張賽陽,栗 燕,郝鵬博,楊秋生
(河南農(nóng)業(yè)大學(xué) 林學(xué)院,鄭州 450002)
?
雜交石竹授粉受精及其胚胎發(fā)育過程的觀察
張賽陽,栗 燕,郝鵬博,楊秋生*
(河南農(nóng)業(yè)大學(xué) 林學(xué)院,鄭州 450002)
以‘雜交石竹’為試驗(yàn)材料,利用熒光顯微鏡觀察其授粉后花粉萌發(fā)、花粉管生長情況,采用石蠟切片法對(duì)其受精及胚胎發(fā)育過程進(jìn)行觀察研究。結(jié)果表明:(1)授粉后1 h母本柱頭上少量花粉開始萌發(fā);授粉后4 h大量花粉萌發(fā),花粉管生長至柱頭中部有胼胝質(zhì)出現(xiàn);授粉后6 h花粉管生長至子房組織并有少量與胚珠結(jié)合;授粉后15 h柱頭中出現(xiàn)大量胼胝質(zhì),花粉管與胚珠結(jié)合數(shù)增多;授粉后24 h胚珠周圍出現(xiàn)多條花粉管,其中1條花粉管進(jìn)入胚珠,部分進(jìn)入胚囊的花粉管卷曲盤繞生長并產(chǎn)生胼胝質(zhì);精細(xì)胞與極核的融合主要發(fā)生在授粉后17~48 h,與卵細(xì)胞融合主要于授粉后1~3 d。(2)雜交石竹胚發(fā)育經(jīng)過原胚、球形胚、棒狀形胚、心形胚、魚雷形胚和子葉形胚階段。(3)雜交障礙表現(xiàn)為:只有游離的胚乳核而無胚發(fā)育的胚囊、合子未分裂、兩極核未融合、球形胚敗育。研究表明,雜交石竹存在受精前和受精后障礙,這是導(dǎo)致其結(jié)實(shí)率低的主要原因。
雜交石竹;花粉萌發(fā);花粉管生長;受精;胚胎發(fā)育;
石竹(Dianthuschinensis)原產(chǎn)中國,是優(yōu)良的庭院花卉,可用于花壇、花境或盆栽觀賞,也是制作花籃花束的切花材料。雜交石竹(Dianthushybridus)為石竹科石竹屬多年生草本植物,由中國石竹(Dianthuschinensis)與美國石竹(Dianthusbarbatus)雜交而成,以種子繁殖為主,生長勢(shì)強(qiáng)、一致性好、花色豐富、開花量大,在園林上應(yīng)用廣泛[1]。目前,國內(nèi)外對(duì)香石竹雜交育種方面研究較多,而對(duì)雜交石竹少見報(bào)道,中國在雜交石竹品種選育方面研究相對(duì)落后,園林綠化應(yīng)用的雜交石竹品種多數(shù)為國外育種公司培育。在雜交石竹制種過程中,經(jīng)常遇到母本結(jié)實(shí)率低,多數(shù)形成空種莢或種莢中種子較少的問題。因此,探究雜交石竹授粉受精特性及胚胎發(fā)育對(duì)提高種子產(chǎn)量有重要意義。
本試驗(yàn)利用熒光顯微鏡對(duì)雜交石竹花粉萌發(fā)、花粉管生長過程進(jìn)行觀察,采用石蠟切片法對(duì)雜交受精及胚胎發(fā)育進(jìn)程進(jìn)行觀察研究,試圖探索其授粉受精特性及雜交不親和原因,提高雜交制種產(chǎn)量,為雜交石竹育種及相關(guān)研究提供理論和技術(shù)依據(jù)。
1.1 材料
供試材料為雜交石竹(Dianthushybridus),田間試驗(yàn)于2015年4~6月在河南省濟(jì)源市雜交石竹制種基地進(jìn)行,室內(nèi)試驗(yàn)在河南農(nóng)業(yè)大學(xué)觀賞植物實(shí)驗(yàn)室進(jìn)行。
