Ethylene production and embyogenesis from anther cultures of barley (Hordeum vulgare)

Summary Ethylene production was measured in cultured barley (Hordeum vulgare L.) anthers. The pattern of ethylene production and the content of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were different among cultivars. Ethylene production appeared to be related to embryogenesis (callus and embryo production). In cultivars in which anthers had low amounts of ACC and produced ethylene slowly, the addition of ethylene promotors (Ethrel or ACC) increased embryogenesis. However, in the cultivar Klages, in which anthers had high amounts of ACC and produced ethylene rapidly, the addition of an ethylene production inhibitor (putrescine) increased embryogenesis. Thus, an optimum level of ethylene production appears to be important for embryogenesis. The differences in anther response and callus production among cultivars may be due to both the capacity to produce ethylene and the sensitivity to high ethylene levels.     

Nuclear genes affecting percentage of green plants in barley (Hordeum vulgare L.) anther culture

Summary The genetics behind response in barley anther culture was studied with 22 reciprocal and one single: cross between three varieties with high and four varieties with low capacity for green plant formation. Effects of genotypes dominated embryo formation and percentages of green plants, accounting for 62 and 76% of total variation, respectively, with almost no genetic effect on the ability to regenerate plants from pollen embryos. Nuclear genes could explain all genotype effects in this plant material, since no reciprocal effects were indicated. The three parents with high and the four parents with low capacity for green plant formation formed two phenotypically homogeneous groups, producing 27–52% and 0–7% green plants, respectively. Genetic variation within hybrids for both embryo and green plant formation could be explained completely by general combining ability (GCA). The results are discussed with respect to a previous similar study in hexaploid wheat and the reported existence of DNA deletions in the plastid genomes in albino plants from anther culture of wheat and barley.     

The influence of genotype and temperature pre-treatment on anther culture response in barley (Hordeum vulgare L.)

The influence of temperature stress pre-treatment on anther culture response has been examined in eight commercially desirable barley cultivars. Spikes were pre-treated in darkness at 4°C for periods of 0, 7, 14, 21 and 28 days. Overall, the optimum pre-treatment period was 21 days, although there were large genotype by pre-treatment interactions. The most responsive cultivar was Igri, with a mean of 38% anthers responding, and relatively little effect of pre-treatment. The greatest effect of pre-treatment was in cv. Heriot, which had 3% response with no pre-treatment and 52% response from 14 days pre-treatment.     

Effect of anther orientation on microspore-callus production in barley (Hordeum vulgare L.)

Barley anthers from cold pretreated spikes produced no or few calluses when plated with both loculi in contact with the medium (flat). When anthers were plated with only one loculus in contact with the medium (up), a high proportion of the anthers produced calluses. The top loculus of the up anthers was most productive. Flat anthers, when compared with up anthers, were not only slower to produce multicellular pollen grains (MCPs) and microcalluses, but also produced fewer of them and ceased production earlier. The MCPs and microcalluses in flat anthers grew more slowly and few developed beyond the 30 cell stage. These results establish the importance of anther orientation for barley anther culture.     

Rapid production of recombinant barley yellow mosaic virus resistant Hordeum vulgare lines by anther culture

Summary In a winter barley breeding program for barley yellow mosaic virus (BaYMV) resistance, the resistant six-rowed cv. Franka was crossed to 17 susceptible and two resistant cultivars, three of which were tworowed. A total of 233,445 anthers of the 19 hybrids and their parents were cultured and 831 green plants regenerated. Anther culture responsiveness varied greatly between genotypes, and the responsiveness of F₁hybrids was generally related to that of the more responsive (high) parent. On average, 3.6 green plants were recovered from 1,000 cultured anthers, almost twice as many as in comparable spring barley experiments. Androgenetic green plants were tested for their reaction to mechanical inoculation of BaYMV. In crosses of resistant parents, all the cross progeny proved to be resistant, which indicates that both parents carry identical gene(s). In the crosses of the resistant cv. Franka to susceptible parents, an average of 62% of the androgenetic progenies were resistant, which indicates that probably more than one gene is responsible for Franka's BaYMV-resistance. From the crosses of Franka to two-rowed cultivars, 282 androgenetic plants were produced. When 132 of these were tested for their reaction to BaYMV, 79 (59.8%) were resistant, and 30 of the latter were shown to be two-rowed recombinant lines. Doubled haploid lines are field-tested for other agronomic characters including grain yield and its components.     

