High temperature enhances ethylene promotion of anther filament growth in Brussels sprouts (Brassica oleracea var. gemmifera)

A study was made of the effect of high temperature on the growth response of Brussels sprout filaments to ethylene. Filaments with or without the anthers attached were incubated continuously at 25 °C or 35 °C for 7 days or for 2 days at 35 °C followed by 5 days at 25 °C. Growth was reduced during both 35 °C treatments compared to that of filaments at continuous 25 °C. Ethylene had little effect on filament growth at continuous 25 °C, whereas with treatment for either 2 or 7 days at 35 °C ethylene promoted filament growth considerably. Thus ethylene effectively overcame the growth inhibition induced by the 35 °C treatment.High temperature treatments reduced ethylene production from filaments alone, and from filaments with anthers attached. The ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) and the ethylene action inhibitor AgNO₃ enhanced filament growth at 25 °C but had little or no effect at 35 °C. The relevance of temperature to ethylene sensitivity is discussed in relation to filament growth and to other plant processes in general.     

Ethylene production during anther culture of Brussels sprouts (Brassica oleracea var gemmifera) and its relationship with factors that affect embryo production

The ethylene inhibitor silver nitrate (AgNO₃) is known to overcome the poor response of the Brussels sprouts cultivar Hal to anther culture. Ethylene production by Hal anthers after 6 h of culture at 35°C was on average 10- and 20-fold greater than from anthers of the highly responsive cultivars Gower and GA1 x RDF2. The initial 24 h period at 35°C necessary for embryogenesis in anther culture of Brussels sprouts generally reduced ethylene production by the anthers after 6, 24, 48 and 72 h of culture, although the effect was not seen in 2 out of 3 Hal experiments until 24 h, and after 6 h was only found with 1 of 3 GA1 x RDF2 experiments. Embryo production was inhibited by the inclusion of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) or the ethylene-releasing compound, ethephon in the media. Silver nitrate (AgNO₃) and the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) promoted embryogenesis but did not substitute for the high temperature treatment. The relevance of ethylene production during anther culture to the effects of genotype and high temperature on anther culture embryogenesis is discussed.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine     

The effects of gibberellic acid,fluridone, abscisic acid and paclobutrazol on anther culture of Brussels sprouts

Both gibberellic acid (GA₃) and fluridone, a non-specific inhibitor of ABA biosynthesis, promoted embryo production in anther cultures of Brussels sprouts cv. Hal, but not in cv. Gower. Abscisic acid (ABA) and the gibberellin-biosynthesis inhibitor paclobutrazol inhibited embryo production in both cultivars.     

Effect of silver nitrate and 2,4-D on anther culture of Brussels sprouts (Brassica oleracea var. gemmifera)

The response to anther culture, of six genotypes of Brussels sprouts was tested on six media which included two levels of 2,4-D and the presence or absence of silver nitrate. The presence of silver was usually beneficial, and with some genotypes had a very large effect. Increasing 2,4-D could be beneficial in the absence of silver nitrate, but was sometimes detrimental in the presence of silver. Replacing agar with highly purified agarose was not particularly beneficial. Genotype, medium and genotype × medium interactions were all significant factors, with genotype being the most important.     

ABA promotion of ethylene production in anther culture of Brussels sprouts (Brassica oleracea var. gemmifera) and its relevance to embryogenesis

Abscisic acid (ABA) inhibited embryogenesis in anther culture of Brussels sprouts. This was accompanied by enhanced ethylene production during the first half of the anther culture period followed by a reduction in ethylene during the latter half, when compared to anthers not treated with ABA. The enhancement of ethylene production by ABA 6 h and 48 h after the start of the culture period was counteracted by the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG). Both AVG and the ethylene antagonist AgNO₃ removed much of the ABA inhibition of embryogenesis, suggesting that at least part of the ABA effect on embryo production is mediated through increased ethylene biosynthesis.
ABA promotion of ethylene production was reduced by high temperature: less ethylene evolved from ABA-treated anthers following a 24 h treatment at 35°C than from ABA-treated anthers incubated continuously at 25°C. A high temperature treatment such as this is invariably necessary for embryogenesis in Brussels sprouts anther culture.     

