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1.
Efficient production of doubled haploid wheat plants by in vitro treatment of microspores with trifluralin or APM 总被引：4，自引：0，他引：4
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 相似文献
2.
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. 相似文献
3.
Hybrids of wheat (Triticum durum) × rye (Secale montanum) (2n = 21) were treated with a 0.06 % solution of colchicine. In the induction of fertile plants, fertile spikes, and seed-set per spike, the root immersion technique was 21.7 %, 14.1 %, and 4.4 % respectively more effective than the use of the inverted vial. Colchicine solutions ranging in pH from 3.5 to 7.5 with the root immersion technique reached the highest frequencies of fertile (or partially fertile) hybrids (77.7 %), and fertile spikes (68.9 %) at a pH 5.5 used for the treatment, although the highest overall percentage of large Q seeds resulted from treatments at low pH (3.5). The overall mean frequency of induced fertile plants was six-fold higher when treated in a growth chamber (19/15 °C day/night temperature and 18 h photoperiod) compared to treatments conducted in a greenhouse. The frequency of euploidy (2n = 42) was found to be highest in the large seed class and lowest, i.e. just about 50 %, in the class of small seeds. 相似文献
4.
The use of doubled haploids improves the efficiency of cultivar development in many crops and can be helpful in genetic and molecular studies. The major problem with this approach is the low efficiency of green plant regeneration. We describe here an efficient method for inducing embryos and regenerating green plants directly from isolated microspores of durum wheat cv. ‘Jennah Khetifa’. Tillers from donor plants were pretreated in 0.3 m mannitol and were stored at 4°C at various times: 3, 5, 6, 7, 8, 10 and 12 days. Our results showed clearly that the novel pretreatment combined mannitol 0.3 m and cold for 7 days had a strong effect on the number of embryos produced and regenerated green plants. Under this condition 13 475 mature embryos were produced from 2 693 500 microspores. Moreover, 85 green plants were obtained. High green plants regeneration frequency was recorded. As an average 11.55 green plants were produced per 100 000 microspores (about the equivalent of six plants per spike). Therefore, this study showed clearly that our results are the best ones published until now in durum wheat. 相似文献
5.
RAPD analysis of sporting and chimerism in chrysanthemum 总被引：12，自引：0，他引：12
Kirsten Wolff 《Euphytica》1996,88(2):159-164
Summary The potential of colchicine and the microtubule depolymerizing herbicides trifluralin, oryzalin, and amiprophosmethyl (APM) for in vitro chromosome doubling during B. napus microspore culture was studied. Colchicine was administered during the first 6, 12 or 24 h of culture with 8 different concentrations up to 3 mM, and herbicides at 6 different concentrations up to 30 M for 12 h.Treatments with moderate concentrations of colchicine (3–100 M) produced a small increase in embryo production, while concentrations above 300 M were toxic. Colchicine treatment for 12 h resulted in higher embryo production than treatment for 6 and 24 h. Duration of treatment and concentration of colchicine both had a significant effect on the chromosome doubling. The highest diploidization rates (94% diploid regenerants) were seen after 24 h treatment with 1 mM colchicine.All three herbicides were similar to colchicine in terms of their effect on embryo formation and chromosome doubling comparable to the one of colchicine, but at concentrations approximately 100 times lower. APM was less toxic than trifluralin and oryzalin, but no significant difference in chromosome doubling efficiency was detected between the compounds. The 12 h treatment resulted in a maximum of approximately 65% diploid regenerants with all three herbicides, but APM may have an advantage because of its less toxic effects. Prolonged treatment with APM (20–24 h) may produce 95–100% diploid regenerants.Abbreviations APM
amiprophos methyl
- DMSO
dimethyl sulfoxide 相似文献
6.
