Wheat Anther Culture Research Articles

Embryo yield in wheat anther culture is influenced by the choice of sugar in the culture medium

The effect of employing different sugars in wheat anther culture has been investigated using four Spring wheat cultivars. The most responsive cultivar, Orofen, gave a three to four-fold increase in embryo yield when maltose was used in place of sucrose, with 50 embryos being produced for every 100 anthers cultured. Measurement of sugar concentrations in the culture media indicated that sucrose was more rapidly hydrolysed than maltose. However, neither the osmotic potential of the medium nor the concentration of glucose appeared to be critical factors in determining embryo yield.

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 Growth Regulator and Genotype on Production of Wheat and Triticale Polyhaploids from Anther Culture*

AbstractHomozygous doubled‐haploid plantlets derived from anther culture of wheat (Triticum aestivum L.) and triticale (×Triticosecale Wittmack) are useful breeding materials. However, efficiency of an‐drogenesis needs improvement. We used media (basic components, are the same as 85DI2) each containing one of the seven auxins [2,4,5‐trichlorophenoxyacetic acid (2,4,5–T), P‐chloraphenoxyacetic acid (pCPA), 3,6‐dichloro‐o‐anisic acid (dicamba), 4‐amino‐3,5,6‐trichloropicolinic acid (picloram), indole‐3‐butrytic acid (IBA), indole‐3‐acctic acid (IAA), and 2,4‐dichlorophenoxyacetic acid (2,4‐D) as a control] in combination with 6‐furturyl‐aminopurine (kinetin). In addition, each of the four cytokinins [6‐benzylaminopurine (6‐HA), 2‐isopenlylnyl adenine (2‐ip), 6‐(4‐hydroxy‐3‐meihylbut‐2‐enylamino) purinc (zeatin), and kinetin as a control] was tested in combination with 1‐naphthalene acetic acid (NAA). Anthers containing microsporcs at miduninucleatc stage from live wheat cultivars (Angus, Centurk, Chris, K.itt, and Pavon 76) and two octoploid trilicale lines (T81, T82) were tested mainly for callus induction and polyhaploid production on each of the 11 media. The cultivar × medium interaction was not significant, When averaged over all growth regulators, Pavon was (he best cultivar which produced 14.4 % calli and 23 % polyhaploid plantlets. Averaged over all cultivars, the medium containing 2, 4‐U produced the highest calli (13.9 %). Undifferentiated calli were regenerated on 87T1 medium, which contained IAA (1 mg/1) and kinetin (2 mg/1).

Microspore development in the anther culture of wheat(Triticum aestivum L.).

A cultivar (Chris) of hard red spring wheat (Triticum aestivum L.) and a one-step haploid generation medium were used to study microspore development and callus formation by using an anther culture technique. Cytological observations on microspore development were made 7, 10, 13 and 16 days after the anther was placed on the medium. The first mitotic division of the uninucleate microspore formed two nuclei, which were similar or different in size. Further divisions of the vegetative nucleus produced multicellular pollen grains that continued to grow and then were released from the exine. This extensive division of the vegetative nucleus could be the source of embryoid or callus generation.

The effects of interactions of culture environment with genotype on wheat (Triticum aestivum) anther culture response

The interactions of genotype and several variables related to culture environment, including temperature pretreatment, conditioned medium and agar concentrations were examined in a series of experiments for their effects on percent anthers producing callus and number of embryoids produced per 100 anthers scored. Significant genotypic interaction was observed for both traits with all environmental variables except methods of medium conditioning. Such interactions involve both changes of response magnitude and changes of rank order of genotypes. The highest response frequencies observed were in excess of 30% of anthers callusing. Most lines examined responded relatively well to a culture regime utilizing a 4°C treatment for 7-14 d prior to anther excision, followed by float culture, without transfer and without preconditioning of the culture medium. The results indicate, however, that particular genotypes may have specific requirements with respect to various environmental conditions so that culture conditions may need to be adjusted, especially for the least responsive genotypes.

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

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.

