Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Embryogenesis and plant regeneration from isolated microspores of Brassica rapa L. ssp. Oleifera

Summary Conditions favourable to embryogenesis from isolated microspores of Brassica rapa L. ssp. oleifera (canola quality) were identified. A population with enhanced responsiveness for microspore embryogenesis (C200) was synthesized by crossing individual plants showing microspore embryogenic potential. For optimal microspore embryogenesis, buds (2–3mm in length, containing mid-late uninucieate microspores) were collected from older plants (2 months old) and microspores isolated and washed in iron-free B5 medium. NLN medium with its iron content reduced to half was beneficial for initial microspore culture. An elevated temperature(33–35°C) during the first day of culture, followed by maintenance at 25°C resulted in dozens of embryos from each isolation (about 100 buds). Seeds were obtained from plants regenerated from microsporederived embryos after colchicine treatment.     

Scanning electron microscopy of microspore embryogenesis inBrassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy     

Optimization of methods for using polyethylene glycol as a non-permeating osmoticum for the induction of microspore embryogenesis in the <Emphasis Type="Italic">Brassicaceae</Emphasis>

The objective of this work was to enhance the quality and quantity of microspore-derived embryos of cruciferous species by using polyethylene glycol (PEG) to replace sucrose in the culture medium. The main advantage in using PEG is that it produces embryos that are morphologically more similar to zygotic embryos and have enhanced germination capabilities. When microspores were cultured in full strength NLN medium supplemented with 25% (w/v) PEG, the addition of 3 ml of full strength NLN with 13% (w/v) sucrose at 14 d was beneficial for embryo quality and quantity. Experiments showed that this PEG system could be used for a number of Brassica napus cultivars, as well as a number of other cruciferous species. PEG enhanced microspore embryogenesis in B. nigra, Crambe abyssinica, and Raphanus oleifera. Microspore-derived embryos were obtained from all cruciferous species evaluated (B. alboglabra, B. carinata, B. juncea, B. rapa, B. nigra, R. oleifera, Crambe abyssinica, Sinapis alba) using either sucrose or PEG as the osmoticum. Microspore embryogenesis was induced in B. napus in PEG-based cultures without a 32°C heat shock (i.e., 4, 15, 18, and 24°C). These temperature conditions were non-inductive when sucrose was used as the osmoticum. Spontaneous chromosome doubling occurred in 64–92% of the regenerated plants when PEG was used in the NLN culture medium, whereas in culture medium containing sucrose, the spontaneous doubling rate was 2–18%.     

Effects of brassinosteroids on microspore embryogenesis in <Emphasis Type="Italic">Brassica</Emphasis> species

Summary Experiments were conducted to determine the effects of brassinosteroids on microspore embryogenesis in Brassica species. Two compounds, 24-epibrassinolide (EBR) and brassinolide (BL), were evaluated. An increase in embryogenesis was observed in all Brassica napus lines evaluated, including Topas 4079 and several recalcitrant cultivars: Garrison, Westar, and Allons. Microspore embryogenesis, calculated as the number of embryos at 21 d of culture, was increased in the recalcitrant cultivars up to 12 times that of the control. An increase in microspore embryogenesis was also observed for B. juncea when EBR or BL was added to the culture medium. In constrast, no significant increases in embryogenesis was observed for several other Brassica species evaluated (i.e. B. carinata, B. nigra, and B. rapa). The addition of brassinosteroids to the induction media did not affect the subsequent conversion of the embryos to plantlets, but did appear to influence chromosome doubling.     

Mapping of RFLP and qualitative trait loci in Brassica rapa and comparison to the linkage maps of B. napus,B. oleracea,and Arabidopsis thaliana

A linkage map of restriction fragment length polymorphisms (RFLPs) was constructed for oilseed, Brassica rapa, using anonymous genomic DNA and cDNA clones from Brassica and cloned genes from the crucifer Arabidopsis thaliana. We also mapped genes controlling the simply inherited traits, yellow seeds, low seed erucic acid, and pubescence. The map included 139 RFLP loci organized into ten linkage groups (LGs) and one small group covering 1785 cM. Each of the three traits mapped to a single locus on three different LGs. Many of the RFLP loci were detected with the same set of probes used to construct maps in the diploid B. oleracea and the amphidiploid B. napus. Comparisons of the linkage arrangements between the diploid species B. rapa and B. oleracea revealed six LGs with at least two loci in common. Nine of the B. rapa LGs had conserved linkage arrangements with B. napus LGs. The majority of loci in common were in the same order among the three species, although the distances between loci were largest on the B. rapa map. We also compared the genome organization between B. rapa and A. thaliana using RFLP loci detected with 12 cloned genes in the two species and found some evidence for a conservation of the linkage arrangements. This B. rapa map will be used to test for associations between segregation of RFLPs, detected by cloned genes of known function, and traits of interest.     

