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    白花玉石籽石榴遺傳轉化體系的建立

    2024-12-31 00:00:00唐靖雯王寧伍程程王麒麟丁文豪葛偉強郭寧錢晶晶
    果樹學報 2024年12期
    關鍵詞:石榴

    摘" " 要:【目的】為更好地研究石榴的基因功能,以白花玉石籽石榴為試材,構建出一種農桿菌介導的石榴遺傳轉化體系?!痉椒ā吭谝延械氖窠M織培養(yǎng)體系的基礎上,通過篩選不同外植體和優(yōu)化激素組合,獲得石榴最佳再分化途徑;探究農桿菌介導法中抗生素濃度、預培養(yǎng)時間、菌液濃度、侵染時間和抑菌劑浸洗時間對白花玉石籽石榴遺傳轉化效率的影響?!窘Y果】終質量濃度為0.22 mg·L-1 6-BA和0.60 mg·L-1 IBA的WPM培養(yǎng)基能顯著提高石榴外植體的再分化率,其中嫩莖分化率達96.30%±5.20%;50 mg·L-1卡那霉素和200 mg·L-1特美汀是篩選抗性芽的最佳質量濃度;白花玉石籽石榴遺傳轉化的最適組合為:預培養(yǎng)3 d、菌液OD600=0.8、侵染10 min、200 mg·L-1特美汀浸洗15 min。最后,通過檢測GFP熒光,驗證上述遺傳轉化體系所獲得的植株,陽性率為26.00%?!窘Y論】成功建立了農桿菌介導的白花玉石籽石榴嫩莖遺傳轉化體系,為石榴基因功能驗證提供有力的技術支持。

    關鍵詞:石榴;再生途徑優(yōu)化;農桿菌介導法;遺傳轉化體系

    中圖分類號:S665.4 文獻標志碼:A 文章編號:1009-9980(2024)12-2621-13

    Establishment of genetic transformation system for Baihuayushizi pomegranate

    TANG Jingwen1, WANG Ning2, WU Chengcheng1, WANG Qilin1, DING Wenhao1, GE Weiqiang2, GUO Ning3, QIAN Jingjing1*

    (1College of Agriculture, Anhui Science and Technology University, Fengyang 233100, Anhui, China; 2Anhui Zhongyi Agricultural Science amp; Technology Co., Ltd., Huaiyuan 233400, Anhui, China; 3Agricultural Technology Extension Center of Jingshan City, Jingmen City, Hubei Province, Jingshan 431800, Hubei , China)

