朱佩佩，羅燚佳，向雯，張明磊，張劍俠

抗寒無(wú)核葡萄雜種胚挽救及分子標(biāo)記輔助選擇

朱佩佩，羅燚佳，向雯，張明磊，張劍俠

西北農(nóng)林科技大學(xué)園藝學(xué)院/旱區(qū)作物逆境生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室/農(nóng)業(yè)部西北地區(qū)園藝作物生物與種質(zhì)創(chuàng)制重點(diǎn)實(shí)驗(yàn)室，陜西楊凌 712100

【】利用胚挽救技術(shù)創(chuàng)制抗寒無(wú)核葡萄新種質(zhì)，為選育抗寒無(wú)核葡萄新品種奠定基礎(chǔ)。分別以抗寒歐美雜交無(wú)核葡萄品種‘Jupiter’和歐山雜種有核優(yōu)系‘00-1-5’（‘玫瑰香’ב山葡萄黑龍江實(shí)生’）為父本，5個(gè)歐亞種無(wú)核品種‘Ruby Seedless’‘秦紅1號(hào)’‘秦紅2號(hào)’‘Crimson Seedless’和‘秦秀’為母本雜交，在各母本最佳胚珠取樣時(shí)期取樣，以固-液雙相培養(yǎng)基MM3和ER分別作為胚發(fā)育培養(yǎng)基進(jìn)行胚珠培養(yǎng)，比較不同母本和2種培養(yǎng)基對(duì)胚發(fā)育率、萌發(fā)率、成苗率及畸形苗率的影響；以WPM+0.2 mg?L-16-BA+0.1 mg?L-1IAA固體培養(yǎng)基作為胚萌發(fā)培養(yǎng)基，獲得胚挽救后代，經(jīng)煉苗后移栽至田間；分別在2MS+0.2 mg?L-16-BA+0.1 mg?L-1IAA培養(yǎng)基中添加0、1.0和1.6 mg?L-1ZnSO4進(jìn)行畸形苗轉(zhuǎn)化正常苗培養(yǎng)，以篩選最適宜轉(zhuǎn)化培養(yǎng)基；利用無(wú)核基因分子標(biāo)記SCF27-2000對(duì)胚挽救后代進(jìn)行無(wú)核性狀分子檢測(cè)。5個(gè)雜交組合的胚珠接種至MM3培養(yǎng)基的數(shù)量為2 158個(gè)，獲得發(fā)育胚175個(gè)和胚挽救苗118株；接種至ER培養(yǎng)基的胚珠數(shù)為894個(gè)，獲得發(fā)育胚74個(gè)和胚挽救苗58株。以‘00-1-5’為父本的2個(gè)雜交組合中，‘秦紅2號(hào)’比‘秦秀’更適合作為母本，其雜種胚的發(fā)育率和成苗率最高，分別為17.04%和7.41%；以‘Jupiter’為父本的3個(gè)雜交組合中，‘Ruby Seedless’בJupiter’效果最好，胚發(fā)育率和成苗率分別為13.71%和10.67%，其次是‘秦紅1號(hào)’בJupiter’，胚發(fā)育率和成苗率分別為14.39%和4.71%，而‘Crimson Seedless’בJupiter’胚發(fā)育率和成苗率最低，分別為9.55%和1.76%。接種至MM3培養(yǎng)基的胚珠，獲得的胚發(fā)育率和成苗率均高于ER培養(yǎng)基?；蚊缏首罡叩氖恰丶t1號(hào)’בJupiter’，而‘Ruby Seedless’בJupiter’未出現(xiàn)畸形苗，ER培養(yǎng)基比MM3培養(yǎng)基形成的畸形苗率高?！丶t2號(hào)’ב00-1-5’組合畸形苗接種至2MS+1.6 mg?L-1ZnSO4+0.2 mg?L-16-BA+0.1 mg?L-1IAA轉(zhuǎn)化培養(yǎng)基效果最好，轉(zhuǎn)化率為40.00%。利用無(wú)核標(biāo)記SCF27-2000對(duì)5個(gè)雜交組合176株雜種后代檢測(cè)表明，159個(gè)株系擁有無(wú)核基因分子標(biāo)記?！甊uby Seedless’‘秦紅1號(hào)’和‘秦紅2號(hào)’適宜作為胚挽救育種的母本，而‘Crimson Seedless’和‘秦秀’不適合作為胚挽救育種的母本，培養(yǎng)基MM3比ER更適宜作為胚發(fā)育培養(yǎng)基，擁有無(wú)核基因分子標(biāo)記的159株胚挽救苗為培育抗寒無(wú)核葡萄新品種提供了種質(zhì)基礎(chǔ)。

