Increasing embryogenesis and doubling efficiency by immediate colchicine treatment of isolated microspores in spring Brassica napus

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.     

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.     

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.     

In vitro induction of tetraploid plants from colchicine-treated diploid daylily callus

Summary Daylily plantlets with tetraploid or octaploid chromosome numbers were induced from colchicine-treated diploid callus (2n=22). Callus tissue of the daylily Hemerocallis flava L. was placed on a morphogenetic induction medium, a modified MS formula supplemented with 1 mg 2,4-D and 1 mg kinetin/liter, plus 0,10, 20, or 40 mg/liter colchicine in the dark at 12°C for 3 days and recuperated for 1 week under the same environmental conditions except that colchicine was left out. The calluses were then returned to the normal growth temperature 25°C. Ploidy was identified by chromosome counts of squashed root-tip cells collected from the newly potted plantlets and later by measurements of stomates and pollen grains. Over 50% of the plants initiated from the colchicine-treated calluses were completely tetraploid. All of the plantlets differentiated from untreated callus had a diploid number of 22. Of the 3 colchicine levels applied, the 20 mg/liter treatment appeared to be the most effective in production of tetraploidy.     

In vitro induction of tetraploids in Phlox subulata L.

Tetraploid plants of Phlox subulata L. were induced successfully by treating shoot tips in vitro with colchicine. Shoot tips excised from in vitro shoots were treated with four different concentrations of colchicine (0.005, 0.01, 0.02, 0.04%) in solid MS medium supplemented with 4.54 μM TDZ and 0.49 μM IBA for 10, 20 or 30 days, respectively. The survival rates of shoots tips were affected by the concentration of colchicine and the duration of treatment. High concentration and longer duration reduced survival of the shoot tips, but the effect of duration of colchicine was more than that of concentration. Tetraploid plants were obtained in all of the treatments, but the percentages of tetraploids varied among different treatments, from 25.0% to 75.0%. The most efficient condition for inducing tetraploids was to treat shoot tips with 0.005% colchicine for 20 days, with 30.0% survival rate of shoot tips and 6 tetraploid plants out of 10 plants examined. The rooted tetraploid plants were transplanted successfully in a solar greenhouse. Under the same growing condition, significant varieties in flower bud and flower sizes were detected between 2x and 4x plants. The flower diameters of tetraploid and diploid plants were 2.91 cm and 2.24 cm, respectively.     

In vitro tetraploid induction via colchicine treatment from diploid somatic embryos in grapevine (Vitis vinifera L.)

A protocol for in vitro induction of tetraploids via colchicine-treated somatic embryos from immature zygotic embryos of diploid grapevine (Vitis vinifera L.) is reported. Embryogenic callus was initiated from immature zygotic embryos cultured on Nitsch and Nitsch (NN) medium supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The callus was transferred to NN medium containing 1.0 mg/l α-naphthalene acetic acid (NAA) and 0.5 mg/l benzyladenine (BA) to establish somatic embryogenesis. The vigorously growing globular embryos were selected and treated by 0, 10 or 20 mg/l colchicine for 1, 2 or 3 days, and then immediately transferred to NN medium supplemented with 0.03 mg/l NAA and 0.5 mg/l BA, for somatic embryo conversion and plant regeneration. The number of surviving embryos and regenerated plantlets following colchicine treatment decreased with increasing colchicine concentration and treatment time. Among 29 randomly investigated plantlets regenerated from colchicine-treated somatic embryos, five solid tetraploids (2n = 4× = 76) were identified by chromosome counting analysis; all others were diploid (2n = 2× = 38). Ploidy level of plant regenerated was also determined from leaves using flow cytometry. No chimeras with both 2C and 4C nuclei was produced from colchicine-treated somatic embryos. Significant differences in leaf stomata parameters were observed between diploid and induced tetraploid plantlets.     

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.     

