RAPD analysis of sporting and chimerism in chrysanthemum

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     

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.     

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.     

Effective Diploidization of Microspore-Derived Haploids of Rape (Brassica napus L.) by In Vitro Colchicine Treatment

A technique for the in vitro colchicine treatment of microspore-derived haploids of rape (Brassica napus L.) is presented. This allows effective and time-saving chromosome doubling. The most effective method consists of an application of 50 mg/1 colchicine in a liquid B5 medium to the rooted micro-spore-derived plantlets for 4–8 days. By this treatment more than 50 % of the plants developed at least diploid sectors. A change of medium after some days may even increase this diploidization rate.     

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.     

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.     

A comparison of traditional and haploid-derived breeding populations of oilseed rape (Brassica napus L.) for fatty acid composition of the seed oil

Summary Microspore embryogenesis technology allows plant breeders to efficiently generate homozygous micros-pore-derived breeding populations of oilseed rape (Brassica napus L.) without traditional generations of inbreeding. This study was conducted to compare the frequency distribution of microspore-derived population and single seed descent populations with respect to fatty acids of seed oil. Both microspore-derived populations and single seed descent populations were produced from each of three crosses made between selected parents containing contrasting amount of erucic, oleic, linoleic and linolenic acids. The fatty acid content of F₃ plants derived lines (F₅ seed) developed by single seed descent was compared to that of microspore-derived populations. The means, ranges and distribution pattern of seed fatty acid contents were similar in both populations for each fatty acid studied, although a few heterozygous lines were observed in the single seed descent populations. The results indicated that microspore-derived population form random, homozygous F₁ plant derived gametic arrays for all fatty acids evaluated. Selection for altered fatty acid composition in microspore-derived and single seed descent homozygous populations should be equally efficient, in the absence of linkage of traits investigated.     

RAPD markers linked to microspore embryogenic ability in Brassica crops

In order to identify the markers linked to microspore embryogenic ability in Brassica crops, RAPD segregation analyses were performed in a microspore-derived (MD) population and a F₂ population derived from F₁between ‘Ho Mei’ (high responsive parent in microspore embryogenesis) and ‘269’ (low responsive parent) in Chinese cabbage, and between ‘Lisandra’ (high responsive parent) and ‘Kamikita’ (low responsive parent) in oil seed rape. After 230 and 143 primers were screened, a total of 148 and 52markers were detected to be polymorphic between the parents in Chinese cabbage and oilseed rape, respectively. Twenty-seven percent of the markers in the MD population showed a significant segregation distortion in both crops. Of the markers showing segregation distortion in the MD population, 71–75% of the markers followed the expected Mendelian segregation ratio in the F₂ population. When the relationships between such distorted markers and microspore embryogenesis of the F₂ population were examined, 7 and 3 markers were identified to be associated with embryogenic ability in Chinese cabbage and oilseed rape, respectively. These markers showed additive effects on embryo yields, and the plants having more alleles of the high responsive parent produced higher embryo yields. These markers maybe useful in marker-assisted selection for improving microspore responsiveness straits in Brassica crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

Intergeneric hybrids and an amphiploid between Elymus canadensis and Psathyrostachys juncea

Summary Fifty four hybrid plants between Elymus canadensis and Psathyrostachys juncea were obtained by handpollination and embryo culture. The average cross compatibility between both species was 31.2 percent. One amphiploid plant was induced by colchicine treatment. The hybrid and amphiploid plants resembled P. juncea in appearance but showed a higher plant height and dry matter yield than the parents. The hybrids showed extremely low pollen stainability and were completely sterile. With the exception of one plant (2n=3x+1=22), all hybrid plants were allotriploids (SHN, 2n=3x=21). The amphiploid plant (SSHHNN, 2n=6x=42) showed 58.9% pollen stainability and 11.6% seed fertility.Mean chromosome associations of the hybrids and amphiploid at metaphase I were 0.02IV+0.06III+2.03II+16.91I and 0.07III+18.00II+5.85I, respectively. Lagging chromosomes, chromosome bridges, abnormal cytokinesis, and micronuclei were occasionally observed at the anaphase, telophase, or tetrad stage.     

