Improvement of green plant regeneration by manipulation of anther culture induction medium of hexaploid wheat

Anthers of three hexaploid wheat (Triticum aestivum L.) genotypes with high frequencies of albino regenerants in anther culture were compared to DH after inoculation on medium supplemented with ficoll, colchicine or maltose separately, pair-wise or combined, in an attempt to increase green plant regeneration. Maltose treatment produced more green regenerated plants than sucrose for all of the genotypes. The three chemicals combined in anther medium either reduced green plant regeneration or did not yield significantly different numbers of green regenerated plants compared to the maltose treatment. With DH fewer embryo-like structures (ELS) were obtained per 100 cultured anthers on all medium containing colchicine but greater frequencies of green plants per 100 ELS were obtained. It appeared that the increase in green regenerated plants per 100 ELS was due to a better quality of embryos that were capable of regenerating into green rather than albino plantlets. Smaller increases in green plants per 100 ELS were observed in ICR 4 and V-15 on colchicine containing medium compared to DH. Genotypic differences in anther culture response were observed for ELS per 100 cultured anthers (increased for V-37, decreased for DH and approx. the same for ICR 4 and V-15 in medium with all three chemicals compared to the sucrose control).     

Formation of multiple embryo-like structures from single microspores during maize anther culture

In a previous study on the RFLP analysis of the maize anther culture response (Wan et al. 1992), some of the anther-derived callus Unes from hybrids H99 x Pa91 (HP) and H99 x FR16 (HF) showed the same RFLP patterns with 58 (for HP Unes) or 35 (for HF lines) RFLP markers used. Since the callus Unes with the identical RFLP pattern were initiated from individual embryo-like structures (ELSs) from the same anther culture plate, these must have originated from the same microspore. Twin embryos which apparently had originated from the same microspore were also observed. Thus in certain cases one microspore must be capable of forming more than one ELS. However, in the case of the callus Unes from a different F1 hybrid (Pa91 x FR16), no identical RFLP patterns were observed. Thus multiple ELS formation from a single microspore may be genetically controlled. Since in some cases the proportion of callus lines resulting from multiple ELS formation can be quite high (about 50% for the HP lines), estimates of gene segregation and anther culture response frequencies can be affected greatly.     

Isolated microspore culture of maize: effects of isolation technique,reduced temperature,and sucrose level

Improvements in ab initio microspore culture of maize are presented using a modified isolation technique, reduced temperature during early stages of culture, and an elevated sucrose level in the culture medium. Blending-isolation, using excised anthers, was less stressful on microspores than pressing anthers against a stainless steel sieve and resulted in a 3-fold increase in the yield of embryo-like structures (ELS). Exposure to reduced temperature (15°C) during the first 4 days of culture improved microspore viability and increased by 2-fold the number of ELS produced. Higher levels of sucrose (8.0–9.5%) also resulted in improved response. Maximum yield in the present study was 92 ELS per 100 anther equivalents, exceeding previously reported values of 15 ELS per 100 anther equivalents for ab initio microspore culture of maize. The increase in the total number ELS produced had no observable effect on their quality as evidenced by the frequency of formation of callus capable of regenerating plants.     

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%.     

Interaction of environment and ABA and GA treatments on the maize anther culture response

