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.     

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.     

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.     

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     

Effect of silver nitrate on long-term culture and regeneration of callus from Brassica oleracea var. gemmifera

Incorporation of AgNO₃ (1–10 mg1^-1) into the culture medium of Brassica oleracea var. gemmifera callus significantly improved growth and allowed long-term callus culture. In the absence of AgNO₃, callus died shortly after removal from the hypocotyl explants. Regeneration of shoots from callus on low-hormone medium was also enhanced by AgNO₃. Significant differences in shoot production were found between the three genotypes examined. Cv. Aries produced large numbers of shoots even in the absence of AgNO₃. Investigation of callus production from the inbred parent lines of cv. Aries indicated that tissue culturability may be determined genetically.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Variations in response to high temperature treatments in anther culture of Brussels sprouts

The level, time of application and duration of the high temperature treatment necessary for embryo production from Brussels sprouts anther culture were examined. The effects of 29, 32, 35, and 38°C given for 24 h immediately following removal of the anthers from the bud, were tested on different cultivars, on different plants within the cultivars and on different occasions for each plant. Most embryos were produced following 32 and 35°C, very few following 30°C and none following 38°C. Although there was a tendency for some cultivars to respond better to one or other of the two more favourable temperatures, this varied considerably between individual plants. Plant to plant variation was also seen in the overall level of the response, although responsiveness tended to decline with successive samplings of the same plant. Experiments with cultivars Hal and Gower suggested that high temperature was required for at least 12 h after anther removal, but beyond that time the optimum period varied from plant to plant. If the excised anthers were held at 25°C for 16 h or more with Hal or 24 h or more with Gower before being exposed to the high temperature treatment, embrogenesis tended to be reduced. It is suggested that apparent non-responsiveness in anther culture may result to a large extent from the specific conditions that are used during the anther culture process.     

In vitro zygotic embryo culture of wild Musa acuminata ssp.malaccensis and factors affecting germination and seedling growth

In vitro zygotic embryo culture of wild banana significantly increased the germination compared to greenhouse grown seeds. Embryo orientation and BAP concentration significantly affected germination rate. These factors together with gelling agent, dark and light conditions and coconut water, also showed variable effects on the number of roots per plant, root length, shoot length, number of days to root emergence and number of days to shoot emergence.     

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.     

In vitro zygotic embryo culture of an endangered forest tree Givotia rottleriformis and factors affecting its germination and seedling growth

Summary This study reports a protocol for germination of Givotia rottleriformis (var. Tel. Thella Poniki) using zygotic embryo culture. A 100% germination was obtained by culturing the embryos on Murashige and Skoog medium containing 30 gl^−1 sucrose. A sucrose concentration lower or higher than 30 gl^−1 resulted in lower germination or promoted callus formation. The seedling growth was promoted by the addition of 100 mgl^−1 tyrosine in the medium. Seedlings germinated in the presence of 0.2–0.4 mgl^−1 α-naphthaleneacetic acid and 0.3–0.5 mgl^−1 indole-3-butyric acid were abnormal, showing a slender stem with slender roots or forming callus with stout roots. Germination also affected embryo orientation in culture; placing embryos upright on the medium was most beneficial for germination. The in vitro-germinated seedlings were acclimatized in soil under shady conditions with a survival rate of 60–70%. These plants were phenotypically normal, healthy, and similar to donor plants. This protocol will be useful for overcoming seed dormancy and for rapid multiplication and conservation of G. rottleriformis using zygotic embryo culture.     

Plant regeneration from microspore-derived embryos ofBrassica Napus: Effect of embryo age,culture temperature,osmotic pressure,and abscisic acid

Summary Microscope cultures ofBrassica napus cv. Topas undergo high frequency embryogenesis in vitro; however, the majority of microspore-derived embryos do not develop directly into plants but usually undergo abnormal development including the formation of secondary embryos on the hypocotyls. The present studies show that older embryos or embryos cultured at higher temperature (25° C) were more likely to undergo secondary embryogenesis whereas embryos cultured at 20° C or pretreated at 5° to 10° C for 28 days developed more readily into normal plants. Compared with embryos cultured at 25° C, those cultured at 20° C gave a threefold increase in normal plant production. Pretreatments at cooler temperatures (5° to 10° C) resulted in an additional two-to threefold increase in the recovery of normal plants. Higher osmoticum during pretreatment improved embryo survival at low temperatures but generally inhibited normal plant development. Abscisic acid was ineffective or deleterious.     

