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.     

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.     

Synthesis and cytogenetic characterization of intergeneric hybrids of Diplotaxis siifolia with Brassica rapa and B. juncea

Intergeneric hybrids involving a wild crucifer, Diplotaxis siifolia (2n = 20; D^sD^s), and two crop Brassica species, namely Brassica rapa (2n = 20; AA) and B. juncea (2n = 36; AABB), were developed through sequential ovary/ovule culture. Hybridization was successful only when D. siifolia was used as the female parent, indicating unilateral cross incompatibility. Hybrids were intermediate between the parents for morphological characteristics but had low male as well as female fertility. Meiotic studies of hybrids revealed partial homoeology between D^s and A/B genomes.     

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.     

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.     

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.     

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.     

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.     

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.     

Genetic diversity in advanced derivatives of Brassica interspecific hybrids

Genetic diversity among the 88 entries including eighty F₄ derivatives i.e., 20 each selected from Brassica crosses viz., B. juncea × B. napus, B. juncea × B. rapa var. toria, B. juncea ×B. rapa var. yellowsarson and B. tournefortii × B. juncea, and eight parent genotypes was assessed through multivariate analysis (D² statistic). Significant differences among the family groupsas well as within the family were recorded for all the 14 characters studied. The D² analysis revealed enormous diversity among the interspecific cross derivatives. The genetic distances calculated among different Brassica species revealed that B. tournefortii had maximumdiversity with B. juncea followed by B. napus, B.rapa var. toria and B. rapa var. yellow sarson.Amongst interspecific crosses, maximum diversity was noticed indescendants of cross B. tournefortii × B. juncea followed byB. juncea × B. napus, B. juncea × B.rapa var. toria and the least in the cross B. juncea ×B. rapa var. yellow sarson. These results indicated that the derivatives selected from cross of diverse parents revealed greater diversity. The clustering pattern showed that many derivatives of the cross fell into the same cluster but in many cases in spite of common ancestry many descendants of the cross spread over different clusters. The characters, namely, plant height, secondary branches per plant, days to flowering and1000-seed weight were contributed maximum towards genetic divergence. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Enarthrocarpus lyratus-based cytoplasmic male sterility and fertility restorer system in Brassica rapa

Substituting the nuclear genome of Brassica rapa into the cytoplasmic background of Enarthrocarpus lyratus through backcross substitution helped in developing cytoplasmic male sterility (CMS). Alloplasmic male sterile plants had pale green leaves, small flowers with narrow petals and rudimentary anthers. Female fertility, low initially, improved considerably with advanced backcross generations. Male sterility expression was stable throughout the growing season. Except for EC 339014, all B. rapa accessions (38) evaluated were partial maintainers of the male sterility. Introgression of gene(s) for fertility restoration from the cytoplasm donor species was facilitated by homoeologous pairing between B. rapa and E. lyratus genomes, as was apparent from the very frequent occurrence of a trivalent in the monosomic addition plants (2n = 10 II + 1 I). Backcrossing of fertile monosomic addition plants with B. rapa led to the recovery of male fertile plants possessing the stable euploid chromosome number (2n = 20). These plants restored male fertility in crosses with different (lyr) CMS B. rapa genotypes, confirming the introgression of fertility restorer gene(s) from E. lyratus, the cytoplasm donor species.     

Interspecific transfer of Brassica juncea-type high blackleg resistance to Brassica napus

Summary Complete resistance to Leptosphaeria maculans, the cause of blackleg of oilseed rape (Brassica napus), was transferred from B. juncea to B. napus through an interspecific cross. B. juncea-type complete resistance (JR) was recognized first in one F₃ progeny (Onap^JR) by the absence of leaf-lesions on seedlings and canker-free adult plants. The commercially important characters of B. napus were retained in advanced lines of Onap^JR, which combined JR with low erucic acid levels (<0.5%), high seed yield and variable maturity dates.JR appeared to be inherited as a major gene or genes. Segregation for resistance and susceptibility contintied to occur during later generations of selection of Onap^JR. JR was readily transferred from Onap^JR to other suitable B. napus cultivars or lines with partial resistance to blackleg and resulted in highly vigorous carly generation selections adapted to cold, wet situations along with complete resistance to blackleg.     

SSR and SCAR markers linked to the fertility-restoring gene for a D2-type cytoplasmic male-sterile line in wheat

‘Yi 4060’ is an elite restorer line of a non‐photoperiod‐sensitive D²‐type cytoplasmic male‐sterile (CMS) line of wheat. Random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were employed to map one major fertility‐restoring gene (D²Rf₁) in ‘Yi 4060′. The sterile and fertile DNA pools were established from individuals in BC₆, based on bulked segregant analysis. One RAPD marker E09, linked to D²Rf₁, was converted to a SCAR marker and designated as E09‐SCAR₈₆₅. The genetic distance between E09‐SCAR₈₆₅ and D²Rf₁ is 9.5 cM. Two SSR markers, Xgwm11 and Xgwm18, were also linked to a D²Rf₁ and co‐segregated with E09‐SCAR₈₆₅. The three molecular markers are useful in marker‐assisted breeding of the elite restorer lines for D² ‐type CMS lines in wheat.     

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.     

Cytological observation of anther structure and genetic investigation of a new type of cytoplasmic male sterile 0A193‐CMS in Brassica rapa L.

Cytoplasmic male sterility (CMS), a maternally transmitted failure in pollen formation, is an effective pollination control system in hybrid rapeseed (Brassica napus) breeding. However, CMS is not widely used in the related oilseed species Brassica rapa. In the past years, several male sterile plants have been isolated from the B. rapa landrace ‘0A193’, collected in Shaanxi, China, in 2011. It is noteworthy that the fertility expression of 0A193‐CMS was affected by temperature. In contrast to pol CMS, fertility tests with 18 B. rapa and 9 B. napus accessions suggest that a different system of maintaining and restoring is responsible for the observed phenotype. Further on, genetic investigation evidenced that fertility of 0A193‐CMS is controlled by both cytoplasmic and one pair of nuclear recessive genes. Interestingly, plants of the 0A193‐CMS type possess a highly specific fragment of the mitochondrial gene orf222, a crucial regulator of male sterility in nap CMS. Our study broadens the CMS resources in B. rapa and provides a highly applicable alternative to pol CMS and ogu CMS for hybrid breeding production.     

Production of a desirable Brassica oleracea CMS line using an alloplasmic B. rapa CMS line carrying Diplotaxis erucoides cytoplasm as a bridge plant

In Brassica oleracea, production of F₁ hybrid seeds mainly makes use of the improved Ogura cytoplasmic male sterile (CMS) line. However, reliance on one particular line is a risk, and it would be advantageous to develop other CMS lines. In this study, we transferred Diplotaxis erucoides cytoplasm to B. oleracea cultivars using an alloplasmic B. rapaCMS line as a bridge plant to avoid incompatibility between donor and recipient plants. The new B. oleraceaCMS lines, which were derived by four generations of backcrossing, had small rudimentary anthers with no pollen grain and showed complete male sterility. There was no functional defect in other floral organs, and the ability to receive normal pollen did not appear to be impaired. Moreover, the B. oleraceaCMS lines carrying D. erucoides cytoplasm had larger leaf areas and a normal plastochron. As a consequence, the B. oleraceaCMS lines carrying D. erucoides cytoplasm have the potential to be valuable alternatives for use in commercial B. oleracea hybrid seed production.     

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.