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.     

Inheritance of rust resistance genes and molecular markers in microspore-derived populations of flax

The inheritance patterns of rust resistance genes and molecular markers in microspore‐derived populations of flax were investigated. Plants were produced from anther culture of F₁ plants from two crosses. Microspore‐derived plants in anther culture of flax were identified using molecular markers. Two rust resistance genes and three out of six molecular markers were inherited in expected Mendelian ratios in microspore‐derived populations. Distorted segregation for the other three molecular markers was shown to be the result of over‐representation of genomic fragments from the more responsive parent in the F₁ donor plant. The implication of this study in relation to androgenesis and flax breeding using anther culture is discussed.     

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。     

QTL mapping and associated marker selection for the efficacy of green plant regeneration in anther culture of rice

Anther culturability of rice is a quantitative trait controlled by nuclear‐encoded genes. The identification of quantitative trait loci (QTL) and associated marker selection for anther culturability is important for increasing the efficiency of green plant regeneration from microspores. QTL associated with the capacity for green plant regeneration in anther culture of rice were mapped on chromosomes 3 and 10 using 164 recombinant inbred (RI) lines from a cross between ‘Milyang 23’ and ‘Gihobyeo’. The quantitative trait locus located on chromosome 10 was detected repeatedly when three anther culture methods were applied and was tightly linked to the markers, RG323, RG241 and RZ400. Associations between these markers and the efficacy of green plant regeneration in 43 rice cultivars and two F₂ populations, ‘MG RI036’/‘Milyang 23’, and ‘MG RI036’;/‘IR 36’ were analysed. One of these markers, RZ400, was able to identify effectively genotypes with good (> 10.0%) and poor (< 3.0%) regenerability, based on the marker genotypes in the cultivars and two F₂ populations. This marker enables the screening of rice germplasm for anther culturability and introgression into elite lines in breeding programmes.     

萝卜花药培养技术研究

通过单倍体育种途径可快速获得纯合材料,增加有益性状的选择几率,加快育种进程。花药培养是获得单倍体的有效途径之一,已在十字花科芸薹属作物上取得巨大成功。但关于萝卜花药培养的报道极少。以15个不同类型的萝卜品种为试材,研究了花药离体培养中胚状体的诱导及再生。结果表明:基因型是限制花药培养成胚的关键因素,15个品种中,有4个品种获得了胚状体及再生苗。同时植株生长条件也对胚状体的形成产生影响。采用流式细胞仪(FCM)鉴定了再生株的倍性,再生株群体的倍性组成比较复杂,以二倍体为主。     

Spike pretreatments, anther culture conditions, and anther culture response of 17 German varieties of spring wheat (Triticum aestivum L.)

The aim of this work was to establish an in vitro regeneration system from anther cultures of different German varieties of spring wheat (Triticum aestivum L.). Using ‘Nandu’ the most widely grown spring wheat cultivar in Germany, different culture conditions were investigated with regard to their influence on anther culture response. The best results were obtained when applying a cold pretreatment to the donor spikes and using the synthetic L3 induction medium, liquid or solidified with gelrite. The highest rates obtained in these experiments with ‘Nandu’ were 8.6% responding anthers, 22.3% embryoid induction, 15.3% albino regeneration and 5.5% green plant regeneration (all rates related to the number of cultured anthers). Of the ‘Nandu’ plants analysed, 51.1% were haploid and 44.3% were diploid, probably as a consequence of spontaneous chromosome doubling. When screening a further 16 commercial German varieties of spring wheat, 10 exhibited good anther culture response and four of these (‘Eta’‘Jondolar’, ‘Mieka’, and ‘Star’) proved to be highly responsive, reaching embryoid induction rates between 4.3 and 10.3% and rates of green plant regeneration between 5.4 and 10.7%.     

Rye doubled haploids as a research and breeding tool – a practical point of view

Doubled haploid (DH) plants were produced using anther culture from out‐crossing rye, including breeders’ lines, cultivars and F₁ plants with DH parents, to examine the feasibility of using the DH technique for breeding and specifically for developing mapping populations. Only 10–36% of green regenerants produced via anther culture were suitable for research or breeding purposes because of low survival rate or low fertility. Spontaneously arising DH regenerants were more often fertile compared with the colchicine‐treated ones. The fertility of spontaneous DHs varied from sterile to half that found in a normal rye population, which has implications for the design of a crossing scheme and subsequent anther culture. In the reciprocal crosses within one DH population, fertility was the lowest observed, probably because of self‐incompatibility factors, whereas in the DH crosses with normal heterozygous cultivars fertility was the highest. Two mapping populations using DHs were established, the first for out‐crossing rye it would seem. These populations will be used for mapping two important traits, the semi‐dwarf growth habit and preharvest sprouting resistance in rye.     

