A unique introgression from Moricandia arvensis confers male fertility upon two different cytoplasmic male-sterile lines of Brassica juncea

A Brassica juncea line carrying an introgression from Moricandia arvensis restored male fertility to two cytoplasmic male‐sterile (CMS) B. juncea lines carrying either M. arvensis or Diplotaxis catholica cytoplasm. Genetics of fertility restoration was studied in the F₁, F₂, F₃ and backcross generations of the cross between CMS and fertility‐restorer lines. No male‐sterile plants were found in F1‐F₃ generations of the cross between CMS [M. arvensis] B. juncea and the restorer. However, a 1: 1 segregation for male sterility and fertility was observed when the F1 was pollinated with non‐restorer pollen from a euplasmic line. These results clearly show that restoration is mono‐genic and gametophytic. In CMS lines carrying D. catholica cytoplasm, the restorer conferred male fertility to the F1 and showed 3: 1 and 1: 1 segregations for male fertility and sterility in F₂ and BC1 generations, respectively, indicating a monogenic, sporophytic mode of fertility restoration. The results were also supported by pollen stainability in the F1 which was about 65% in M. arvensis‐based CMS and >90% in D. catholica‐based CMS. The above results are discussed in the light of previous molecular studies which showed association between CMS and atpA in both systems.     

An improved cytoplasmic male sterile (Diplotaxis berthautii) Brassica juncea: identification of restorer and molecular characterization

We report the development of an improved cytoplasmic male sterile (CMS) system of Brassica juncea carrying cytoplasm of the wild species Diplotaxis berthautii. Flowers of the CMS line are smaller than the euplasmic line but have improved nectaries. Anthers are slender and fail to extend to the level of stigma. Female fertility of the CMS line is comparable to the euplasmic line. Fertility restorers of Moricandia arvensis and D. catholica-based alloplasmic CMS systems of B. juncea were found capable of restoring male fertility to this new CMS line. The fertility restoration is monogenic and gametophytic. Southern analysis showed that the cytoplasm of the CMS line is different from euplasmic B. juncea and other CMS systems restored by the same restorer lines. Northern analysis of the CMS, fertility restored and euplasmic lines using eight mitochondrial gene probes revealed altered atpA expression associated with male sterility. Cleaved amplified polymorphic sequence (CAPS) markers were identified for the plastid gene psbB, which could be useful for a quick identification of this CMS line. S.R. Bhat and P. Kumar contributed equally to this work.     

Moricandia arvensis cytoplasm based system of cytoplasmic male sterility in Brassica juncea: Reappraisal of fertility restoration and agronomic potential

Cytoplasmic male sterility (CMS) system based on the cytoplasm from Moricandia arvensis (mori) was investigated for fertility restoration and agronomic potential. Fertility restorer gene for mori CMS was introgressed from cytoplasm donor species as all the evaluated Brassica juncea genotypes (155) acted as sterility maintainers. The allosyndetic pairing between M^a and the A/B genome chromosomes in the monosomic addition plants (2n= 18II + 1M^a) facilitated the gene introgression. Partial fertility restoration (43–52% pollen grain stainability) in F₁ hybrids and absence of segregation for male sterility in F₂ progenies suggested gametophytic control of fertility restoration. The pollen fertility in the F₁ hybrids was, however, sufficient to ensure complete seed set upon bag selfing. Introgression from M. arvensis also helped in correction of chlorosis associated with mori cytoplasm in CMS and fertile alloplasmic B. juncea plants. Yield evaluation of thirty F₁ hybrids having the same nuclear genotype but varied male sterilizing cytoplasms (mori, oxy, lyr, refined ogu), in comparison to respective euplasmic hand bred control hybrids, allowed an estimate of yield penalty associated with different CMS systems. It ranged from 1.8% to 61.6%. Hybrids based on cytoplasmically refined ogu were most productive followed by those based on cytoplasmically refined mori CMS. The male sterility systems emanating from somatic hybridization were found superior than those developed from sexual hybridization.     

