Nuclear genes affecting albinism in wheat (Triticum aestivum L.) anther culture

Summary Inheritance of the ability to respond in wheat anther culture was studied from 6×2 reciprocal crosses between six varieties with high and two varieties with low capacity for green plant formation and their parents, replicated in two environments. Effects of genotypes dominated embryo formation and percentages of green plants, accounting for 78.4% and 85.4% of total variation, respectively, while smaller genetic effects were indicated for regeneration. Nuclear genes could explain almost all the genotype effects in this material. Embryo formation showed heterosis over high parent for 5 of the 12 hybrids, while percentages of green plants from the hybrids were intermediate to the parents. General Combining Ability (GCA) could explain 78.8% of the variation for embryo formation among the hybrids, whereas differences in percentage of green plants were dominated by Specific Combining Ability (SCA), accounting for 67.9% of hybrid variation. A positive correlation (r=0.81^**) was observed between the genetic capacity for regeneration and green plant formation. Analysis of covariance indicated that effects causing GCA for green plant formation were mainly responsible for this correlation. A regression model with two parallel lines divided the six parent lines with high green plant formation into three groups with respect to their reactions with the two testers. The results are discussed with regard to possible involvement of two sets of nuclear genes affecting the percentage of green plants obtained in wheat anther culture: one set consisting of mainly additive effects affecting green plant percentage through an initial effect on regeneration ability, and another set of two or a few more major genes with dominance or epistatic effects uncorrelated with regeneration.     

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 the 1B/1R translocation on anther culture ability in wheat (Triticum aestivum L.)

Using two varieties, their reciprocal hybrids, F₈ lines and doubled haploids, results confirmed that three genetic components are involved in wheat anther culture ability, viz embryo induction frequency, regeneration ability and the frequency of albinism. In these experiments, no significant maternal effects were noticed. For embryo yields, transgressive lines were obtained from hybrids between distant genotypes. Regeneration of green plants depended upon two independent traits: regeneration ability and the frequency of albinos. F₈ lines and two doubled haploids equaled the 50% regeneration rate of the hybrids, but they only regenerated green plants. Based upon cytological examination and gliadin patterns, it is suggested that genes favoring regeneration ability could be linked to the 1BL-1RS translocated chromosome from Aurora.Abbreviations DH doubled haploids - MS Murashige and Skoog - MPG multicellular pollen grains     

Inheritance of anther culture derived green plantlet regeneration in wheat (Triticum aestivum L.)

A study was set up to determine the inheritance and combining ability of the factors anther culture response and green plant regeneration. Reciprocal crosses were made between cultivar Ringo Sztar, showing high anther culture response and the cultivars Ciano 067 and Benoist H77022, showing a high level of green plant regeneration. Averaged over all genotypes, 23.0% of the anthers responded and a callus induction frequency of 77.8% was observed. Of all the embryos, 43.0% developed into plantlets, 25.6% of the regenerants being green, the result being that 3.3 green plants per 100 anthers were formed. Genotypic effects accounted for 57.7%, 86.3% and 77.5% of the total variance of anther culture response, callus induction frequency and embryo induction frequency, respectively. Additive and dominant gene action was detected for all characteristics, including green plant regeneration. No reciprocal differences were found for anther culture response, embryo induction frequency and green plant regeneration, indicating no cytoplasmic effects. A small but significant reciprocal difference was found for callus induction frequency. Embryo production was primarily correlated with anther culture response and not with the number of embryos produced per plated anther or per responding anther. Possible mechanisms for the inheritance of green plant regeneration are discussed.Abbreviations CIRA callus induction frequency per responding anther - ERA embryo induction frequency per responding anther - FHB fusarium head blight - MS-medium Murashige & Skoog (1962) medium - REML residual maximum likelihood     

Improved plant regeneration from wheat anther and barley microspore culture using phenylacetic acid (PAA)

Summary The effect of the auxin phenylacetic acid (PAA) on wheat anther and on barley anther/microspore culture was investigated. With PAA the induction response was not usually significantly different from controls but a significantly higher number of green plants were produced in wheat anther and barley microspore culture. For wheat anther culture 100 mg/L PAA was beneficial. For barley microspore culture the optimum levels were from 1 to 100 mg/L, depending on genotype. In barley anther culture there were no improvements using PAA. In wheat anther culture, 145 green plants/100 anthers were obtained with cultivar VeeryS, while the average response from twelve F₁ hybrids in the breeding program was 332 green plants/100 anthers. At least 1000 green plants were obtained using isolated microspores from 100 anthers in barley cv. Igri. With cv. Bruce, regeneration occurred only when 100 mg/L PAA was used. The influence of PAA appears at the embryogenic phase of the culture system. The possible mechanisms by which PAA may improve regeneration are discussed.     