無(wú)核葡萄；抗寒性；胚挽救；新種質(zhì)；分子標(biāo)記輔助選擇

0 引言

【研究意義】無(wú)核葡萄是育種的一個(gè)重要目標(biāo)。目前世界上栽培的無(wú)核葡萄品種大多屬于歐亞種葡萄（L.），品質(zhì)優(yōu)良，但抗寒性差，生產(chǎn)上缺乏抗寒無(wú)核葡萄品種，限制了其在寒冷地區(qū)的發(fā)展。原產(chǎn)中國(guó)的野生山葡萄（Rupr.）可耐-40℃的低溫，是葡萄屬中極為重要的抗寒種質(zhì)資源[1-2]，此外，原產(chǎn)北美洲的美洲葡萄（L.）也是重要的抗寒種質(zhì)資源[2]。利用抗寒的歐山雜種（×）或歐美雜種（×）作父本，與種子敗育型歐亞種無(wú)核葡萄品種作母本雜交，借助胚挽救技術(shù)，在雜種胚敗育前提供種胚正常發(fā)育所需的營(yíng)養(yǎng)物質(zhì)使其萌發(fā)成苗，是創(chuàng)制抗寒無(wú)核葡萄新種質(zhì)的有效途徑，將為進(jìn)一步選育抗寒無(wú)核葡萄新品種奠定基礎(chǔ)。【前人研究進(jìn)展】1982年，美國(guó)葡萄育種家RAMMING和EMERSHAD[3]首次以無(wú)核葡萄品種的胚珠進(jìn)行離體培養(yǎng)，獲得了2株雜種，標(biāo)志著無(wú)核葡萄胚挽救技術(shù)的創(chuàng)立。由于傳統(tǒng)的無(wú)核葡萄育種只能以有核品種作母本與無(wú)核品種作父本雜交，雜交后代中無(wú)核雜種的幾率極低，育種效率低，周期長(zhǎng)；而胚挽救技術(shù)以無(wú)核葡萄作母本與無(wú)核或有核品種作父本雜交，擴(kuò)大了母本選擇的范圍，提高了育種的效率，從而使該技術(shù)在許多國(guó)家得到廣泛的研究和應(yīng)用[4-7]。迄今，國(guó)內(nèi)外已通過(guò)該技術(shù)選育出‘Melissa’‘Autumn Crisp’‘Early Sugar’‘秦紅無(wú)核’和‘秦紅1號(hào)’等47個(gè)無(wú)核葡萄新品種[8]。作為一項(xiàng)新的無(wú)核葡萄育種技術(shù)，影響其成功的因素很多，如父母本基因型、花前噴施激素、取樣時(shí)期、基本培養(yǎng)基及其形態(tài)、添加有機(jī)物種類與濃度、培養(yǎng)條件等[9-13]。親本基因型是影響胚挽救效果的一個(gè)關(guān)鍵因素，特別是母本基因型，前人研究認(rèn)為‘Flame Seedless’[4]、‘Ruby Seedless’[4,9]、‘Perlette’[9]、‘秦紅2號(hào)’[9]和‘Askari’[13]等適宜作胚挽救母本。徐海英等[6]研究認(rèn)為胚過(guò)早發(fā)生敗育的品種不適宜作胚挽救的母本，如‘Thompson Seedless’和‘Himrod’等，但Pommer等[12]認(rèn)為早熟和中熟品種比晚熟品種作母本的胚挽救效果更好。此外，父本對(duì)胚挽救效果也有一定的影響[9-10]。培養(yǎng)基及其形態(tài)是決定胚挽救成敗的另一關(guān)鍵因素。Razi等[13]認(rèn)為NN培養(yǎng)基培養(yǎng)效果好，也有使用ER培養(yǎng)基作為胚發(fā)育培養(yǎng)基[10]，Pommer等[12]使用Cain培養(yǎng)基作為胚發(fā)育培養(yǎng)基，但筆者課題組研究獲得的MM3培養(yǎng)基（改良ER培養(yǎng)基）被認(rèn)為更適合以中國(guó)野生葡萄作為父本的胚挽救育種[9,14]。關(guān)于培養(yǎng)基的形態(tài)，Spiegel-Roy等[11]研究認(rèn)為固相培養(yǎng)基最好，但RAMMING等[15]認(rèn)為液體培養(yǎng)基效果好。TIAN等[16]認(rèn)為固-液雙相培養(yǎng)基最適合作為胚發(fā)育培養(yǎng)基。由此可見(jiàn)，胚發(fā)育培養(yǎng)基及其形態(tài)有必要做進(jìn)一步的研究。對(duì)于通過(guò)胚挽救技術(shù)獲得的雜種，一般是在樹(shù)體結(jié)果后通過(guò)田間鑒定是否為無(wú)核性狀，來(lái)確定取舍，育種速度較慢，而利用分子標(biāo)記技術(shù)可以實(shí)現(xiàn)對(duì)目標(biāo)性狀在幼苗期的鑒定，極大縮短品種選育時(shí)間[17]。目前已獲得的葡萄無(wú)核基因分子標(biāo)記主要有SCAR標(biāo)記SCC8-1018[18]、SCF27-2000[19]，RAPD標(biāo)記GSLP1-569[20]，SSR標(biāo)記p3-VvAGL11-216[21]、VMC7F2-198[22]和VvSD10[23]。其中以SCF27-2000對(duì)無(wú)核葡萄品種檢測(cè)具有較廣泛的適用性[14,24]?！颈狙芯壳腥朦c(diǎn)】針對(duì)目前無(wú)核葡萄胚挽救中胚發(fā)育率、萌發(fā)率和成苗率仍較低，以及生產(chǎn)上缺乏抗寒無(wú)核葡萄品種的問(wèn)題，本研究對(duì)胚挽救的主要影響因素（母本和胚發(fā)育培養(yǎng)基）進(jìn)行研究，配置無(wú)核品種×抗寒歐山雜種、無(wú)核品種×抗寒歐美雜種雜交組合，以篩選出適宜作胚挽救的母本，比較2種胚發(fā)育培養(yǎng)基MM3和ER的胚挽救效果，并篩選出適宜畸形苗轉(zhuǎn)化為正常苗的培養(yǎng)基，同時(shí)對(duì)胚挽救后代進(jìn)行早期無(wú)核性狀的分子標(biāo)記輔助選擇?！緮M解決的關(guān)鍵問(wèn)題】通過(guò)比較母本基因型、篩選胚發(fā)育培養(yǎng)基、篩選畸形苗轉(zhuǎn)化正常苗培養(yǎng)基及分子標(biāo)記輔助選擇，為提高無(wú)核葡萄胚挽救效率提供參考依據(jù)，同時(shí)創(chuàng)制抗寒無(wú)核葡萄新種質(zhì)，為進(jìn)一步選育抗寒無(wú)核葡萄新品種奠定材料基礎(chǔ)。