Treatments designed to influence abscisic acid (ABA) or gibberellin (GA) concentrations were applied to developing tassels of maize (Zea mays L.) plants in different environments or to anthers in culture to determine the effect on formation of embryo-like structures (ELS). Production of ELS was significantly affected in certain environments when ABA, GA₃, ancymidol, or fluridone solutions were pipetted into whorls of field-grown plants approximately 3 days before tassel harvest. In 1996 anthers from 10 M ancymidol-treated plants were most responsive, producing 35 ELS/100 anthers and 50 M GA₃-treated plants were least responsive, producing 12 ELS/100 anthers. In 1997 under hotter, drier conditions, anthers from 50 M GA₃-treated plants were most responsive, producing 20 ELS/100 anthers and those from 50 M ABA-treated plants were least responsive, producing 2.4 ELS/100 anthers. Anthers from growth chamber plants were significantly more responsive when grown in a 16-h than a 12-h photoperiod. With the 16-h photoperiod the response was significantly greater with a 250 M ABA whorl treatment. With the 12-h photoperiod there was no significant effect from whorl treatments. Modification of the culture medium with added ABA, GA₃, ancymidol, or fluridone was generally ineffective, except in 1997 when the response was significantly higher with 1 M ABA added to the culture medium. The results suggest that the maize anther culture response may be influenced by environmental conditions that interact with ABA and GA treatments to donor plants during tassel development.     

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%.     

RFLP analysis to identify putative chromosomal regions involved in the anther culture response and callus formation of maize

Summary RFLP analysis was performed with anther culture-derived callus lines developed from the maize F₁ hybrids Pa91 x FR16 (PF), H99 x Pa91 (HP) and H99 x FR16 (HF). Relatively evenly spaced RFLP markers were selected to cover the maize genome with 52, 58 and 35 RFLP markers for the PF, HP and HF callus lines, respectively. The results from populations PF and HP combined with limited information from HF showed that six chromosomal regions on chromosomes 1, 2 (two regions), 3, 6 and 8 appear to be associated with the formation of embryo-like structures (ELSs) from microspores or the subsequent formation of regenerable callus from the ELSs. Regions at the end of the long arm of chromosome 2 and on the long arm of chromosome 8 appear to be associated with ELS formation, and the other regions appear to be associated with either ELS or regenerable callus formation or both. Certain regions that we have identified are the same as those found in other studies to be important for friable, embryogenic callus formation (chromosomes 1 and 3 and near the centromere of 2) and for ESL formation (chromosomes 1 and 3). This study has provided evidence for the genetic basis of the maize anther culture response and callus formation.     

大麦不同基因型游离小孢子的直接培养再生及培养体系的优化

以微搅拌法建立了小孢子直接游离的预处理和培养程序。在大田生长的４个对培养反应不同的大麦基因型上,以新鲜幼穗游离小孢子进行直接培养,均成功地诱导了胚状体并获得再生绿色植株。小孢子的发育进程说明,直接游离的小泡子在预处理过程中的发育要慢于在花药中预处理的小孢子,而且其培养效率也较低。直接游离小孢子的培养密度以０．８～１．０×１０５／ｍｌ较理想,至少应不低于６×１０４／ｍｌ．８％－１０％的糖浓度可明显提高小孢子分裂频率和胚状体诱导频率。实验结果也表明两种培养基ＦＨＧ和ＭＮ６无明显差异,均适宜于直接游离的小孢子培养,并对游离小孢子直接培养在理论和应用上的意义进行了讨论     

The influence of culture conditions on anther culture response of commercial varieties of Solanum tuberosum L.

The effect of media manipulatioss, temperature pretreatment, carbohydrate source, and seasonal variation on tetraploid potato anther cultures was investigated. The anther culture responses of three commercial Nordic potato varieties from Scandinavia and two from Germany were compared on different media manipulations. With most of the varieties, solid MS media gave better yields than other published media manipulations. Pretreatments at +6°C and at +30°C were studied on Pito and Danva varieties. The +6°C pretreatment and no pretreatment had the same effect on the anther culture response of cv. Pito, while with cv. Danva pretreatment at +6°C promoted embryogenesis. The +30°C pretreatment had no positive effect on anther culture response on either cultivar. The effect of maltose, melibiose and mannitol individually and in combination with sucrose were compared to normal sucrose medium in cv. Pito anther cultures. Anthers incubated on normal sucrose medium gave the highest embryoid and plant yields; the second highest plant yields were obtained on pure maltose medium. Strong seasonal variation was observed throughout the year in cv. Pito anther cultures. The percentage of anthers producing embryoids ranged from 15–20% during September and October to just 1–3% from February through May. The annual average embryoid production rate was 6.18%.     

