羅燦燦，沈家葆，黃小芳，郭 媛,胡勝武

(1.旱區(qū)作物逆境生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室，陜西楊凌 712100；2.西北農(nóng)林科技大學(xué) 農(nóng)學(xué)院，陜西楊凌 712100)

甘藍(lán)型油菜MSL不育系花藥發(fā)育的細(xì)胞學(xué)研究

羅燦燦1,2，沈家葆2，黃小芳2，郭 媛2,胡勝武1,2

(1.旱區(qū)作物逆境生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室，陜西楊凌 712100；2.西北農(nóng)林科技大學(xué) 農(nóng)學(xué)院，陜西楊凌 712100)

為了揭示MSL (male sterility lembke) 的小孢子敗育時(shí)期及其特點(diǎn)，從而促進(jìn)中國(guó)及世界油菜雜種優(yōu)勢(shì)利用的步伐。選用MSL衍生系的不育材料和可育材料為試驗(yàn)材料，采用常規(guī)壓片和石蠟切片技術(shù)，觀察其花藥發(fā)育過程。通過對(duì)MSL不育材料的細(xì)胞學(xué)觀察，發(fā)現(xiàn)MSL可發(fā)育到四分體時(shí)期，四分體不同程度皺縮，絨氈層徑向伸長(zhǎng)，并含有一個(gè)特別大的液泡，液泡將細(xì)胞核和細(xì)胞質(zhì)擠向細(xì)胞的一邊，胞質(zhì)濃縮。隨著發(fā)育的進(jìn)行，絨氈層進(jìn)一步伸長(zhǎng)，向內(nèi)擠壓四分體，腔室體積變小。緊接著液泡化并且伸長(zhǎng)了的絨氈層和濃縮的四分體溶解在一起，花粉囊橫切面的形狀發(fā)生改變，由圓形變成長(zhǎng)條形，最后整個(gè)花藥室萎縮。結(jié)果表明MSL的敗育時(shí)期為四分體時(shí)期，由于絨氈層的異常而導(dǎo)致敗育。

甘藍(lán)型油菜；細(xì)胞核雄性不育；MSL；花藥敗育；細(xì)胞學(xué)觀察

油菜作為重要的油料作物，雜種優(yōu)勢(shì)顯著，油菜雜種優(yōu)勢(shì)利用的主要授粉控制系統(tǒng)包括細(xì)胞核雄性不育、細(xì)胞質(zhì)雄性不育、化學(xué)殺雄、自交不親和，其中細(xì)胞核雄性不育，是油菜雜種優(yōu)勢(shì)育種中一個(gè)重要的授粉控制系統(tǒng)[1]。隨著大家對(duì)核不育的重視，核不育在高粱[2]、大豆[3]、油菜[4]、小麥[5]和棉花[6]上取得成功應(yīng)用。

目前，油菜中已報(bào)道的核不育類型有顯性核不育[7]、雙隱性核不育[8-10]和3對(duì)基因控制的隱性核不育[11-12]。油菜MSL (male sterility lembke) 系統(tǒng)是德國(guó)NPZ (Norddeutsche Pflanzenzucht Hans-Georg Lembke) 育種公司1984年發(fā)現(xiàn)的一種天然突變體，是目前歐洲雜交油菜生產(chǎn)的主要授粉控制系統(tǒng)[13-14]。經(jīng)典遺傳學(xué)和分子遺傳學(xué)對(duì)MSL進(jìn)行遺傳分析和驗(yàn)證，認(rèn)為MSL由1對(duì)隱性不育基因和1對(duì)隱性上位抑制基因互作控制[15]，到目前為止，有關(guān)MSL花藥敗育的細(xì)胞學(xué)特征還未見報(bào)道。