Plant regeneration capacity of mesophyll protoplasts from Brassica napus and related species

Protoplasts from a total of thirty-six genotypes of Brassica species – B. napus, B. campestris (syn. B. rapa), B. juncea, and three distant relatives, Orychophragmus violaceus, Isatis indigotica and Xinjiang wild rape – were analysed for shoot regeneration using a feeder culture system. With the exception of B. campestris and Xinjiang wild rape, some genotypes of all the species could regenerate plants with high efficiency (above 20% of isolated calli initiating shoots). Several genotypes with high regeneration ability were elite breeding lines. Culture conditions as well as genotype had a significant impact on shoot regeneration frequency. In particular, silver nitrate added to the regeneration medium at doses of 6 and 30 μM improved shoot regeneration frequency to 25.4% and 52.2% of isolated calli, respectively, compared to 7.3% percent shoot regeneration without silver nitrate in seven responsive genotypes. Addition of silver nitrate to the regeneration medium also induced shoot regeneration in non-responsive genotypes. Intact plants could be obtained within three months from protoplast isolation in the regenerative genotypes using the current culture system. Advantages of mesophyll protoplasts as compared to protoplasts isolated from hypocotyls for genetic manipulation in Brassica species are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetics of Clubroot Resistance in <Emphasis Type="Italic">Brassica</Emphasis> Species

Clubroot disease, caused by the obligate plant pathogen Plasmodiophora brassicae Wor., is one of the most economically important diseases affecting Brassica crops in the world. The genetic basis of clubroot resistance (CR) has been well studied in three economically important Brassica species: B. rapa, B. oleracea, and B. napus. In B. rapa, mainly in Chinese cabbage, one minor and seven major CR genes introduced from European fodder turnips have been identified. Mapping of these CR genes localized Crr1 on R8, Crr2 on R1, CRc on R2, and Crr4 on R6 linkage groups of Chinese cabbage. Genes Crr3, CRa, CRb, and CRk mapped to R3, but at two separate loci, CRa and CRb are independent of Crr3 and CRk, which are closely linked. Further analysis suggested that Crr1, Crr2, and CRb have similar origins in the ancestral genome as in chromosome 4 of Arabidopsis thaliana. Genetic analysis of clubroot resistance genes in B. oleracea suggests that they are quantitative traits. Twenty-two quantitative trait loci (QTLs) were mapped in different linkage groups of B. oleracea. In B. napus, genetic analysis of clubroot resistance was found to be governed by one or two dominant genes, whereas resistance conferred by two recessive genes is reported. The quantitative analysis approach, however, proved that they are polygenic. In total, at least 16 QTLs have been detected on eight chromosomes of B. napus, N02, N03, N08, N09, N13, N15, N16, and N19. The chromosomal location of the other six QTLs is not clear. Cloning of any of these QTLs or resistance loci was not, however, possible until recently. Progress in genomics, particularly the techniques of comparative mapping and genome sequencing, supplements cloning and allows improved characterization of CR genes. Further development of DNA markers linked to CR genes will in turn hasten the breeding of clubroot-resistant Brassica cultivars.     

Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs)

Summary RFLPs were used to study genome evolution and phylogeny in Brassica and related genera. Thirtyeight accessions, including 10 accessions of B. rapa (syn. campestris), 9 cultivated types of B. oleracea, 13 nine-chromosome wild brassicas related to B. oleracea, and 6 other species in Brassica and allied genera, were examined with more then 30 random genomic DNA probes, which identified RFLPs mapping to nine different linkage groups of the B. rapa genome. Based on the RFLP data, phylogenetic trees were constructed using the PAUP microcomputer program. Within B. rapa, accessions of pak choi, narinosa, and Chinese cabbage from East Asia constituted a group distinct from turnip and wild European populations, consistent with the hypothesis that B. rapa had two centers of domestication. A wild B. rapa accession from India was positioned in the tree between European types and East Asian types, suggesting an evolutionary pathway from Europe to India, then to South China. Cultivated B. oleracea morphotypes showed monophyletic origin with wild B. oleracea or B. alboglabra as possible ancestors. Various kales constitute a highly diverse group, and represent the primitive morphotypes of cultivated B. oleracea from which cabbage, broccoli, cauliflower, etc. probably have evolved. Cauliflower was found to be closely related to broccoli, whereas cabbage was closely related to leafy kales. A great diversity existed among the 13 collections of nine-chromosome wild brassicas related to B. oleracea, representing various taxonomic states from subspecies to species. Results from these studies suggested that two basic evolutionary pathways exist for the diploid species examined. One pathway gave rise to B. fruticulosa, B. nigra, and Sinapis arvensis, with B. adpressa or a close relative as the initial ancestor. Another pathway gave rise to B. oleracea and B. rapa, with Diplotaxis erucoides or a close relative as the initial ancestor. Raphanus sativus and Eruca sativus represented intermediate types between the two lineages, and might have been derived from introgression or hybridization between species belonging to different lineages. Molecular evidence for an ascending order of chromosome numbers in the evolution of Brassica and allied genera was obtained on the basis of RFLP data and phylogenetic analysis.     