Histology of embryogenic responses in soybean anther culture

In order to clarify the embryogenic responses in soybean anther culture, anthers of four cultivars were cultured under known conditions to trigger androgenic response. A histological study was performed with anthers in vivo and with approximately 100 explants sampled after 9, 12, 15, 18, 21, 30 and 45 days of culture. In vitro culture triggered the frequent accumulation of phenolic compounds on the locular and anther surfaces, and also caused the destruction of cells and tissues in complex structure such as the tapetum, microspores and pollen grains. Somatic embryogenesis of unicellular origin was observed from the epidermis and the middle layer, and of multicellular origin from connective calluses. No androgenic response could be observed in the anthers of these four soybean genotypes, in the medium and conditions indicated. We point out to the need of changing the approach to the study of androgenesis in soybean, either by using culture conditions unfavourable to the proliferation of diploid tissues, or by culturing isolated microspores.     

Inheritance of competencies for leaf disc regeneration,anther culture,and protoplast culture in Solanum phureja and corrleations among them

Competence for leaf disc regeneration, anther culture, and protoplast culture was examined in the parental, F₁, and F₂ generations of a population of the diploid, cultivated, primitive potato, S. phureja (2n=2x=24). The parental pair consisted of AM3-8, an anther culture derived homozygous diploid, and NBP2, a heterozygous, field selected line. AM3-8 produced embryos in anther culture, and shoots on cultured leaf discs, but its cells did not divide after protoplast isolation. Cells of NBP2 divided to form calli and shoots in protoplast culture, but the clone did not respond to anther culture or leaf disc regeneration. All the individual plants in the F₁ generation were responsive to both anther and protoplast culture; however, there was segregation for the ability to regenerate shoots from leaf discs. The F₂ population, the result of a sib-cross, segregated for all three tissue culture competencies. Segregation data fit a one gene model for anther culture competence with the homozygous dominant genotype expressing the highest response, the heterozygote resulting in a marginal response, and the homozygous recessive resulting in no response. A two-gene model applied to the protoplast culture data, with a dominant allele at both loci required for division to occur after protoplast isolation. Leaf disc regeneration data could only be explained by a two gene model with recessive alleles at each locus required for the highest response, a dominant allele at either of the loci resulting in a marginal response, and dominant alleles at both loci resulting in no response. No significant correlation was found among these traits, implying three separate genetic mechanisms which segregate independently.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Improved androgenesis of interspecific potato and efficiency of SSR markers to identify homozygous regenerants

Three interspecific diploid potato hybrids between selections of Solanum phureja Juz. & Buk. and S. chacoense Bitt. were used in anther culture experiments to construct a monoploid family. Different aspects of the anther culture process were affected by the treatments, such as: growing conditions of donor plants, ways of preparing the anther culture medium, and conditions of anthers in culture. Genotype and date of culture initiation were among the most significant sources of variation. Significant improvements in anther culture response were achieved by growing plants at 30°C and by a heat shock of 35°C for 12 h given to anthers in culture, which gave an increase of up to 40% in embryo yield. However, the heat shock reduced the plant regeneration rate. The majority of regenerated plants was diploid, suggesting that there were several recessive lethal alleles in heterozygous status in the anther-donor. Among the regenerants, the homozygotes could be successfully identified by simple sequence repeat analysis, using eight polymorphic primer pairs. This revised version was published online in June 2006 with corrections to the Cover Date.     

Wheat anther culture: effect of genotype and environmental conditions

Twenty-two cultivars and lines of winter and spring wheat (Triticum aestivum L.) were studied, most for the first time, for their anther culture response. The response was genotype dependent. Plants grown in the field gave higher callus induction frequency than those grown in the greenhouse and the controlled environment chamber. Donor plants grown in a season of low drought stress as compared to a season of severe drought stress resulted in a higher frequency of callus induction. Spherical microcalli were observed in two wheat genotypes in some of only those anthers that were placed with only one loculus in contact with the medium. Wheat lines that were more responsive to anther culture were identified.     

