments, the optimal medium for the regenertion of LH05-143 from the callus was MS+6-BA 0.5mg/L+NAA 0.1mg/L, and SN04-31 was MS+6-BA 1.0mg/L+NAA 0.1mg/L.
4. Optimal concentrations of 6-BA and NAA and their combinations were tested with quadratic regressive orthogonal design for their effect on the introduction for the adventitious shoots of Poplar clone LH05-143.We get a regression equation: y=2.5699+0.4101x1-0.2694x2+0.0093x1x2-0.9183x12-0.2537x22 Results showed that the best combination of the 6-BA and NAA was 1.75mg/L and 0.30mg/L. On the study of adventitious buds regeneration of SN04-31,the results showed that the optimal medium was MS+6-BA 0.5mg/L+NAA 0.1mg/L.
5.In the experiment of adventitious bud multiplication,the optimal medium for LH05-143 is MS+sucrose 30g/L+agar 6g/L+6-BA 0.1mg/L+NAA 0.05mg/L, and the optimal medium for SN04-31 is MS+sucrose 30g/L+Agar 7g/L+6-BA 0.1mg/L+ NAA 0.02mg/L.
6.The optimal medium of rooting for LH05-143 is 1/2MS+IBA 0.5mg/L, and the mudium for SN04-31 is 1/2MS+IBA 0.2mg/L.
7.Kanamycin sensitive experiments showed that the critical kanamycin sensitive concentrantions for inducing shoots and rooting of the poplar clone LN05-51 were 20mg/L and 15 mg/L.
8.The best anti-bacterial methods for the transformation by Agrobacterium:selective medium with cefotaxime 400mg/L, and covered with a layer in the medium surface, and also need to chage the medium in time.
9.By using Agrobacterium tumefaciens mediated to transformate the insect-resistant gene Cry1C+9C,there were no significant effects with acetosyringone.
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