Influence of 2,4-D, IAA, and duration of callus induction in anther cultures of spring wheat

Culture media and environmental factors may significantly influence the yield of haploid plants from anther cultures. Our objectives were to identify a combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA) concentrations which produce the maximum number of haploid plants, and to evaluate the effects of duration in induction medium on calli induction, plant regeneration, and green plant production from anther cultures in spring wheat. Significant (P ≤ 0.01) plant growth regulator concentration effects (2,4-D and IAA) were observed on the number of calli, green plants and albino plants produced, and on direct plant regeneration. Addition of 2,4-D to the induction medium resulted in significantly (P ≤ 0.01) higher means for all anther culture components compared to IAA> While addition of 2,4-D significantly (P ≤ 0.01) reduced plant regeneration, it substantially increased green plant percentage at a 0.3-mg l⁻¹ concentration of IAA. Use of response functions to estimate the maximum effective 2,4-D × IAA combination implied that higher 2,4-D levels in the induction medium should be investigated, and that the optimum hormone combination differs for plant regeneration and green plant percentage. Significant (P ≤ 0.01) effects of duration on callus induction medium were observed for plant regeneration and green plant percentage.     

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.     

Ploidy of small individual embryo-like structures from maize anther cultures treated with chromosome doubling agents and calli derived from them

Summary In order to determine the ploidy of individual embryo-like structures (ELSs) following chromosome doubling treatments, a method was developed to determine the DNA content (ploidy level) of nuclei from single ELSs weighing as little as 1 mg using flow cytometry. About half (53%) of the ELSs which formed during anther culture of the maize inbred line used in control medium were haploid, 27% mixoploid and 20% diploid. Gibberellic acid (GA₃) increased the diploid percentage to 52% without affecting the mixoploid frequency (26%). A four day treatment with the chromosome doubling agent colchicine (50M) increased chromosome doubling while oryzalin eliminated the diploidy and mixoploidy. When regenerable callus cultures were initiated from the ELSs none were found to be mixoploid but the haploid and diploid proportions were similar to that of the ELSs analyzed. Regenerable cultures could not be initiated from the colchicine treated ELSs, however. These studies show that with the genotype used here, GA₃ and colchicine increased the amount of chromosome doubling of the ELSs while oryzalin and pronamide did not. The mixoploidy which existed in about 25% of the ELSs was never observed in calli apparently because these structures do not initiate callus or cells of only one ploidy level grew.Abbreviations ELS embryo-like structure - GA₃ gibberellin A₃     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Androgenesis,gynogenesis, and parthenogenesis haploids in cucurbit species

Haploids and doubled haploids are critical components of plant breeding. This review is focused on studies on haploids and double haploids inducted in cucurbits through in vitro pollination with irradiated pollen, unfertilized ovule/ovary culture, and anther/microspore culture during the last 30 years, as well as comprehensive analysis of the main factors of each process and comparison between chromosome doubling and ploidy identification methods, with special focus on the application of double haploids in plant breeding and genetics. This review identifies existing problems affecting the efficiency of androgenesis, gynogenesis, and parthenogenesis in cucurbit species. Donor plant genotypes and surrounding environments, developmental stages of explants, culture media, stress factors, and chromosome doubling and ploidy identification are compared at length and discussed as methodologies and protocols for androgenesis, gynogenesis, and parthenogenesis in haploid and double haploid production technologies.     

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     

Application of trifluralin to embryogenic microspore cultures to generate doubled haploid plants in Brassica napus

Microspore or anther culture has been used to produce desirable meiotic recombinants in numerous species. However, the utilization of these recombinants relies on inefficient genome doubling procedures to obtain fertile doubled haploid plants. This study presents a simple and rapid procedure to generate fertile doubled haploids in Brassica napus cv. Topas using trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl- p -toluidine), a plant specific microtubule inhibitor. The effects of trifluralin on microtubule depolymerization and chromosome doubling in embryogenic microspore cultures of B. napus were examined and compared with those of colchicine. Indirect immunofluorescence labeling of isolated microspores indicated that microtubules were depolymerized within 30 min of trifluralin treatment and after 3–8 h of colchicine treatment. The direct application of these microtubule inhibitors to microspore cultures resulted in the recovery of fertile doubled haploid plants. Continuous culture in the presence of colchicine, was more effective than 18-h treatments for fertile plant production but resulted in abnormal embryo formation and recalcitrant plant regeneration. The application of 1 or 10 μ M trifluralin during the first 18 h of microspore culture was found to be the superior method for doubled haploid production. The embryos generated after trifluralin treatment developed normally, germinated readily and of the plants produced, close to 60% were fertile. The use of trifluralin to double chromosomes very early in microspore cultures is a simple process requiring minimal manipulation and should be very useful for genetic studies and breeding programs of B. napus and possibly other species.     

