植物花粉培养研究进展

花粉培养又称为游离小孢子培养,指将发育到一定阶段的花粉从花药中游离出来成为分散或游离状态,通过培养使花粉粒脱分化,进而发育成完整植株的过程。花粉培养的主要目的是获得单倍体植株,进而得到双单倍体(double haploid,DH)植株,最终获得纯合系物种。本文对花粉培养形成植株的物种信息进行了收集整理,概述了国内外花粉培养的一些最新研究进展,包括影响花粉培养形成胚的因素以及提高花粉胚产量的措施,并对花粉培养的前景进行了展望。     

辐射花粉授粉和胚培养诱导产生黄瓜单倍体植株

以5个基因型的黄瓜(Cucumis sativus L．)为试材,通过γ射线辐射花粉授粉并结合胚培养,从3个基因型中获得了单倍体植株。与正常二倍体植株相比,单倍体植株生长缓慢,花器异常。研究发现辐射剂量、亲本基因型、授粉组合对座果率和单倍体产率有一定影响。     

花粉单倍体的遗传品质——花药培养是否降低了育种材料的农艺价值

自S.Guha和S.C.Maheshwari 1964年以曼陀罗(Datura)花药为材料,首次成功地诱导出离体幼胚以来,人们经过不断的发展和完善使之形成了一套完整的花药培养技术,即通过植物花药(或小孢子)的培养,大批创制单倍体并经染色体加倍进而获得双单倍体(Doubled haploid)植株。该技术被迅速而广泛地应用于作物育种程序之中。目前已有不少的栽培作物成功地获得了花粉植株,如:烟草、小麦、大麦、水稻、黑麦、小黑麦、玉米、甘     

小黑杨花粉植株H_1株系间变异及其选择利用的研究

小黑杨(Populus xiaohei T.S.Hwang ex C.Wang et Tung)花药离体培养获得单倍体植株,经14年的生长观察,发现花粉植株间表现出明显分离。选出19个株系进行田间比较试验,统计分析,结果发现材积生长差异显著。可从中选出优良株系加以利用,并提出杨树单倍体育种程序。     

柑桔花粉植株的诱导

目前在国内外已有数十种植物利用花药培养获得单倍体植株,但在果树花药培养方面的研究,至今尚未见到诱导出花粉单倍体植株的报道。我们自1974年3月开始,对柑桔类花药培养进行了一系列探索,经过多次反复试验,于1979年5月首次在四季桔(Citrus microcarpa Bunge)花药培养中获得了具有根、茎、叶的完整植株,经镜检胚状体和根尖细胞染色体为单倍体(2n=9)(图版1,7),表明是花粉发育的单倍体植株。现将试验结果报告于后。     

菸草(Nicotiana tabacum)和辣椒(Capsicum annuum)花药离体培养的研究

本文报道关于菸草和辣椒花药离体培养的研究结果。对于菸草花药离体培养曾进行四种培养基的对比试验,结果以 NH 效果最好,Blaydes 次之。10—20%(体积/体积)椰乳能提高花药成胚的百分比,对单倍体幼苗也有促进作用。蔗糖浓度从1—4%都能产生“胚状体”,以3%的蔗糖浓度产生“胚状体”的百分比最高,长出的幼苗也比较健壮。对花粉不同发育时期进行了比较试验,花药中花粉单核靠边时期进行培养的出苗率最高。“胚状体”的形成主要由单核花粉粒均等分裂或由营养核分裂两种方式形成“胚状体”。生殖核只进行几次分裂而最后逐渐退化消失。对菸草花粉植株进行根尖或茎尖制片,观察其染色体数目为24,证明是单倍体。用不同浓度的秋水仙碱对单倍体植株进行染色体加倍,得到许多二倍体后代,其中少数是四倍体。本文对花药离体培养和组织培养中产生“胚状体”问题进行了讨论。辣椒采用花粉单核靠边时期的花药,培养在 NT 及 MS 加有补充物质的培养基上,花药既产生“胚状体”又产生愈伤组织。观察到单核花粉最初分裂发育成多细胞“原胚”到最后形成幼苗的过程。“胚状体”的发育与合子胚的发育过程相似。在花粉发育成幼苗的各个发育时期都可能停止发育,长成幼苗的只是少数。由单核花粉粒所形成的多细胞“原胚”可突出花粉壁外但仍与花粉粒相连,花粉粒内细胞大而染色淡,花粉壁外的多细胞“原胚”的细胞排列紧密,体积小而有分生能力,有的已开始分化。小苗的根尖和愈伤组织细胞,用醋酸洋红压片检查,其染色体数目为12,证明是单倍体。     

