植物单倍体的产生、鉴定、形成机理及应用

单倍体(Haploid)是指含有配子染色体数目的个体,对其进行基因组加倍可以快速获得纯合双单倍体(Doubled haploid, DH)。单倍体和双单倍体在植物品种选育、突变体筛选、基因功能鉴定、细胞学研究、遗传群体构建等方面具有重要作用,是近年来植物领域的一大研究热点。本文从单倍体和双单倍体的产生途径、鉴定、形成机理以及应用等方面较全面地综述了单倍体的最新研究进展,为单倍体的研究利用作一定的参考。     

植物单倍体诱导技术发展与创新

单倍体育种是培育作物新品种的主要育种技术之一,提高单倍体诱导频率和简化诱导程序是单倍体育种技术的关键。随着单倍体诱导技术的发展与改进,单倍体育种技术已被广泛应用于许多重要植物的育种研究中,展现出基因纯合快速、育种年限缩短、育种效率提高等优势。单倍体诱导技术与杂交育种、诱变育种、反向育种和分子标记辅助选择育种等技术相结合,在作物品种改良上的作用更加显著。单倍体和双单倍体在遗传群体构建、基因功能鉴定、转基因研究、细胞学研究等方面具有重要应用价值。本文从单倍体诱导技术、单倍体和双单倍体应用等方面综述了植物单倍体诱导技术的发展,尤其是近年来利用基因组编辑技术创制主要作物单倍体诱导系的进展,并分析了目前研究中存在的问题和今后的发展方向,以期促进单倍体诱导技术尤其是利用基因编辑创造诱导系技术在作物育种中的应用。     

单倍体小麦染色体加倍的研究

从七十年代利用花药培养技术获得单倍体植株以来,单倍体植物在育种应用上的潜力日益显示出来。但是这种潜力只有使单倍体植物加倍成为纯合二倍体才有可能发挥。因此,对于单倍体植物的染色体加倍,许多植物育种工作者进行过多方面的试验研究,提出过多种加倍方法,其中秋水仙碱法应用最为广泛,但加倍成功率并不理想,植株死亡率也较高。近年来有人用秋水仙碱和DMSO结合处理单倍体植株,加倍效果显著提高。就小麦来说,最高成功率有达9.0％     

从大麦雌配子体诱导单倍体植株

近年来,应用离体培养花药的方法诱导花粉单倍体植物的研究得到迅速发展,但从雌配子体系统诱导单倍体植物的研究却作得很少,进展也不大。在裸子植物上,Tulecke曾离体培养未受精的银杏雌配子体,得到单倍体愈伤组织,但没有分化成植株。在被子植物上,用未受精的胚珠或子房进行培养,Uchimica 得到茄子胚珠的单倍体愈伤组织,Jensen 则在棉花的胚珠中看到极核的融合及二倍体胚乳的分裂,但都没有进一步的结     

菊科植物单倍体研究进展

单倍体培养是快速获得菊科纯合系的重要途径。目前已进行单倍体研究的菊科植物共有13个种,其中9个已成功获得单倍体植株。菊科中诱导单倍体的途径有花药培养、小孢子培养、离体雌核培养、远源杂交和辐射花粉诱导单倍体。本文详细论述了不同外植体发育时期、预处理、培养基、培养条件等因素对单倍体植株诱导再生的影响。对菊科植物单倍体诱导的几种途径进行对比总结,指出研究中存在的问题并提出思路和建议。     

从白魔芋未授粉子房培养出单倍体植株（简报）

通过花药培养已从20多个科的一百多种植物中得到单倍体植物。未授粉子房离体培养人工诱导单倍体的研究,已从二棱大麦、小麦和烟草、水稻、玉米、普通大麦、向日葵、百合、青稞等植物的未授粉子房培养出单倍体植物。1987年我们进行了白魔芋未授粉子房的离体培养,并获得单倍体植物,现将实验初步结果报道如下。     

离体染色体加倍的新技术

利用花药衍生的单倍体植株进行植物育种取决于这些植株染色体数目加倍而获得双单倍体植株的能力,染色体加倍可自然发生,也可通过抗有丝分裂试剂如秋水仙碱处理完整单倍体植株或单倍体组织培养物来诱导发生。秋水仙碱用于进行多种作物染色体加倍已达50余年。然而,秋水仙碱可以促使植物诱变,同时也对人类具有剧毒,因此,应用秋水仙碱进行研究难度较大。荷兰 Wageningen 植物育种与繁殖研究中心的 J.M.Van.Tuyl 和同事研究发现,除草剂 Oryzal-in 可用作低浓度秋水仙碱的替代品。他们用0.0001～0.01% Oryzalin 离体处理 Nerine 和 Lilium 的不育     