Induced parthenogenesis as a possible method of haploidization in sunflower (H. annuus L.) was tested in previous investigations
(Todorova et al., 1994) and conditions for reproducible regeneration of gynogenic doubled haploids were established by the
present work. Forty eight treatments were studied, involving four pollen donors and four recipient hybrids. Pollen was irradiated
with doses of 300 Gy, 600 Gy and 900 Gy. In total, 2279 embryos were cultivated in vitro of which 1107 plants were obtained
and 582 of them produced seeds after selfing. The ploidy level of the regenerants was evaluated at the two – three leaf stage
and 296 of the plantlets obtained were haploids. Some of them underwent spontaneous diploidization, the others were treated
with colchicine solution for chromosome doubling. The diploid plantlets were checked for their gynogenic origin by genetical
and biochemical methods. The effectiveness of the method expressed as the number of agronomically useful DH lines to the number
obtained that were fertile and resistant to downy mildew, branched and unbranched plants was 8,6% on average.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
7.
Efficient production of doubled haploid Brassica napus plants by colchicine treatment of microspores 总被引：7，自引：0，他引：7
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. 相似文献
8.
For the efficient application of haploid induction procedures in cabbage breeding, a sufficient number of regenerants should be achieved in a broad spectrum of genotypes. However, the majority of genotypes are somewhat recalcitrant. The efficiency of microspore culture was tested by crossing a responsive (28.7 embryos per Petri dish) and a non- responsive (0.1 embryo) cabbage cultivar. The embryo yield of one progeny was intermediate (18.9) while two were superior to the best parent cultivar (52.9 and 64.0 embryos). Thus, genes for haploid embryogenesis, present in responsive lines, can be effectively transmitted to responsive × non-responsive hybrids. Abscisic acid-induced desiccation of embryos was used for the efficient regeneration of plants. High germination percentages (54.7-70.6%) followed by normal plantlet development were achieved. Spontaneous genome doubling measured at the plantlet stage differed markedly in untreated genotypes. The percentage of diploids ranged from 21 to 67%. The effects of two antimitotic drugs applied to freshly isolated microspores were determined in two experiments. In the first experiment, trifluralin (0.5 and 1.0 mg:l) had no effect on embryo induction while oryzalin partly (0.125-0.25mg/l) or completely (0.5.mg/l) inhibited the formation of embryos. In the second experiment, higher concentrations of trifluralin increased the proportion of diploidized plants. Application of anti-mitotic drugs to microspores did generally not improve the overall production of haploid plants, which was higher in an untreated control. 相似文献
9.
Efficient Production of Doubled Haploid Plants through Chromosome Doubling of Isolated Microspores in Brassica napus 总被引：12，自引：0，他引：12
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. 相似文献
10.
The effect of colchicine on induction of embryogenesis andchromosome doubling during microspore culture was evaluated in twoF1 hybrids of spring oilseed rape (Brassica napus L.). Immediatecolchicine treatment of isolated microspores with the concentrations 50 and500 mg/L for 15 h stimulated embryogenesis and produced largeamounts of healthy-looking embryos. These normal embryos germinatedwell at 24 °C after being transferred to solid regeneration mediumand an initial period of low temperature (2 °C) for 10 days, andcould directly and rapidly regenerate vigorous plants. A high doublingefficiency of 83–91% was obtained from 500 mg/L colchicinetreatment for 15 h with low frequency of polyploid and chimeric plants.The present experiment showed that a treatment duration of 30 h revealedless positive effects on embryogenesis and doubling efficiency, especially athigher colchicine concentration (1000 mg/L). Poor embryogenesis andembryo germination were observed from ordinary microspore culturewithout change of induction medium and colchicine treatment, and severalsubcultures were required for induction of secondary embryogenesis andplant regeneration. 相似文献
11.
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. 相似文献
12.