Induction of microspore embryos in a CMS line of Brassica juncea and formation of the androgenic plantlets

Anther culture of two wide hybrids (Diplotaxis erucoides × Brassica campestris) × B. juncea and (D. berthautii × B. campestris) × B. juncea, their CMS lines and the parent species elicited a range of responses highlighting the importance of the genotype. Androgenesis was expressed in cultured anthers of CMS (D. erucoides) B. juncea (22.8%), in restored pollen fertile plants of this CMS line (1.66%), and in the parent, B. juncea cv Pusa Bold (13.02%). AgNO₃ was essential for androgenic response in the CMS lines, and it markedly increased the frequency of androgenesis in the cultivated species. Multiple crops of microspore embryos were obtained from responsive anthers of CMS plants in anther recultures. As high as92% microspore embryos of the CMS line germinated on basal B₅medium and formed normal plantlets. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of two new cytoplasmic male-sterile lines in Brassica juncea through wide hybridization

Two new cytoplasmic male-sterile (CMS) lines have been developed in Brassica juncea using the bridgecross hybrids (Diplotaxis erucoides×Brassica campestris)×B. juncea and (Diplotaxis berthautii×B. campestris)×B. juncea. These were backcrossed ×ve times with pollen of B. juncea. The CMS line (D. erucoides) B. juncea segregated into tall and short true breeding types; both resembled the cultivar B. juncea in vegetative and floral morphology and in cytology, except for a greater number of secondary branches and smaller anthers with empty sterile pollen in the CMS line. Female fertility was as good in the CMS line as in the cultivar. The other CMS line (D. berthautii) B. juncea resembled the cultivar in vegetative morphology and cytology. Four true breeding floral types were isolated as follows: (1) smaller and indehiscent anthers containing empty, sterile pollen, (2) all six stamens petaloid, (3) one petaloid stamen and five stamens antherless, and (4) apetalous flowers with all six stamens antherless.     

Identification of the Putative Cytoplasmic Donor of a CMS System in Brassica juncea

Chloroplast (cp) and mitochondrial (mt) DNA restriction profiles of a cytoplasmic male sterile (CMS) line of Brassica juncea and its maintainer line were compared and found to be markedly different. Comparison of cpDNA restriction profiles of fifty different species of genus Brassica and some allied genera showed that the cpDNA profiles of CMS lines were similar to that of B. tournefortii for twenty different restriction endonucleases. This CMS system is thus not of spontaneous origin as reported earlier, but is alloplasmic in nature. Comparison of restriction profiles of mtDNA of B. tournefortii and CMS lines revealed some differences which might either be due to changes in DNA pattern during the transfer, or, due to the cytoplasm coming from a B. tournefortii line different from the one used in this study.     

Chromosomal structural changes in Capsicum annuum L. and C. chinense Jacq.

Summary Wide hybridizations between M. arvensis and Brassica amphidiploid species (B. napus and B. juncea) were carried out in order to incorporate desirable traits of M. arvensis into Brassica crops. Crossing barriers between them were present without the use of in vitro techniques. F₁ hybrids have been produced through ovary culture, when M. arvensis were used as a female parent. Higher hybrid embryo productivity (3.07 embryos per pollination) was obtained in the cross of M. arvensis x B. napus than in that of M. arvensis x B. juncea (0.79 embryos). The hybridity was confirmed by morphology, cytology, isozyme and Southern analyses. The first backcrossing progenies and open pollinated ones were produced.     