Effects of nucleus, cytoplasm and male sterile nucleus-cytoplasm combinations on callus initiation in anther culture of wheat

Nuclear and cytoplasmic factors affect tissue culture response in wheat (Triticum aestivum), and cytoplasmic male sterility may enhance callus initiation in anther culture. Three wheat nuclear genotypes, each in normal and two alien cytoplasms conferring cytoplasmic male sterility, were evaluated for callus initiation frequency in anther culture. Nuclear genotype had the greatest effect on callus initiation, but cytoplasm and nucleus X cytoplasm interaction also produced significant effects. The nuclear genotype of ‘Chris’ outperformed ‘Butte’ and ‘Coteau’ in all cytoplasms. Ordinary wheat and Triticum timopheevi cytoplasms outperformed Aegilops speltoides cytoplasm. Cytoplasmic male sterility did not increase callus initiation. This suggests manipulating the nuclear genotype is the best strategy for improving the capacity of wheat to initiate callus in anther culture.

Evidence for cytoplasmic control of in vitro microspore embryogenesis in the anther culture of wheat (Triticum aestivum L.)

Anthers were cultured from two sets of seven lines of hexaploid wheat (Triticum aestivum L.) with different cytoplasms, the euplasmic nucleus donors, 'Siete Cerros 66' and 'Penjamo 62', as well as their six alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. Significant cytoplasmic and nuclear effects but no cytoplasmic-nuclear interaction were found for embryogenic anther response, with the best performance of 'Penjamo 62' in Ae. kotschyi cytoplasm. Plant regeneration was not affected significantly by the cytoplasmic background of the lines cultured. The possible genetic implications of the observed cytoplasmic and nuclear influences on the in vitro haploid induction of wheat are discussed.

Fertility of Triticum aestivum plants derived from anther culture

The fertility of plants from wheat anther culture was studied. It was found that one half of the 36 plants with diploid root tips didn't set seeds at all, and that 41 of the 42 plants with haploid root tips were completely sterile. It was surprising that sterility was so widely distributed even among the plants with diploid root tips.

Improvement of Anther Culture Methods for Haploid Production in Wheat

The yield of haploid production in wheat (Triticum aestivum L.) anther culture has been extremely low with the currently employed methods. The objective of this study was to determine whether variation in anther culture media would affect the yield of haploids. After a 48‐h cold pretreatment, anthers from six spring wheat cultivars and one winter wheat cultivar were cultured on liquid and agar‐solidified media, and in some cases the liquid medium was supplemented with Ficoll (Sigma Chemical Co., St Louis, MO). Liquid culture significantly increased callus induction, which averaged 98.3 calli per 100 anthers for the seven cultivars tested, compared with 5.1 calli per 100 anthers for agar culture. Although the calli from liquid media usually had lower plantlet regeneration than calli from agar‐solidified media, regeneration ability was improved by adding 200 g/L Ficoll 400 to the liquid induction medium, or by changing the plant growth regulator composition of the regeneration medium. By employing the liquid culture method and the modified regeneration medium, it is now more feasible to use doubled haploids in plant breeding practice and genetic research.

Gametic selection during wheat anther culture

Gametic selection was investigated in a monosomic 1D line of wheat. Comparison of chromosome numbers in progenies from cross- or self-pollination and anther culture indicated the frequency of the different types of gametes acting in zygote formation. The progenies obtained from reciprocal crosses (monosomic × euploid) were different in terms of frequencies of monosomic plants. All the pollen grains do not have an equal probability of fertilization since a strong gametic selection for euhaploid male gametes was observed when the monosomic line was used as male parent; only 13% of the nullisomic survived to embryogenesis and seed germination. Regenerants from anther culture also demonstrated that the percentage of nullihaploids was reduced, indicating a strong selection for euhaploid gametes. A comparison between the frequency of male nullihaploid gametes in the cross disomic × monosomic and in the anther culture revealed that the two processes generate the same gamete transmission. The in vitro cultures do not induce more selection pressure than the embryogenic development even if they are together biased samples of the male gametic population.

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

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.

Substitution analysis of callus induction and plant regeneration from anther culture in wheat (Triticum aestivum L.)

The genetic determination of callus induction, total plant regeneration and green plant regeneration from anther culture were studied using a "Chinese Spring"/"Cheyenne" substitution series. All the three characteristics were found to be polygenically determined, but their inheritance was independent from one another. The 7A and 18 chromosomes had a considerable effect on callus induction. In the case of total plant regeneration the most influential chromosome "as the 3A while the 2D chromosome showed a definite influence on green plant regeneration. The interaction between the genetic background of the recipient plant and the substituted chromosome plays an important role in the manifestation of the studied features.