1 材料與方法

2018年4月至2019年5月在西北農(nóng)林科技大學(xué)葡萄種質(zhì)資源圃、旱區(qū)作物逆境生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室與園藝學(xué)院葡萄育種與分子生物學(xué)實(shí)驗(yàn)室進(jìn)行。

1.1 親本材料

共設(shè)計(jì)5個(gè)雜交組合，分別是‘秦紅2號(hào)’ב00-1-5’、‘秦秀’ב00-1-5’、‘Ruby Seedless’בJupiter’、‘秦紅1號(hào)’בJupiter’和‘Crimson Seedless’בJupiter’。其中父本‘Jupiter’為抗寒歐美雜交無(wú)核品種，引自美國(guó)；另一父本‘00-1-5’（‘玫瑰香’ב山葡萄黑龍江實(shí)生’）為抗寒歐山雜種有核優(yōu)系，由筆者課題組選育。父、母本均為二倍體品種（系）。

1.2 方法

1.2.1 葡萄田間雜交 在父本植株上一個(gè)花序中有5%小花開(kāi)放時(shí)取花穗，制備新鮮花粉，然后干燥、低溫保存。母本在開(kāi)花前3 d，選擇健壯植株上發(fā)育一致的花穗進(jìn)行人工去雄（圖1-A）。2—3 d后，于上午7:00—9:00進(jìn)行授粉，連續(xù)授粉3 d以保證授粉效率。

1.2.2 胚珠培養(yǎng) 5個(gè)母本品種‘秦秀’‘Crimson Seedless’‘秦紅2號(hào)’‘秦紅10號(hào)’和‘Ruby Seedless’的取樣時(shí)期分別為花后42 d[9]、51 d[9]、51 d[24]、51 d[24]和61 d[9]。對(duì)采集的幼果先用自來(lái)水進(jìn)行表面沖洗，然后在超凈工作臺(tái)中依次用濃度為75%乙醇和1%次氯酸鈉進(jìn)行表面消毒，再用無(wú)菌水沖洗3次，最后用手術(shù)刀剝?nèi)∨咧?，接種于胚發(fā)育培養(yǎng)基中。

將5個(gè)組合‘秦秀’ב00-1-5’、‘秦紅2號(hào)’ב00-1-5’、‘Ruby Seedless’בJupiter’、‘Crimson Seedless’בJupiter’和‘秦紅1號(hào)’בJupiter’的胚珠接種在MM3固-液雙相培養(yǎng)基中，研究不同母本對(duì)胚挽救效果的影響。

將材料較多的2個(gè)組合‘秦紅2號(hào)’ב00-1-5’和‘Ruby Seedless’בJupiter’的胚珠，分別接種在固-液雙相培養(yǎng)基ER和MM3中，比較不同培養(yǎng)基對(duì)胚挽救效果的影響。

兩種培養(yǎng)基添加物均為0.5 g?L-1水解酪蛋白+1 mmol?L-1絲氨酸+0.5 mg?L-1GA3+1.0 mg?L-1IAA+ 0.5 mg?L-16-BA+60 g?L-1蔗糖+3 g?L-1活性炭+7 g?L-1瓊脂（pH 6），其中液相部分不加瓊脂。采用容量為200 mL的培養(yǎng)瓶，每瓶接種20個(gè)胚珠。培養(yǎng)瓶上覆蓋黑布進(jìn)行遮光培養(yǎng)，溫度為（25±1）℃，培養(yǎng)9—10周。

