本文關(guān)鍵詞： 南瓜 外植體 愈傷組織 子葉節(jié)離體再生 遺傳轉(zhuǎn)化 農(nóng)桿菌介導(dǎo)　出處：《河南科技學(xué)院》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：南瓜(Cucurbita spp.)種質(zhì)資源豐富,用途廣泛,在世界范圍內(nèi)普遍種植。隨著南瓜資源產(chǎn)業(yè)化利用的不斷深入,篩選出抗逆性強(qiáng)的新品種已成為當(dāng)前主要的育種目標(biāo),而現(xiàn)有的南瓜資源已經(jīng)很難完全滿(mǎn)足新品種選育的不同需求。傳統(tǒng)的育種方法不僅周期長(zhǎng),難度系數(shù)大,而且遺傳性狀難以穩(wěn)定,越來(lái)越多的育種工作者借助基因工程進(jìn)行種質(zhì)創(chuàng)新以提高南瓜的抗逆性。建立高效穩(wěn)定的南瓜離體再生體系是開(kāi)展南瓜基因工程的重要前提,也為進(jìn)一步開(kāi)展基因功能研究和遺傳轉(zhuǎn)化等相關(guān)育種工作奠定基礎(chǔ)。本研究以印度南瓜‘北觀’(C.maxima‘Beiguan’)自交系為試驗(yàn)材料,研究不同外植體愈傷組織的誘導(dǎo)及愈傷組織繼代培養(yǎng)的條件,探究利用誘導(dǎo)的愈傷組織建立植株再生體系的方法;研究了以南瓜子葉節(jié)為外植體的再生體系建立中無(wú)菌苗的培養(yǎng)方式、苗齡、子葉節(jié)外植體大小、不同植物激素濃度配比等因素對(duì)南瓜離體再生的影響,優(yōu)化了南瓜的離體再生體系;同時(shí)研究了南瓜子葉節(jié)再生中不同抑菌劑濃度、不同抗生素濃度、超聲波輔助農(nóng)桿菌介導(dǎo)法等因素對(duì)南瓜遺傳轉(zhuǎn)化的影響,優(yōu)化了南瓜的遺傳轉(zhuǎn)化體系,確立了南瓜的最佳再生和農(nóng)桿菌介導(dǎo)StNHX1基因轉(zhuǎn)化南瓜的條件,以期為南瓜遺傳轉(zhuǎn)化研究奠定基礎(chǔ)。研究的主要內(nèi)容和結(jié)果如下:(1)南瓜愈傷組織的誘導(dǎo)及其建立再生體系的探究:以印度南瓜‘北觀’無(wú)菌幼苗為試驗(yàn)材料,其下胚軸、子葉、真葉、莖段作為外植體均可誘導(dǎo)出愈傷組織,其中子葉和莖段較易誘導(dǎo)出愈傷組織;愈傷組織在MS+1.0 mg/L 6-BA(或2.0 mg/L KT)+0.2mg/L NAA培養(yǎng)基上進(jìn)行繼代培養(yǎng)效果較好,可獲得質(zhì)量較好的愈傷組織。將誘導(dǎo)出的愈傷組織轉(zhuǎn)接到含有不同植物激素種類(lèi)、濃度組合的培養(yǎng)基以誘導(dǎo)愈傷組織的再生,但均未誘導(dǎo)出不定芽。(2)南瓜子葉節(jié)離體再生體系的優(yōu)化:將剝?nèi)シN殼的種子,先在超凈工作臺(tái)上用75%酒精消毒30s,再用0.1%升汞消毒9min,無(wú)菌水沖洗4-5次,再用無(wú)菌水浸泡5h后,接種于MS培養(yǎng)基中,28℃暗培養(yǎng),待胚根長(zhǎng)至0.5cm左右時(shí),再暗培養(yǎng)24h,然后進(jìn)行光照培養(yǎng),可獲得優(yōu)質(zhì)的無(wú)菌苗;通過(guò)篩選,以5d苗齡的子葉節(jié)(1/4子葉+2 mm下胚軸)作為外植體,以MS+1.5 mg/L 6-BA+4 mg/L AgNO3為子葉節(jié)誘導(dǎo)培養(yǎng)基,以MS+0.5 mg/L 6-BA+0.05 mg/L GA3為不定芽伸長(zhǎng)培養(yǎng)基,以MS+0.1 mg/L NAA為生根培養(yǎng)基,可以作為南瓜子葉節(jié)再生的最佳培養(yǎng)體系。(3)南瓜子葉節(jié)的遺傳轉(zhuǎn)化條件的優(yōu)化:用農(nóng)桿菌EHA105菌株侵染南瓜子葉節(jié),培養(yǎng)基中添加500mg/L的抑菌劑羧芐青霉素,可有效抑制農(nóng)桿菌過(guò)度增殖,且不會(huì)對(duì)外植體分化產(chǎn)生抑制作用;篩選抗生素雙丙氨膦的最適濃度為5mg/L。當(dāng)應(yīng)用超聲波輔助農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化南瓜子葉節(jié)時(shí),超聲波60W功率,處理3min,可在外植體損傷最輕的情況下,較大程度的提高轉(zhuǎn)化率。
[Abstract]:Cucurbita spp.is rich in germplasm resources and widely used in the world. Screening new varieties with strong resistance to stress has become the main breeding target at present, but the existing pumpkin resources have been difficult to fully meet the different needs of breeding new varieties. The traditional breeding methods not only have a long period, but also have a large coefficient of difficulty. And hereditary traits are difficult to stabilize. More and more breeders are using genetic engineering to carry out germplasm innovation to improve the resistance of pumpkin. It is an important prerequisite to develop pumpkin genetic engineering to establish an efficient and stable pumpkin regeneration system in vitro. This study also laid a foundation for further research on gene function and genetic transformation. In this study, the inbred line of Indian pumpkin'C. Maxima Beiguanli 'was used as experimental material. To study the induction of callus from different explants and the conditions of callus subculture, and to explore the method of establishing plant regeneration system by callus induction. The effects of culture methods, seedling age, cotyledon explant size and hormone concentration on regeneration of pumpkin cotyledons were studied. The regeneration system of pumpkin in vitro was optimized. At the same time, the effects of different concentrations of antimicrobial agents, different concentrations of antibiotics and ultrasonic assisted Agrobacterium tumefaciens on the genetic transformation of pumpkin were studied, and the genetic transformation system of pumpkin was optimized. The optimal regeneration of pumpkin and the conditions of Agrobacterium tumefaciens mediated transformation of StNHX1 gene into pumpkin were established. In order to lay a foundation for the study of genetic transformation of pumpkin, the main contents and results of the study are as follows: 1). Study on Callus Induction and Regeneration system of Pumpkin: the sterile Seedling of Indian Pumpkin 'Beiguan' was used as experimental material. Its hypocotyls, cotyledons, true leaves and stem segments can be used as explants to induce callus, among which cotyledons and stem segments are easy to induce callus. The callus was subcultured on MS 1.0 mg/L 6-BA (or 2.0 mg/L KT) 0.2 mg / L NAA medium. Good quality callus could be obtained. The callus could be transferred to the medium containing different plant hormones and concentration to induce callus regeneration. But none induced adventitious bud. 2) Optimization of in vitro regeneration system of pumpkin cotyledon: peeling off the seeds of the seed shell, sterilizing with 75% alcohol for 30s on the super-clean table. After sterilizing with 0.1% dimercuric acid for 9 minutes, washing with sterile water for 4-5 times, then soaking in sterile water for 5 hours, inoculating in MS medium at 28 鈩，

本文編號(hào)：1465400