A study of factors affecting anther culture of cauliflower (Brassica oleracea var. botrytis)

Experiments on three autumn-heading cauliflower genotypes (2 hybrids and a genotype selected from a population) were conducted to study different factors affecting anther culture. Culture conditions of the donor plants proved to be important: the best results were obtained during spring in a greenhouse where the temperature was maintained between 10 and 20°C. Overall winter and spring seemed more suitable than summer and early autumn for culture establishment. The optimal bud development stage depended on the genotype: for the hybrid 702, the greatest number of embryos for 100 plated anthers was obtained at the uninucleate pollen stage of the microspores; for V23.2 and 703, the optimal stage of the buds corresponded to the first mitotic division. Sucrose proved to be the best carbon supply for embryogenesis with an optimal concentration of 140 g l^-1. The addition of a cytokinin (BAP) in the medium led to lower embryo production, and this negative effect increased when the hormone concentration in the medium increased. The use of liquid medium and a dark incubation period immediately after the high temperature treatment were favourable for embryogenesis.     

Heat shock response during anther culture of broccoli (Brassica oleracea var italica)

Embryo formation from microspores of Brassica oleracea var Italica (Broccoli) and other Brassica species is greatly enhanced by an initial incubation at elevated temperatures (eg 35°C) followed by continued incubation of 25°C. In the present study we observed that a three hour high temperature treatment induced the formation of heat shock proteins in cultured anthers. These were identified in two dimensional gels by silver staining, and labelled heat shock proteins were synthesised in vitro from isolated anther RNA. The appearance of heat shock proteins in anthers followed a similar pattern and displayed similar characteristics to that from leaves. Comparison of the heat shock proteins induced in isolated cultured anthers of known highly embryogenic and less embryogenic plans did not reveal obvious qualitative differences.     

Histology of embryogenic responses in soybean anther culture

In order to clarify the embryogenic responses in soybean anther culture, anthers of four cultivars were cultured under known conditions to trigger androgenic response. A histological study was performed with anthers in vivo and with approximately 100 explants sampled after 9, 12, 15, 18, 21, 30 and 45 days of culture. In vitro culture triggered the frequent accumulation of phenolic compounds on the locular and anther surfaces, and also caused the destruction of cells and tissues in complex structure such as the tapetum, microspores and pollen grains. Somatic embryogenesis of unicellular origin was observed from the epidermis and the middle layer, and of multicellular origin from connective calluses. No androgenic response could be observed in the anthers of these four soybean genotypes, in the medium and conditions indicated. We point out to the need of changing the approach to the study of androgenesis in soybean, either by using culture conditions unfavourable to the proliferation of diploid tissues, or by culturing isolated microspores.     

The response of anther culture to culture temperature in Triticum aestivum

Summary The response of anther culture to culture temperature was studied in detail using many varieties, F₁ hybrids and pollen-derived lines of wheat (Triticum aestivum) as materials. The suitable culture temperature for inducing pollen callus (or embryoids) in wheat anther culture ranged from 26 °C to 30 °C, varying with genotypes. But for the great majority of wheat genotypes the suitable culture temperatures lay between 28 °C and 30°C. The most significant genotypic variation in the response to culture temperature was observed in the comparison between the culture at 33 °C for eight days followed by culture at 25 °C (or 26 °C) and the continuous culture at 25 °C (or 26 °C). This genotypic variation in the response to culture temperature is a heritable character which may be controlled by multiple genes. The effect of culture at 30 °C for eight days followed by culture at 26 °C was similar to, or in some cases, better than that of continuous culture at 28 °C, and the effect of culture at 32 °C for eight days followed by culture at 28 °C was similar to that of continuous culture at 30 °C. In the range from 26 °C to 32 °C, the overwhelming majority of pollen calli emerged before the 40th day after anther inoculation, and the higher the culture temperature, the earlier and more concentrated the emerging period of the pollen callus. The pollen callus obtained at high temperatures above 28 °C should be transferred in time onto the regeneration medium at 25°–27°C to induce shoots.     

