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.     

Chromosome doubling of pear haploid plants and homozygosity assessment using isozyme and microsatellite markers

The improvement of perennial fruit trees through traditional breedingmethods is a long-term effort because of their long generation time. Theproduction of haploid and doubled haploid plants should offer newpossibilities for genetic studies and breeding work. In this study, haploidclones of pear were treated in vitro by oryzalin for chromosomedoubling; the level of ploidy was assessed by flow cytometry. Oryzalinappeared to be an efficient agent for chromosome doubling, the optimalconcentrations range from 200 to 300 M. For homozygosityassessment, analyses of isozyme markers were carried out, together withmicrosatellite markers PCR-amplified with primers initially developed forapple. The use of isozyme markers confirmed homozygosity of all thedoubled haploid clones except for one. The microsatellite markers can beused earlier than isozymes for checking homozygosity during theprogramme of haploid and doubled haploid clones production. Truedoubled haploid clones of pear were obtained in less than one year andtheir acclimatisation in greenhouse has already started.     

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.     

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.     

Influence of timing of cross-versus self-pollination on self-incompatibility in Brussels sprout inbreds

Summary The impact of cross pollination 30 h, 4 h, and immediately prior to self-pollination, and self-pollination 2, 4, 8, 12, 24 and 30 h prior to cross-pollination was assessed for pollen tube number per style, seed number per siliqua, and proportion of self-seed (sibs) per siliqua, in two inbred lines of brussels sprout. Pollination procedure had a marked effect on the amount of sib-and hybrid seed produced. Cross pollination 30 h prior to self-pollination produced significantly greater numbers of sib progeny, as did self-pollination 8–12 h before cross pollination. Prior application of self pollen reduces the number of pollen tubes in the style. The results of this experiment suggest that self-incompatibility in brussels sprout could better be assessed by self-pollination with a subsequent cross pollination 8 h later, and the determination of amounts of sib and F₁ hybrid seed per siliqua.     

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.     

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.     

Increased atmospheric humidity post pollination: A possible aid to the production of inbred line seed from mature flowers in the Brussels sprout (Brassica oleracea var. Gemmifera)

Summary In two highly self incompatible inbred lines of Brussels sprouts the effect of increased atmospheric humidity post pollination was examined immediately following 1) hand pollination of green buds and open flowers, and 2) blowfly pollination of open flowers. Data were obtained for mean number of seeds set per pollination, mean number of fruits setting seed, and mean number of seeds produced per fruit which set for both varieties. Measured as number of seeds produced per minute spent pollinating, it was clear that open flower pollination followed by high humidity conditions was a much more efficient method of producing inbred line seed (46 seeds/minute) than green bud pollination (27 seeds/minute).     

Chromosome doubling in Tripsacum: the production of artificial, sexual tetraploid plants

A collection of embryogenic diploid calli of Tripsacum was established and treated with colchicine to induce chromosome doubling. Sections containing duplicated cells in calli were identified using flow cytometry and ploidy level was determined in the regenerated plantlets. Tetraploid plants from several origins were obtained. In contrast to wild polyploid plants, which show apomictic development, the regenerated tetraploid plants reproduced sexually. By hybridizing these plants with wild tetraploid apomicts, various populations were established; these will allow a study of the inheritance of apomixis in Tripsacum.     

Chromosome doubling of haploid maize seedlings using nitrous oxide gas at the flower primordial stage

In maize, inbred lines are used for the production of hybrid varieties. Corn breeders and researchers have considered using haploids to develop inbred lines; however, this procedure has not been practically applied because of the inefficiency of chromosome doubling of maize haploid seedlings. In this report, a procedure has been developed to overcome this difficulty. Maize haploid seedlings obtained from eight different genotypes were treated with nitrous oxide gas (2 days at 600 kPa). Treatment at the six‐leaf stage (flower primordia formation stage) significantly increased the occurrence of fertile sectors on both tassels and ears so that approximately half (44%) of the treated haploids produced kernels after self‐pollination. In the control, only 11% of haploids produced selfed kernels owing to spontaneous chromosome doubling. A strong genotypic effect on the occurrence of fertile sectors after the treatment was observed. This procedure can be used for inbred line development in maize breeding programmes.     

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.     

Carbon dioxide treatment as an effective aid to the production of selfed seed in kale and Brussels sprouts

Summary The use of carbon dioxide to overcome the self-incompatibility mechanisms in marrow-stem kale and Brussels sprouts (Brassica oleracea L.) is discussed. Results are presented to show the effectiveness of the treatment, and a method is described of applying it as a routine procedure for the production of selfed seed in quantily.     

Development of novel chromosome doubling strategies for wheat × maize system of wheat haploid production

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.     