不同组织培养途径对小麦再生能力的研究

从现在推广的小麦优良品种和有苗头的新品系中选用10个小麦基因型品种进行组织培养,从愈伤组织诱导率、绿苗分化率等方面比较了幼穗培养、花药培养、幼胚培养三种培养方式的培养效果。结果表明,幼胚培养效果最好,基因型间差异小,都能获得足够数量的再生植株。幼穗的培养效果最差,愈伤组织分化生根和绿芽十分容易,但分化成完整植株则较为困难。花药培养在基因型间差异非常明显而且有较多白化苗。此外,本研究还分析了影响小麦再生能力的因素,建立了一套高效、可靠的小麦组培再生系统,为小麦的转基因技术提供优良的受体材料。     

Genetic analysis of anther culture response in wheat using aneuploid,chromosome substitution and translocation lines

Summary Marked effects of genotype on wheat anther culture response have been observed. Genetic factors have been recognised to be one of the major contributors to in vitro responses of cultured wheat tissues. In wheat anther culture, embryo induction, plant regeneration and albina/green ratio have been determined to be heritable traits. Using Chinese Spring (CS) monosomic 1D, single chromosome substitution lines of chromosome 5B or chromosome arm 5BL from Chinese Spring into six varieties, and F₁ hybrids heterozygous for the 1B chromosome structure (1BL-1BS/1BL-1RS), the anther culture response was studied: genes on CS1D chromosome and 5BL chromosome arm increases the embryo frequency; gene(s) involved in regeneration ability are located on the 1RS chromosome arm; a gene increasing albina frequency is located on Chinese Spring 5B chromosome. Our results support the fact that without gametic selection, a differential development occurred from the particular classes of microspores carrying genes for higher regeneration ability. Moreover, in some crosses, a few genes with major effects were involved in determination of anther culture response.     

The effect of osmotic potential on anther culture in spring wheat (Triticum aestivum)

This study was conducted to determine the effect of osmotic potential in a modified 85D12 medium on both callus induction and plant regeneration in the anther culture of two wheat genotypes, cv. Chris and cv. Pavon. Altering the medium osmotic potential by changing the carbohydrate source and concentration or by adding a non-metabolized osmoticum appeared to have the greatest potential for improving anther-derived green plant production. The medium osmotic potentials were varied (-0.67 to –2.30 MPa) by altering sucrose and PEG concentration. Both osmotica affected callus production, with –0.9 to –1.4 MPa media producing the most calluses. Callus production depended on genotype and osmoticum. Only PEG concentration affected green plant regeneration. The greatest number of green plants (11.5 plants per 100 anthers in cv. Chris) was obtained with 0.0125 M of PEG. This experiment suggested that a low level of PEG in the medium was beneficial for producing green plants from wheat anthers.     

Immature embryo and anther culture of chromosome addition lines of rye in chinese spring wheat

Significant variation among Chinese Spring wheat (Triticum aestivum L.) and a set of seven addition lines in which chromosomes from rye (Secale cereale L.) were incorporated into the Chinese Spring background was observed for callus formation and plant regeneration from anther cultures and for plant regeneration from immature embryo cultures. Callus initiation from immature embryo cultures was uniformly high. Rye chromosome 4 contains factors which significantly increase both anther culture responses relative to Chinese Spring. Rye chromosomes 6 and 7 both contain positive factors for regeneration from immature embryo culture. While no correlation was found between anther culture and embryo culture responses, a positive correlation was observed between the two anther culture response variables.     