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.     

Flavonoids from cabbage are feeding stimulants for diamondback moth larvae additional to glucosinolates: Chemoreception and behaviour

In caterpillars two styloconic contact chemoreceptors on the maxillary galea are assumed to contain the main taste receptors involved in host plant selection. The diamondback moth, Plutella xylostella L. is a specialist feeder of plants in the Brassicaceae, a plant family characterized by the biosynthesis of glucosinolates. We used pea (Pisum sativum L., Leguminosae) as a neutral non-host for a dual-choice leaf disc assay to quantify feeding stimulation by glucosinolates and flavonoids. Increasing concentrations of sinigrin resulted in significant preferences for sinigrin-treated leaf discs, with a threshold between 1 and 3 M. Millimolar concentrations of four of the five flavonol triglucosides likewise elicited a significant preference for flavonoid-treated leaf discs. A mixture of four flavonoids and sinigrin was significantly preferred over sinigrin-treated leaf discs alone. Vigorous unicellular electrophysiological responses of medial maxillary styloconic taste sensilla were observed in response to five glucosinolates (glucocapparin, sinigrin, glucobrassicin, glucoiberin, and gluconasturtiin). This medial taste neuron responded in a dose-dependent manner to a concentration series of sinigrin, with a threshold of response of ca. 1 M. The lateral sensillum styloconicum contained a neuron sensitive to sucrose, glucose, and fructose. However, no responses in the two types of maxillary styloconic sensilla to the phagostimulatory flavonoids could be detected, suggesting that other taste organs mediate chemoreception of flavonoids. We conclude that diamondback moth larvae employ a combination of biosynthetically distinct categories of feeding stimulants which allows for a higher degree of discriminatory ability than when this would be based on glucosinolates alone.     

Transformation of rapid cycling cabbage (Brassica oleracea var. capitata) with Agrobacterium rhizogenes

Summary Genetically transformed cabbage (Brassica oleracea var. capitata) roots were obtained after inoculation with two engineered Agrobacterium rhizogenes strains, each harbouring a plant selectable marker gene in their T-DNA. Axenic root clones resistant to kanamycin or hygromycin B were established, most of which did not exhibit the phenotypic characteristics of Ri-transformed roots. Shoot regeneration was induced from roots after treatment with 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting plants exhibited various phenotypes: some looked normal, while others showed the transformed phenotype observed in other species. Direct evidence for genetic transformation was obtained by molecular hybridization. The trait was transmitted to the progeny. Transformed cabbage plants can be obtained within 6 months using this approach.     

Response of cabbage head weight to simulated Lepidoptera defoliation

Studies were conducted to determine the effects of continuous constant amounts of artificial defoliation through the preheading and heading growth stages on head weight of cabbage. High levels of continuous artificial defoliation caused a reduction in head weight in all three years of the study, but the highest yield was always attained at some low level of preheading or heading defoliation. These results demonstrate that cabbage is tolerant to some levels of continuous defoliation before and after head formation. Results from this study are incorporated into a cost-benefit analysis to estimate an economic threshold.

---

Résumé L'étude a porté sur le poids des pommes de choux (Brassica oleracea) soumis à différentes intensités de défoliations répétées avant et pendant la formation des pommes. Pendant les trois années de l'étude, huit intensités de défoliation continue ont réduit le poids des pommes, mais le poids le plus élevé a toujours été obtenu avec une faible défoliation avant et pendant la formation des pommes. Ceci montre que le chou tolère une certaine défoliation avant et pendant la formation des pommes. Les résultats de cette étude ont été utilisés dans une analyse coût-bénéfice pour estimer le seuil économique de défoliation.