前人對(duì)油菜核不育材料花藥敗育的細(xì)胞學(xué)觀察發(fā)現(xiàn)，大多花藥敗育發(fā)生于花粉母細(xì)胞至單核花粉時(shí)期。比如隱性核不育材料S45A和117A在四分體至單核小孢子階段發(fā)生敗育，表現(xiàn)為小孢子不能形成花粉壁并且絨氈層細(xì)胞徑向肥大和液泡化[16]。隱性核不育材料9012A[11]及其衍生材料1740A[17]花藥敗育于四分體時(shí)期，9012A敗育是由于絨氈層細(xì)胞未能成功轉(zhuǎn)化為分泌型細(xì)胞，無(wú)法分泌胼胝質(zhì)酶降解胼胝質(zhì)，導(dǎo)致四分體不能從胼胝質(zhì)中釋放出來(lái)[16,18]；1740A敗育是由于胼胝質(zhì)不能解體和絨氈層細(xì)胞的徑向肥大所致[17]。顯性核不育株Rsl046A[19]、Shaan-GMS的衍生材料0A30A[20]、溫敏核不育系TE5A[21]和核不育系宜3A[22]花藥敗育原因都是減數(shù)分裂異常導(dǎo)致無(wú)法形成二分體及四分體。溫敏核不育系H50S[23]和373S[24]，光溫敏核不育系Huiyou50S[25]均為單核花粉敗育型。

本研究以MSL的衍生系5A135AB為試驗(yàn)材料，采用常規(guī)壓片和石蠟切片，觀察其花藥發(fā)育過程，明確MSL花藥敗育時(shí)期和特點(diǎn)，為揭示該材料的雄性不育機(jī)理及深入利用奠定基礎(chǔ)。本試驗(yàn)結(jié)果在油菜雜種優(yōu)勢(shì)利用方面具有重要的理論與實(shí)踐意義。

1 材料與方法

1.1 材 料

供試材料為兩型系5A135AB，其中不育株為5A135A，可育株為5A135B。5A135AB是從雜交種Marthow自交后代中分離而來(lái)，該雜交種是基于德國(guó)NPZ育種公司的油菜MSL雄性不育系配置而成。Marthow為捷克作物研究所Miroslav Klima博士惠贈(zèng)。供試材料于2015年9月中旬播種于西北農(nóng)林科技大學(xué)北校區(qū)農(nóng)學(xué)院標(biāo)本園，田間管理同一般大田常規(guī)管理。

1.2 方 法

1.2.1 常規(guī)壓片 2016年4月初，取初花期不育株5A135A和可育株5A135B主花序以及上部分枝的花蕾，立刻放入卡諾固定液[V(無(wú)水乙醇)∶V(冰乙酸)=3∶1]中固定1 h，100%(體積分?jǐn)?shù)，下同)、95%、85%梯度酒精洗滌各2 h，然后轉(zhuǎn)入75%的酒精中，于4 ℃冰箱保存?zhèn)溆?。使用時(shí)先取花蕾6枚雄蕊中的1枚雄蕊，放在1 mol/L的鹽酸中解離20 min，然后經(jīng)1%醋酸洋紅染色壓片，在Olympus BX51 光學(xué)顯微鏡下鏡檢花藥發(fā)育時(shí)期并照相[20]。

1.2.2 石蠟切片 將保存在75%酒精中的已確定發(fā)育時(shí)期的材料經(jīng)85%、95%、100%、100%梯度酒精脫水各2 h，經(jīng)酒精和二甲苯體積比分別為1∶1、0∶1、0∶1透明各2 h，60 ℃浸蠟3次，每次2 h，56～58 ℃進(jìn)行石蠟包埋，將修好的花蕾包埋塊橫切，切片厚度為6～8 μm，40 ℃的蒸餾水粘片，放入60 ℃的烘箱2 h烘干，3次二甲苯各25 min脫蠟，100%、95%、85%、70%、50%、30%梯度酒精復(fù)水各5 min，蒸餾水水洗5 min，0.1%甲苯胺藍(lán)水溶液染色5 s，阿拉伯樹膠封片，Olympus BX51 光學(xué)顯微鏡觀察、照相[26]。

