Production of doubled haploid rice plants (Oryza sativa L.) by anther culture

The results of anther culture of F₂ pollen issued from 23 single crosses are presented. A relation between the morphology of the panicle and the microspore stage was established. After cold-pretreatment (8 days at 4°C), the anthers were cultured on the callus-induction medium N6 supplemented with 1 mg l^–1 naphthaleneacetic acid. The calli were transferred to MS plant regeneration medium supplemented with 3 mg l^–1 kinetin + 0.5 mg l^–1 naphthaleneacetic acid. The induction frequency varied from 0.22% to 29% and the regeneration frequency from 0% to 144.4%, dependent upon the crosses used. On average, 27% of the plants obtained were albinos and 59% of the green plants underwent spontaneous chromosome doubling. Thirtynine doubled haploid lines were evaluated and multiplied in the field. Lines with an excellent behaviour in upland culture conditions were selected from two crosses.     

In vitro production of haploid and doubled haploid plants from pollinated ovaries of maize (Zea mays)

Haploid induction has potential application for maize breeding. This paper reports that maize haploid plants have been induced by in vitro culture of pollinated ovaries. From a total of 26,400 cultured ovaries, 24 haploid plants were obtained and two of them were doubled after colchicine treatment. The maximum frequency of gynogenesis was 0.17% at 19.5 h post-pollination (HPP). The results showed that HPP was an important factor affecting plant induction from ovaries. Regenerated diploid R₀ plants were then subjected to genetic analysis using SSR molecular markers. One R₀ plant, whose progeny revealed a high level of homogeneity for several agro-morphological traits, was homozygous at 20 loci tested, with 11 showing paternal and 9 maternal banding pattern. This demonstrates that it is feasible to induce maize haploid plants by in vitro culture of pollinated ovaries.     

Comparison of frequency distributions of doubled haploid and single seed descent lines in barley

Summary Both doubled haploid (DH) and single seed descent (SSD) methods were used to derive homozygous lines from two crosses of barley. The frequency distributions of grain yield, heading date, and plant height of the DH and SSD lines were compared by the Mann-Whitney U test, Kolmogorov-Smirnov twosample test and Wald-Wolfowitz runs test. It was found that the DH lines distributed in the same manner as the SSD lines with respect to the three characters. The results indicated that although the SSD method had more opportunity for recombination than the DH method, it did not produce a sample of recombinants which differed significantly from the DH sample; thus both methods were equally efficient for use in deriving homozygous lines from F₁ hybrids in a relatively short time.Contribution no. 455 Charlottetown Research Station, Agriculture Canada, P.E.I. (Canada)     

Increased doubled haploid plant regeneration from rice (Oryza sativa L.) anthers cultured on colchicine-supplemented media

Plating rice anthers on a semisolid induction medium containing 250 or 500 mg/l colchicine for 24 or 48 h-incubations followed by transfer to colchicine-free medium and standard anther culture procedures resulted in overall 1.5- to 2.5- fold increases in doubled haploid green plant productions compared to control anther cultures. The addition of colchicine had no detrimental effects on the different anther culture efficiency parameters, but in some treatments led to significant enhancement of anther callusing frequency or callus green plant regenerating ability. The most efficient treatment raised doubled haploid plant recovery from 31% to 65.5%. These results suggest that post-plating colchicine treatment of anthers, since it was found to improve both anther culture efficiency and doubled haploid plant recovery frequency, could be integrated into rice doubled haploid plant production programmes.Abbreviations DH doubled haploid - NAA naphthalenacetic acid - PAS periodic acid Schiff     

Significant improvement of androgenetic haploid and doubled haploid induction from wheat plants treated with a chemical hybridization agent

Summary A very significant improvement of the total yield of androgenetic green plants after anther culture is presented. The process involves treatment of the donor plants by spraying at different stages around the meiosis with a chemical hybridization agent (CHA) solution, fenridazon-potassium. When harvested at the normal uninucleate pollen grain stage, anthers have shown during in vitro culture very significant increases in embryo production. Compared to the control, we observed up to a 20-fold increase in the production. Moreover, when cultivated later, anthers still remained embryogenic. Therefore the process appears to be very efficient and to allow a broadening of the target period for androgenesis in vitro. The regeneration was not disturbed by the CHA treatment and, as shown in this paper, the technique seems to be applicable to a large range of genotypes. Cytological observations revealed both a low frequency of aneuploidy among the regenerated plants and peculiar features in the pollen grain walls after treatments; a triploid plant was observed. Hypotheses to explain the phenomenon are presented and related to previous observations on the effects of gametocide substances like ethrel, male sterility and pollen dimorphism on androgenesis.     

