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.     

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     

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.     

The influence of anther cold pretreatments and donor plant genotypes on in vitro androgenesis in wheat (Triticum aestivum L.)

Cold pretreatments applied to excised anthers in liquid potato 2 medium proved to be unnecessary. Generally, cold pretreatments inhibited anther response and productivity as the duration was lengthened or as the pretreatment temperature was lowered. There were significant differences in response attributed to the anther donor genotype. Green and albino plants as well as roots only have been regenerated from the spring wheat cultivars Sinton, Neepawa, Pitic 62 and DW 50. Most plants were haploid.     

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.     

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25°C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25–27°C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.Communicated by W. Harwood     

Effects of donor plant environment and light during incubation on anther cultures of some spring wheat (Triticum aestivum L.) cultivars

Four different growth environments (field, two phytotron greenhouses and one growth chamber) were compared, using two genotypes of spring wheat, one recalcitrant and one responsive. Field-grown plants gave inferior results. Large improvements could be made by improving the conditions, embryoid frequencies in the two genotypes reaching 77.1% and 183.9% per 100 anthers, respectively. High light intensity during the induction phase strongly suppressed induction in both genotypes, but stimulated regeneration of green plants in the recalcitrant genotype, which had the lowest regeneration ability. Weak, diffuse light did not inhibit induction while the positive effect on regeneration was maintained. Also, another recalcitrant genotype was grown in the field, together with two F₁-hybrids (recalcitrant x recalcitrant and recalcitrant x responsive). Evidence for a three-factor system was obtained.     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

Arabinogalactans and arabinogalactan-proteins induce embryogenesis in wheat (Triticum aestivum L.) microspore culture

The objective of this study was to improve induction of embryogenesis in wheat microspore culture in order to obtain a high number of regenerable embryos. The arabinogalactan (AG) Larcoll and the arabinogalactan-protein (AGP) from gum arabic were tested on two spring genotypes to see if they could increase microspore viability and induce embryogenesis in the microspore culture. Adding Larcoll significantly decreased microspore mortality in both genotypes regardless of the presence or absence of ovaries in the culture. Similarly, gum arabic had a strong effect on the number of embryos produced and regenerated green plants. In fact, by using only gum arabic we were able to obtain green plants from wheat microspore cultures without the presence of ovaries. In addition to preventing a high mortality rate of the cells, our results show that the induction of embryogenesis in wheat microspore cultures is strongly affected by the use of both AG or AGP.An erratum to this article can be found at     

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     

Plant growth-inhibitory pseudomonads colonizing winter wheat (Triticum aestivum L.) roots

Summary Pseudomonads, which inhibit root extension, can be present in the winter wheat rhizosphere in large numbers, but they are not detectable until late winter or early spring. Their presence was not related to the presence of wheat straw residues or type of tillage, although they were present on the wheat residues when they appeared in the rhizosphere. Wheat seedlings were more sensitive to the bacteria at 15° C than at 20° C during bioassays. The type of agar used in the bioassay can affect the results obtained. The inhibitory factor expressed by the pseudomonads is quite variable and is radically affected by transfer of isolates.Contribution from Agric. Res. Serv. U.S. Dept. of Agric., in cooperation with the College of Agric. Res. Center. Washington State Univ., Pullman, WA 99164 (Scientific Paper No. 6743); and Agricultural Research Council, Letcombe Laboratory, Wantage, Oxon OX12 9JT, England.     

Accelerated production of transgenic wheat (Triticum aestivum L.) plants

We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 g of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.     

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.     

Zinc deficiency and the formation of white structures in immature embryo cultures of wheat (Triticum aestivum L.)

The effect of microelements on the induction of embryogenic callus from epiblast and scutellum of wheat (Triticum aestivum L.) embryos was studied by the sequential omission of each of the microelements from Murashige & Skoog medium. Omission of iron caused a marked decrease in yield and poor shoot formation from embryogenic callus. The yield of embryogenic callus on medium without added manganese was also reduced. Omission of boron, copper-cobalt, iodine, and molybdenum had little effect on the induction of embryogenic epiblast callus. By contrast there was a marked increase in the formation of white structures on the medium without any microelements or, specifically without addition of zinc. Since the formation of typical embryoids of wheat is associated with the formation of white structures, our result highlights the importance of certain microelements on somatic embryogenesis of wheat.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium Murashige & Skoog 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     

Effects of auxin and cytokinin on induction of sister chromatid exchanges in cultured cells of wheat (Triticum aestivum L.)

Summary In order to know the mutagenic effects of synthetic auxins (NAA, 2,4-D, and 2,4,5-T) and a cytokinin (kinetin) in vitro, sister chromatid exchanges (SCEs) were analyzed in cultured cells of a hexaploid wheat (Triticum aestivum L.). In the MS medium supplemented with 2.0 mg/l 2,4-D, the mean number of SCEs per cell was 15.2, and per pg of DNA, 0.42. No significant effect was found in the treatments of NAA or 2,4-D at concentrations of 0.5–10.0 mg/l, whereas more than 2.0 mg/l of 2,4,5-T induced dramatic increases of SCEs. Kinetin itself had no significant effect on SCE induction, but there was a tendency that SCEs induced by 2,4,5-T were suppressed by kinetin.     

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.     

Carbon balance assessment of a Belgian winter wheat crop (Triticum aestivum L.)

The carbon balance of a winter wheat crop in Lonzée, Belgium, was assessed from measurements carried out at different spatial and temporal scales between November 2004 and August 2005. From eddy covariance measurements, the net ecosystem exchange was found to be ?0.63 kg C m^?2 and resulted from the difference between gross primary productivity (GPP) (?1.58 kg C m^?2) and total ecosystem respiration (TER) (0.95 kg C m^?2). The impact of the u_* threshold value on these fluxes was assessed and found to be very small. GPP assessment was partially validated by comparison with an estimation scaled up from leaf scale assimilation measurements. Soil respiration (SR), extrapolated from chamber measurements, was 0.52 kg C m^?2. Net primary productivity, assessed from crop sampling, was ?0.83 kg C m^?2. By combining these fluxes, the autotrophic and heterotrophic components of respiration were deduced. Autotrophic respiration dominated both TER and SR. The evolution of these fluxes was analysed in relation to wheat development.     

Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.)

Summary Regenerable embryogenic cell suspensions initiated from immature embryo-derived friable, fast growing, embryogenic calli of GK Ságvári winter wheat (Triticum aestivum L.) served as sources of protoplasts, which were cultured in different liquid or agarose-solidified media. Protocallus formation was best on KM_8p (Kao and Michayluk 1975) and GM (Li and Murai 1990) media, and protocallus growth on MS (Murashige and Skoog 1962) callus growing medium. Green shoot/plant regeneration occurred on MS regenerating medium, and rooting on MS or N6M (Mórocz et al. 1990) hormone-free media. Protocalli maintained their morphogenic capacity over 4 months, and with multiple subcultures on half-strength MS regenerating medium, the total number of regenerants could be increased. Approximately 1000 shoots/plants were regenerated and over 500 plants were transplanted in the greenhouse. The majority of them had an abnormal chromosome number and low viability, however, one plant grew to maturity and set seed.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - GA₃ gibberellic acid - GM General medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid - RECS regenerable embryogenic cell suspension