Characterization of different cytoplasmic male sterility systems in Indian mustard (Brassica juncea L. Czern & Coss)

Six cytoplasmic male sterility (CMS) systems, viz. moricandia, ogura, oxyrrhina, siifolia, tournefortii and trachystoma of Indian mustard (Brassica juncea L.) were characterized for agronomic and floral characteristics. Introgression of alien cytoplasm caused alterations in different floral traits in ogura, siifolia, tournefortii and trachystoma CMS systems. Varied response to different genetic backgrounds of CMS lines indicated the presence of cytoplasmic–nuclear interaction in alteration of floral traits. On the basis of floral characteristics, CMS systems could be grouped into distinct classes. Siifolia, tournefortii and trachystoma CMS lines had narrow petals, while moricandia, ogura and oxyrrhina had wider petals, which were distinguishable on the basis of visual observations. The ratio between length and width of petals were >2.0 in wide petal group but <2.0 in narrow petal group. Further, the relative position of anther and stigma, which was estimated as the ratio between stamen and style length could differentiate the CMS systems. Stamens were longer than styles in oxyrrhina, equal in moricandia and shorter in ogura, siifolia, tournefortii and trachystoma male sterile lines. Non‐viable pollen grains were present in moricandia and oxyrrhina systems, but absent in other systems. In tournefortii and trachystoma, few flowers showed petaloid corolla. All male sterile lines, except trachystoma, which showed crooked siliqua formation were at par with their respective maintainers for flower initiation, plant height, primary branches, seeds per siliqua, seed yield, harvest index, oil and protein content. In general, flower senescence and maturity occurred earlier in male sterile lines than in their respective maintainer lines.     

Heat stress during seed development affects forage brassica (Brassica napus L.) seed quality

In two consecutive seasons, forage rape (Brassica napus L.) plants were exposed to short periods (240°C hr) of heat stress (30°C day/25°C night) during seed filling (80% seed moisture content = S1), at physiological maturity (50% seed moisture content = S2) and at both S1 plus S2 (=S3) in a Biotron before being returned to the field until seed harvest. Seeds were hand harvested at 14% seed moisture content and their quality assessed by measuring germination, seed vigour (using the accelerated ageing and conductivity tests) and seed mass (as determined by thousand seed weight). Heat stress at both S1 and S2 caused a small (<10%) but significant reduction in germination in both seasons. There was a significant heat stress timing interaction in the first but not the second season. Reductions in germination were a result of increased abnormal seedling production not seed death. All three heat stress treatments significantly reduced seed vigour, with S3>S2>S1. Seed mass was reduced by heat stress at S1 but not at S2. Variable seed vigour in high‐germinating New Zealand‐produced forage rapeseed lots is most likely explained by short periods of heat stress during seed development.     

Testing for Tolerance to Aphids in Indian Mustard, Brassica juncea (L.) Czern and Coss.

Brassica juncea plants were screened for aphid tolerance using three methods. Seven and 14 days old seedlings grown in wooden trays in a glasshouse were kept between infector trays with heavy aphid infestation. There were no surviving plants in the susceptible genotypes 50 days after sowing but survival rates ranging between 80 and 90 % were observed for selection 44. The progenies of the surviving plants were tested under natural infestation in the field. Plants were scored for leaves without aphids 40 days after sowing, healthy plants at harvest and seed yield in g/m² For most of these lines, the aphids’ survival, life span and fecundity (nymphs produced per adult) were recorded after introducing five newly-born nymphs on excised fifth leaves cultured in petri dishes. Selections showing greater tolerance to aphids than the check cultivars were identified in the progenies of a cross RLM-514 × T-6342. It was concluded that the excised leaf method can provide a reliable measure of the lines’ reaction to aphids.     