Production of doubled-haploid plants from tritordeum anther culture

The effects of different media and cold pretreatment of spikes on the androgenic response and regeneration capacity from anther culture of tritordeum was studied. L5 medium gave the highest frequency of anther response. The frequency of cultures regenerating green or albino plantlets was not affected by the composition of the medium tested. Cold pretreatment of the spikes significantly increased the frequency of anther response and also the percentage of cultures giving albino plantlets. A mean of four green plants was obtained per 100 subcultured calli/embryos. The percentage of spontaneous chromosome doubling was only 1%. The addition of colchicine at 0.02% to the induction medium significantly increased the frequency of doubled haploids regenerated without any effect on regeneration capacity. This technique proved more efficient than a conventional chromosome-doubling method.     

Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus

Summary A chromosome doubling technique, involving colchicine treatment of an embryogenic, haploid callus line of maize (Zea mays L., derived through anther culture), was evaluated. Two colchicine levels (0.025% and 0.05%) and three treatment durations (24, 48, and 72 h) were used and compared to untreated controls. Chromosome counts and seed recovery from regenerated plants were determined. No doubled haploid plants were regenerated from calli without colchicine treatment. After treatment with colchicine for 24 h, the callus tissue regenerated about 50% doubled haploid plants. All of the plants regenerated from the calli treated with colchicine for 72 h were doubled haploids, except for a few tetraploid plants. No significant difference in chromosome doubling was observed between the two colchicine levels. Most of the doubled haploid plants produced viable pollen and a total of 107 of 136 doubled haploid plants produced from 1 to 256 seeds. Less extensive studies with two other genotypes gave similar results. These results demonstrate that colchicine treatment of haploid callus tissue can be a very effective and relatively easy method of obtaining a high frequency of doubled haploid plants through anther culture.     

Plant regeneration from embryogenic cell suspensions derived from anther cultures of barley (Hordeum vulgare L.)

Summary We have established embryogenic cell suspension cultures of barley (Hordeum vulgare L. cultivars Igri, Gimpel, Princesse, and Baronesse) from anther-derived embryogenic callus. Suspension cultures of cultivars Igri and Gimpel were regenerable. The most successful cultivar was Igri, from which a number of independent cell lines producing plantlets were established. Plants could be transferred to soil; up to now, 50% of more than 200 regenerated plants were morphologically normal and fertile. The relative frequency of sterile plants increased as suspensions aged. Suspensions older than 1 year produced embryogenic callus but only albino plantlets could be regenerated.     

Regeneration of anther-derived plants ofAvena sterilis

Anther culture response of accessions of several hexaploid Avena species was studied with respect to requirement for auxin. The highest callus induction frequency was achieved withA. sativa L. cv. Stout (17.3%). The three most responsive genotypes, two of A. sativa and one of A. nuda L., had two-fold higher anther culture responses on growth regulator -free medium than on a medium containing 2,4-d. The A. sterilis L. accession CAV 2648 was the only genotype which consistently produced white, embryogenic structures (on solid MS+ 10% sucrose) and did so irrespective of the presence of 2,4-d. When transferred onto medium with lower sucrose concentration and an auxin transport inhibitor (TIBA), three green (two diploid [2t n=6t x=42] and one haploid [1t n=3t x=21]) and two albino plantlets were regenerated. The diploid regenerants set seed in the greenhouse. This first report of plants recovered from anther culture in the wild oat A. sterilis may provide an avenue to understand better and possibly overcome problems associated with androgenesis in cultivated oat. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro production of haploid plants

Although several methods have been developed for producing haploid plants, the in vitro techniques are much more efficient than inter-specific hybridization or treatment with plant-growth regulators, temperature or irradiation. Androgenesis is the most universal of these techniques but ovule culture and the bulbosum method could complement or replace anther culture in those species or genotypes with less responsive male gametes. Genotype, environment, physiological status of the donor plant, and culture conditions and components all need to be taken into account when developing procedures for producing haploid and dihaploid plants. Suitable methods are already well established for a number of important crops. However, many problems, related to regeneration frequency, gametoclonal variation and albinism, are still unsolved. It is now clear that haploids and dihaploids form the ideal system for genetic manipulation in plants. Their key role in producing new theoretical and applied knowledge in plant science is an important aspect of our review.A. Atanassov is with the De Montfort University Norman Borlaug Center for Plant Science Research, Institute of Genetic Engineering, 2232 Kostinbrod, Bulgaria; N. Zagorska is with the Institute of Genetics, 1113 Sofia, Bulgaria. P. Boyadjiev is with the Institute of Plant Introduction and Genetic Resources, 4122 Sadovo, Bulgaria D. Djilianov is with the Institute of Genetic Engineering, 2232 Kostinbrod-2, Bulgaria.     

