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     

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.     

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     

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.     

Combining abilities and heritability of callus formation and plantlet regeneration in wheat (Triticum aestivum L.) anther cultures

Summary Frequency of callus formation in wheat (Triticum aestivum L. em. Thell) anthers cultured in vitro and the frequency of subsequent plantlet formation from such calli were examined in a diallel population produced from five inbred spring wheat cultivars. Two of the five cultivars were believed to possess relatively high frequencies of response and the other three relatively low response frequencies, based on previous studies. General and specific combining abilities were estimated and found to be highly significant for both traits. Reciprocal effects were also estimated and were highly significant for both traits. Of the 25 entries, the largest mean callus formation frequency was observed on anthers of Kitt x Olaf, while the largest mean plantlet formation frequency was observed using anthers of the cultivar, Fielder. No significant correlation was observed between the two traits. Heritability estimates in the range of 0.6–0.7 suggested, however, that both traits were highly heritable, so that rapid gain from selection for these traits should be possible. Current limitations due to genetic variation in responses therefore may not constitute a major obstacle to application of in vitro techniques by wheat breeders.Scientific Article No. 3710 Contribution No. 6686 of the Maryland Agric. Exp. Stn., Dept. of Agronomy, College Park, MD 20742, and USDA-ARS, Beltsville, MD 20705, USA     

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.     

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.     

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.     

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.     

Effects of donor plant genotype and growth environment on anther culture of soft-red winter wheat (Triticum aestivum L.)

The effects of donor plant growth temperature and photoperiod on embryo formation and plant regeneration from cultured anthers in five genotypes of soft-red winter wheat (Triticum aestivum L.) were examined. There were no significant differences between the three environments studied (15°C - 16/8 h light/dark, 20°C - 16/8 h light/dark, and 20°C - 12/12 h light/dark) when frequencies were averaged over genotypes; however, significant genotype and genotype x environment interactions were observed for embryo formation. When averaged over environments, highest embryo and plant production frequencies were exhibited by a line derived from the cross IL 72-2219-1/Amigo. A mean of 8.6 embryos per 100 anthers plated was observed for this genotype grown in the 20°C - 16/8 h light/dark environment. The cultivar Scotty averaged 4.2 plants produced per 100 anthers plated when grown in the 15°C - 16/8 h light/dark environment. The results from this study suggest a potential for increasing embryo and plant production in this material and point toward the need to optimize donor plant growth environmental conditions to maximize response frequencies for specific genotypes of interest.     

Plant regeneration from coleoptile tissue of wheat (Triticum aestivum L.)

Plant regeneration was achieved from coleoptile tissue of wheat (Triticum aestivum L. cv. Kharachia-65). Coleoptiles (1.0 - 3.5 cm long) were excised from 2- to 5-d-old seedlings and cultured on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D - 0.5, 2.5, and 5.0 mg dm^-3). Cream, friable callus was obtained after 6 weeks of inoculation. This callus was sub-cultured on MS medium supplemented with 2,4-D (2.5 mg dm^-3) and 5 % coconut water. After 6 weeks of sub-culturing white, cream or pale, friable, nodular callus was obtained. Plant regeneration occurred when this callus was sub-cultured on MS medium supplemented with 0.2 mg dm^-3 1-naphthalene acetic acid + 1.0 mg dm^-3 6-benzylaminopurine. For rooting, regenerated shoots or plantlets were transferred on MS medium supplemented with 0.5 mg dm^-3 indole-3-acetic acid. Rooted plantlets were directly transferred into pots and grown under field conditions. Seed setting invariably occurred in all plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Monosomic analysis of tissue culture response in wheat (Triticum aestivum L.)

Summary The ability of immature embryos of wheat (Triticum aestivum L.) to respond in cell culture was examined in crosses between the Wichita monosomic series and a highly regenerable line, ND7532. Segregation in disomic controls and 13 monosomic families showed a good fit to a monogenic ratio indicating a qualitative mode of inheritance. Segregation in the cross involving monosomic 2D showed a high frequency of regeneration (93.6%) and high callus growth rate (1.87 g/90 days) indicating that 2D is a critical chromosome. Modifying genes may be located on other chromosomes. Substitution of chromosomes from a low regenerable cultivar Vona further indicated that the group 2 chromosomes, in particular chromosome 2D, possess genetic factors promoting callus growth and regeneration.     

Identification of callus types for long-term maintenance and regeneration from commercial cultivars of wheat (Triticum aestivum L.)