Heterosis breeding in Indian mustard (Brassica juncea L. Czern & Coss): Analysis of component characters contributing to heterosis for yield

Summary Divergence of 25 accessions of Brassica juncea of Indian, CIS (Commonwealth of Independent States, former USSR) and synthetic origin was studied by D² analysis. On the basis of divergence, ten accessions were selected and crossed in a diallel fashion without reciprocals to study the combining ability and heterosis. None of the accessions was found to be a good general combiner for all the nine quantitative characters that were studied. Significant heterosis over better parent for single plant yield was recorded in CIS x Indian and synthetic x CIS crosses (5 each) followed by Indian x synthetic types (3). The analysis of component characters showed that the mean performance of the majority of hybrids was intermediate for five out of six yield attributing traits, thus exhibiting dominance or partial dominance effect. To estimate the contribution of such yield attributing traits towards heterosis for yield, a comparison was made among three parameters viz. heterosis over mid parent (MP), better parent (BP) and better yielding parent (BYP) of the concerned hybrid. It was observed that estimation of heterosis from BYP was a more accurate method to determine the contribution of component characters towards yield heterosis than the analysis based on MP and BP. From the component character analysis, it was concluded that characters like number of primary and secondary branches, number of siliqua per plant and siliqua density contributed significantly towards heterosis in yield. Plot level yield trials of two selected hybrids (Skorospieka II x RH30 and Donskaja IV x Varuna) over two growing seasons revealed 29.4 to 91.8% heterosis over BYP.     

Development of Strains with Yellow Seedcoat in Indian Mustard (Brassies juncea Czern. & Coss.)

A mutant with yellow seedcoat colour was isolated by Nayar (1968) in the mustard variety‘Rai-5′. This mutant was crossed to the national check cultivar ‘Varuna’ in order to develop improved strains with yellow seedcoat. Four such strains with yellow seeds were evaluated for their seed yield, yield components and percent oil. Two strains TM-9 and TM-17 were more productive than ‘Varuna’ in seed yield. All the yellow seeded strains showed higher oil percentage as compared to ‘Varuna’. The seedcoat in the yellow seeded strains accounts for 14-15% of the seed weight as compared to 18% in the black seeded ‘Varuna’. The higher proportion of the cotyledons and embryo accounts for the increased oil percentage in the yellow seeded types.     

Effect of alien cytoplasm and fertility restorer genes on agronomic and physiological traits of Brassica juncea (L.) Czern

To assess the effect of alien cytoplasm and fertility restorer genes on agronomic and physiological traits in Brassica juncea, cytoplasmic male sterile (CMS) and fertility restorer lines involving five alloplasms in three nuclear backgrounds were constituted through repeated backcrossing. These lines were evaluated along with euplasmic lines for agronomic traits in field experiments. Respiration efficiency of in vitro cultured hypocotyls, and chlorophyll content in leaves were also estimated. Significant adverse effects of trachystoma and catholica cytoplasms on yield‐contributing traits and yield were noticed. The restorer gene corrected the floral defects in CMS trachystoma and restored yield to the level of euplasmic line, while it did so only partly in CMS catholica. Restorer genes had no adverse effect on traits in any of the systems. On the contrary, the Rf gene of moricandia CMS system exerted a positive effect on leaf chlorophyll content. Likewise, in vitro assay indicated the moricandia CMS system as vigorous. The moricandia CMS plants gave higher seed yield perhaps due to overall better vigour and higher leaf chlorophyll content.     

A Simple Method of Inducing Somatic Embryogenesis in Brassica juncea (L.) Czern & Coss.

A single-step method for the induction and development of somatic embryoids from hypocotyls explains of Brassica juncea is reported. On modified MS medium containing 2 % sucrose, 0.25 mgl ¹ 2,4-D, 0.5 mgl ¹ each of NAA and BaP-R, each explant calluses at both of and at its best, 31% of explants produce embryoids. In the variety RLM-198, the number of embryoids ranges from 8–21 per culture. Each embryoid, upon proliferation, developed up to the 25 shoots. The method is rapid; the time La ken from inoculation to the development of intact plantlets is 8–10 weeks. Regenerated plants have flowered normally and have set seed. The system can profitably be used for in vitro mutant selection and early bulking in mustard.     

