早实薄皮核桃“绿岭”的无融合生殖特性研究
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摘要
为探明早实薄皮核桃“绿岭”无融合生殖能力的稳定性、发生时期和倍性及其果实性状。本试验以“绿岭”核桃为试材,通过显微观察、运用石蜡切片和组织培养技术,观察了“绿岭”核桃的开花物候期和雌花开放过程,调查了雌花套袋、切柱头和聚乙烯醇封柱头3种处理方法的核桃无融合生殖坐果情况,对无融合生殖过程中胚珠、胚囊、胚胎的发育、果实性状和胚的倍性进行了研究。主要研究结果如下:
“绿岭”核桃雌花柱头的开放过程为:开花前2 d(2009年4月14日和4月15日),“绿岭”核桃雌花的柱头闭合,无粘液分泌;开花第1 d(4月16日),柱头向两侧稍张开且分泌少量的粘液;开花第3 d(4月18日),柱头张开呈‘Y’字形,柱头表面分泌物增多,可持续到开花第6 d(4月21日);开花第7 d(4月22日),柱头表面分泌物减少,开始干涸;开花第9 d(4月24日),柱头表面无分泌物、明显干涸且局部枯萎。综上所述,“绿岭”核桃从柱头微张、有粘液分泌到张开180度、粘液减少、柱头开始干涸,需要8 d,柱头颜色变化依次为嫩绿色或粉红色、浅黄绿色、黄绿色、黄白色、黄褐色。自然授粉的子房明显膨大期比无融合生殖早4 d～5 d。
“绿岭”核桃为单子房直立胚珠,成熟胚囊属于蓼型胚囊。无融合生殖胚是由卵细胞或者与其中的一个助细胞融合的卵细胞发育而来。胚乳细胞是由两个中央极核融合并分裂而形成的。
3月21日,“绿岭”核桃雌花芽膨大时形成胚珠原基;随着雌花芽开绽,珠被原基逐渐出现;4月1日,在结果梢伸出时,孢原细胞进行平周分裂分化出的造孢细胞直接发育为大孢子母细胞;从4月2日到4月15日,结果梢继续伸长,渐渐露出子房和嫩绿色或粉红色的柱头,柱头闭合,无受粉能力,胚囊逐渐发育成熟;从4月16日到4月21日(开花第1~6 d),在柱头分泌粘液、有受粉能力时,由于柱头被聚乙烯醇封住或套袋,无法接受花粉,胚囊内无明显变化。4月22日(开花第7 d),当柱头稍微干涸后,可以发现卵细胞与其中一个助细胞融合形成一个细胞。4月23日到4月26日(开花第8~11 d),柱头明显干涸,子房缓慢膨大时,减数分裂的胚囊中的卵细胞或与一个助细胞融合的卵细胞开始有丝分裂依次形成2细胞原胚、4细胞原胚、8细胞原胚、球状原胚、早心形原胚、晚心形原胚,并进一步发育为无融合生殖胚。
套袋、切柱头和聚乙烯醇封柱头3种柱头处理方法的无融合生殖生理落果期后(5月14日)的坐果率分别为42.5%、26.7%、46.7%,套袋和聚乙烯醇封柱头处理的坐果率差异不显著,且均显著高于切柱头处理的坐果率。
“绿岭”核桃的无融合生殖坚果的纵径、横径、侧径、三径均值、单果重、种仁重、出仁率分别为3.362 cm、2.986 cm、3.020 cm、3.123 cm、9.12 g、5.64 g、61.85%,自然授粉坚果的纵径、横径、侧径、三径均值、单果重、种仁重、出仁率分别为3.534 cm、3.102 cm、3.142 cm、3.259 cm、10.59 g、6.76 g、63.89%,两者间差异显著。“绿岭”核桃自然授粉果实胚培养的组培苗根尖的染色体数目为32条,占计数细胞的93%,因此“绿岭”核桃体细胞染色体数目为2n=32条。而无融合生殖胚观察统计的染色体数有16条和32条两种,即“绿岭”核桃无融合生殖胚既有单倍体也有二倍体。单倍体占25.81%,二倍体占74.19%。接种后,可观察到二倍体组培苗的萌发情况明显优于单倍体组培苗。
In order to prove up the stability, occur period, ploidy and fruit character of apomixes ability in“LvLing”walnut., "LvLing" walnut was used as material in this experiment, " LvLing " walnut flowering phenology and female flowers opening process were studied with the method of microexamination, paraffin slice up and tissue culture techniques, fruit setting instance of walnut apomixis were investigated through 3 treatment of female flower baging, cutting stigma and envelop stigma with PVA. Ovule, embryo, embryonic development and fruit characters were also studied.The main results were as follows:
The opening process of female stigma in“LvLing”walnut:Before blossom 2 d(from April 14th to April 15th in 2009), the female stigma was closed and no mucus;The female stigma opened slightly and secreted little mucus in the 1st day(April 16th); From the 3rd day (April 18th) to the 6th day (April 21st), there were abundant secretion when the female stigma puff and took on“Y”tape ; The secretion on the stigma began to dry and reduced in 7th day of blossom. with the appearance of stigma began drying obviously and parts of them withering in the 9th day (April 24th). Above all, It was need eight days during the development of stigma in“LvLing”walnut. The color change of stigma was peak green or pink, light Kelly, Kelly, yellow-white,and filemot in turn. It was concluded that ovary swollened of natural pollination was obvious earlier 4 ~ 5 d than that of the apomixis .