Microspore culture of Hordeum vulgare L.: the influence of density and osmolality

Summary Donor plants of Hordeum vulgare L. cv. Igri were grown in a conditioned environment to minimise fluctuations in the composition of the microspore population. After isolation different types of microspores were identified within each population, amongst others an embryogenic subpopulation. It was shown that the optimum plating density is achieved by adjusting the density to 2×10⁴ embryogenic microspores per ml, with a lower threshold at 5×10³ per ml. By increasing the osmolality of the pretreatment solution to 440 mOs.kg^–1 and that of the culture medium to 350 mOs.kg^–1, up to 15% of the population developed into embryo-like structures. When microspores of cv. Igri were cultured under the optimized conditions, the ratio of green/albino plants increased from 11 to 341, and 50 green plants per anther were formed.     

Optimizing somatic embryogenesis and particle bombardment of barley (Hordeum vulgare L.) immature embryos

Summary A highly regenerable target tissue and a high-frequency DNA delivery system are required for the routine production of transgenic barley. This project separately optimized tissue culture and particle bombardment parameters. Immature zygotic embryos (0.7 to 1.2 mm) were excised and culture on B5L solid medium. Klages and H930-36 cultivars regenerated significantly more green plants than Sabarlis and Bruce. The regeneration pathway shifted from organogenesis to somatic embryogenesis when maltose was used as the medium carbohydrate source instead of sucrose. More somatic embryos were induced on 5 mg/liter 2,4-dichlorophenoxyacetic acid than 2 mg/liter. Gene delivery was optimized using anthocyanin regulatory genes as a transient marker. A 3-mm rupture disc-to-macrocarrier gap distance, a 1-day prebombardment embryo culture period, and a maltose carbohydrate source were each significantly better than other treatments. Double bombardments per plate, a 6-mm macrocarrier fly distance, and 650-psi rupture discs each had the highest number of transiently expressing cells in individual experiments, although the results were not statistically significant compared to the other treatments. Using the optimized parameters, over 200 cells routinely expressed anthocyanin in a bombarded immature embryo. In tissue culture experiments, 350 to 400 green plants regenerated per 100 immature embryos. The improvement of green plant regeneration and gene delivery forms a strong basis to develop a practical barley transformation system.     

Production of gynogenic haploids of Hordeum vulgare L.

Haploid plants were regenerated from cultured unfertilized ovaries of Hordeum vulgare L. (barley). Optimal response was obtained by the addition of 0.6 M 4-chloro-2-methylphenoxyacetic acid (MCPA), 2.8 M indole-3-acetic acid (IAA) and 4.4 M 6-benzyladenine (BA) in the N₆ medium. Further increase in the rate of callus formation and the number of green plants produced was possible with the addition of 90 g/l sucrose and 100 g/l coconut water. The stage of development of the ovaries at the time of culture was critical; the largest number of plants being produced by ovaries from flowers at the trinucleate stage of pollen.Abbreviations (BA) 6-benzyladenine - (MCPA) 4-chloro-2-methylphenoxyaceticacid - (2,4-D) 2,4-dichlorophenoxyaceticacid - (GA₃) gibberellic acid - (IAA) indole-3-acetic acid     

Genome relationships between Hordeum vulgare L. and H. bulbosum L.

Interrelationships between H. vulgare (2x=14) and H. bulbosum (2x=14; 4x=28) were estimated on the basis of the karyotypes and the pairing behaviour of the chromosomes in diploid, triploid and tetraploid hybrids obtained with the aid of embryo culture. — A comparison of the karyotypes of the two species revealed similarities as well as differences. It was concluded that at least 4 or more of the chromosomes were similar in morphology and probably closely related. — Diploid and tetraploid hybrids are rarely obtained and their chromosome numbers tend to be unstable whereas triploid hybrids (1 vulgare + 2 bulbosum genomes) were stable and relatively easy to produce. In the diploid hybrid only 40% of the meiotic cells contained 14 chromosomes while the numbers ranged from 7 to 16 in other cells. All hybrids exhibited pairing between the chromosomes of the two species. Diploid hybrids had a mean of 5.0 and a maximum of 7 bivalents per cell in those cells having 14 chromosomes. Triploid hybrids from crosses between 2x H. vulgare and 4x H. bulbosum exhibited a mean of 1.5 and a maximum of 5 trivalents per cell. In a hexaploid sector found following colchicine treatment of a triploid the mean frequencies of chromosome associations per cell were: 5.5_I+8.0_II+0.7_III+3.7_IV+0.3_V+0.4_VI. One unstable 27 chromosome hybrid obtained from crosses between the autotetraploid forms had a mean of 1.1 and a maximum of 4 quadrivalents per cell. The chromosome associations observed in these hybrids are consistent and are taken as evidence of homoeologous pairing between the chromosomes of the two species. Interspecific hybridization between these two species also reveals that chromosome stable hybrids are only obtained when the genomes are present in a ratio of 1 vulgare2 bulbosum. Based upon the results obtained, the possibility of transferring genetic characters from H. bulbosum into cultivated barley is discussed.     