Factors inducing regeneration response in oat (Avena sativa L.) anther culture

The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’. Despite the high resistance of oat to the process of androgenesis, all tested cultivars produced embryo-like structures and only two of them, ‘Akt’ and ‘Chwat’, produced fertile doubled haploid plants. A strong cultivar dependency was observed during induction of androgenesis. Further, cold pretreatment together with high temperature shock enhanced the efficiency of this technique. The highest number of embryo-like structures and haploid plants was obtained from cv. ‘Chwat’ (3.6% and 0.8%, respectively). Embryo-like structure formation also depended on the distance from the base of the flag leaf to the penultimate leaf of the panicle. Most of them were observed on anthers harvested from panicles of which the distance from the base of the flag leaf to the penultimate leaf was less than 4 cm. The presence of the induction medium supplemented with different plant growth regulators was essential for the induction of embryo-like structures but did not increase the production of haploid plants and doubled haploid lines. The highest number of embryo-like structures and plants was obtained on W14 medium with the addition of 2.0 mg/dm³ 2,4-dichlorophenoxyacetic acid and 0.5 mg/dm³ kinetin (2.7%). The low haploid plant regeneration rate (from 0.03 to 0.05%) still limits the practical application of anther culture for the production of doubled haploid lines in oat.

     

Haploid plants from anther cultures of poplar (Populus × beijingensis)

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial woody plants using conventional breeding techniques is currently a challenge due to a long juvenile period, high heterozygosity, and substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In the present study, we report the regeneration of haploid lines of poplar (Populus × beijingensis) via anther culture. Anthers at the uninucleate stage were induced to produce callus using three basic media. Two auxins (naphthalene acetic acid [NAA] and 2,4-dichloro-phenoxyacetic acid [2,4-D]), and two cytokinin (kinetin [KT] and 6-benzyladenine [BA]) were tested to explore the influence of plant growth regulators on callus response. H medium (Bourgin and Nitsch 1967) supplemented with 1.0 mg/L NAA and 1.0 mg/L KT induced the highest rate of callus formation. When callus obtained from anthers were subcultured in MS medium containing 1.0 mg/L BA and 0.2 mg/L NAA, followed by transfer to half-strength MS medium supplemented with indole-3-butyric acid (0.2–0.5 mg/L), the formation of regenerated plantlets increased dramatically. Inclusion of gibberellic acid (0.02–0.2 mg/L) in addition to a combination of BA (0.6 mg/L)-NAA (0.2 mg/L) in the culture medium resulted in enhanced frequency of shoot development, as well as greater internode elongation. Ploidy analysis of 580 regenerated plants, using both flow cytometry and chromosome counting, revealed 10.3 % haploid and 1.0 % triploid plantlets. The remaining plantlets were all diploid.     

Effects of colchicine on anther and microspore culture of bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The aim of this work was to study the effects of colchicine application on chromosome doubling and androgenic response in anther and microspore culture of different bread wheat genotypes. Colchicine was applied during a mannitol stress pretreatment or during the first 48 h of culture at concentrations of 0, 150 and 300 mg l⁻¹. When colchicine was applied during stress pretreatment, the percentage of doubling depended on genotype and concentration. A significant increase in doubling was observed with 300 mg l⁻¹ in the low androgenic responding cv. Caramba. Colchicine incorporation during the first hours of culture improved percentage of doubling in all genotypes, in both anther and microspore culture. Application of 300 mg l⁻¹ colchicine improved the percentage of doubling in the two low responding genotypes, to 118% of control in DH24033, and 75% in Caramba in microspore and anther culture, respectively. Concerning the androgenic response, the effect of colchicine on embryo formation and percentage of green plants depended on the genotype and on the culture method. In cv. Pavon, a 2- and a 3-fold increase in percentage of embryogenesis and green plants, respectively, were obtained with 300 mg l⁻¹ colchicine in microspore culture. However, no significant differences in these two variables were observed in anther culture. The number of green doubled haploid (DH) plants reflects the index of success of the procedure. Regardless of the culture method, when colchicine was incorporated during the first hours of culture, the number of green DH plants increased significantly in three of four genotypes. These results confirm the usefulness of colchicine application during the first hours of culture in wheat breeding programs.     