1.2 方法
1.2.1 花粉萌發(fā)及花粉管生長過程觀察 于2015年5月盛花期,上午10:00采集當(dāng)天散粉的父本花粉,授在母本柱頭上,于授粉后1、4、6、15和24 h分別采集4~6個(gè)雌蕊,用FAA固定液(38%甲醛5 mL∶冰醋酸5 mL∶70%酒精90 mL)固定。取固定好的待觀察材料,首先依次用70%酒精、50%酒精、30%酒精、蒸餾水分別浸泡2 h,然后用8 mol/L NaOH水浴(56 ℃)3~12 h,蒸餾水沖洗2~3次,每次10~20 min,之后用0.1%苯胺藍(lán)溶液染色。Nikon熒光顯微鏡觀察花粉萌發(fā)、花粉管生長情況。
1.2.2 受精過程及胚胎發(fā)育觀察 于2015年5月盛花期,上午10:00采集當(dāng)天散粉的父本花粉,授在母本柱頭上,于授粉后17 h、1 d、2 d、3 d、5 d、12 d、15 d分別采集4~6個(gè)雌蕊,用FAA固定液固定。采用常規(guī)石蠟切片法,切片厚度8~10 μm,番紅固綠染色,中性樹膠封片,在Motic數(shù)碼顯微鏡下觀察受精和胚胎發(fā)育情況。
2.1 花粉萌發(fā)及花粉管生長過程觀察
利用熒光顯微鏡對(duì)雜交石竹花粉萌發(fā)及花粉管生長過程進(jìn)行觀察研究,結(jié)果顯示:授粉后1 h柱頭附著大量花粉并有少量花粉開始萌發(fā)(圖版Ⅰ,1);授粉后4 h大量花粉開始萌發(fā),花粉管穿過柱頭乳突細(xì)胞進(jìn)入柱頭(圖版Ⅰ,2),授粉后4 h花粉管生長至柱頭1/2處并出現(xiàn)胼胝質(zhì)塞,同時(shí)觀察到異?;ǚ哿3霈F(xiàn)較大胼胝質(zhì)塊,花粉管停止生長(圖版Ⅰ,3);授粉后6 h花粉管已生長至子房組織并有少量與胚珠結(jié)合(圖版Ⅰ,4);授粉后15 h柱頭中出現(xiàn)大量胼胝質(zhì)(圖版Ⅰ,5),授粉后15 h花粉管與胚珠結(jié)合數(shù)增多(圖版Ⅰ,6);授粉后24 h胚珠周圍出現(xiàn)多條花粉管,其中一條花粉管進(jìn)入胚珠(圖版Ⅰ,7),授粉后24 h部分進(jìn)入胚珠的花粉管卷曲盤繞生長并產(chǎn)生胼胝質(zhì)反應(yīng)(圖版Ⅰ,8、9)。觀察結(jié)果表明雜交石竹花粉萌發(fā)和花粉管生長均無明顯阻礙,受精前障礙主要在于進(jìn)入胚囊內(nèi)的花粉管卷曲盤繞生長并產(chǎn)生胼胝質(zhì)而無法完成受精。
2.2 受精過程觀察
利用石蠟切片對(duì)雜交石竹受精過程進(jìn)行觀察,結(jié)果顯示:授粉后17 h花粉管穿過珠心組織,經(jīng)珠孔進(jìn)入胚囊并與助細(xì)胞結(jié)合釋放內(nèi)容物,助細(xì)胞中內(nèi)含物較多,染色呈現(xiàn)較深的紅色(圖版Ⅰ,10);授粉后1 d其中1個(gè)精細(xì)胞貼附在1個(gè)極核核膜上(圖版Ⅰ,11),并與二極核細(xì)胞融合形成3核的初生胚乳核(圖版Ⅰ,12);授粉后1 d在珠孔端觀察到1個(gè)精細(xì)胞向卵核移動(dòng),進(jìn)入卵細(xì)胞的細(xì)胞質(zhì)(圖版Ⅰ,13);精核貼附在卵核核膜上,進(jìn)行核的融合(圖版Ⅰ,14);授粉后2 d在胚囊中觀察到合子和初生胚乳細(xì)胞,合子靠近珠孔端,初生胚乳核在胚囊的中心位置,受精卵的核比初生胚乳核小(圖版Ⅰ,15)。通過大量石蠟切片觀察發(fā)現(xiàn),雜交石竹配子融合速度快,融合時(shí)間很短,但配子融合時(shí)間段相對(duì)較長,雜交石竹精細(xì)胞與極核的融合主要發(fā)生在授粉后17~48 h,與卵細(xì)胞融合過程主要于授粉后1~3 d。