Summary Immature embryos, inflorescences, and anthers of eight commercial cultivars of Triticum aestivum (wheat) formed embryogenic callus on a variety of media. Immature embryos (1.0–1.5 mm long) were found to be most suitable for embryogenic callus formation while anthers responded poorly; inflorescences gave intermediate values. Immature embryos of various cultivars showed significant differences in callus formation in response to 11 of the 12 media tested. No significant differences were observed when the embryos were cultred under similar conditions on MS medium with twice the concentration of inorganic salts, supplemented with 2,4-D, casein hydrolysate and glutamine. Furthermore, with inflorescences also no significant differences were observed. Explants on callus formation media formed two types of embryogenic calli: an off-white, compact, and nodular callus and a white compact callus. Upon successive subcultures (approximately 5 months), the nodular embryogenic callus became more prominent and was identified as aged callus. The aged callus upon further subculture, formed an off-white, soft, and friable embryogenic callus. Both the aged and friable calli maintained their embryogenic capacity over many subculture passages (to date up to 19 months). All embryogenic calli (1 month old) from the different callus-forming media, irrespective of expiant source, formed only green shoots on regeneration media that developed to maturity in the greenhouse. There were no significant differences in the response of calli derived from embryos and inflorescences cultured on the different initiation media. Also, the shoot-forming capacity of the cultivars was not significantly different. Anther-derived calli formed the least shoots. Aged and friable calli on regeneration media also formed green shoots but at lower frequencies. Plants from long-term culture have also been grown to maturity in soil.Florida Agricultural Experiment Station Journal Series No. R-00494     

Improved production of doubled haploids by colchicine application to wheat (Triticum aestivum L.) anther culture

The aim of this study was to optimize the in vitro chromosome-doubling procedure in wheat anther culture. Colchicine, at concentrations of 100–5000 mg/l, was added to the induction medium for 1–5 days. Beneficial effects were obtained with concentrations of 100 and 1000 mg/l colchicine. With time, significant reductions in embryo–like structures as well as higher doubling indices were found. Similar results were obtained with the high- and low-responding genotypes. Colchicine (100 mg/l), added 5 and 20 days after inoculation for 1 and 3 days increased the induction response, but this value was reduced when colchicine was added 10 or 15 days after inoculation. The doubling effect was similar to the control, except for a significant increase with the 3-day application 20 days after inoculation. The highest success index was reached when colchicine was added to the culture medium after 20 days.     

Genetic analysis of anther culture response and identification of QTLs associated with response traits in wheat (Triticum aestivum L.)

Molecular Biology Reports - Anther culture is the most effective tool for doubled haploid production of wheat. This investigation was conducted to estimate genetic parameters of anther culture...     

Somatic embryogenesis and plant regeneration from protoplasts isolated from embryogenic cell suspensions of wheat (Triticum aestivum L.)

We describe the early formation of somatic embryos followed by plant regeneration from protoplasts isolated from an embryogenic wheat cell suspension, which was initiated from small granular (0.2 to 1 mm in size) embryogenic calli. These granular calli formed embryogenic cell suspensions within 20 days in liquid culture, and were selected gradually from young inflorescence-derived nodular embryogenic calli of the winter wheat cv. Kehong 1041. The division frequency of protoplasts was 11 to 16%, and the frequency of differentiation into plants was about 0.001% (number of plants formed divided by the total number of protoplasts plated). About 20% of somatic embryos present in the culture formed directly from protoplast-derived cells within 15 days of cultures.     

In vitro development of wheat (Triticum aestivum L.) from zygote to plant via ovule culture

Ovules of the wheat breeding line Veery #5 were excised and transferred to culture within 24 h after pollination. When ovules were cultured on Phytagel-solidified medium, and the pericarp removed exclusively at the micropylar tip and the abaxial side, zygotes from up to 79.2% of the ovules underwent embryogenesis with the same developmental pattern as found in planta. Embryos from more than 50% of the cultured ovules germinated when transferred to regeneration medium. More than 100 plantlets were randomly chosen for transfer to soil, all of which developed to phenotypically normal and fertile plants. With this system, the entire process of zygotic embryogenesis can be studied using living material. Furthermore, the method could be used as an embryo rescue technique for plant breeding purposes. Received: 17 June 1996 / Revision received: 22 October 1996 / Accepted: 15 December 1996     

Suspension and protoplast culture of hexaploid wheat (Triticum aestivum L.)

Suspension cultures have been initiated from embryogenic callus of hexaploid wheat (Triticum aestivum L.). Most commonly, these suspensions are composed of callus-like clusters (up to 2 mm in diameter). Two rapidly-growing lines (MBE6 and C82d) have been obtained, which consist of smaller aggregates of cytoplasmic cells, and these have been maintained for more than 4 years. These lines show very limited morphogenetic capacity and only a single plantlet has been regenerated, from line MBE6, after 9 months in culture. Protoplasts isolated from line MBE6 are unable to divide, but protoplasts from line C82d consistently undergo sustained divisions to form callus or secondary cell suspensions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Control of tissue culture response in wheat (Triticum aestivum L.)

Summary The ability of immature embryos of wheat (Triticum aestivum L.) to respond to tissue culture has been shown to involve the group 2 chromosomes. The available group 2 ditelosomic and nullisomic-tetrasomic lines of Chinese Spring wheat were used to determine the chromosome arm location and chromosome dosage effect associated with the expression of tissue culture response (TCR). Significant differences were found between the aneuploid lines and the euploid control for the expression of both regenerable callus formation and callus growth rate. A model is proposed suggesting that a major TCR gene is located on 2DL and that 2AL and 2BS possess minor TCR genes. Furthermore, a major regulatory gene controlling the expression of TCR genes may be located on chromosome 2BL.