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.     

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.     

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.     

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.     

Stimulation of androgenesis in white cabbage (Brassica oleracea var. capitata) anthers by low temperature and anther dissection

Anther culture was performed on two local cultivars, Ljubljansko and Varadinsko, and the F₁ cv. Krautman (Bejo-Zaden). The effects on androgenesis of hot and cold temperature treatments and different dissections of anthers were evaluated. In contrast to cv. Krautman, cvs. Ljubljansko and Varadinsko produced more embryos after cold pretreatment of flower buds (4°C, 48 h) than after standard treatment (35°C, 24h). Simultaneous cutting of the anther tip and removal of the filament gave the best results in comparison to other tested dissections. Microscopical observations of sectioned anthers revealed enhanced embryo development near the cut ends of the anthers. Ploidy analysis revealed the presence of haploids among embryos resulting from cold treatment (4°C, 48 h), treatment at elevated temperature (35°C, 24 h), and among embryos resulting from dissections of anther tips.     

Effects of support medium on embryo and plant production from cultured anthers of soft-red winter wheat (Triticum aestivum L.)

The present study was designed to examine the effects of media support on the frequency of embryo and plant production from cultured anthers of soft-red winter wheat. Approximately twice as many embryos were produced when anthers were cultured in a liquid as compared to an agar-solidified medium. Upon transfer to regeneration medium, a significantly lower percentage of the embryos produced in liquid regenerated plants. The addition of activated charcoal to an agar-solidified medium resulted in a considerable increase in embryo production, however, plant regeneration from embryos produced on charcoal-containing medium was significantly lower than those produced on agar only. Embryo production frequencies ranged from 2.4–13.2 and 2.5–32.2 embryos per 100 anthers on media with and without charcoal, respectively. Plant regeneration frequencies from embryos produced in the presence of activated charcoal ranged from 0–5.5% as compared to 0–39.1% from embryos produced in the absence of charcoal. More than twice as many embryos produced on Ficoll-containing liquid medium regenerated plants when compared to embryos produced in liquid only. The results from this study suggest that cultural modifications designed to maximize embryo production must take into account the quality of the resulting embryos as they relate to plant regeneration.     

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     

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.     

DNA Synthesis in Microspores in Anther Culture of Wheat (Triticum aestivum L.)

Anthers with mid-unlnucleate microspores were cultured on W5 medium supplemented with 0.5 mg/l kinetin, 2 mg/l 2,4-D and 9% or 3% sucrose. At a series of interval (0, 1, 1.5, 2, 14 days) after cultured, the anthers were labelled with 3H-thymidine (4 MCi/mi) for 24 h, fixed, and then performed autoradiography according to conventional method. Results show that after cultured for 24 h, 3H-thymidine was incorporated into some late-uninucleate microspores (see Plate I, 3), and after for 2.5 days, vegetative nuclei in pollen grains were la- belled (see Plate I, 4). Usually, vegetative nuclei were labelled frequently and generative ones were labelled rarely. Sometimes generative cell which could synthesis DNA might develop suspensor-like structure individually (see Plate I, 13). During early stage of development of a multicellular pollen grain, the DNA synthesis in the cells were synchronized. With pollen development, the synchronism of DNA synthesis was destroyed. When anthers cultured on medium with 3% sucrose, DNA in microspores could be synthesized normally, and the number of labelled microspores was more than that of anthers cultured on medium with 9% sucrose. However, on medium with 3% sucrose, the nuclei in microspores stopped dividing after one or two divisions and the cell wall of them could not be formed and multicellular pollen was not observed. It seems that the absence of multicellular pollen on medium with 3% sucrose was primarily due to the block of cell division and cell wall formation, not due to the interruption of DNA synthesis.     

