Induction of Hydrolytic Enzymes in Brassica campestris in Response to Pathovars of Xanthomonas campestris

Inoculation of mature leaves of turnip (Brassica campestris) with the incompatible Xanthomonas campestris pv vitians resulted in the induction of β-1,3-glucanase and chitinase/lysozyme (CHL) activity. No increase in the basal activity of β-1,3-glucanase was observed after inoculation of leaves with heat- or rifampicin-killed X. c. vitians, Escherichia coli, or sterile water. Inoculation with the compatible X. campestris pv campestris resulted in a slower induction of glucanase than that seen with X. c. vitians. In contrast, all bacteria caused an induction of CHL activity. One major β-1,3-glucanase (molecular mass 36.5 kilodaltons, isoelectric point [pl] ~8.5) was purified from both inoculated and untreated leaves by ion-exchange chromatography. The enzyme degraded laminarin by an endo-glycolytic mechanism. Two major CHL isozymes (CHL 1 and CHL 2, molecular mass 30 kilodaltons and pl 9.4 and 10.2, respectively) were purified from X. c. vitians inoculated leaves by affinity chromatography on a chitin column followed by ion-exchange chromatography. Both enzymes degraded chitin by an endo-glycolytic mechanism although the ratio of lysozyme to chitinase specific activities for CHL 1 and CHL2 were different. The induction of CHL 1 was associated with the hypersensitive reaction caused by X. c. vitians whereas all other treatments induced largely CHL 2.     

Induced polyploidization in Brassica campestris L. (Brassicaceae)

Present experimental design has been made up to obtain crop with higher ploidy level via synthetic polyploidization. Since ploidy manipulation is generally associated with the obtainment of some increased enviable traits of the crop and also provides them greater adaptability to unfavorable or harsh circumstances as compared to its diploids counterparts. Thus, herein present research autotetraploids of Brassica campestris L. have been lucratively achieved by the application of colchicine. Two methods of treatment were utilized i.e. seed treatment and seedling treatment. No polyploidy could be obtained through seed treatment while seedling treatment responded well towards polyploidy. However, the status of autotetraploidy has been confirmed by cytomorphological investigations of treated plants as against its diploids counterparts. For the purpose, morphological parameters such as increased stomata size, pollen diameter, flower size, reproductive organs whereas reduction in plant height, leaf length, leaf breadth, stomata frequency, number of flowers/inflorescence etc. were appraised. Further, cytological observations were made that had clearly revealed the doubling of genome in the autotetraploids as compared to diploids. Meanwhile, pollen fertility and size of pollen grains were evaluated as well.     

Ethylene-induced Fine Structure Alterations in Cotton and Sugarbeet Radicle Cells

Electron microscopic examination of cotton (Gossypium hirsutum L.) and sugarbeet (Beta vulgaris L.) radicles subjected to ethylene at 1, 10, 100 microliters per liter and a saturated ethylene atmosphere showed distinct ultrastructural differences compared to aerobic, control tissue and to anaerobic, nitrogen-treated radicles. Short term ethylene treatments of 1 hour induced the formation of cisternal stacks of rough endoplasmic reticulum in undifferentiated radicle cells. Similar profiles of rough endoplasmic reticulum were not observed in control radicles, but were found in nitrogen-treated tissues. Additionally, ribosomes in all ethylene-treated radicles were larger than ribosomes in control and nitrogen-treated cells. By ribosome density determinations it was found that the ethylene-treated ribosomes were swollen and less dense. The cell walls of the ethylene-treated tissue were also expanded and less dense than those of control tissue.     

Inheritance of oilseed rape (Brassica napus) RAPD markers in a backcross progeny with Brassica campestris

Different cultivars/transgenic lines of oilseed rape (Brassica napus) were crossed (as females) with different cultivars/populations of Brassica campestris. All cross combinations produced seed, with an average seed set per pollination of 9.8. Backcrossing of selected interspecific hybrids (as females) to B. campestris resulted in a much lower seed set, average 0.7 seed per pollination. In the single backcross progeny where a large enough population (92 plants) was obtained for analysis, 33 B. napus specific RAPD markers were investigated to determine the extent of transfer of oilseed rape genetic material into this population. Markers were transferred to the backcross generation with frequencies ranging from 26% to 91%. Almost all of the markers (30/33) were transferred in a frequency not significantly different from 50%. Analysis of the pairwise segregation of markers revealed that 23 markers could be assigned to six linkage groups, most probably reflecting six B. napus C-chromosomes. The presence of backcross plants with recombinant genotypes suggests that complex genetic processes can take place during interspecific hybridisation and backcrossing in these Brassica species. The implications of our results for the possible choice of integration sites of transgenes in oilseed rape are discussed.     

