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.     

Microspore embryogenesis in anther cultures of two Indian cultivars of Brassica juncea (L.) Czern.

Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained. Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis. Embryogenesis was also stimulated by an initial culture at 5°C for 3 days. Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos. All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets. The androgenic plants were haploid (2n=18).     

Rapid plant regeneration through organogenesis and somatic embryogenesis from cultured protoplasts of Brassica juncea

Protoplasts derived from hypocotyls of seedlings grown on half-strength MS medium containing 1% sucrose were cultured at a density of 5×10⁴ ml^-1 in Kao's medium supplemented with 1.0 mgl^-12,4-D, 0.1 mgl^-1 NAA and 0.5 mgl^-1 zeatin riboside. After three days of culture in darkness at 25±1°C, cultures were transferred to light (70 Em^-2s^-1) in a 16/8 h ligø ht/dark cycle. Cultures were diluted on the 7th, 10th and 13th day with Kao's medium containing 3.4% sucrose, 0.1 mgl^-1 2,4-dichlorophenoxyacetic acid and 1.0 mgl^-1 benzyladenine. On the fifteenth day, microcalli were plated on K₃ medium gelled with 0.5% agarose (Type 1, low EEO, Sigma). After a further period of two weeks, transfers were made to specific media to achieve either organogenesis or somatic embryogenesis. Time taken from plating protoplasts to obtaining plantlets is 8–10 weeks. Using this procedure, several hundred regenerated plants have been hardened in a growth chamber and transferred to soil.     

植物游离小孢子胚胎形成机理

植物小孢子胚胎的形成是基于具有单套染色体的小孢子在外界胁迫条件下通过脱分化和再生而形成一个完整植株的过程,是产生双单倍体的常用方式.它体现了植物细胞的全能性,为植物组织培养和胚胎发育的基础研究提供了一个独特的模式.从细胞学水平、代谢水平和分子水平3个方面综述在植物游离小孢子胚胎形成机制方面已取得的研究进展,并提出目前尚待解决的问题,以及对未来的展望,为进一步研究小孢子胚胎发生机制提供理论依据和奠定技术基础.     

A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea

Summary An efficient culture system has been developed for repeated cycles of somatic embryogenesis in microspore-derived embryos of Brassica juncea without a callus phase. Haploid embryos produced through anther culture showed a high propensity for direct production of somatic embryos in response to 2 mgL^–1 BA and 0.1 mgL^–1 NAA. The embryogenic cultures which comprised the elongated embryonal axis of microspore-derived embryos when explanted and grown on the medium of same composition produced a large number of secondary embryos. These somatic embryos in turn underwent axis elongation and produced more somatic embryos when explanted and cultured. This cycle of repetitive somatic embryogenesis continued with undiminished vigour passage after passage and was monitored for more than a year. Somatic embryos from any passage when isolated at cotyledonary stage and grown on auxin-free medium for 5 days and then on a medium containing NAA (0.1 mgL^–1), developed into complete plants with a profuse root system and were easily established in the soil. The cytology of the root tips of these plants confirmed their haploid nature. The total absence of callus phase makes the system ideal for continuous cloning of androgenic lines, Agrobacterium-mediated transformation and mutation induction studies.     

Morphogenesis in Isolated Microspore Cultures of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Androgenesis is a phenomenon in which microspores are made to bypass the sexual pathway and follow the sporophytic mode of development to generate new plants without the intervention of fertilization under specialized in vitro conditions. Microspore culture provides an ideal system, with a large, relatively uniform population of haploid cells, for use in mutant selection, genetic transformation and in studies on the molecular mechanism of induction of androgenesis and embryogenesis. This paper involves a study on establishing a reproducible and efficient protocol for microspore embryogenesis in various varieties of Brassica juncea. The genotype had a pronounced effect on androgenic response in microspore cultures. The cultivar Rajat exhibited the most response, producing around 3500 embryos/100 buds. The microspores of B. juncea cv. PR-45 from ed plants maintained at a day/night temperature of 10 °C/5 °C form embryos with suspensors with varied morphology. The microspore embryos germinated to produce plants with frequencies. These plants exhibited 52% survival and 74% fertility.     