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.     

Potential use of doubled haploid lines for the screening of resistance to yellow rust (Puccinia striiformis) in hexaploid wheat

Doubled haploid lines derived from anther culture of two Iranian spring wheat genotypes‘Ghods’susceptible and‘9106’resistant to yellow rust in Iranian field conditions, and their F₁ hybrids were used in this study. Seedlings of 36 doubled haploid lines, selected out of 96 according to their agronomic traits and the two parental genotypes were inoculated with eight races of yellow rust. The parental genotypes (‘Ghods’and‘9106’) were segregating for some of the races but their doubled haploid lines were either resistant or susceptible to them.‘Ghods’was susceptible to three of the races studied but three doubled haploid lines derived from it were resistant to them. Five selected doubled haploids from the‘9106’genotype and six from F₁ hybrid plants were resistant to all eight races tested. After further investigations in Iranian field conditions it was found that some of these lines can be used as donor genotypes for resistance to yellow rust in wheat breeding programmes. Use of these genotypes should be possible if the French yellow rust races used for selection also represent the dominant races in Iran. It can be concluded that anther culture provides an efficient method for fixing genes of resistance to yellow rust and desirable doubled haploids from F₁ plants can be derived.     

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。     

Anther and isolated microspore culture response of wheat lines from northwestern and eastern Europe

Hexaploid wheat genotypes from north-western Europe show low responses to current anther culture techniques. This phenomenon was investigated on 145 north-western European wheat lines. Twenty-seven lines from eastern Europe were included to observe the response pattern of wheat from an area, where the technique has been used successfully. On average, eastern European wheat lines produced 3.6 green plants per 111 anthers, while only 1.4 green plants per 111 anthers were obtained in north-western European lines. This difference was due to the high capacity for embryo formation among the eastern European lines, while the ability to regenerate green plants was widespread in both germplasm groups. Isolated wheat microspore culture performed on 85 of these wheat lines gave an average 3.7-fold increase in green plants per anther compared with the anther culture response. The increased recovery of green plants was due to improved plant regeneration and increased green plant percentage from embryos derived from isolated microspore culture.     

Comparative analysis of in vitro anther- and isolated microspore culture in hexaploid Triticale (X Triticosecale Wittmack) for androgenic parameters

Two haploid induction media (190-0 and W14mi) were tested in isolated microspore culture of two triticale (X Triticosecale Wittmack) genotypes. The W14mi medium proved superior for the production of green plantlets in both genotypes. This basic medium (W14) was used to compare two doubled haploid production methods (isolated microspore culture and anther culture) with the same genotypes. The induction of androgenesis was more effective in isolated microspore culture than in anther culture. The number of embryo-like structures was 9.2 times higher in microspore culture (511.0/100 anthers) compared to anther culture (55.5/100 anthers) and the number of regenerant plantlets was also 3.4 times higher (anther culture—20.15/100 anthers; isolated microspore culture—67.6/100 anthers). However, the regenerant plantlets from isolated microspore culture were mainly albinos while predominantly green plantlets were regenerated from anther culture. The production of green plantlets from anther culture (16.8/100 anthers) was 2.9 times higher than from isolated microspore culture (5.8/100 anthers). The efficiency of anther culture was tested with eight winter triticale genotypes. The phenomenon of albinism did not hinder the green plant production in anther culture. Mean green plantlet production was 10.87/100 anthers. This value was two times higher than the number of albinos (5.01/100 anthers) and higher than previously published reports. The anther culture protocol described in this study is an efficient tool for the production of microspore-derived green plantlets in triticale.     

