Efficient callus induction and plant regeneration from mature embryo culture of winter wheat (Triticum aestivum L.) genotypes

Immature and mature embryos of 12 common winter wheat (Triticum aestivum) genotypes were cultured in vitro to develop an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of both embryo cultures. Fifteen days after anthesis, immature embryos were aseptically dissected from seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of Murashige and Skoog (MS) and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were moved slightly in the imbibed seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg/l 2,4-D for callus induction. The developed calli and regenerated plants were maintained on 2,4-D-free MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Regenerated plantlets all maintained the hexaploid chromosome number. A strong genotypic effect on the culture responses was found for both explant cultures. Callus induction rate, regeneration capacity of callus and number of plants regenerated were independent of each other. Mature embryos had a high frequency of callus induction and regeneration capacity, and therefore, being available throughout the year, can be used as an effective explant source in wheat tissue culture. Received: 4 February 1997 / Revision received: 1 April 1997 / Accepted: 5 May 1997     

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.     

Reciprocal substitutions analysis of embryo induction and plant regeneration from anther culture in wheat (Triticum aestivum L.).

Reciprocal substitutions for all chromosomes between the hard red winter wheat cultivars Wichita and Cheyenne were used to investigate the effects of individual chromosomes, as well as their interactions with the genetic background, on androgenesis. Duplicate lines for each chromosome were included to check background homogeneity. Six experiments, two for each genome, were performed. In each experiment, 14 substitution lines, their 14 duplicate lines, and the two parental genotypes ('Cheyenne' and 'Wichita') were studied. The experimental design was a randomized block with three replications. 'Wichita' and 'Cheyenne' differed significantly in embryo yield and green plant regeneration (except green plant regeneration for the B-genome tests) and were equal for albino and total plant regeneration. Embryogenesis was influenced by some chromosomes of the A, B, and D genomes; green plant production was influenced by all chromosomes of the A and D genomes except 5D; albino and total plant regeneration were affected by some chromosomes of the B and D genomes. Reciprocal effects were obtained with chromosomes 1A, 7A, 1B, 5B, 1D, and 2D for embryogenesis, chromosomes 2D and 7D for green plant regeneration, and chromosome 2D for total plant regeneration. Reciprocal substitution lines revealed reciprocal effects of homologous chromosomes, as well as interactions between substituted chromosomes and their specific genetic background.     

In vitro mature embryo culture protocol of einkorn (Triticum monococcum ssp. monococcum) and bread (Triticum aestivum L.) wheat under boron stress

Mature embryos of einkorn (Triticum monococcum ssp. monococcum) and bread (Triticum aestivum L.) wheat were used for callus induction on media containing four different doses (0, 1, 2 and 4 mg L^?1) of 2,4-D and dicamba supplemented with five different boron concentrations (0, 6.2, 12.4, 24.8, and 37.2 mg L^?1). The obtained callus was transferred to culture media with three (0, 0.5, and 2 mg L^?1) different BAP doses with five boron concentrations for further regeneration. The maximum callus weight in einkorn wheat was in culture media with 1 mg L^?1 dicamba and 6.2 mg L^?1 (3.71?±?0.13 g). Bread wheat had the maximum callus weight on culture media with 4 mg L^?1 dicamba and 12.4 mg L^?1 (3.46?±?0.40 g). The highest plantlet numbers were in only 2 mg L^?1 BAP (2.92?±?0.88) for einkorn wheat and 0.5 mg L^?1 BAP supplemented with 6.2 mg L^?1 boron (3.71?±?1.12) for bread wheat. This indirect regeneration protocol using mature embryos of einkorn and bread wheat under boron stresses expected to be useful for future wheat breeding studies.

     

Cefotaxime stimulates callus growth,embryogenesis and regeneration in hexaploid bread wheat (Triticum aestivum L em. thell)

The tissue culture responses of wheat are known to be affected by the type and concentration of growth regulator in the medium and the genotype of the tissues in culture. In this report the effects of the cephalosporin antibiotics, carbenicillin and cefotaxime, on the growth, organogenesis, embryogenesis and regeneration of wheat calli in culture are described. Cefotaxime significantly improved the performance of wheat in culture as measured by these characters. The potential uses of cefotaxime for stimulating regeneration and inducing embryogenesis are discussed.     

A new,endosperm-supported callus induction method for wheat (Triticum aestivum L.)

