Efficient Production of Doubled Haploid Plants through Chromosome Doubling of Isolated Microspores in Brassica napus

Three methods of chromosome doubling to produce doubled haploid plants from microspore cultures of Brassica napus were compared: colchicine treatment of microspore-derived plants, microspore-derived embryos, and isolated microspores. In the whole plant treatment, 53% of the treated plants set seed, but the treatment delayed plant growth and reduced seed set. When microspore-derived embryos were treated with colchicine, the doubling frequency was 32% (compared to 15% for spontaneous doubling). Direct colchicine treatment of isolated microspores resulted in a doubling efficiency of 70 % of the whole plants. This treatment also stimulated embryogenesis in microspore culture, leading to increased plant regeneration. Thus, direct chromosome doubling of isolated microspores is efficient and more than 10 000 doubled haploid plants have been produced in this manner in the past three years in order to accelerate the plant-breeding process.     

甘蓝型油菜小孢子培养影响因素的研究

本实验对四川成都生态区甘蓝型油菜进行游离小孢子培养,对小孢子培养的影响因素作了系统研究,结果表明：小孢子形成阶段的温度及昼夜温差、花蕾消毒液、小孢子培养浓度及株龄都是影响小孢子胚产量的因素,小孢子形成阶段的温度以10-15℃,昼夜温差5℃左右胚产量最高达到300枚/蕾,温度低于5℃或高于20℃胚产量都较低甚至不出胚;花蕾消毒液以0.1%的Hgcl2效果较好,培养浓度以3-4蕾/皿较好;2009年成都生态环境下,株龄从125天到150天,胚产量随着株龄增加而增加,到141天达到最高,后逐渐下降。     

Microspore culture is successful in most crop types of Brassica oleracea L.

Summary Microspore culture was shown to be applicable to a broad range of accessions belonging to six horticulturally important crop types of Brassica oleracea: broccoli, white cabbage, cauliflower, savoy cabbage, Brussels sprouts and curly kale. Of 64 accessions tested 86% were responsive. Large genotypic differences were found in number of embryos produced per flower bud, and in frequency and mode of regeneration of plants from embryos. B. oleracea was characterized by a strong asynchrony of microspore development within single buds. Microspore populations optimal for culture contained a large proportion (10–40%) of binucleate pollen. An initial high temperature treatment was essential for microspore embryogenesis. Growth conditions of the donor plants during inflorescence formation were less critical.     

Functional genomics of microspore embryogenesis

Isolated plant microspores, when stressed and cultured in vitro, can be diverted from their normal gametophytic pathway towards sporophytic development, with the formation of haploid embryos and ultimately doubled-haploid plants. This process is called androgenesis or microspore embryogenesis, and is widely used in plant breeding programmes to generate homozygous lines for breeding purposes. Protocols for the induction of microspore embryogenesis and the subsequent regeneration of doubled haploid (DH) plants have been successfully developed for more than 200 species. These practical advances stand in stark contrast to our knowledge of the underlying molecular genetic mechanism controlling this process. The majority of information regarding the genetic and molecular control of the developmental switch from gametophytic to sporophytic development has been garnered from four intensely studied (crop) plants comprising two dicotyledonous species, rapeseed (Brassica napus) and tobacco (Nicotiana tabacum), and two monocotyledonous species, wheat (Triticum aestivum) and barley (Hordeum vulgare). In these species the efficiency of microspore embryogenesis is very high and reproducible, making them suitable models for molecular studies. In the past, molecular studies on microspore embryogenesis have focussed mainly on the identification of genes that are differentially expressed during this developmental transition and/or early in embryo development, and have identified a number of genes whose expression marks or predicts the developmental fate of stressed microspores. More recently, functional genomics approaches have been used to obtain a broad overview of the molecular processes that take place during the establishment of microspore embryogenesis. In this review we summarise accumulated molecular data obtained in rapeseed, tobacco, wheat and barley on embryogenic induction of microspores and define common aspects involved in the androgenic switch.     