1.2.3 胚萌發(fā)培養(yǎng) 胚珠培養(yǎng)9—10周后，在超凈工作臺(tái)中借助解剖鏡剝?nèi)÷闩?，接種到胚萌發(fā)培養(yǎng)基中（圖1-B），置于（25±1）℃、每日光照16 h的培養(yǎng)室培養(yǎng)。40 d后，胚萌發(fā)成苗（圖1-C—E）。萌發(fā)培養(yǎng)基為WPM固體培養(yǎng)基，培養(yǎng)基成分為：WPM+0.2 mg?L-16-BA+0.1 mg?L-1IAA+20 g?L-1蔗糖+1.5 g?L-1活性炭+7 g?L-1瓊脂（pH 6）。

幼苗在煉苗移栽前需要進(jìn)行繼代培養(yǎng)，培養(yǎng)基為1/2 MS+0.2 mg?L-1IBA+30 g?L-1蔗糖+7 g?L-1瓊脂（pH 6）。

1.2.4 畸形苗觀察及轉(zhuǎn)化培養(yǎng)基篩選 胚萌發(fā)培養(yǎng)5—6周后，觀察不能正常萌發(fā)的畸形苗，包括子葉扭曲無(wú)根苗、白化苗、玻璃化苗、矮小苗等，比較不同母本組合及同一組合分別在MM3和ER固-液雙相培養(yǎng)基上的畸形苗率。

將‘秦紅2號(hào)’ב00-1-5’組合產(chǎn)生的畸形苗，分別接種至添加0、1.0和1.6 mg?L-1ZnSO4的2MS+0.2 mg?L-16-BA+0.1 mg?L-1IAA培養(yǎng)基中，培養(yǎng)30 d后統(tǒng)計(jì)畸形苗轉(zhuǎn)化為正常苗的百分率。

1.2.5 無(wú)核基因分子標(biāo)記輔助選擇 采用CTAB法提取雜交后代株系基因組DNA，利用葡萄無(wú)核基因SCAR標(biāo)記SCF27-2000的引物（F：5′-CAGGTGGGA GTAGTGGAATG-3′；R：5′-CAGGTGGGAGTAAGA TTTGT-3′）對(duì)雜交組合親本及后代進(jìn)行PCR檢測(cè)，凡能擴(kuò)增出2 000 bp大小的條帶即初步確定為具有無(wú)核性狀。

A：去雄后的花穗；B：胚；C—E：幼胚萌發(fā)成苗；F：胚挽救苗移栽至田間

PCR反應(yīng)程序?yàn)椋?4℃預(yù)變性4 min；95℃變性30 s，62℃復(fù)性90 s，72℃延伸1 min，共35個(gè)循環(huán)；72℃延伸1 min，12℃終止。

1.2.6 數(shù)據(jù)統(tǒng)計(jì)與分析 記錄各組合胚的發(fā)育數(shù)、萌發(fā)數(shù)、成苗數(shù)和畸形苗數(shù)，統(tǒng)計(jì)胚的發(fā)育率、萌發(fā)率、成苗率、畸形苗率及畸形苗轉(zhuǎn)化率，計(jì)算方法如下：

胚的發(fā)育率（%）=從胚珠中剝?nèi)∨叩膫€(gè)數(shù)/胚珠數(shù)×100

胚的萌發(fā)率（%）=萌發(fā)胚的個(gè)數(shù)/胚珠數(shù)×100

胚的成苗率（%）=成苗數(shù)/胚珠數(shù)×100

畸形苗率（%）= 畸形苗數(shù)/胚珠數(shù)×100

畸形苗轉(zhuǎn)化率（%）=轉(zhuǎn)化成的正常苗數(shù)/畸形苗數(shù)×100

使用SPSS 22.0軟件，對(duì)同一雜交組合3次生物

學(xué)重復(fù)中胚的發(fā)育率、萌發(fā)率、成苗率、畸形苗率和畸形苗轉(zhuǎn)化率進(jìn)行差異顯著性分析（＜0.05）。

2 結(jié)果

2.1 不同母本對(duì)胚挽救效果的影響

5個(gè)雜交組合中不同母本的胚發(fā)育率、萌發(fā)率和成苗率的結(jié)果（表1）顯示，以MM3為胚發(fā)育培養(yǎng)基，當(dāng)以‘00-1-5’為父本，‘秦紅2號(hào)’為母本的雜交組合的胚發(fā)育率和成苗率顯著高于‘秦秀’ב00-1-5’，分別為17.04%和7.41%。以‘Jupiter’為父本，‘Ruby Seedless’為母本胚挽救效果最好，發(fā)育率和成苗率為13.71%和10.67%；其次是‘秦紅1號(hào)’為母本的雜交組合，而‘Crimson Seedless’作母本胚的發(fā)育率和成苗率最低，與其他兩個(gè)組合均具有顯著性差異（＜0.05）。

表1 不同母本對(duì)胚挽救效果的影響

不同小寫(xiě)字母代表在＜0.05水平上差異顯著。下同

Different lowercase letters represent significant differences at<0.05 level. The same as below

2.2 不同發(fā)育培養(yǎng)基對(duì)胚挽救效果的影響

將‘Ruby Seedless’בJupiter’和‘秦紅2號(hào)’ב00-1-5’的胚珠分別接種至MM3和ER基本培養(yǎng)基。對(duì)于‘Ruby Seedless’בJupiter’組合，接種至MM3培養(yǎng)基所獲得的胚發(fā)育率和成苗率顯著高于ER培養(yǎng)基（＜0.05）（表2）；組合‘秦紅2號(hào)’ב00-1-5’在MM3培養(yǎng)基中，胚的發(fā)育率和成苗率比ER培養(yǎng)基高，兩種培養(yǎng)基中胚的發(fā)育率和成苗率之間差異顯著（＜0.05）。