Path Analysis of Important Growth Functions of Indian Mustard (Brassica juncea L. Czern and Coss)

Field experiment conducted at the Viswavidyalaya Farm during the winter seasons of 1980—81 to 1983—84 to study the effects of major six productive functions viz. number of inflorescence bearing primary branches, number of inflorescence bearing secondary branches, leaf area indices at flowering, number of seeds/siliqua, number of siliqua/m² and test weight of grains on the grain yields of eight elite varieties of Indian mustard ( Brassica juncea L. Czern and Coss) when sown on three different times revealed that correlations between them were close and positive in all the varieties. Path analysis of the data, further, showed that except the inflorescence bearing primary branches and the seeds/siliqua all other attributes showed high direct influences on grain yield; the maximum direct influence was obtained from number of siliqua/m².     

Interactive Effect of Nitrogen and Sulphur on Growth and Yield of Rape-seed-Mustard (Brassica juncea L. Czern. and Coss. and Brassica campestris L.) Genotypes

A simple randomized field experiment was conducted to assess the growth and yield of rape-seed-mustard in relation to sulphur and nitrogen interaction. Three levels of sulphur (0, 40 and 60 kg ha⁻¹) in combination with three levels of nitrogen (60, 100 and 150 kg ha⁻¹) were tested as treatments, T₁, T₂, T₃, T₄, and T₅. Results indicated significant favourable effects of sulphur and nitrogen, when applied together, on yield components, seed and oil yield. Maximum response was observed with treatment T₃ (having S and N of 40 and 100 kg ha¹, respectively). Percentage oil content of seed was maximal at T₄ (having S and N of 60 and 100 kg ha¹) in both cultivars. The increase in N dose from 100 to 150 kg ha⁻¹ without any change in applied S, i.e. 60 kg ha¹ (T₅), decreased the percentage oil content. The seed and oil yield, however, were similar to T₃. Favourable responses of S and N interaction on leaf area index, rate of photosynthesis and biomass production were also observed.     

Physiological Investigation of the Impact of Nitrogen and Sulphur Application on Seed and Oil Yield of Rapeseed (Brassica campestris L.) and Mustard (Brassica juncea L. Czern. and Coss.) Genotypes

Field experiments were conducted to determine the physiological basis of the effects of N and S interactions on seed and oil yield of Brassica species. Five combinations of N and S (in kg ha⁻¹) 0S+100N (T₁), 40S+60N (T₂), 40S+100N (T₃), 60S+100N (T₄) and 60S+150N (T₅), were used for this purpose. Nitrate reductase (NR) activity and ATP-sulphurylase activity in the leaves were measured at various growth stages, as the two enzymes catalyse rate-limiting steps of the assimilatory pathways of nitrate and sulphate, respectively. The activities of these enzymes were strongly correlated with seed and oil yield. The highest nitrate reductase activity, ATP-sulphurylase activity and yield were achieved with the combination T₃ in both species. Any variation from this combination decreased the activity of these enzymes, resulting in a reduction of the seed and oil yield of Brassica species. The higher seed and oil yield achieved in these species at T₃ could be due to optimization of leaf soluble protein and photosynthetic rate, as these parameters are influenced by N and S assimilation.     

Potential involvement of rapeseed (Brassica napus L.) metallothioneins in the hydrogen peroxide‐induced regulation of seed vigour

Germination is a crucial process in crop plant life cycle that determines future yield. Hydrogen peroxide plays a significant role in both dormancy release and seed ageing. The potential of the rapeseed metallothioneins (BnMT1‐4) as reactive oxygen species scavengers was investigated. In the presence of H₂O₂, bacterial cells expressing BnMT1‐4 had higher growth rate than control cells. However, such effect was not observed in an oxidative stress oversensitive yap‐1Δ yeast mutant strain expressing BnMT1‐4. Next, it was shown that H₂O₂, up to 100 and 10 mM, promotes rapeseed germination rate and seedling growth, respectively. Moreover, it was demonstrated that in seeds germinating in the presence of H₂O_2, the expression of seed‐specific BnMT4 decreases to a high extent in a dose‐ and time‐dependent manner. It was shown that the decline in BnMT4 mRNA level, accelerated by up to 10 mM H₂O₂ treatment, correlates positively with increased rapeseed germination and early growth, underscoring the plausible role of BnMT4 as a H₂O₂ scavenging protein that level has to be downregulated in conditions where the compound is needed. Finally, it was demonstrated that H₂O₂ could be used as a priming agent of rapeseeds.     

