In vitro culture of isolated microspores and regeneration of plants in Brassica campestris

A protocol previously developed for B. napus microspore culture was modified to produce embryos from several lines of Brassica campestris. Bud size, genotype, media constituents, and incubation time and temperature were examined. Donor plants were grown in a growth cabinet at a day/night temperature of 10/5°C. Microspores were isolated from buds 2.0 – 2.9 mm in length and cultured in modified Lichter (1982) medium containing 17% sucrose, pH 6.2. After 48 h at 32°C, the incubation medium was replaced with NLN (Lichter 1982) medium containing 10% sucrose. Microspores were cultured at 24°C in darkness and embryos developed after three weeks. More than 1000 plants have thus far been regenerated. Genotypic differences were observed for microspore embryogenesis. The majority of the regenerants were haploid, however colchicine could be effectively used to achieve chromosome doubling.     

Cytological study of interspecific hybrid between Brassica campestris x B. hirta (Sinapis alba)

Interspecific hybrids from the crossing Brassica campestris x B. hirta are reported in our study for the first time. F₁ plants were obtained by using ovary culture. The phenotype of hybrids was similar to B. napus; the plants were self-fertile. Investigation of meiotic division and nuclear DNA content measurements showed the amphidiploid origin of these hybrids. The relationship between genome A and D, as well as the spontaneous amphidiploidization of the hybrids, are discussed.     

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.     

Regeneration of transgenic vegetable brassicas (Brassica oleracea andB. campestris) via Ri-mediated transformation

A procedure for the production of fertile transgenic brassicas via Ri-mediated transformation is reported in this paper. Transgenic hairy root lines were selected for 12 vegetable brassica cultivars and lines representing six varieties: broccoli, Brussels sprouts, cabbage, cauliflower, rapid-cycling (allBrassica oleracea) and Chinese cabbage (B. campestris). Leaf explants or petioles of intact cotyledons were co-cultivated withAgrobacterium strain A4T harbouring various binary vectors. The T-DNA region of all binary vectors contained a neomycin phosphotransferase II gene for kanamycin resistance, in addition to other genes. Hairy root lines grew prolifically on hormone-free medium containing kanamycin. Transgenic shoots were regenerated from all cultivars either spontaneously or after transfer of hairy roots to a hormone-containing medium. Southern analysis confirmed that the plants were transgenic. Plants from all brassica types were successfully transferred to greenhouse conditions. Plants were fertile and segregation analysis confirmed transmission of traits to progeny.Abbreviations BA 6-Benzylaminopurine - GUS -Glucuronidase - LS Linsmaier and Skoog medium - NAA I-Naphthaleneacetic acid - NPTII Neomycin phosphotransferase II - TDZ thidiazuron     

Molecular marker analysis of genes controlling morphological variation in Brassica rapa (syn. campestris)

Construction of a detailed RFLP linkage map of B. rapa (syn. campestris) made it possible, for the first time, to study individual genes controlling quantitative traits in this species. Ninety-five F₂ individuals from a cross of Chinese cabbage cv Michihili by Spring broccoli were analyzed for segregation at 220 RFLP loci and for variation in leaf, stem, and flowering characteristics. The number, location, and magnitude of genes underlying 28 traits were determined by using an interval mapping method. Zero to five putative quantitative trait loci (QTL) were detected for each of the traits examined. There were unequal gene effects on the expression of many traits, and the inheritance patterns of traits ranged from those controlled by a single major gene plus minor genes to those controlled by polygenes with small and similar effects. The effect of marker locus density on detection of QTL was analyzed, and the results showed that the number of QTL detected did not change when the number of marker loci used for QTL mapping was decreased from 220 to 126; however, a further reduction from 126 to 56 caused more than 15% loss of the total QTL detected. The detection of putative minor QTL by removing the masking effects of major QTL was explored.     