2.3 不同母本及發(fā)育培養(yǎng)基對(duì)畸形苗率的影響

對(duì)5個(gè)雜交組合幼胚在MM3培養(yǎng)基中的畸形苗率的調(diào)查結(jié)果（表3）表明，畸形苗率最高的是‘秦紅1號(hào)’בJupiter’，為5.96%，而組合‘Ruby Seedless’בJupiter’未出現(xiàn)畸形苗，其他3個(gè)組合介于1.17%—5.03%。

不同發(fā)育培養(yǎng)基對(duì)不同雜交組合畸形苗率的影響結(jié)果見(jiàn)表4。對(duì)于‘秦紅2號(hào)’ב00-1-5’組合，在ER培養(yǎng)基上獲得的畸形苗率更高，顯著高于MM3培養(yǎng)基，為4.15%。而‘Ruby Seedless’בJupiter’在MM3和ER培養(yǎng)基上均未出現(xiàn)畸形苗。

2.4 畸形苗轉(zhuǎn)化培養(yǎng)基的篩選

‘秦紅2號(hào)’ב00-1-5’組合的45株畸形苗轉(zhuǎn)化結(jié)果（表5）表明，畸形苗轉(zhuǎn)接至2MS+1.6 mg?L-1ZnSO4+0.2 mg?L-16-BA+ 0.1 mg?L-1IAA轉(zhuǎn)化培養(yǎng)基上效果較好，轉(zhuǎn)化率為40.00%，顯著高于對(duì)照組。

表2 胚發(fā)育培養(yǎng)基MM3與ER對(duì)胚挽救成苗的影響

表3 母本對(duì)畸形苗形成的影響

表4 MM3和ER培養(yǎng)基對(duì)畸形苗率的影響

表5 不同ZnSO4濃度對(duì)畸形苗轉(zhuǎn)化率的影響

2.5 利用無(wú)核基因分子標(biāo)記對(duì)雜交后代的無(wú)核性狀鑒定

利用無(wú)核基因分子標(biāo)記SCF27-2000對(duì)‘秦紅2號(hào)’ב00-1-5’組合的53個(gè)雜種株系進(jìn)行檢測(cè)，表明51個(gè)株系擴(kuò)增出特異條帶（圖2），該組合無(wú)核率為96.23%。

M：Marker (2k Plus DNA marker) 1：秦紅2號(hào)；2：00-1-5；3—55：‘秦紅2號(hào)’ב00-1-5’組合雜交后代?！?”表示具有2000 bp特異帶；“-”表示無(wú)2000 bp特異帶。下同

利用無(wú)核標(biāo)記SCF27-2000對(duì)‘秦紅1號(hào)’בJupiter’組合的19個(gè)雜種株系進(jìn)行檢測(cè)，所有株系均擴(kuò)增出特異條帶（圖3），該組合無(wú)核率為100%。

利用無(wú)核基因分子標(biāo)記SCF27-2000分別對(duì)‘秦秀’ב00-1-5’‘Ruby Seedless’בJupiter’和‘Crimson Seedless’בJupiter’組合的雜種株系進(jìn)行檢測(cè)，無(wú)核率分別為83.33%、90.59%和100%。

3 討論

SAHIJRAM等[7]認(rèn)為，利用傳統(tǒng)育種方法對(duì)種子敗育型無(wú)核葡萄作親本進(jìn)行雜交，很難獲得無(wú)核雜種后代。而胚挽救技術(shù)能夠解決這一問(wèn)題，但影響胚挽救成功的因素很多，如親本類型、激素處理、取樣時(shí)期、培養(yǎng)基、培養(yǎng)條件等[9,24-26]。NOTSUKA等[5]研究認(rèn)為，由于無(wú)核葡萄胚挽救中選用的母本不同，雜交親和性不同，形成合子胚的比率不同，合子胚敗育時(shí)間也不相同，用不同品種作母本進(jìn)行雜交，胚的發(fā)育率不同。本試驗(yàn)結(jié)果表明，‘Ruby Seedless’‘秦紅1號(hào)?’和‘秦紅2號(hào)?’較適合作母本，這與LIU等[14]和VALDEZ[25]及筆者課題組[9]前期的結(jié)果相一致。而‘秦秀’和‘Crimson Seedless’作母本的胚挽救效果較差，但LIU等[14]和SIMIN等[26]認(rèn)為‘Crimson Seedless’較適合作胚挽救母本，可能是由于每年物候期不同、取樣時(shí)期不同和選用的培養(yǎng)基不同影響胚挽救的效果。不同母本由于基因型不同，胚珠內(nèi)營(yíng)養(yǎng)狀況不同，受精卵發(fā)育程度不同，幼胚離體培養(yǎng)后進(jìn)一步發(fā)育的程度也不同，造成部分幼胚不能萌發(fā)成正常植株。當(dāng)‘Crimson Seedless’‘秦紅1號(hào)’和‘秦秀’作母本時(shí)，畸形苗率較高，而‘Ruby Seedless’‘秦紅2號(hào)’作母本時(shí)，畸形苗率較低。以上說(shuō)明‘Ruby Seedless’‘秦紅1號(hào)’和‘秦紅2號(hào)’較適合作胚挽救母本。