2 結(jié)果與分析

2.1 5A135A花藥敗育的壓片觀察

根據(jù)常規(guī)壓片結(jié)果，甘藍(lán)型油菜對(duì)照可育株5A135B花藥發(fā)育的主要細(xì)胞學(xué)時(shí)期包括減數(shù)分裂期、四分體時(shí)期、小孢子時(shí)期、花粉后期、成熟花粉期。減數(shù)分裂期花粉母細(xì)胞在胼胝質(zhì)的包裹下進(jìn)行減數(shù)分裂(圖1-A)，減數(shù)分裂結(jié)束后形成四分體(圖1-B)，胼胝質(zhì)降解，小孢子從四分體中釋放出來(lái)，合成花粉壁，細(xì)胞核被液泡擠向一邊，小孢子三邊加厚(圖1-C)，隨著發(fā)育的進(jìn)行，小孢子體積增大(圖1-D)，花粉進(jìn)一步發(fā)育，形成2個(gè)生殖核，花粉粒發(fā)育成熟(圖1-E)。不育材料5A135A減數(shù)分裂時(shí)期發(fā)育正常，花粉母細(xì)胞在胼胝質(zhì)內(nèi)經(jīng)歷2次減數(shù)分裂(圖1-F)、形成正常的四分體(圖1-G)，但是在四分體發(fā)育后期，包裹在胼胝質(zhì)內(nèi)的四分體被擠壓變形(圖1-H)，四分體不斷皺縮(圖1-I)，最后四分體傾向于解體(圖1-J)。不育材料花藥發(fā)育只能發(fā)育到四分體時(shí)期，被胼胝質(zhì)包裹的四分體不能被成功釋放，擠壓變形，不斷皺縮直至解體。所以，5A135A敗育時(shí)期發(fā)生在四分體時(shí)期。

2.2 5A135A花藥敗育的石蠟切片觀察

從石蠟切片結(jié)果來(lái)看，可育株5A135B花藥發(fā)育主要經(jīng)歷花粉母細(xì)胞時(shí)期、減數(shù)分裂期、四分體時(shí)期、單核期、雙核期和成熟期?；ǚ勰讣?xì)胞早期粘連在一團(tuán)(圖2-A)，隨后以多邊形分離，開始減數(shù)分裂(圖2-B)，形成四分體(圖2-C)，接著四分體周圍的胼胝質(zhì)解體，釋放出小孢子，隨著小孢子外壁的孢粉素和脂類物質(zhì)的積累，單核小孢子三邊加厚(圖2-G)；單核小孢子第一次有絲分裂，形成雙核花粉粒，此時(shí)絨氈層發(fā)生解體(圖2-H)；雙核花粉粒再進(jìn)行一次有絲分裂，形成成熟花粉粒，絨氈層幾乎完全降解(圖2-I)。

不育材料5A135A在花粉母細(xì)胞時(shí)期發(fā)育正常,花粉母細(xì)胞形狀規(guī)則、排列緊密，絨氈層細(xì)胞整齊地排列在花粉母細(xì)胞的外圈(圖2-D)；減數(shù)分裂時(shí)期絨氈層細(xì)胞體積變大，內(nèi)含大液泡，減數(shù)分裂體由胼胝質(zhì)包裹進(jìn)行減數(shù)分裂(圖2-E)；和可育材料相比，不育材料在四分體時(shí)期出現(xiàn)明顯差異，絨氈層徑向伸長(zhǎng)向內(nèi)擠壓四分體，絨氈層細(xì)胞內(nèi)含有一個(gè)特別大的液泡，液泡將細(xì)胞核和細(xì)胞質(zhì)擠向細(xì)胞的一邊，胞質(zhì)濃縮(圖2-F)。隨著發(fā)育的進(jìn)行，未發(fā)現(xiàn)形成小孢子，并且絨氈層進(jìn)一步伸長(zhǎng)，向內(nèi)擠壓四分體，腔室體積變小(圖2-J)；花粉囊的橫切面開始變形，變成長(zhǎng)條形(圖2-K)，最后，絨氈層和皺縮了的四分體全部解體，整個(gè)花粉囊萎縮(圖2-L)。根據(jù)石蠟切片結(jié)果，判斷5A135A不育材料在四分體以后小孢子無(wú)法釋放出來(lái)，同時(shí)絨氈層異常膨大，向內(nèi)擠壓四分體，花藥室變形，最終導(dǎo)致敗育。