The result showed that:“LvLing”walnut belong to single ovary and erection ovule in the female flower , the mature embryo sac was a typical eight-nuclei Polygonumembryo sac. Apomictic embryos of walnut came from egg cell or amalgamation with one of synergid. The endosperm cell was formed through amalgamation and division of two central polar nucleus.
On March 21st, the ovule primordia formed when the female buds swollened; The integument primordium was appered gradually along with the female bud rending; On April 1st, the sporogenous cells carried through periclinal differentiated and differentiated archesporial cells which developed the megaspore mother cell when the tip of branch stretched out. From April 2nd to April 15th, the tip of branch continued elongation, light green or pink stigma exposed ovary gradually .At the same time, the stigma was closed and had no pollination ability, the embryo matured gradually; From blomming 1st day to 6th day, the stigma began to secret mucus and owned pollination ability but it was unable to accept pollen due to it by polyvinyl alcohol (pva) sealed or bagged, So that embryo sac had no significant changes. It is concluded that egg cell fusion with one of synergid and formed a cell when the stigma slightly dry On April 22nd. When the ovary swelled slowy and the stigma dried cleary , embryo sac egg cell or syncretic synergid began to mitosis, and formed two cell embryos, 4-cell embryos, 8-cell embryos, globular embryos, early heart-shaped embryos, late heart-shaped embryos, and further developed the apomictic embryo in turn.
The apomixes fruit setting rate were 42.5%、26.7%、46.7% by way of bagging, cutting stigma and sealing stigma by polyvinyl alcohol. There had no significant difference between bagging and polyvinyl alcohol treatment,but both of them were significantly higher than that of the cutting stigma.
The longitudinal diameter, side tracks, three tracks average, fruit weight, kernel weight, kernel percentage were 3.362 cm, 2.986 cm, 3.020 cm, 3.123 cm, 9.12 g, 5.64 g and 61.85% leave each other in apomixes nut, and that of natural pollination were 3.534 cm, 3.102 cm, 3.142 cm, 3.259 cm, 10.59 g, 6.76 g and 63.89%, there had no significant difference in the two treatment.
