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.     

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.     

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 hybridization between Diplotaxis siettiana and crop brassicas for the production of alloplasmic lines

Summary Intergeneric hybrids were produced between Diplotaxis siettiana and Brassica campestris through embryo rescue. The hybrids were completely pollen sterile and backcrosses with pollen of B. campestris did not yield any seeds. Induction of colchiploidy restored pollen fertility and backcross pollinations yielded viable seeds. Cytological details of the hybrid, amphidiploid and backcross progenies were studied. Both pollen-sterile and pollen-fertile plants have been obtained in backcross 2 progeny. This hybrid (D. siettiana x B. campestris) was used as a bridge cross to transfer the cytoplasm of D. Siettiana to two other incompatible cultivars of Brassica — B. juncea and B. napus. Pollinations of the amphidiploid (D. siettiana x B. campestris, 2n = 36) with pollen of B. juncea/B. napus readily produced seeds without embryo rescue. These hybrids were grown to flowering and their cytological details were studied. Seeds have been produced from backcross pollinations of both these hybrids with the pollen of the respective cultivars. The results clearly show the feasibility of producing alloplasmic lines in all the three oilseed brassicas.     

Comparison of the karyotypes ofPsathyrostachys juncea andP. huashanica (Poaceae) studied by banding techniques

The karyotypes ofP. juncea (Elymus junceus) andP. huashanica (both outbreeders) were investigated by Feulgen-staining and by C-, N-, and Agbanding, based on a single plant in cach case. Both species have 2n=2x=14 and large chromosomes, possibly a generic character. The karyotype ofP. juncea has 8 metacentrics and 6 SAT-chromosomes with minute, heterochromatic satellites while that ofP. huashanica has 9 metacentrics and 5 SAT-chromosomes only, 2 of which with small, heterochromatic satellites. The C-banding patterns ofP. juncea chromosomes comprise from one to five, mostly small, bands at distal, and terminal positions, while those ofP. huashanica chromosomes are characterized by large telomeric bands in most arms. Banding patterns and chromosome morphology allow identification of the homologues of the seven chromosome pairs inP. juncea, but of two pairs inP. huashanica only. The patterns of both taxa are polymorphic, supporting that both taxa are outbreeders. The karyotypic characters suggest thatP. juncea is more closely related toP. fragilis than either is toP. huashanica. N-banding stains weakly. Silver nitrate staining demonstrates that nucleolus organizers of both species have different nucleolus forming capacities. The presence of micronucleoli suggests that both species have an extra unidentified chromosome with nucleolus forming capacity.     

Effects of waterlogging on growth and some physiological parameters of four Brassica species

Waterlogging tolerance of four Brassica species, Brassica campestris L., B. carinata A. Br., B. juncea (L.) Czern and Coss., and B. napus L. was assessed after 4 weeks growth in greenhouse at two waterlogging treatments, unflooded control soil, and fully waterlogged soil.Shoot fresh and dry biomass, in both mean and relative terms, was highest in B. juncea and lowest in B. napus at waterlogging treatment. B. carinata was as good as B. juncea in mean shoot fresh and dry matter but it had almost same relative shoot fresh matter as that in B. campestris, but was second highest in relative shoot dry weight.Waterlogging treatment caused a marked reduction in chlorophyll content in all four species but the species difference was not evident. However, B. juncea and B. napus had lower relative total chlorophyll than the other species.A marked increase in soluble protein content of B. juncea and a significant increase in total amino acids in B. carinata was observed under waterlogged conditions as compared to the other species.At the waterlogging regime, an increase in iron content in both shoots and roots was observed in all four species. B. juncea accumulated lower amount of iron in both shoots and roots as compared to the other species, whereas B. carinata had also lower iron in the roots. The species did not differ for shoot manganese content but B. carinata had significantly higher manganese in the roots as compared to the other species.     