水稻胚囊植株染色体倍性及其它性状的研究

对水稻未传粉子房培养获得的“胚囊植株”作了染色体观察与性状考查,并与花粉植株进行比较。胚囊植株多数为单倍体,少数为二倍体,未见到多倍体;其单倍体比例较花粉植株为高。与花粉植株相反,胚囊植株多数为绿苗,白化苗很少。对胚囊植株与花粉植株及其二代株系的性状表现进行了初步观察。     

黑麦(Secale cereale L.)花药培养及其雄核发育

离体培养黑麦花药,单核花粉可以通过不等分裂和均等分裂由营养性质的核发育成花粉胚状体或愈伤组织,并且最后形成了单倍体植株。接种前对穗子进行适当的低温处理,可以有效地提高胚状体和愈伤组织的生成率。根据多核和多细胞花粉的败育情况,认为新壁的形成和花粉外壁的适时破裂是已经启动分裂的小孢子能否发育成胚状体和愈伤组织的两个关键问题。     

辣椒游离小孢子细胞团培养的胚状体形成

从预培养15天后的花药中机械游离小孢子及其细胞团,经28℃液体悬浮暗培养．30天后,获得了自球形期胚到子叶期胚发育程度不等的各类胚状体。从12个花药中可以形成高达22个胚状体,且子叶期胚的比例约为23％。显微镜检表明,这些胚状体来自游离的小孢子细胞．经核的对称分裂形成多核细胞或者早期形成多细胞团,最后经细胞的分裂分化形成。胚状体体表具毛,活力有差异。在适当培养基上,具活力的鱼雷期及子叶期胚状体均能发育成正常植株。7℃、32℃、35℃8天的胁迫处理均能诱导小孢子胚状体发生。但花药培养中7℃、35℃处理下的出胚率较32℃下高,而游离小孢子细胞团培养中以35℃、32℃下较好。7℃处理下获得的胚状体数很少．对产生这种现象的原因进行了探讨。出胚率在基因型间,不同胁迫处理温度间表现明显差异。而在温度处理的不同天数间差异不明显。流式细胞仪对再生株真叶的DNA含量分析表明．获得的再生株中具有单倍体、双单倍体以及单倍一双倍嵌合体植株。本结果为进一步开展辣椒雄性生殖途径的胚状体发育研究。提高辣椒成熟胚状体的频率提供了实验体系。     

从玉米花药培养中诱导胚状体的发生

从植物花药离体培养中诱导花粉植株,可以通过两种途径获得。一种是由花粉细胞先形成愈伤组织,再分化成植株。另一种是直接由花粉细胞形成胚状体—花粉胚—发育成植株。后一种途径引起了许多研究者的兴趣。这是因为胚状体成苗速度快,成苗率高。     

Isolated pollen culture: plant reproductive development in a nutshell

Abstract

Isolated pollen can develop into two different directions when cultured in vitro. In a rich medium, microspores and young pollen grains develop into mature pollen that is fertile. When pollen is treated by a stress treatment such as a hunger treatment in a sucrose - and/or nitrogen - free medium, embryogenic pollen is formed that, after transfer to a rich medium, develops into embryos and haploid plants. This system of isolated pollen culture offers an opportunity to study two developmental processes, i.e. pollen development and embryogenesis, as well as a basic regulatory event, i.e. the transition from the gametophytic to the sporophytic phase in the alternation of generations in higher plants. In addition, both systems offer various application-oriented possibilities, such as production of doubled haploids, to overcome self-incompatibility, to rescue sterile pollen, pollen selection and pollen transformation. An understanding of the cell biological and molecular events during embryogenic induction may promote a wider application of doubled haploid breeding and the use of such plants for gene transfer.     