Two chromosome doubling strategies were evaluated for producing wheat doubled haploids from wheat x maize crosses: (i) in vitro colchicine application to haploid embryos and (ii) colchicine treatment through postpollination tiller injections. In the in vitro approach the haploid embryos were rescued on medium containing colchicine (at concentrations of 0.2, 0.3, 0.4 and 0.5%) and moved to a colchicine‐free regeneration medium 48 h later. Embryos exposed to 0.5% colchicine had 91.67% of their regenerated plants showing chromosome doubling. In the tiller injection approach, different concentrations (0.5, 0.75 and 1.0%) of colchicine solution, which also contained 2,4‐D (100 ppm), were injected into the uppermost inter‐node of crossed tillers 48 and 72 h after pollination. The chromosome doubling efficiency varied from 33 to 100%, with 1% treatment being the most effective. No chimeras of doubled/haploid sectors were observed in the case of the tiller injection treatment and all the florets showed seed set in the doubled plants. Stomatal guard cell length provided rapid, early‐stage and unambiguous analysis of ploidy level on the basis of 10 guard cell observations per plant. 相似文献
13.
剪秋罗种子催芽及秋水仙碱诱导多倍体研究 总被引：1，自引：0，他引：1
为了解决剪秋罗种子发芽率不高的问题,以剪秋罗种子为试验材料,比较种子新旧、播种基质以及不同浓度赤霉素(GA3)对剪秋罗种子发芽势和发芽率的影响,并在此基础上利用不同浓度秋水仙碱和不同时间处理剪秋罗萌动种子诱导多倍体。通过形态鉴定、气孔鉴定以及细胞学鉴定变异植株。结果表明：存放1年的剪秋罗种子发芽指标均优于当年新种子,当年种子可能具有初生休眠特性。以150 mg/L的GA3预处理存放1年的剪秋罗种子24 h并播种于培养皿中能获得最高的发芽势(76.33%)和最高的发芽率(85.33%)。经过秋水仙碱处理的种子普遍存活率低,但每个处理浓度下都获得了变异植株,秋水仙碱浓度0.1%为筛选出的最适诱导浓度。变异植株表现出了与对照植株在形态上、气孔分析上和细胞学检测上的差异。经鉴定,初选出1株多倍体。研究为培育剪秋罗多倍体新品种探索出了一条新的育种途径。 相似文献
14.
Regeneration of fertile doubled haploid plants from colchicine-supplemented media in wheat anther culture 总被引：6，自引：0，他引：6
I. Zamani G. Kovács E. Gouli-Vavdinoudi D. G. Roupakias B. Barnabás 《Plant Breeding》2000,119(6):461-465
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. 相似文献
15.
Components of Response in Barley Anther Culture 总被引：2，自引：0，他引：2
Anther culture response with 17 widely-grown varieties and one model variety of barley was studied with one replication from field-grown donor plants and one replication from a growth-chamber. Plants were regenerated from all 18 varieties and green plants were obtained from 16 of them. On average, 1.6 green plants were obtained per 100 cultured anthers from all the material. Estimated variance components for the formation of embryos/callus from the anthers were dominated by the effects of the genotypes and interactions between plant material and environments which together accounted for 60.1 and 17.0 % of the total variation respectively, while environments were nonsignificant for this character. Plant regeneration from embryos/callus were not significantly influenced by either genotype or environments. Components of variance for green plant formation were dominated by the effects of the genotypes, accounting for 73.2 % of the total variation, and a smaller effect from environments accounting for 11.2% of the total variation. Main effects from genotypes on the percentage of green regenerants divided 7 varieties into two distinct groups, indicating that major genetic factors were involved. The genetic basis for green plant regeneration seems different from that governing embryo formation. The results are discussed with respect to the possible prediction of anther culture response for new barley hybrids, as a means for directing the use of barley anther culture towards material that responds well. 相似文献
16.
S. Tuvesson A. Ljungberg N. Johansson † K.E. Karlsson L. W. Suijs J.P. Josset 《Plant Breeding》2000,119(6):455-459
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. 相似文献
17.