Radicle ofEchinocactus platyacanthus (Cactaceae)

Radicle of matureEchinocactus platyacanthus embryo is approximately 320 m long and represents less then 1/7 of the embryonal axis length. The radicle-hypocotyl boundary can be distinguished according to the striking difference in the size and shape of cells in protoderm and procambium, as well as discontinuity and different number of the cell files in the ground meristem. The root cap is small, consists of 4 layers of cells covering the apex of the radicle. The upper limit of the root cap is approximately 100 m closer towards the radicle tip than the radicle-hypocotyl boundary. Ultrastructure of radicle cells showed numerous lipid bodies as is typical for other oily seeds. Protein bodies of variable structure were also present together with other cell structures. Striking differences in protein body structure were found when protoderm and ground meristem were compared. Several small globoid crystals were present in each protein body of the protoderm, while protein bodies in the radicle ground meristem mostly contained one large globoid crystal. X-ray microanalysis revealed presence of P, K and Mg in all analyzed globoid crystals. Fe, Ca and Zn were detected in some of them.Abbreviations EDX microanalysis energy-dispersive X-ray microanalysis - GC(s) globoid crystals - ICP spectroscopy ion-coupled plasma spectroscopy - LM light microscopy - PB(s) protein bodies - SEM scanning electron microscopy - TEM transmission electron microscopy     

Meiosis in double trisomic Brassica campestris

Meiosis in a double trisomic Brassica campestris (2n=20+1+1) found among the progeny of autotriploid B. campestris (3n=30) was studied to detect homologies of duplicate types of chromosomes in the a genome of Brassica. The two extra chromosomes paired with the corresponding homologues and formed two trivalents in 10.5% of nuclei revealing the double trisomic nature. They formed a separate bivalent in 15.6% of nuclei proving the homology of these two chromosomes in the a genome. Anaphase I and II segregations revealed a normal disjunction of chromosomes.Emeritus Professor of Botany & Emeritus Scientist, Indian Council of Agricultural Research.     

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.     

Effect of sodium dodecyl sulphate on the high molecular weight protein fraction of mustard (Brassica juncea) and rapeseed (Brassica campestris)

The effect of sodium dodecyl sulphate on mustard and rapeseed 12S protein has been monitored by the techniques of ultracentrifugation, viscosity, difference spectra and fluorescence spectrophotometry. At low concentration of sodium dodecyl sulphate (<3.47 mM) mustard protein undergoes aggregation and at higher concentrations it dissociates to 1.8 S protein, the dissociation being complete at 17.3 mM sodium dodecyl sulphate. The rapeseed protein, on the other hand, undergoes dissociation at all the concentrations of sodium dodecyl sulphate. The reduced viscosity values of mustard protein in the presence of the denaturant are higher than those of rapeseed protein. Similarly in difference spectra change in absorbance values of mustard protein are higher.’ The relative fluorescence intensity of the mustard protein increases with sodium dodecyl sulphate concentration, upto 0.87 mM and this is followed by fluorescence quneching at higher denaturant concentrations. However, with the rapeseed protein fluorescence quenching was observed at all concentrations of sodium dodecyl sulphate.     

Molecular cloning and characterization of nucleoside diphosphate (NDP) kinases from Chinese cabbage (Brassica campestris)

Nucleoside diphosphate kinases (NDPKs) are key metabolic enzymes that catalyze the synthesis of non-adenosine nucleoside triphosphates (NTP) by transfer of the terminal phosphate between NDP and NTP. Recently we isolated three NDPK cDNAs from Chinese cabbage cDNA library. BcNDK1 has 636 bp and encodes a putative 17.4 kDa protein, BcNDK2 has 854 bp and encodes a putative 25.5 kDa protein, and BcNDK3 is 986 bp long and encodes a putative 25.7 kDa protein. The precursor proteins of BcNDK2 and BcNDK3 have long N-terminal extensions containing putative chloroplast or mitochondrial targeting sequences. A phylogenic tree showed that the 3 BcNDKs are highly homologous to other plant NDPK genes, especially those of Arabidopsis. Expression of the BcNDK genes depended on the developmental stage and the conditions of seed germination. Most notably, expression of BcNDK2 increased dramatically in seedlings transferred to the light after germinating in the dark. In addition, BcNDK3 differed from BcNDK1 in being highly expressed in the hooks and cotyledons of seedlings. Although all BcNDKs were highly expressed in petals, BcNDK1 was also expressed in pistils. Expression of each of the BcNDKs increased as the flower bud matured. These results indicate that NDPKs are involved in physiological pathways activated by a variety of environmental conditions and at different developmental stages.     