The chromosome number of the radicle tip was 32 in“LvLing”wulnut through normal pollination ,wich was 93% in total cell. Thereby, somatic chromosome number were 2n = 32 in " LvLing " walnut. There had two kinds of chromosome wich was 16 and 32 in apomixes embryo. That was, apomictic " LvLing " walnut had both haploid and diploid embryos. The haploid accounted for 25.81% and the diploid embryos accounted for 74.19%. The germination of diploid tissue culture seedling was superior than that of haploid after inoculation.
“绿岭”核桃雌花柱头的开放过程为:开花前2 d(2009年4月14日和4月15日),“绿岭”核桃雌花的柱头闭合,无粘液分泌;开花第1 d(4月16日),柱头向两侧稍张开且分泌少量的粘液;开花第3 d(4月18日),柱头张开呈‘Y’字形,柱头表面分泌物增多,可持续到开花第6 d(4月21日);开花第7 d(4月22日),柱头表面分泌物减少,开始干涸;开花第9 d(4月24日),柱头表面无分泌物、明显干涸且局部枯萎。综上所述,“绿岭”核桃从柱头微张、有粘液分泌到张开180度、粘液减少、柱头开始干涸,需要8 d,柱头颜色变化依次为嫩绿色或粉红色、浅黄绿色、黄绿色、黄白色、黄褐色。自然授粉的子房明显膨大期比无融合生殖早4 d～5 d。
“绿岭”核桃为单子房直立胚珠,成熟胚囊属于蓼型胚囊。无融合生殖胚是由卵细胞或者与其中的一个助细胞融合的卵细胞发育而来。胚乳细胞是由两个中央极核融合并分裂而形成的。
3月21日,“绿岭”核桃雌花芽膨大时形成胚珠原基;随着雌花芽开绽,珠被原基逐渐出现;4月1日,在结果梢伸出时,孢原细胞进行平周分裂分化出的造孢细胞直接发育为大孢子母细胞;从4月2日到4月15日,结果梢继续伸长,渐渐露出子房和嫩绿色或粉红色的柱头,柱头闭合,无受粉能力,胚囊逐渐发育成熟;从4月16日到4月21日(开花第1~6 d),在柱头分泌粘液、有受粉能力时,由于柱头被聚乙烯醇封住或套袋,无法接受花粉,胚囊内无明显变化。4月22日(开花第7 d),当柱头稍微干涸后,可以发现卵细胞与其中一个助细胞融合形成一个细胞。4月23日到4月26日(开花第8~11 d),柱头明显干涸,子房缓慢膨大时,减数分裂的胚囊中的卵细胞或与一个助细胞融合的卵细胞开始有丝分裂依次形成2细胞原胚、4细胞原胚、8细胞原胚、球状原胚、早心形原胚、晚心形原胚,并进一步发育为无融合生殖胚。
套袋、切柱头和聚乙烯醇封柱头3种柱头处理方法的无融合生殖生理落果期后(5月14日)的坐果率分别为42.5%、26.7%、46.7%,套袋和聚乙烯醇封柱头处理的坐果率差异不显著,且均显著高于切柱头处理的坐果率。
“绿岭”核桃的无融合生殖坚果的纵径、横径、侧径、三径均值、单果重、种仁重、出仁率分别为3.362 cm、2.986 cm、3.020 cm、3.123 cm、9.12 g、5.64 g、61.85%,自然授粉坚果的纵径、横径、侧径、三径均值、单果重、种仁重、出仁率分别为3.534 cm、3.102 cm、3.142 cm、3.259 cm、10.59 g、6.76 g、63.89%,两者间差异显著。“绿岭”核桃自然授粉果实胚培养的组培苗根尖的染色体数目为32条,占计数细胞的93%,因此“绿岭”核桃体细胞染色体数目为2n=32条。而无融合生殖胚观察统计的染色体数有16条和32条两种,即“绿岭”核桃无融合生殖胚既有单倍体也有二倍体。单倍体占25.81%,二倍体占74.19%。接种后,可观察到二倍体组培苗的萌发情况明显优于单倍体组培苗。
In order to prove up the stability, occur period, ploidy and fruit character of apomixes ability in“LvLing”walnut., "LvLing" walnut was used as material in this experiment, " LvLing " walnut flowering phenology and female flowers opening process were studied with the method of microexamination, paraffin slice up and tissue culture techniques, fruit setting instance of walnut apomixis were investigated through 3 treatment of female flower baging, cutting stigma and envelop stigma with PVA. Ovule, embryo, embryonic development and fruit characters were also studied.The main results were as follows:
The opening process of female stigma in“LvLing”walnut:Before blossom 2 d(from April 14th to April 15th in 2009), the female stigma was closed and no mucus;The female stigma opened slightly and secreted little mucus in the 1st day(April 16th); From the 3rd day (April 18th) to the 6th day (April 21st), there were abundant secretion when the female stigma puff and took on“Y”tape ; The secretion on the stigma began to dry and reduced in 7th day of blossom. with the appearance of stigma began drying obviously and parts of them withering in the 9th day (April 24th). Above all, It was need eight days during the development of stigma in“LvLing”walnut. The color change of stigma was peak green or pink, light Kelly, Kelly, yellow-white,and filemot in turn. It was concluded that ovary swollened of natural pollination was obvious earlier 4 ~ 5 d than that of the apomixis .