Genetic and environmental effects on in vitro shoot regeneration from cotyledon explants of Brassica juncea

Twenty-one lines of Brassica juncea were screened for frequency of in vitro shoot formation from cultured cotyledons of 8-day old seedlings. All brown-seeded Indian lines showed good shoot regeneration with 20–50% of cotyledons giving rise to shoots. Very poor shoot regeneration (0–12%) was observed for the predominantly yellow-seeded Chinese/European lines, including two erucic acid-free lines. Germination of seedlings in hydroponic nutrient solution markedly enhanced subsequent shoot regeneration frequency from cotyledons of three Indian lines but had no effect on shoot formation from the recalcitrant Chinese/European lines.     

甘蓝型油菜游离小孢子培养的胚胎发生

以生长在非控温控光下的４个冬性甘蓝型油菜（ＢｒａｓｓｉｃａｎａｐｕｓＬ．）品种为供体,进行游离小孢子培养。研究发现,多数品种在开花３－７天取材最为适宜。在甘蓝型油菜小孢子培养中只有单核晚期的小孢子才可能发育成胚状体,而花药培养时处于单核早期的小孢子易于发育成胚状体。在适当花期选取发育比较一致的单核晚期小孢子培养,经数小时后,部分小孢子便开始膨大,这是小孢子发育成胚的最早标志,膨大的小孢子中,有部分形成多细胞球并进一步发育成胚。用春性甘蓝型油菜为材料进行蔗糖浓度的实验结果表明：培养３天后,在１６％蔗糖培养基中存活的小孢子最多,达１６．１３％;培养３０天后,胚状体诱导频率则以１３％蔗糖浓度为最高,每花蕾可达１４４个胚状体。如果在１６％蔗糖培养基中培养３天后,添加等体积的１３％蔗糖培养基,能够大大提高胚状体的诱导频率,为仅用１３％蔗糖培养基培养的３．７倍。这一实验体系正在用于抗菌核病的诱变与筛选,并作为外源基因导入的实验体系。     

Plant regeneration from stem cortex expiant and protoplast cultures of Brassica juncea (mustard)

Summary Plant regeneration from stem cortex explants of 13 genotypes of Brassica juncea was assessed. Regeneration was strongly affected by genotype, as up to 50.6 shoots were produced per 100 calli of the most responsive line (Blaze), whereas no shoots were obtained from less responsive lines (Zeml, Vniimk351). Blaze was chosen for B. juncea stem cortex protoplast isolation. After one week of culture, 11–14% of the cells had divided, and about 0.002% produced 1–2 mm colonies within 6 weeks. Up to 7% of these colonies gave rise to shoots upon transfer to plant regeneration medium.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - BAP 6-benzylaminopurine - FDA fluorescein diacetate - IAA indole acetic acid - IBA indole butyric acid - 2ip 2-isopentenyl-adenine - NAA 1-naphthaleneacetic acid - TD thidiazuron     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

The genetics of adult-plant blackleg (Leptosphaeria maculans) resistance from Brassica juncea in B. napus

The genetic control of adult-plant blackleg (Leptosphaeria maculans) resistance in a Brassica napus line (579NO48-109-DG-1589), designated R13 possessing Brassica juncea-like resistance (JR), was elucidated by the analysis of segregation ratios in F₂ and F₃ populations from a cross between R13 and the highly blackleg-susceptible B. napus cultivar Tower. The F₂ segregration ratios were bimodal, demonstrating that blackleg resistance in R13 was controlled by major genes. Analysis of the segregation ratios for 13 F₃ families indicated that blackleg resistance in these families was controlled by three nuclear genes, which exhibited a complex interaction. Randomly sampled plants of F₃ progeny all had the normal diploid somatic chromosome number for B. napus. The similarities between the action of the three genes found in this study with those controlling blackleg resistance in B. juncea is discussed.     

Properties of partially purified leaf ornithine aminotransferase of Brassica juncea under salt stress

Properties of the partially purified L-ornithine: 2-oxoacid aminotransferase (EC 2.6.1.13) of leaves of Brassica juncea salt tolerant somaclone SR3P6-2 and its parent cv. Prakash were studied. The enzyme from the somaclone SR3P6-2 was relatively more efficient in terms of its Km, Vmax, and Ea (activation energy) and required higher levels of chlorides for inhibition as compared to the enzyme from the parent cv. Prakash. These results suggest some salt-stress related changes in the enzyme.     