2.3 胚胎發(fā)育觀察
利用石蠟切片對(duì)雜交石竹胚胎發(fā)育進(jìn)行觀察,結(jié)果顯示:授粉后2 d卵核受精形成合子,初生胚乳核開始分裂形成多個(gè)游離胚乳自由核(圖版Ⅱ,1);授粉后3 d可以觀察到合子分裂形成原胚,以及正在分裂的原胚和大量的游離胚乳核(圖版Ⅱ,2、3);授粉后5 d原胚細(xì)胞繼續(xù)分裂形成球形胚,球形胚周圍存在游離胚乳自由核(圖版Ⅱ,4);授粉后12 d,球形胚體積增大,細(xì)胞緊密排列,周圍存在大量胚乳自由核(圖版Ⅱ,5);授粉后12 d,在部分胚囊中觀察到棒狀形胚和心形胚(圖版Ⅱ,6、7);授粉后15 d,觀察到大量魚雷形胚和子葉形胚(圖版Ⅱ,8、9)。觀察結(jié)果表明雜交石竹胚胎發(fā)育經(jīng)歷原胚、球形胚、棒狀形胚、心形胚、魚雷形胚和子葉形胚階段。
同時(shí)對(duì)異常胚胎發(fā)育進(jìn)行觀察,結(jié)果顯示:授粉后2 d觀察到只有游離的胚乳核而無胚發(fā)育的胚囊(圖版Ⅱ,10);授粉后3 d,觀察到部分胚囊中有合子未分裂的現(xiàn)象,而在這個(gè)時(shí)期正常的胚胎已發(fā)育到原胚階段(圖版Ⅱ,11),同時(shí)觀察到有兩極核未融合的現(xiàn)象(圖版Ⅱ,12),和只有游離的胚乳發(fā)育而無胚發(fā)育的胚囊(圖版Ⅱ,13);授粉后5 d,敗育的球形胚(圖版Ⅱ,14);授粉后12 d,開始解體的球形胚,核膜界限變模糊(圖版Ⅱ,15)。觀察結(jié)果表明雜交石竹受精后障礙發(fā)生在受精過程和胚胎發(fā)育時(shí)期,表現(xiàn)為:合子未分裂、兩極核未融合、只有游離的胚乳發(fā)育而無胚發(fā)育的胚囊、球形胚敗育。
雜交親和性的障礙發(fā)生在生殖過程的不同階段,主要包括受精前障礙和受精后障礙。被子植物中,授粉后花粉在柱頭上的附著量、萌發(fā)及花粉管生長情況和受精后胚胎發(fā)育情況對(duì)雜交結(jié)實(shí)率影響很大,其中任何一個(gè)環(huán)節(jié)出現(xiàn)異常都有可能導(dǎo)致較低的結(jié)實(shí)率[2]。
王沖等[3]通過對(duì)君子蘭種間雜交及自交親和性研究,熒光顯微觀察發(fā)現(xiàn)在君子蘭種間雜交花粉萌發(fā)與花粉管伸長均存在胼胝質(zhì)現(xiàn)象,母本柱頭表面出現(xiàn)大量胼胝質(zhì),而這些胼胝質(zhì)阻礙花粉萌發(fā)和花粉管向下伸長。周旭紅等[4]通過對(duì)不同倍性香石竹雜交花粉管生長熒光顯微觀察,發(fā)現(xiàn)花粉管先端沉積胼胝質(zhì)而停止生長。櫟屬的2個(gè)種進(jìn)行雜交時(shí),柱頭中出現(xiàn)的大量胼胝質(zhì)使花粉管不能到達(dá)胚囊,導(dǎo)致雜交失敗[5]。本試驗(yàn)觀察到花粉能夠在柱頭上大量萌發(fā),少量花粉粒中出現(xiàn)較大胼胝質(zhì)塊花粉管停止生長,小部分花粉管頂端胼胝質(zhì)異常積累、膨大,母本柱頭中觀察到花粉管壁內(nèi)出現(xiàn)大量胼胝質(zhì)塞,花粉管能夠順利生長至柱頭基部,并未觀察到胼胝質(zhì)阻礙花粉管生長的現(xiàn)象。