3 討 論

油菜MSL系統(tǒng)是德國(guó)NPZ育種公司發(fā)明的一種油菜不育系統(tǒng)，是目前歐洲油菜雜種生產(chǎn)的主要授粉控制系統(tǒng)[13-14]。本研究通過對(duì)MSL衍生系5A135A花藥敗育的壓片及石蠟切片觀察結(jié)果顯示，其敗育時(shí)期為四分體時(shí)期。主要特征是，四分體不同程度皺縮，絨氈層徑向伸長(zhǎng)，并含有一個(gè)特別大的液泡，液泡將細(xì)胞核和細(xì)胞質(zhì)擠向細(xì)胞的一邊，胞質(zhì)濃縮。隨著發(fā)育的進(jìn)行，絨氈層進(jìn)一步伸長(zhǎng)，向內(nèi)擠壓四分體，腔室體積變小。緊接著液泡化并且伸長(zhǎng)了的絨氈層和濃縮的四分體溶解在一起，花粉囊的形狀發(fā)生改變，由圓形變成長(zhǎng)條形，最后整個(gè)花藥室萎縮。本研究結(jié)果顯示，MSL敗育時(shí)期和隱性核不育材料9012A[16,18]及其衍生材料1740A[17]相同，均為四分體時(shí)期。敗育特征也很相似，絨氈層細(xì)胞大液泡化并且徑向伸長(zhǎng)，向內(nèi)擠壓四分體。有研究發(fā)現(xiàn)在白菜[27]、辣椒[28]、馬鈴薯[29]、紅麻[30]花藥敗育特點(diǎn)中也存在類似的現(xiàn)象。在花藥發(fā)育的過程中，輸送花藥小孢子發(fā)育所需的營(yíng)養(yǎng)物質(zhì)是絨氈層主要的功能之一。已有研究表明，異常發(fā)育的絨氈層會(huì)引起花藥敗育[31]。分為2種形式，分別是絨氈層細(xì)胞的程序化死亡進(jìn)程異常[32]和絨氈層細(xì)胞自身的發(fā)育異常[33]。本研究的MSL敗育原因?qū)儆诤笳?，是絨氈層細(xì)胞在四分體時(shí)期異常徑向伸長(zhǎng)，無(wú)法正常提供營(yíng)養(yǎng)物質(zhì)，反而向內(nèi)擠壓小孢子，侵占小孢子生長(zhǎng)所必需的空間，最終使得花藥敗育。在油菜中，已發(fā)現(xiàn)一些基因與絨氈層細(xì)胞的發(fā)育相關(guān)，如曾芳琴[34]報(bào)道的 BnCYP704BJ基因和夏秀云[35]研究的 BnATA20基因， BnCYP704BJ基因如果突變，會(huì)以影響絨氈層分泌合成脂類物質(zhì)的方式引起敗育， BnATA20基因的突變體以絨氈層異常膨大的形式敗育。因此，隨著生物科技的發(fā)展，已經(jīng)可以人為地通過基因工程方式獲得雄性不育系。

A.可育、減數(shù)分裂期 Fertile,meiosis stage；B.可育、四分體時(shí)期 Fertile,tetrad stage；C.可育、小孢子時(shí)期 Fertile,microspore stage；D.可育、花粉后期 Fertile,later pollen stage；E.可育、成熟花粉期 Fertile,mature pollen stage；F.不育、減數(shù)分裂期 Sterile,meiosis stage；G.不育、四分體時(shí)期 Sterile,tetrad stage；H，I，J.不育、敗育的四分體形態(tài) Sterile,deformed tetrad morphology；標(biāo)尺為10 μm Scale bars=10 μm

圖1甘藍(lán)型油菜可育株5A135B和不育株5A135A花藥發(fā)育的主要細(xì)胞學(xué)時(shí)期
Fig.1Majorcytologicaleventsduringantherdevelopmentinfertileplants5A135Bandsterileplants5A135AinB.napus