    Abstract: 【Objective】Pomegranate is a favorate among consumers because of its high economic, nutritional and medicinal values. The rapid development of molecular biology has made genetic transformation an important means to obtain new excellent germplasm of crops and carry out gene function verification, as well as apply an effective supplement to traditional breeding. At present, the genetic transformation system of pomegranate is incomplete, resulting in the lagging of gene function research and molecular breeding in pomegranate compared with other fruit crops. This experiment aimed to build a stable and efficient genetic transformation system for Baihuayushizi pomegranate, with a view to providing satisfactory technical support for gene function verification of pomegranate and the improvement of its germplasm resources. 【Methods】In this study, sterile seedlings of Baihuayushizi pomegranate were used. On the basis of the tissue culture system in early stage, the redifferentiation system of pomegranate and the concentrations of kanamycin and timentin were screened, followed by a discussion on the related influencing factors concerning genetic transformation such as pre-culture time, concentration of Agrobacterium, infection time and socking time of antibacterial agents. Finally, the optimal genetic transformation system for Baihuayushizi pomegranate mediated by Agrobacterium was established. 【Results】The addition of 0.22 mg·L-1 6-BA and 0.60 mg·L-1 IBA into the WPM medium significantly improved the redifferentiation of pomegranate implants, which was manifested as higher differentiation rates of leaves and tender stems compared to other treatment portfolios. The differentiation rates were 83.93%±2.52% and 96.30%±5.20%, respectively, and the differentiation rate of tender stems increased by 14.74% compared with that of leaves. In addition, tender stems with a high differentiation rate were used as the receptor, and both induction rate and differentiation rate were significantly higher than other treatments without addition of kanamycin. When kanamycin was 50 mg·L-1, the induction rate of the callus of tender stem was reduced from 80.00% to 56.67%, and the differentiation of adventitious buds was as weak as only 8.33%±0.02%. When kanamycin was >60 mg·L-1, the callus induced by tender stems was severely browned or even died with a differentiation rate of zero, which indicated that this concentration was not suitable for screening pomegranate seedlings transformed from tender stems. When timentin was used as the antibacterial agent, if the concentration was 50 mg·L-1, the differentiation rate of tender stems was the highest at 67.22%±0.03%, but the contamination rate of the implant was also significantly higher than that of other treatments. When the concentration of timentin increased by 200 mg·L-1, while the growth of Agrobacterium was basically inhibited, the differentiation rates of tender stems and buds could reach more than 50%. Although 250-300 mg·L-1 timentin completely inhibited the growth of Agrobacterium, the excessively high concentration was also had a certain inhibitory effect on the growth of implants, and the differentiation rate was less than 50%. In addition, the study on the four important factors of Agrobacterium-mediated genetic transformation of pomegranate showed that the transformation rate varied greatly among different treatment portfolios, and their effect on the genetic transformation rate was manifested as follows: pre-culture time > concentration of Agrobacterium>infection time > socking time of antibacterial agents. Further single-factor analysis of variance showed that the genetic transformation rate with pre-culture for 3 d was the highest at 19.33%, which was significantly higher than that of other treatment portfolios. When concentration of Agrobacterium OD600=0.7, the transformation rate was 12.17%, which was not much different from that when OD600=0.6 and 0.8 but was significantly different from that when OD600=0.5. The genetic conversion rate with 10 min infection and 15 min immersion in antibacterial agent was higher than that of other treatments. 【Conclusion】The addition of 0.22 mg·L-1 6-BA and 0.60 mg·L-1 IBA into the WPM medium significantly improved the redifferentiation of pomegranate implants, with a differentiation rate of tender stems of 96.30%±5.20%. 50 mg·L-1 kanamycin and 200 mg·L-1 timentin were optimal for screening resistant bud. Pre-culture for 3 d, concentration of Agrobacterium OD600=0.8, 10 min infection time and 15 min immersion in 200 mg·L-1 timentin were the most suitable portfolio for genetic transformation of pomegranate. GFP fluorescence detection was performed for verification on the plants obtained under the above-mentioned genetic transformation system, and the positive plant acquisition rate was 26.00%. In this study, genetic transformation system of tender stems that were Agrobacterium-mediated was successfully established, laying the foundation for verifying pomegranate gene function.

    Key words: Pomegranate; Regeneration pathway optimization; Agrobacterium-mediated method; Genetic transformation system

    石榴(Punica granatum L.)為千屈菜科(Pomegranate)石榴屬(Punica L.)植物,是具有較高營養(yǎng)、經(jīng)濟和醫(yī)藥價值的水果[1]。2021年中國石榴栽培面積(約8.67萬hm2)和產(chǎn)量(約130萬t)穩(wěn)居世界首位[2]。目前,石榴產(chǎn)業(yè)在全球經(jīng)濟市場中逐漸興起[3],因經(jīng)濟效益顯著,已成為助力中國鄉(xiāng)村振興的特色產(chǎn)業(yè)[4]。然而石榴產(chǎn)業(yè)發(fā)展仍然存在效率低、品質劣等問題[5]。目前關于石榴基因功能的研究局限于異源表達,包括擬南芥(Arabidopsis thaliana)[6]、番茄(Solanum lycopersicon Mill.)[7]等植物,這在一定程度上導致石榴的基因功能研究和分子育種進度相對落后,致使石榴產(chǎn)業(yè)高質量發(fā)展緩慢[8]。因此,建立白花玉石籽石榴遺傳轉化體系有助于了解石榴基因功能,并為后續(xù)石榴遺傳改良奠定基礎[9]。