Mutant acetolactate synthase gene is an efficient in vitro selectable marker for the genetic transformation of Brassica juncea (oilseed mustard)

We report in this study, the successful deployment of a double mutant acetolactate synthase gene (ALSdm, containing Pro 197 to Ser and Ser 653 to Asn substitutions) as an efficient in vitro selection marker for the development of transgenic plants in Brassica juncea (oilseed mustard). The ALS enzyme is inhibited by two categories of herbicides, sulfonylureas (e.g. chlorsulfuron) and imidazolinones (e.g. imazethapyr), while the mutant forms are resistant to the same. Three different selection agents (kanamycin, chlorsulfuron and imazethapyr) were tested for in vitro selection efficiency in two B. juncea cultivars, RLM198 and Varuna. For both the cultivars, higher transformation frequencies were obtained using chlorsulfuron (3.8 +/- 0.6% and 4.6 +/- 0.9% for RLM198 and Varuna, respectively) and imazethapyr (10.2 +/- 0.7% for RLM198 and 7.8 +/- 1.2% for Varuna) as compared to that obtained on kanamycin (3.1 +/- 0.2% and 2.8 +/- 0.5% for RLM198 and Varuna, respectively). Additionally, transformation frequencies were higher on imazethapyr than on chlorsulfuron for both the cultivars indicating that imidazolinones are better selective agents than sulfonylureas for the selection of mustard transgenics.     

Growth of Albugo candida (race unidentified) on Brassica juncea callus cultures

An obligate fungus Albugo candida (Pers. ex Lév.) Ktze. (race unidentified) was successfully grown on host callus tissues of Brassica juncea cv. Varuna. Of the various type of diseased explants used, young (green) hypertrophied inflorescence axis bearing non-erumpent zoosporangial blisters allowed the fungus to multiply asexually over the host calli on modified MS-medium (Murashige and Skoog, 1962). The dual cultures were maintained up to 6–8 subcultures without loss of viability of zoosporangia on MS-medium supplemented with 10.0 mg L^–1 IBA, 0.05 mg L^–1 kinetin, 25.0 mg L^–1 AA, 1.0 mg L^–1 biotin, 1.0 mg L^–1 thiamine-HCl and 1.0 g L^–1 casein hydrolysate. The fungus grew only on the callus cells and not axenically on the medium. Pathogenicity test and histopathology of cultures proved the existence of the viable fungus in vitro.Abbreviations AA ascorbic acid - BAP 6-benzyl aminopurine - CH casein hydrolysate acid hydrolysed - 2,4-D-2,4 dichlorophenoxy acetic acid - FAA formaldehyde acetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - HgCl₂ mercuric chloride - Kinetin 6-furfuryl aminopurine - MS Murashige and Skoog (1962) - NAA alpha naphthalene acetic acid - rh relative humidity - sdw sterile distilled water - wt. weights     

Microspore development inBrassica napus and the effect of high temperature on division in vivo and in vitro

Summary Brassica napus cv. Topas microspores isolated and cultured near the first pollen mitosis and subjected to a heat treatment develop into haploid embryos at a frequency of about 20%. In order to obtain a greater understanding of the induction process and embryogenesis, transmission electron microscopy was used to study the development of pollen from the mid-uninucleate to the bicellular microspore stage. The effect of 24 h of high temperature (32.5 °C) on microspore development was examined by heat treating microspore cultures or entire plants. Mid-uninucleate microspores contained small vacuoles. Late-uninucleate vacuolate microspores contained a large vacuole. The large vacuole of the vacuolate stage was fragmented into numerous small vacuoles in the late-uninucleate stage. The late-uninucleate stage contained an increased number of ribosomes, a pollen coat covering the exine and a laterally positioned nucleus. Prior to the first pollen mitosis the nucleus of the lateuninucleate microspore appeared to be appressed to the plasma membrane; numerous perinuclear microtubules were observed. Microspores developing into pollen divided asymmetrically to form a large vegetative cell with amyloplasts and a small generative cell without plastids. The cells were separated by a lens-shaped cell wall which later diminished. At the late-bicellular stage the generative cell was observed within the vegetative cell. Starch and lipid reserves were present in the vegetative cell and the rough endoplasmic reticulum and Golgi were abundant. The microspore isolation procedure removed the pollen coat, but did not redistribute or alter the morphology of the organelles. Microspores cultured at 25 °C for 24 h resembled late-bicellular microspores except more starch and a thicker intine were present. A more equal division of microspores occurred during the 24 h heat treatment (32.5 °C) of the entire plant or of cultures. A planar wall separated the cells of the bicellular microspores. Both daughter cells contained plastids and the nuclei were of similar size. Cultured embryogenie microspores contained electron-dense deposits at the plasma membrane/cell wall interface, vesicle-like structures in the cell walls and organelle-free regions in the cytoplasm. The results are related to embryogenesis and a possible mechanism of induction is discussed.Abbreviations B binucleate - LU late uninucleate - LUV late uninucleate vacuolate - M mitotic - MU mid-uninucleate - RER rough endoplasmic reticulum - TEM transmission electron micrograph     