     

Isolated microspore culture of Chinese flowering cabbage (Brassica campestris ssp. parachinensis)

Microspores of several genotypes of Brassica campestris ssp. parachinensis have been cultured in vitro and induced to undergo embryogenesis and plant formation. Conditions favourable for embryogenesis in this species include a bud size of 2–2.9 mm, NLN-13 culture medium (Nitsch and Nitsch 1967; Lichter 1981, 1982; Swanson 1990), and an induction through exposure to 32°C for a period of 48 h. Longer periods of an elevated temperature for induction of embryogenesis resulted in embryo abortion at early developmental stages. With the protocol developed here, microspores of 60–80% of donor plants could be induced to produce embryos, although embryo yields were low, i.e. 2–5 embryos per 10 buds. Some genotypes responded to culture conditions with high numbers of embryo formation (100–150 embryos per 10 buds) but most of these subsequently failed to mature. The pattern of cell division and morphological changes of the microspores in culture were studied using various microscopic techniques.     

Studies of protoplast culture and plant regeneration from commercial and rapid-cyclingBrassica species

Protoplasts were isolated from aseptic shoot cultures of commercial cultivars ofBrassica napus, B. oleracea andB. campestris, and from the six rapid-cycling brassica species. Of the rapid-cycling species, onlyB. napus responded well to the culture conditions used; 2% of protoplasts formed calli and up to 5% of calli regenerated shoots. Regeneration was also achieved from commercial cultivars ofB. napus andB. oleracea. For these two species the plating density, time of dilution with fresh medium and the composition of the shoot-inducing medium were all found to have an important influence on the efficiency of plant regeneration. Both responded better to maltose than to sucrose-based media. Under the optimum conditionsB. napus showed a plating efficiency of 7.8% and shooting efficiency of 17%; forB. oleracea the figures were 2% and 56%, respectively.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Application of a rapid glucose-6-phosphate isomerase assay in white cabbage breeding

In the studied conditions of cellulose acetate electrophoresis only glucose-6-phosphate isomerase (PGI, EC 5.3.1.9) appeared to be a polymorphic isozyme. The PGI analysis of 23 cabbage (Brassica oleracea var. capitata) inbred lines, carried out in two years, allowed detecting off-type individuals in four lines. Seed contamination caused by sib-pollination was detected in 10 out of 19 F₁ hybrids. In five of them the contamination did not exceed 5 %, and in the remaining five, ranged from 27–52 %. Also homozygosity of 67 plants obtained through anther culture in vitro was confirmed. The results indicate that PGI electrophoresis in cellulose acetate matrix is very fast and can be useful in the assessment of genetic purity in cabbage breeding materials.     

Brassinolide promotes adventitious shoot regeneration from cauliflower hypocotyl segments

When hypocotyl segments of cauliflower (Brassica oleracea var. boturytis L.) were cultured on MS medium containing brassinolide (BR) in the light, a significant stimulation of adventitious shoot regeneration was observed. Cytokinins (zeatin and iso-pentenylaminopurine) also promoted shoot regeneration. When BR was added together with these cytokinins, the maximal regeneration was strongly improved and the dose–response curve of cytokinin was shifted to the left. Regeneration was much lower in the dark. This was not due to a possible increased ethylene synthesis in the dark.     

The improvement in regenerated doubled haploids from anther culture of wheat by anther transfer

This study was conducted to determine the most suitable method of regeneration by comparing two approaches: transfer of anthers (with and without embryo-like structures) to regeneration conditions after a period of two to four weeks on induction medium (= anther-transfer treatment) and transfer of embryo-like structures to regeneration conditions after five to eight weeks on induction medium. The early transfer of anthers brought about a significant reduction in the number of embryos formed, but nevertheless significantly improved the frequency of plant regeneration. Combining an optimal date of anther transfer with the early addition of colchicine to the induction medium (100 mg l⁻¹ for 1 and 3 days) led to an increase in the number of doubled haploid regenerants. The results indicate that transferring the anthers after 28 days and adding 100 mg l⁻¹ colchicine to the induction medium on one day only caused a significant improvement in the ability of green plants to regenerate (7.0 compared to 0.50) as well as in chromosome doubling (success index: 4.0 compared to 0.33). This revised version was published online in August 2006 with corrections to the Cover Date.     

Rescue of interspecific lens hybrids by means of embryo culture

A two-stage in vitro technique was established for the development of interspecific hybrid embryos in the genus Lens. The culture of 14-day-old fertilized ovules on MS medium supplemented with zeatin, followed by the release of the embryos from the ovular integuments, allowed the development of viable and vigorous plants.