    目前,由農桿菌介導的遺傳轉化技術較為常用,已在柑橘(Citrus reticulata Blanco)[10]、蘋果(Malus pumila Mill.)[11]、梨(Pisum salivum)[12]等果樹中得到運用[13-14]。但是,該方法在石榴中的報道相對缺乏。趙玉潔[15]以石榴胚培苗的子葉、葉片、上下胚軸為外植體,經(jīng)共培養(yǎng)后,GUS瞬時表達率約70.00%,建立了突尼斯軟籽石榴遺傳轉化體系;劉真真[16]以胚培苗的子葉和葉片為外植體,優(yōu)化了突尼斯軟籽和豫大籽石榴遺傳轉化體系,成功獲得5株抗性芽嫁接苗;吳亞君[17]和Verma等[18]均以胚培養(yǎng)苗為試材,初步建立了石榴遺傳轉化體系。石榴許多品種為雜合體,種子的遺傳背景不清晰,其有性后代會出現(xiàn)性狀分離等現(xiàn)象,對基因功能驗證等相關研究有較大影響。因此,石榴遺傳轉化技術雖已開展了一些研究,但大多以胚培苗為試材,無法更高效地為后續(xù)石榴基因功能驗證及育種提供技術支持;同時不同的石榴品種遺傳轉化所需培養(yǎng)基、激素濃度等存在差異,導致建立的遺傳轉化體系無法適用于所有石榴品種。針對上述現(xiàn)象,劉真真等[19]認為,建立高效穩(wěn)定的再生體系是石榴遺傳轉化體系建立的首要條件。

    目前尚未見關于以莖尖為試材,建立白花玉石籽石榴遺傳轉化體系的相關研究。采用農桿菌介導的白花玉石籽石榴遺傳轉化技術,不僅可以解決多種問題,包括遺傳轉化外植體材料要求多、遺傳背景不清晰、轉化困難等,也能夠為石榴的基因功能研究提供良好的技術支持。因此,筆者以白花玉石籽石榴莖尖脫毒獲得的無菌苗為試材,擬在已建立的組織培養(yǎng)體系的基礎上[20],通過優(yōu)化石榴再分化體系、篩選卡那霉素(kanamycin,Kan)和特美?。╰imentin,TMT)濃度并對預培養(yǎng)時間、菌液濃度、侵染時間、抑菌劑浸洗時間等影響遺傳轉化的相關因素進行探討,以期建立穩(wěn)定的石榴遺傳轉化體系,為后續(xù)石榴的基因功能驗證和種質資源改良提供良好的技術支撐。

    1 材料和方法

    1.1 試驗材料

    1.1.1" " 植物材料" " 白花玉石籽石榴材料由安徽省懷遠縣中以農業(yè)科技有限公司種質圃提供,莖尖脫毒苗的獲得參考Qian等[20]的方法。繼代培養(yǎng)2次后,用于本試驗。

    1.1.2" " 載體構建、農桿菌的獲得及菌液制備" " 植物表達載體pRI101-35S::GFP受贈于南京農業(yè)大學,載體圖譜如圖1所示,帶有卡那霉素抗性標簽。利用同源重組的方法[21]將目的基因PgCYP85A1與表達載體連接,獲得重組質粒。采用凍融法[22]將重組質粒pRI101-35S::PgCYP85A1-GFP轉入具有Ach5型背景的農桿菌感受態(tài)細胞LBA4404[購自Takara(日本)公司]。農桿菌侵染液制備參考王民炎等[23]的方法,待菌液OD600為0.5~0.8時,3000 r·min-1、28 ℃條件下離心5 min,收集菌體,用等體積的侵染液(WPM+30 g蔗糖+0.22 mg·L-1 6-BA+0.60 mg·L-1 IBA+0.1 mol·L-1乙酰丁香酮(Acetosyringone,AS)重懸菌體,室溫孵育2 h備用。

    1.2 試驗方法

    1.2.1" " 不同質量濃度的激素組合對石榴外植體再分化的影響" " 以無菌苗葉片和嫩莖作為外植體,分別接種于含不同激素組合的WPM培養(yǎng)基中(表1)。外植體接種程序為:葉片剪成0.50 cm×0.50 cm的小塊,背面劃口后接種于培養(yǎng)基上;嫩莖去除生長點后剪成0.50 cm小節(jié),接種于培養(yǎng)基上。其中每個外植體為1次試驗重復,共計進行12次重復。每一激素濃度和外植體的組合為1個試驗組,共進行48組平行試驗。培養(yǎng)35 d后觀察記錄并計算分化率。