王文鵬等[6]在夏蠟梅與光葉臘梅屬間雜交未觀察到胼胝質(zhì)阻礙花粉管生長的現(xiàn)象,花粉萌發(fā)和花粉管生長階段均無明顯阻礙。花粉管中胼胝質(zhì)栓塞的存在將花粉管后部內(nèi)腔堵死,使內(nèi)腔成為不連續(xù)系統(tǒng),同時(shí)防止管內(nèi)原生質(zhì)倒流,從而保證花粉管的頂端生長[7]。當(dāng)花粉管生長到達(dá)一定長度后,在后端形成胼胝質(zhì),當(dāng)花粉管延長時(shí),在一定的間隔位置多次形成胼胝質(zhì)塞,因此胼胝質(zhì)塞區(qū)隨著花粉管生長而延長[8]。
胚珠周圍出現(xiàn)多條花粉管,其中1條與胚珠結(jié)合,在雙受精作用中,通常有阻止多條花粉管進(jìn)入胚囊的機(jī)理,因此僅有1條花粉管攜帶的1對(duì)精細(xì)胞到達(dá)胚囊[8]?;ǚ酃茉谒M(jìn)入的助細(xì)胞中停止生長和釋放精子,是由于當(dāng)花粉管進(jìn)入助細(xì)胞后,它周圍的環(huán)境發(fā)生變化而觸發(fā)的[8]。周旭紅等[9]在不同倍性香石竹雜交受精過程及胚胎發(fā)育研究中,認(rèn)為受精的子房頻率低,是不同倍性香石竹雜交結(jié)實(shí)率低的重要原因。楊鳳君等[10]在歐洲櫻桃與草原櫻桃雜交時(shí),認(rèn)為花粉管不能進(jìn)入胚囊,雌雄配子不能相遇而導(dǎo)致不能結(jié)實(shí)。玉米等作物遠(yuǎn)緣雜交中發(fā)現(xiàn),花粉管雖然能夠進(jìn)入胚囊,但并不一定能完成受精作用,出現(xiàn)胚囊不親和現(xiàn)象[11]。Williams等[12]在杜鵑花種間雜交研究中發(fā)現(xiàn),花粉管經(jīng)珠孔進(jìn)入胚囊后,因卷曲盤繞等過度生長情況,而無法完成受精。本試驗(yàn)觀察到經(jīng)珠孔進(jìn)入胚囊的一條花粉管在珠孔附近卷曲盤繞生長并出現(xiàn)胼胝質(zhì),這一現(xiàn)象表明,雖然花粉管能夠到達(dá)子房并進(jìn)入胚囊,但不能完成雙受精,存在受精前障礙,這可能是導(dǎo)致雜交石竹結(jié)實(shí)率低的重要原因之一。
Daston等[13]在玄參科龍面花屬種間雜交時(shí),觀察到父本花粉管進(jìn)入子房和胚珠,但授粉后子房并沒有膨大,最終沒有得到雜交種子,存在受精后障礙,受精后障礙也是導(dǎo)致不同倍性香石竹雜交結(jié)實(shí)率低的原因之一[9]。胚胎發(fā)育不良是引起種子敗育的主要原因[14],較高比例的胚敗育是影響結(jié)實(shí)率的另一個(gè)重要因素[15],Deng等[16]在菊花和細(xì)裂亞菊的屬間雜交中發(fā)現(xiàn)較高比率的胚胎敗育是結(jié)實(shí)率低的主要原因,而花粉活力和花粉管萌發(fā)對(duì)結(jié)實(shí)率的影響不大。本試驗(yàn)通過石蠟切片法對(duì)授粉后不同時(shí)間的雜交石竹子房進(jìn)行觀察,發(fā)現(xiàn)雜交障礙發(fā)生在受精過程和胚胎發(fā)育時(shí)期,表現(xiàn)為:兩極核未融合、胚囊中合子未分裂、只有游離的胚乳核無胚發(fā)育的胚囊、球形胚敗育。因此,受精后障礙也是導(dǎo)致雜交石竹結(jié)實(shí)率低的重要原因之一。