Chromosome doubling of Allium fistulosum × A. cepa interspecific F1 hybrids through colchicine treatment of regenerating callus

Regenerating calluses of Allium fistulosum × A. cepa interspecific F₁ hybrids were treated in vitro with colchicine. A factorial experiment was designed to test the effects of colchicine concentration and time on the recovery of tetraploid plants from in vitro-colchicine-treated calluses. Shoot production of regenerating calluses following in vitro colchicine treatment decreased with increasing colchicine concentration and treatment time. Cytological analyses of root tip cells from regenerated plantlets showed that chromosomes of control plantlets (not treated with colchicine) were not doubled. Chromosome number of some plantlets regenerated from in vitro-colchicine-treated calluses were doubled, resulting in tetraploids. Calluses treated with 0.1 or 0.2% colchicine in BDS liquid medium for 48 or 72 hours yielded the highest numbers of tetraploid plantlets. Chromosome bridges at anaphase or early telophase were observed in diploid and tetraploid plants; their potential use is discussed. These results demonstrate that in vitro-colchicine treatment of regenerating calluses of interspecific F₁ hybrids is effective in recovering tetraploid plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicine in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses

Summary We cultured colchicine-treated hybrid ovules in vitro to produce fertile amphidiploids of C. persicum (2n=2x=48. referred to as AA) × C. purpurascens (2n=2x=34, referred to as BB). Seedlings and mature plants were obtained from the ovules without colchicine and those exposed to 50 mg/l colchicine for 5, 10 and 15 days, whereas they were not obtained from the ovules exposed to 50 mg/l colchicine for 20 days and 500 mg/l for 5, 10, 15 and 20 days. Although 8 mature hybrids derived from the ovules without colchicine produced a few fertile pollen grains, they failed to produce viable seeds by self-fertilization. The hybrids had 41 somatic chromosomes. Four and 3 mature plants were derived from ovules exposed to 50 mg/l colchicine for 10 and 15 days, respectively. One each among 4 and 3 mature plants showed a high frequency of pollen grain fertility, produced several seeds by self-fertilization, and had 82 somatic chromosomes which is twice the number of hybrid chromosomes (2n=41, AB). These findings indicated that these plants are amphidiploids (2n=82, AABB) between C. persicum and C. purpurascens. Three and 2 viable seeds were derived by the conventional crosses of diploid C. persicum × the amphidiploid and the amphidiploid × C. purpurascens, respectively. Flowering plants that developed from the seeds of diploid C. persicum × the amphidiploid were barely fertile and had 65 somatic chromosomes (2n=65, AAB), whereas those that developed from the seeds of the amphidiploid × C. purpurascens were barely fertile and had 58 somatic chromosomes (2n=58, ABB). The somatic chromosomes indicated that these plants are probably sesquidiploids between the amphidiploid and either C. persicum or C. purpurascens. The interspecific cross-breeding of cyclamen using the amphidiploids and the sesquidiploids is discussed.     

Resynthesizing <Emphasis Type="Italic">Brassica napus</Emphasis> from interspecific hybridization between <Emphasis Type="Italic">Brassica rapa</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis> through ovary culture

Using three varieties of Brassica rapa, cv. Hauarad (accession 708), cv. Maoshan-3 (714) and cv. Youbai (715), as the maternal plants and one variety of B. oleracea cv. Jingfeng-1 (6012) as the paternal plant, crosses were made to produce interspecific hybrids through ovary culture techniques. A better response of seed formation was observed when ovaries were cultured in vitro at 9–12 days after pollination on the basal MS and B5 media supplemented with 6-benzylaminopurine (BA) and naphthylacetic acid (NAA). The best response was observed for cross 714×6012 with the rate of seeds per ovary reaching 43.0%. Seeds for cross 715×6012 showed the best germination response (66.7%) on the regeneration medium (MS+1.0 mg l^–1 BA+0.05 mg l^–1 NAA). In all three cross combinations, good response in terms of root number and length of plants was observed on the root induction medium (MS+1.0 mg l^–1 BA+0.1 mg l^–1 NAA). A better response was observed for the regenerated plants cultured for 14 days than for 7 days. The ovary-derived plants with well-developed root system were hardened for 8 days and their survival rate reached over 80%. Cytological studies showed that the chromosome number of all plants tested was 19 (the sum of both parents), indicating that these regenerated plants were all true hybrids of B. rapa (n = 10) × B. oleracea (n = 9). The regenerated plants were doubled with colchicine treatment, and the best response in the crosses 708×6012, 714×6012 and 715×6012 was observed when treated with 170 mg l^–1 colchicine for up to 30 h and their doubling frequency reached 52, 56 and 62%, respectively.     

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.     