    分化率/%=分化出芽的外植體數(shù)/接種外植體數(shù)×100。

    1.2.2" " Kan質量濃度的篩選" " 以1.2.1獲得的分化率高的植物組織為外植體(下同),將未經(jīng)農桿菌侵染的石榴外植體分別接種于含0、30、40、50、60和70 mg·L-1 Kan的分化培養(yǎng)基(1.2.1篩選的最佳激素濃度組合,下同)中,每個濃度處理接種60個外植體,其中10個外植體·皿-1,3次重復。35 d后統(tǒng)計不同處理中外植體分化率。

    1.2.3" " TMT質量濃度的篩選" " 將用農桿菌侵染(OD600=0.6~0.8)后的外植體接種至含50、100、150、200、250、300 mg·L-1共6個質量濃度梯度TMT的分化培養(yǎng)基中,每個濃度處理接種60個外植體,其中10個外植體·皿-1。3次重復。35 d后統(tǒng)計外植體分化率及污染率,確定適宜的TMT質量濃度。

    污染率/%=污染的外植體數(shù)/接種外植體數(shù)×100。

    1.2.4" " 不同遺傳轉化因子對農桿菌介導的白花玉石籽石榴遺傳轉化效率的影響" " 根據(jù)遺傳轉化的基本步驟[24-25],為了篩選白花玉石籽石榴遺傳轉化4個轉化因子的最佳水平,設置了預培養(yǎng)時間(A)梯度為1、2、3、4 d;設置農桿菌菌液OD600(B)梯度為0.5、0.6、0.7、0.8,將預培養(yǎng)的外植體放入不同濃度的侵染液中;設置侵染時間(C)為5、10、15、20 min,在不同濃度的農桿菌侵染液中侵染固定時間;設置抑菌劑浸洗時間(D)梯度為15、20、25、30 min,將共培養(yǎng)結束后的外植體放入添加了200 mg·L-1 TMT的無菌水中浸洗。按照L16(45)進行四因素四水平的正交試驗設計,共16個處理組合(表2),每個處理組合均接種50個外植體,每組設3次重復。

    1.2.5" " 轉基因植株的鑒定" " 培養(yǎng)35 d后,待抗性芽長到2~3 cm時,切下芽點置于生根培養(yǎng)基中(添加0.8 mg·L-1 IBA和1.2.3、1.2.4中最適Kan、TMT質量濃度)培養(yǎng)成苗。將生根培養(yǎng)35 d的抗性植株與未轉化的植株,利用倒置熒光顯微鏡(Thermo Fisher Scientific,美國)觀察根系GFP熒光,統(tǒng)計并記錄熒光苗率。

    熒光苗率/%=熒光苗數(shù)量/接種外植體數(shù)×100。

    1.3 數(shù)據(jù)分析

    采用Excel 2019錄入和整理數(shù)據(jù),采用SPSS 18.0軟件進行方差分析。

    2 結果與分析

    2.1 不同激素組合和外植體對石榴再生途徑優(yōu)化的影響

    將石榴無菌苗葉片和嫩莖接種到含不同激素組合的WPM培養(yǎng)基中,培養(yǎng)35 d后進行觀察和數(shù)據(jù)統(tǒng)計。由表3可知,添加不同質量濃度6-BA和IBA組合均可誘導葉片和嫩莖分化出芽。當添加0.22 mg·L-1 6-BA時,葉片和嫩莖的分化效果較好,與添加0.50、0.60、0.70 mg·L-1 IBA配比組合后顯著高于添加0.40 mg·L-1 IBA的激素組合;其中,IBA為0.60 mg·L-1時,葉片和嫩莖的分化率最高,分別為83.93%和96.30%,嫩莖分化率較葉片提高了14.74%(圖2)。綜合分析可得,石榴再生體系中不定芽分化最適外植體為嫩莖,分化培養(yǎng)基的最佳激素配比組合為0.22 mg·L-1 6-BA和0.60 mg·L-1 IBA。

