番茄游离小孢子培养研究进展

雄核发育是小孢子或未成熟花粉细胞沿孢子体发育途径形成植株的过程，也是创制育种材料的有效途径之一。为建立重复性好、再生频率高的番茄小孢子诱导方法，文章通过回顾国内外研究进展，总结了番茄雄核番茄雄核发育各阶段（胚胎发生能力的获得、诱导小孢子分化形成胚状体以及胚状体再生形成单倍体植株）的主要影响因素，并简要归纳了现有的游离小孢子分离培养技术。分析表明，基因型是制约番茄小孢子诱导体系的关键因素，且需与发育时期、培养条件等多个影响因素同期发生，才能使雄核发育偏离配子体途径。最后，展望了番茄小孢子培养未来的研究方向与发展趋势，以期为番茄双单倍体技术的深入研究提供参考。     

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.     

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.     

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.     

Effect of donor plant age and inflorescence age on microspore culture of Brassica napus L.

Summary Effect of age of donor plants and age of inflorescence on embryogenesis in microspore culture of B. napus was examined. Microspores isolated from buds of older plants had a higher embryo yield than those of younger ones. The effect of the age of inflorescence showed a different pattern. In older plants, a higher embryogenesis response was observed in microspores isolated from buds of new inflorescences, while in young plants, microspores isolated from buds of old inflorescences showed high embryo yield. These different responses were considered to be attributable to a difference in the developmental stage of pollen at the time of microspore isolation. Our results indicated that microspores collected from older inflorescences and older plants have sufficient embryogenic potential when the optimum developmental stage of pollen was used. Frequency of embryo to plant conversion was influenced by the size of embryos subcultured, but not by donor plant age or the age of the inflorescence.     

Efficient production of doubled haploid wheat plants by in vitro treatment of microspores with trifluralin or APM

Isolated microspore cultures from two doubled haploid (DH) lines of wheat, Triticum aestivum L., were used to develop an in vitro chromosome-doubling protocol. During the initial 24 h or 48 h of culture the microspores were treated with either of the two antimicrotubule herbicides trifluralin or amiprophos-methyl (APM) in concentrations ranging from 0.1 μM to 10μM. Untreated control cultures yielded 209 embryos per 100000 microspores, which is the equivalent of one spike. Among the regenerated plantlets 67% were green, and 15% of the flowering plants were spontaneously chromosome doubled. Treatments with both the herbicides had a significant effect on chromosome doubling, measured as the percentage of fertile regenerants. With the best combination of treatment duration (48 h) and herbicide concentration (10/μM) the percentage of fertile plants among regenerants could be increased up to 74% with APM and up to 65% with trifluralin. The largest numbers of DH plants per spike could be obtained with herbicide concentrations at 1–3 μM. Treatments with either herbicide at these concentrations resulted in an estimated average between the two genotypes of 27 DH plants per 100 000 microspores. These results demonstrate the high potential of APM and trifluralin as chromosome-doubling agents in isolated microspore cultures. The in vitro treatment integrated into tissue culture procedures will constitute an efficient method for chromosome doubling in future wheat breeding     

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.     

The Effect of Carbohydrate Composition and Concentration on Anther Culture Response in Barley (Hordeum vulgare L.)

Culture medium composition is critical for the successful induction of microspore division in vitro. The present experiments have focused on a relatively neglected area of cell and tissue culture research, namely the carbohydrate component used in the medium. Three spring barley genotypes were cultured on a medium which was modified by replacing sucrose with the following carbohydrates (6 % w/v): maltose, fructose, malt extract, galactose and a glucose (3 % w/v)/fructose (3 % w/v) mixture. Both maltose and malt extract were superior to sucrose in their capacity to induce green plantlet differentiation from microspores. The concentrations of both sucrose and maltose were also varied. Overall the response of anthers on maltose based media was higher than on sucrose based media. Furthermore, a concentration of maltose m the range 6—12 % w/v produced a higher frequency of green plants than a low concentration (1—3 % w/v). The effect of maltose based media on germplasm of direct relevance to barley breeders was also tested. The cultivar ‘Blenheim’ was shown to be very responsive and this genetic factor was transmitted to the F₁ hybrid. The frequency of haploid to diploid regenerants was not consistent over genotypes, but in general there were more haploid than diploid regenerants. The implications of these results for barley breeding are discussed.     

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.     

