本文選題：獼猴桃 + 組織培養(yǎng)　； 參考：《河南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：獼猴桃(Actinidia chinensis Planch.)是世界上一種重要的水果,該屬有54個(gè)種,21個(gè)變種,共約75個(gè)分類單元,其中中華獼猴桃(A.chinensis Planch.var.chinensis,果肉綠色)和美味獼猴桃(A.chinensis var.deliciosa,果肉金黃色)是栽培最廣泛的的兩個(gè)商業(yè)種類。而軟棗獼猴桃(Actinidia arguta Planch)目前僅有零星栽培,因其果面無(wú)毛,風(fēng)味獨(dú)特,是一種具有發(fā)展前景的果樹(shù)資源。其中‘天源紅’軟棗獼猴桃果皮、果肉和果心全為紅色,酸甜適口,光潔無(wú)毛,可帶皮直接食用,具有重要推廣經(jīng)濟(jì)價(jià)值。為了建立‘天源紅’軟棗獼猴桃快繁體系,本試驗(yàn)以組培苗葉片及莖尖為材料,探討了植物生長(zhǎng)調(diào)節(jié)劑對(duì)葉片愈傷再生的影響。獼猴桃細(xì)菌性潰瘍病,是一種毀滅性細(xì)菌病害,對(duì)世界獼猴桃產(chǎn)業(yè)的發(fā)展造成了嚴(yán)重的威脅。目前尚無(wú)有效方法抑制獼猴桃潰瘍病害的發(fā)生,而采取基因工程技術(shù)培育抗病性強(qiáng)的品種是避免潰瘍病帶來(lái)?yè)p失的有效方法之一。本研究以轉(zhuǎn)抗菌肽D的中華獼猴桃為材料,誘導(dǎo)生根和移栽獲得了大量的獼猴桃盆栽苗,通過(guò)噴施獼猴桃細(xì)菌性潰瘍病病原菌,分析獼猴桃噴施菌液前后相關(guān)酶活性的變化,預(yù)測(cè)轉(zhuǎn)基因植株的抗病性變化。主要研究結(jié)果如下:(1)不同濃度的6-BA和NAA對(duì)‘天源紅’軟棗獼猴桃莖段增殖的影響分析結(jié)果表明,莖段增殖倍數(shù)隨著6-BA的濃度增加而增大,在6-BA濃度為3.0 mg/L,NAA濃度為0.5 mg/L時(shí),莖段增殖倍數(shù)達(dá)到最大,為3.29±0.03。(2)不同濃度的ZT和6-BA對(duì)葉片形成愈傷的結(jié)果表明,6-BA和ZT對(duì)愈傷形成的影響有很大的不同。6-BA濃度從0.5 mg/L到3.0 mg/L,葉片均不能形成愈傷組織,褐化率高,而ZT能明顯誘導(dǎo)愈傷組織的形成,且褐化率較低。(3)不同濃度的ZT和IAA對(duì)葉片再生率的影響結(jié)果表明,當(dāng)ZT濃度為2.5 mg/L,IAA濃度為0.5 mg/L時(shí),葉片再生率達(dá)到最高,為45.00%。(4)不同時(shí)間的暗培養(yǎng)對(duì)葉片再生率的影響。以MS+ZT 2.5mg/L+IAA 0.5 mg/L為基本培養(yǎng)基,分別進(jìn)行15 d,20 d,25 d,30 d的暗處理,結(jié)果表明,當(dāng)葉片進(jìn)行30d的暗處理,葉片再生率最高,為81.67%。(5)以轉(zhuǎn)抗菌肽D的中華獼猴桃抗性芽為材料,通過(guò)誘導(dǎo)生根處理和移栽獲得了大量的獼猴桃盆栽苗,生根結(jié)果顯示,使用1.0 g/L的IBA處理15 s生根效果最好,形成的愈傷較少,且根系較多。(6)轉(zhuǎn)基因獼猴桃的PCR和RT-PCR檢測(cè)結(jié)果表明,外源抗菌肽D基因已經(jīng)整合到獼猴桃基因組中,并在獼猴桃基因組中進(jìn)行轉(zhuǎn)錄,且不同株系間的外源基因的表達(dá)量有差異。(7)對(duì)噴施菌液前后獼猴桃苗葉片中的過(guò)氧化氫酶(CAT)、過(guò)氧化物酶(POD)和苯丙氨酸解氨酶(PAL)的活性分析結(jié)果表明,轉(zhuǎn)抗菌肽D基因在獼猴桃基因組中的過(guò)量表達(dá)對(duì)獼猴桃葉片CAT、POD、PAL活性變化影響不明顯。但是在噴施病原微生物(菌液濃度為1.2×107~108,OD值為0.2左右)的條件下,轉(zhuǎn)基因植株的CAT和POD活性比對(duì)照植株明顯降低,PAL活性比對(duì)照植株明顯升高。
[Abstract]:Actinidia chinensis Planch.Actinidia A. chinensis Planch.var. chinensis (green pulp) and A. chinensis var. deliciosa are the two most widely cultivated commercial species. Actinidia arguta Planche is only cultivated sporadically, because of its hairless surface and unique flavor, Actinidia arguta Planche is a promising fruit resource. Among them, 'Tianyanhong' soft jujube kiwifruit peel, pulp and heart are all red, sweet and sour palatability, smooth and clean hairless, can be eaten directly with skin, has important economic value. In order to establish the rapid propagation system of Actinidia chinensis, the effects of plant growth regulators on leaf callus regeneration were studied. Kiwifruit bacterial canker is a kind of destructive bacterial disease, which poses a serious threat to the development of kiwifruit industry in the world. At present, there is no effective method to inhibit the occurrence of ulcer disease