    2.2 不同質量濃度Kan對石榴嫩莖分化的影響

    將石榴無菌苗嫩莖(去生長點,不包含芽原基部分)接種到含不同質量濃度Kan的分化培養(yǎng)基中培養(yǎng)35 d。表4和圖3結果顯示,不添加Kan時愈傷組織分化的不定芽生長狀況最佳,分別表現(xiàn)在嫩莖誘導的愈傷組織為嫩綠色,不定芽的分化率達到88.33%,顯著高于其他處理,分化能力旺盛;隨著Kan質量濃度的增加,石榴嫩莖愈傷組織誘導率及分化率逐漸下降;當Kan質量濃度為50 mg·L-1時,嫩莖愈傷組織誘導率由80.00%降至56.67%,且愈傷組織出現(xiàn)黃化現(xiàn)象,不定芽分化能力較弱;當Kan質量濃度增加至60~70 mg·L-1時,對外植體毒害作用逐漸加重,表現(xiàn)為石榴嫩莖愈傷組織誘導率低至30%以下,愈傷組織褐化嚴重甚至死亡,分化率為0。因此,采用50 mg·L-1 Kan作為石榴嫩莖愈傷組織分化的臨界質量濃度進行轉化苗的篩選。

    2.3 不同質量濃度TMT對石榴嫩莖分化的影響

    將農桿菌菌液侵染后的嫩莖接種于含不同TMT質量濃度的分化培養(yǎng)基35 d后進行觀察,結果如表5所示,不同質量濃度的TMT均能夠較好抑制農桿菌生長,但隨著TMT質量濃度的增加,石榴嫩莖分化率和污染率呈逐漸下降的趨勢。在50 mg·L-1 TMT處理下,嫩莖分化率最高為67.22%,但外植體污染率顯著高于其他處理;當TMT質量濃度增加至200 mg·L-1時,在基本抑制農桿菌生長的同時,嫩莖不定芽分化率能超過50%;250~300 mg·L-1 TMT雖完全抑制了農桿菌的生長,但外植體分化率逐漸降低,表明TMT質量濃度過高會對芽點的生長造成一定的影響。因此綜合考慮,選取200 mg·L-1 TMT作為遺傳轉化的抑菌質量濃度。

    2.4 農桿菌介導石榴遺傳轉化條件的影響

    方差分析結果表明,4個轉化因子中預培養(yǎng)時間和菌液濃度對轉化率有極顯著影響,而侵染時間和抑菌劑浸洗時間的影響不顯著(表6)。通過正交試驗結果直觀分析(表7),在16組不同水平處理的轉化因子組合試驗中組合間差異顯著,各處理組合轉化率差異較大,極差結果看出預培養(yǎng)時間>菌液濃度>侵染時間>抑菌劑浸洗時間。比較均值發(fā)現(xiàn)轉化率隨著預培養(yǎng)時間和菌液濃度的增加呈“S”形曲線上升(預培養(yǎng)時間的轉化率均值依次為9.67%、2.50%、19.33%、3.33%;菌液濃度的轉化率均值依次為5.00%、7.83%、12.17%、9.83%),預培養(yǎng)時間3 d和菌液OD600=0.7時的轉化率達到了最高值。侵染時間和抑菌劑浸洗時間對轉化率雖沒有顯著影響,但侵染10 min和抑菌劑浸洗15 min的轉化率均值高于其他處理。進一步的單因素方差分析和多重比較(表8)可知,不同預培養(yǎng)時間的轉化率具有顯著差異,預培養(yǎng)3 d的轉化率與預培養(yǎng)1、2和4 d的轉化率之間有顯著差異,預培養(yǎng)1 d的轉化率與預培養(yǎng)4 d的轉化率之間沒有顯著差異;不同菌液濃度水平下OD600=0.7的轉化率與OD600=0.6和0.8的轉化率間差異不顯著,與OD600=0.5之間差異顯著;侵染時間和抑菌劑浸洗時間各水平間差異不顯著。綜合各因子試驗結果,得到農桿菌介導石榴嫩莖遺傳轉化的最佳轉化因子組合是A3B4C2D1(組合12),即預培養(yǎng)3 d、菌液OD600=0.8、侵染時間10 min和抑菌劑浸洗時間15 min為白花玉石籽石榴遺傳轉化體系建立的最佳條件組合(圖4)。

    2.5 石榴轉基因植株的鑒定

    通過熒光顯微鏡能夠在特定波長的藍色激發(fā)光源下,觀察到GFP在細胞中綠色熒光的表達情況。筆者取篩選后的石榴健康植株根系為樣本進行熒光顯微鏡檢測(圖5)。經(jīng)過統(tǒng)計,在最優(yōu)體系條件下,接種50株外植體,獲得熒光苗13株,轉化效率為26.00%。