In vitro vegetative propagation of Chinese cabbage

Explants from cotyledons, axillary buds, inflorescence stems and flower buds of Brassica campestris ssp. pekinensis (Lour.) Olsson (Chinese cabbage, cv. Wongbok) were cultured on MS medium with growth regulators. Multiple shoots were obtained from cotyledons, axillary buds and flower buds but not from inflorescence stems. Propagation of shoots from cotyledons was more successful than from axillary buds and flower buds. The vegetative propagation rates varied amongst clones derived from cotyledons of the same cultivar and seed lot. The propagation rates of the cotyledon-derived material followed a normal distribution with an average propagation rate of 2.6 shoots per two weeks subculture when cultured on MS media plus 44.4 m benzyladenine (BA) and 14.8 m -indolebutyric acid (IBA). Shoots from three clones were cultured on MS medium with nine different concentrations of BA. The concentration of BA which promoted the highest rate of shoot propagation varied for the three clones and was in the range 44.4 to 177.6 m.     

Evaluation of Brassica rapa L. genotypes for microspore culture response and identification of a highly embryogenic line

Isolated microspore culture techniques are being widely used in Brassica breeding programs to generate haploid and doubled haploid plants. A number of factors influence regeneration response in vitro including genotype. In order to assess the effect of genotype on microspore embryogenesis in B. rapa L. var. oleifera, 17 cultivars and breeding lines were evaluated. Embryos developed from all but one genotype when using NLN medium with 17% sucrose, followed by a reduction in sucrose concentration to 10%, 48 h later. The number of embryos /100 buds differed between genotypes, ranging from 0 to 70. Further studies indicated that sucrose concentration and incubation time influenced embryogenesis. Selection studies carried out with an Agriculture and Agri-Food Canada breeding line have resulted in the identification of a highly embryogenic B. rapa line. This line produced thousands of microspore-derived embryos /100 buds and will be useful in mutant selection and gene transfer as well as biochemical and developmental studies.     

In vitro regeneration of plantlets in Brassica alboglabra

Different vegetative parts of Brassica alboglabra seedlings and mature plants were used as explants in culture.A high frequency (60–100%) of shoot regeneration was obtained from hypocotyl explants, nodal stem segments, internodal segments and shoot apices cultured on Murashige-Skoog basal medium. Addition of 6-benzylaminopurine and kinetin increased the average number of shoots per explant. When detached and transferred to basal medium, the shoots readily developed roots. Regenerated plantlets could be successfully transplanted in soil.     

In vitro mutation and selection of doubled-haploid Brassica napus lines with improved resistance to Sclerotinia sclerotiorum

This paper describes a new protocol to develop doubled-haploid (DH) Brassica napus lines with improved resistance to Sclerotinia sclerotiorum. In this protocol, haploid seedlings derived from microspore cultures of B. napus were used to produce haploid calli for in vitro mutation-selection. For routine screening, mutation was induced by EMS (ethylmethane sulfonate) or occurred spontaneously, and screening for resistant mutants occurred on media with added oxalic acid (OA) as a selection agent. In tests with selected lines, the optimal concentration of EMS for mutation was determined to be 0.15%, and the optimal concentration of OA for in vitro screening was 3 mmol/l (half lethal dose was 3.1 mmol/l) for the first cycle of screening. There was an accumulated effect of OA toxicity on calli over two cycles of screening, but the growth and capacity of the surviving calli for regenerating seedlings were not affected by OA. Of the 54 DH lines produced from the in vitro mutation-selection, two DH lines of resistant mutants, named M083 and M004, were selected following seedling and glasshouse tests. The resistance of M083 and M004 to S. sclerotiorum following tests with both mycelial inoculum and OA was greater than that of their donor lines and the resistant control Zhongyou 821. In both glasshouse and field disease nurseries, disease indices on M083 and M004 were less than 50% of those of the control. The time required for M083 and M004 to mature was 14 days and 10 days shorter, respectively, than that of their donor lines. Furthermore, M083 had more pods per inflorescence, a greater 1,000 seed weight and higher yield than its donor line. Random amplified polymorphic DNA characterisation showed that M083 had DNA band patterns that differed from its donor line.     