Effect of genotype and medium on wheat (Triticum aestivum L.) anther culture

Four winter wheat (Triticum aestivum L.) and two spring wheat cultivars were evaluated in anther culture on three to four different media for their ability to initiate callus and green plants. Five media were used in the experiment: stored-potato medium with Ficoll 400, fresh-potato medium with Ficoll 400, fresh-potato medium with agar, fresh-potato liquid medium without agar or Ficoll 400, and a one tep 85D12-3 medium. Greatly different frequencies of calli and/or green plants were obtained from different cultivars and media. The callus initiation frequency varied from 2.7% for Arapahoe to 52% for Pavon, both on the stored potato medium with Ficoll 400. The frequency of green plant regeneration ranged from 0% for Arapahoe and Siouxland on the stored-potato medium with Ficoll 400 and 0% for Redland and Arapahoe in the fresh-potato medium with Ficoll 400 to 12% for Chris in the 85D12-3 medium (one-step procedure). Chris and Centurk 78, previously reported as having high levels of response, had significantly higher (P < 0.05) frequencies of green plant regeneration on the 851312-3 medium than the other cultivars. An unexpected observation is that wet MSC^– medium enhanced callus regeneration more than a drier MSC^– medium.     

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.     

Plant regeneration from protoplasts of indica rice: genotypic differences in culture response

Summary Fourteen varieties of indica rice (Oryza saliva L.) were examined for their capacity for plant regeneration from protoplasts using the nurse culture methods developed for japonica rice. Calli induced from germinating seeds were grouped into two types: type I, white and compact; type II, yellow and friable. In four varieties producing type II callus, colony formation (2%–4.5%) and plant regeneration (2%–35%) were observed. The inability to develop suspension cultures was a major obstacle in regenerating plants from protoplasts of the remaining rice varieties studied.     

Genetic and non-genetic factors affecting anther culture of Brussels sprouts (Brassica oleracea var. gemmifera)

Summary Eight inbred lines of Brussels sprouts and ten F₁ hybrids derived from them were tested for their response to anther culture. From 5–19 plants per genotype were tested, and each plant was tested on 3–6 separate occasions. Results from the inbred lines were broadly similar to those from the F₁ hybrids, despite the inbreds producing fewer buds and having a higher frequency of anther deformities. The maximum embryo yield from an inbred line was 215 embryos per 100 anthers, and from a hybrid was 275. From estimation of the variance components it was calculated that, for both inbreds and hybrids, about half the total variation was genetic whereas variation due to plants within genotypes and to occasions within plants were each about 13% of the total. The narrow sense heritability of responsiveness to anther culture (estimated by the proportion of variation between inbred lines which was genetic) was 0.48, and there was partial dominance for this character. In three cases the hybrid outyielded the better inbred, and this heterosis may well be due to dispersed dominant genes.     

Regeneration of Fertile Barley Plants from Mechanically Isolated Protoplasts of the Fertilized Egg Cell

A simple procedure is described for the mechanical isolation of protoplasts of unfertilized and fertilized barley egg cells from dissected ovules. Viable protoplasts were isolated from ~75% of the dissected ovules. Unfertilized protoplasts did not divide, whereas almost all fertilized protoplasts developed into microcalli. These degenerated when grown in medium only. When cocultivated with barley microspores undergoing microspore embryogenesis, the protoplasts of the fertilized egg cells developed into embryo-like structures that gave rise to fully fertile plants. On average, 75% of cocultivated protoplasts of fertilized egg cells developed into embryo-like structures. Fully fertile plants were regenerated from ~50% of the embryo-like structures. The isolation-regeneration techniques may be largely genotype independent, because similar frequencies were obtained in two different barley varieties with very different performance in anther and microspore culture. Protoplasts of unfertilized and fertilized eggs of wheat were isolated by the same procedure, and a fully fertile wheat plant was regenerated by cocultivation with barley microspores.     

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%.     

Factors affecting anther culturability of recalcitrant barley genotypes

One major problem encountered with cereal anther culture is that some genotypes are low or non-responders to the technique. The objective of this study was to improve anther culture efficiency of recalcitrant barley (Hordeum vulgare L.) genotypes. Reciprocal F₁s between the two low responsive cultivars, Morex and Steptoe, were used. These were chosen because doubled haploids (DH) were required from these genotypes for the North American Barley Genome Mapping project. Ficoll 400 at 200 g l^–1 in the induction medium significantly increased green plant production compared to four other media formations containing different gelling/viscosity modifying agents. Cold pretreatment of donor spikes of 28 vs 14 d resulted in an increase in embryoid, total plant and green plant production. Anther culture response in these experiments was little influenced by donor plant growth conditions. Indole-3-acetic acid (1 mg l^–1) or 1-naphthaleneacetic acid (2 mg l^–1) in the induction medium did not affect anther culturability or plant regeneration. Based on this research, the negative genotypic effect for doubled haploid production could be diminished, which is desirable for practical application.Abbreviations BAP 6-benzylaminopurine - IAA Indole-3-acetic acid - LS Linsmaier & Skoog - NAA 1-naphthaleneacetic acid - DH doubled haploid     