Lead (Pb)-induced biochemical and ultrastructural changes in wheat (Triticum aestivum) roots

The focus of the present study was to explore lead (Pb)-induced metabolic alterations vis-à-vis ultrastructural changes in wheat roots to establish Pb toxicity syndrome at a structural level. Pb (50–500 μM) enhanced malondialdehyde (an indicator of lipid peroxidation) and hydrogen peroxide content, and electrolyte leakage, thereby suggesting reactive oxygen species-induced disruption of membrane integrity and oxidative stress in wheat roots. The activities of superoxide dismutases and catalases enhanced upon Pb exposure, whereas those of ascorbate and guaiacol peroxidases declined. Pb-induced metabolic disruption was manifested in significant alterations in wheat root ultrastructure as analyzed by transmission electron microscopy. Pb caused thinning of cell wall (at 50 μM), formation of amoeboid protrusions and folds and intercellular spaces, and appearance of lesions and nicks/breaks (at ≥250 μM Pb). Pb was deposited along the cell walls as dark precipitates. At ≤250 μM Pb, the number of mitochondria increased significantly, whereas structural damage in terms of change of shape and disintegration was observed at ≥ 250 μM Pb. Pb reduced the size of nucleoli and induced puff formation (at 250 μM), resulting in complete disintegration/disappearance of nucleolus at 500 μM. The study concludes that Pb inhibited wheat root growth involving an ROS-mediated oxidative damage vis-à-vis the ultrastructural alterations in cell membrane and disruption of mitochondrial and nuclear integrity.     

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.     

Developmental instability in Brassica campestris (Cruciferae): fluctuating asymmetry of foliar and floral traits

The degree of fluctuating asymmetry of bilateral traits provides a measure of developmental instability, which can be influenced by genetic as well as environmental stress. We studied genetic variation between and within two populations of the mustard Brassica campestris for asymmetry of foliar (cotyledon width) and floral (petal length and width) traits as well as for phenological (germination and flowering) and performance (biomass and flowering) traits. The two populations differed in mean expression of most traits, including asymmetry. However, within-population estimates of genetic variability tended to be lower for asymmetry than other traits. Asymmetry was greater in the population that had lower biomass accumulation and flower production, which supports the idea that population-level asymmetry may be indicative of population-level performance. However, within each population, evidence that performance was negatively correlated with asymmetry was equivocal. Within populations there was little or no concordance among estimates of asymmetry based on different structures, i.e., plants that had highly asymmetrical cotyledons did not tend to have highly asymmetrical petals. The lack of a general buffering capacity at the individual level may be explained by developmental processes (e.g., action of different genes or morphogens) as well as evolutionary processes (e.g., selection on asymmetry of different traits).     

铅胁迫对不同品种小白菜种子萌发的影响

采用4个有代表性的小白菜(Brassica campestris ssp.chinensis L.)品种(‘抗热605’,‘矮抗青’,‘绿优星’,‘七宝青)’为材料,用0.3mmol醋酸铅进行重金属胁迫处理,比较了这4个小白菜品种的抗重金属能力差异。结果表明,Pb胁迫下,‘抗热605’的发芽率、发芽指数、发芽势、胚长以及胚鲜重等均高于其他品种,且抑制指数低于其他品种。说明,‘抗热605’的耐Pb胁迫能力最强,其他3个品种依次为:‘绿优星’、‘矮抗青’、‘七宝青’。     

A protein kinase activated by darkness phosphorylates nitrate reductase in Komatsuna (Brassica campestris) leaves

Although it has been shown that leaf nitrate reductase (NR: EC 1.6.6.1) is phosphorylated by subjecting plants to darkness, there is no evidence for the existence of dark-activated or dark-induced NR kinase. This study was undertaken to investigate the occurrence of a protein kinase phosphorylating NR in response to dark treatments. Immediately after transferring Komatsuna (Brassica campestris L.) plants to darkness, we observed rapid increases in the phosphorylating activity of the synthetic peptide, which is designed for the amino acid sequence surrounding the regulatory serine residue of the hinge 1 region of Komatsuna NR, in crude extracts from leaves. The activity reached a maximum after 10 min of darkness. Inactivation states of NR estimated from relative activities with or without Mg2+ were correlated to activities of the putative dark-activated protein kinase. Using the synthetic peptide as a substrate, we purified a protein kinase from dark-treated leaves by means of successive chromatographies on Q-Sepharose, Blue Sepharose, FPLC Q-Sepharose, and ATP-gamma-Sepharose columns. The purified kinase had an apparent molecular mass of 150 kDa with a catalytic subunit of 55 kDa, and it was Ca2+-independent. The purified kinase phosphorylated a recombinant cytochrome c reductase protein, a partial protein of NR, and holo NR, and inactivated NR in the presence of both 14-3-3 protein and Mg2+. The kinase also phosphorylated synthetic peptide substrates designed for sucrose phosphate synthase and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase. Among inhibitors tested, only K252a, a potent and specific serine/threonine kinase inhibitor, completely inhibited the activity of the dark-activated kinase. The activity of the purified kinase was also specifically inhibited by K252a. Taken together with these findings, results obtained suggest that the putative dark-activated protein kinase may be the purified kinase itself, and may be responsible for in vivo phosphorylation of NR and its inactivation during darkness.     

