本文選題：百合 + 遺傳轉化�。� 參考：《山西農(nóng)業(yè)大學》2016年碩士論文

【摘要】：百合(Lilium spp.),百合科百合屬多年生草本球根植物,是一類我國資源豐富,兼具營養(yǎng)藥用價值,觀賞價值和商業(yè)價值的栽培花卉品種。傳統(tǒng)的百合育種方式受到育種周期長,后代分離復雜等因素限制,使得百合定向選育困難。而分子育種方式可以彌補其不足,在短時間內(nèi)實現(xiàn)新品種的定向選育。但是由于百合基因組龐大,外源基因與受體基因間互作方式不清楚,常導致外源基因沉默不表達,轉化效率低。本研究以東方百合‘索邦’(Lilium Oriental'Sorbonne')鱗片為試材,通過三種不同的轉化方式,即以噴霧式植物反應器(開放式培養(yǎng))為基礎的遺傳轉化方式,以組織培養(yǎng)為基礎及以花粉管通道法為基礎的遺傳轉化方式,分別建立了相應的高效無抗生素標記的百合遺傳轉化體系,對百合鱗片進行外源基因AtCKX3的轉化,并成功獲得了轉基因植株,以期為提高百合抗逆性研究提供參考,對尋求可提高百合基因轉化效率的新的轉化系統(tǒng)做出嘗試,獲得的研究結果如下：1.基于噴霧式植物反應器的AtCKX3基因轉化索邦的研究(1)對‘索邦’幼苗進行不同除草劑草丁膦(PPT)濃度的噴施,通過噴施后百合幼苗的植株葉色、形態(tài)變化等表型特征,篩選出PPT噴施的臨界濃度為8.5 mg·L-1。(2)首次建立了將噴霧式植物反應器與農(nóng)桿菌介導的遺傳轉化手段相結合的新的遺傳轉化體系。研究結果表明噴霧式反應器具有操作簡便,成本低廉,對操作要求低等優(yōu)點,更易于實現(xiàn)外源基因轉化工作的大量操作。并且通過分子檢測,初步證明了轉基因植株的獲得,轉化率為0.29%左右。此外,對轉化植株進行移栽后發(fā)現(xiàn),植株移栽的成活率也有很大提高,可達75%,證明了該遺傳轉化體系的可行性。2.基于組織培養(yǎng)的4tCKX3基母轉化索邦的研究(1)將以兩種不同培養(yǎng)基(MS+0.01 mg·L-1 TDZ+0.2 mg·L-1NAA; MS+2.0 mg·L-16-BA+0.2ng·L-1NAA)培養(yǎng)的百合鱗片的分化效率和生長狀態(tài)進行比較,結果表明培養(yǎng)基激素配比：0.01 mg·L-1 TDZ+0.2 mg·L-1 NAA較后者可更高效直接地誘導出鱗莖苗,更利于百合‘索邦’外源基因轉化受體系統(tǒng)的建立。(2)對‘索邦’離體再生鱗片分別進行了相應的抑菌劑羧芐(Cb)濃度以及除草劑草丁膦(PPT)濃度篩選。結果表明,‘索邦’組培鱗片對抗菌劑羧芐(Cb)的耐受濃度為500 mg·L-1,草丁膦篩選的臨界濃度為2.5 mg·L-1。(3)通過農(nóng)桿菌介導法對‘索邦’無菌苗鱗片進行了轉化,并對抗性植株進行了PCR檢測,初步證明目的基因AtCKX3已整合到百合基因組中,轉化效率為0.17%。3.基于花粉管通道法的AtCKX3基因轉化索邦的研究對以花粉管通道法為基礎的遺傳轉化方式進行了初探。通過對東方百合雜交系的4個栽培品種和鐵炮百合(雷山1號)開花植株為材料,進行最優(yōu)雜交組合和合適的DNA穿膜肽溶液導入方式的篩選。結果表明,雜交組合(鐵炮×索邦)結實率,種子的有胚率均為最高,分別為94.74%,18.31%。而直接對柱頭進行涂抹,注射與涂抹相結合的兩種方式對百合植株造成的傷害較小,適宜DNA穿膜肽溶液的導入。
[Abstract]:Lilium (Lilium spp.), Lilium Lilium perennial herb, is a kind of cultivated flower species, which is rich in resources and has both medicinal and medicinal value, ornamental and commercial value in our country. The traditional breeding method of lily is limited by long breeding cycle and complicated separation of offspring, which makes it difficult to select Lilium orientated breeding. The method can make up for the deficiency and realize the directional breeding of new varieties in a short time. However, because of the huge genome of Lilium, the interaction mode between the foreign gene and the receptor gene is not clear, which often leads to the silence of the foreign gene and the low conversion efficiency. The three different transformation methods, namely, the genetic transformation based on the spray plant reactor (open culture), based on the tissue culture and the pollen tube pathway based genetic transformation, established the corresponding efficient and non antibiotic labelled Lilium relic transformation system, and the exogenous gene AtCKX was carried out on the lily scales. 