Virulence of Agrobacterium tumefaciens strains with Brassica napus and Brassica juncea

Summary Brassica napus and Brassica juncea were infected with a number of Agrobacterium tumefaciens strains. Tumourigenesis was very rapid and extremely efficient on B. juncea with all but one of the strains. Tumourigenesis on B. napus varied widely. It was very efficient with the nopaline strains, was reduced with the succinamopine strain A281 and was very weak with the octopine strains. The latter observation was confirmed with six different B. napus rapeseed cultivars. The selectivity was due to differences in the virulence of Ti plasmids with B. napus, rather than the tumourigenicity of the T-DNA or virulence of the chromosomal genes associated with the strains. An exception was strain LBA4404. The virulence of the octopine strains was increased by coinfection with more virulent disarmed strains and by induction with acetosyringone.     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

Role of polyamines on de novo shoot morphogenesis from cotyledons of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro

Summary The promotive effect of ethylene inhibitors (Els), i.e. AgNO₃ and aminoethoxyvinylglycine (AVG) on de novo shoot regeneration from cultured cotyledonary explants of Brassica campestris ssp. pekinensis cv. Shantung in relation to polyamines (PAs) was investigated. The endogenous levels of free putrescine and spermidine in the explant decreased sharply after 1–3 days of culture, whereas endogenous spermine increased, irrespective of the absence or presence of Els. AgNO₃ at 30 M did not affect endogenous PAs during two weeks of culture. In contrast, explants grown on medium containing 5 M AVG produced higher levels of free putrescine and spermine which increased rapidly after three days and reached a peak at 10 days. An exogenous application of 5 mM putrescine also resulted in a similar surge of endogenous free spermine of the explant. More strikingly, shoot regeneration from explants grown in the presence of 1–20 mM putrescine, 0.1–2.5 mM spermidine, or 0.1–1 mM spermine was enhanced after three weeks of culture. However, exogenous PAs generally did not affect ethylene production, and endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC of the explant. This study shows the PA requirement for shoot regeneration from cotyledons of B. campestris ssp. pekinensis in vitro, and also indicates that the promotive effect of PAs on regeneration may not be due to an inhibition of ethylene biosynthesis.Abbreviations PAs polyamines - AVG aminoethoxyvinylglycine - SAM S-adenosylmethionine - ACC 1-aminocyclopropane-1-carboxylate - Els ethylene inhibitors     

A simple method to estimate the percentage of hybridity in canola (Brassica napus) F1 hybrids

We have developed an efficient PCR-based system that uses RAPD markers for the certification of F₁ hybrids of canola. These markers were selected by screening five parental lines used in three crosses X, Y and Z with 131, 131 and 322 primers respectively. Stable DNA fragments that were homozygous and specific to the male inbreds were used to certify F₁ hybrid populations. The hybrid production system was based on self-incompatibility (SI) alleles that prevent self-pollination of the female parent. The efficiency of two S-alleles was compared under both field and greenhouse conditions. The percentage of hybridity was estimated in different F₁ populations. We found a significant difference between the two alleles for their efficiency in controlling selfing; both alleles were stable under greenhouse conditions, one allele appeared less reliable under field conditions.     

Effects of molybdenum on ascorbate-glutathione cycle metabolism in Chinese cabbage (Brassica campestris L. ssp. pekinensis)

A hydroponic trial was conducted to investigate effects of molybdenum (Mo) on ascorbate-glutathione cycle (AsA-GSH cycle) metabolism in Chinese cabbage (Brassica campestris L. ssp. pekinensis). Mo was applied at four rates: 0, 0.01, 0.15 and 1.5 mg l⁻¹. The concentrations of ascorbate, dehydroascorbate, reduced- and oxidized- glutathione, and activities of five key enzymes in the AsA-GSH cycle were studied. The results showed that appropriate Mo application increased the fresh weight of Chinese cabbage, but excess application of Mo (1.5 mg l⁻¹ Mo) decreased the fresh weight. Total ascorbate and reduced ascorbate concentrations in the Chinese cabbage increased with Mo application rates. Although no significant differences existed in DHA concentration between the different Mo regimes, but it has an increase trend with the 0.01 mg l^-1 Mo treatment, and then decreased with the Mo level increasing. No significant difference in GSH concentration was found between the different Mo treatments. Compared with the control, the GSSG concentration decreased significantly in the 0.01 mg l⁻¹ Mo treatment. The activities of APX, MDHAR, DHAR and GR increased due to Mo application. But the activity of AAO decreased with increasing Mo application rates. It is hypothesized that Mo may promote the redox process and regeneration of ascorbic acid, and affect the ability of anti-oxidation in the Chinese cabbage. Responsible Editor: Jian Feng Ma.     