The production and behaviour of hexaploid hybrids between Hordeum vulgare and H. Bulbosum

Summary To produce hexaploid (or other polyploid) hybrids, diploid or tetraploid Hordeum vulgare was crossed with hexaploid or octoploid H. bulbosum, and perennial triploid hybrids between the two species were treated with colchicine. The crosses did not yield viable plants: seedset was low, the seed aborted and embryo culture was unsuccessful. The colchicine treatments geve rise to plants in which hexaploid chromosome numbers were observed. At the hexaploid level chromosomal instability occurred, resulting in chromosome elimination.The colchicine-treated triploid hybrids showed in the first years after the treatment better fertility after open flowering than untreated plants, but the level of fertility remained very low. The offspring consisted of haploid, diploid and approximately triploid plants like H. vulgare, tetraploid and approximately tetraploid plants like H. bulbosum, and plants with hybrid morphology and unstable chromosome number, which were highly sterile. Thus the crossing barrier between H. vulgare and H. bulbosum could not be broken down at higher ploidy level.     

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.     

Efficient in vitro Chromosome Doubling During Beta vulgaris Ovule Culture

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.     

Improved techniques for the induction and isolation of polyploids in the genus Fragaria

Summary The sensitivity of Fragaria seedlings to colchicine is dependent on the plant organ that is treated. Complete immersion of seedlings in a 1.5% colchicine solution results in total lethality, whereas the survival rates were more than 75% even at concentrations of 3.0% when only shoot apices were treated. High proportions of polyploids were isolated by treating shoot apices of seedlings with a 2.0% colchicine solution for 24–28 h. The dropper method is preferred to the tube method as it involves a minimum of manipulation and requires simple equipment. A differential response to colchicine was observed within and between different diploid species, diploid and tetraploid hybrids.     

Evaluation of microspore-derived embryos as models for studying lipid biosynthesis in seed of rapessed (Brassica napus L.)

Summary Microspore culture of rapeseed (Brassica napus L.) has provided a powerful tool not only for breeding but also in developmental studies. In this study, microspore-derived embryos (MDE) of B. napus were evaluated as a model in seed for studying accumulations of triacylglyceride (TAG) fatty acids in both a low and high erucic acid rapeseed line; and accumulations of TAG and free fatty acids (FFA) in a high erucic acid rapessed line. The accumulation patterns confirmed that MDE had a similar TAG fatty acid profile to seed during the embryo development within each genotype. The oil accumulation in MDE after 36 days in culture (DIC) approached levels similar to those in zygotic seed 25 days after flowering (DAF). Significant differences were detected in contents of both total free fatty acids and specific free fatty acids between MDE and seed. During the developmental period, total free fatty acids changed from 16% to 2.1% in MDE, but from 10.5% to 0.1% in seed. MDE had much higher percentage of free linolenic and erucic acids than seed, particularly during the late developmental stages. The current study indicated that MDE can be used as a model to study TAG and TAG fatty acids in seed but caution must be taken to study free fatty acid metabolism.     

The assessment of in vitro characters and their influence on the success rates of doubled haploid production in barley

Summary A assessment of in vitro characters: embryo size, differentiation stage and germination time of haploid embryos, was carried out with a view to developing a selection procedure to increase the efficiency of doubled haploid (DH) production using the Hordeum bulbosum system. The differentiation stage of haploid embryos at the time of excision was found to be the most promising character for selection. In the two spring crosses examined 55% of excised embryos were undifferentiated and 45% were differentiated, with overall success rates, in terms of the percentage of DHs produced, of 7.91% and 25.16%, respectively. 17% of excised embryos produced callus. These embryos performed poorly in terms of regeneration and subsequent growth. It is suggested that only differentiated non-callusing embryos are maintained in order to maximize the efficiency of DH production. It is also shown that the overall success rate depends not only on initial stages such as seed setting but also on genotypic differences in response to colchicine treatment at later stages.     