通過對(duì)雜交石竹授粉后花粉管生長及胚胎發(fā)育情況進(jìn)行觀察研究,發(fā)現(xiàn)雜交石竹存在受精前和受精后障礙,花粉管進(jìn)入胚珠后卷曲盤繞生長并產(chǎn)生胼胝質(zhì)、受精后合子未分裂、兩極核未融合、胚敗育,這幾個(gè)因素共同作用,是導(dǎo)致結(jié)實(shí)率低的主要原因,但引起雜交石竹雜交生殖障礙的機(jī)理還不清楚。今后在雜交石竹制種過程中,應(yīng)選擇在花粉活力和柱頭可授性較強(qiáng)時(shí)期內(nèi)進(jìn)行授粉,增加受精胚珠的概率,以期獲得更多、更飽滿的雜交種子,提高雜交結(jié)實(shí)率和制種生產(chǎn)效率。
[1] 宋利娜,許 超,辛海波,等.雜交石竹雄性不育株離體快繁技術(shù)研究[J].北京農(nóng)學(xué)院學(xué)報(bào),2015,30(3):92-95.
SONG L N,XU C,XIN H B,etal. A study on in vitro rapid propagation technique for male sterile lines ofDianthushybridus[J].JournalofBeijingUniversioyofAgriculture,2015,30(3):92-95.
[2] 孫春青,陳發(fā)棣,房偉民,等.野菊與菊花雜交中花粉活力和柱頭可授性及胚胎發(fā)育研究[J].西北植物學(xué)報(bào),2009, 29(7):1 335-1 341.
SUN C Q,CHEN F D,F(xiàn)ANG W M,etal. Pollen viability, pistil receptivity and embryogenesis in the cross betweenDendranthemaindicumandD.grandiflorum[J].ActaBotanicaBoreali-OccidentaliaSinica,2009,29(7):1 335-1 341.
[3] 王 沖,雷家軍,姜 闖,等.君子蘭種間雜交及自交親和性[J]. 中國農(nóng)業(yè)科學(xué),2011,44(18):3 822-3 829.
WANG C, LEI J J,JIANG C,etal. Study on cross-compatibility of interspecific hybridization and selfing inCliviaLindl[J].ScientiaAgriculturaSinica,2011,44(18):3 822-3 829.
[4] 周旭紅,桂 敏,王繼華,等.不同倍性香石竹雜交花粉管生長熒光顯微觀察及結(jié)實(shí)研究[J].西北植物學(xué)報(bào),2012,32(1):67-74.
ZHOU X H,GUI M,WANG J H,etal. Pollen Tube growth after crossing between different ploidsDianthuscaryophyllusby Fluorescence Microscopy and seed setting[J].ActaBotanicaBoreali-OccidentaliaSinica,2012,32(1):67-74.