Chlorophyllian durum wheat plants obtained by isolated microspores culture: importance of the pre-treatments

In Triticum turgidum subsp. durum (Desf.) Husn., the utilization of in vitro anther culture is hampered by the very high frequency of albinism of the regenerated plants reaching in most cases 100%. Only in vitro ovary culture or intergeneric crosses with maize produce gynogenetic green haploid and doubled haploid plants. This paper is concerned with another very interesting method of androgenetic doubled haploid plant production, the in vitro isolated microspore culture. It is shown that this method, associated with cold alone or cold plus mannitol pre-treatments, of the spikes kept within their sheath leaves, during different times, have significant positive effects, not only on embryo production, but also on chlorophyllian plant regeneration. All pre-treatments and control taken together, a total of 16 490 embryos was obtained from 17.4 x 10(6) microspores of two T. durum varieties, among which 9320 embryos were transferred to regeneration medium and developed 150 chlorophyllian plants. Thus a long-term (five weeks) 4 degrees C cold pre-treatment of the microspores could be promising for green regeneration in durum wheat.     

Efficient production of wheat-barley hybrids and preferential elimination of barley chromosomes

Summary Intergeneric hybridization between four common wheat cultivars, Triticum aestivum L. cultivars Chinese Spring, Norin 12, Norin 61, and Shinchunaga, and cultivated barley, Hordeum vulgare L. cultivars Betzes, Nyugoruden, Harunanijou, and Kinai 5 were carried out in a greenhouse under 15 – 20 °C and long-day (15 h) photoperiod conditions. Two days prior to pollination, a 100 mg/1 2,4-D solution was injected into wheat stems. Among wheat cultivars, Norin 12, Norin 61, and Shinchunaga showed higher crossabilities than that of Chinese Spring, suggesting the presence of crossability gene(s) other than the kr system of Chinese Spring. Variation was also found among the barley cultivars as male parents. Betzes barley showed the highest crossability with wheat. Thus, the cross Norin 12×Betzes showed the highest crossability (8.25%), followed by Norin 61 ×Betzes (6.04%), Shinchunaga×Betzes (5.00%), and Shinchunaga×Kinai 5 (5.00%). The embryos were rescued by culture at 15–20 days after pollination. Seventyfour plants were obtained from 82 embryos. The morphology of the hybrid plants resembled that of wheat parents. Among 60 seedlings observed, 28 had 28 chromosomes, 8 had 21, 23 had aneuploid numbers of chromosomes (22–27), and 1 had 29 chromosomes. About half of the aneuploid hybrids showed mosaicism for chromosome number. By analyzing five isozyme markers of barley chromosomes, the chromosome constitutions of the aneuploid hybrids were determined. Barley chromosomes 1 and 5 were found to be preferentially eliminated in the hybrids, while chromosomes 2 and 4 were eliminated infrequently. The conditions and genetic factors for high crossability and the tendency of barley chromosome elimination are discussed.     

Effects of n-butanol on barley microspore embryogenesis

Doubled haploid (DH) production is an efficient tool in barley breeding, but efficiency of DH methods is not consistent. Hence, the aim of this study was to study the effect of n-butanol application on DH barley plant production efficiency. Five elite cultivars of barley and thirteen breeding crosses with different microspore embryogenesis capacities were selected for n-butanol application in anther and isolated microspore cultures. Application of 0.1 % n-butanol after a mannitol stress treatment in anther culture significantly increased the number of embryos (up to almost twice) and green plants (from 1.7 to 3 times) in three low-responding cultivars: Albacete, Astoria and Majestic. No significant differences on microspore embryogenesis efficiency were observed in medium and high responding cultivars. The application of n-butanol treatment to isolated microspores from cold treated spikes in thirteen spring breeding crosses with a low or very low androgenetic response did not have a significant effect on the overall number of green plants. Nevertheless, an increase in the number of green plants was observed when 0.2 % n-butanol was applied in four out of seven low-responding crosses. Therefore, application of n-butanol could be routinely applied to anther cultures using mannitol treatment, in low-responding material. However, further studies are needed to determine optimal conditions in protocols using cold treatment and isolated microspore cultures.     

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.