Intergeneric hybridization between Diplotaxis siifolia,a wild species and crop brassicas

Summary Attempts were made to obtain intergeneric hybrids between Diplotaxis siifolia, a wild species, and cultivars of Brassica (B. campestris, B. juncea, and B. napus). The crosses showed unilateral incompatibility. When the wild species was used as female parent, pollen germination and pollen tube growth were normal, but hybrid seeds aborted due to post-fertilization barriers. Reciprocal crosses (cultivars as female parent) showed strong pre-fertilization barriers; although pollen grains showed germination, pollen tubes failed to enter the stigma. Hybrids were realized in two of the crosses, D. siifolia x B. juncea and D. siifolia x B. napus, through ovary culture. The hybrids were multiplied in vitro by multiplication of axillary shoots, or somatic embryogenesis. Detailed studies were carried out on the hybrid D. siifolia x B. juncea. F₁ hybrids had shrivelled anthers and were pollen sterile. Amphiploids of this hybrid showed 60% pollen fertility and produced seeds upon self-pollination as well as backcross pollination with the pollen of B. juncea.     

Intergeneric (intersubtribe) hybridization between Moricandia arvensis and Brassica A and B genome species by ovary culture

Summary Intergeneric hybrids between Moricandia arvensis (C₃–C₄ intermediate species) and Brassica A and B genome species (B. campestris and B. nigra) were produced via ovary culture. When M. arvensis was used as a female parent, the hybrid embryo yield (0.25–0.45 embryo per pollination) was similar between two genomes, regardless of the male parent. The reciprocal hybrid using B. campestris as a female was also obtained, although yield of embryo was lower (0.02 embryo per pollination). On the other hand, no hybrids were obtained without the in vitro technique. As most hybrid embryos could not develop normal shoots, plants were regenerated by inducing shoots on the cultured hypocotyl. The hybrid nature of the regenerated plant was confirmed morphologically and cytogenetically. A certain amount of bivalents (2.52-2.71) in the hybrids indicated the existence of partial chromosome homology between two genera. The present results indicate that ovary culture is an effective technique for overcoming the crossing barrier between M. arvensis and Brassica cultivated species.     

Effect of boron on microspore embryogenesis and direct embryo to plant conversion in <Emphasis Type="Italic">Brassica napus</Emphasis> (L.)

The NLN-medium has been successfully used, since 1982, for microspore culture in Brassica napus and other Brassica species. Changes to the media composition were restricted to carbohydrate and nitrogen sources and growth regulators while micro-nutrients have not been optimized. The NLN-medium contains boron at a concentration of 162 µM. Boron is required for diverse physiological and metabolic processes in the cell. This study investigated the effect of seven- and 13-fold increased boron concentration on the induction of embryos in microspore cultures of four genotypes of B. napus. A significant improvement of microspore embryogenesis was achieved by both enhanced boron concentrations in the NLN medium. No effect on the regeneration of embryo to plant conversion was observed.     

A Complex Recombination Pattern in the Genome of Allotetraploid Brassica napus as Revealed by a High-Density Genetic Map

Polyploidy plays a crucial role in plant evolution. Brassica napus (2n = 38, AACC), the most important oil crop in the Brassica genus, is an allotetraploid that originated through natural doubling of chromosomes after the hybridization of its progenitor species, B. rapa (2n = 20, AA) and B. oleracea (2n = 18, CC). A better understanding of the evolutionary relationship between B. napus and B. rapa, B. oleracea, as well as Arabidopsis, which has a common ancestor with these three species, will provide valuable information about the generation and evolution of allopolyploidy. Based on a high-density genetic map with single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers, we performed a comparative genomic analysis of B. napus with Arabidopsis and its progenitor species B. rapa and B. oleracea. Based on the collinear relationship of B. rapa and B. oleracea in the B. napus genetic map, the B. napus genome was found to consist of 70.1% of the skeleton components of the chromosomes of B. rapa and B. oleracea, with 17.7% of sequences derived from reciprocal translocation between homoeologous chromosomes between the A- and C-genome and 3.6% of sequences derived from reciprocal translocation between non-homologous chromosomes at both intra- and inter-genomic levels. The current study thus provides insights into the formation and evolution of the allotetraploid B. napus genome, which will allow for more accurate transfer of genomic information from B. rapa, B. oleracea and Arabidopsis to B. napus.     

