Relative Efficiency of Anther Culture and Chromosome Elimination Techniques for Haploid Induction in Triticale × Wheat and Triticale × Triticale Hybrids

Summary The study was undertaken to evaluate the relative efficiency of anther culture and chromosome elimination (by crosses with maize) techniques of haploid induction in intergenotypic triticale and triticale × wheat hybrids. For this, 15 triticale × wheat and 8 triticale × triticale F₁ hybrids were subjected to anther culture and were also simultaneously crossed with the `Madgran Local' genotype of maize (Zea mays L.) to induce haploids through the chromosome elimination technique. The haploid embryo formation frequency through the chromosome elimination technique was significantly higher in both, triticale × wheat (20.4%) and triticale × triticale (17.0%) F₁ genotypes, as compared to the calli induction frequencies through anther culture (1.6 and 1.4%, respectively). Further, four triticale × wheat and three triticale × triticale F₁ genotypes failed to respond to anther culture, whereas, all the F₁ genotypes formed sufficient number of haploid embryos through the chromosome elimination technique with no recovery of albino plantlets. The haploid plantlet regeneration frequencies were also significantly higher through the latter technique in both triticale × wheat (42.7%) and triticale × triticale (49.4%) F₁s as compared to anther culture (8.2 and 4.0%, respectively), where the efficiency was drastically reduced by several constraints like, high genotypic specificity, low regeneration frequency and albinism. The overall success rates of obtaining doubled haploids per 100 pollinated florets/anthers cultured were also significantly higher through the chromosome elimination technique (1.1% in triticale × wheat and 1.5% in triticale × triticale hybrids), proving it to be a highly efficient and economically more viable technique of haploid induction as compared to anther culture, where the success rates were only 0.2% and 0.1%, respectively.     

Androgenetic response of heterozygous triticale populations using a greenhouse hydroponic system

Production of haploids, followed by chromosome doubling to produce doubled haploids (DH) represents the most rapid means of achieving complete inbreeding. In order to improve the androgenetic responses and maximize the production of green regenerants from selected plants of twenty-five triticale (× Triticosecale, Wittmack) populations (BC₁F₁, TC₁F₁, and F₂) we used a uniform and optimal growth environment for anther donor plants within a greenhouse hydroponic system. Non-orthogonal analysis of deviance showed highly significant differences (p < 0.001) among populations for both induction and regeneration. The overall induction response of the populations was very high with a mean of 50.4 embryoids per 100 anthers plated. Among all tested-populations, M86-6068/TW179//EP80 (TC₁F₁) was the most responsive for both induction and regeneration which could be associated to its Triticum timopheevii cytoplasm. On the other hand, although populations 80465/II83-194 both as BC₁F₁ and F₂ had a high level of induction response, only a few green plants were regenerated. These populations probably possess a partial Secale montanum genome, which could be contributing to the low regeneration ability. In conclusion, the need for optimization of donor plant growth conditions to effectively assess the androgenetic ability of individual populations/lines would be emphasized. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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%.     

Die Nutzung der Antherenkulturmethode im Zuchtprozeß von Winterweizen

Anther culture in the breeding process of winter wheat. III. Ability of winter wheat F₁ populations with the two heterozygous 1AL–IAS/1AL–IRS and 1BL–1BS/1BL–IRS chromosome pairs Application of anther culture to four F₁ hybrids between the IBL–IRS (‘Amigo’) and several 1BL–IRS wheat-rye translocation forms yielded 129 green pollen plants in an average embryo induction frequency of 17.6 %. A total of 2632 anthers was inoculated. 25 % and 42 % of the regenerated plants were haploid and spontaneously doubled haploid, and 33 % had abnormal chromosomal structure. After chromosome doubling treatment 87% of all pollen plants set seeds. By means of multiple peroxidases and Giemsa C-banding patterns, the anther culture progeny could be further classified into 16 plants without the short arm of IR-chromosome of rye, 21 IAL–IRS and 50 1BL–IRS translocation lines and into 16 IAL–IRS, IBL–IRS double translocation lines according to the four possible characteristic types of F₂ gametes of the tested F₁ hybrids. Advantages of the haploid technique for the selection of desirable traits and the meaning of the IRS genes in wheat are discussed.     