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.     

Anther culture response of F1 durum × bread wheat hybrids after colchicine application

The effect of colchicine on the androgenic response of durum × bread wheat F₁ hybrids was studied. For this, three Greek durum wheat cultivars, exhibiting good yield and pasta quality, were crossed to two bread wheat cultivars with good response to anther culture. Spikes of the resulting hybrids, containing microspores in the mid to late uninucleate stage, were selected and cultured on two different media (W₁₄ and solid potato‐2) with and without colchicine. A negative effect on anther culture response was observed when colchicine was added to the W₁₄ induction medium. A similar effect was observed in two of the hybrids when they were cultured on to potato‐2 induction medium. In both induction media, green plant production was influenced negatively by colchicine treatment. The same was observed in albino plant production. The results of the present study support the view that anther culture response is strongly genotype dependent. Finally, potato‐2 induction medium was the most suitable one for the material studied.     

Short-duration colchicine treatment for in vitro chromosome doubling during ovule culture of Beta vulgaris L.

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.     

Single and combined effects of colchicine, L-proline and post-inoculation low temperature on anther culture of wheat, Triticum aestivum L.

This investigation was undertaken to study the effects of colchicine, L-proline and a post-inoculation cold temperature of 14°C for 7 days, either as a single or as combined treatments, on the parameters of anther culture of the DH83Z118.32 wheat genotype. Results indicate that the addition of 100 mg/1 colchicine to the induction medium for a period of 3 days and at an incubation temperature of 28°C yielded the highest success index. This treatment, although it significantly reduced embryogenesis, improved embryoid quality so that not only the total regeneration, but also the percentage of green plantlets increased significantly, In addition, it raised the percentage of doubled haploids and, consequently, led to a significantly better final success index. Combining the colchicine treatment with a reduced post-inoculation temperature did not intensify its beneficial effect, although a treatment of reduced temperature alone was superior to normal temperature (28°C) for most parameters studied. The beneficial effects of adding L-proline (200 mg/1) to the induction medium, combined with a low temperature (10°C for 4 days) treatment, were diminished when this treatment was combined with a colchicine treatment.     

Mutation breeding of Streptocarpus

Summary Leaves of Streptocarpus Constant Nymph have been irradiated with X-rays and fast neutrons. Considering the production of plantlets on the rooted leaf-halves as well as the frequency of mutants among them, the optimal dose for X-rays was found to be 3 krad.Like Saintpaulia ionantha, Streptocarpus produces solid mutants after irradiation, a fact which indicates that each plantlet, developing from an adventitious bud, originates from only one cell. Moreover, a high mutation frequency has been observed as well as a wide variability among the 857 mutants obtained.Application of a heavy X-ray dose (4–5 krad) a few days after colchicine treatment resulted in a frequency of tetraploids which was twice the one obtained after colchicine treatment alone.A number of mutants and (auto)tetraploids have been propagated and tested for their commercial value. Sofar, 5 mutants have been commercialized, viz. Purple Nymph, Mini Nymph, Blue Nymph, Netta Nymph and Cobalt Nymph.     

Improved doubled haploid production protocol for Brassica napus using microspore colchicine treatment in vitro and ploidy determination by flow cytometry

A rapid and efficient microspore culture protocol was applied to produce homozygous progeny of crosses between low erucic canola and high erucic resynthesized rapeseed (Brassica napus L.). Microspores of Canadian cultivars ‘Excel’ and ‘Profit’ as well as three F₁ hybrids with the resynthetic line ‘RS239’ were treated with colchicine immediately after isolation. Flow cytometry was applied for early identification of doubled haploid (DH) regenerants. The diploidization rate was subsequently verified by scoring flower morphology. In vitro colchicine treatment had a positive effect on induced diploidization, and was associated with the frequency of preliminary spontaneous diploidization which was, however, determined by the genotype. In addition, the effects of colchicine treatment on embryoid formation and regeneration have been evaluated. The method presented is feasible for commercial large‐scale production of DHs in rapeseed as the genotype‐specific diploidization can be efficiently balanced by in vitro colchicine treatment. In addition, the use of flow cytometry immediately after in vitro culture allows efficient selection for DHs, thus saving labour and cost and in the laboratory and subsequent greenhouse phase.     

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.     

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。     

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.