Expression of male sterility in alloplasmic Brassica juncea with Erucastrum canariense cytoplasm and the development of a fertility restoration system

An alloplasmic mustard, Brassica juncea, has been synthesized by placing its nucleus into the cytoplasm of the related wild species Erucastrum canariense to express cytoplasmic male sterility. To achieve this, the sexual hybrid E. canariense (2n=18, E^cE^c) ×Brassica campestris (2n= 20, AA) was repeatedly backcrossed to B. juncea (2n= 36, AABB). Cytoplasmic male‐sterile (CMS) plants were recovered in the BC₄ generation. These plants are a normal green and the flowers have slender, non‐dehiscing anthers that contain sterile pollen. Nectaries are well developed and female fertility is > 90%. The fertility restoration gene was introgressed to CMS B. juncea from the cytoplasmic donor E. canariense through pairing between chromosomes belonging to B. juncea with those of the E. canariense genome. The restorer plants have normal flowers, with well‐developed anthers containing fertile pollen. Meiosis proceeds normally. Pollen and seed fertility averaged 90% and 82%, respectively. F₁ hybrids between CMS and the restorer are fully pollen fertile and show normal seed set. Preliminary results indicate that restoration is achieved by a single dominant gene. The constitution of the organelle genomes of the CMS, restorer and fertility restored plants is identical, as revealed by Southern analysis using mitochondrial and chloroplast probes atp A and psb D, respectively.     

Gene flow from transgenic oilseed Brassica juncea (L.) Czern. into weedy Sinapis arvensis L. (wild mustard)

Gene flow from acetolactate synthase‐resistant (HR) Brassica juncea oilseed canola to related weed, Sinapis arvensis (density 1 plant/m²) was assessed in a 100 m² field plot of HR B. juncea. Two HR F₁ hybrids (H1 and H2) were detected among 109 951 seedlings screened with imazethapyr (hybridization frequency – 1.8 × 10^?5). Hybridity was confirmed using flow cytometry, B. juncea‐specific amplified fragment length polymorphisms (AFLPs) markers, genomic in situ hybridization (GISH) and PCR‐based detection of B. juncea's HR gene. H1 and H2 had 2n = 27 and 2n = 45 chromosomes, corresponding 3x (SrAB) and 5x (SrAABB) genomic structures and reduced male fertility, 3.2 and 16.6% pollen viability, respectively. H1 was self‐incompatible, whereas H2 set seed when selfed (B. juncea trait). Selfed F₂, F₃ and F₄ plants showed HR trait persistence and vigorous growth and high (80–100%) pollen fertility in 22% and 39% of the F₂ and F₃ plants, respectively. No progeny were obtained from F_1, F₂ or F₃ hybrids × S. arvensis backcrosses, suggesting the likelihood of introgression of traits is low to negligible.     

A stable cytoplasmic male-sterile line of Brassica juncea carrying restructured organelle genomes from the somatic hybrid Trachystoma ballii+B. juncea

A cytoplasmic male-sterile (CMS) line of Brassica juncea has been developed by combining the cytoplasm originating from the somatic hybrid Trachystoma ballii+B. juncea, and the nucleus of B. juncea cv. Pusa Bold by repeated backcrossing. Male-sterile plants closely resembled the normal fertile B. juncea in general morphology, but had delayed flowering (5–7 days) when compared with fertile ‘Pusa Bold’ which flowered in 45 days. Stamens of the male-sterile line were transformed into petaloid structures. Pollen abortion occurred after tetrad formation. Female fertility of the male-sterile line was normal. Molecular analysis of organelle genomes indicated extensive mitochondrial DNA recombinations in the CMS line. Preliminary analysis of the chloroplast genome of the CMS line also indicated chloroplast DNA recombination.     

Introgression of a gene restoring fertility to CMS (Trachystoma) Brassica juncea and the genetics of restoration

A dominant gene restoring fertility to a cytoplasmic male sterile (CMS) line of Brassica juncea was derived from the somatic hybrid Trachystoma ballii+B. juncea. Its introgression resulted from forced pairing between chromosomes of the cultivar ‘Pusa Bold’ and chromosomes of the fusion hybrid. Segregation ratios of this fertility restorer gene followed a monogenic pattern. The introgression of the fertility restorer gene did not cause any abnormalities, such as reduced fertility; pollen and seed fertilities of the restored plants were over 90%. Restored fertile and CMS plants exhibited similar Southern hybridization patterns when probed with the mitochondrial probe atp6.     