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。     

A cytological study on aluminium-treated wheat anther cultures resulting in plants with increased Al tolerance

The in vitro selection of microspores and microspore‐derived structures under Al stress is one way to improve the Al tolerance of crops. In our study, cytological alterations caused by Al were examined in anther cultures of a commercial wheat (Triticum aestivum L.) variety ‘Mv Pálma’, and the efficiency of in vitro selection was demonstrated. Although the anther walls retarded the appearance of toxicity symptoms, cytological changes similar to those observed in root cells (inhibition of cell division, intense vacuolisation, occurrence of micronuclei and cell wall thickening) were detected in the microspores. The severity of Al toxicity and the efficiency of selection depended on the Al concentration and the mode of treatment. Single Al treatments (0.6 and especially 1.6 mM) allowed DH lines with increased Al tolerance to be selected. Repeated Al treatment severely inhibited the cell division of the microspores and it was lethal even at a concentration as low as 0.6 mM. The results show that microspore embryogenesis can be exploited for studying the cytological effect of Al and for increasing the Al tolerance of wheat.     

Application of in vivo and in vitro mutation techniques for crop improvement

Summary Conventional mutation techniques have often been used to improve yield, quality, disease and pest resistance in crops, or to increase the attractiveness of flowers and ornamental plants. More than 1700 mutant varieties involving 154 plant species have been officially released. In some economically important crops, e.g. barley, durum wheat and cotton, mutant varieties occupy the majority of cultivated areas in many countries. Mutation techniques have become one of the major tools in the breeding of ornamentals such as alstroemeria, begonia, chrysanthemum, carnation, dahlia and streptocarpus. The use of in vitro techniques such as anther culture, shoot organogenesis, somatic embryogenesis and protoplast fusion can overcome some of the limitations in the application of mutation techniques in both seed and vegetatively propagated crops. In vitro culture in combination with induced mutations can speed up breeding programmes, from the generation of variability, through selection, to multiplication of the desired genotypes. The expression of induced mutations in the pure homozygote obtained through microspore, anther or ovary culture, can enhance the rapid recovery of the desired traits. In some vegetatively propagated species, mutations in combination with in vitro culture technique, may be the only method of improving an existing cultivar. Currently, many molecular studies rely on the induction and identification of mutants in model species for construction and subsequent saturation of genetic maps, understanding of developmental genetics and elucidation of biochemical pathways. Once identified and isolated, the genes that encode agronomically-important features can be either introduced directly into crop plants or used as probes to search for similar genes in crop species. It seems most likely that the recent developments based on these technologies will soon provide improved methods for selection of desired mutants.     

Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Production of doubled haploids (DHs) through androgenesis induction is an important biotechnological tool for plant breeding. In some species, DHs are efficiently obtained through embryogenesis from isolated microspore cultures. In eggplant, however, this process is still at its infancy, despite the economic relevance of this important agricultural crop. To date, only two studies have focused previously on this process, suggesting that in eggplant microspore cultures, the only morphogenic response is callus formation. Given the notable lack of studies on eggplant microspore cultures, in this work we explored this process with different experimental approaches. We studied the response of different cultivars and characterized the development of microspores induced to divide and proliferate. We demonstrated that microspore-derived embryos (MDEs) can be produced in eggplant; however, MDEs stopped at the globular stage, to turn into euploid and principally mixoploid calli. From these calli, 60 % of DH plants could be regenerated. In order to promote microspore induction we evaluated the effect of polyethylene glycol (PEG) and mannitol. PEG, but not mannitol, significantly increased induction of microspore embryogenesis. We also tested the ability of eight different media compositions to promote efficient plant regeneration from calli. In order to test it in a genotype-independent manner, we previously developed a method to generate clonal callus populations derived from single microspore-derived calli. Together, the results presented hereby constitute an efficient way to produce eggplant DHs through microspore culture. In addition, they contribute significant insights into the knowledge of the particularities of androgenesis induction in this species.     

Microspore mutagenesis of Brassica species for fatty acid modifications: a preliminary evaluation

A microspore mutagenesis protocol was developed for Brassica rapa, Brassica napus and Brassica juncea for the production of double haploid lines with novel fatty acid profiles in the seed oil. Freshly isolated Brassica microspores were first cultured with ethyl methane sulphonate (EMS) for 1.5 h. The EMS was removed and the microspores were then cultured according to the standard Brassica microspore culture protocol. This protocol was used to generate over 80 000 Brassica haploid/double haploid plants. Field evaluation of B. napus and B. juncea double haploids was conducted between 2000 and 2003. Fatty acid analysis of the B. napus double haploid lines showed that saturated fatty acid proportions ranged from 5.0% to 7.7%. For B. juncea, saturate proportions ranged from 5.4% to 9.5%. Of the 7000 B. rapa lines that were analysed, 197 lines had elevated oleic acid (>55%), 69 lines had reduced α‐linolenic acid (<8%) and 157 lines had low saturated fatty acid proportions (<5%), when compared with the parental lines.     