[5] BOAVIDA L C,SILVA J P,F(xiàn)EIJO J A. Sexual reproduntion in the cork oak (QuercussuberL).II.Crossing intra-and interspecific barriers[J].SexualPlantReproduction,2001,14:143-152.
[6] 王文鵬,周莉花,劉華江,等. 夏蠟梅與美國蠟梅屬間雜交障礙的組織學(xué)機(jī)理[J].園藝學(xué)報(bào),2013,40(10):1 943-1 950.
WANG W P ,ZHOU L H,LIU H J,etal. Histological reproductive barriers for intergeneric cross betweenSinocalycanthuschinensisandCalycanthusfloridusvar.oblongifolius[J].ActaHorticulturaeSinica,2013,40(10):1 943-1 950.
[7] ALEXANDER KRICHEVSKY, STANISLAV V. KOZLOVSKY, GUO W T,etal. How pollen tubes grow[J].DevelopmentBiology, 2007,(303):405-420.
[8] 胡適宜,朱 澂.,被子植物有性生殖圖譜[M].北京:科學(xué)出版社,2000.
[9] 周旭紅,桂 敏,陳 敏,等.不同倍性香石竹雜交受精過程及胚胎發(fā)育研究[J].西北植物學(xué)報(bào), 2013, 33(1):1-6.
ZHOU X H,GUI M,CHEN M,etal. Fertilization and development of embryo in different interploidal crosses ofDianthuscaryophyllus[J].ActaBotanicaBoreali-OccidentaliaSinica,2013, 33(1):1-6.
[10] 楊鳳軍,臧忠婧,吳 瑕. 草原櫻桃種內(nèi)和種間授粉親和性的熒光顯微觀察[J]. 果樹學(xué)報(bào), 2015,(5):909-913.
YANG F J,ZANG Z J,WU X. Fluorescent microscope observation on pollination compatibility of Ground cherry[Cerasusfruticosa(Pall.)]crossing with different cherry cultivars[J].JournalofFruitScience,2015,(5):909-913.
[11] 段桃利.玉米與其近緣種屬雜交花粉管行為研究[D].四川雅安:四川農(nóng)業(yè)大學(xué),2008.
[12] WILLIAMS E G, KAUL V, ROUSE J L,etal. Overgrowth of pollen tubes in embryo sacs of Rhododendron following interspecific pollinations[J].AmerJBot,1986,(34):413-423.
[13] DASTON P M, MURRAY B G, HAMMETT K R W. Pollination systems hybridization barriers and meiotic chromosome behavior in Namesia hybrids[J].Euphytia,2006,151:173-185.
[14] 陳曉月,王金鑫,裴艷梅,等. 無核小棗與金絲小棗授粉受精過程的觀察[J]. 河北農(nóng)業(yè)大學(xué)學(xué)報(bào). 2014, 37(6):28-32.
CHEN X Y, WANG J X, PEI Y M,etal. Observation of pollination and fertilization ofZiziphusjujubaMill.‘Jinsixiaozao’ andZ.jujubaMill.‘Wuhexiaozao’[J].JournalofAgriculturalUniversityofHebei, 2014, 37(6):28-32.
[15] 胡適宜.被子植物生殖生物學(xué)[M].北京:高等教育出版社,2005.
[16] DENG Y M, TENG N J, CHEN S M,etal. Reproductive barriers in theintergeneric hybridization betweenChrysanthemumgrandiflorum(Ramat)Kitam.andAjaniaprzewalskiiPoljak.Euphytica,2010,174(1):41-50.
