Regeneration of fertile doubled haploid plants from colchicine-supplemented media in wheat anther culture

The effect of colchicine added to induction medium for the production of fertile doubled haploid plants after in‐vitro anther culture was studied in wheat, Triticum aestivum L. For this, one winter and two spring wheat varieties were used. Anther cultures of the three genotypes were treated with 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine had no significant effect on anther response and embryoid production of the genotypes examined. However, in the winter wheat genotype ‘Mv Szigma’, colchicine caused a significant reduction in microspore‐derived structures. A significant decrease was also observed in plant regeneration ability of two genotypes (‘Vergina’ and ‘Acheloos’) after colchicine treatment. In addition, a significant reduction of the albinos produced was observed in all genotypes after olchicine treatment. In contrast, the regenerants obtained from the colchicine‐supplemented induction media produced significantly higher percentages of fertile plants in all genotypes. However, the level of fertility, was significantly different among the fertile plants obtained. This, together with the observation that in the case of the winter wheat variety the colchicine treatment resulted in 100% completely fertile plants with a high seed‐setting ability indicate that there is space for further improvement of the method when it is applied to spring cultivars. Finally, the increased number of seeds per 100 plated anthers obtained from all three genotypes after colchicine treatment, clearly demonstrates that the addition of colchicine to induction medium was superior to the conventional anther culture method and it could therefore be introduced into wheat breeding programmes.     

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.     

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。     

活性炭对甘蓝型油菜(Brassica napus L.)小孢子胚胎发生的影响

试验以大田环境下的多份甘蓝型油菜的品种或品系为材料,研究了活性炭对游离小孢子胚胎发生的影响.结果表明:活性炭对甘蓝型油菜游离小孢子培养胚状体发生有很好的促进作用,不仅提高了胚产量,而且有利于小孢子胚的正常发育,添加活性炭的固液双层培养基效果尤为显著.     

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.     

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     

Glucosinolates Determined by HPLC in the Seeds of Microspore-Derived Homozygous Lines of Rapeseed (Brassica napus L.)*

Microspore culture was employed to measure the relative efficiencies of anther culture and isolated microspore culture for the regeneration of embryoids and plants of Brassica napus. The yield of embryoids and plants was at least 10-fold greater from isolated microspores than from anther cultures. Approximately 1400 microspore-derived homozygous line's, the parental varieties and the corresponding F₂ plants were grown in a field trial. Important agricultural characteristics, such as morphological homogeneity, growth rate, onset of flowering and seed setting were evaluated subjectively and seed yield and glucosinolate content of individual plants were determined. The relative concentrations of up to S different glucosinolates in these seeds were measured via an automated high performance liquid chromatography (HPLC) system. The alkenyl and indole glucosinolates, the two most important categories of glucosinolates, were found in varying proportions and were independently determined in these line's. Our results do not support the previously suggested connection between low concentrations of glucosinolates and weak growth and/or poor seed yield. Additionally, no evidence was found that the lines derived from isolated microspore culture were subjected to unexpected selection pressures that might adversely affect the diversity of the lines obtained. These results demonstrate that microspore culture is a powerful tool not only for genetic analysis bur also for practical plant breeding.     

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.     

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。     

油菜小孢子培养技术体系研究

小孢子培养在油菜的基础研究和应用研究中均具有十分重要的意义。自1982年Lichter首次在甘蓝型油菜中进行小孢子培养获得成功以来,国内外在油菜小孢子培养技术方面已取得大量研究成果,包括油菜小孢子胚状体发生的影响因素,小孢子植株的再生、成苗、大田移栽、染色体加倍等,近年来又对一些关键技术环节加以了改进,笔者在对这些研究成果进行总结的基础上针对中国国情建立了大田条件下油菜高效小孢子培养技术体系。用该体系对甘蓝型油菜和新疆野生油菜的体细胞杂种后代进行小孢子培养的出胚率达到300枚/皿以上,采用小孢子苗直接移栽大田技术,成活率达到89.0%。此外还成功构建了含127个DH系的黄籽油菜DH群体及含115个DH系的粒重分离群体。     

Effect of parental genotypes and colchicine treatment on the androgenic response of wheat F1 hybrids

The effect of the parental genotypes and colchicine treatment on the androgenic response of wheat (Triticum aestivum L.) F1 hybrids was studied. For this, anthers from three F1 hybrids and their parents were cultured on W14 initiation medium and W14 supplemented with 0.03% colchicine. The number of responding anthers, microspore‐derived structures/100 anthers, green plants/embryos cultured, green plants/100 anthers and albino plants/100 anthers were recorded. It was observed that embryo formation and plant regeneration ability were genetically controlled and genotype dependent. In both treatments the variety Kavkaz had a significantly higher percentage of responding anthers, microspore‐derived structures and green plants/100 anthers than the other genotypes. On the other hand, the variety Myconos also demonstrated high microspore‐derived structure production and green plant regeneration when treated with colchicine. The good response observed in these two varieties indicates the importance of colchicine treatment only for certain genotypes. Green plant production capacity of the hybrids was intermediate to that of the parental varieties. As one parent with a high or even an intermediate response to anther culture could lead to the production of sufficient (for breeding purposes) green plants from the F₁ hybrids, it was concluded that screening the inbred lines for the response to anther culture with and without colchicine treatment could contribute to utilization of breeding material with a low response to anther culture via the proper hybrid combinations.     

Development of B-Genome Chromosome Addition Lines of B. napus Using Different Interspecific Brassica Hybrids

By interspecific hybridization within the genus Brassica, trigenomic haploids were produced and back-crossed four times with B. napus, variety ‘Andor’. From this material, monosomic B-genome chromosome addition lines were selected with the extra chromosome derived from three different B-genome sources, i.e., B. nigra (BB), B. carinata (BBCC), and B. juncea (AABB). After selfing and/or microspore culture, disomic addition lines were obtained. Meiotic behavior was studied of the trigenomic hybrids, the pentaploid BC₁ plants, and the monosomic addition lines. The addition lines were shown to possess cytological stability and good fertility.     

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.     

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.     

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.     

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.     

Ploidy of broccoli regenerated from microspore culture versus anther culture

The use of microspore or anther culture to generate doubled-haploids (DH) is an important adjunct to broccoli breeding) Regenerated populations from broccoli anther culture are usually mixtures of ploidy. However, ploidy composition of populations derived from microspore culture has not been reported. The purpose of the present study was to characterize regenerants derived from microspore culture, to evaluate factors influencing these characteristics and to compare results with those from anther culture. Eight populations, four from each culture method, were generated simultaneously using the same four F₁ hybrids as donor parents. The ploidy level of all regenerants was determined by DNA flow cytometry: the majority of them were diploid. As in anther culture, a mixture of ploidy was observed in all populations derived from microspore culture. Ploidy variation was more frequent among clonal families from anther culture (10%) than microspore culture (5%)‘Everest’ was the most productive donor parent with both methods, while ‘Greenbelt’ and ‘Major’ were least productive in anther and microspore culture, respectively. Genotype specificity for the total number of regenerated plants and ploidy composition occurred in both culture methods.     

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.     

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.