in kiwifruit. Genetic engineering is one of the effective methods to avoid the damage caused by canker disease. In this study, a large number of kiwifruit plantlets were obtained by rooting and transplanting with antimicrobial peptide D, and the changes of enzyme activity before and after spraying kiwifruit bacterial canker were analyzed. The change of disease resistance of transgenic plants was predicted. The main results were as follows: (1) the effects of different concentrations of 6-BA and NAA on the growth of Actinidia chinensis stem segments were analyzed. The results showed that the multiplication ratio of stem segments increased with the increase of 6-BA concentration, when the concentration of 6-BA was 3.0 mg / L, the concentration of NAA was 0.5 mg/L. The effects of ZT and 6-BA on callus formation of leaves were significantly different from 0.5 mg/L to 3.0 mg / L in different concentrations of ZT and ZT. All leaves could not form callus and the browning rate was high. The results showed that when ZT concentration was 2.5 mg 路L ~ (-1) IAA concentration was 0.5 mg/L, leaf regeneration rate was the highest. The effect of dark culture at different time on leaf regeneration rate. MS ZT 2.5mg/L IAA 0.5 mg/L was used as the basic medium, respectively, for 15 days, 20 days and 25 days for 30 days. The results showed that when leaves were treated with dark for 30 days, the regeneration rate of leaves was the highest, which was 81.67. 5) the resistant buds of Actinidia chinensis transferred with antimicrobial peptide D were used as materials. A large number of potted kiwifruit seedlings were obtained by inducing rooting and transplanting. The rooting results showed that IBA of 1.0 g / L for 15 s had the best rooting effect and less callus formation. The results of PCR and RT-PCR analysis of transgenic kiwifruit showed that the exogenous antimicrobial peptide D gene had been integrated into the genome of kiwifruit and transcribed in the genome of kiwifruit. The activity of catalase (CAT), peroxidase (POD) and phenylalanine ammonia-lyase (pal) in leaves of kiwifruit seedlings were analyzed before and after spraying with different strains. The overexpression of transgenic antimicrobial peptide D gene in the genomes of kiwifruit had no significant effect on the change of the activity of POD pal in the leaves of kiwifruit (Actinidia deliciosa L.). However, the CAT and POD activities of transgenic plants were significantly lower than those of control plants under the condition of spraying pathogenic microorganisms (the OD value was about 1.2 脳 10 ~ (7) C ~ (10 ~ 8) O _ (2), which was significantly higher than that of the control plants.
【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S663.4

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