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.     

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.     

小麦与玉米杂交产生小麦单倍体与双单倍体的稳定性

小麦与玉米杂交是诱导小麦单倍体最有效的途径之一, 但单倍体和双单倍体产生频率不稳定影响了该技术的应用。选用13个小麦杂种F₁代单交组合与玉米杂交, 研究了不同小麦生长环境、生长素处理、培养基和壮苗处理对单倍体及双单倍体产生频率的影响。小麦生长在大田, 去雄后割穗培养与玉米杂交平均得胚率为23.9%, 每个杂交穗平均得胚数6.8个, 均是返青后从大田移回冷温室盆栽的3倍以上;不同小麦杂交组合间胚产生频率存在明显差异。生长素Dicamba蘸穗处理平均得胚率是21.5%, 与2,4-D处理得胚率(21.1%)无显著差异, 但不同杂交组合间差异显著。B5培养基幼胚萌发率为70.9%~88.3%, 平均82.0%;1/2 MS培养基胚萌发率为70.0%~86.0%, 平均76.6%;两种培养基平均胚萌发率无显著差异。试管苗经壮苗培养基壮苗处理与试管苗经移栽壮苗处理后加倍效率分别是67.6%和8.6%。移栽壮苗处理的苗分蘖少, 生长较弱, 加倍处理后存活率低和加倍率低是其单倍体加倍效率低的原因。     

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.     

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     

The effect of colchicine on triticale anther-derived plants: Microspore pre-treatment and haploid-plant treatment using a hydroponic recovery system

In cereals, chromosome doubling of microspore-derived haploid plants is a critical step in producing doubled haploid plants. This investigation was undertaken to study the effect of incorporation of colchicine in the induction medium for anther culture, and the effect of colchicine on anther culture-derived plants of triticale grown under controlled greenhouse conditions. In the latter case, chromosome doubling of adult sterile plants derived from anther culture of fourteen triticale populations was attempted, where androgenetic plants with non-dehiscent anthers were cloned and subjected to the colchicine treatment, and then grown with the aid of hydroponics. The hydroponic system provided optimal conditions for recovery of the affected haploids from the toxic effects of colchicine treatment and all colchicine-treated plants survived. A topcross-F₁ (TC₁F₁) population with timopheevii cytoplasm produced the highest percentage of plants with seed-set either due to chromosome doubling by colchicine (98%) or spontaneous doubling of chromosome number (15%). Colchicine-treated anthers performed inferior than control in both induction and regeneration phases. One of the key observation of this study was the reversal from reproductive stage back to the vegetative stage which in turn enabled further cloning of haploid plants under hydroponic conditions once they were identified as sterile. The one hundred percent survival rate of in vitro-derived plants, 100% survival rate of colchicine treated haploid plants and the high chromosome doubling success rate (X = 82.3) observed in this study imply that a temperature-controlled greenhouse with an hydroponic system provides an efficient environment for inducing chromosome doubling of haploid plants in cereals. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Doubled haploid production in winter wheat and triticale genotypes,using the Hordeum bulbosum system

Summary An attempt was made to produce doubled haploids on 16 winter wheat and six spring and winter triticale genotypes thought to carry genes for interspecific incompatibility. The potential for haploid production was maximized by the use of Hordeum bulbosum genotypes selected for high crossability on crossable wheat genotypes, the use of two post-pollination applications of gibberellic acid and by the pollination of immature florets.A low frequency of seed was set on both the wheat and the triticale genotypes, having mean seed sets of 0.20 per cent and 0.27 per cent respectively. Although the frequency of embryos (seed quality) was high, doubled haploid production was further limited by poor embryo differentiation and regeneration. Haploid plantlets were obtained from the wheat cultivars Moulin and Renard, although successful chromosome doubling and doubled haploid production was achieved in Moulin only.     

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.     

Dicamba and growth condition effects on doubled haploid production in durum wheat crossed with maize

Doubled haploid plants are useful in genetic studies and plant breeding, but a consistent and satisfactory frequency of production has been difficult to achieve in durum wheat. Triticum turgidum L., using the maize pollen method. The objective of this study was to develop an objective method of producing doubled haploids in durum wheat. Plant growing and handling conditions, aspects of hormone treatments, wheat genotype and pollen source were considered. The number of caryopses, embryos, haploids, doubled plants and doubled plants that set seed were measured. Although growth conditions, pollen source, method of handling plants and wheat genotype are important considerations, the type of hormone was found to be most significant in the production of doubled haploid plants. When 50mg/l dicamba was substituted for 100 mg/l 2,4‐D the number of doubled haploids per spike increased from 0.2 for the best 2,4‐D treatment to 1.3 for the dicamba treatment. This increased frequency was largely attributed to an increase in the number of caryopses generated for each spike emasculated and from an increased frequency of germination of embryos to haploid plantlets. The best production of caryopses was 0.41 caryopses per florest with 2,4‐D. The best production of haploids per 100 florets was 12 with dicamba and 1.65 with 2,4‐D. The frequency of one doubled haploid per emasculated spike through the use of dicamba is a practical level for generating populations for genetic studies.     