Agrobacterium-mediated transformation protocol to overcome necrosis in elite AustralianBrassica juncea lines

In recent years,Brassica species have acquired an important position in the oilseed industry. Even thoughBrassica transformation protocols are well established,there is still a need for the development of new transformation protocols for elite AustralianB. juncea lines,because regeneration inB. juncea is highly genotype-dependent and in addition, their hypocotyl explants are susceptible to necrosis.Agrobacterium-mediated transformation protocol to overcome necrosis in elite AustralianB. juncea lines is described here. To overcome necrosis, we have adopted 2 strategies: extension of precultivation time of hypocotyl explants, and use of a 2-stage hygromycin selection process.The frequency of recovery of transformants from AustralianB. juncea andBrassica napus lines was 1.7% and 0.9%, respectively. Polymerase chain reaction tests confirmed that allBrassica plants that survived through stringent screening procedures were positive for the inserted hygromycin resistance gene,hph. Progeny from 6Brassica lines tested segregated for thehph gene, and χ² analysis suggested a 3:1 segregation ratio.This is in line with a tDNA integration into a single locus, which is an important feature of a transformation protocol for subsequent breeding purposes. Although the scientific content of this article has been reviewed,the full-text Web publication has not been edited in detail.     

Interaction of silicon and cadmium in <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

The objective of this study was to determine the effect of silicon (Si) and cadmium (Cd) on root and shoot growth and Cd uptake in two hydroponically cultivated Brassica species (B. juncea (L.) Czern. cv. Vitasso and B. napus L. cv. Atlantic). Both species are potentially usable for phytoextraction. Inhibitory effects of Cd on root elongation were diminished by the impact of Si. Primary roots elongation in the presence of Cd + Si compared with Cd was stronger and the number of lateral roots was lower in B. juncea than in B. napus. Cd content per plant was higher in B. napus roots and shoots compared with B. juncea. Suberin lamellae were formed closer to the root apex in Cd + Si than in Cd treated plants and this effect was stronger in B. napus than in B. juncea. Accelerated maturation of endodermis was associated with reduced Cd uptake. Cd decreased the content of chlorophylls and carotenoids in both species, but Si addition positively influenced the content of photosynthetic pigments which was higher in B. napus than in B. juncea. Si enhanced more substantially translocation of Cd into the shoot of B. napus than of B. juncea. Based on our results B. napus seems to be more suitable for Cd phytoextraction than B. juncea because these plants produce more biomass and accumulate higher amount of Cd. The protective effect of Si on Cd treated Brassica plants could be attributed to more extensive development of suberin lamellae in endodermis.     

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

Summary Restriction fragment length polymorphisms (RFLPs) of nuclear DNAs have been used to explore the origin and evolution of the six cultivated Brassica species. Extensive RFLP variation was found at the species, subspecies and variety levels. Based on RFLP data from Brassica and related genera, a detailed phylogenetic tree was generated using the PAUP microcomputer program, which permits a quantitative analysis of the interrelationships among Brassica species. The results suggested that 1) B. nigra originated from one evolutionary pathway with Sinapis arvensis or a close relative as the likely progenitor, whereas B. campestris and B. oleracea came from another pathway with a possible common ancestor in wild B. oleracea or a closely related nine chromosome species; 2) the amphidiploid species B. napus and B. juncea have evolved through different combinations of the diploid morphotypes and thus polyphyletic origins may be a common mechanism for the natural occurrence of amphidiploids in Brassica; 3) the cytoplasm has played an important role in the nuclear genome evolution of amphidiploid species when the parental diploid species contain highly differentiated cytoplasms. A scheme for the origins of diploid and amphidiploid species is depicted based on evidence gathered from nuclear RFLP analysis, cpDNA RFLP analysis, cytogenetic studies and classical taxonomy.     

A Brassica campestris-alboglabra addition line and its use for gene mapping,intergenomic gene transfer and generation of trisomics

