本文選題：菊花 + CBL基因　； 參考：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：菊花(Chuysanthemum morifolium)是菊科菊屬(Chrysanthemum)的多年生草本花卉,因其種類繁多、色彩豐富、形態(tài)各異而深受世人喜愛。本研究首先以菊花品種'C008'的頂芽為外植體獲得了該品種的無菌苗;其次,以菊花品種'C008'和'A'的無菌苗葉片為試驗材料,建立了高頻再生體系;然后在篩選并分析影響遺傳轉(zhuǎn)化因素的基礎(chǔ)上,建立了兩個菊花品種穩(wěn)定的遺傳轉(zhuǎn)化體系,利用農(nóng)桿菌介導(dǎo)的方法將CBL基因成功轉(zhuǎn)入了菊花品種'C008'中。本研究為獲得菊花轉(zhuǎn)基因新品種及抗性研究奠定了基礎(chǔ),主要試驗結(jié)果如下:1.菊花品種'C008'無菌苗的獲得:用0.1%升汞消毒5 min,萌發(fā)率最高,可達(dá)92%。菊花品種'C008'的莖段增殖培養(yǎng)基為MS+0.3mg·L-16-BA+0.3mg·L-1NAA。2.菊花品種'C008'和'A'再生體系的建立:菊花品種'C008'和'A'不定芽分化最適培養(yǎng)基分別是 MS+3.0 mg·L-1 6-BA+0.1 mg·L-1NAA 和 MS+1.0 mg·L-16-BA+0.3 mg.L-1NAA;生根的最適培養(yǎng)基為 1/2MS+0.2 mg.L-1NAA 和 1/2MS+0.3mg.L-1NAA;煉苗移栽成活率分別為84%和80%;抑制菊花品種'A'內(nèi)生菌產(chǎn)生的最適頭孢霉素濃度為50mg·L-1。3.菊花品種'C008'和'A'遺傳轉(zhuǎn)化體系的建立:菊花品種'C008'潮霉素篩選不定芽濃度為3.0 mg.L-1,不定芽生根的篩選濃度為3.0 mg.L-1,頭孢霉素的抑制濃度為300 mg·L-1;菊花品種'A'潮霉素不定芽篩選濃度為1.0 mg.L-1,不定芽生根的篩選濃度為3.0mg.L-1,頭孢霉素的抑制濃度為300mg·L-1。菊花品種'C008'的遺傳轉(zhuǎn)化體系為:預(yù)培養(yǎng)2 d,菌液濃度OD600=0.5-0.6,稀釋倍數(shù)為50倍,侵染時間8 min,共培養(yǎng)2d、延遲培養(yǎng)2d,該品種共獲得抗性植株41株,PCR檢測表明,陽性植株為5株,證明CBL基因已轉(zhuǎn)化到菊花'C008'的基因組DNA中,轉(zhuǎn)化率為12.2%。菊花品種'A'的遺傳轉(zhuǎn)化體系為:預(yù)培養(yǎng)1d,菌液濃度OD600=0.5-0.6,稀釋倍數(shù)為50倍,侵染時間10 min,共培養(yǎng)1 d、延遲培養(yǎng)2 d,該品種雖有抗性芽產(chǎn)生,但并未獲得抗性植株。
[Abstract]:Chuysanthemum morifolium is a perennial herbaceous flower of Chrysanthemum of Compositae. In this study, the apical buds of chrysanthemum cultivar C008 'were used as explants to obtain the sterile seedlings of this variety, and the leaves of aseptic plantlets of Chrysanthemum chrysanthemum cultivars C008' and A' were used as experimental materials to establish a high frequency regeneration system. On the basis of screening and analyzing the factors affecting genetic transformation, the stable genetic transformation system of two chrysanthemum varieties was established, and the CBL gene was successfully transferred into the chrysanthemum cultivar C008'by Agrobacterium tumefaciens. This study laid a foundation for the study of transgenic varieties and resistance of chrysanthemum. The main results are as follows: 1. The aseptic seedling of chrysanthemum variety C008 'was obtained: the germinating rate was the highest, up to 92%, when 0.1% mercuric chloride was sterilized for 5 min. The stem segment proliferation medium of chrysanthemum cultivar C008 'was MS 0.3mg L-16-BA 0.3mg L-1NAA.2. Establishment of Regeneration system of Chrysanthemum cultivars C008 'and A': the most suitable medium for adventitious bud differentiation of chrysanthemum cultivars C008' and A' was MS 3.0 mg L-1 6-BA 0.1 mg L-1NAA and MS 1.0 mg L-16-BA 0.3 mg 路L -1 NAA, respectively, and the optimum medium for rooting was 1/2MS 0.2 mg.L-1NAA and 1/2MS 0.3 mg 路L -1 NAA, respectively, and the optimal medium for seedling transplantation was MS 3.0 mg L -1 6-BA 0.1 mg L-1NAA and MS 1.0 mg L-16-BA 0.3 mg 路L -1 NAA, respectively. The survival rate was 84% and 80%, respectively, and the optimal concentration of ceftomycin was 50mg L-1.3. Establishment of genetic Transformation system for Chrysanthemum cultivars C008 'and A': the concentration of adventitious buds, rooting and cefomycin were 3.0 mg 路L ~ (-1), 3.0 mg 路L ~ (-1) and 300 mg 路L ~ (-1), respectively. The concentration of shoot screening was 1.0 mg 路L ~ (-1), the screening concentration of adventitious bud rooting was 3.0 mg 路L ~ (-1), and the inhibitory concentration of cefamycin was 300mg L ~ (-1). The genetic transformation system of chrysanthemum cultivar C008 'was as follows: Pre-culture for 2 days, concentration of OD600N 0.5-0.6, dilution multiple of 50 times, infection time of 8 min, co-culture for 2 days and delayed cultivation for 2 days. PCR analysis of 41 resistant plants showed that there were 5 positive plants. The results showed that CBL gene had been transformed into the genomic DNA of chrysanthemum chrysanthemum C008 'and the transformation rate was 12.2%. The genetic transformation system of chrysanthemum cultivar was as follows: 1 day of pre-culture, 0.5-0.6 of bacterial solution concentration, 50 times of dilution, 10 min of infection time, 1 day of co-culture and 2 days of delayed cultivation. Although resistant buds were produced, resistant plants were not obtained.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S682.11