Polyploidization and early screening of Rhododendron hybrids

Polyploid induction represents a useful tool for breeders of floral crops as larger flowers, longer flowering period and deeper colors can be achieved through chromosome doubling. This study aimed at testing the efficiency of colchicine and oryzalin in inducing polyploidy in three Rhododendroncultivars grown in vitro. The chemicals were used in two concentrations with 24 h and 48 h treatment durations. The survival of the plants was better in colchicine than in oryzalin solutions. The higher concentration of both chemical skilled more plantlets. The treatment duration in oryzalin did not affect the survival, but 48 h in colchicine was more destructive than 24 h. The low survival rate may not be a disadvantage, if the treatment induces desired ploidy. The ploidy levels were screened with flow cytometry. Oryzalin was more efficient than cochicine in inducing polyploidy, the treatment duration and the concentration did not have significant effects as main factors. The biggest proportion of solid tetraploids (18.2% of the survived plants) was obtained from the 24 h treatment in 0.005% oryzalin. Immediately after the treatment the polyploids grew very slowly, whereas most of the unaffected diploids were vigorous from the very beginning. More mixoploids than solid tetraploids were obtained in all treatments. Most of the mixoploids retained their chimerism, one third shifted todiploidy and one single plant to tetraploidy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosome numbers of Saintpaulia ionantha plantlets regenerated from leaves cultured in vitro with caffeine and colchicine

Summary Parts of Saintpaulia ionantha leaves were cultured on a medium supplemented with different concentrations of colchicine or caffeine. The explants were transferred to a control medium without these chemicals after different periods of time. The chromosome number of the regenerated plantlets was counted. In all the experiments, polyploid plantlets were found, but the percentage of them was higher with colchicine. There were no differences in frequency of polyploid plantlets among treatments with the same chemical. In both cases a high percentage of cytochimeric plantlets was observed and possible explanations of their occurrence are given.     

Aneuploid Analysis of Anther Culture Response in Wheat

Genetic stocks of Triticum aestivum including the disomic, 8 ditelosomic and 3 nullisomic-tetrasomic ‘Chinese Spring’ wheat lines were employed to ascertain the chromosomal arm locations of the genes acting on the production of embryos from micro-spores and on the regeneration capacity (green and albina) of the microspore-derived embryos. All these aneuploid lines differed significantly from the parental line ‘Chinese Spring’ for embryo production. Our results confirmed or in most cases established that genes affecting embryo production are located on several chromosomal arms: IBS, 1BL, 3AS, 3AL, 5AS, SAL, 5BS, 5BL, 7DS, 7DL. Whereas most of the chromosomal arms stimulate the production of embryos from the microspores, IBS and 1BL reduce it. The results of plant production from microspore-derived embryos suggest that the genes increasing regeneration ability are located on CS5A chromosome and are likely associated to a gene increasing green plant frequency. On the contrary, the 1BL arm increases the albina frequency.     

High frequency spontaneous production of doubled haploid plants in microspore cultures of Brassica rapa ssp. chinensis

Brassica rapa (syn. Brassica campestris) ssp. chinensis is an important vegetable crop, but it is relatively recalcitrant to microspore culture. One genotype each of B. rapa ssp. chinensis var. communisand var. utilis were used formicrospore culture. Embryo production of3.8–42.4 embryos/bud was obtained. A high rate of plant regeneration directly from microspore-derived embryos without subculture was achieved by an improved protocol involving replacement of culture media and reduction of sucrose concentrations after 48 h of induction,among other modifications. More than 70%of regenerated plants were spontaneous diploids. Some spontaneous tetraploid plants were also obtained from isolated microspores of both genotypes tested. These tetraploids may be directly exploited a snew varieties in a Brassica rapabreeding programme. This revised version was published online in July 2006 with corrections to the Cover Date.     

Introgression of Cajanus platycarpus genome into cultivated pigeonpea, C. cajan

Summary Cajanus platycarpus, an incompatible wild species from the tertiary gene pool of pigeonpea (C. cajan (L.) Millspaugh), has many desirable characteristics for the improvement of cultivated varieties. To necessitate such transfers, embryo rescue techniques were used to obtain F₁ hybrids. The F₁ hybrids were treated with colchicine to obtain tetraploid hybrids, that were selfed to obtain F₂, F₃ and F₄ progenies. All of the hybrids and subsequent progenies had an intermediate morphology between the two parents. Backcrossing of the tetraploid hybrids with cultivated pigeonpea was not possible given embryo abortion, with smaller aborted embryos than those obtained in the F₀ parental cross.As a route of introgression, diploid F₁ hybrids were backcrossed with cultivated pigeonpea and BC₁ progeny obtained by in vitro culture of aborting embryos. BC₂ plants were obtained by normal, mature seed germination. Although embryo rescue techniques had to be used to obtain F₁ and BC₁ plants, it was possible to produce BC₂ and subsequent generations through direct mature seed. Every backcross to cultivated pigeonpea increased pollen fertility and the formation of mature seeds.Special project assistant till December, 2003.