Anther Factor(s) in Barley Anther Culture

The effects of anther tissues were studied systematically on microspores forming multicellular units and furthermore on pollen callus formation in the anther culture of Hordeum vulgare (cv. Sabarlis). Anther productivity was found to be greatly enhanced by use of medium previously conditioned by anthers. In 15 experiments observed, anthers produced 26 times on average more calli in the conditioned medium than in control, in a few cases, even more than 80 times more calli formed. According to this, the authors supposed that cultured anthers released some components, anther factor (s) (AF), which is important to androgenesis in the culture. To achieve high yields of callus, culture was restricted to anthers which had been subpected to cold pretreatment. The temperature stress could not be replaced by the AF. However, for conditioning medium, anthers at binuclear stage were found to be more effective than the test anthers either with or without the pretreatment. Anthers from other 8 barley varieties were also effective for conditioning, as the difference of anther productivity still existed in the culture with conditioned medium between various genotypes tested. Anther response and callus yield were increased in both the culture of anthers at mid and late-uninuclear stage by use of conditioned medium. AF interacted synergistically with m-inositol. Cytological observation showed that AF increased apparently the formation of MPGs, while m-inositol mainly stimulated callus formation from MPGs. To some extent, the effect of exogenous hormone(s) could be replaced by AF. The anther response and pollen callus yield could be much enhanced by increasing anther inoculation density, which also raised the AF level in the culture. Thus, by use of the temperature stress prior to anther culture and culture of test anthers in conditioned medium with m-inositol, or at higher inoculation density, a very high production of pollen callus could be obtained in barley anther culture. For meeting the more specialized requirements of less responsive species or genopypes, the principles given here may be provide some basic information.     

Evidence for microspore embryogenesis in wheat anther culture

Summary In wheat, plants may be regenerated from microspores via direct embryogenesis or organogenesis or embryogenesis from callus. Light and scanning electron microscopy were used to carefully study morphogenesis of microspore-derived plants from anther culture on modified 85D12 starch medium and to determine whether the plants were formed via organogenesis or embryogenesis. Our results indicate that plants are formed via embryogenesis from microspores. Evidence for embryogenesis included the formation of the epidermis and a suspensorlike structure (21 days after culture), followed by initiation of an apical meristem, differentiation of the scutellum, and embryo elongation. At 28 days in culture, the embryo possessed a well-developed scutellum and axis with suspensor. Embryogenesis was further confirmed by coleoptile and radicle elongation during germination when the embryos were cultured on medium supplemented with kinetin with or without coconut water. In this system, an average 67 microspores per responsive anther began cell division but only 3.69 embryos were formed per responsive anther after 6 wk. Adventitious embryos could be induced if the embryos, once formed, remained on initiation medium for 10 wk instead of being transferred to regeneration medium. Developmental stages which may be amenable to changes that could enhance plant production were identified. The potential to use this information to enhance plant production is discussed.     

Screening South African wheat germplasm for androgenic competence

Microspore and anther cultures provide an opportunity to create haploid and doubled haploid plants within a single season, thereby reducing the time and cost of cultivar development. Microspore and anther culture has been widely used and incorporated into wheat breeding programs in many countries, but little is known about the effectiveness of these techniques on South African germplasm. By using two responsive genotypes, isolated microspore culture was shown as more effective at revealing androgenic competence, and was used to evaluate the response of four South African inbred lines and two hybrids. Inbred lines A and B were highly responsive (336 and 207 embryo-like structures [ELS] per 100 anthers, respectively), line D was slightly responsive (5.1 ELS per 100 anthers) while line C was recalcitrant. The hybrid A × C was highly responsive (274 ELS per 100 anthers), and B × D did not respond at all. Green plant regeneration in a local genotype was very low (1% for line B) compared to that of foreign genotype (17% for Pavon 76). Similarly to other wheat genotypes grown around the world, the responsiveness of the South African varieties is also very variable. Thus, more efforts are needed so that isolated microspore culture can become a general tool in breeding programs.     