Production of Doubled Haploids by Anther Culture and Wheat X Maize Method in a Wheat Breeding Programme

Utilization of the doubled haploid method of breeding usually shortens the time to cultivar release, and methods of haploid production need evaluation in a breeding programme. Thirty-eight different three-way crosses were tested for anther culture response. On average 5.8 percent of the anthers cultured produced calli. Three crosses were found recalcitrant for callus induction. Overall, the anther culture method produced 0.6 plantlet per 100 anthers cultured. Five crosses with an average of 5.8 and 2.8 percent of anthers producing calli and plantlets, respectively, were compared using anther culture and wheat × maize crosses. Non-responsive genotypes for callus induction and plantlet formation in the anther culture method proved to be good parental material in wheat × maize crosses. The average percentages of embryo formation and plantlet production in wheat × maize crosses were 10.3 and 4.7, respectively. Anther-derived plants were cytologically unstable, whereas all the plants regenerated from wheat × maize crosses were haploids (n = 21 chromosomes). The chromosome numbers of the polyhaploids were doubled with a colchicine treatment. Improvement of the two haploid production methods to facilitate their efficient use in a breeding programme is discussed.     

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.     

Die Nutzung der Antherenkulturmethode im Zuchtprozeß von Winterweizen

Anther culture in the breeding process of winter wheat. I. Ability of 1B—-1R wheat-rye translocation forms for androgenesis 45 winter wheat varieties or F₁ hybrids, F₂ populations and lines with 1B—1R wheat-rye translocation were tested for their anther culture ability. A total of 48058 anthers was cultured on Potato-2 medium. When averaged over all genotypes and the two experimental years frequencies of embryoid formation of 5.4—6.8 per 100 anthers were observed. Plant regeneration efficiency from embryoids ranged from 5.3—9.1 % or a mean of 4—5 green plants per 1000 anthers plated. The results confirmed the preferential regeneration frequency of gametes with the 1BL—1RS chromosome compared to the gametes with the 1BL—IBS chromosome. Multiple peroxidase were used as marker. The effect of cold pretreatment or of media on the androgenetic response and productivity was not important. On the contrary the variability between the anther response from single ears of the same genotype was noticeable. Examples are presented for the transferability of the androgenetic ability to breeding material. Most green plants obtained were haploid or spontaneous doubled haploid. By cloning it was guaranteed, that progenies were obtained from most of the haploids after colchicine treatment.     

Assessment of different anther culture approaches to produce doubled haploids in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Cucumber is one of the most important vegetable crops worldwide, which makes it a good candidate to produce doubled haploid (DH) lines to accelerate plant breeding. Traditionally, these approaches involved induction of gynogenesis or parthenogenesis with irradiated pollen, which carries some disadvantages compared to androgenesis. Despite this, studies on anther/microspore cultures in cucumber are surprisingly scarce. Furthermore, most of them failed to unambiguously demonstrate the haploid origin of the individuals obtained. In this work we focused on anther cultures using two cucumber genotypes, different previously published protocols for anther culture, different in vitro culture variants to make it more efficient, and most importantly, a combination of flow cytometry and microsatellite molecular markers to evaluate the real androgenic potential and the impact of anther wall tissue proliferation. We developed a method to produce DH plants involving a bud pretreatment at 4 °C, a 35 °C treatment to anthers, culture with BAP and 2,4-D, and induction of callus morphogenesis by an additional 35 °C treatment and sequential culture first in liquid medium in darkness and second in solid medium with light. We also found that factors such as genotype, proliferation of anther wall tissues, orientation of anthers in the culture medium and growth regulator composition of the initial anther culture medium have a remarkable impact. Our rate of chromosome doubling (81%) was high enough to exclude additional chromosome doubling steps. Together, our results present androgenesis as an improvable but yet more convenient alternative to traditional gynogenesis and parthenogenesis-based approaches.     

