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.     

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.     

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.     

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.     

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.     

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.     

Development of a cytoplasmic male‐sterile line NJCMS4A for hybrid soybean production

Cytoplasmic male‐sterile (CMS) lines are being used to produce hybrid seeds. Thus far, four CMS sources in soybean [Glycine max (L.) Merr.] have been reported in China. However, they are not sufficient or efficient in meeting the requirements of commercial soybean hybrid seed production. In this study, 33 varieties were tested for CMS using 45 crosses among 37 landraces and 17 annual wild soybean accessions (Glycine soja Sieb. et Zucc.). The cross of N23661 × N23658 showed partial to complete male sterility in backcross generations, while the corresponding reciprocal cross showed normal male fertility. Thus, the cytoplasm of N23661 is male‐sterile, the continuously backcrossed line is a male‐sterile line (designated NJCMS4A), and N23658 is its maintainer (designated NJCM4B). The male fertility of NJCMS4A was restored by another accession, Nansheng9403. Accordingly, NJCMS4A along with its maintainer and restorer composes a complete set of three lines for producing hybrid soybean. Using mitochondrial markers and sequence analyses, NJCMS4A is a CMS line with its cytoplasm not identical to the four previously reported CMS sources in soybean.     

Characterization for agronomic use of cytoplasmic male-sterility in sunflower (Helianthus annuus L.) introduced from H. resinosus Small

A backcrossing programme was carried out both to assess the stability of a cytoplasmic male‐sterility (CMS) source from Helianthus resinosus, designated RES1, and to incorporate it into inbred sunflower lines (HA89, RHA271, RHA801). All the progenies, grown in different environments, were completely male‐sterile. This suggests that the expression of this cytoplasm is stable. Female‐fertility of lines HA89, RHA271 and RHA801 carrying CMS RES1 were compared with those of the corresponding fertile inbred lines. There were no differences in the number of seeds per head. This indicates that female‐fertility is not affected by RES1 cytoplasm. Cytological studies showed that meiosis proceeds normally until the tetrad stage; consequently, the absence of pollen is caused by alterations that take place during postmeiotic stages. With the aim of identifying male‐fertility restorer genotypes, crosses were made between HA89 (CMS RES1) plants and different annual diploid and perennial hexaploid Helianthus species. All the diploid germplasm evaluated behaved as a CMS RES1 maintainer. However, the hexaploid species, H. resinosus, H. x laetiflorus, H. pauciflorus and H. tuberosus, restored pollen fertility in CMS RES1 plants.     

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.     

Comparative genetic analysis and molecular mapping of fertility restoration genes for WA,Dissi, and Gambiaca cytoplasmic male sterility systems in rice

The genetic relationship among three cytoplasmic male sterility (CMS) systems, consisting of WA, Dissi, and Gambiaca, was studied. The results showed that the maintainers of one CMS system can also maintain sterility in other cytoplasmic backgrounds. The F₁ plants derived from crosses involving A and R lines of the respective cytoplasm and their cross-combination with other CMS systems showed similar pollen and spikelet fertility values, indicating that similar biological processes govern fertility restoration in these three CMS systems. The results from an inheritance study showed that the pollen fertility restoration in all three CMS systems was governed by two independent and dominant genes with classical duplicate gene action. Three F₂ populations, generated from the crosses between the parents of good-performing rice hybrids, that possess WA, Dissi, and Gambiaca CMS cytoplasm, were used to map the Rf genes. For the WA-CMS system, Rf3 was located at a distance of 2.8 cM from RM490 on chromosome 1 and Rf4 was located at 1.6 cM from RM1108 on chromosome 10. For the Dissi-CMS system, Rf3 was located on chromosome 1 at 1.9 cM from RM7466 and Rf4 on chromosome 10 was located at 2.3 cM from RM6100. The effect of Rf3 on pollen fertility appeared to be stronger than the effect of Rf4. In the Gambiaca-CMS system, only one major locus was mapped on chromosome 1 at 2.1 cM from RM576. These studies have led to the development of marker-assisted selection (MAS) for selecting putative restorer lines, new approaches to alloplasmic line breeding, and the transfer of Rf genes into adapted cultivars through a backcrossing program in an active hybrid rice breeding program.     

Identification and Inheritance of Fertility Restorer Genes for 'tour' CMS in Rapeseed (Brassica napus L.)

Eighteen genotypes of Brassica napus were crossed to a cytoplasmic male sterile (CMS) line of B. napus BO 15 carrying B. tournefortii cytoplasm (‘tour’ cytoplasm). Fourteen genotypes were found to be stable maintainers of the ‘tour’ CMS. Of the remaining four genotypes, GSL-1 and ‘Asahi-natane’ were found to be heterozygous and ‘Mangun’ and ‘Yudal’ were homozygous for the restorer gene. Analysis of the F₁ and F₂ progenies of (CMS) BO 15 בMangun’ and (CMS) BO 15 בYudal’ showed that fertility restoration is controlled by a single dominant gene. The availability of a number of stable maintainer lines and the simple inheritance pattern of fertility restorer gene makes ‘tour’ CMS a useful system for hybrid seed production in rapeseed.     