Enhanced anther culture efficiency of indica rice (Oryza sativa L.) through modification of the culture media

The production of microspore-derived green plants from anther culture of indica rice is generally very low compared with japonica cultivars. A modified anther culture medium, consisting of a higher KNO₃ content (31 mm ) and casein hydrolysate (CH, 500 mg/1) but without ammonium salts, was tested in comparison with a medium consisting of the widely-used N6 medium nitrogen background, using four indica × indica F₁ hybrids as test materials. Green plant regeneration frequency was at least three-fold higher in the microspore-calli derived from the former medium than in those derived from the modified N6 medium. More than 700 microspore-derived plants were raised in the field. Another study was carried out using indica × japonica and indica × javanica F₁ hybrids. The results indicated that a medium with higher (3.5 mm ) ammonium sulphate may induce a higher frequency of anthers with microspore-calli but not necessarily lead to a larger number of green-plant regenerating calli. Subsequently, using the indica cv. ‘IR-43’ as the test material, use of a lower level (1.75 mm ) of (NH₄)₂SO₄, in addition to KNO₃ (31 mm ), was found to be better than CH (500 mg/l) for anther-response as well as green plant regenerability of the derived microspore-calli. Nitrate-nitrogen or ammonium-nitrogen alone elicited poor response. Twenty-five media involving combinations of KNO₃ (20–34 mm ) and (NH₄)₂SO₄ (1–3 mm ) were tested for their effects on anther response. Combinations involving KNO₃ (31–34 mM) and (NH₄)₂SO₄ (2.0–2.5 mm ) were found superior not only for achieving greater anther response but also, for subsequent green-plant regeneration. This contrasts with the 28 mm of KNO₃ and 3.5 mm of (NH₄)₂SO₄ in the widely-used N6 medium developed for japonica rice. Other than potassium nitrate and ammonium sulphate, and potassium phosphate to some extent, the levels of other inorganic salts tested did not make any significant difference to the process of anther response. Based on these results, modified media with three levels of ammonium sulphate were tested for anther culture efficiency of indica × japonica and indica × javanica derivatives (F₃s). Microspore-calli derived from a medium of a lower (1.75 mm ) level of (NH₄)₂SO₄ showed a higher regeneration potential overall than those derived from a higher (3.5 mm ) level. A revised medium has been suggested, on the basis of these results, for the realization of improved anther culture efficiency and, consequently, improved feasibility of using doubled haploids in genetic and breeding research with indica rice.     

Segregation distortion for agronomic traits in doubled haploid lines of barley

Four barley doubled haploid populations were produced by anther culture from the reciprocal crosses between two six‐row barley cultivars, ‘Plaisant’ and ‘Orria’; the doubled haploid lines (DHLs) derived from each cross were subsequently assigned to weak or vigorous populations according to the weak or vigorous nature of the originating embryos. Well‐formed embryos at day 25 on the induction medium were considered vigorous, whereas embryos maturing later were considered weak. The classification of vigorous and weak was closely associated with the ratio of green to albino plantlets regenerated. A random set of 25 DHLs from each of the four populations were selected for field testing in a replicated trial. Furthermore, a second set consisting of a total of 454 unreplicated DHLs from the four populations were also field assessed for grain yield. Distortion during in vitro culture may impede regeneration of a random array of microspores from a given cross, and may bias genetic estimates of specific trait/marker association in genetic studies. However, no significant differences were detected in this study among the four populations for days to heading, height, grain yield and thousand‐kernel‐weight when measured on the replicated trial of 100 DHLs, nor for grain yield in the second collection of 454 entries. This suggests that the likelihood of producing improved agronomic pure lines is independent of the direction of crossing and, more importantly, independent of the time when embryos matured in the induction media, at least for these particular six‐row cultivars and for the anther culture method used.     

Androgenetic plants of Anemone coronaria derived through anther culture

The genus Anemone (Ranunculaceae) includes many species cultivated for ornamental purposes. Most cut flower cultivars belong to A. coronaria L. and are multiplied by seed and sold for cultivation as 1‐year‐old tubers. As cultivars represent a population of hybrid individuals derived from crosses between heterozygous parents, the use of a true F₁ hybrid would improve the uniformity and quality of the product. As a first step towards the development of pure‐breeding lines, anther cultures were established from elite cultivars of A. coronaria. Somatic embryos and plantlets were regenerated from five elite cultivars, and up to 16.9 regenerants per 100 cultured anthers were obtained. Cytological analysis identified that 11 of 19 regenerants had either a 2x = 16 karyotype, or were mixoploids. RAPD‐based DNA fingerprinting showed that all the regenerants tested differed genetically from their anther donor, confirming their androgenetic origin. The shortening to 15 months for the time required to produce homozygous lines may convince seed companies to invest in F₁ hybrid breeding.     