Fine mapping of qSS‐9, a major and stable quantitative trait locus,for seed storability in rice (Oryza sativa L.)

Seed storability in rice (Oryza sativa L.) is an important agronomic trait. We previously showed a quantitative trait locus of seed storability, qSS‐9, on chromosome 9 in a backcross population of ‘Koshihikari’ (japonica) / ‘Kasalath’ (indica) // ‘Koshihikari’. In this study, fine mapping of the chromosomal location of qSS‐9 was performed. Effect of ‘Kasalath’ allele of qSS‐9 was validated using a chromosome segment substitution line, SL36, which harboured the target quantitative trait loci (QTL) from ‘Kasalath’ in the genetic background of ‘Nipponbare’ under different ageing treatments in different environments. Subsequently, an F₂ population from a cross between ‘Nipponbare’ and SL36 was used for fine mapping of qSS‐9. Simultaneously, four subnear isogenic lines (sub‐NILs) that represented different recombination breakpoints across the qSS‐9 region were developed from F₃ progeny. Finally, the qSS‐9 locus was located between the Indel markers Y10 and Y13, which delimit a region of 147 kb in the ‘Nipponbare’ genome. These results provide a springboard for map‐based cloning of qSS‐9 and possibilities for breeding rice varieties with strong seed storability.     

Interactive Effect of Sulphur and Nitrogen on the Oil and Protein Contents and on the Fatty Acid Profiles of Oil in the Seeds of Rapeseed (Brassica campestris L.) and Mustard (Brassica juncea L. Czern. and Coss.)

In long-term field experiments on loamy sand and sandy loam, legumes (pea and lupine) stimulated microbial activity in the rhizosphere more than cereals (winter rye, winter wheat and spring barley), maize and oil flax. In the rhizosphere of winter wheat and maize, microbial activity and the bacteria species Pseudomonas , Agrobacterium and Xanthomonas were more stimulated by organic manuring than by mineral fertilization. A positive correlation between the stimulated bacteria species and the growth of young plants was found. Various mineral nitrogen applications had no influence on the rhizosphere microflora. The leghemoglobin content of pea nodules – an indicator of nitrogen fixation activity – was reduced by high nitrogen application in crop rotation.     

Generation of tribenuron‐methyl herbicide‐resistant OsCYP81A6‐expressing rapeseed (Brassica napus L.) plants for hybrid seed production using chemical‐induced male sterility

Chemical‐induced male sterility (CIMS) is a method for hybrid rapeseed (Brassica napus L.) production. Some sulphonylurea herbicides such as tribenuron‐methyl (TBM) are used as chemical hybridization agents (CHAs) in CIMS systems. However, the male parents must be protected from herbicide injury with a shield during spraying of the female parents with CHAs to induce male sterility. Thus, using herbicide‐resistant rapeseed lines as the male parents can significantly simplify the seed production procedure and reduce the cost in hybrid seed production. A rice cytochrome P450 hydroxylase, OsCYP81A6, has been previously characterized to confer resistance to bentazon and sulphonylurea herbicides. We demonstrate here that the introduction of OsCYP81A6 renders rapeseed plants resistant to TBM. Compared with wild‐type plants, the transgenic plants displayed normal stamen development and male fertility when treated with 0.05 mg/l of TBM, the dose used for inducing male sterility in hybrid seed production. These results indicate that the OsCYP81A6‐expressing rapeseed plants can be used as the male parents for hybrid rapeseed production using CIMS.