    3 討 論

    高效遺傳轉化技術是進行基因功能驗證最直接、最廣泛的方法。目前,石榴遺傳轉化體系的研究存在不同品種差異較大、外植體遺傳背景不清晰、轉化率低等問題,其根本原因是石榴組織培養(yǎng)技術體系建立得不完善。建立穩(wěn)定、高效的再生體系,是遺傳轉化的關鍵環(huán)節(jié)[26]。通常石榴組織培養(yǎng)體系建立不完善會出現(xiàn)材料褐化[27]、再生率低[28]等問題。筆者以此為切入點,在已克服外植體褐化問題、建立石榴組織培養(yǎng)體系的基礎上,優(yōu)化外植體再生途徑;進行篩選劑、抑菌劑的濃度確定;最后通過探究預培養(yǎng)時間、菌液濃度、侵染時間等影響白花玉石籽石榴遺傳轉化的重要因子,得到遺傳轉化率為26.00%。研究表明,紅腺忍冬(Lonicera hypoglauca Miq.)遺傳轉化率最高為8.56%[29];甜瓜(Cucumis melo L.)則僅為6.70%[30];通過對柑橘遺傳轉化體系進行優(yōu)化,其轉化率為13.00%~36.00%[31]。白城小黑楊(Populus simonii×P. nigra ‘Baicheng’)[32]、萬壽菊(Tagetes erecta)[33]等植物轉化效率均低于5.00%。因此,筆者建立了相對完善的白花玉石籽石榴遺傳轉化體系,為后續(xù)石榴基因功能及調控、分子育種等方向的研究提供堅實的技術支撐。

    不定芽的發(fā)生方式一般可以分為2種,即直接發(fā)生型和間接發(fā)生型[34]。直接發(fā)生型操作過程簡化且能夠縮短育種周期。在研究白術(Atractylodes macrocephala)胚軸和胚根直接分化不定芽的再生體系的過程中,梁玉玲等[35]發(fā)現(xiàn)采用間接發(fā)生型的方式獲得植株,極容易出現(xiàn)褐變、體細胞變異等情況,這與朱雅靜等[36]對紫果西番蓮(Passiflora edulis)子葉再生體系的建立結果相似,最終均采用了直接發(fā)生型的方式獲得不定芽。因此,筆者通過對石榴再生途徑的優(yōu)化,獲得石榴最佳直接發(fā)生型的不定芽誘導途徑,在一定程度上避免了遺傳轉化過程中嵌合體的出現(xiàn),提高了遺傳轉化率。筆者采用嫩莖和葉片為外植體進行不定芽分化,發(fā)現(xiàn)嫩莖分化率顯著高于葉片,分化率達96.30%。因此,選用嫩莖為白花玉石籽石榴遺傳轉化體系的受體材料,為成功建立遺傳轉化體系奠定了基礎。

    在遺傳轉化過程中,需要兩種類型的抗生素,分別為轉化受體材料的篩選劑及抑制農桿菌生長的抑菌劑。因此,選擇適宜的抗生素種類及濃度是遺傳轉化體系建立的重要環(huán)節(jié)[37]。Kan作為遺傳轉化中廣泛使用的抗生素,其質量濃度會對遺傳轉化效率產(chǎn)生關鍵影響。研究發(fā)現(xiàn),添加50 mg·L-1 Kan的篩選效果較好,能穩(wěn)定地進行石榴遺傳轉化,大巖桐(Sinningia speciosa Benth)[38]和甜椒(Capsicum annuum)[39]遺傳轉化相關因素優(yōu)化均證明50 mg·L-1 Kan可提高遺傳轉化效率。在抑制農桿菌生長中,周賡等[40]關于黃瓜(Cucumis sativus L.)對不同濃度TMT的抑菌作用研究表明,共培養(yǎng)后用脫菌處理可顯著抑制農桿菌的生長。本研究也發(fā)現(xiàn),適宜的抑菌劑濃度有助于在植物分化與抑制農桿菌生長中找到平衡點,從而成功建立遺傳轉化體系。