Effect of IAA content Modulators on peroxidase activity and on endogenous IAA during cold pretreatment of maize anthers prior to androgenesis

A cold pretreatment is usually applied to induce maize androgenesis. Peroxidase activity, including indole-3-acetic acid (IAA) oxidase activity, and endogenous IAA concentrations were followed during a cold pretreatment (14 days, 7°C) in anthers of two maize genotypes, Seneca 60 and DH5×DH7, respectively with a low or high androgenetic response. The most prominent result was the absence of a detectable IAA oxidase activity in DH5×DH7. Adding effectors of IAA-oxidase activity or IAA transport did not affect significantly the crude peroxidase activity of DH5×DH7 anthers while inducing a clear inhibition of androgenesis at higher concentrations. No strict correlation was found between IAA level and physiological response, the low responding variety having as much IAA as DH5×DH7. However, for DH5×DH7, every treatment that lowered the IAA level after 14 days of cold resulted in a decrease in androgenetic response.     

Microspore embryogenesis in anther cultures of two Indian cultivars of Brassica juncea (L.) Czern.

Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained. Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis. Embryogenesis was also stimulated by an initial culture at 5°C for 3 days. Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos. All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets. The androgenic plants were haploid (2n=18).     

Cytological aspects of in vitro androgenesis in wheat (Triticum aestivum L.) using fluorescent microscopy

Summary Two winter wheat genotypes (Diószegi 200 and Mv 15) were compared for their in vitro androgenic capacity. On average, the induction frequency of embryogenic structures was 71.7% in Diószegi 200 and only 4.3% in Mv 15. The haploid induction ability of the two genotypes differed considerably, with Diószegi 200 being much higher. The difference in the in vitro inductability of the microspores may result from genetic differences which are manifested in the survival rate of the microspores during the culture period and their adaptability to in vitro conditions. Special DNA fluorochrornes were suitable for studying the different pathways of in vitro androgenesis. Our data indicate that the repeated equal divisions of the microspore nucleus might lead to pollen embryo formation, and subsequent divisions of the vegetative portion of the pollen grain after the first asymmetric microspore mitosis can result in pollen callus formation.     

Silicon absorption by wheat (Triticum aestivum L.)

Although silicon (Si) is a quantitatively major inorganic constituent of higher plants the element is not considered generally essential for them. Therefore it is not included in the formulation of any of the solution cultures widely used in plant physiological research. One consequence of this state of affairs is that the absorption and transport of Si have not been investigated nearly as much as those of the elements accorded 'essential' status. In this paper we report experiments showing that Si is rapidly absorbed by wheat (Triticum aestivum L.) plants from solution cultures initially containing Si at 0.5 mM, a concentration realistic in terms of the concentrations of the element in soil solutions. Nearly mature plants (headed out) 'preloaded' with Si absorbed it at virtually the same rate as did plants grown previously in solutions to which Si had not been added. The rate of Si absorption increased by more than an order of magnitude between the 2-leaf and the 7-8 leaf stage, with little change thereafter. This revised version was published online in June 2006 with corrections to the Cover Date.     

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     

Ethylene production and involvement during the first steps of durum wheat (Triticum durum) anther culture

The role of ethylene in anther culture of durum wheat ( Triticum durum Desf. cv. Ardente) was analyzed by testing the effects of 2-chloroethylphosphonic acid (ethrel) silver thiosulfate (Ag⁺), a -aminooxyacetic acid (AOA) and 1-aminocyclopropane-l-carboxylic acid (ACC) on microspore division observed after 21 days of culture and on development of calli estimated at day 45. The use of ethrel and Ag⁺ indicated a positive effect of ethylene on microspore division, whereas the use of AOA, and to a lesser extent ACC, snowed a negative effect. In contrast, the addition of ethrel or Ag⁺ indicated that ethylene inhibits the development of microspore-derived calli. AOA gave contradictory results. Ethylene production by anthers was about 7 pl anther⁻¹h⁻¹ and decreased during culture. ACC content in the anthers was maximal at day 9, whereas malonyl ACC (MACC) increased sharply from day 0 to day 3 and then decreased. The addition of AOA or ACC to the culture medium decreased or increased, respectively, ethylene production of anthers and the ACC and/or MACC content, but at concentrations higher than those that modified the formation of calli. This formation seems to occur in two successive phases: induction and initiation of microspore division, which was promoted by ethylene, followed by callus development, which was inhibited by ethylene.     

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     

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     

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.     

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