Response of flax genotypes to doubled haploid production

Forty-four flax genotypes with a diverse genetic background were evaluated for anther culture response using a standard anther culture protocol in order to determine the feasibility to initiate a routine haploid production system in applied breeding programs. A strong genotype effect on callus induction and shoot regeneration in anther culture was found in this study. A number of genotypes, including two low cadmium content lines 96-11785 and 96-11826, a high oil content line 96-22109 and a high linolenic acid content line M 4919 were identified as highly responsive. The impact of the findings in this study on flax breeding was discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Efficient production of doubled haploid plants by immediate colchicine treatment of isolated microspores in winter Brassica napus

The effect of colchicine on embryogenesis induction and chromosomedoubling during microspore culture was evaluated in two F₁ hybridsofwinter oilseed rape (Brassica napus L.). Colchicinetreatment (50 and 500 mg/L) of isolated microspores during thefirst 15 h in culture stimulated embryogenesis and produced large amounts ofhealthy-looking embryos. These normal embryos germinated well at 24°C after being transferred to solid regeneration medium and aninitial period of low temperature (2 °C) for 10 days, andcoulddirectly and rapidly regenerate vigorous plants. A high doubling efficiency of84–88% was obtained from 500 mg/L colchicine treatment for15h with low frequency of polyploid and chimeric plants. Acolchicinetreatment duration of 6 h was less effective on embryogenesis anddoubling efficiency. The present experiment also showed that changing of induction medium 15h after microspore isolation produced higher spontaneous doublingefficiency, as compared with medium change 6 h after isolation.     

Combination of reversible male sterility and doubled haploid production by targeted inactivation of cytoplasmic glutamine synthetase in developing anthers and pollen

Reversible male sterility and doubled haploid plant production are two valuable technologies in F₁-hybrid breeding. F₁-hybrids combine uniformity with high yield and improved agronomic traits, and provide self-acting intellectual property protection. We have developed an F₁-hybrid seed technology based on the metabolic engineering of glutamine in developing tobacco anthers and pollen. Cytosolic glutamine synthetase (GS1) was inactivated in tobacco by introducing mutated tobacco GS genes fused to the tapetum-specific TA29 and microspore-specific NTM19 promoters. Pollen in primary transformants aborted close to the first pollen mitosis, resulting in male sterility. A non-segregating population of homozygous doubled haploid male-sterile plants was generated through microspore embryogenesis. Fertility restoration was achieved by spraying plants with glutamine, or by pollination with pollen matured in vitro in glutamine-containing medium. The combination of reversible male sterility with doubled haploid production results in an innovative environmentally friendly breeding technology. Tapetum-mediated sporophytic male sterility is of use in foliage crops, whereas microspore-specific gametophytic male sterility can be applied to any field crop. Both types of sterility preclude the release of transgenic pollen into the environment.     

Efficient tissue culture and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of haploid poplar derived from anthers

Calli were induced from anthers of Populus simonii × P. nigra. Haploid plants were then regenerated from the callus and multiplied efficiently by culturing leaf explants. The presence of both haploid and diploid cells in the same plant revealed spontaneous chromosome doubling in haploid cells. The haploid plants were transformed with the nptII gene by Agrobacterium-mediated method using leaf explants, and five independent kanamycin-resistant lines were obtained, with a transformation frequency more than 6%. Further PCR test indicated that the exogenous betA gene was transferred into these kanamycin-resistant lines, which were still haploid. Thus, the efficient tissue culture system and transformation of haploid poplar plants were achieved. Our study will contribute to forest improvement via the haploid culture and transgenic technology. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 629–633. The text was submitted by the authors in English.     

The relationship between in vitro performance of haploid embryos and the agronomic performance of the derived doubled haploid lines in barley

Summary The relationship between in vitro performance of haploid embryos and the agronomic performance of the derived doubled haploid (DH) lines during the stages of field evaluation was investigated. The results showed a positive correlation between coleoptile height of haploid plantlets in culture and final plant height of their DH progeny lines in the field. These results illustrate that in vitro selection for plant height and other linked quantitative or pleiotropic characters can be carried out at the initial stages of a DH breeding programme.     