A new, endosperm-supported callus induetion method was developed using mesocotyls of mature wheat embryos. After seed germination under aseptic condition, most of the germ tissues were cut off and only a few mm of the mesocotyl tissue with the scutellum was used for callus induction. The seeds were placed furrow downwards in 2,4-D solution (6–8 mg l^-1). Proliferating callus tissues were already observed on the cut surface of the mesocotyls on the 2nd day after inoculation. On the MS nutrient medium, callus formation from the isolated scutella with attached mesocotyls was negligible even after 6 days. For shoot and root regeneration, the calli produced up to 10 days were removed from the seeds and transferred onto a hormone-free MS medium. As shown by histological methods, the plantlets regenerated via organogenesis.     

Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat (Triticum aestivum L.)

 Anthers from a doubled-haploid line of spring wheat (Triticum aestivum L.) cv. Pavon 76 were plated in liquid P-4 medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at four concentrations (0.5, 1.0, 2.0, 4.0 mg/l) for 5, 10, 15, and 25 days before being transferred to another medium with the same or reduced 2,4-D concentrations for the remainder of the induction phase for a total of 45 days. Incubation with 0.5 mg/l 2,4-D for 45 days produced lower callus yield and plant regeneration, indicative of insufficient auxin for callus induction. Callus yield and regeneration frequencies were higher with 1.0 mg/l 2,4-D. With 2.0 or 4.0 mg/l 2,4-D, an induction period of 10 or 15 days was sufficient for initiation of callus development. The extended presence of 2–4 mg/l 2,4-D in the medium beyond the initiation phase was detrimental to plant regeneration. Thus optimal callus induction and plant regeneration could be obtained through manipulating the 2,4-D concentration and the duration of its presence in the induction medium. Received: 1 December 1997 / Revision received: 15 February 1999 / Accepted: 26 February 1999     

Cytoplasmic effects on the tissue culture response of wheat (Triticum aestivum) callus

Summary Calli were initiated from immature embryos of eight lines of hexaploid wheat (Triticum aestivum L. em. Thell) with different cytoplasms, the euplasmic nuclear donor Chinese Spring and seven alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. The calli were found to differ in their initial growth rates, their sensitivity to 2,4-D and their ability to organise shoot primordia, demonstrating that the cytoplasm can significantly affect the behaviour of tissues in culture. The potential for improving the responses of tissues in culture by cytoplasmic changes is noted.     

小麦根愈伤组织胚胎发育过程研究

实验通过对６个人工合成小麦品系和对照品种“中国春”种子根愈伤组织分化形成再生植株的过程进行形态和组织切片观察,发现分化初期有２种途径,一种是从愈伤组织先形成不定胚,然后再发育成不定芽和不定根,另一种途径是直接从愈伤组织中分化发育成不定根和不定芽;分化后期不定芽和不定根生长发育有３种类型：一种是不定芽发育先于不定根,一种是不定芽与不定期不定芽和不定根生长发育有种类型：一种是不一定芽发育先于不定根,一     

Agrobacterium-mediated In-planta transformation of bread wheat (Triticum aestivum L.)

Journal of Plant Biochemistry and Biotechnology - Agrobacterium-mediated in-planta transformation method allows efficient plant transformation without tissue culture. In the present study, a tissue...     

The interaction of genotype and culture medium on the tissue culture responses of wheat (Triticum aestivum L. em. thell) callus

The effect of the interaction of genotype and culture medium on the initiation of callus from immature embryos and subsequent plant regeneration was investigated in eight hexaploid wheat lines. Intervarietal differences in culture response and interaction of the genotype with coconut milk are reported. The relative contributions of media and genotype effects to culture performance are assessed. The observation that primordia and shoot development was promoted by coconut milk in some lines and inhibited in others is particularly significant given that coconut milk is widely used to try to improve culture response. This report shows that this effect is dependent on the genotype of the tissues in culture.     