Oryzalin as an Efficient Agent for Chromosome Doubling of Haploid Apple Shoots in vitro

In an outbreeding species such as apple, haploid plants may be especially useful in breeding programmes for the production of homozygous material. However, methods must be available to induce chromosome doubling in the haploid plants. Two antimitotic agents, colchicine and oryzalin, were compared as regards their efficiency in inducing chromosome doubling of in vitro haploid apple shoots. Three colchicine levels (0.025, 0.25 and 1.25 mM) and three oryzalin levels (5, 15 and 30 μM) were evaluated. Three techniques were also used and compared. Survival rate and chromosome counts were determined. Differences were observed between the two antimitotic agents and between the three techniques. This study demonstrates that oryzalin could be a better choice than colchicine for chromosome doubling on haploid apple shoots in vitro.     

Large-scale production of wheat and triticale double haploids through the use of a single-anther culture method

A total of 257 parental wheat and 38 triticale lines were used for anther culture. On average, 2.1 green wheat haploids were obtained per spike. This response occurred irrespective of the origin of the material (Germany, France, Sweden or UK) and 5 years of testing. Triticale responded with 5.3 green haploids per spike. Using the criterion that one parental line should give at least one green haploid per spike in the screening experiment, green haploids were produced from 88 out of 91 F₁ wheat breeding combinations and from each of 21 F₁ and F₂ triticale breeding combinations. An average of 4.7 green plants were obtained per spike from the wheat production programme, while the triticale programme gave an average of 6.2 green plants per spike. A single medium supplemented with different hormones for anthers and embryos was used for culture of both crops.     

基因型和环境条件对小麦花药培养效果的影响

为进一步提高小麦花培育种效率,明确花药培养力的遗传控制基础,以11个小麦品种及其组配的20个F₁杂种为材料,探讨了基因型、培养基和环境条件对愈伤组织诱导率的影响。在W14D、W14gD、W14GD培养基上,Alondra、Verry、石4185、新春9号和百农3217的花药易被诱导产生愈伤组织,诱导率为25.3%~51.9%,其中石4185是目前公认的花培育种优良亲本,新春9号为新发现的优良花培基因型。以宁春4号配制的部分F₁杂种的愈伤组织诱导率较高,大多数组合高于10.0%,表明宁春4号与供试品种间具有较高的花药培养配合力。小麦花培育种技术要求亲本之一具有较高的花药愈伤组织诱导率或较高的花药培养配合力。小麦花药培养力的遗传控制复杂,表现为数量性状遗传,亲本花药培养力很高,其F₁组合花药培养力不一定很高,这与双亲配合力有关。小麦花药培养中,供体植株生长和愈伤组织诱导的适宜条件为较长的营养生长期、适宜的前期(分蘖期)温度和较高的中期(拔节后期)温度。在添加低浓度生长素和葡萄糖的液体培养基中发现小麦花药直接成苗现象,2,4-D诱导花药直接成苗效果优于Dicamba。随着年度间气候升高的影响,相同基因型花药愈伤组织诱导率呈现增加趋势。     

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.     

The production of homozygous diploid plants of Solanum verrucosum by tissue culture techniques

Summary Monohaploid plants of S. verrucosum (2n=x=12) were induced in anther culture. Axillary buds from these plants were treated with colchicine in shoot tip culture for 48 hours and then transferred to a colchicine free medium. The resulting plantlets were scored for diploidy by stomatal chloroplast counts and root tip cytology and it was found that doubling of the chromosome number had occurred.     

由染色体重组制造小麦族的染色体组——Ⅱ.筛选高花培效率的单倍体小黑麦无性系

用花药培养选择具有高诱导率(a)和高分化率(b)的高花培效率(a×b)的八倍体小黑麦(2n=8x=56)和单倍体小黑麦(n=4x=28)是很有效的。八倍体小黑麦的花培效率同普通小麦一样,每百个花药可出2.5株绿苗。由八倍体小黑麦(A 类)花培所得的花粉植株(B 类)再进行一次花培,在所获得的 C 类植株中就能选出花培效率达到八倍体小黑麦水平的     

Addition of Colchicine to Wheat Anther Culture Media to Increase Doubled Haploid Plant Production