菊科植物单倍体研究进展

单倍体培养是快速获得菊科纯合系的重要途径。目前已进行单倍体研究的菊科植物共有13个种,其中9个已成功获得单倍体植株。菊科中诱导单倍体的途径有花药培养、小孢子培养、离体雌核培养、远源杂交和辐射花粉诱导单倍体。本文详细论述了不同外植体发育时期、预处理、培养基、培养条件等因素对单倍体植株诱导再生的影响。对菊科植物单倍体诱导的几种途径进行对比总结,指出研究中存在的问题并提出思路和建议。     

Production of ethanol from mannitol by Zymobacter palmae

Extracts from brown seaweeds could possibly be fermented to ethanol, particularly seaweeds harvested in the autumn, which contain high levels of easily extractable laminaran and mannitol. Few microorganisms are able to utilise mannitol as a substrate for ethanol production and Zymobacter palmae was tested for this purpose. Bacterial growth as well as ethanol yield depended on the amount of oxygen present. Strictly anaerobic growth on mannitol was not observed. At excessive aeration, a change in the fermentation pattern was observed with high production of acetate and propionate. Under oxygen-limiting conditions, the bacteria grew and produced ethanol in a synthetic mannitol medium with a yield of 0.38 g ethanol (g mannitol)⁻¹. Z. palmae was also successfully applied for fermentation of mannitol from Laminaria hyperborea extracts. Journal of Industrial Microbiology & Biotechnology (2000) 24, 51–57. Received 27 June 1999/ Accepted in revised form 23 September 1999     

Plant regeneration through callus initiation from anthers and ovules of Scabiosa columbaria

Anthers and ovules of Scabiosa columbaria L. were cultured in vitro to determine whether gametophytic cells would proliferate and/or a protocol for plant regeneration could be developed. Several factors were tested, including explant type, donor plant, cold pre-treatment, and medium composition. Callus induction frequency varied among treatments, indicated by significant effects of explant type, medium composition, and their interactions. Histological analysis revealed numerous sites of callus induction, however, gametophytic cells did not proliferate. Stepwise removal of growth regulators and simultaneous lowering of sucrose from the nutrient medium, resulted in initiation of embryogenesis or shoot organogenesis, and allowed plant regeneration. Under the conditions tested, regeneration capacity was donor related, because only material of one donor responded. Regenerants were diploid (except one mixoploid individual), but showed various types of flower heads. They were probably of sporophytic origin. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mapping quantitative and qualitative disease resistance genes in a doubled haploid population of barley (Hordeum vulgare)

Stripe rust, leaf rust, and Barley Yellow Dwarf Virus (BYDV) are important diseases of barley (Hordeum vulgare L). Using 94 doubled-haploid lines (DH) from the cross of Shyri x Galena, multiple disease phenotype datasets, and a 99-marker linkage map, we determined the number, genome location, and effects of genes conferring resistance to these diseases. We also mapped Resistance Gene Analog Polymorphism (RGAP) loci, based on degenerate motifs of cloned disease resistance genes, in the same population. Leaf rust resistance was determined by a single gene on chromosome 1 (7H). QTLs on chromosomes 2 (2H), 3 (3H), 5 (1H), and 6 (6H) were the principal determinants of resistance to stripe rust. Two- locus QTL interactions were significant determinants of resistance to this disease. Resistance to the MAV and PAV serotypes of BYDV was determined by coincident QTLs on chromosomes 1 (7H), 4 (4H), and 5 (1H). QTL interactions were not significant for BYDV resistance. The associations of molecular markers with qualitative and quantitative disease resistance loci will be a useful information for marker-assisted selection. Received: 2 February 1999 / Accepted: 30 December 1999     

Variations in green plant regeneration response from anthers of indica rice and their hybrids with japonica cv. Taipei 309

The green plant regeneration ability from anthers of BR-7, a high yielding indica cultivar, Binnatoa (BA), a salt tolerant indica land race and IR-43 was tested in N6, M8, He2 and R2 media. The response was calculated on the basis of number of anthers producing green plants. The number of green plants per responding anther was also recorded. The response of BR-7 and BA was poor compared to the indica cultivar IR-43 in three of the media that were tested. In N6 medium, green plant regeneration of BA and BR-7 was respectively 10-fold and 100-fold less than the japonica cultivar Taipei 309 (T-309). No anther-derived green regenerant was obtained from another salt tolerant indica land race, Rajashail (RAJ). The N6 medium was selected to test green plant regeneration frequency from anthers obtained from the F1 crosses of T-309 × BR-7, T-309 × BA, T-309 × RAJ and T-309 × BR-7 AC regenerants backcrossed with BA. Our objective was to combine the salt tolerant trait of BA and the high yield of BR-7 in a single line. The intermediate crossing step with T-309 was performed to increase the green plant regenerability of the anthers. All F1 progeny from the crosses with T-309 showed significantly increased callus induction compared to the indica parent although the values were lower than the midparent means. Green plant regeneration compared to their respective indica parents either increased or decreased but never approached the level of T-309. This revised version was published online in June 2006 with corrections to the Cover Date.