Efficient production of doubled haploid Brassica napus plants by colchicine treatment of microspores

Summary The effect of colchicine on isolated microspore cultures of Brassica napus was evaluated in order to combine a positive effect of colchicine on the induction of embryogenesis with the possibility to induce chromosome doubling at an early developmental stage, thus avoiding the production of haploid or chimeric plants. Colchicine was added to the culture medium immediately after isolation of B. napus microspores. The cultures were incubated from 6 to 72 h with various concentrations of colchicine. Samples were taken from the regenerating embryoids after 6 weeks for ploidy determination by flow-cytometry.The highest diploidization rate was obtained after a 24 h treatment of microspores with 50 mg/l colchicine, leading to 80–90% diploid embroids. A concentration of 100 mg/l colchicine applied for the same duration resulted in a lower diploidization rate (76–80%). Treatment durations of 6 h were not long enough to induce a high rate of diploidization, whereas the application of 10 mg/l for 72 h was also very effective.A sample of the plants regenerated from the colchicine treated microspores was transferred to the greenhouse. The plants looked similar to normal diploid rapeseed plants and showed reasonable pod and seed set. Thus, an additional generation for seed increase in the greenhouse is rendered unnecessary. The advantage of applying a minimum volume of colchicine under controlled in vitro conditions means a considerable saving of time and labour in DH-breeding programs.     

中国白菜小孢子离体培养和双单倍体植株再生

摘 要: 采用略加修改的NLN培养基（无机大量元素1/2）和甘蓝型油菜小孢子的胚状体诱导方法,培养了5个中国菜用小白菜品种和1个中国大白菜品种的小孢子,进行了双单倍体的诱导。结果表明,不同品种的小孢子产胚量差异很大,在接种的6个基因型中,有5个诱导出胚,诱导成功率83.33％ 。其中的中萁青抗热605产胚量最高,是8.676个胚／10蕾。这些胚在添加了NAA和PP333（多效唑）的B5培养基发育成健壮的小植株。关于6个基因型的小孢子胚胎发生能力,还有待进一步研究。     

油菜小孢子培养和双单倍体育种研究 Ⅰ.供体植株和小孢子密度对小孢子培养的影响

在油菜小孢子培养中,通过对供体植株两种生长状况和在NLN液体培养基中三种小孢子密度的研究表明,来自生长室的供体植株,每天摘除将开放的花朵,使其停留在蕾期阶段,油菜花蕾中处于单核期至双核期的小孢子同步化程度高,单核期小孢子核质比大,增长速度快,小孢子成胚的百分率高。平均每毫升NLN液体培养基中接种1个花蕾的小孢子的处理,成胚率最高,而且胚的分化较快。     

Increasing embryogenesis and doubling efficiency by immediate colchicine treatment of isolated microspores in spring Brassica napus

The effect of colchicine on induction of embryogenesis andchromosome doubling during microspore culture was evaluated in twoF₁ hybrids of spring oilseed rape (Brassica napus L.). Immediatecolchicine treatment of isolated microspores with the concentrations 50 and500 mg/L for 15 h stimulated embryogenesis and produced largeamounts of healthy-looking embryos. These normal embryos germinatedwell at 24 ^°C after being transferred to solid regeneration mediumand an initial period of low temperature (2 ^°C) for 10 days, andcould directly and rapidly regenerate vigorous plants. A high doublingefficiency of 83–91% was obtained from 500 mg/L colchicinetreatment for 15 h with low frequency of polyploid and chimeric plants.The present experiment showed that a treatment duration of 30 h revealedless positive effects on embryogenesis and doubling efficiency, especially athigher colchicine concentration (1000 mg/L). Poor embryogenesis andembryo germination were observed from ordinary microspore culturewithout change of induction medium and colchicine treatment, and severalsubcultures were required for induction of secondary embryogenesis andplant regeneration.     

Microspore culture of white cabbage, Brassica oleracea var. capitata L.: Genetic improvement of non-responsive cultivars and effect of genome doubling agents

For the efficient application of haploid induction procedures in cabbage breeding, a sufficient number of regenerants should be achieved in a broad spectrum of genotypes. However, the majority of genotypes are somewhat recalcitrant. The efficiency of microspore culture was tested by crossing a responsive (28.7 embryos per Petri dish) and a non- responsive (0.1 embryo) cabbage cultivar. The embryo yield of one progeny was intermediate (18.9) while two were superior to the best parent cultivar (52.9 and 64.0 embryos). Thus, genes for haploid embryogenesis, present in responsive lines, can be effectively transmitted to responsive × non-responsive hybrids. Abscisic acid-induced desiccation of embryos was used for the efficient regeneration of plants. High germination percentages (54.7-70.6%) followed by normal plantlet development were achieved. Spontaneous genome doubling measured at the plantlet stage differed markedly in untreated genotypes. The percentage of diploids ranged from 21 to 67%. The effects of two antimitotic drugs applied to freshly isolated microspores were determined in two experiments. In the first experiment, trifluralin (0.5 and 1.0 mg:l) had no effect on embryo induction while oryzalin partly (0.125-0.25mg/l) or completely (0.5.mg/l) inhibited the formation of embryos. In the second experiment, higher concentrations of trifluralin increased the proportion of diploidized plants. Application of anti-mitotic drugs to microspores did generally not improve the overall production of haploid plants, which was higher in an untreated control.     