M：Marker (2k Plus DNA marker)；1：秦紅1號(hào)；2：Jupiter；3—21：‘秦紅1號(hào)’בJupiter’組合雜交后代

胚發(fā)育培養(yǎng)基為離體胚珠提供了幼胚體外發(fā)育所需的生長(zhǎng)環(huán)境和營(yíng)養(yǎng)物質(zhì)，是影響胚挽救效果的關(guān)鍵因素之一。前人對(duì)胚發(fā)育培養(yǎng)基和萌發(fā)培養(yǎng)基進(jìn)行了大量研究[5-7,11-13]，目前應(yīng)用較多的胚發(fā)育培養(yǎng)基主要有Bouquet and Davis（BD）培養(yǎng)基（1989）[27]、Nitsch and Nitsch（NN）培養(yǎng)基（1969）[28]、Cain培養(yǎng)基（1983）[12]、Emershad and Ramming（ER）培養(yǎng)基（1984）和MM3培養(yǎng)基等[29-32]。EBADI等[33]以‘Flame Seedless’自然授粉胚珠為研究對(duì)象，認(rèn)為NN培養(yǎng)基比ER培養(yǎng)基效果更好。BURGER等[29]認(rèn)為采用BD培養(yǎng)基并添加活性炭效果最好。PONCE等[34]認(rèn)為MS培養(yǎng)基效果最好。KHOSHANDAM等[32]認(rèn)為NN培養(yǎng)基適合作為胚發(fā)育培養(yǎng)基。趙雅楠等[35]認(rèn)為MM3培養(yǎng)基效果比ER培養(yǎng)基效果好。本研究發(fā)現(xiàn)‘Ruby Seedless’בJupiter’接種在MM3培養(yǎng)基中，胚挽救效果最好，胚的成苗率最高，這與LIU等[14]、LI[24]等的結(jié)果相一致。MM3比ER培養(yǎng)基中多添加了半胱氨酸鹽酸鹽，半胱氨酸鹽酸鹽具有抗氧化和防止非酶褐變的作用，推測(cè)其可以減輕剝?nèi)∨咧闀r(shí)造成的損傷，促進(jìn)胚的進(jìn)一步發(fā)育，減少畸形苗的形成。

發(fā)育培養(yǎng)基選定后，在培養(yǎng)基中添加適量激素配比也會(huì)顯著促進(jìn)胚的發(fā)育。KUMAR等[30]認(rèn)為植物激素在植物發(fā)育各個(gè)方面的調(diào)節(jié)中具有重要作用，因此被廣泛研究。LI等[4]認(rèn)為在WPM培養(yǎng)基中添加0.1 μmol?L-16-BA作胚發(fā)育培養(yǎng)基效果最好。RAZI等[13]認(rèn)為IAA的濃度對(duì)胚萌發(fā)具有顯著影響，‘Askari’בRuby Seedless’和‘Askari’בBidane Sefid’組合在NN培養(yǎng)基上添加1 mg?L-1IAA效果最好。KHOSHANDAM等[32]認(rèn)為在NN培養(yǎng)基中添加0.35 mg?L-1GA3和1 mg?L-1IAA效果最好。GRAY等[36]發(fā)現(xiàn)培養(yǎng)基中添加BA能顯著提高胚萌發(fā)率。本研究表明，當(dāng)以MM3為胚發(fā)育培養(yǎng)基時(shí)，除添加IAA濃度與RAZI等[13]和KHOSHANDAM等[32]相同外，還應(yīng)添加0.5 mg?L-1GA3和0.5 mg?L-16-BA，適宜作為抗寒無(wú)核葡萄胚挽救的胚發(fā)育培養(yǎng)基。KHOSHANDAM等[32]認(rèn)為NN培養(yǎng)基也可以作為胚萌發(fā)培養(yǎng)基。POMMER等[12]用WPM液體培養(yǎng)基在濾紙橋上培養(yǎng)幼胚。EBADI等[33]也認(rèn)為WPM培養(yǎng)基作為胚萌發(fā)培養(yǎng)基效果較好。

有研究表明，在胚挽救過(guò)程中，部分萌發(fā)的胚畸形生長(zhǎng)，不能形成正常幼苗[25]。LESHEM等[37]認(rèn)為培養(yǎng)基中添加激素后能促進(jìn)和誘導(dǎo)畸形苗的產(chǎn)生，可能是由于生長(zhǎng)素和細(xì)胞分裂素的比例不協(xié)調(diào)所造成。LIU等[38]認(rèn)為胚挽救過(guò)程中畸形苗的出現(xiàn)可能與胚發(fā)育水平相關(guān)。Ji等[39]認(rèn)為2MS+0.4 mg?L-16-BA+0.1 mg?L-1IBA+10 μmol?L-1ZnSO4作轉(zhuǎn)化正常植株的培養(yǎng)基，轉(zhuǎn)化率最高，為27.9%，本研究與此結(jié)果類似。