单倍体的形成及其育性

介绍了单倍体的种类,综述了单倍体的自然形成及植物上人工获得单倍体的方法,并分析了不同生物单倍体的育性及其产生原因。     

单倍体胚胎干细胞的获得与应用前景

单倍体细胞在遗传筛选、生物发育与辅助生殖等研究中都具有重要的应用价值。近年来,有实验室建立了哺乳动物的单倍体胚胎干细胞系,但这些单倍体胚胎干细胞是否真的具有多能性以及转基因的单倍体胚胎干细胞能否直接从细胞水平传递到动物水平,这些问题并不清楚。以自然科学基金委重大研究计划为依托,周琪实验室开展了系统的研究,获得了一系列前沿性进展:获得了具有多能性的单倍体胚胎干细胞;利用单倍体胚胎干细胞获得基因修饰动物;利用单倍体胚胎干细胞替代精、卵结合获得后代;利用单倍体胚胎干细胞进行同性生殖;利用单倍体胚胎干细胞研究物种间杂交。这一系列自主创新的研究成果推进了整个领域对单倍体胚胎干细胞的认识,大大拓展了单倍体胚胎干细胞研究的理论价值和应用前景。现主要对自然科学基金委重大研究计划中周琪实验室的原创性工作进行综述。     

植物和动物的殊途同归(2)

动物从单鞭毛的领鞭毛虫演化而来,最初的动物都是二倍体的;而植物却是从双鞭毛的绿藻演化而来,最初的植物都是单倍体的。二倍体的生物由于细胞内含有双份遗传物质,生活力强于单倍体的生物,所以后来的动物都继承了最初动物二倍体的优点,一直保持二倍体的身体状态。植物从单倍体开始,一开始就处于不利地位,因此在亿万年的时间中通过几个步骤逐渐变为二倍体:从绿藻的单倍体生命,到苔藓植物以单倍体的配子体为主,二倍体的孢子体寄生在配子体上,到蕨类植物以二倍体孢子体为主,单倍体的配子体相对弱小的生命,再到种子植物(包括裸子植物和被子植物)中占绝对优势的孢子体"收容"高度退化的配子体的生命,植物完成了遗传物质份数上的"华丽转身",在繁殖方式上几乎与动物相同,即都是二倍体的生物体产生二倍体的下一代,原来单倍体的形式几乎退化干净。植物二倍体的出现需要新的身体发展蓝图,以及"规定"二倍体细胞必须采用新的发展蓝图的控制机制,而这是由KNOX蛋白实现的。     

Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus

Summary A chromosome doubling technique, involving colchicine treatment of an embryogenic, haploid callus line of maize (Zea mays L., derived through anther culture), was evaluated. Two colchicine levels (0.025% and 0.05%) and three treatment durations (24, 48, and 72 h) were used and compared to untreated controls. Chromosome counts and seed recovery from regenerated plants were determined. No doubled haploid plants were regenerated from calli without colchicine treatment. After treatment with colchicine for 24 h, the callus tissue regenerated about 50% doubled haploid plants. All of the plants regenerated from the calli treated with colchicine for 72 h were doubled haploids, except for a few tetraploid plants. No significant difference in chromosome doubling was observed between the two colchicine levels. Most of the doubled haploid plants produced viable pollen and a total of 107 of 136 doubled haploid plants produced from 1 to 256 seeds. Less extensive studies with two other genotypes gave similar results. These results demonstrate that colchicine treatment of haploid callus tissue can be a very effective and relatively easy method of obtaining a high frequency of doubled haploid plants through anther culture.     