SPECIAL PAPER: HAPLOIDS FROM POLLEN GRAINS—RETROSPECT AND PROSPECT

The production of large numbers of haploid plants from pollen grains in aseptic culture of anthers or isolated pollen grains has now been demonstrated in many angiosperms. It is a technique which holds much promise for creating desired variability by mutations for biochemical and applied genetics and for producing pure lines for crop improvement. This paper reviews the research on the production of haploids from pollen grains. The response is dependent on a variety of factors such as media formulation (especially the hormone component), genotype and physiological status of the donor plant, developmental stage of the pollen grain, anther culture environment and anther wall itself—all of which influence greatly the successful production of haploid plants. Although still in its infancy, the culture of isolated pollen is seen as a promising line of further research as it has the potential of offering several advantages over the culture of whole anthers.     

Gametophytic vs. sporophytic control of pollen aperture number: A generational conflict

In flowering plants, the haploid phase is reduced to the pollen grain and embryo sac. These reproductive tissues (gametophytes) are actually distinct individuals that have a different genome from the plant (sporophyte), and are more or less independent. The morphology of pollen grains, particularly the openings permitting pollen tube germination (apertures), is crucial for determining the outcome of pollen competition. Many species of flowering plants simultaneously produce pollen grains with different aperture numbers in a single individual (heteromorphism). In this paper, we show that the heteromorphic pollen aperture pattern depends on the genetic control of pollen morphogenesis. This points out a conflict of interest between genes expressed in the sporophyte and genes expressed in the gametophyte. More generally, such a conflict should exist whenever heteromorphism is an ESS resulting from a bet-hedging strategy. For pollen aperture, heteromorphism has been observed in about 40% of angiosperm species, suggesting that conflicting situations are the rule. In this context, the sporo-gametophytic conflict could be one of the factors that led to the reduction of the haploid phase in plants.     

玉米花粉单倍体植株染色体上异染色质的变异

我们用Giemsa BSG C-带技术检查了玉米花药培养获得的花粉单倍体植株根尖细胞染色体上异染色质的变异,观察结果表明,有的植株所显示的C-带数目是与供体植株的相一致,有的植株所显示的C-带数目则发生了显著变化,其中有的增加,有的减少。并讨论了异染色质发生变异的可能原因。还相应地观察到间期核中染色中心的变化是与中期染色体上C-带数目的变化相一致。     

Pollen—Tiny and ephemeral but not forgotten: New ideas on their ecology and evolution

Ecologists and evolutionary biologists have been interested in the functional biology of pollen since the discovery in the 1800s that pollen grains encompass tiny plants (male gametophytes) that develop and produce sperm cells. After the discovery of double fertilization in flowering plants, botanists in the early 1900s were quick to explore the effects of temperature and maternal nutrients on pollen performance, while evolutionary biologists began studying the nature of haploid selection and pollen competition. A series of technical and theoretic developments have subsequently, but usually separately, expanded our knowledge of the nature of pollen performance and how it evolves. Today, there is a tremendous diversity of interests that touch on pollen performance, ranging from the ecological setting on the stigma, structural and physiological aspects of pollen germination and tube growth, the form of pollen competition and its role in sexual selection in plants, virus transmission, mating system evolution, and inbreeding depression. Given the explosion of technical knowledge of pollen cell biology, computer modeling, and new methods to deal with diversity in a phylogenetic context, we are now more than ever poised for a new era of research that includes complex functional traits that limit or enhance the evolution of these deceptively simple organisms.     