Pt.花粉管;Pg.花粉粒;C.胼胝質(zhì);Sy.助細(xì)胞;EC.卵細(xì)胞;SN.精核;PEN.初生胚乳核;Zy.合子1.柱頭附著大量花粉、授粉后1 h少量花粉開始萌發(fā),×100;2. 授粉后4 h大量花粉開始萌發(fā),少量花粉粒出現(xiàn)較大胼胝質(zhì),花粉管穿過柱頭乳突細(xì)胞進(jìn)入柱頭,×100;3.授粉后4 h花粉管生長至柱頭1/2處并出現(xiàn)胼胝質(zhì)塞,×100;4. 授粉后6 h花粉管已生長至子房組織并有少量與胚珠結(jié)合,×200; 5. 授粉后15 h柱頭中出現(xiàn)大量間斷胼胝質(zhì)反應(yīng),×200;6. 授粉后15 h大量花粉管進(jìn)入子房組織,花粉管與胚珠結(jié)合數(shù)增多,×200; 7. 授粉后24 h胚珠周圍出現(xiàn)多條花粉管,其中1條花粉管進(jìn)入胚珠,×200;8、9. 授粉后24 h部分進(jìn)入胚囊的花粉管卷曲盤繞生長并產(chǎn)生胼胝質(zhì),×400;10.授粉后17 h花粉管經(jīng)珠孔進(jìn)入胚囊并進(jìn)入助細(xì)胞釋放內(nèi)容物;11. 授粉后1 d1個(gè)精細(xì)胞貼附在1個(gè)極核核膜上;12. 授粉后1 d1個(gè)精細(xì)胞與二極核細(xì)胞融合形成3核的初生胚乳核;13. 授粉后1 d1個(gè)精細(xì)胞向卵核移動(dòng),進(jìn)入卵細(xì)胞的細(xì)胞質(zhì);14. 授粉后1 d精核貼附在卵核核膜上;15. 授粉后2 d合子和初生胚乳細(xì)胞圖版Ⅰ ‘雜交石竹’花粉萌發(fā)、花粉管生長及受精過程Pt. pollen tube; Pg. Pollen grain; C. Callose response; Sy. Synergid; EC. Egg cell; SN. Sperm nuclei; PEN. Primary endosperm nucleus; Zy. ZygoteFig.1. Stigma attached to a large number of pollens a small amount of pollens began to germinationed in 1 h after pollination, ×100; Fig.2. Many of pollen grains on the stigma began to germinationed in 4 h after pollination, pollen grains appear larger callose, pollen tube through the stigma papilla cells into stigma, ×100; Fig.3. The pollen tubes grew to stigma 1/2 and callose plug in 4 h after pollination, ×100; Fig.4. Pollen tubes grew to the ovary tissue and a small amount of them entered the ovule in 6 h after pollination, ×200; Fig.5. Stigma callose reactions occurred in 15 h after pollination, ×200;Fig.6. the large number of pollen tubes into the ovary tissue, pollen tubes increased within ovule, in 15 h after pollination, ×200; Fig.7.Many of pollen tubes had arisen ovule around, one of the pollen tube entered the ovule, ×200; Fig.8,9. Some pollen tubes had formed coiled overgrowth and generated callose reaction in the embryo sac, ×400; Fig.10. The pollen tube entered into one synergid and released contents 17 h after pollination; Fig.11. Sperm nucleus adhered to the nuclear membrane of the polar nuclei 1 d after pollination; Fig.12. Sperm nucleus fused with two polar nuclei and the primary endosperm nucleus 1 d after pollination; Fig.13. Sperm nucleus moved towards egg nucleus and entered the cytoplasm of egg nucleus 1 d after pollination; Fig.14. Sperm nucleus adhered to the nuclear membrane of the egg cell 1 d after pollination; Fig.15. Zygote and primary endosperm nucleus 2 d after pollinationPlate Ⅰ ‘Dianthus hybridus’ pollen germination, pollen-tube growth and fertilization
Zy.合子;EFN.胚乳自由核;PN.極核1.授粉后2 d,合子和游離胚乳核;2.授粉后3 d合子分裂形成原胚;3.正在分裂的原胚細(xì)胞和胚乳核;4.授粉后5 d,原胚細(xì)胞分裂形成球形胚;5.授粉后12 d,球形胚體積增大;6.授粉后12 d,棒狀形胚;7. 授粉后12 d,心形胚;8.授粉后15 d,魚雷形胚;9. 授粉后15 d,子葉形胚;10.授粉后2 d,無合子只有游離胚乳核;11.授粉后3 d,合子未分裂;12.授粉后3 d,兩極核未融合;13.授粉后3 d,無胚發(fā)育的胚囊;14.授粉后5 d,敗育的球形胚;15.授粉后12 d,正在解體的球形胚。