In vitro plant regeneration of leguminous trees (Albizia spp)

Hypocotyl explants of three leguminous forest tree species, Albizia amara, A. lucida and A. richardiana, have differentiated shoot buds on B₅ basal medium. Maximum number of shoots per explant developed on basal medium augmented with 2,4-D (0.1 M) in A. amara (2) and BA (10 M) for both A. lucida (2) and A. richardiana (1.6). Higher concentrations of auxins in the medium, in general, enhanced rooting and callusing but cytokinins promoted the growth of green calli. BA enchanced the differentiation of shoots in the three species. The in vitro grown shoots of A. amara and A. richardiana, after subculturing on B₅+1 M IAA developed roots (up to 30–40%). These plants have been successfully transferred to the field.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - BM Gamborg's B₅ medium with 0.9% agar+3% sucrose - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - NAA -naphthaleneacetic acid     

Genetic analysis of novel intra-species unilateral incompatibility in Brassica rapa (syn. campestris) L.

Plants have evolved many systems to prevent inappropriate fertilization. Among them, incompatibility is a well-organized system in which pollen germination or pollen-tube growth is inhibited in pistils. Self-incompatibility (SI), rejecting self-pollen, promotes outbreeding in flowering plants. On the other hand, inter-species incompatibility, preventing gene flow among species to restrict outbreeding, usually occurs unilaterally, and is known as unilateral incompatibility (UI). In Brassicaceae, little is known about the molecular mechanism of UI, although S-locus genes involved in recognition of self-pollen have been characterized in the SI system. In the present study, we characterized novel UI observed between members of the same species, Brassica rapa; pollen of Turkish SI lines was specifically rejected by pistils of the Japanese commercial SI variety Osome. The incompatible phenotype of this intra-species UI closely resembled that of SI. Segregation analysis revealed that the pollen factor of this UI was not linked to the S-locus.The revised version was published online in December 2004 with corrections to figure 1.     

In vitro pollen selection in Brassica napus L. for resistance to phytotoxic compounds from Alternaria brassicicola (Schw.) Wilts

Summary Pollen samples from Brassica napus cvs Arran and Herkules were incubated for 1 h in a germination medium or in a medium to which 20 mg ml^–1 of a toxic extract from Alternaria brassicicola had been added. The pollen samples were then used to pollinate cv Primor. A number of the plants, obtained from pollinations using pollen incubated in the toxic extract, produced pollen with a significantly increased ability to germinate in medium containing 10 mg ml^–1 of the extract, evidence that some selection for resistance to the toxic compounds produced by A. brassicicola had occurred. The potential application of in vitro pollen selection and conditions necessary for its success are discussed.     

In vitro multiplication of a sterile interspecific hybrid,Brassica fruticulosa x B. campestris

In vitro methods for plant multiplication of a sterile interspecific hybrid between Brassica fruticulosa and B. campestris through either micropropagation or callus regeneration is described. Shoot-tip, single-node and leaf explants, obtained from in vitro-grown hybrids, regenerated on media containing NAA and BA. In vitro application of colchicine induced chromosome doubling in in vitro-regenerated shoots resulting in the production of fertile amphidiploids. Comparative studies on regeneration potential of the hybrid and its parents were also carried out using callus from leaf explants. The explants of B. fruticulosa and the hybrid were capable of shoot and root formation while those of B. campestris failed to form shoots but produced profuse roots. The results demonstrate the efficacy of an in vitro method in producing a large number of hybrid plants and fertile amphidiploids from incompatible crosses that yield very few hybrid seeds/seedlings.Abbreviations BA benzyladenine - CMS cytoplasmic male sterile - AA diploid genome of B. campestris - FF diploid genome of B. fruticulosa - NAA -naphthaleneacetic acid     

Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro

Summary A system has been developed for efficient regeneration of shoots from Brassica campestris in vitro. Using 4-day old cotyledons with petioles as expiants and a combination of BA and NAA in the regeneration media, up to 70% of expiants produced shoots after 2 weeks in culture. The optimal conditions for regeneration were found to include a BA concentration of 2mgL^–1 and NAA concentration of 1mgL^–1. Light intensity had a profound effect on regeneration potential. The use of silver ions as an inhibitor of ethylene action reduced regeneration rates in this system. Rooting occured simultaneously with shoot formation on these media and the resultant shoots could be rooted readily on minimal medium. The genotype dependency was investigated and indicated that this method would be widely applicable to B. campestris cultivars. Regeneration of one cultivar, a high erucic acid type (R-500), was inefficient in the system described here. Histological studies indicated the development of multiple shoot primordia from the petiolar cut ends of the expiants after the initiation of meristematic activity in the cells about 100m from the cut site within 2 days of culture initiation. The system described is compatible with previously reported Agrobacterium — mediated transformation protocols involving cotyledonary petioles.     

Bioassembly of acyl lipids in microspore-derived embryos of Brassica campestris L.

The native lipid composition and the capacity of cell-free extracts to biosynthesize acyl lipids in vitro were determined for the first time using the recently reported microspore-derived (MD) embryo system from the Brassica campestris low erucic acid line BC-2 (Baillie et al. 1992). The total lipid fraction isolated from midcotyledonary stage MD embryos (21 days in culture) was composed primarily of triacylglycerol (76%) with an acyl composition quite similar to that of mature BC-2 seed. When incubated in the presence of glycerol-3-phosphate, ¹⁴C 181-CoA, and reducing equivalents, homogenates prepared from 21-day cultured MD embryos were able to biosynthesize glycerolipids via the Kennedy pathway. The maximum in vitro rate of triacylglycerol biosynthesis could more than account for the known rate of lipid accumulation in vivo. The homogenate catalyzed the desaturation of 181 to 182 and to a lesser extent, 183. The newly-synthesized polyunsaturated fatty acids initially accumulated in the polar lipid fraction (primarily phosphatidic acid and phosphatidylcholine) but began to appear in the triacylglycerol fraction after longer incubation periods. As expected for a low erucic acid cultivar, homogenates of MD embryos from the BC-2 line were incapable of biosynthesizing very long chain monounsaturated fatty acyl moieties (201 and 221) from 181-CoA in vitro. Nonetheless, embryo extracts were still capable of incorporating these fatty acyl moieties into triacylglycerols when supplied with ¹⁴C 201-CoA or ¹⁴C 221-CoA. Collectively, the data suggest that developing BC-2 MD embryos constitute an excellent experimental system for studying pathways for glycerolipid bioassembly and the manipulation of this process in B. campestris.Abbreviations CPT sn-1,2-diacylglycerol cholinephosphotransferase - DAG diacylglycerol - DGAT diacylglycerol acyltransferase - DGDG digalactosyldiacylglycerol - G-3-P glycerol-3-phosphate - G-3-PAT glycerol-3-phosphate acyltransferase - LPA lyso-phosphatidic acid - LPAT lyso-phosphatidic acid acyltransferase - LPC lyso-phosphatidylcholine - LPCAT acyl-CoA: lyso-phosphatidylcholine acyltransferase - LPE lyso-phosphatidylethanolamine - MGDG monogalactosyldiacylglycerol - PA phosphatidic acid - PA Phosphatase, phosphatidic acid phosphatase - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - TAG triacylglycerol - 181-CoA oleoyl-Coenzyme A - 181 oleic acid, cis-9-octadecenoic acid - 182 linoleic acid, cis-9,12-octadecadienoic acid - 183 -linolenic acid, cis-9,12,15-octadecatrienoic acid - 201 cis-11-eicosenoic acid - 221 erucic acid, cis-13-docosenoic acid; all other fatty acids are designated by number of carbon atoms: number of double bonds National Research Council of Canada Publication No. 35896