The use of seed acid phosphatases in the determination of the purity of F1 hybrid brussels sprout seed

Summary Starch gel electrophoresis of single seeds of five different F₁ hybrids of Brussels sprouts showed that they possessed three cathodal acid phosphatases. By comparison with cathodal acid phosphatases present in their inbred parents these have been interpreted as the two parental types plus a hybrid enzyme. All of the parental material could be classified into two groups depending upon whether or not their cathodal acid phosphatase was fast or slow moving. It was shown that these acid phosphatases are suitable for the determination of sibs in F₁ hybrid sprout seed provided that one of the parents possesses the slow moving cathodal acid phosphatase and the other the fast moving one. A survey of 35 different F₁ hybrids showed that 18 could be analysed for sibs using this method, those which could not were assumed to have had parents who possessed cathodal acid phosphatases of the same mobilities.     

Somatic cell genetic studies inBrassica species. II. Production of androgenetic haploid plants inBrassica nigra (L.)Koch

Summary Callus growth and its subsequent regeneration into complete plantlets was achieved from in vitro cultured anthers ofBrassica nigra (L.)Koch. Callus was induced on a modified N6 medium containing trace elements, organics of B5 medium and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Morphogenesis of callus in the form of shoots on MS medium containing indole-3-acetic acid (IAA) and N⁶-benzyladenine (BA) 0.5 mg/l each and embryoids on MS medium containing 0.5–1.0 mg/l IAA and 3.0–5.0 mg/l BA could be accomplished. Chromosomal analysis revealed presence of 41% haploids (n=8) amongst the regenerated plants.     

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.     

Early diagnosis of ploidy status in doubled haploid production of maize by stomata length and flow cytometry measurements

Reliable discrimination of haploid (H), doubled haploid (DH) and crossing (C) plants in early growth stages could streamline DH production in maize. By detecting in early growth stages undesirable sterile H plants and undesirable heterozygous C plants, a large proportion of resources required for DH production could be saved. The goal of this study was to evaluate the effectiveness of early classification of plants in growth stages V3‐V4 as H, DH or C in the context of DH production using flow cytometry and stomata length. As the reference classification, we used a field score based on plant phenotype commonly applied in DH production and research. Our results show that identification of misclassified C seeds is possible because the overlap in distributions of stomata length between H&DH and C plants is small and the association between flow cytometry and the reference field score is high. In contrast, overlap between H and DH distributions is substantial. Consequently, the main application we see for these classification methods in early growth stages is the identification of C seedlings.     

The effect of alternating temperatures on the self-incompatibility of some clones of Brussels sprouts (Brassica oleracea L. var. Gemmifera (DC.) Schulz)

Summary The influence of temperature on self-incompatibility of six S-homozygous clones of Brussels sprouts was studied. The clones were treated with constant temperatures of 14°, 17° and 20°C and alternating day and night temperatures of 17/14°, 20/14°, 23/14° and 26/14°C. To determine the degree of incompatibility the mean number of pollen tubes per style after selfing was calculated.The clone with the weak S-allele S-5 was less self-incompatible in the 23/14° and 26/14°C treatments than in the other treatments. The other clones with the weak S-alleles S-15 and S-45 remained sufficiently self-incompatible during all treatments. The clone with the rather strong S-39 showed the highest level of self-incompatibility at 26/14°C.In the two weakly self-incompatible clones with S-5 and S-39 a clear difference in self-incompatibility was found between young and old flowers. The strongly SI clones with S-39 showed no difference and in the two clones with S-15 the differences were small and significant in one case only.     

Routine large-scale electrophoresis for plant breeding. An example with F1 seeds of Brussels sprouts (Brassica oleracea var.gemmifera)

Summary A routine method of large-scale electrophoresis is proposed for use in plant breeding. The method can be applied both with research and teaching and does not require sophisticated apparatus. A skilled laboratory technician can thus handle 768 samples a day. Within a series of electrophoretic investigations the individual electrophoresis can be stopped at any time, while the other investigations in the series continue.     

Chromosome doubling through adventitious shoot formation on in vitro cultivated leaf explants from diploid interspecific potato hybrids

Summary Nearly 450 plantlets were produced from 51 diploid Solanum etuberosum x S. pinnatisectum F₁ hybrids through adventitious shoot formation on in vitro cultivated rachis and petiole explants.On the basis of phenotypical assessment of the ploidy level of 425 plants, 84.7% of the plants were scored as doubled or doubled twice. A cytological analysis of ploidy in the three layers L₁.L₂ and L₃ of 112 plants revealed 83.9% complete doubling: periclinal ploidy chimeras were not found and only two sectorial ploidy chimeras were detected. Doubled plants were obtained from all 51 clones.Various flower colours and epinastic leaves (in 1 clone) may be indications of mutagenesis through the treatment.