Summary Brassica campestris-alboglabra monosomic addition lines were developed from a trigenomic Brassica hybrid (2 n=3 x=29, AAC) obtained by backcrossing a resynthesized B. napus (2 n=4 x=38, AACC) line to its parental B. campestris (2 n=2 x=20, AA) line. One addition line was characterized genetically with three loci specific for the alien chromosome and cytologically by meiotic analysis. The following results were obtained. (1) The same chromosome in the B. alboglabra (2 n= 2 x=18, CC) genome carried the three loci, E _c, W _c and Lap-1 C ^c, which control the biosynthesis of erucic acid, white flower colour and the faster migrating band of leucine aminopeptidase, respectively. The linear order and possible positions of the three loci were inferred. The meiotic behaviour of the alien chromosome was documented and its transmission frequency was assessed. (2) Intergenomic recombination frequently occurred in the monosomic addition line, resulting in the introgression of one or two loci from the alien chromosome into the B. campestris genome. (3) B. campestris trisomics were found in the progeny of the monosomic addition line. (4) The removal of the other eight C-genome chromosomes from the trigenomic Brassica hybrid led to a dramatic increase in the erucic acid content of the monosomic addition line. (5) No offspring of the trigenomic Brassica hybrid showed evidence of intergenomic recombination and introgression of the W _c locus into the B. campestris genome. It is questioned whether such a difference might be due to a possible regulating mechanism for homoeologous chromosome pairing.     

Inter- and intra-genomic homology of the Brassica genomes: implications for their origin and evolution

In order to determine the homologous regions shared by the cultivated Brassica genomes, linkage maps of the diploid cultivated B. rapa (A genome, n = 10), B. nigra (B genome, n = 8) and B. oleracea (C genome, n = 9), were compared. We found intergenomic conserved regions but with extensitve reordering among the genomes. Eighteen linkage groups from all three species could be associated on the basis of homologous segments based on at least three common markers. Intragenomic homologous conservation was also observed for some of the chromosomes of the A, B and C genomes. A possible chromosome phylogenetic pathway based on an ancestral genome of at least five, and no more than seven chromosomes, was drawn from the chromosomal inter-relationships observed. These results demonstrate that extensive duplication and rearrangement have been involved in the formation of the Brassica genomes from a smaller ancestral genome.     

Male sterility caused by cytoplasm of Brassica oxyrrhina in B. campestris and B. juncea

Summary Synthetic alloploid Brassica oxyrrhina (2n = 18, OO) x B. campestris (2n = 20, AA) was repeatedly backcrossed with B. campestris to place B. campestris nucleus in the cytoplasm of B. oxyrrhina. Alloplasmic plants, obtained in BC₅ generation, were stably male sterile but mildly chlorotic during initial development. Synthetic alloploid B. oxyrrhina-campestris was also hybridized with B. juncea to transfer B. oxyrrhina cytoplasm. Segregation for green and chlorotic plants was observed in BC₁ and BC₂ generations. By selection, however, normal green male sterile B. juncea was obtained in BC₃. Pollen abortion in both B. campestris and B. juncea is post-meiotic.     

In vitro breeding of Brassica juncea L. to enhance metal accumulation and extraction properties

In vitro breeding and somaclonal variation were used as tools to improve the potential of Indian mustard (Brassica juncea L.) to extract and accumulate toxic metals. Calli from B. juncea were cultivated on a modified MS medium supplemented with 10–200 μM Cd or Pb. Afterwards, new B. juncea somaclones were regenerated from metal-tolerant callus cells. Three different phenotypes with improved tolerance of Cd, Zn and Pb were observed under hydroponic conditions: enhanced metal accumulation in both shoots and roots; limited metal translocation from roots to shoots; reduced accumulation in shoots and roots. Seven out of thirty individual variants showed a significantly higher metal extraction than the control plants. The improvement of metal shoot accumulation of the best regenerant (3× Cd, 1.6× Zn, 1.8× Pb) and metal extraction (6.2× Cd, 3.2× Zn, 3.8× Pb) indicated a successful breeding and selection of B. juncea, which could be used for phytoremediation purpose.     

Unique chromosome behavior and genetic control in Brassica×Orychophragmus wide hybrids: a review

Researchers recognized early that chromosome behavior, as other morphological characters, is under genetic control and gave some cytogenetical examples such as the homoeologous chromosome pairing in wheat. In the intergeneric sexual hybrids between cultivated Brassica species and another crucifer Orychophragmus violaceus, the phenomenon of parental genome separation was found under genetic control during mitosis and meiosis. The cytogenetics of these hybrids was species-specific for Brassica parents. The different chromosome behavior of hybrids with three Brassica diploids (B. rapa, B. nigra and B. oleracea) might contribute to the different cytology of hybrids with three tetraploids (B. napus, B. juncea and B. carinata). The finding that genome-specific retention or loss of chromosomes in hybrids of O. violaceus with B. carinata and synthetic Brassica hexaploids (2n=54, AABBCC) is likely related to nucleolar dominance gives new insight into the molecular mechanisms regarding the cytology in these hybrids. It is proposed that the preferential expressions of genes for centromeric proteins from one parent (such as the well presented centromeric histone H3) are related with chromosome stability in wide hybrids and nucleolar dominance is beneficial to the production of centromere-specific proteins of the rRNAs-donor parent and to the stability of its chromosomes.     