The result showed that:“LvLing”walnut belong to single ovary and erection ovule in the female flower , the mature embryo sac was a typical eight-nuclei Polygonumembryo sac. Apomictic embryos of walnut came from egg cell or amalgamation with one of synergid. The endosperm cell was formed through amalgamation and division of two central polar nucleus.
On March 21st, the ovule primordia formed when the female buds swollened; The integument primordium was appered gradually along with the female bud rending; On April 1st, the sporogenous cells carried through periclinal differentiated and differentiated archesporial cells which developed the megaspore mother cell when the tip of branch stretched out. From April 2nd to April 15th, the tip of branch continued elongation, light green or pink stigma exposed ovary gradually .At the same time, the stigma was closed and had no pollination ability, the embryo matured gradually; From blomming 1st day to 6th day, the stigma began to secret mucus and owned pollination ability but it was unable to accept pollen due to it by polyvinyl alcohol (pva) sealed or bagged, So that embryo sac had no significant changes. It is concluded that egg cell fusion with one of synergid and formed a cell when the stigma slightly dry On April 22nd. When the ovary swelled slowy and the stigma dried cleary , embryo sac egg cell or syncretic synergid began to mitosis, and formed two cell embryos, 4-cell embryos, 8-cell embryos, globular embryos, early heart-shaped embryos, late heart-shaped embryos, and further developed the apomictic embryo in turn.
The apomixes fruit setting rate were 42.5%、26.7%、46.7% by way of bagging, cutting stigma and sealing stigma by polyvinyl alcohol. There had no significant difference between bagging and polyvinyl alcohol treatment,but both of them were significantly higher than that of the cutting stigma.
The longitudinal diameter, side tracks, three tracks average, fruit weight, kernel weight, kernel percentage were 3.362 cm, 2.986 cm, 3.020 cm, 3.123 cm, 9.12 g, 5.64 g and 61.85% leave each other in apomixes nut, and that of natural pollination were 3.534 cm, 3.102 cm, 3.142 cm, 3.259 cm, 10.59 g, 6.76 g and 63.89%, there had no significant difference in the two treatment.
The chromosome number of the radicle tip was 32 in“LvLing”wulnut through normal pollination ,wich was 93% in total cell. Thereby, somatic chromosome number were 2n = 32 in " LvLing " walnut. There had two kinds of chromosome wich was 16 and 32 in apomixes embryo. That was, apomictic " LvLing " walnut had both haploid and diploid embryos. The haploid accounted for 25.81% and the diploid embryos accounted for 74.19%. The germination of diploid tissue culture seedling was superior than that of haploid after inoculation.
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