Analysis of airborne pollen fall in Sakarya, Turkey

In this study, pollen grains were identified using Durham sampler in the atmosphere of Sakarya in 2000 and 2001. During these two years, a total of 10 805 pollen grains were recorded. A total of 5 386 pollen grains per cm² were recorded in 2000 and a total of 5 419 pollen grains per cm² in 2001. Pollen fall in the years 2000–2001 comprised grains belonging to 40 taxa and some unidentified pollen grains. Of these taxa, 22 belonged to arboreal and 18 taxa to non arboreal plants. Total pollen grains consisted of 69.45% grains from arboreal plants, 28.11% grains from non-arboreal plants and 2.44% unidentified pollen grains. In the region investigated, Gramineae, Pinus sp., Quercus sp., Cupressaceae/Taxaceae, Salix sp., Platanus sp., Populus sp., Carpinus sp., Fagus sp., Chenopodiaceae/Amaranthaceae, Xanthium sp., Moraceae, Corylus sp., Fraxinus sp., and Urticaceae released the greatest amount of pollen. The season of maximum pollen fall was from March to May, with a prevalence of arboreal pollen in the first months, and of pollen from non-arboreal plants in the last months of the year.     

Analysis of Airborne Pollen Fall in Edirne,Turkey

In the atmosphere of Edirne 12 691 pollen grains belonging to 42 taxa were identified by using of Durham sampler in 2000 and 2001. A total of 6 189 pollen grains per cm2 were recorded in 2000 and a total of 6 502 pollen grains per cm2 in 2001. Total pollen grains consisted of 71.81% grains from arboreal plants, 25.88% grains from non-arboreal plants and 2.31% unidentified pollen grains. Pollen from the following taxa were also found to be prevalent in the atmosphere of Edirne: Gramineae, Pinus sp., Quercus sp.,Cupressaceae/Taxaceae, Platanus sp., Salix sp., Morus sp., Populus sp., Carpinus sp., Juglans sp.,Chenopodiaceae/Amaranthaceae, Fraxinus sp., Fagus sp., Ulmus sp., Ailanthus sp., Alnus sp., Ostrya sp.,Helianthus sp. The season of maximum pollen fall was from April to June, with a prevalence of arboreal pollen in the first month, and of pollen from non-arboreal plants in the last months of the year.     

雄性不育相关基因msLTP的反义RNA验证

根据普通白菜雄性不育相关的脂质转移蛋白基因(msLTP)的cDNA序列设计引物,从普通白菜花蕾的cDNA中扩增出312bp的片段,然后将该片段连接至双元载体pBI12l中,得到反义RNA植物表达载体并导入农杆菌LBA4404菌株中,通过农杆菌介导法转化菜心;利用PCR和Southern blot分析检测得到了25株转基因植株,转基因植株的花粉部分畸形或空瘪,花粉离体萌发率为38.56%,较未转化植株的萌发率(76.32%)降低了37.76个百分点.研究表明,反义RNA技术使msLTP基因沉默而导致了菜心转基因植株的部分花粉发育不良,说明msLTP基因在普通白菜和菜心等花粉发育中具有重要作用.     

Analysis of Airborne Pollen Fall in Edirne, Turkey

In the atmosphere of Edirne 12691 pollen grains belonging to 42 taxa were identified by using of Durham sampler in 2000 and 2001. A total of 6 189 pollen grains per cm~2 were recorded in 2000 and a total of 6502 pollen grains per cm~2 in 2001. Total pollen grains consisted of 71.81% grains from arboreal plants, 25.88% grains from non-arboreal plants and 2.31% unidentified pollen grains. Pollen from the following taxa were also found to be prevalent in the atmosphere of Edirne: Gramineae, Pinus sp., Quercus sp., Cupressaceae/Taxaceae, Platanus sp., Salix sp., Morus sp., Populus sp., Carpinus sp., Juglans sp., Chenopodiaceae/Amaranthaceae, Fraxinus sp., Fagus sp., Ulmus sp., Ailanthus sp., Alnus sp., Ostrya sp., Helianthus sp. The season of maximum pollen fall was from April to June, with a prevalence of arboreal pollen in the first month, and of pollen from non-arboreal plants in the last months of the year.