Phenylacetic acid stimulation of direct shoot formation in anther and somatic tissue cultures of rice (Oryza saliva L.)

The effect of phenylacetic acid (PAA) on rice (Oryza saliva L.) anther culture was investigated with six genotypes, using 2,4-D as control. In the two-step culture protocol, replacing 2, 4-D with PAA in the induction medium did not influence callus induction but significantly improved the shoot differentiation from callus, particularly in the indica cultivar Teqing. The anther-derived calli of all genotypes regenerated shoots directly on the callus induction medium containing PAA. Most of the directly-regenerated plantlets had well-developed root systems and were therefore readily transplanted into soil. The improved shoot differentiation potential and the frequency of direct regeneration depended on genotype, basal medium and PAA concentration. The one-step green shoot regeneration frequencies obtained were 1.98% with the indica cultivar ‘129’, 1.5% with the indica × japonica hybrid ‘Teqing/02428’ (F₁), and 1.98% with the indica × indica hybrid ‘Waiyin 2/C.B.’ (F₁). The PAA-based one-step method was most effective on the anther culture of indica genotypes. Three DH populations have been constructed from hybrids (F₁) via one-step culture. PAA also enhanced the one-step plantlet formation in rice somatic tissue culture.     

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.     

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.     

Characterization of anther culture-derived cell suspensions exclusively regenerating green plantlets in wheat (Triticum aestivum L.)

A reproducible procedure for deriving highly regenerable cell suspensions that can readily and consistently regenerate green plantlets in wheat is described. Initiation and selection of the right type of callus from anther cultures, which consisted of friable early embryogenic portions that can easily disperse in liquid medium was important for the establishment of rapidly growing embryogenic suspensions. Using this type of inoculum no significant variation between three different independent replications was noted when cell suspensions from eleven specially recombined doubled haploid lines were maintained on General medium supplemented with dicamba and a predominance of amino acid nitrogen. This approach also enhanced a long-term embryogenic competence of the cell cultures, with some of the suspensions retaining their morphogenic capacity over a period of more than 15 months. Depending on the medium composition high frequencies of embryogenesis (over 70%) and green plantlet regeneration (repeatedly producing 90–100% of green regenerants) were obtained from the cell aggregates for most of the embryogenic cell lines. Potential advantages of anther culture-derived embryogenic cell suspensions for transformation purposes are the high number of cell lines which can be established routinely and the apparent maintenance of a stable haploid genome by the regenerants in culture. It is anticipated that an increased use of anther or microspore derived doubled haploid techniques in future wheat breeding programmes may favour selection in the breeding material of plant types generally responsive to such protocols. This revised version was published online in July 2006 with corrections to the Cover Date.     

Selection in vitro for erucic-acid content in segregating populations of microspore-derived embryoids of Brassica napus

Microspore culture of Brassica napus under optimized conditions leads to the regeneration of microspore-derived embryoids that, at the late cotyledonary stage, contain large amounts of storage lipids, equal or similar in composition to those found in seeds of the homozygous donor plants. At that stage, the microspore-derived embryoids are large enough to allow the dissection of one cotyledon under aseptic conditions and the determination of its fatty-acid composition. The remaining part of the embryoid can be cultured further and regenerated to give a plant. This offers the possibility of early selection for fatty-acid composition in segregating populations of microspore-derived embryoids. In order to verify this hypothesis, embryoids were generated from microspores of F| plants derived from a cross between doubled haploid lines of the low-erucicacid cv. ‘Duplo’ and the high-erucic-acid cv. ‘Janetzki’. The contents of eicosenoic acid (C20: 1) and erucic acid (C22: 1) in the cotyledons and in the seeds derived from plants regenerated from the remaining parts of the embryoids were highly correlated (rs = 0.85**, P = 0.01). This indicates that, in breeding programmes for high erucic acid, the majority of the microspore—derived embryoids can be discarded at an early stage in vitro. Only microspore-derived embryoids with a high content of C20: 1+C22:1 in the cotyledons need to be transferred to the greenhouse. This report also deals with the addition of abscisic acid (ABA) to the embryoid culture medium to increase the correlation, and discusses the possible application of this system for the selection of high-oleic or low-linolenic types in corresponding microspore-derived embryoid populations.