Optimization of culture conditions for improved plant regeneration efficiency from wheat microspore culture

The production of haploid plants through microspore culture is a very important tool for plant breeding. However, progress in microspore culture for many species has been hampered by a number of factors that have resulted in low recovery of regenerated green plants. In this study, a series of experiments were conducted to increase the regeneration of haploid green plants from isolated wheat microspores. The use of different basal media and variations in media components resulted in the increased recovery (approximately double) of regenerated haploid wheat plants. Our findings demonstrate that CHB medium, in combination with 2,4-d, was a better medium for embryoids induction and plant regeneration than medium MC17 with either 2,4-d or PAA growth hormones. Wheat microspores cultured without ovary co-cultivation did not respond. Furthermore, high efficiency of microspore derived embryoids (up to 296 MDEs per 100 anthers) and green plant regeneration (up to 71 green plants per 100 anthers) were achieved by the use of gelrite instead of agarose as a gelling agent, and by the addition of media additives such as spent medium or MET.     

甘蓝类蔬菜小孢子培养研究进展

综述了甘蓝类蔬菜小孢子培养中有关取样、选样、胚胎发生以及影响胚胎发生的关键因素、植株再生等方面的研究进展,并指出了目前甘蓝类小孢子培养所存在的问题及解决方法。     

Response of different genotypes of Brassica carinata to microspore culture

Sixteen lines of Brassica carinata were evaluated for microspore culture and plant regeneration. The highest values of cell division and embryo yield resulted from buds between 2.5 and 3.5 mm long with 3 days of pretreatment at 32°C and plating densities of 100 000-150 000 microspores/ml. Ten out of 16 lines tested (63%) responded positively to microspore culture. In all breeding and F₁ lines, both cell division and embryo yield varied over a wide range, but embryogenic response was higher in F₁ lines than in the breeding lines.     

辣椒花药培养研究进展

归纳了辣椒花药培养过程中影响雄性胚胎发生的诸多因素,包括供试材料、小孢子发育时期、基本培养基、植物生长调节剂、培养基添加物质和温度胁迫处理,总结了小孢子胚胎发生的细胞学观察、再生株的倍性水平和单倍体基因组加倍研究,指出尽管人类已从细胞和分子方面对辣椒花药培养中诱导小孢子胚胎发生和形成胚状体有了进一步的认识,但尚未完全了解小孢子是如何被激发进入孢子体发育途径。当小孢子胚胎发生之谜完全揭开后,辣椒花药培养技术将会应用于更加广泛的领域。     

甘蓝型油菜离体小孢子胚胎发生能力的遗传分析

对一套甘蓝型油菜6×6半双列杂交组合和1个F2群体进行了离体小孢子培养。 不同基因型(组合)间产胚率差异极显著, 小孢子产胚率的广义遗传力为0.85。 F1小孢子产胚率具有明显的杂种优势, 平均优势率为10.52%。 小孢子产胚率的一般配合力和特殊配合力方差均达到极显著。 根据一般配合力的大小, 可将供试亲本分为高和低配合力     

青花菜小孢子发育时期与花器形态的相关性

对青花菜小孢子发育时期细胞学特征及其与花器外部形态的相关性展开研究,试图从花器的外部形态特征来推断小孢子的发育时期,最终目的是为花药或游离小孢子培养研究提供科学依据。结果表明,青花菜小孢子发育经历四分体时期、单核早中期、单核靠边期和双核期共4个时期,供试3个青花菜各时期细胞学特征存在明显差异。青花菜小孢子发育时期与花蕾大小和花药颜色等指标密切相关。供试青花菜花蕾纵径为10.52~11.05 mm,花蕾横径为3.64~4.25 mm,花药长度为2.85~2.87 mm,花药宽度为0.95~0.96 mm,且花瓣微露出花萼,花瓣和花药均为淡黄色时,80%以上的小孢子发育至单核靠边期。     

换培养液和秋水仙碱处理对白菜型油菜小孢子胚胎发生的影响

选用对小孢子培养反应较好的白菜型油菜地方品种（茅山种）为供体材料,研究了换培养液和秋水仙碱直接处理分离小孢子对胚产量的影响,并分析了秋水仙碱直接处理小孢子所获再生植株的染色体倍性水平。结果表明,分离小孢子在10％蔗糖浓度的NLN-10培养基上培养2 d后换成新鲜培养液,能显著提高出胚产量,比不换培养液的胚产量     