圖版Ⅱ ‘雜交石竹’胚胎發(fā)育情況(標(biāo)尺=200 μm)Zy. Zygote; EFN. Endosperm free nuclei; PN. Polar nucleiFig.1. Formation of a zygote and endosperm free nuclei 2 days after pollination; Fig.2. Zygote division formed proembryon 2 days after pollination; Fig.3. Division of proembryo and endosperm free nuclei 2 days after pollination; Fig. 4. Proembryo division formed globular embryo 5 days after pollination; Fig.5. Globular embryo volume increase 12 days after pollination; Fig.6. Club-shaped embryo 12 days after pollination; Fig. 7. Heart embryo 12 days after pollination; Fig.8. Torpedo embryo 15 days after pollination; Fig.9. Cotyledonary embryo 15 days after pollination; Fig.10. Only the endosperm without embryo sac 2 days after pollination; Fig.11. Undivision of zygote 3 days after pollination; Fig.12. Degenerated endosperm free nuclei 3 days after pollination; Fig.13. No embryos of embryo sac 3 days after pollination; Fig.14. Degenerated globular embryo 5 days after pollination; Fig.15. The dissolution of globular embryo 12 days after pollination.Plate Ⅱ Embryogenesis after pollination in ‘Dianthus hybridus’(Bars=200 μm)
(編輯:潘新社)
Pollination,Fertilization and Embryonic Development of Dianthus hybridus
ZHANG Saiyang, LI Yan, HAO Pengbo, YANG Qiusheng*
(College of Forestry, Henan Agricultural University, Zhengzhou 450002,China)
With ‘Dianthushybridus’ as experiment material, we used fluorescence microscope to observe the pollen germination and pollen tube growth after pollination, to study the process of fertilization and embryo development with paraffin section method. The results showed that: (1) at 1 h after pollination, a small amount of pollens germinationed; At 4 h after pollination, a large number of pollens germinationed, pollen tube tips were found in the style middle, and callose response was found in pollen tubes; 6 h after pollination pollen tube grow to capital base and with a small amount of ovules; After 15 h stylar cannal has callose response, the pollen tube with the ovule number increased; At 24 h, many of pollen tubes have arisen around, one of the pollen tube enters the ovule, some pollen tubes have formed coiled overgrowth and generate callose reaction in the embryo sac. Sperm nucleus fused with polar nuclei mainly in 17-48 h after pollination, and the egg cell fusion process is mainly in 1-3 d after pollination. (2) The development of embryo through the proembryo, globular embryo, rod shaped embryo, heart-shaped embryo, torpedo embryo and cotyledon embryo stages. (3) Cross obstacle characterized by: only free endosperm nuclear and no embryo developmental embryo sac, zygote did not divided, sperm did not fused with polar nucleus, only free endosperm development without embryos of embryo sac, globular embryo aborted. The pre-fertilization and post-fertilization, obstacle are the main cause of low seed setting rate.
Dianthushybridus; pollen germination; pollen tube growth; fertilization; embryo abortion
1000-4025(2016)10-1984-06
10.7606/j.issn.1000-4025.2016.10.1984
2016-07-14;修改稿收到日期:2016-08-16
張賽陽(1990-),男,在讀研究生,主要從事園林植物種質(zhì)資源創(chuàng)新及育種。E-mail:zhangsaiyang1990@126.com
*通信作者:楊秋生,博士生導(dǎo)師,主要從事園林植物栽培教學(xué)和研究。E-mail:qsyang@henau.edu.cn
Q321+.8; Q944.58
A