High efficiency plant regeneration by somatic embryogenesis from callus of mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor)

Summary Tissue culture methods were developed for reproducible induction and maintenance of embryogenic (E) callus established from developmentally mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor). Embryogenic callus was obtained by culturing seeds and mature embryos of wheat on Linsmaier and Skoog’s (LS) medium containing 5 or 2 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D), respectively, and for sorghum mature embryos on LS medium containing 2 mg/1 2,4-D plus 0.5 mg/liter kinetin. Plant regeneration from E callus was achieved for several months and quantified on a fresh-weight basis of E callus. Phenotypically normal plants were regenerated from E callus cultured on LS medium supplemented with 0.1 mg/liter IAA plus 0.5 mg/liter benzyladenine (BA) for wheat and 1.0 mg/liter IAA plus 0.5 mg/1BA for sorghum. Wheat research was funded by the United States Agency for International Development, Washington, DC, cooperative agreement DNA-4137-A-00-4-53-00. Sorghum research was supported by the Gas Research Institute, Chicago, IL, contract 5084-260-0973. Expert technical asistance was provided by Nitschka S. ter Kuile, Barbara J. Ashton, Laurie Osborne, Erin Scott, and Kathleen M. Petersen.     

Somatic embryogenesis and morphogenesis in callus derived from the epiblast of immature embryos of wheat (Triticum aestivum)

Factors affecting the formation of embryogenic callus from the epiblast of immature embryos of wheat (Triticum aestivum L.) are described. Embryos were incubated on a modifed Murashige and Skoog medium with the scutellum in contact with medium. Callus formation from the epiblast was affected by the type of cultivar used, the stage of embryo development, and the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium. The number of embryos forming embryogenic callus ranged from 10% to 72%, depending on the cultivar. Embryo development was classified into five distinct morphological stages and higher yields of callus were produced using embryos excised at stages II and III. Concentrations of 2,4-D higher than 1 μM were required for the formation of embryogenic callus. Epiblast callus gave rise to embryoids which differentiated into multiple plants at a high frequency when placed on hormone-free medium.     

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     

Auxin and sugar effects on callus induction and plant regeneration frequencies from mature embryos of wheat (Triticum aestivum L.)

Summary Genetic engineering of cereals currently depends on the use of tissue culture and plant regeneration systems. In wheat (Triticum aestivum L.), immature embryos are the most widely used explant to initiate cultures, but they are inconvenient due to their temporal availability and production requirements. Mature embryos are easily stored and are readily available as mature seeds. However, plant regeneration frequencies from cultures derived from mature embryos are generally low. This research was undertaken to improve callus induction and plant regeneration from wheat mature embryos of cultivar ‘Bobwhite’. The effects of four auxins [2,4-dichlorophenoxyacetic acid (2,4-D): 3,6-dichloro-o-anisic acid (dicamba); 4-amino-3,5,6-trichloropicolinic acid (picloram): and 2-(2-methyl-4-chlorophenoxy) propionic acid (2-MCPP)], and the effect of maltose vs. sucrose under filter sterilized and autoclaved conditions were evaluated. All auxin treatments resulted in callus induction except 2 MCPP. A highly significant effect of auxin type on both callus and plantlet production was detected, though interactions were observed. The effect of sugar type was dependent on the type of auxin used. Substitution of sucrose by maltose enhanced the regenration ability of callus from embryos cultured on media containing 2,4-D and picloram, but caused an opposite effect on media containing dicamba. Picloram significantly enhanced callus growth, however, embryogenic response and plant regenerability were low. Relative to 2.4-D, dicamba (18μM) resulted in a twofold increase in the number of plants regenerated per embryo and reduced the amount of time required for plant regeneration by 3–4 wk. Mention of a trademark or proprietary product does not constitute a guarantce or warranty by the University of Wisconsin and does not imply its approval to the exclusion of other products that may be suitable.     

Comparison of two methods for callus culture and plant regeneration in wheat (Triticum aestivum)

Callus growth and development involve a complex relationship between the explants used to initiate callus, the constituents of the medium and the environmental conditions during culturing. Use of high molecular weight osmotica such as polyethylene glycol (PEG-4000) results in non-solidification of agar medium used for culturing and selection. Thus, a new filter paper bridge technique was compared with the existing agar medium for callus initiation, multiplication, and plant regeneration of wheat. The yield of both total and embryogenic callus was doubled and significantly higher number of regenerants was obtained on filter paper bridges compared to agar medium.     

Nonrandom chromosome variation and morphogenic potential in cell lines of bread wheat (Triticum aestivum L.).