Improved Techniques for Haploid Production in Wheat using Chromosome Elimination

Wheat × maize and wheat × pearl millet crosses have proved efficient for haploid production using various genotypes of wheat; 22 and 27 % of florets produced embryos. In favourable conditions 6—9 haploid plants per spike were produced. The following simplifications or improvements in technique are recommended: 1. Only a single treatment with an aqueous solution of dicamba or 2,4-D (50–100 ppm) for embryo stimulation in vivo; 2. Application by spraying or dipping the spikes; 3. Application time two to four days after pollination; 4. Embryo rescue 15 to 18 days after pollination; 5. Crosses without emasculation are possible if pollination occurs 1–2 days before anthesis. More than 450 haploids and some doubled haploid (DH) lines (after colchicine treatment in vitro) were produced using these methods. No hybrid plants, chromosome additions or substitutions were found.     

Review of doubled haploid production in durum and common wheat through wheat × maize hybridization

Production of doubled haploids (DHs) is an important methodology to speed the process of breeding and development of mapping populations in crops. The procedure for DH production includes two major steps: haploid induction and chromosome doubling. In recent years, wide hybridization between wheat and maize has become a main approach for haploid production in wheat. In this method, the maize chromosomes are completely eliminated during the early development of the hybrid seeds after wheat spikes were pollinated with maize pollen. Numerous wheat cultivars and mapping populations have been developed using wheat–maize hybridization. In this study, we review the procedures of DH production of durum and common wheat via wide hybridization with maize, the factors which affect the efficiency of DH production, and the mechanism of selective elimination of the maize genome during the early development of the hybrid embryos. We also report a highly efficient protocol for DH production in durum and common wheat, which was established based on the optimal conditions for each of the factors that affect the efficiency of DH production.     

Genetic characterization of spontaneous diploid androgenetic wheat and triticale plants

The homozygosity of spontaneous hexaploid plants derived from anther culture was evaluated in wheat and triticale by means of 18 and 22 microsatellite markers, respectively. Most of the spontaneous hexaploid plants were homozygous for all the loci tested and had chromosomes recombining for parental alleles. Only 12% of the spontaneous hexaploid wheat plants, 11% of the artificially doubled wheat plants and 4.4% of the spontaneous hexaploid triticale plants were heterozygous at one to three of the loci studied. This showed that spontaneous hexaploid plants mostly came from normal haploid cells, obtained after meiosis, that underwent spontaneous chromosome doubling. However, first or second division restitution, producing unreduced gametes, cannot be completely excluded to explain the origin of the spontaneous hexaploid plants. Cytomixis, involving only one chromosome, or development of aneuploid trisomic gametes, followed by chromosome doubling for the spontaneous hexaploid plants, or transposition events could also explain some of the results obtained.     

Segregation for isozymes and HMW glutenins in a doubled-haploid cross of winter wheat carrying 1BL.1RS and 5DL.5RS translocations from rye

The doubled haploid (DH) wheat line ‘dh 5841’ carrying two translocations from rye, 5DL.5RS and 1BL.1RS, has been crossed to the subline of wheat cultivar ‘Amadeus 7143’ with a 1BL.1RS translocation. The resulting F₁ hybrid IJ 98 with a heterozygous 5DL.5DS‐5DL.5RS chromosome pair has been used to produce doubled haploids. A total of 57 DH lines were obtained from plantlets regenerated in anther culture after successful colchicine treatment and seed set. These lines were identified regarding the constitution of chromosome 5D (5DL.5DS or 5DL.5RS) by means of isoenzyme marker analysis. Thirty DH lines possessed the 5DL.5DS chromosome, while the remaining 27 lines carried the 5DL.5RS translocation. For some of these lines, the 5DL.5RS chromosome was cytologically confirmed by C‐banding. Furthermore, the DH lines were evaluated for their high molecular weight glutenin subunit composition. All possible combinations for the four independent loci —Skdh, Glu‐Al, Glu‐B1 and Glu‐D1— were detected in only 57 DH lines and no segregation distortion was observed.     

Doubled haploid production in Turkish durum wheats using crosses with maize

Durum wheats cultivated in the Middle Anatolia region of Turkey (Triticum durum cvs ‘Kunduru’, ‘Berkmen’ and ‘Cakmak’) were crossed with maize to evaluate their capacity for haploid embryo and doubled haploid (DH) plant production. A total of 2960 florets were crossed with maize and 13.7% of the florets produced haploid embryos across the three varieties. Haploid embryo and plant regeneration frequencies were highest in ‘Kunduru’, but colchicine doubling was less successful with this genotype. There were statistically significant differences between ‘Cakmak’ and other genotypes for embryo yield (P < 0.01). Overall, 52.3% of all embryos differentiated, but there were no geno-typic differences in differentiation frequencies.     