胚挽救苗獲得后，需要對(duì)其進(jìn)行無(wú)核性狀檢測(cè)，保留具有目的性狀的后代。田間鑒定方法準(zhǔn)確有效，但等待結(jié)果的時(shí)間較長(zhǎng)。分子標(biāo)記輔助選擇實(shí)現(xiàn)了對(duì)基因型的直接選擇，是早期輔助選擇的有效手段[17]。目前應(yīng)用較多的葡萄無(wú)核基因分子標(biāo)記有SCAR標(biāo)記SCC8-1018、SCF27-2000和RAPD標(biāo)記GSLP1- 569[9,14,35]。前期研究表明，SCF27-2000標(biāo)記可以有效區(qū)分無(wú)核與有核親本，被許多研究者使用[9,14,24]，利用該分子標(biāo)記鑒定的無(wú)核率與田間測(cè)定的實(shí)際無(wú)核率相關(guān)性達(dá)81%[19]。而SCC8-1018和GSLP1-569只能區(qū)分部分無(wú)核與有核品種，特別是GSLP1-569僅對(duì)‘無(wú)核白’及其親緣關(guān)系較近的無(wú)核品種具有良好的適用性[9,14]。因此，本研究采用SCF27-2000對(duì)親本及其雜交后代進(jìn)行無(wú)核性狀鑒定。由于分子標(biāo)記與無(wú)核基因的連鎖程度不同，連鎖程度越高，檢測(cè)無(wú)核性狀的準(zhǔn)確率越高，因此，篩選連鎖緊密的無(wú)核基因分子標(biāo)記仍然是本領(lǐng)域研究的重要內(nèi)容。對(duì)于已獲得的胚挽救后代，除了采用無(wú)核基因分子標(biāo)記SCF27-2000進(jìn)行早期鑒定外，仍需要與傳統(tǒng)的田間鑒定相結(jié)合，以篩選出真正的無(wú)核雜種單株。

胚挽救雜種后代無(wú)核性狀的遺傳較為復(fù)雜，目前已有許多無(wú)核性狀遺傳的假說(shuō)，包括單顯性基因假說(shuō)[40]、單隱性基因假說(shuō)[41]、數(shù)量性狀假說(shuō)[42]、2隱性基因假說(shuō)[43]、3互補(bǔ)顯性基因假說(shuō)[44]、1顯性3隱性假說(shuō)[45]與1主效基因3微效基因假說(shuō)[46]等。由于葡萄無(wú)核機(jī)理尚未研究清楚，現(xiàn)有的假說(shuō)都不夠全面[8,47]。RAMMING等[15]認(rèn)為無(wú)核品種×無(wú)核品種無(wú)核后代比率理論上可達(dá)100%，本研究中無(wú)核品種×無(wú)核品種雜交后代無(wú)核率在90.59%—100%，與RAMMING等[15]的假說(shuō)基本一致。但是，無(wú)核品種×有核品種雜交后代無(wú)核率達(dá)到83.33%—96.23%，與筆者前期研究結(jié)果一致[9]，但高于NOTSUKA等[5]、LIU等[14]和趙雅楠等[35]的結(jié)果，推測(cè)不同組合對(duì)無(wú)核性狀的遺傳力不同，還可能與細(xì)胞質(zhì)遺傳有關(guān)[48]。因此，選擇無(wú)核性狀傳遞力強(qiáng)的無(wú)核葡萄品種作為胚挽救的母本，有利于獲得無(wú)核雜種。

4 結(jié)論

母本基因型對(duì)胚挽救效果有顯著影響。以‘00-1-5’作父本時(shí)，‘秦紅2號(hào)’作母本的胚挽救效果較好；以‘Jupiter’作為父本時(shí)，‘Ruby Seedless’作母本的胚挽救效果較好。以‘Ruby Seedless’和‘秦紅2號(hào)’作母本時(shí)，雜種胚的成苗率較高且畸形苗率較低，說(shuō)明‘Ruby Seedless’和‘秦紅2號(hào)’適宜作胚挽救母本。‘秦紅1號(hào)’作母本雜種胚的成苗率較高，但是畸形苗率也比較高；而‘Crimson Seedless’和‘秦秀’作母本時(shí)，雜種胚的成苗率低且畸形苗率較高，說(shuō)明‘Crimson Seedless’和‘秦秀’不適合作母本。對(duì)于‘Ruby Seedless’בJupiter’和‘秦紅2號(hào)’ב00-1-5’2個(gè)組合，在MM3固-液雙相培養(yǎng)基上的成苗率均比ER固-液雙相培養(yǎng)基高，畸形苗率比ER固-液雙相培養(yǎng)基低，表明MM3比ER培養(yǎng)基更適合作為胚發(fā)育培養(yǎng)基。利用無(wú)核基因分子標(biāo)記SCF27-2000對(duì)胚挽救后代進(jìn)行無(wú)核性狀早期鑒定，初步判定獲得的159個(gè)胚挽救苗是潛在的無(wú)核株系。

