本文選題：蝴蝶蘭　切入點(diǎn)：花粉管通道法　出處：《西南科技大學(xué)》2017年碩士論文

【摘要】：蝴蝶蘭(Phalaenopsis ssp.)素有“蘭花王后”的美譽(yù),為熱帶蘭珍品,具有極高的觀賞價(jià)值和經(jīng)濟(jì)價(jià)值,在世界花卉產(chǎn)業(yè)中占有重要的地位�，F(xiàn)階段蝴蝶蘭的育種主要以雜交為主,但育種周期漫長(zhǎng),世代選擇復(fù)雜。因此進(jìn)行花粉管通道法介導(dǎo)的蝴蝶蘭轉(zhuǎn)基因研究,為創(chuàng)造特異性優(yōu)異新種質(zhì)開辟途徑。本文較系統(tǒng)地開展了花粉管通道法介導(dǎo)轉(zhuǎn)cbf 1抗寒性基因蝴蝶蘭研究,主要研究結(jié)果如下:(1)對(duì)蝴蝶蘭花粉活力與柱頭可授性研究表明,花粉母細(xì)胞減數(shù)分裂進(jìn)程與其它植物有一定差別,其生殖發(fā)育過程較為特殊,雄配子發(fā)育完成于花蕾期,開花后的蝴蝶蘭花粉母細(xì)胞已進(jìn)入明顯的四分體時(shí)期,生殖細(xì)胞與營(yíng)養(yǎng)核均已形成,花粉活力率(染色率)大小為:完全開放1d花蕾期花蕾展開期,在花蕾展開期的花粉粒活性最強(qiáng),之后隨時(shí)間的增加,活性逐漸降低;柱頭可授性測(cè)定顯示,蝴蝶蘭完全開花10d~30d內(nèi)柱頭保持較高的活性,在10d~15d內(nèi)具有較高的可授性。取花蕾展開期的花粉對(duì)完全開花10d~15d的柱頭授粉,可獲得較高的雜交成功率。(2)對(duì)蝴蝶蘭花粉管通道介導(dǎo)的遺傳轉(zhuǎn)化受體系統(tǒng)進(jìn)行了研究,蝴蝶蘭種胚非共生萌發(fā)形成原球莖的最適培養(yǎng)基為Hyponex(3g/L)+瓊脂7.5g/L+香蕉泥100g/L+蔗糖25g/L+蛋白胨1.5g/L+活性碳2.0g/L;原球莖增殖培養(yǎng)基為Hyponex(3g/L)+瓊脂7.5g/L+香蕉泥100g/L+蔗糖25g/L+蛋白胨2.0g/L+NAA1.0mg/L+6-BA10.0mg/L;由原球莖實(shí)現(xiàn)植株再生的最適培養(yǎng)基為1/2MS+瓊脂7.0g/L+香蕉泥100g/L+蛋白胨2.0g/L+活性碳2.0g/L+蔗糖25g/L。抗生素敏感性試驗(yàn)表明,蝴蝶蘭原球莖及再生植株在卡那霉素選擇壓為0~200mg/L時(shí),抑制生長(zhǎng)不明顯,在300~400mg/L處理組中,生長(zhǎng)明顯受到抑制,當(dāng)達(dá)到500mg/L及更高濃度時(shí),存活率為0%。可確定500mg/L的Kan為轉(zhuǎn)化株陽(yáng)性植株篩選的選擇壓力。(3)以攜帶目的基因cbf 1的p BI121質(zhì)粒和轉(zhuǎn)化了cbf 1表達(dá)載體的農(nóng)桿菌液,分別采用花粉粒攜帶法和子房注射法進(jìn)行遺傳轉(zhuǎn)化。結(jié)果表明,花粉攜帶法比子房注射法有更高的結(jié)實(shí)率,但子房注射法的轉(zhuǎn)化率更高;質(zhì)粒直接侵染比農(nóng)桿菌轉(zhuǎn)化侵染,有更高的結(jié)實(shí)率,質(zhì)粒侵染的最適濃度為100ng/μL,農(nóng)桿菌為OD600=1.0。對(duì)Kan篩選的陽(yáng)性植株進(jìn)行PCR檢測(cè),證明外源cbf 1基因已整合到Kan抗性植株的基因組中。
[Abstract]:Phalaenopsis sp.)Known as "the queen of orchids", it is a tropical orchid treasure with high ornamental value and economic value, and plays an important role in the world flower industry.At present, the breeding of Phalaenopsis is dominated by cross, but the breeding cycle is long and the selection of generations is complicated.Therefore, the transgene research of Phalaenopsis mediated by pollen tube channel method will open the way for the creation of new germplasm with excellent specificity.In this paper, pollen-tube pathway mediated transformation of cbf _ 1 cold resistance gene Phalaenopsis was carried out systematically. The main results were as follows: 1) the pollen viability and stigma viability of Phalaenopsis were studied.The process of meiosis of pollen mother cells is different from that of other plants, and its reproductive development process is special. The male gametes develop at the bud stage, and the pollen mother cells of Phalaenopsis after anthesis have entered the obvious tetrad stage.Both germ cells and vegetative nuclei had been formed, and the pollen vigor rate (staining rate) was as follows: at the stage of full opening for 1 day, the activity of pollen grains was the highest at the stage of flower bud development, and then decreased with the increase of time.The determination of stigma affordability showed that Phalaenopsis had higher activity in 10d~30d and higher viability in 10d~15d.Pollen pollination on the stigma of fully blooming 10d~15d was used to obtain a high success rate of hybridization. The transgenetic transformation receptor system mediated by pollen tube channel of Phalaenopsis was studied.The optimum medium for plant regeneration was 1/2MS Agar 7.0g/L banana mud 100g/L peptone 2.0g/L active carbon 2.0g/L sucrose 25 g / L.The antibiotic sensitivity test showed that the growth inhibition of Phalaenopsis protocorms and regenerated plants was not obvious when kanamycin was selected as 0~200mg/L. In 300~400mg/L treatment group, the growth was obviously inhibited, and the survival rate of Phalaenopsis was 0 when 500mg/L and higher concentration were reached.It was confirmed that the Kan of 500mg/L was the selection pressure for the selection of the positive plants of the transformed strain. The p BI121 plasmid carrying the target gene cbf 1 and Agrobacterium tumefaciens transformed into the cbf 1 expression vector were used to carry out the genetic transformation by pollen grain carrying method and ovary injection method, respectively.The results showed that pollen carrying method had higher seed setting rate than ovary injection method, but the transformation rate of ovary injection method was higher than that of Agrobacterium tumefaciens, and plasmid direct infection had higher seed setting rate than Agrobacterium tumefaciens. The optimum concentration of plasmid infection was 100ng/ 渭 L and Agrobacterium tumefaciens OD600N 1.0.PCR analysis of Kan positive plants showed that exogenous cbf 1 gene had been integrated into the genome of Kan resistant plants.
【學(xué)位授予單位】：西南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S682.31

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