Rapid introduction of disease resistance from rye into common wheat by anther culture of a 6x triticale × nulli-tetrasomic wheat

Powdery mildew (caused by Erysiphe graminis) and yellow rust (caused by Puccinia striiformis) are the two most serious wheat diseases found in China. Rye chromosomes, carrying genes for resistance to these diseases, were introduced into common wheat in two generations using chromosome engineering and anther culture. The F₁ hybrids from a cross involving a hexaploid triticale (×Triticosecale Wittmack) בChinese Spring’ nulli‐tetrasomic N6DT6A wheat aneuploid line were anther cultured and doubled‐haploid plants were regenerated. Using genomic in situ hybridization, C‐banding and biochemical marker analyses, one of the anther‐cultured lines (ZH‐1)studied in detail, proved to be a doubled‐haploid with one rye chromosome pair added (1R) and a homozygous 6R/6D substitution (2n= 44). The line was tested for expression of disease resistance and found to be highly resistant to powdery mildew and moderately resistant to yellow rust.     

开发实用的染色体加倍体系构建成烟草DH群体

通过对4种染色体加倍方法(烟草浸花药法、浸花培苗法,叶片再生法及浸腋芽法等)的比较研究表明：以4g／L浓度的秋水仙碱浸泡花药法加倍率最高达47．5％～75％,其次为叶片再生法(30％～36．67％)和浸苗法(16．67％～32．35％),而浸腋芽法较低(17．78％)。前两种方法加倍率虽高,但有较高畸形苗比率,叶片再生法工序较繁琐。作者认为烟草加倍单倍体产生,应以采用浸苗法为主。在使用秋水仙碱浸苗加倍时,添加DMSO可明显促进秋水仙碱的加倍效率,且促进作用随时间延长而提高：从高效、快捷和节约等原则考虑,我们开发了烟草有效的染色体加倍体系,以4g／L的秋水仙碱 20g／LDMSO溶液浸苗48h的效果最好。对两个组合烟草单倍体苗浸12h以上,其染色体加倍效率达到对照的2．30-2．93倍。本试验用较低浓度秋水仙碱添加DMSO有利于节约实验费用。通过完善染色体加倍技术程序已构建成2个DH群体,供基因定位研究。     

Spontaneous and induced chromosome doubling in gynogenic lines of onion (Allium cepa L.)

The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) and 6-benzyladenine (6—BA) in the medium is required to induce an acceptable yield of gynogenic embryos from unfertilized ovary and flower cultures in onion. Four different exposure times of ovary and flower cultures to exogenous growth regulators (15, 30, and 45 days, and the entire culture period) were assayed. The objective was to ascertain the effect of these substances and of their period of application on the formation of gynogenic embryos and on the yield of haploids. An exposure of 15 days was sufficient for ovaries and flowers to be stimulated towards the gynogenic response, whereas, for the remaining period of 30—80 days, the pro-embryos could easily grow on a growth-regulator-free medium. In the gynogenic material obtained, phenotypic differences were visible between genetically independent lines but not between plants within each line, even when they had a different ploidy level (n or 2n). Almost all lines obtained by gynogenesis were sterile. Only a small percentage (1%) became fertile through spontaneous chromosome doubling, and these produced 2—20 seeds each, giving normal plants. The recovery of fertility occurred more often during the generations of bulbils. To exploit this natural propensity towards diploidization in this phase, different amounts and numbers of applications of colchicine were evaluated in two experiments. The treatments corresponding to 10 and 100 mg/1 of colchicine applied for 24h gave the highest number of diploid cells in root tips but no diploidization occurred in the shoot apices. Three days of colchicine treatment at 10 mg/1 produced 46% of plants completely diploid in the shoot apex. The flower fertility of these doubled haploid plants is being evaluated.     

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.     

芦笋单倍体染色体加倍技术研究

研究了芦笋单倍体染色体加倍的方法。在离体培养条件下,以秋水仙素为诱变剂,分别用浸泡法和培养基添加法处理芦笋单倍体幼苗的茎尖,比较了秋水仙素不同浓度、不同处理时间的诱导效果。结果表明,培养基添加法的诱导效果好于浸泡法,当在培养基中添加0.3%秋水仙素并处理7天时诱导效果最佳,染色体加倍频率与成活率分别可达82.50%和80%。加倍后的二倍体植株与单倍体植株相比,茎干变粗,气孔与保卫细胞增大,保卫细胞内的叶绿体数增多。