Studies of the Genetic Relationship between Anther Culture and Somatic Tissue Culture Abilities in Wheat

AbstractA population of thirty‐eight doubled haploid lines, developed from the F1 between two wheat parents differing in anther culture and somatic tissue culture responses, ‘was used to examine the genetical control of responses to these in vitro systems.During anther culture genetic variation between lines was exhibited for frequencies of callus induction., embryo production and embryo regeneration rates. In addition the relative frequencies of green and albino plants was shown to be genotype dependent. However, there was no correlation, between the frequencies of embryo production and the regeneration rate of those embryos suggesting an independent genetic control of these two components. Transgressive segregation for performance was observed for all components indicating that at least two genes are involved in the response of each, and lines for improved performance, combining high ernoryo production rates and good regeneration capacity were identified.No genetic variation for frequencies of callus induction from immature embryos was observed in this cross. However, genetic variation for the regeneration frequencies of plants was observed. Lines with an improved tissue culture response over the two parents were identified.There was no correlation between the performance of lines in anther culture and somatic tissue culture, indicating separate genetical control, and lines with alternative levels of response to the two systems were identified.

Varietal Differences in Pollen Embryoid Formation in Wheat Anther Culture

Varietal Differences in Pollen Embryoid Formation in Wheat Anther Culture

Plant Regeneration of Pollen Embryoids from Wheat Anther Culture

Plant Regeneration of Pollen Embryoids from Wheat Anther Culture

The effects of KNO3 concentration in callus induction medium for wheat anther culture

KNO3 concentration was found to significantly affect the anther culture of wheat (Triticum aestivum L.). When KNO3 was increased from 0 to 15 mM (in cultivar Jinghua 1) or from 10 to 15 mM (in cultivars 2531-10, Xiaoyan 759 and Norin 10), the callus induction frequency increased significantly. When KNO3 was increased further above 20 mM, the callus induction frequency decreased significantly in all the tested cultivars. The subsequent frequency of green plantlet regeneration increased significantly, and the ratio of green to albino regenerants increased sharply when KNO3 concentration increased. Further experiments found that the decrease of callus induction frequency in the medium with too much KNO3 might be caused by NO3 - ion alone, while the effect of KNO3 on green plantlet regeneration might be caused by both K+ and NO3 - ions, and that the effects of NO3 - concentration were independent of NH4 + concentration in the medium.

Effect of a Modified Potato Medium on Anther Culture of Wheat1

Modification of a potato extract medium was undertaken to increase pollen embryogenesis from anthers of a hard red spring wheat (Triticum aestivum L. cv. Chris). Whole potato tubers (Solatium tuberosum L. cv. Kennebec) were separated into peeled tubers and peel from which extracts were prepared. A 3 ✕ 3 latin square design with three replications in time was used to compare media prepared from: (i) extract of whole potato tubers, (ii) extract of the peel only, and (iii) extract of peeled tubers. A 60% increase in anther induction resulted from the peeled tuber extract medium compared to the whole potato medium. Removal of the peel from tubers probably reduces the concentration of compounds that may be inhibitory to wheat embryogenesis during anther culture. Anther source, defined by spike section (basal, middle, and distal), was a second variable in the experiment but did not significantly affect embryogenesis.

Effects of incubation temperature on microspore callus production and plant regeneration in wheat anther cultures

Anthers of wheat cultivars Orofen and Pitic 62 were incubated for 8 days at 15, 20, 25, 30, 35 and 40°C before transfer to 25°C. Compared with anthers cultured at 25°C constantly, anthers treated at 30°C produced 40% more microspore callus and green plants in both cultivars whereas those treated at 35°C produced 2–3 fold more green plants. Treatment at 40°C was deleterious. Possible modes of action of high temperature on callus production and albinism were discussed.

Two regeneration systems for the production of haploid plants from wheat anther culture

Haploid wheat (Triticum aestivum L.) plants can be regenerated from microspores via two different systems. The first step for both systems is the formation of embryoids from microscopes cultured on P1 media. In one system embryoids can be induced to germinate: embryogenesis. The second system produces plants via green centers: organogenesis. The two system are comparable in the number of green plants produced and the frequency of aneuploid production. The embruogenic system requires half the time in culture and two fewer transfers when compared to the organogenic system, making embrygenesis the more efficient choice for plant production via anther culture.