Plant regeneration from embryogenic cell suspensions derived from anther cultures of barley (Hordeum vulgare L.)

Summary We have established embryogenic cell suspension cultures of barley (Hordeum vulgare L. cultivars Igri, Gimpel, Princesse, and Baronesse) from anther-derived embryogenic callus. Suspension cultures of cultivars Igri and Gimpel were regenerable. The most successful cultivar was Igri, from which a number of independent cell lines producing plantlets were established. Plants could be transferred to soil; up to now, 50% of more than 200 regenerated plants were morphologically normal and fertile. The relative frequency of sterile plants increased as suspensions aged. Suspensions older than 1 year produced embryogenic callus but only albino plantlets could be regenerated.     

Somatic embryogenesis and plant development from anther culture of Vitis vinifera (L.)

Anthers from Frumoasa alba (White beauty), Otilia, Valerien, Mission and Siegfried Rebe (FS₄) cultivars were cultured at the uninucleate stage of the microspore on Murashige and Skoog (1962) and Nitsch and Nitsch (1969) media supplemented with 2,4-dichlorophenoxyacetic acid (4.9 M) and benzyladenine (4.4 M). The primary calli were subcultured on MS medium with 6.6 M BA and 1.1 M indolylacetic acid, in order to induce their growth and plant regeneration. After seven months, vegetative buds were obtained with Frumoasa alba (2.7%), Otilia (0.3%), Valerien (4.5%), embryogenic callus was obtained with Mission and plant regeneration with Siegfried Rebe. Long term embryogenesis was maintained in Mission cv. for four years, by selection and regular transfer of the embryogenic areas of anther-derived calli. The embryogenic calli have the ability to generate abnormal somatic embryos with one, two or three cotyledons and cup or trumpet-shaped with fused cotyledons. In parallel with the embryogenic process, organogenesis with buds, leaf and shoot differentiation was regularly observed.     

Synthesis and processing of thionin precursors in developing endosperm from barley (Hordeum vulgare L.)

Thionin is a lysine-rich polypeptide (mol. wt. 5000) which is synthesized in developing barley endosperm from ˜8 days to ˜30 days after anthesis. Two thionin precursors (THP1 and THP2) have been identified using monospecific antibodies (A-TH) prepared against the mature protein. THP1, which is the only polypeptide recognized in vitro by A-TH, is encoded by a 7.5S mRNA obtained from membrane-bound polysomes, and its alkylated derivative has an apparent mol. wt. of 17 800. THP2, which is selected together with mature thionin by A-TH among labelled proteins in vivo, differs from THP1 in apparent mol. wt. (17 400 alkylated) and in electrophoretic mobility at pH 3.2. Both THP1 and THP2 are competed out of the antigen-antibody complex by purified thionin. The conversion of THP2 into thionin, which has been demonstrated in a pulse-chase experiment in vivo, is a post-translational process. As it has not been possible to detect THP1 in vivo it is assumed that it is converted co-translationally into THP2. Final deposition of thionin as an extrinsic membrane protein, possibly associated with the endoplasmic reticulum, has been tentatively established on the basis of subcellular fractionation experiments.     