Complete mitochondrial genome sequence of Brassica oxyrrhina and comparative analysis of the region containing orf108, a male sterility gene for mustard (Brassica juncea), among B. oxyrrhina,Diplotaxis erucoides and Sinapis species

To date, several cytoplasms of wild species have been introduced to Brassica juncea, for inducing cytoplasmic male sterility (CMS). One of the causal genes of CMS is orf108, which is widely distributed in Brassicaceae including Brassica oxyrrhina. To further understand the origin of orf108, we assembled the complete mitochondrial genome sequence of B. oxyrrhina. We also determined the DNA sequences upstream of atp1 including orf108 for D. erucoides and five Sinapis species. The orf108 was definitively placed in the mitochondrial genome of B. oxyrrhina consisting of 247,936 bp. In S. alba, previously reported to possess orf108, the gene was not present, whereas Sinapis arvensis and Sinapis turgida contained orf108. The orf108 sequence in the two Sinapis species showed a novel nucleotide substitution compared to D. erucoides. Northern hybridization suggested, furthermore, that orf108 mRNA was processed in the two species similarly to that in B. oxyrrhina. The results clarified the interspecific differentiation of orf108‐apt1 region in Sinapis.     

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.     

Hybridizations among Brassica napus, B. rapa and B. juncea and their two weedy relatives B. nigra and Sinapis arvensis under open pollination conditions in the field

The rate of natural crosses occurring among the cultivated Brassica species B. napus, B. rapa, and B. juncea and their two weedy relatives B. nigra and Sinapis arvensis was studied in co-cultivation experiments under field conditions in Saskatchewan, Canada, with special reference to evaluation of the possibility of transgene escape from the cultivated to the weedy species. Natural crosses occurred among B. napus, B. rapa, and B. juncea, indicating that hybridizations among these three species do occur under field conditions. On the other hand, no natural crosses occurred between the cultivated species and B. nigra or S. arvensis. It is concluded that the crosses between the cultivated and weedy species are practically impossible under field conditions in Saskatchewan, and that the escape of transgenes from transgenic cultivars of B. napus, B. rapa and B. juncea into B. nigra and S. arvensis is basically zero in this region.     

Establishing Cytoplasmic Male Sterility in Brassica napus by Mitochondrial Recombination with B. tournefortii

Fusion experiments between B. napus and X-ray-treated B. tournefortii protoplasts were carried out to develop cytoplasmic male sterility (ems) in B. napus. From the regenerants, six lines containing male sterile plants were selected; five lines segregated for male sterility, but one line (25–143) was completely male-sterile from the beginning. Molecular analyses of mitochondrial (mt) and chloroplast (cp) DNA of B. napus, B. tournefortii, B. juncea and cms juncea indicated that the original cytoplasmic donor of the cms juncea-system in B. napus was a B. tournefortii form, while the B. napus genotype used for the fusion experiments had a B. campestris cytoplasm. By analysis (it regenerated plants, line 25–143 was identified as possessing mt-DNA recombined between B. campestris and B. tournefortii. with the major part derived from B. campestris. No differences were detected between epDNAs from H. campestris and from line 25—143. The other five lines were similar to B. campestris with all the probes used. The low frequency of sterile lines from the fusion experiments and the inheritance of the cms in segregating progenies are both discussed.     

Development of a New Cytoplasmic Male-Sterility System in Brassica juncea through Wide Hybridization

A new cytoplasmic male-sterility system was developed in an oilseed Brassica, viz. B. juncea var. ‘Pusa Bold’ with the cytoplasmic background of a wild species, Diplotaxis siifolia, obtained through wide hybridization. The synthetic alloploid (D. siifolia×B. juncea: 2n = 56, D³D³AABB) was repeatedly backcrossed to B. juncea to achieve cytoplasmic substitution. The CMS plants resembled the cultivar in growth and morphology. The flowers had narrow sepals and petals and short, shrivelled anthers which failed to dehisce. The meiotic process appeared to be normal. The microspores degenerated at an early stage after tetrad formation. Female fertility in the CMS plants was as good as in the cultivar. Female transmission of sterility confirmed it to be cytoplasmically encoded.     