Reciprocal effects in anther cultures of wheat hybrids

This study was conducted to determine the reciprocal effects for anther culture response in wheat (Triticum aestivum L.) using a set of 4 × 4 full diallel crosses. Both reciprocal and nuclear genetic effects were highly significant for anther culture response and useful for selection and breeding purposes. General combining ability (GCA) effects were predominant for all investigated anther culture traits. Also, significant differences for specific combining ability (SCA) effects were detected between reciprocal crosses. Although significant reciprocal differences for responding anther, callus number and green plant regeneration were recorded in some reciprocal crosses, there were no significant reciprocal differences for albino plant regeneration. The use of one parent as male or female could lead to change at the production of green plants from the F₁ hybrids and screening of inbred lines for response to anther culture, without reciprocal effects, could decrease the utilization of breeding material.     

Effect of genome, induction medium and temperature pretreatment on green plant production in durum ( Triticum turgidum var. durum ) x bread wheat ( Triticum aestivum L. em Thell) crosses

The recalcitrancy of durum wheat (Triticum turgidum var. durum) to anther culture, was attempted to be overcome by transferring the responsible genes form bread wheat B-genome to the respective on durum wheat, determining an appropriate induction medium and clarifying the necessity of cold pretreatment. For this, three durum wheat cultivars were crossed to two bread wheat (Triticum aestivum L. em Thell) cultivars. The resulting F₁ plants and their original cultivars were grown in the field and anthers at the appropriate microspore stage were cultured on potato-2 and W₁₄ media with and without low temperature pretreatment. No green plants were produced from the parental durum wheat cultivars. In contrast, green plants were produced from the F₁ plants. The best results in three of the four F₁ hybrids were recorded when potato-2 was used as induction medium. A more variable response of the examined genotypes was noticed with respect to temperature pretreatment. Regarding green plant production, a negative effect of cold pretreatment was observed in two of the F₁ hybrids when they were cultured on potato-2. Chromosome counts on root tips from the resulting green plants revealed that they all carried D-genome chromosomes. The last observation could suggest that D-genome chromosomes are necessary for anther culture response in wheat. Yet, the production of one green plant with 15 chromosomes may indicate that the development of extracted durum genotypes from bread wheat genotypes with good response to in vitro anther culture might be possible. Further work however, is needed for this to be verified.     

Anther and microspore culture ofLupinus albus in liquid culture medium

Embryo-like structures (ELS) with clearly defined cotyledons and radicles have been regenerated fromLupinus albus L. microspores. ELS induction from microspores in liquid medium was successful, with a maximum of over 3,500 ELS per anther being produced from microspores predominantly at the early binucleate stage of development. Cytological analysis revealed that first pollen grain mitosis occurred in closed buds with maximum ELS production being obtained from buds at the point of first petal emergence. Generally there was a lack of synchronisation of microspores within anthers from the less mature bud germination from ELS has not been observed; recurrent somatic embryogenesis occurred following internal cleavage within the ELS or on the surface of the ELS. Methods of increasing the level of mature ELS capable of germination are under investigation.Abbreviations BA 6-benzyladenine - 24-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - 2iP N-isopentyl-adenine - GA₃ gibberellic acid - S&H Schenk & Hildebrandt medium (1972) - M&S Murashige & Skoog medium (1962) - N&N Nitsch & Nitsch medium (1969) - B5 Gamborg's B5 medium (1968) - NNB5 N&N salts plus B5 vitamins - ELS Embryo Like Structures - DAPI 4, 6-Diamidino-2-Phenylindole     

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.