Inheritance of seed colour in turnip rape (Brassica campestris L.)

Summary A high gene frequency for ps (parallel spindles) is expected in cultivated tetraploid potatoes, S. tuberosum Group Tuberosum, if 2n pollen produced by ancestral diploid plants which were psps was involved in the origin and evolution of the potato. Fifty-six North American cultivars (varieties and advanced selections) were pollinated by diploid clones, either W 5295.7 or W 5337.3 which are homozygous recessive for ps. The segregation ratios in regard to 2n pollen production in derived tetraploid progenies, from 4x×2x crosses, reveal the genotype of ps in the cultivars. Microsporogenesis of 2n pollen producing 4x progeny was observed to avoid an overestimation of the frequency of 2n pollen producing plants due to mechanisms other than parallel spindles. More than 50% of the 56 cultivars are simplex (Pspspsps), since in each of these cultivars about 50% of their progeny produced 2n pollen. The ps gene frequency in the 56 cultivars was estimated as high as 0.69. The high frequency of ps in the tetraploid cultivars clearly supports the hypothesis that 2n pollen produced by plants homozygous recessive for ps have been involved in the origin of cultivated tetraploid potatoes, since a higher frequency of ps in the tetraploid than in the ancestral diploid population can be expected from sexual polyploidization but not from somatic doubling. The importance of meiotic mutants such as ps for the successful evolution of polysomic polyploids is emphasized.     

S-Locus-Specific Glycoproteins Associated with Self-Incompatibility in Brassica campestris

Specific S-glycoproteins were isolated from three Brassica campestriscultivars homozygous with respect to the S-alleles S₈, S₉ andS₁₂. Amino acid sequences of various peptide fragments of theS-proteins were determined using a gas-phase protein sequencer,and a nearly complete amino acid sequence of the S₈-glycoproteinwas determined on the basis of the revised cDNA sequence ofthe B. oleracea S-specific glycoprotein. The lysyl endopeptidasefragments of S₉ and S₁₂-glycoproteins were aligned in comparisonwith the sequence of the S₈-glycoprotein. Although extensivesequence homology was evident among the three S-glycoproteins,the sequences of the middle part were relatively different fromeach other. The numbers and positions of N-glycosylation alsodiffered among the S-glycoproteins of Brassica species. (Received April 20, 1987; Accepted July 29, 1987)     

Presevvation of Xanthomonas campestris in Brassica oleracea seeds

Summary Brassica oleracea seeds were sterilized by gamma radiation and sodium hypochlorite washing. Xanthomonas campestris was inoculated into the seeds by incubating, under vacuum, a suspension of the bacteria with the seeds. After thorough washings with sterile distilled water, the seeds retained about 13 000 cells per seed. The seeds were maintained at 4°C during 21 months, during which the viability of the bacteria and their capacity to produce xantham gum in shake flasks, were evaluated. Bacterial viability showed oscillations but after 20 months it was 10% of the initial. When these seeds were used as a pre-inoculum for a culture to produce xanthan, the final polymer concentration increased slightly with time of seed storage and the final broth viscosity was fairly constant. The specific polymer production (per weight of final bacterial cells) increased about three-fold after 21 months of experimentation. The method, besides being able to produce xantham in quantity and quality, has the advantages of an easy inoculation procedure, no need for transfers, less contamination risk and improved growth rate of the bacteria in the inoculation medium. Correspondence to: E. Galindo     

Structural investigation of oxalate-soluble rapeseed (Brassica campestris) polysaccharides. 3. An arabinan

An arabinan isolated from rapeseed was shown by sedimentation studies to be essentially homogeneous, and methylation analysis revealed a highly branched structure. Hydrolysis of the methylated polysaccharide yielded 2,3,5-tri-O-methyl-L-arabinose (11 mol.), 2,3-di-O-methyl-L-arabinose (7 mol.), 3-O-methyl-L-arabinose (trace), 2-O-methyl-L-arabinose (7 mol.), and L-arabinose (2 mol.). Periodate-oxidation data substantiate the methylation results. The general, structural features of the arabinan are discussed.     

不结球白菜种质资源形态性状多样性分析

分析了125份不结球白菜的形态多样性.结果表明,我国不结球白菜具有丰富的形态多样性,以江苏的平均多样指数最高,达0.972,性状中以叶柄长的变异系数最大,达59.77%.通过多变量的主成分分析,第一主成分和第二主成分代表了不结球白菜形态多样性的48.4%.通过系统聚类,把125份不结球白菜种质资源聚成6类,普通白菜不同程度地分别与塌菜、菜心、分蘖菜和薹菜聚在一起,说明普通白菜与其它种类间存在一定的亲缘关系.