油菜小孢子培养影响因素及黄籽油菜双单倍体群体的构建

针对我国油菜小孢子培养供体材料一般种植在大田,受外界环境影响较大,出胚率较低的现状,对大田环境条件下小孢子培养的主要影响因素进行了研究。同时,为了构建黄籽油菜双单倍体群体,研究了黄籽油菜的出胚情况。结果表明,不同取样时间对小孢子胚状体再生频率的影响很大;秋水仙素处理游离小孢子对胚状体的诱导有一定的负面影响,浓度越高,影响越大;黄籽油菜的出胚率较低(0.56枚/皿),同样条件下,不到黑籽油菜的1/8。不同黄籽单株间出胚率差异较大,变异幅度在0.04~1.97枚/皿。采用小孢子苗直接移栽大田技术,移栽成活率达89.0%,经染色体加倍后,最终构建了含有127个株系的黄籽油菜双单倍体群体。     

甘蓝型油菜小孢子培养影响因素研究及再生苗早期倍性鉴定

对不同年份、不同地点种植的油菜育种材料进行小孢子产胚率鉴定,并以11种不同基因型的甘蓝型油菜品系为供体材料,在武汉田间,分别于初花期和盛花期分离培养小孢子,研究基因型、生态条件、开花时间对小孢子产胚率的影响及秋水仙碱处理的加倍效果,通过采用流式细胞仪测定小孢子再生苗的DNA含量鉴定其倍性,并种植验证.结果表明:相同基因...     

Improving androgenesis‐mediated doubled haploid production efficiency of FCV tobacco (Nicotiana tabacum L.) through in vitro colchicine application

Production of doubled haploid plants through androgenesis in flue‐cured Virginia (FCV) tobacco is a promising and convenient alternative to conventional selfing techniques for the generation of absolute homozygous lines. Here, we show a robust in vitro haploid and doubled haploid development protocol in FCV tobacco with major emphasis on improving the efficiency of chromosome doubling using in vitro colchicine treatment. We used five FCV tobacco hybrids for comparison of colchicine treatments. The anther culture response varied with developmental stages of the buds, and the highest response was observed in stage 2 buds. The effect of cold pretreatment was significant, and 4 days of pretreatment was optimum for gametic embryogenesis. Among the methods used for determining the ploidy status of plants, flow cytometry was found to be easy, fast and reliable for high‐throughput screening of haploids. Doubled haploids regeneration percentage varied from 6.77 to 11.95 in in vivo treatment, while the range of variation was 22.11% to 28.40% in in vitro colchicine treatment. We observed a pronounced increase in plant survival and the proportion of doubled haploid plants in in vitro treatment compared with the standard in vivo approach.     

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.     

Efficient Yield of Embryoids by Culture of Isolated Microspores of Different Brassicaceae Species

Microspore culture of rapeseed (Brassica napus) is well established in practical plant breeding as a potent method to produce homozygous plants. Except for B. carinata the method has not yet been applied to other Brassicaceae species. The present study describes successful microspore culture of B. campestris, B. nigra, B. oleracea and Raphanus sativus, overcoming the break down of such microspore cultures possibly arising from the release and accumulation of toxic substances into the medium. The modified procedure is as follows: exchange of the incubation medium after one day of culture, addition of activated charcoal to the medium and aerating the three day old microspore culture by agitating the petri dishes on a specially constructed shaker.     

In vitro chromosome doubling with colchicine during microspore culture in wheat (Triticum aestivum L.)

Isolated microspores of two DH lines of wheat were treated with 8 different colchicine concentrations up to 3 mM for either 24 h or 48 h during microspore culture. Untreated control cultures produced on average 220 embryos per spike (100,000 microspores), 68% of the regenerated plantlets were green, and 15% of the flowering plants were fertile. The colchicine treatments had a significant effect on chromosome doubling as measured by the percentage of fertile regenerants. Using colchicine concentrations around 1 mM the percentage of fertile plants among the regenerants was increased up to 53%. The highest number of embryos and regeneration rates were observed after 24 h colchicine treatment, while the highest frequencies of green plants and fertile plants were obtained with 48 h colchicine treatments. The highest number of DH plants per spike was found after treatment with colchicine concentrations of 300 to 1000 μM. Such treatments resulted in an estimated average between the two genotypes of 23 doubled haploid plants per spike. This revised version was published online in July 2006 with corrections to the Cover Date.