Modified complementation test of male sterility mutants in pepper (Capsicum annuum L.): preliminary study to convert male sterility system from GMS to CMS

The male sterility system in hybrid seed production can eliminate the cost of emasculation and ensure seed hybridity through avoidance of self pollination. GMS and CMS are two types of male sterility system that currently employed in pepper breeding. Conversion from GMS to CMS will increase the male sterility proportion of female parent from 50 to 100%. In this study, segregation analysis of four male sterile mutants consisting of one CMS mutant (CA1) and three GMS mutants (GA1, GA3 and GA4) showed that each had single recessive gene inheritance. A modified complementation test was performed by replacing male sterile mutants with their maintainer line as male parent. The nuclear restorer gene for CMS was independent of all nuclear restorer genes for GMS and all nuclear restorer genes for GMS were independent each other. Further observation on CMS and GMS male sterility loci revealed that GA1 and GA3 had mutated in both nuclear restorer genes for CMS and GMS, while CA1 and GA4 each carried mutation in single male sterility system of nuclear restorer gene for CMS and GMS, respectively. Conversion from GMS to CMS in the case of lines carried mutations in both sterility systems required only S-type cytoplasm donor, while lines carried mutation in single nuclear restorer gene for GMS required not only S-type cytoplasm but also rf allele donors. The important finding is the broader function of maintainer line in certain male sterility system that can be used as a maintainer or restorer line for other male sterility systems. We also confirmed that line CC1 is the general restorer for both CMS and GMS 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.     

Development of cytoplasmic-genic male sterility in safflower

An interspecific cross was made between Carthamaus oxyacantha and the cultivated species C. tinctorius to develop a cytoplasmic‐genic male sterility (CMS) system in safflower. C. oxyacantha was the donor of sterile cytoplasm. The 3: 1 segregation pattern observed in BC₁F₂ suggested single gene control with dominance of male‐fertility over male‐sterility. The information obtained from crossing male sterile X male fertile plants in BC₁F₃ and BC₁F₄ generations showed statistically significant single gene (1: 1) segregation for male sterility vs. male fertility. The results demonstrated that C. tinctorius possesses a nuclear fertility restorer gene and that a single dominant allele restored fertility (Rf) in progeny carrying CMS cytoplasm of C. oxyacantha. Male sterility occurred with the homozygous recessive condition (rfrf) in a sterile C. oxyacantha cytoplasm background and not in the normal cytoplasm of C. tinctorius. The genetic background of different restorer lines of C. tinctorius having normal cytoplasm did not effect fertility restoration. The absence of male sterile plants in C. tinctorius populations ruled out the possibility of genetic male sterility. Normal meiosis in F₁ and BC₁F₂ ruled out a cytogenetic basis for the occurrence of male sterility.     

Alloplasmic effects of <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">B. juncea</Emphasis> on seed characteristics of <Emphasis Type="Italic">B. carinata</Emphasis>

Effects of Brassica napus (N) and B. juncea (J) cytoplasm on seed characteristics of B. carinata (C) were examined. Alloplasmic lines of B. carinata were produced from N × C and J × C hybrids by recurrent backcrossing to the BC₈ generation. Fourteen sets of reciprocal crosses were used. Compared with their euplasmic sibs, alloplasmic B. carinata line seeds with B. napus cytoplasm showed reduced dormancy, higher seed weight, lower germination rate at high temperatures, higher germination rate at low temperatures, and had lower erucic acid and higher linoleic acid contents. Alloplasmic B. carinata line seeds with B. juncea cytoplasm had higher seed weight but lower germination rate than their corresponding euplasmic sibs. These results showed a cytoplasmic effect on seed development, and an influence on seed weight, dormancy, and fatty acid composition. B. carinata was more deleteriously affected by cytoplasm from B. napus than by cytoplasm of B. juncea.     

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.     

Effect of wild Helianthus cytoplasms on agronomic and oil characteristics of cultivated sunflower (Helianthus annuus L.)

Sunflower (Helianthus annuus L.) productions reliance on a single source of cytoplasmic male sterility, PET1, derived from H. petiolaris Nutt., makes the crop genetically vulnerable. Twenty diverse cytoplasmic substitution lines from annual and perennial wild species were compared with the inbred line HA89 over four environments. Pairwise comparisons of alloplasmic male fertile (MF) with euplasmic MF lines suggested that cytoplasm of perennial H. angustifolius increased lodging while perennial cytoplasms of H. mollis, H. grosseserratus, and H. divaricatus reduced head size and consequently reduced yield and will need to be used with caution. Lines having annual species, cytoplasms had no effects on agronomic traits. Comparisons of CMS HA 89 with 10 CMS lines suggested yield‐reducing cytoplasmic effects of perennial H. maximiliani and annual H. annuus PI 413178 and PI 413024. Cytoplasmic effects on oil percentage and fatty acid composition were minimal and should not be of concern for sunflower breeders. In general, most cytoplasms of wild annual Helianthus species accommodate cultivated nuclear genes without drastic adverse interactions, and are potential sources of cytoplasmic diversity for sunflower breeding.     

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.