Linked epistasis for six quantitative traits in Indian mustard (Brassica juncea (L.) Czern & Coss)

Summary Gene effects, and interactions, and associations between days-to-flower initiation and maturity, number of secondary branches and siliquae per plant, and 1,000-seed weight and yield per plant were studied in a cross of Indian mustard (Brassica juncea (L.) Czern & Coss) using the parents and F₁, F₂, F₃, B₁, B₂, B₁₁, B₁₂, B₂₁, B₂₂, B_1S, B_2S, B₁F₁, B₂F₁, B_1bip, B_2bip, F₂P₁, F₂F₁, and F_2bip generations. A linked digenic model was adequate for all characters studied. According to this model, the main effects, additive and interactions between linked pairs of genes, were present in varying proportions for days-to-flower initiation and maturity and number of siliquae per plant. The contribution of linked epistatic effects, however, was much greater than that of additive effects. Dominance effects contributed significantly to the inheritance of days-to-flower initiation. Duplicate epistasis was observed for all traits except 1,000-seed weight where epistasis was of the complementary type. A complete association among the genes of similar effect (increasing or decreasing) was observed for number of secondary branches and siliquae, and yield per plant. Coupling phase linkage was observed for days-to-flower initiation whereas repulsion phase linkage was observed for daysto-maturity and 1,000-seed weight.     

Isozyme studies in Indian mustard (Brassica juncea L.)

Summary A comparative study of peroxidase and esterase isozymes was carried out at five developmental stages of siliqua in order to characterize twelve genotypes of Indian mustard. The studies showed nearly the same number of isozyme bands at every stage for peroxidase and a varying number of isozyme bands for esterase. The appearance and disappearance of bands, along with their intensity scores, indicated the role of different isozymes at different stages of siliqua development. It has been ascertained that these patterns, especially the intensity scores, can be successfully used to characterize different Indian mustard genotypes.     

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.     

Artificial synthesis of interspecific chimeras between tuber mustard (Brassica juncea) and cabbage (Brassica oleracea) and cytological analysis

Interspecific chimeras between tuber mustard and red cabbage were obtained by in vitro graft-culture method. Before grafting, 6-day-old seedlings of tuber mustard and red cabbage were vertically half-cut and treated with different concentrations of 6-BA and NAA for 1 min, then, they were symmetrically fit together. As a result, sectorial chimeras were initially produced from the united shoot tips. The maximum frequency of chimeral bud formation reached 6.33% when the vertical sections of tuber mustard and cabbage were treated with 2 mg/l 6-BA and 1 mg/l NAA. When sectorial chimeras were propagated on MS medium containing 1 mg/l 6-BA, periclinal and mericlinal chimeras gradually developed. Chimeral shoots were rooted on half-strength MS medium containing 0.1 mg/l NAA. The rooted chimeras were acclimatized and transferred to the field for cytological and morphological analysis. The results showed that stomata density in the chimeras was significantly higher than that of their parents, while chloroplast size, starch grain size and number were intermediate between the two parents. The chimeras were further analyzed by flow cytometry, and the results indicated that they contained both sets of parental chromosomes. Moreover, chimeral plants possessed valuable characters from the two parents.