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。     

phlb基因诱导小麦ABD染色体组部分同源染色体配对的研究

通过花药培养首次获得了“中国春”phlb突变体单倍体,同时也获得了“中国春”单倍体。对其细胞学观察表明,前者花粉母细胞减数分裂中期Ⅰ每个细胞染色体交叉为5.08个,后者为1.30个。证明了phlb基因在单倍体状态下具有强的诱导ABD染色体组部分同源染色体间的配对作用。     

葫芦科植物幼嫩子房壁制片技术的优化及其染色体倍性鉴定

该研究利用黄瓜、甜瓜、西瓜和西印度黄瓜这几种葫芦科植物的幼嫩子房壁作为材料进行染色体制片,探索子房材料的样品大小、预处理时间和酶解时间对染色体制片的影响及其优化,并用该制片方法对黄瓜候选单倍体植株的子房壁进行倍性鉴定和荧光原位杂交实验。结果发现:(1)黄瓜、甜瓜、西瓜和西印度黄瓜的幼嫩子房壁最佳预处理时间分别为1 h 30 min、1 h、55 min和45 min,子房长度为0.2～1 cm,子房壁材料切成边长为1～1.5 mm小块,酶解时间为1 h 10 min～1 h 20 min时,用该优化制片方法均可观察到较多的分裂相。(2)利用该方法鉴定结果显示,葫芦科植物黄瓜、甜瓜、西瓜和西印度黄瓜的染色体分别为14、24、22和24条,黄瓜候选单倍体植株的体细胞染色体数为7条。(3)将该制片方法获得的染色体装片用于荧光原位杂交结果显示,在二倍体黄瓜染色体中有3对明亮的45S rDNA杂交信号和1对5S rDNA杂交信号,而单倍体黄瓜中相应信号数量均减半;在甜瓜、西瓜和西印度黄瓜中均有2对45S rDNA杂交信号和1对5S rDNA杂交信号。研究认为,利用葫芦科植物子房壁作为制片材料,不仅可以获得良好的分裂相,还具有易于取材、制片效率高等优点,因此子房壁制片法是研究植物染色体数目和鉴定倍性的有效方法,且该制片方法也适用于进一步的荧光原位杂交分析。     

Improved chromosome doubling of parthenogenetic haploid plants of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) using colchicine,trifluralin, and oryzalin

An effective chromosome doubling protocol was established in essential garden crop of cucumber (Cucumis sativus L.) Cv. Hi Power. The different concentrations of colchicine (0, 250, 500, 750, and 1500 mg/L), oryzalin (0, 5, 15, 25, 50, 75, and 150 mg/L) and trifluralin (0, 5, 15, 25, 50, 75, and 150 mg/L) were applied on parthenogenesis-induced haploid nodal and shoot tip explants of cucumber for 18 and 38 h in three independent factorial experiments. Increasing concentrations of applied antimitotic agents led to the significant reduction in the survival rate of both shoot tip and nodal explants, especially in longer exposure duration. Three ploidy levels including haploid, mixoploid, and doubled haploid were regenerated form both explant types treated with colchicine, oryzalin, and trifluralin. Flow cytometry analysis proved successful chromosome doubling of haploid plants. Based on the results obtained, the highest number of regenerated doubled haploid plants (92.31%) and fruit set (86.21%) were related to immersion of nodal explants in 50 mg/L oryzalin for 18 h. The highest doubled haploid regeneration for colchicine and trifluralin antimitotic agents were 58.33 and 83.33%, respectively. The leaf size of doubled haploid plants was larger than their correspond haploids. The optimized chromosome doubling protocol would be applicable for doubled haploid production in garden crops of Cucurbitaceae family, which is recalcitrant to the spontaneous doubling, and also for in vitro polyploidy induction studies.     

Production of haploid and doubled haploid plants of melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) for use in breeding for multiple virus resistance

We have developed improved procedures for recovery of haploid and doubled haploid (DH) melon plants, using hybrids derived from crosses of lines with multiple virus resistance. Seeds formed after pollination with irradiated pollen were cultured in liquid medium for 10 days before excision of the embryos for further culture. This made it easier to identify the seeds containing parthenogenetic embryos, thereby reducing the effort required and increasing the percentage of plants recovered. The plants obtained (approximately 175) were transferred to a greenhouse for evaluation. Three fertile lines were identified, and selfed seeds were obtained for evaluating virus resistance. Flow cytometry of leaf tissues showed that two of these lines were spontaneous DH and the third was a mixoploid containing haploid and diploid cells. The other plants remained sterile through the flowering stage. Flow cytometry of 20 sterile plants showed that all were haploid. Attempts to induce chromosome doubling by applying colchicine to greenhouse-grown plants were unsuccessful. Shoot tips from the haploid plants were used to establish new in vitro cultures. In vitro treatment of 167 micropropagated haploid shoots with colchicine produced 10 diploid plants as well as 100 mixoploid plants. Pollen from male flowers that formed in vitro on the colchicine-treated plants was examined. High percentages of viable pollen that stained with acetocarmine were found not only in the diploids but also in >60% of the plants scored as mixoploid or haploid by flow cytometry. Efficient recovery of DH from hybrid melon lines carrying combinations of important horticultural traits will be a valuable tool for melon breeders.     