The effect of starch and ineubation temperature in anther culture of potato

Three Andean tetraploid potato genotypes (2n=48) and 7 anther-derived dihaploids (2n=24) originating from two of the tetraploids were used in anther culture. Relative number of embryos/vial was significantly higher when the anther culture media was gelatinized with 3% potato starch than when Gelrite or wheat starch (3%) were used as gelatinizing agents. The degree of anther culture response varied between tetraploids but also within a group of related dihaploids. Additionally, the embryo production of individual genotypes, tetraploids as well as dihaploids, was dependent on the incubation temperature (10, 15, 20, 25, 30°C) of the anther culture. The incubation temperature of the anther culture was also important for the regeneration rate. Direct regeneration was mostly stimulated when the anther culture was incubated at 20°C.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid     

Effects of temperature on the mode of pollen development in anther culture of Brassica campestris

In anther culture of Brassica campestris L., the yield of pollen-derived embryoids is greatly stimulated by a high-temperature (35°C) tratment for the first 1 to 3 days of the culture. We have inestigated the effects of the high-temperature treatment on the mode of pollen division in cultured anthers.
Anthers containing late uninucleate pollen were cultured on modified B5 medium. High-temperature treatment for the first 24 h inhibited the normal development of the pollen and induced abnormal symmetrical division. Which is the first step in androgenesis. This symmetrical division was rarely observed in pollen developed in vivo. In anthers cultured without high-temperature treatment, the mode of pollen development was similar to that in vivo. This suggests that the normal differentiation of the pollen is blocked by high temperatures, and sporophtic growth is induced. Sucrose (0.29 M) was essential for the induction of this symmetrical division, though neither plant growth regulator nor any other nurient was necessary. Pollen division could not be induced effectively if sucrose was replaced by either mannitol or sorbitol plus a lower concentration of sucrose. Therefore, it seems that sucrose actively influences the embryogenic division of pollen, and does not have only an osmotic effect.     

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.     

Genotype-specific response of cultured broccoli (Brassica oleracea var. italica) anthers to cytokinins

Anther culture medium was prepared with different types and concentrations of cytokinins to gain greater insight into the control of embryo formation during Brassica oleracea L. var. italica (broccoli) anther culture. The independent addition of the four cytokinins tested had widely divergent effects dependent upon cytokinin concentration and the genetic background of the test plants. All cytokinins were generally inhibitory at high concentrations, however, individual plants showed significant stimulation of embyro formation at typical physiological levels. The influence of cytokinins was highly cultivar-specific, some lines were stimulated, others inhibited and still other test lines were largely unaffected. Although the addition of cytokinins was needed for embryo formation for some plants, in no instance were cytokinins able to replace the inductive effect of high-temperature treatments.     

The influence of various physical parameters on anther culture of broccoli (Brassica oleracea var. italica)

Embryo formation by cultured broccoli (Brassica oleracea L. var. italica) anthers was best in the pH range of 5.5 to 5.8. Manipulation of the initial medium pH showed, however, that embryos could be recovered throughout the entire pH range tested. Experiments designed to test the influence of anther density on embryo production exhibited an apparent population effect. Comparison of anthers cultured with and without filaments showed a significantly lower level of embryo formation with filaments attached. The importance of anther orientation with the adaxial surface up was also demonstrated. Detailed studies of the effect of temperature on anther response showed the importance of 35°C treatments. Other temperatures and a variety of temperature manipulations were either comparatively ineffective or inhibitory. The duration of 35°C exposure required for optimal response varied widely between 18 and 48 h. Wide variation in plant to plant response was observed despite attempts to optimize the manipulation of physical parameters. Individual plants were identified that reliably formed many thousands of embryos, whereas other plants failed to form embryos under all tested conditions.     