Resynthesis of Brassica carinata by protoplast fusion and recovery of a novel cytoplasmic hybrid

Brassica carinata (2n=34, BBCC), was synthesized by fusing dark grown etiolated hypocotyl protoplasts of B. nigra (2n=16, BB) with green mesophyll protoplasts of B. oleracea (2n=18,CC) using polyethylene glycol. Heterokaryons could be microscopically distinguished from the parental types by their dark green chloroplasts in the colourless hypocotyl protoplast background. The mean heterokaryotic fusion frequency estimated on the basis of this morphological distinction was about 16%. A total of 626 calli were obtained, of which 92 regenerated shoots after transfer to zeatin (2 mg/l) supplemented MS medium. Of these, 81 calli differentiated into plants morphologically similar to naturally occurring B. carinata and 11 calli yielded plants resembling parental types. Meiosis in seven hybrid plants showed the chromosome number to be 2n=34 the sum of B. nigra and B. oleracea chromosomes. Molecular confirmation of the amphidiploid nature of hybrids was obtained by probing with a B. juncea derived genomic clone. The use of chloroplast and mitochondrial specific gene probes, revealed that one plant was a cytoplasmic hybrid having cp DNA sequences of both B. oleracea and B. nigra and mt DNA sequences of B. nigra.Abbreviations PEG Polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - IBA Indole-3-butyric acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962)     

芥菜型油菜脂肪酸含量的变异、相关性分析及芥酸调控基因FAE1特异引物设计

芥菜型油菜是我国芸苔属的三大油料作物之一,具有耐旱、抗病虫等优良特性;而我国是芥菜型油菜的重要起源中心,具有丰富的种质资源.该研究从全国各地搜集了34份芥菜型油菜,在贵阳环境条件下种植,其脂肪酸含量(芥酸、油酸、硬脂酸、亚麻酸和亚油酸)表现出丰富的变异,并呈正态分布.结果表明:这些芥菜型油菜种质资源的不同脂肪酸含量间的相关性发现,芥酸和油酸间呈极显著负相关,亚麻酸和硬脂酸呈极显著的正相关,亚麻酸和亚油酸呈现负相关.利用这些材料的脂肪酸含量进行主成分分析,发现绝大部分材料(30份,占88.2%)集中在二维图的特定区域,只有少数其它材料散落在图中其它区域,他们分别是SL63、棱角油菜、T6342和长阳黄芥,这些变异较大的材料在芥菜型油菜的育种中可以发挥特殊作用.此外,运用来自甘蓝型油菜和甘蓝的芥酸调控基因FAE1的已知序列,并设计了FAE1特异引物,而引物在全部34份芥菜型油菜种质资源中均表现出了较好的扩增效果.因此证实芥菜型油菜中至少含有一个FAE1拷贝.该研究结果对于芥菜型油菜育种在我国的开展及其未来的分子育种具有重要的指导意义.     

A PCR based B-genome-specific marker in <Emphasis Type="Italic">Brassica</Emphasis> species

Previous hybridisation studies showed that the repetitive DNA sequence pBNBH35 from Brassica nigra (genome BB, 2n=16) bound specifically to the B-genome and not to the A- or C-genomes of Brassica species. We amplified a sub-fragment of pBNBH35 from B. nigra by PCR, cloned and sequenced this sub-fragment, and confirmed that it was a 329-bp sub-fragment of pBNBH35. PCR and hybridisation techniques were used to confirm that the pBNBH35 sub-fragment was Brassica B-genome-specific. Fluorescence in situ hybridisation (FISH) in B. nigra, B. juncea (AABB, 2n=36) and B. napus (AACC, 2n=38) showed that the pBNBH35 sub-fragment was present on all eight Brassica B-genome chromosomes and absent from the A- and C-genome chromosomes. The pBNBH35 repeat was localised to the centromeric region of each B-genome chromosome. FISH clearly distinguished the B-genome chromosomes from the A-genome chromosomes in the amphidiploid species B. juncea. This is the first known report of a B-genome repetitive marker that is present on all B-genome chromosomes. It will be a useful tool for the detection of B chromosomes in interspecific hybrids and may prove useful for phylogenetic studies in Brassica species.     