Two methods of haploidization in pear,Pyrus communis L.: greenhouse seedling selection and in situ parthenogenesis induced by irradiated pollen

Seedlings of 12 crosses involving pear varieties or hybrids were observed for the presence of haploid plants. On the basis of phenotypic characteristics, 17 plants corresponded to the haploid condition and, of these, 12 were determined by chromosome counting to be haploid (2n=x=17). In addition, and in order to induce in situ parthenogenesis, several pear varieties were pollinated with a selected clone carrying a homozygous dominant marker gene for the colour of red. This pollen had previously been irradiated with -rays of cobalt 60 at 0, 200, 250 and 500 Grays. The immature embryos were cultured in vitro, whereby 1 haploid and two mixoploid plants were obtained. Numerous diploid plants with the maternal phenotype were also obtained, and their genetic origin was subsequently studied by means of isozyme analysis.     

Efficient tissue culture and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of haploid poplar derived from anthers

Calli were induced from anthers of Populus simonii × P. nigra. Haploid plants were then regenerated from the callus and multiplied efficiently by culturing leaf explants. The presence of both haploid and diploid cells in the same plant revealed spontaneous chromosome doubling in haploid cells. The haploid plants were transformed with the nptII gene by Agrobacterium-mediated method using leaf explants, and five independent kanamycin-resistant lines were obtained, with a transformation frequency more than 6%. Further PCR test indicated that the exogenous betA gene was transferred into these kanamycin-resistant lines, which were still haploid. Thus, the efficient tissue culture system and transformation of haploid poplar plants were achieved. Our study will contribute to forest improvement via the haploid culture and transgenic technology. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 629–633. The text was submitted by the authors in English.     

The auxins centrophenoxine and 2,4-D differ in their effects on non-directly induced chromosome doubling in anther culture of wheat (T. aestivum L.)

Doubled haploid technologies have become key tools for plant breeding. Using these techniques, the speed and efficiency of plant improvement processes can be significantly enhanced. Anther culture-based technologies have the potential to regenerate large numbers of doubled haploid plants without colchicine treatment. In an attempt to elucidate the influence of phytohormones on non-directly induced chromosome doubling, two synthetic auxins, 2,4-D and centrophenoxine, were tested in a wheat anther culture approach. Whereas the induction of androgenic embryo-like structures (ELSs) was efficient for both auxins, we observed a significantly higher frequency of chromosome doubling when using 2,4-D than when using centrophenoxine. When 2,4-D was added to the induction medium, a positive correlation between the size of ELSs and their ploidy level was detected by flow cytometry. The morphological selection of ELSs, a process that was included in routine operations of the method without significantly extending the input of time and effort, facilitates the production of fertile DH plants with a frequency of 60 %. Our findings may contribute to a more efficient production of doubled haploid wheat plants using a colchicine-free anther culture approach.     

Measuring Meiotic Crossovers via Multi-Locus Genotyping of Single Pollen Grains in Barley

The detection of meiotic crossovers in crop plants currently relies on scoring DNA markers in a segregating population or cytological visualization. We investigated the feasibility of using flow-sorted haploid nuclei, Phi29 DNA polymerase-based whole-genome-amplification (WGA) and multi-locus KASP-genotyping to measure meiotic crossovers in individual barley pollen grains. To demonstrate the proof of concept, we used 24 gene-based physically mapped single nucleotide polymorphisms to genotype the WGA products of 50 single pollen nuclei. The number of crossovers per chromosome, recombination frequencies along chromosome 3H and segregation distortion were analysed and compared to a doubled haploid (DH) population of the same genotype. The number of crossovers and chromosome wide recombination frequencies show that this approach is able to produce results that resemble those obtained from other methods in a biologically meaningful way. Only the segregation distortion was found to be lower in the pollen population than in DH plants.