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.     

Male sterility in alloplasmic Brassica rapa L. carrying Eruca sativa cytoplasm

For the development of a new cytoplasmic male sterility (CMS) system in Brassica rapa (2n= 20, AA), intergeneric hybridization was performed in Eruca sativa (2n= 22, EE)×B. rapa. The original amphihaploid F₁ plant (2n= 21, EA) generated via embryo rescue produced a sesquidiploid F₁ plant (2n= 31, EAA), from which the alloplasmic F₃ plants were generated. In F₃, some progenies with malformed anthers were maintained as male-sterile lines up to the F5 generation. In the F₆ and F₇ generations, the alloplasmic male-sterile plants were bred by backcrossing to several B. rapa genotypes and were then classified into the three distinctive types: petaloid, antherless and brown anther, in addition to three intermediate types between them. By southern blot analysis, each plant of the petaloid and antherless types was shown to carry the cytoplasm genome of E. sativa. These male-sterile plants produced as many viable seeds as the corresponding male-fertile plants, although their nectary gland development was minimal. Consequently, CMS lines of the petaloid and antherless types with enhanced seed fertility and nectary gland development could provide promising plant materials for F₁ seed production in B. rapa.     

Development and primary genetic analysis of a fertility temperature-sensitive polima cytoplasmic male sterility restorer in Brassica napus

Over the past decade, the polima cytoplasmic male sterility ( pol CMS) three-line and two-line systems have been developed for the production of hybrid seed in Brassica napus oilseed rape in China. The discovery of the novel pol CMS restorer line FL-204 is described here. It restores male fertility of hybrid plants in the pol CMS system, but hybrid seed production can only be carried out under autumn sowing in Wuhan in south China under moderate temperatures at flowering. The restorer cannot be used as a male for hybrid seed production in northwestern China (Gansu) under spring sowing conditions, because there it is more or less male sterile due to high temperatures at flowering. Because of this behaviour, it is referred to as a fertility temperature-sensitive restorer (FTSR) in this paper. F₂, BC₁ as well as double haploid populations were constructed to determine the inheritance of fertility restoration of FL-204 in the autumn at Wuhan and under spring sowing conditions at Gansu, respectively. Deviations from Mendelian genetics were observed. It was hypothesized that the change of fertility was the result of the interaction between nuclear genes [restoring gene ( Rf ) and temperature-sensitive genes ( ts )] and the cytoplasm. The Rf gene in FL-204 was incapable of restoring male fertility of pol CMS lines under spring sowing conditions at Gansu where it is inactivated by the recessive ts gene present in FL-204. However, the ts gene(s) could be non-functional under moderate temperature conditions at flowering at Wuhan which allows full expression of male fertility in FL-204. The recessive ts gene(s) can only be expressed in plants containing the pol sterile cytoplasm. A method for the utilization of the FTSR pol CMS restorer FL-204 for the production of hybrid seed in B. napus oilseed rape is proposed.     

Tournefortii male sterility system in Brassica napus. Identification, expression and genetic characterization of male fertility restorers

A germplasm collection of 152 diverse rapeseed accessions from Canada, China, France, India, Poland and South Korea was assayed for identifying new fertility restorers and sterility maintainers for a Tournefortii (tour) cytoplasmic male sterility (CMS) system in rape‐seed. Only 16 (10.5%) genotypes showed complete fertility restoration following hybridization with tour CMS line NE 409A. Notable among these were GSL 8851, GSL 8953, Mokpo # 9, Mali, Buk‐wuk‐13, Kuju‐27 and Mokpo # 84. As many as 78 (51.3%) genotypes were perfect maintainers of sterility, the remaining 58 (38.2%) genotypes were classified as partial maintainers. To study the inheritance of fertility restoration, 20 CMS (tour) rapeseed lines were crossed with the four best fertility restorers, namely GSL 8851, GSL 8953, Kuju‐27 and Mokpo # 9, to obtain F₂ and test cross populations. Segregation data indicated that fertility restoration for tour CMS was governed by two genes, of which, one is stronger than the other (χ²_12:3:1). Differences in gene interactions were also observed (χ²_9:3:4) which could be explained on the basis of influence of female parent genotypes/or modified expression of the restorer gene(s) in different genetic backgrounds. Tests of allelism indicated that the restorer genes present in the four restorers evaluated were allelic.     