Pod shatter resistance evaluation in cultivars and breeding lines of Brassica napus, B. juncea and Sinapis alba

Pod shatter susceptibility was investigated in Brassica napus germplasm and shatter resistant species of B. juncea and Sinapis alba. The comparisons were made by measuring seed yield in field plots, detached pod rupture energy (RE) and the half‐life of pod‐opening. Pod shatter resistance was significantly greater in B. napus lines derived from interspecific hybridizations of B. napus with B. rapa, B. carinata and B. juncea, than common B. napus cultivars. While these lines exhibited no significant difference in resistance to pod shatter than B. juncea, an entry of S. alba had no yield loss caused by pod shatter. Resistance to pod shatter was characterized in the field as little or no yield loss after full maturity, delayed shattering in time, and stable yield performance under variable climatic conditions during pod maturity. Yield loss caused by pod shatter ranged from a low of 4% for the B. juncea cv. ‘AC Vulcan’ to a high of 61% for the black seeded B. napus line DH12075 in 2‐year field trials after 1 month maturity. Pod shatter resistance was not significantly associated with specific plant and pod morphological traits, except pod length (P = 0.005) in tested materials. Field visual scores of pod shatter through inspections of average pod shatter per plant within plots were highly correlated with plot yield loss. Indoor quantitative evaluations of pod strength using a pendulum machine to measure pod RE and random impact test to measure half‐life of pod‐opening resistance were highly correlated with field yield loss. Multiple evaluations of pod shatter in method and in time after pod maturity are recommended for reliable evaluation of pod shatter resistance.     

Transgene introgression from Brassica napus to different varieties of Brassica juncea

Transgene introgression from transgenic rapeseed (Brassica napus) to different varieties of B. juncea was assessed in this study. Crossability between a transgenic rapeseed line Z7B10 (pollen donor) and 80 cultivars of 16 B. juncea varieties (including two wild accessions) was estimated by artificial pollination in a greenhouse. As a result, interspecific crossability between the transgenic Z7B10 line and the 80 B. juncea cultivars varied considerably, with seeds per flower from 0.00–10.67. Seed germination rates of the interspecific F₁ hybrids ranged from 49.0%–89.3%. The estimated frequencies of natural gene flow from the transgenic Z7B10 line to 10 B. juncea cultivars with different uses in the experiment field varied from 0.08% to 0.93%. The natural F₁ hybrids were highly sterile, with seeds per silique ranging from 0.27 to 1.03. In addition, seeds per flower of hybrid descendants varied from 0.02 to 0.22 when F₁ hybrids were self‐pollinated, and those ranged from 0.03 to 0.30 when F₁ hybrids were backcrossed with their corresponding B. juncea parents. Results of this study suggest a low level of transgene introgression from transgenic rapeseed to different B. juncea varieties, which provides a sound scientific basis for the safety management of coexisting transgenic B. napus and B. juncea varieties in China.     

Obtaining an Ogura-type CMS line from asymmetrical protoplast fusion between cabbage (fertile) and radish (fertile)

Cytoplasmic male sterile (CMS) plants were obtained by asymmetrical protoplast fusion between red cabbage (fertile) and normal radish (fertile). The CMS plants showed restriction fragment length polymorphism (RFLP) patterns of mitochondrial (mt) DNA that were different from those of both parental lines. PCR analysis of mtDNA of the CMS plants and though Southern blot analysis showed that the mtDNA of the CMS line had the characteristics of the ogura CMS type. The results suggested that the orf138 gene and the ogura type atp6 gene are present in normal fertile cabbage and radish at a low copy level. This revised version was published online in July 2006 with corrections to the Cover Date.