Spontaneous versus colchicine-induced chromosome doubling in maize anther culture

The range of genetic variation of spontaneous chromosome doubling frequency of maize haploid plantlets derived from in vitro anther culture was evaluated. When regeneration is obtained by direct embryo-genesis, bypassing the callus phase, it appears that the frequency of spontaneous doubling may exceed 40 of the regenerated plantlets. This high frequency may be one consequence of the use of doubled haploid lines derived from anther culture and spontaneous chromosome doubling. We also report an increase, by more than 50, of the productivity of diploid fertile regenerated plantlets produced by colchicine supplemented medium during the cold shock pretreatment of the microspores inside the anthers. Optimization of the treatments and the anther culture procedure are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

芍药花药愈伤组织诱导及体细胞胚发生

以芍药(Paeonia lactiflora)品种粉玉奴花药为外植体,研究不同浓度2,4-D对愈伤组织诱导、体胚发生及植株再生的影响,采用组织细胞学方法观察愈伤组织以及体细胞胚发育过程,采用根尖染色体法鉴定再生植株倍性。结果表明,芍药花药愈伤组织诱导的最适培养基为MS+1 mg·L–1 2,4-D+1 mg·L–1 N...     

Differences among auxin treatments on haploid production in durum wheat × maize crosses

Three doubled haploid lines of durum wheat [Triticum turgidum ssp. durum (Desf.) Husn.] were crossed with maize (Zea mays L.), and five hormone treatments were applied to test their effect on the production of caryopses, embryos and haploid plants. The auxin treatments consisted of 100 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mg/l or 50 mg/l dicamba and two combination mixtures of 95/5 mg/l and 50/50 mg/l 2,4-D plus dicamba, respectively. Hormones were added to the culture medium of the detached tillers. Differences were not observed among the four hormone treatments that contained dicamba, nevertheless, these treatments significantly increased the production of caryopses, embryos and haploid plants. On average, 8.9 caryopses, 2.6 embryos and 1.3 haploid plants per spike were obtained following the treatment with 100 mg/l 2,4-D, and 15.0 caryopses, 6.0 embryos and 3.0 haploid plants per spike were obtained following the various treatments with dicamba. We propose the application of dicamba alone, or dicamba plus 2,4-D, as a means for improving the yield of haploid plants of durum wheat through crosses with maize.     

Haploid plants from in vitro anther culture of the leguminous tree,Peltophorum pterocarpum (DC) K. Hayne (Copper pod)

The development of haploid callus, embryos and plantlets from cultured anthers and the various factors affecting androgenesis in Peltophorum pterocarpum (Copper pod), a tropical legume tree is reported. A pretreatment of flower buds at moderate temperature of 14°C for 8 days was most effective for callus production. The colour of the anther was found to be a reliable and efficient indicator for identification of suitable stage of anther for culture. The frequency of anthers which produced callus and shoots was highest when anthers were cultured at mid or late-uninucleate stage. A high sucrose concentration of 10% is a specific media requirement for androgenesis. The haploid nature of the embryos, callus and regenerated plants (n=14) were confirmed by chromosome count.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KN kinetin - NAA naphthaleneacetic acid - BAP bezylaminopurine     

Ovary co-culture improves embryo and green plant production in anther culture of Australian spring wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

A simple anther culture protocol for Australian spring wheat cultivars was developed using ovary co-culture. The inclusion of ovaries in the induction medium significantly increased the production of embryo-like structures (ELS), green and albino plants in two spring wheat cultivars tested. When five ovaries were added to the induction medium, the mean number of ELS per spike increased from 7.6 to 50.1 and green plants per spike increased from 0.6 to 8.9. The addition of 10 ovaries, however, did not further increase the production of ELS or green plants. The growth regulator combination of 2,4-D and KIN was compared with IAA and BA. There were no significant differences in the numbers of ELS or green plants although significantly fewer albino plants were produced with IAA and BA. Eight additional cultivars were screened using the protocol with either 5 or 10 ovaries in the induction medium. Green plants were obtained from nine varieties at frequencies ranging from 0.3 to 33.0 green plants per spike. Regenerant plants at maturity exhibited chromosome fertility rates in different cultivars ranging from 15% to 100%.     

Colchicine-mediated chromosome doubling during anther culture of maize (Zea mays L.)