Modification of cell development in vitro: The effect of colchicine on anther and isolated microspore culture in Brassica napus

In an attempt to discover the biological basis of microspore derived embryogenesis, the effect of the antimicrotubule agent colchicine on anther and free microspore embryogenesis was investigated. The microtubule inhibitor colchicine promoted embryogenesis from cultured anthers, both with regard to the number of anthers responding and the number of embryos being produced per anther. A similar promotional response was also observed with cultured microspores. Although the parameters for cultured anthers and free microspores differed, administration of the drug for a short period immediately prior to pollen mitosis I seems to exert the maximum promotional effect. Of the five cultivars of Brassica napus studied, all responded to colchicine treatment. However, the drug did release more embryogenic potential in poor-responding varieties (i.e. Lirawell and Optima) than in the highest responding variety (Topas). Colchicine also resulted in increased embryogenic response in microspores cultured at lower temperatures.These results are considered in terms of models proposed to explain the switch in microspore development from a gametophytic to a sporophytic pathway. The use ofcolchicine as agent to promote embryogenesis in previously recalcitrant species other than Brassica is also discussed.     

Genetic and non-genetic factors affecting anther culture of Brussels sprouts (Brassica oleracea var. gemmifera)

Summary Eight inbred lines of Brussels sprouts and ten F₁ hybrids derived from them were tested for their response to anther culture. From 5–19 plants per genotype were tested, and each plant was tested on 3–6 separate occasions. Results from the inbred lines were broadly similar to those from the F₁ hybrids, despite the inbreds producing fewer buds and having a higher frequency of anther deformities. The maximum embryo yield from an inbred line was 215 embryos per 100 anthers, and from a hybrid was 275. From estimation of the variance components it was calculated that, for both inbreds and hybrids, about half the total variation was genetic whereas variation due to plants within genotypes and to occasions within plants were each about 13% of the total. The narrow sense heritability of responsiveness to anther culture (estimated by the proportion of variation between inbred lines which was genetic) was 0.48, and there was partial dominance for this character. In three cases the hybrid outyielded the better inbred, and this heterosis may well be due to dispersed dominant genes.     

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.     

Performance of recombinant inbred lines in Brussels sprouts (Brassica oleracea var. gemmifera)

Summary Performance of a random array of recombinant inbred lines derived by single seed descent from five different source populations of Brussels sprouts (Brassica oleracea var. gemmifera) is presented. A total of 2,356 lines were tested in trials during 1985 and 1986. Three of the source populations were derived from double crosses between F₁ hybrids. These hybrids show a considerable heterotic advantage over their inbred parents for the most important agronomic traits. The recombinant inbred lines performed, on average, less well than the parental inbred material, indicating that additive x additive genie interactions may make a significant contribution to the performance of current inbred material. Nevertheless, the very large variation among the recombinant inbred lines permitted many lines to be identified which outperformed the best parental inbred for all traits. Two lines outperformed the reference F₁ hybrid, Gower, for an index that included marketable yield and quality. Consideration was also given to the dangers of misinterpreting phenotypically based proportions. Accordingly, response equations were used to ascertain the real genetic progress that was made. Advance seemed small when compared with the large heterotic effect, which is consistent with the segregation of a large number of loci. The distribution of the recombinant inbred lines was compared to predictions made from early generation trials. There was broad agreement but significant discrepancies existed which, it is suggested, may arise from the effects of genotype-environment interactions.     

Isolation of reduced genotypes of Hieracium pilosella using anther culture

Plants of reduced ploidy were derived from anther tissue of a pentaploid, apomictic biotype of Hieracium pilosella (mouse-ear hawkweed). Callusing was observed from both the somatic and generative tissue of the anther. Capitulum diameter was used as a correlative character to determine the best stage for harvesting the tissue. The highest degree of callusing coincided with the early uninucleate stage of microsporogenesis. Regenerants included tetraploids, triploids and two classes of aneuploids. Segregation for apomixis was observed among the tetraploid regenerants, an indication of the dominant inheritance of apomixis in this species.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid     

Wheat anther culture: effect of genotype and environmental conditions

Twenty-two cultivars and lines of winter and spring wheat (Triticum aestivum L.) were studied, most for the first time, for their anther culture response. The response was genotype dependent. Plants grown in the field gave higher callus induction frequency than those grown in the greenhouse and the controlled environment chamber. Donor plants grown in a season of low drought stress as compared to a season of severe drought stress resulted in a higher frequency of callus induction. Spherical microcalli were observed in two wheat genotypes in some of only those anthers that were placed with only one loculus in contact with the medium. Wheat lines that were more responsive to anther culture were identified.