Chromosome doubling is critical for obtaining doubled-haploid plants from wheat (Triticum aestivum L.) anther culture. The most common doubling method applies colchicine to the plant. However, colchicine is phytotoxic and can induce a high frequency of plant death. In this experiment, anthers from two wheat genotypes (“Pavon 76” and ‘Centurk’) were placed on nine embryoid initiation media having three sugar sources (maltose, sucrose, and maltose + glucose) with three colchicine concentrations (0.0, 0.1, and 0.2 g · l^-1). Wheat starch was used as a gelling agent. After three days, the anthers were washed and moved to fresh media without colchicine. Increasing the colchicine concentration decreased the number of embryoids produced from 77.4 embryoids/100 anthers to 29.9 embryoids/100 anthers, but did not significantly affect the frequency of plant regeneration (0.49 green plants/embryoid to 0.40 green plants/embryoid), and increased the frequency of doubled-haploid plants (19.0 doubled-haploid plants/100 green plants to 72.3 doubled-haploid plants/100 green plants). Considering the total number of doubled-haploid plants produced, low levels of colchicine added to the initiation media were very effective.     

Chromosome doubling of androgenic haploid plantlets of rice (Oryza sativa) using antimitotic compounds

The regeneration of haploid plantlets is considered as a bottleneck in rice anther culture. In this study, an antimitotic chromosome doubling method, simple and efficient, of androgenic haploid plantlets resulted in an efficient doubled haploid obtainment. Through chromosome doubling capacity comparison of the three antimitotic compounds (colchicine, trifluralin and oryzalin), colchicine at 500 and 625 mg/L without supplementing with DMSO was found to be the best antimitotic treatment, with a chromosome doubling capacity of 40%. Furthermore, the in vitro growth of plantlets was followed to analyse the effects of antimitotic compounds. Colchicine treatments were more toxic than dinitroanilines, and colchicine DMSO-supplemented treatments had significant lower values on shoot growth. On the other hand, dinitroaniline compounds impeded root growth, provoked helical growth of shoot and caused the apparition of white nodules in the base of the plantlet due to sprouting abortion. In this study, a protocol for doubled haploid plant recovery was established taking advantage from androgenic haploid plantlets in order to increase the number of doubled haploid plantlets produced after an anther culture protocol.     

Early spontaneous diploidization of maternal maize haploids generated by in vivo haploid induction

The production of doubled haploid (DH) lines has become a key technology in maize (Zea mays L.) research and breeding. However, most of the haploid plants are sterile and in many cases artificial chromosome doubling involves the use of costly and toxic chemicals. Here, we report a special kind of doubled haploid named the early doubled haploid (EH) that was generated directly by in vivo haploid induction. We found 83 EH plants induced from the hybrid Zhengdan958, 55 families of its F_2:3 population and the parental lines, all of which were confirmed to be homozygous diploids via flow cytometry and 104 SSR markers. The progeny of EH₀ (EH₁) behaved in the same manner and showed the same potentialities as the parents of Zheng58 and Chang7-2. EH plants were also detected in other genetic backgrounds at a frequency of 1–3.5 % based on the total number of haploid plants. Because the EH lines exhibited completely fertility and were obtained from induction directly in one step, they could be used in DH breeding as a new breeding strategy. According to our observations, it is likely that spontaneous doubling in EH occurred during embryo development when haploid induction. The possible mechanism of EH is also discussed.     

Effect of genotype,environment and carbohydrate on anther culture response in head cabbage (Brassica oleracea L. convar. capitata (L.) Alef.)

Summary The effect of genotype, growing conditions for donor plants and type and concentration of carbohydrate in the culture medium was investigated for anther culture of head cabbage (white cabbage, savoy cabbage, pointed-headed cabbage). Strong genotypic effects on embryo formation from the cultured anthers were shown as well as superior embryo formation from anthers of field grown donor plants compared to plants grown in the greenhouse. When comparing 7, 10 and 13% sucrose in the medium, embryo response increased with increasing sucrose concentration. With maltose, which was generally inferior to sucrose as carbohydrate source for anther culture, the embryo response did not increase with maltose concentration above 10 per cent.     

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.