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     

Production of hybrids, amphiploids and backcross progenies between a cold-tolerant wild species, Erucastrum abyssinicum and crop brassicas

Three intergeneric hybrids were produced between a cold-tolerant wild species, Erucastrum abyssinicum and three cultivated species of Brassica, B. juncea, B. carinata and B. oleracea, through ovary culture. The hybrids were characterized by morphology, cytology and DNA analysis. Amphiploidy was induced in all the F₁ hybrids through colchicine treatment. Stable amphiploids and backcross progenies were obtained from two of the crosses, E. abyssinicum x B. juncea and E. abyssinicum x B. carinata. The amphiploid, E. abyssinicum x B. juncea was successfully used as a bridge species to produce hybrids with B. napus, B. campestris and B. nigra. These hybrids and backcross progenies provide useful genetic variability for the improvement of crop brassicas.     

Cryopreservation of isolated micropores of spring rapeseed (Brassica napus L.) for in vitro embryo production

Microspore cryopreservation is a potentially powerful method for long-term storage of germplasm for in vitro embryo production in plant species. In this study, several factors influencing embryo production following the ultra-low temperature (–196 °C in liquid nitrogen) storage of isolated microspores of rapeseed (Brassica napus L.) were investigated. Microspores were prepared in cryogenic vials and subjected to various cooling treatments before immersion in liquid nitrogen for varying periods. Efficiency of microspore cryopreservation was reflected by in vitro embryo production from frozen microspores. Of all the cooling treatments, microspores treated with a cooling rate of 0.25% °C/min and a cooling terminal temperature of –35 °C before immersion in liquid nitrogen produced the highest embryo yields (18% and 40% of unfrozen controls in two genotypes, respectively). Fast thawing in a 35 °C water bath was necessary to recover a high number of embryos from microspore samples being frozen at a higher cooling rate, while thawing speed did not affect samples after freezing at a slower cooling rate. The storage density of cryopreserved microspores affected embryo production. Storage at the normal culture density (8×10⁴ microspores/ml) was less efficient for embryo production than at high densities (4×10⁶ microspores/ml and 1.6×10⁷ microspores/ml), although no significant difference was found between the high densities. Evaluation of plant lines derived from frozen microspores indicated no variation in isozyme pattern and no enhanced cold tolerance of these lines. Isolated microspores of B. napus could be stored for extended period for in vitro embryo production.     

Effects of genotypes and culture conditions on microspore embryogenesis and plant regeneration in several subspecies of Brassica rapa L.

A number of factors influencing microspore embryogenesis and plant regeneration were examined in five subspecies (rapa, oleifera, niposinica, perviridis, broccoletto) of B. rapa. Addition of 6-benzylaminopurine (BA) in 1/2 NLN-10 medium improved the embryo yield by 2?C12 fold. Addition of activated charcoal (AC) in the medium was not effective for microspore embryogenesis. Moreover, AC canceled the positive effect of BA, when the medium containing both BA and AC was used. Of 24 genotypes examined for microspore embryogenesis, 22 genotypes of all five subspecies produced embryos ranging from 0.02 to 15.0 per 2?×?10⁵ microspores, but two genotypes were not responsive. Low temperature pretreatment of flower buds significantly improved the microspore embryogenesis. When cotyledonary embryos were subcultured on a filter paper placed on top of 0.8?% agar-solidified B5-2 medium and 1.6?% agar B5-2 medium, plant regenerations were increased 4?C8 fold compared to 0.8?% agar medium. The ploidy levels of regenerated plants in three genotypes were determined by flow cytometry, revealing that 66?C100?% of them were diploid. The results enable the advancement of breeding programs and genetic studies in B. rapa.     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

Enhanced post-germinative growth of encapsulated somatic embryos of Siberian ginseng by carbohydrate addition to the encapsulation matrix

Based on a protocol for microspore culture in apple (Malus domestica Borkh.), the embryo induction phase has been improved with regard to pretreatment of microspores for initiation of microspore embryogenesis, the concentration of carbon source in the induction medium and the microspore density in the suspension. Furthermore, the effect of the genotype was studied. To determine the efficiency of in vitro androgenesis, both methods, via anther and microspore culture, were investigated using the same bud material. A comparison of the efficiency of embryo induction in anther and microspore cultures showed that microspore culture resulted in an increase up to 10 times, depending on the genotype. The regeneration route in microspore culture is similar to that of androgenic embryos via anther culture and showed adventitious shoot formation in most cases after a long period of secondary embryogenesis.Communicated by H. Lörz