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Rescue and Molecular Marker Assisted-Selection of the Cold-Resistant Seedless Grape Hybrid Embryo

ZHU PeiPei, LUO YiJia, XIANG Wen, ZHANG MingLei, ZHANG JianXia

College of Horticulture, Northwest Agriculture and Forestry University/State Key Laboratory of Crop Stress Biology in Arid Areas/Key Laboratory of Horticultural Plant Germplasm Resource Utilization in Northwest China, Ministry of Agriculture, Yangling 712100, Shaanxi

【】The traditional methods of breeding seedless grape can only use the seeded variety as the female parent and the seedless variety as the male parent to cross, and the rate of the seedless hybrid is extremely low. As a new breeding technology, the embryo rescue can use seedless grapes as female parents, which expands the selection range of female parents, and greatly improves the efficiency of seedless grape breeding. However, the embryo development rate, germination rate and seedling formation rate in seedless grape embryo rescue are still low, which restricts the application of this technique. 【】The main objective of this paper was to screen suitable female parents and embryo development medium for embryo rescue, and to create new cold-resistant seedless grape germplasm by embryo rescue and molecular marker assisted-selection, so as to provide a basis for improving the effect of seedless grape embryo rescue and for further breeding new cold-resistant seedless grape varieties.【】Using the cold-resistant seedless grape cultivar Jupiter (Euro-American hybrid) and the seeded hybrid 00-1-5 (×) as the male parents and five seedless grape cvs. (Ruby Seedless, Qinhong No.1, Qinhong No.2, Crimson Seedless and Qinxiu) as the female parents, respectively, the hybridization was carried out between seedless grape and cold-resistant grape. The immature fruits of each female parent at the suitable sampling time were collected for ovule culture. The solid-liquid biphasic MM3 and ER medium were used as the embryo development medium to compare the effects of different medium and female parents on the rate of embryo development, germination, normal seedlings and abnormal seedlings. WPM + 0.2 mg?L-16-BA + 0.1 mg?L-1IAA solid medium was used as embryo germination and seedling formation medium. The embryo-rescue seedlings obtained after domestication in the greenhouse were transplanted to the field in spring. Inaddition, 2MS + 0.2 mg?L-16-BA + 0.1 mg?L-1IAA medium was used for transforming deformed seedlings into normal seedlings, which was added with 0, 1.0 and 1.6 mg?L-1ZnSO4, respectively. Finally, the molecular marker SCF27-2000 linked to the seedless gene was employed to detect the seedless hybrids for embryo-rescue seedlings. 【】For five cross combinations, 175 embryos from 2 158 ovules cultured in MM3 were obtained, which were further germinated and formed 118 hybrid plants. 74 embryos from 894 ovules cultured in ER medium were obtained and further germinated and formed 58 hybrid plants. Using 00-1-5 as male parent, seedless cv. Qinhong No.2 was much more suitable to be the female parent than Qinxiu, its embryo development rate and seedling rate were the highest, reaching 17.04 % and 7.41%, respectively. Using Jupiter as the male parent, Ruby Seedless × Jupiter had the best effect, with embryo development and seedling rate of 13.71% and 10.67%; the next was Qinhong No.1 × Jupiter, the embryo development rate and seedling rate were 14.39% and 4.71%, respectively. However, Crimson Seedless × Jupiter had the lowest embryo development rate and seedling rate, reached to 9.55% and 1.76%, respectively. The rates of embryo development and seedling formation of ovules inoculated into the MM3 medium were higher than that of the ER medium. The highest rate of deformed seedlings was Qinhong No.1 × Jupiter, while Ruby Seedless × Jupiter had no deformed seedlings. The rate of deformed seedlings in the ER medium was higher than that in the MM3 medium. The deformed seedlings of combination Qinhong No.2 × 00-1-5 were transformed to 2MS+1.6 mg?L-1ZnSO4+0.2 mg?L-16-BA+0.1 mg?L-1IAA, producing the best results with a conversion rate of 40.00%. Using the molecular marker SCF27-2000 linked to seedless character, 176 hybrid strains from 5 cross combinations were detected and there were 159 strains with the seedless molecular markers. 【】 Ruby Seedless, Qinhong No.1 and Qinhong No.2 were suitable as the seedless female parents for embryo rescue in vitro, but Crimson Seedless and Qinxiu were not relevant. MM3 medium was more appropriate for embryo rescue in vitro than the ER medium. The 159 plants from embryo rescue with the molecular marker linked to the seedless gene were potential seedless hybrids.

seedless grape; cold-resistance; embryo rescue; new germplasm; molecular marker- assisted selection

10.3864/j.issn.0578-1752.2021.06.012

2020-06-30；

2020-08-28

陜西省重點(diǎn)項(xiàng)目-農(nóng)業(yè)（2017ZDXM-NY-026）、楊凌示范區(qū)科技計(jì)劃項(xiàng)目（2018NY-29）

朱佩佩，E-mail：825965951@qq.com。通信作者張劍俠，E-mail：zhangjx666@126.com

（責(zé)任編輯 趙伶俐）