【相似文獻(xiàn)】

相關(guān)期刊論文 前4條

1 李利斌;劉開昌;王殿峰;方志軍;倪中福;張義榮;;玉米CBL基因的生物信息學(xué)分析[J];玉米科學(xué);2010年01期

2 趙晉鋒;余愛麗;田崗;杜艷偉;郭二虎;刁現(xiàn)民;;谷子CBL基因鑒定及其在干旱、高鹽脅迫下的表達(dá)分析[J];作物學(xué)報;2013年02期

3 張永濤;劉立峰;李化銀;張一卉;王煥芹;王鳳德;高建偉;李利斌;;三個新的大白菜CBL基因的鑒定和特征分析[J];山東農(nóng)業(yè)科學(xué);2012年12期

4 陳偉;范華;劉賢嫻;李利斌;王淑芬;;兩個甘藍(lán)CBL基因的基因組解析[J];山東農(nóng)業(yè)科學(xué);2011年03期

相關(guān)博士學(xué)位論文 前1條

1 鄧小敏;小麥CBL基因和CIPK基因的克隆及在非生物脅迫中的功能研究[D];華中科技大學(xué);2013年

相關(guān)碩士學(xué)位論文 前2條

1 李剛波;杜梨CBL基因家族三成員序列特征、表達(dá)特點及功能初探[D];南京農(nóng)業(yè)大學(xué);2014年

2 賈紅梅;農(nóng)桿菌介導(dǎo)CBL基因轉(zhuǎn)化菊花的研究[D];沈陽農(nóng)業(yè)大學(xué);2017年

，

本文編號：1811106