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.     

Variations in chlorosis and potential usefulness of alloplasmic Brassica rapa with the cytoplasm of male sterile Brassica juncea

To select superior seed parents for vegetable hybrid seed production, we conducted interspecific crosses between male sterile Brassica juncea (2n = 36, AABB) and eight inbred lines of Brassica rapa (2n = 20, AA). Alloplasmic lines of B. rapa with the cytoplasm of B. juncea were developed from B. juncea × B. rapa hybrids by repeated backcrossing using B. rapa as the recurrent male parent until the BC₃ generation. Seed fertility, male sterility and chlorophyll content were investigated in these plants cultivated under four different temperature conditions (5, 10, 12 and 20°C). At 10°C, the alloplasmic lines of B. rapa with the cytoplasm of B. juncea were male sterile with partly chlorotic leaves. The alloplasmic B. rapa had lower chlorophyll a, chlorophyll b and carotenoid contents than those of the original B. rapa. The leaves recovered from chlorosis when the plants were cultivated at 20°C. An alloplasmic line of B. rapa (A6) is available as a seed parent for vegetable hybrid seed production and contributes seed fertility, slight chlorosis and stable male sterility.     

Characterization of tribenuron-methyl-induced male sterility in Brassica juncea L.

Significant heterosis has been documented in Brassica juncea L. that are grown as agriculturally important oilseeds, vegetables and condiments crops. Male sterility induced by chemical hybridizing agents is an important pollination control system in hybrid crop breeding. Herein, we show that tribenuron-methyl (TBM), a sulfonylurea herbicide, is an effective male gametocide in B. juncea when used at a very low dosage. In the present study, foliar application of various rates of TBM induced a significant increase in pollen sterility in B. juncea (90.57–100%). TBM-treated plants exhibited reductions in size of floral organ and yield components; however, lower dose of TBM (0.075 g a.i. ha^–1) did not cause a significant reduction in seed yield per plant. Tapetum cells of TBM-treated plants were hypertrophied and degenerated earlier, and abnormal meiosis was observed at the meiotic stage. A significant decrease of acetohydroxyacid synthase (AHAS) activities was detected in buds of plants treated with 0.10 g a.i. ha^–1 TBM, and RT-qPCR analysis showed that TBM exposure perturbed AHAS expression in small buds, which support that TBM induces male sterility in B. juncea by targeting AHAS expression. Our results suggest that TBM could be used as an efficient chemical hybridization agent in B. juncea, which has practical implications for the application of hybrid breeding in B. juncea.     

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.     

Male sterility in populations of Daucus and the development of alloplasmic male-sterile lines of carrot

A collection of Daucus species and subspecies, as well as alloplasmic carrot lines were evaluated for expression of male sterility. More than 100 male‐sterile plants were identified and their phenotypes and partial genetic background were characterized. Three potentially new cytoplasmic male‐sterile systems based on the cytoplasm of Daucus carota (D. c.) gummifer, D. c. maritimus and D. c. gadecaei were selected. Genetic analyses suggested that a nuclear‐cytoplasmic interaction determined the male sterility of the three sources. A clear differentiation between the new sources and carrot cultivars was possible based on the phenotypic and molecular genetic relationships.     

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.     