A cDNA clone encoding Brassica calmodulin

A 834 bp cDNA encoding calmodulin (CaM) has been isolated from Brassica juncea. On Northern analysis this cDNA hybridises this cDNA to mRNAs of about 0.9 kb in leaf, silique and peduncle. Genomic Southern analysis indicates the presence of a CaM multigene family in Brassica juncea. Comparison of the predicted amino acid sequence of Brassica CaM with that of Arabidopsis CaM ACaM-2 and ACaM-3 showed 100% homology, which is not unusual, since both plants belong to the family Cruciferae. In situ hybridisation studies on Brassica seedlings using a digoxigenin-labelled RNA probe showed that high levels of CaM mRNA were detected in the leaf primordia and the shoot apical meristem, and to a lesser degree, in the zone of root elongation of the root tip. The occurrence of a higher rate of cell division and growth in these regions than its surrounding tissue may possibly be related to higher levels of CaM mRNA.     

Production and cytogenetics of the intergeneric hybrids Brassica juncea×Orychophragmus violaceus and B. carinata×O. violaceus

 Intergeneric hybrids between Brassica juncea (2n=36), B. carinata (2n=34) and Orychophragmus violaceus (2n=24) were produced when B. juncea and B. carinata cultivars were used as female parents. The hybrids between B. juncea and O. violaceus had an intermediate morphology except for petal colour and were partially fertile. The hybrids between B. carinata and O. violaceus had a matroclinous morphology and were nearly fertile. Cytological analysis of the hybrids and their progenies gave the following results. (1) In the hybrids between B. juncea and O. violaceus, the somatic tissues of the roots, leaves and styles were mixoploid (2n=12–42), and cells with 24, 30 or 36 chromosomes were the most frequent. Based on the recorded numbers and behaviour of the mitotic and meiotic chromosomes, complete and partial separation of the parental genomes was proposed to have occurred during mitosis. This resulted in the occurrence of cells with possibly complete and incomplete complements of the parental species and cells with parental complements and some additional chromosomes from the other parent. (2)  Pollen mother cells (PMCs) possibly with both parental chromosome complements, only B. juncea chromosomes or a complete B. juncea complement with additional O. violaceus chromosomes were more competitive in entering meiosis. The majority of fertile gametes were deduced to have been produced by PMCs with a B. juncea complement with or without additional O. violaceus chromosomes. (3) The progeny plants from selfed hybrids between B. juncea and O. violaceus were morphologically either of a B. juncea, hybrid or variable type. Cytologically they were grouped into six types according to the frequencies of cells with various chromosome numbers. All of the plants except 2 which constituted two types, were mixoploids, composed of cells with various chromosome numbers, mainly in a certain serial range. (4) The hybrid plants between B. carinata and O. violaceus were mixoploids with chromosome numbers in the range of 12–34, and cells with 2n=34 were the most frequent. The main categories of PMCs with 17 bivalents at metaphase I and 17 : 17 segregations at anaphase I contributed to the high fertility of the hybrids and the fact that their progeny after selfing were mainly plants with 2n=34. Somatic and meiotic separation of the parental genomes was proposed to have occurred in the hybrids between B. carinata and O. violaceus. (5) Mitotic and meiotic elimination of what could be O. violaceus chromosomes might also have contributed to the observed mitotic and meiotic cell types in the two kinds of hybrids studied. Finally, the possible mechanisms behind these cytological observations and their potential in the production of Brassica aneuploids were discussed. Received: 4 February 1997/Accepted: 29 July 1997     

Fertile somatic hybrids between Petunia hybrida and a wild species,Petunia variabilis

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F₂ and BC₁ seedlings were obtained.