A cytogenetical analysis of 18 cell lines, 9 microspore derived, 6 anther derived, and 3 immature-embryo derived, of bread wheat (Triticum aestivum L.) varying in their morphogenic potential was undertaken. Chromosome variation, both structural and numerical, was detected in all lines studied. Variation was present and, in some cases quite extensive, in the earliest samples taken (only 12 weeks after initiation of the suspensions). Within any culture, the pattern and extent of variation changed throughout the course of the study and cells with a euploid constitution generally decreased in frequency with culture age. Among the nine microspore-derived suspensions, morphogenic lines generally showed a more restricted range of chromosome numbers and higher proportions of euploid cells than nonmorphogenic lines. The patterns of distribution of chromosome numbers among the anther-derived cultures were similar to those of the microspore-derived lines but the correspondence between instability and regenerative capacity was less. The immature embryo derived lines, which were neither regenerable nor morphogenic, were all unstable. The anther-derived lines were sampled over several months to determine whether loss of morphogenic potential was related to changes in chromosome instability of specific lines. Analysis of the "elite" line Fl.7, initially capable of regenerating green plants, showed that substantial decreases in the frequencies of normal euploid cells (from 45 to 5%) occurred over the period when morphogenic capacity was lost. However, whether the chromosome instability resulted in loss of morphogenicity or vice versa was not clarified. C-banding analyses of lines Fl.7 and C82d indicated that instability was not random with respect to the three genomes (A, B, and D) of wheat nor to the different chromosomes within the genomes. Chromosomes of the B genome were most often lost or involved in rearrangements, with breakpoints located at, or near, the heterochromatic blocks. Because of the heterogeneity of the cell lines, extensive analyses of large numbers of cells would be required before it would be possible to determine whether loss of morphogenic potential arises as a result of specific chromosome loss(es).     

Distribution of gluten proteins in bread wheat (Triticum aestivum) grain

Background and Aims

Gluten proteins are the major storage protein fraction in the mature wheat grain. They are restricted to the starchy endosperm, which forms white flour on milling, and interact during grain development to form large polymers which form a continuous proteinaceous network when flour is mixed with water to give dough. This network confers viscosity and elasticity to the dough, enabling the production of leavened products. The starchy endosperm is not a homogeneous tissue and quantitative and qualitative gradients exist for the major components: protein, starch and cell wall polysaccharides. Gradients in protein content and composition are the most evident and are of particular interest because of the major role played by the gluten proteins in determining grain processing quality.

Methods

Protein gradients in the starchy endosperm were investigated using antibodies for specific gluten protein types for immunolocalization in developing grains and for western blot analysis of protein extracts from flour fractions obtained by sequential abrasion (pearling) to prepare tissue layers.

Key Results

Differential patterns of distribution were found for the high-molecular-weight subunits of glutenin (HMW-GS) and γ-gliadins when compared with the low-molecular-weight subunits of glutenin (LMW-GS), ω- and α-gliadins. The first two types of gluten protein are more abundant in the inner endosperm layers and the latter more abundant in the subaleurone. Immunolocalization also showed that segregation of gluten proteins occurs both between and within protein bodies during protein deposition and may still be retained in the mature grain.

Conclusions

Quantitative and qualitative gradients in gluten protein composition are established during grain development. These gradients may be due to the origin of subaleurone cells, which unlike other starchy endosperm cells derive from the re-differentiation of aleurone cells, but could also result from the action of specific regulatory signals produced by the maternal tissue on specific domains of the gluten protein gene promoters.     

The origin of the B-genome of bread wheat (Triticum aestivum L.)

Understanding the origin of cultivated wheats would further their genetic improvement. The hexaploid bread wheat (Triticum aestivum L., AABBDD) is believed to have originated through one or more rare hybridization events between Aegilops tauschii (DD) and the tetraploid T. turgidum (AABB). Progenitor, of the A-genome of the tetraploid and hexaploid wheats has generally been accepted to be T. urartu. In spite of the large number of attempts and published reports about the origin of the B-genome in cultivated wheats, the donor of the B-genome is still relatively unknown and controversial and, hence, remains open. This genome has been found to be closely related to the S-genome of the Sitopsis section (Ae. speltoides, Ae. longissima, Ae. sharonensis, Ae. searsii, and Ae. bicornis) of the genus Aegilops L. Among Sitopsis species, the most positive evidence has been accumulated for Ae. speltoides as the progenitor of the B-genome. Therefore, one or more of the Sitopsis species were proposed frequently as the B-genome donor. Although several reviews have been written on the origin of the genomes of wheat over the years, this paper will attempt for the first time to review the immense literature on the subject, with a particular emphasis on the B-genome which has attracted a huge attention over some 100 years. The ambiguity and conflicting results in most of the methods employed in deducing the precise B-genome donor/s to bread wheat are also discussed.