Improving male fertility restoration of common wheat for Triticum timopheevii cytoplasm

The fertile pure line R3‐37 of common wheat with cytoplasm of Triticum timopheevii Zhuk. is an R‐line (restorer) that can restore the male fertility of A‐lines (male sterile lines) with T. timopheevii cytoplasm. In breeding hybrid wheat, the hybrid of the cross R3‐37/ Baimian3 was found to be completely male sterile, indicating that Baimian3 has some genes that are epistatic to the Rf genes in R3‐37. In order to elucidate the essence of this phenomenon, the male fertilities of the hybrids of 27 crosses including R3‐37 and/or Baimian3 were studied. The results show that inheritance of male fertility of the hybrid R3‐37/Baimian3 involves interactions among Rf alleles, male fertility‐inhibiting genes and genetic background. Although more than 70 different kinds of male sterile cytoplasm to common wheat have been discovered, the systems of hybrid wheat production based on male sterile cytoplasm are all the A‐line/R‐line type and all have similar problems of hybrid fertility restoration. This study confirmed that there is a new model (A‐line/R*‐line//R‐line) for producing hybrid wheat with high fertility restoration. In the new model, the completely male sterile hybrids of A‐line/R*‐line can act as common A‐line.     

Establishment of a random-mating population of 'Polima' CMS restorers in Brassica napus by use of dominant genie male sterility

Five restorers of ‘polima’ cytoplasmic male sterility (pol CMS) cannot restore the fertility in dominant genie male sterility (DGMS). A dominant male sterility gene from both, a DGMS line Rs l046AB and DGMS hybrid ‘Zhongza No. 3’, was successfully introduced into Polima cytoplasm. A random-mating population of pol CMS restorers was established by using many double-low pol CMS restorers as pollinators to cross continuously to the DGMS plants which had Polima cytoplasm.     

Cytoplasmic male sterility in barley. III. Maintenance of sterility and restoration of fertility in the msml cytoplasm

Summary 58 varieties, strains or mutants of barley were tested in the msml cytoplasm in Finland, location ca. 61 N. Three (5%) were found to be partial restorers, the rest being maintainers of sterility. 31 Israeli strains of wild barley (ssp. spontaneum) were tested. About one third were maintainers of sterility, the majority partial restorers, and two strains were full restorers. There is probably polymorphism of the restorer gene in Israeli wild barley. Partial restoration displays an environmental response. The physiology of partial restoration is discussed.     

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.     

Temperature related instability of a naturally induced cytoplasmic male sterility in industrial chicory (Cichorium intybus L.)

Natural alloplasmic cytoplasmic male sterile (CMS) clones of industrial chicory were obtained after crossing wild chicory with selected breeding lines. We investigated the CMS stability of 10 clones in various environmental growing conditions. CMS was stable under cool growing conditions in most of them. Fertility restoration, based on pollen production scores, was observed in all clones after a period of hot temperatures. The early flower bud stage was sensitive, resulting in fertile flowers 12–17 days after exposure to high temperatures. Experiments under controlled growing conditions at 15°C demonstrated that a heat shock of 2 days at 25 or 30°C was sufficient to restore fertility. Sterile flowers were formed when plants were again grown at lower temperatures. Significant differences between individual clones were observed, indicating the potential of genetic selection to obtain stable CMS parent lines.     

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.     

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.     

Genetics of fertility restoration in A2-based cytoplasmic genetic male sterility system in pigeonpea (Cajanus cajan)

The three short duration cytoplasmic genetic male sterility (CGMS) hybrids developed using A₂ (Cajanus scarabeoides) cytoplasm-based male sterile lines (CORG 990047A, CORG 990052A and CORG 7A) and the restorer inbred AK 261322 and their segregating populations (F₂ and BC₁F₁) were subjected to the study of inheritance of fertility restoration in pigeonpea. The fertility restoration was studied based on three different criteria, namely, anther colour, pollen grain fertility and pollen grain morphology and staining. The F₂ and BC₁F₁ populations of the three crosses, namely, CORG 990047A × AK 261322, CORG 990052A × AK 261322 and CORG 7A × AK 261322, segregated in the ratio of 3:1 and 1:1, for anther colour (yellow:pale yellow), pollen grain fertility (fertile:sterile) and for pollen grain morphology and staining. The above study confirmed that the trait fertility restoration was controlled by single dominant gene. This finding can be utilized for the identification of potential restorers, which can be further used in the development of CGMS-based hybrids in pigeonpea.