3 transformation, and successfully obtained transgenic plants, in order to provide reference for improving the resistance research of Lilium, and try to find a new transformation system which can improve the transformation efficiency of Lilium. The results are as follows: 1. the study on the AtCKX3 gene conversion of the spray plant reactor (1) to the seedlings of Sorbonne By spraying PPT concentration of different herbicides, the critical concentration of PPT spraying was selected as 8.5 mg. L-1. (2) by spraying the leaf color and morphological characteristics of Lilium seedlings after spraying, and the new genetic transformation system combining the spray plant reactor with Agrobacterium mediated genetic transformation was established for the first time. It shows that the spray reactor has the advantages of simple operation, low cost and low operating requirements. It is easier to achieve a large number of operation of exogenous gene transformation. And through molecular detection, it has been proved that the conversion rate of transgenic plants is about 0.29%. In addition, the survival rate of transplanting is also found after transplanting. A great increase, up to 75%, proves the feasibility of the genetic transformation system.2. based on the tissue culture 4tCKX3 base mother transformation Sorbonne (1) will compare the differentiation efficiency and growth state of the lily scales in two different cultures (MS+0.01 mg. L-1, TDZ+0.2 mg. L-1NAA; MS+2.0 mg. L-16-BA+0.2ng L-1NAA). 0.01 mg. L-1 TDZ+0.2 mg. L-1 NAA can be more efficient and direct induction of bulb seedlings than the latter, which is more beneficial to the establishment of exogenous gene transformation receptor system in Lilium. (2) the concentration of bacteriostat carboxylic benzyl (Cb) and herbicide butylphosphine (PPT) concentration sieves are carried out respectively. The results showed that the tolerance concentration of "Sorbonne" tissue culture scale against bacterial agent carboxybenzyl (Cb) was 500 mg. L-1, and the critical concentration of chlorinyl phosphine screening was 2.5 mg. L-1. (3) through agrobacterium mediated transformation of "Sorbonne" aseptic seedlings, and the antagonistic plants were detected by PCR. It was preliminarily proved that the target gene AtCKX3 had been integrated into the lily base. In the group, the conversion efficiency was 0.17%.3. based on the pollen tube channel method of AtCKX3 gene transformation Sorbonne. The genetic transformation method based on the pollen tube pathway was studied. 4 varieties of oriental lily hybrid and the flowering plant of Lilium Lilium (Lilium 1) were used as the material, and the optimal hybrid combination and appropriate DN were carried out. The results showed that the seed setting rate of the hybrid combination (iron gun, SAC) was the highest, and the seed embryo rate was the highest, which was 94.74%, 18.31%. respectively, which was applied directly to the stigma, and the injections of the two methods combined with the injection and smearing were less harmful to the lily plants and suitable for the introduction of DNA membrane peptide solution.
【學位授予單位】：山西農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S682.29

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