Efficient methods of chromosome doubling are critical for the production of microspore-derived, doubled-haploid (=DH) plants, especially if, as in maize anther culture, spontaneous chromosome doubling occurs infrequently. In the present study, colchicine (5–1000 mg/l) was added to the induction medium and maize anthers were incubated in the colchicine-containing medium for different durations (1–7 days). In order to improve overall anther culture response, the culture temperature was adjusted to 14°C during the first 7 days. Colchicine applied at low concentration, i.e. 5 mg/l (7 days), or for short duration, i.e. 1–3 days (250 mg/l), showed beneficial effects on the formation of embryolike structures (=ES) and thus led to increased plant production, but was comparatively ineffective regarding chromosome doubling. Optimal doubling effects were observed when anthers had been exposed to culture medium containing 250 and 1000 mg/l of colchicine (7 days); in these treatments the doubling index (=DI), defined as the quotient of the number of DH plants and the number of totally regenerated plants in a specific treatment, rose to 0.56 and 0.53, respectively, compared to 0.20 in the untreated control. However, colchicine administered at concentrations higher than 250 mg/l seemed to be detrimental to general plant production; thus, in spite of a high DI, the overall DH plant production was even lower than in the control treatment. Maximum DH plant production for three different genotypes was accomplished with culture medium containing 250 mg/l of colchicine (7 days). With the best-responding genotype (ETH-M 36) a DH plant production of 9.9 DH plants/100 anthers was accomplished, i.e. a 7-fold increase compared to the non-treated anthers. This is the first report on efficient chromosome doubling in anther culture by subjecting anthers to colchicinecontaining induction medium during a post-plating cold treatment. Chromosome doubling as described here becomes an integral part of the maize anther culture protocol and thus represents a rapid and economical way to produce DH plants.     

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.     

Anther culture as an effective tool in winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) breeding

The aim of this study was to determine the effect of genotype and induction medium in anther culture of wheat (Triticum aestivum L.). Ten F1 winter wheat genotypes were tested in anther culture (AC) to compare the two most frequently applied induction media (W14mf and P4mf). Androgenesis was induced during the treatment of each tested genotypes and green plants were produced from them using both media. Based on statistical analysis, the genotypes significantly influenced (at the 0.001 probability level) the efficiency of AC (embryo-like structures (ELS), albinos, green plantlets and transplanted plantlets) and the media also had a significant effect on the number of ELS and albino plantlets. Both media can be used for AC in wheat doubled haploid (DH) plant production. The production of ELS and green plantlets was higher in P4mf medium (48.84 ELS/100 anthers, 4.82 green plantlets/100 anthers) than in W14mf medium (28.14 ELS/100 anthers, 4.59 green plantlets/100 anthers). However, the green plant regeneration efficiency of the microspore-derived structures was 16.9% when using W14mf medium, while this value was 9.6% in the case of ELS induced with P4mf medium. The application of W14mf medium thus proved to be time- and labour-saving medium in the large-scale production of DH wheat plants. In our experiments, 267 DH plants were produced for our winter wheat breeding program. The spontaneous rediploidization rate was 32.72%.     

Direct regeneration of haploid or doubled haploid plantlets in cucumber (Cucumis sativus L.) through ovary culture

This paper reports a simple and effective method of directly producing cucumber plants through unfertilized ovary culture. The paper involves an experiment investigating factorial interactions between TDZ treatment, cold pretreatment, genotypes of cucumber, to improve plant induction. Cold pretreatment was effective in stimulating the ovary. The results showed that cold pretreatment for 4 days, TDZ for 0.06 mg·L^?1, the interaction with genotype can be used as an effective strategy to improve the efficiency of gynogenesis. The plant regeneration induction rate was highest (79.3%). In addition, we observed the process of cucumber megasporogenesis and plant regeneration. The plants obtained from ovary culture of cucumber were identified as diploid or haploid by flow cytometry, consistent with the results of chromosome counting. The diploid plants were further identified as pure doubled haploid using simple sequence repeats (SSR). The doubling treatment we used was one of the simplest and most effective methods, completed in a short time (1 h) with a doubling rate of 75%. The acclimation rate for the surviving was 70%. This work provides a basis for promoting haploid breeding in cucumber.

     

Isolated wheat microspore culture

The use of doubled haploid plants in a wheat breeding program requires an efficient haploid production system. While the techniques for producing doubled haploids from anther culture are well established, those for isolated microspores are complicated and inefficient. Four methods of isolating microspores from anthers (blending, stirring, macerating, and floating) were compared. Isolated microspores were washed and cultured in liquid medium. The effects of pre-isolation mannitol conditioning, cell density, culture dilution, and sucrose centrifugation on microspore viability were evaluated. Isolation by blending gave the highest initial microspore viability (75%). Mannitol conditioning and purification by sucrose centrifugation had a detrimental effect on initial viability. An initial microspore density of 2 × 10⁵ microspores per ml was necessary for continued microspore viability. One hundred and nine haploid or spontancously doubled haploid plants were regenerated from microspores isolated without mannitol conditioning using the blending method. Based on this research, blender isolation with an initial density of 2 × 10⁵ microspores per ml is recommended for isolated microspore culture.Abbreviations LSmean least square mean - MES 2-N-morpholinoethane sulfonic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphtaleneacetic acid