A.可育、花粉母細(xì)胞時(shí)期 Fertile,pollen mother cell stage；B.可育、減數(shù)分裂期 Fertile,meiosis stage；C.可育、四分體時(shí)期 Fertile,tetrad stage；D.不育、花粉母細(xì)胞時(shí)期 Sterile,pollen mother cell stage；E.不育、減數(shù)分裂期 Sterile,meiosis stage；F.不育、四分體時(shí)期 Sterile,tetrad stage；G.可育、單核期 Fertile,uninuclear microspore stage；H.可育、雙核期 Fertile,binuclear microspore stage；I.可育、成熟期 Fertile,mature pollen stage；J.不育、四分體時(shí)期的敗育花粉囊 Sterile,degenerated anther sac at tetrad stage；K-L.不育、敗育花粉囊 Sterile,degenerated anther sac;T.絨氈層 Tapetum；PMCs.花粉母細(xì)胞 Pollen mother cells；MC.減數(shù)分裂體 Meiotic cell；Tds.四分體 Tetrads；Msp.小孢子 Microspores；標(biāo)尺在A、B、D、E、F、J中為10 μm，在C、G、H、I、K、L中為20 μm Scale bars in A,B,D,E,F,J were 10 μm and in C,G,H,I,K,L were 20 μm

圖2甘藍(lán)型油菜可育株5A135B和不育株5A135A花藥發(fā)育主要時(shí)期的石蠟切片觀察
Fig.2Paraffinsectionofmajoreventsduringantherdevelopmentoffertileplants5A135Bandsterileplants5A135AinB.napus

綜上，本研究揭示了MSL的敗育時(shí)期和敗育特征，豐富了油菜核不育材料MSL的理論內(nèi)容，為日后深入利用該材料奠定了基礎(chǔ)，同時(shí)對(duì)促進(jìn)中國(guó)及世界油菜雜種優(yōu)勢(shì)利用的步伐，具有深遠(yuǎn)的意義。

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CytologicalObservationonAntherDevelopmentofGenicMaleSterileLineMSLinBrassicanapusL.

LUO Cancan1,2,SHEN Jiabao2,HUANG Xiaofang2,GUO Yuan2and HU Shengwu1,2

(1.State Key Laboratory of Crop Stress Biology in Arid Areas,Yangling Shaanxi 712100,China;2.College of Agronomy,Northwest A&F University,Yangling Shaanxi 712100,China)

To characterize MSL (male sterility lembke) anther abortion and promote its utilization in breeding of hybrid rapeseed (BrassicanapusL.) in China,even in the world,cytological observations of the anther development in sterile line which developed on the basis of MSL were employed in the study. The results revealed that anther development of the MSL had been abnormal at tetrad stage,and some tetrads had concentrated. The tapetal cell extended radially and contained a particularly large vacuole. The vacuole squeezed the nucleus and cytoplasm to one side of the cell,which it lead to the cytoplasm concentrated. The tapetum occupied the space of the tetrads. Along with the tapetum continued to swelled,the locule was getting smaller and smaller. Sequentially,the vacuolized,elongated tapetum and the concentrated tetrads dissolved together,and the shape of the anther sac changed from a circle to a long strip shape,and the anther sac atrophied finally. The results showed that anther of MSL aborted at tetrad stage,because it could not form functional tapetal cells.

BrassicanapusL. ; Genetic male sterility; MSL; Anther abortion; Cytological observation

2017-04-04

2017-06-01

Earmarked Fund for China Agricultural Research System(No.CARS-13).

LUO Cancan,female,master student. Research area:genetics and breeding of rapeseed. E-mail: 18956141956@163.com

S565.4

A

1004-1389(2017)10-1470-07

日期：2017-10-18

網(wǎng)絡(luò)出版地址：http://kns.cnki.net/kcms/detail/61.1220.S.20171018.1733.016.html

2017-04-04

2017-06-01

現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(xiàng)資金(CARS-13)。

羅燦燦，女，碩士研究生，從事油菜遺傳育種研究。E-mail: 18956141956@163.com

胡勝武，男，博士，教授，博士生導(dǎo)師，主要從事油菜遺傳育種研究。E-mail:swhu83251@nwsuaf.edu.cn

CorrespondingauthorHU Shengwu,male,Ph.D,professor,doctoral supervisor.Research area:genetics and breeding of rapeseed.E-mail:swhu83251@nwsuaf.edu.cn

(責(zé)任編輯：成敏Responsibleeditor:CHENGMin)