本文選題：紅豆越橘 + 組織培養(yǎng)　； 參考：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：本試驗(yàn)以3個(gè)紅豆越橘品種‘艾達(dá)’、‘桑那’和‘羅西’試管苗為材料,研究了培養(yǎng)基與5種植物生長(zhǎng)物質(zhì)對(duì)葉片誘導(dǎo)不定芽的影響,初步建立了紅豆越橘離體葉片不定芽誘導(dǎo)體系。初步建立葉盤(pán)法將VcANS基因轉(zhuǎn)至紅豆越橘‘艾達(dá)’的遺傳轉(zhuǎn)化的實(shí)驗(yàn)條件,旨在為紅豆越橘離體葉片不定芽誘導(dǎo)和遺傳轉(zhuǎn)化技術(shù)體系提供依據(jù)。研究結(jié)果如下:1.以不同品種的紅豆越橘莖段葉片為實(shí)驗(yàn)材料,通過(guò)比較WPM、MS、1/2MS和1/4MS四種培養(yǎng)基中,最適培養(yǎng)基為WPM培養(yǎng)基;‘艾達(dá)’、‘桑那’和‘羅西’三個(gè)品種離體葉片再生體系生長(zhǎng)調(diào)節(jié)物質(zhì)最優(yōu)組合為:WPM+ZT 4.0 mg/L+IBA 0.1 mg/L�！_(dá)’在TDZ 2.0 mg/L時(shí)誘導(dǎo)率最高為99%,在ZT 5.0 mg/L的處理?xiàng)l件下再生頻率最高為35.2個(gè)/葉;‘桑那’在TDZ 2.0 mg/L時(shí)誘導(dǎo)率最高為93%,在ZT 4.0 mg/L+IBA 0.1 mg/L時(shí)生頻率最高為28.3個(gè)/葉;‘羅西’在ZT 4.0 mg/L+NAA 0.5 mg/L時(shí)誘導(dǎo)率最高為87.5%,在ZT 5.0 mg/L處理?xiàng)l件下再生頻率最高為29.9個(gè)/葉。2.紅豆越橘‘艾達(dá)’在各類(lèi)生長(zhǎng)調(diào)節(jié)物質(zhì)中生長(zhǎng)狀態(tài)最佳,因此,以‘艾達(dá)’繼代苗葉片為受體材料進(jìn)行后續(xù)遺傳轉(zhuǎn)化研究。3.成功構(gòu)建植物表達(dá)載體pBASTA-VcANS。4.為紅豆越橘遺傳轉(zhuǎn)化體系篩選的抑菌劑頭孢噻肟鈉(Cef)最適濃度為250 mg/L;紅豆越橘遺傳轉(zhuǎn)化陽(yáng)性植株篩選試劑為除草劑草銨膦,最適使用濃度為0.9 mg/L,使用以農(nóng)桿菌介導(dǎo)的葉盤(pán)法進(jìn)行遺傳轉(zhuǎn)化試驗(yàn),將VcANS基因轉(zhuǎn)化到‘艾達(dá)’基因組中,轉(zhuǎn)化體系為:將培養(yǎng)25~30 d的‘艾達(dá)’中部葉片,用解剖刀在葉片的近軸面上用橫向劃兩刀處理以后平鋪于預(yù)培養(yǎng)基中暗培養(yǎng)3 d后,將葉片小心取出侵入含植物表達(dá)載體pBASTA-VcANS的農(nóng)桿菌菌液中,浸染10 min,浸染結(jié)束后將葉片撈出吸干多余菌液后,平鋪于共培養(yǎng)基上,25℃暗培養(yǎng)2 d;用液體培養(yǎng)基(WPM+ZT 4.0 mg/L+IBA 0.1 mg/L+AS100 mg/L+3%蔗糖)漂洗后,將葉片轉(zhuǎn)接入選擇培養(yǎng)基(WPM+ZT 4.0 mg/L+IBA 0.1 mg/L+8g/L瓊脂粉+3%蔗糖+250mg/LCef+草銨膦0.9 mg/L)中,暗培養(yǎng)2 d后轉(zhuǎn)至光下培養(yǎng),30 d后將長(zhǎng)出的不定芽接入抗性培養(yǎng)基(WPM+ZT 4.0 mg/L+IBA 0.1 mg/L+8 g/L瓊脂粉+3%蔗糖+250 mg/L Cef)中繼代培養(yǎng)。5.使用農(nóng)桿菌介導(dǎo)的葉盤(pán)法遺傳轉(zhuǎn)化法中,在選擇條件草銨膦存在條件下,得到20棵草銨膦抗性植株,經(jīng)過(guò)對(duì)它們分別進(jìn)行PCR檢測(cè),結(jié)果得到一株轉(zhuǎn)化植株。
[Abstract]:The effects of culture medium and five plant growth substances on adventitious buds induced by leaves were studied by using three red bean blueberry varieties' Adaganthus' sauna 'and' Rossi'in vitro.The adventitious bud induction system of red bean blueberry leaves in vitro was established.The experiment condition of transferring VcANS gene to 'Ada' by leaf disk method was established to provide the basis for adventitious bud induction and genetic transformation of red bean blueberry leaves in vitro.The results are as follows: 1.The stem leaves of different varieties of red bean blueberry were used as experimental materials. By comparing the four kinds of media, WPMN MS 1 / 2 MS and 1/4MS, the most suitable medium was WPM medium.The optimum combination of growth regulators of 'sauna' and 'Rossi' leaves in vitro was: 1: WPM ZT 4.0 mg/L IBA 0.1 mg / L.Under the condition of ZT 5.0 mg/L, the highest induction rate of 'Ida' was 93.30 at TDZ 2.0 mg/L, and that of ZT 4.0 mg/L IBA 0.1 mg/L was 28.3 / leaf.The highest induction rate of 'Rossi' was 87.5 at ZT 4.0 mg/L NAA 0.5 mg/L, and the highest regeneration frequency was 29.9 leaves / leaf under the condition of ZT 5.0 mg/L.Red bean blueberry 'Ada' is the best growth regulator among all kinds of growth regulators. Therefore, the following genetic transformation study was carried out with the leaves of 'Ada' subculture seedling as the recipient material. 3.The plant expression vector pBASTA-VcANS.4was successfully constructed.The optimum concentration of cefotaxime sodium cefin for screening genetic transformation system of red bean blueberry was 250 mg / L.The optimal concentration was 0.9 mg / L, and Agrobacterium tumefaciens-mediated leaf disc method was used to carry out genetic transformation test. The VcANS gene was transformed into the genome of 'Ada'. The transformation system was as follows: the middle leaf of 'Ida' was cultured for 2530 days.The leaves were treated with two transversal blades on the paraxial surface of the leaves and cultured in a dark culture medium for 3 days. The leaves were carefully removed and invaded into Agrobacterium tumefaciens solution containing plant expression vector pBASTA-VcANS.After soaking for 10 minutes, the leaves were removed from the superfluous bacteria solution after soaking, and then spread on the co-culture medium at 25 鈩，

本文編號(hào)：1761372