Fertile maize lines obtained from isolated microspores

Microspore response of three- way cross maize hybrid genotype 3AL/95 (Zea mays L.) was studied under simplified isolation and culture conditions. Fertile plant production was achieved through abundant plant regeneration. As a total, microspores of 160 tassels were inoculated and five sustainable microspore derived callus cultures (SMC) were obtained. Hybrid seeds (ML SC), which were produced by crossing of regenerates from two SMCs, gave rise to subsequent vigorous and fertile progeny. The response of the 3AL/95 and ML SC microspores was studied in three liquid culture media in order to improve the early viability of microspores. Them N6M medium provided better survival of cultured microspores (p = 5%) than the ppN6M/89 and the YPM-G media. The pH 5.8 in mN6M medium revealed significant increase (p = 1%) in microspore viability as compared to pH 3.0. The ML SC microspores showed higher viability(30%) on the first day of culture in the mN₆M than those of the3AL/95 (19%) but without improved rate of callus formation and plant regeneration. This revised version was published online in August 2006 with corrections to the Cover Date.     

High frequency spontaneous production of doubled haploid plants in microspore cultures of Brassica rapa ssp. chinensis

Brassica rapa (syn. Brassica campestris) ssp. chinensis is an important vegetable crop, but it is relatively recalcitrant to microspore culture. One genotype each of B. rapa ssp. chinensis var. communisand var. utilis were used formicrospore culture. Embryo production of3.8–42.4 embryos/bud was obtained. A high rate of plant regeneration directly from microspore-derived embryos without subculture was achieved by an improved protocol involving replacement of culture media and reduction of sucrose concentrations after 48 h of induction,among other modifications. More than 70%of regenerated plants were spontaneous diploids. Some spontaneous tetraploid plants were also obtained from isolated microspores of both genotypes tested. These tetraploids may be directly exploited a snew varieties in a Brassica rapabreeding programme. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficient doubled haploid production in microspore culture of loose-curd cauliflower (Brassica oleracea var. botrytis)

We present an improved protocol for highly efficient production of doubled haploid loose-curd cauliflower plants (Brassica oleracea var. botrytis) via microspore culture. Our experiment explored factors such as donor plant treatment, flower bud pretreatment, embryo germination medium, and ploidy characterization of regenerated plants. Our technique efficiently produced embryos from both tight- and loose-curd donor plants, although the embryo yields were genotype dependent. We achieved a germination rate of around 30 % by employing a hormone combination of zeatin, indole-3-acetic acid, and 6-benzylaminopurine pretreatment culture. We also used 1–4 days of cold pretreatment of the flower buds, which were submerged into NLN-13 medium, to induce microspore embryogenesis. Analysis using an FCM Ploidy Analyzer showed that more than 50 % of regenerated plants were spontaneously doubled haploids, more than 25 % were tetraploids, and fewer than 7 % were haploid. Visual examination of plants in the field revealed that they had distinct phenotypic characteristics relating to their ploidy level. The efficient production of double haploids using our improved microspore culture technique is a promising approach that can be applied in loose-curd cauliflower breeding programmes and genetic research.     

Effect of thidiazuron on microspore embryogenesis and plantlet regeneration in Chinese flowering cabbage (Brassica rapa. var. parachinenis)

Traditional breeding methods require more than 6 years to obtain homozygous inbred lines, while isolated microspore culture (IMC) is an effective way to cultivate double haploid homozygous lines in only 2 years. However, low embryogenesis induction frequency in Chinese flowering cabbage remains a key obstacle to the practical application of this technique. Thidiazuron was added at different concentrations to NLN‐13 medium to estimate its effects on microspore embryogenesis and plantlet regeneration. Results showed that three genotypes responded positively. Optimum thidiazuron concentrations produced embryo yields of up to 14.67 embryos per bud and increased microspore embryogenesis frequency with up to 100% survival. Plantlet regeneration rates were up to 81.67%, and the treatment groups showed lower callus formation. We obtained up to 552 diploid plants from the tested genotypes, and the percentage of doubled haploid at different TDZ concentrations showed slight differences, and doubled haploid rates in the three genotypes were above 70%. They showed a high uniformity and can be directly used for hybrid breeding. This method accelerates microspore application in Chinese flowering cabbage hybrid breeding.