The effect of in vitro colchicine treatment of sugar beet ovules, after 7 days culture, on embryo formation, regeneration and ploidy of regenerated plants was studied with 5 concentrations of colchicine and 5 durations of treatment arranged as a 5 × 5 factorial in incomplete blocks. The best results were obtained with the shortest duration of treatment (5 hours) and the highest concentration of colchicine (0.4 %) giving 5.0 diploid plants per 100 ovules with 62.1 % of regenerated plants being diploid. Statistical analysis revealed that treatment effects could be separated into a toxic effect reducing embryo formation and a chromosome doubling effect affecting percentages of diploid regenerated plants. Toxic effects on embryo formation could be explained by simple exponential decay models, toxicity of the drug (decay constant) increasing linearly with duration of treatment. Duration of treatment had no effect on chromosome doubling percentages. The effects of colchicine concentration on chromosome doubling were explained by an exponential saturation model with spontaneous chromosome doubling of 8.1 % and saturation at 51.4 % diploid plants at 0.2 % and higher colchicine concentrations. In addition, treatments increased percentages of 4N and 6N plants from 0 % without colchicine to 10 % on average for treated ovules. A response surface model fitted to the total yield of diploid plants per ovule indicated that shorter durations of treatment and higher colchicine concentration may improve results. 相似文献
18.
Doubled haploids (DH) have become a standard tool in breeding and genetic studies of many crops and in most cases androgenesis is the only available route of their production. It has been recently observed that some populations of DH lines obtained via androgenesis contain high proportions of clones. This seriously reduces the efficiency of breeding and may jeopardize genetic studies. This study was designed to determine at which stage of androgenesis these clones are created, using samples set aside during routine production of DH lines in breeding of hexaploid triticale. The fate of each androgenic structure was carefully followed through the entire regeneration process, and all obtained plants were genotyped using DNA markers. Overall, 189 plants were regenerated forming 33 families, each originating from a single original androgenic structure (callus, polyembryos). In ca. 80 % of cases all members of a family were genetically identical. However, in about 20 % of cases the families of regenerants were genetically heterogeneous, showing that not all androgenic structures originate from single microspores. The evidence shown here demonstrates that retention of single plants from each original structure guarantees the production of only unique genotypes but it reduces the total output of plants. If maximum output is desired, multiple regenerants from single callus can be retained but must be genotyped using at least 10 polymorphic markers to identify clones. 相似文献
19.
Effect of parental genotypes and colchicine treatment on the androgenic response of wheat F1 hybrids
I. Zamani E. Gouli-Vavdinoudi G. Kovacs I. Xynias D. Roupakias B. Barnabas 《Plant Breeding》2003,122(4):314-317
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. 相似文献
20.
Short-duration colchicine treatment for in vitro chromosome doubling during ovule culture of Beta vulgaris L. 总被引：1，自引：0，他引：1
Colchicine uptake into ovules of sugar beet after 7 days of culture and its chromosome-doubling effect on ovule-derived plants were studied with high colchicine concentrations (0.4–6.0%) and short treatment duration (0–5 h). The best result of 4.2 diploid plants per 100 ovules was produced by treatment with 0.4% colchicine for 2.5 h. Both colchicine concentration and treatment time of ovules showed toxic effects on embryo formation, but it was stabilized at a low level with short exposure. The chromosome-doubling effect, by contrast, was unchanged with the colchicine concentrations used, but highly affected by the duration of exposure studied. A maximum percentage of 60% diploid plants was obtained after 3–5 h of uptake, which corresponds to only 31–39% of the total capacity for colchicine uptake in the ovules. Further uptake of the drug produced mainly toxic effects. Flow-cytometric measurements of the ploidy level in plantlets in vitro and of the same plants before flowering in soil were similar in about 80% of cases. Thus, flow-cytometric selection of diploid plants in vitro may be an efficient tool. 相似文献