Anther culture of Hordeum vulgare L.: a genetic study of microspore callus production and differentiation

Summary The inheritance of the ability of barley anthers to produce microspore-derived callus in vitro was investigated. The genotypes selected were the two spring cultivars Dissa (D) and Sabarlis (S), the two F₁ hybrids (DxS, SxD), the two backcross generations [Dx(DxS), Sx(DxS)], and an F₂ generation derived from DxS. From a number of individuals of each generation, the first five spikes were harvested sequentially and after pre-treatment the anthers were removed and placed in culture. Cultures were scored for microspore callus production and plantlet differentiation. Although Dissa gave a significantly higher level of callus production than Sabarlis, the overall frequencies of green and albino plant production were higher from Sabarlis. There was no significant difference between reciprocal F₁ hybrids. Analysis of variance revealed significant differences in response between the spikes sampled from the plants. This was the major source of variation in the experiment. Spike to spike variation also appeared to be a heritable character.     

Pollen embryogenesis in anther cultures of Solanum carolinense L.

The direct differentiation of bicellular pollen grains of Solanum carolinense L. (Horse-nettle; Solanaceae) into embryoids and plantlets was induced by culturing whole anthers on Murashige and Skoog's medium supplemented with IAA. The highest frequency of embryogenic induction occurred at 10 mg/l IAA. Developmentally, both the generative and vegetative cells of the pollen grain contributed to embryoid formation whose pattern of development was similar to that of zygotic embryos. In a previous study, it was show that 2,4-D promoted callus formation by pollen grains in cultured anthers of S. carolinense. It appears then that there are two distinct pathways of androgenesis in this species that are determined by the type of auxin present in the medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - BA benzyladenine - KIN kinetin - MS Murashige and Skoog     

Cloning and mapping of telomere-associated sequences from Hordeum vulgare L.

Summary We present a novel approach to the efficient cloning of telomere-associated sequences and demonstrate its application to the cloning and mapping of these sequences from barley. The method is a modification of the Vectorette PCR technique and allows specific amplification and cloning of subtelomeric sequences. Telomere-associated sequences isolated from barley include hypervariable, repetitive sequences. Polymorphisms detected by subtelomeric markers behaved as Mendelian factors and were mapped to the most distal positions of barley chromosomes 1, 2, and 4.     

Evidence for microspore embryogenesis in wheat anther culture

Summary In wheat, plants may be regenerated from microspores via direct embryogenesis or organogenesis or embryogenesis from callus. Light and scanning electron microscopy were used to carefully study morphogenesis of microspore-derived plants from anther culture on modified 85D12 starch medium and to determine whether the plants were formed via organogenesis or embryogenesis. Our results indicate that plants are formed via embryogenesis from microspores. Evidence for embryogenesis included the formation of the epidermis and a suspensorlike structure (21 days after culture), followed by initiation of an apical meristem, differentiation of the scutellum, and embryo elongation. At 28 days in culture, the embryo possessed a well-developed scutellum and axis with suspensor. Embryogenesis was further confirmed by coleoptile and radicle elongation during germination when the embryos were cultured on medium supplemented with kinetin with or without coconut water. In this system, an average 67 microspores per responsive anther began cell division but only 3.69 embryos were formed per responsive anther after 6 wk. Adventitious embryos could be induced if the embryos, once formed, remained on initiation medium for 10 wk instead of being transferred to regeneration medium. Developmental stages which may be amenable to changes that could enhance plant production were identified. The potential to use this information to enhance plant production is discussed.     

Plant regeneration from mesocotyl callus of Hordeum vulgare L.

Callus tissue was induced on barley mesocotyl explants of germinated seven-day-old seedlings on MS medium supplemented with 2,4-D or 2,4,5-T in high concentrations. Two morphologically different tissue cultures were maintained in vitro for a long time: a callus tissue without organogenesis and a culture with high rhizogenic capacity. Shoots and plantlets were generated when the auxin-media induced callus was transferred to medium supplemented with 3 M TIBA. In 62% of cultures, during the first five subcultures, four to twentyeight plants per single mesocotyl were obtained. Some cultures produced shoots even in the 9th subculture, being in culture for nearly 14 months. The largest number of plants obtained per one mesocotyl was forty. Plantlets rooted well on MS with 5.7 M IAA and survived transplantation to soil in high percentages.Abbreviations IAA indole-3yl-acetic acid - BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - MS Murashige and Skoog (1962) medium     

Anatomical studies of Ustilago galls on Hordeum vulgare L.

The present paper deals with the histopathological studies of galls of Ustilago hordei (Pers.) on Hordeum vulgare L. The gall formation in the present case is the result of both hyperplastic and hypertrophic activities of the infected host cells. Also the gall formation by U. hordei on. H. vulgare is being reported for the first time through present. communication.