    預培養(yǎng)時間[41]、農桿菌濃度與侵染時間[42]及抑菌劑浸洗時間[43]是遺傳轉化研究中討論較多的影響因素。一般來講,預培養(yǎng)時間過短,細胞未進入最佳分化階段,外源基因的整合較少;時間過長,外植體傷口會產(chǎn)生保護層,阻礙農桿菌與分裂細胞的接觸。筆者以石榴嫩莖為受體材料,發(fā)現(xiàn)遺傳轉化過程中預培養(yǎng)3 d時轉化率最高。本研究結果與趙玉潔[15]以突尼斯軟籽石榴子葉和葉片為受體材料建立遺傳轉化體系得到的預培養(yǎng)時間一致;唐伶俐等[30]以甜瓜子葉為受體材料進行遺傳轉化研究,結果表明子葉進行3 d預培養(yǎng)可明顯提高轉化率,這說明不同植物材料對預培養(yǎng)時間的要求基本一致。合適的菌液濃度和最佳的侵染時間有利于提高轉化效率,如果農桿菌菌液濃度過低或侵染時間過短,導致侵染后受體內農桿菌含量過低,轉化效果不理想;反之,會使農桿菌過量生長,不利于受體材料的恢復及脫菌困難。筆者發(fā)現(xiàn)嫩莖侵染條件為菌液OD600=0.8、侵染時間為10 min效果較好,與李躍霞[44]以實生苗葉片為受體材料進行遺傳轉化得到的最佳侵染時間相同,但菌液濃度差異較大,原因可能是進行遺傳轉化的石榴品種不同、受體材料不同或受體材料來源不同造成結果有差異;郭利軍等[45]以菠蘿[Ananas comosus (L.) Merr.]愈傷組織為受體材料進行遺傳轉化時侵染時間差異較大,分析其原因可能是外植體不同造成結果有差異,愈傷組織作為外植體較難受到農桿菌的侵染,而導致試驗結果有較大差異。研究發(fā)現(xiàn),選擇培養(yǎng)前受體材料在抗生素中的浸洗時間對植物生長分化也有不同的影響,通過前期試驗發(fā)現(xiàn),200 mg·L-1 TMT對農桿菌的抑制作用比較明顯,對受體材料的轉化率影響較小。但共培養(yǎng)后植株會染菌,因此對受體材料表面的農桿菌進行浸洗,能夠避免因農桿菌脫菌困難造成遺傳轉化率低等問題,郭紅艷等[43]結果同樣表明,適宜的TMT溶液浸洗時間能抑制農桿菌的生長,是外植體轉化的關鍵,這一結論與本研究結果相似。

    4 結 論

    筆者以白花玉石籽石榴無菌苗為材料,對石榴再生體系中的外植體類型和激素組合進行了再優(yōu)化,篩選了適宜石榴嫩莖分化的Kan、TMT質量濃度,并探究了影響農桿菌介導的白花玉石籽石榴嫩莖遺傳轉化因子。結果表明,WPM培養(yǎng)基中加入0.22 mg·L-1 6-BA和0.60 mg·L-1 IBA能顯著促進石榴外植體的再分化,分化率超過96.00%;以分化率高的石榴嫩莖為受體材料,50 mg·L-1 Kan、200 mg·L-1 TMT為篩選抗性芽的最佳質量濃度。白花玉石籽石榴嫩莖最適的遺傳轉化步驟為:(1)石榴嫩莖預培養(yǎng)3 d后,置于菌液OD600=0.8中侵染10 min,侵染后用無菌濾紙吸干菌液,接種于添加0.1 mol·L-1 AS的分化培養(yǎng)基中,于(25±2)℃條件下暗培養(yǎng)2 d;(2)暗培養(yǎng)后,將嫩莖放入添加200 mg·L-1 TMT的無菌水中浸洗,浸洗后使用無菌濾紙吸干多余水分,接種于含添加50 mg·L-1 Kan、200 mg·L-1 TMT的分化培養(yǎng)基中進行培養(yǎng);(3)培養(yǎng)35 d后,待抗性芽長到2~3 cm時,切下芽點置于生根培養(yǎng)基中(WPM+0.8 mg·L-1 IBA+50 mg·L-1 Kan+200 mg·L-1 TMT)培養(yǎng)成苗。通過以上方法獲得的植株通過GFP熒光檢測,陽性植株獲得率為26.00%。綜上所述,筆者成功建立了以嫩莖為受體材料的白花玉石籽石榴遺傳轉化體系,為石榴基因功能驗證和品種改良提供技術支撐和理論依據(jù)。

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