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.     

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.     

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.     

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.     

Development of new sources of cytoplasmic male-sterile lines in rice

Two male-sterile lines, KalashreeA and PadminiA, with a Miz.21 cytoplasm source were developed through indica/indica hybridization followed by repeated backcrossing with their respective recurrent male parents (Kalashree and Padmini) up to the BC₆ generation. These two cytoplasmic male-sterile lines are suitable for use in the development of hybrids for lowland situations owing to their intermediate to semi-tall stature, late flowering duration, good grain quality and easy fertility restoration ability.     

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.     

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.     

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.     

Development of Polima temperature-sensitive cytoplasmic male sterile lines of Brassica napus through isolated microspore culture

A novel Pol CMS named Polima temperature‐sensitive cytoplasmic male sterile (Pol TCMS) is sterile at high temperatures and partially fertile at low temperatures. Its temperature sensitivity is controlled by several minor genes, and probably influenced by the genetic background, making it quite difficult to breed a stable Pol TCMS line through the conventional breeding methods. The present study aimed to obtain stable homozygous Pol TCMS lines by microspore culture. The highest frequency of embryogenesis was induced from cultures of buds sampled at 4–7 days before first blossom. The second method of microspore cultures mentioned in this study could significantly increase the production of embryos. Analysis of fatty acid composition, glucosinolate content and genetic diversity by simple sequence repeat (SSR) markers indicated that microspore culture was effective for selecting Pol TCMS lines.     

Molecular mapping of a fertility restorer gene for cytoplasmic male sterility in soybean

In this study, we report the mapping of the Rf locus in soybean by microsatellite simple sequence repeat (SSR) genetic markers. A cross was made between cytoplasmic male sterility (CMS) line JLCMS82A and restorer line JIHUI 1 based on the DNA polymorphisms revealed by 109 SSR markers. A F₂ population derived from a single F₁ plant containing 103 individuals was used for mapping the Rf locus. The Rf gene of JIHUI 1 gametophytically restores male fertility to JLCMS82A. Fertile and semi-fertile DNA bulks and parental DNAs were screened with 219 SSR markers, and Satt215 which was previously mapped to soybean LG J, was found linked to the Rf gene. Five additional polymorphic SSR markers from LG J were used for analysis and a regional linkage map around the Rf locus was established. SSR markers, Sctt011 and Satt547, flanked the Rf locus at 3.6 cM and 5.4 cM, respectively. The availability of these SSR markers will facilitate the selection of restorer lines in hybrid soybean breeding.     

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 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.     

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.     

Inheritance and gene tagging of male fertility restoration of cytoplasmic-nuclear male-sterile line NJCMS1A in soybean

The F₁, F₂ and F_2:3 of the NJCMS1A × 'Zhongdou 5' cross were used to analyse the inheritance of the male fertility restoration of the cytoplasmic-nuclear male-sterile line NJCMS1A in soybean. The results of genetic analysis showed two pairs of dominant genes conferring the male fertility restoration of NJCMS1A, which further confirmed previous results. The F₂ population from the NJCMS1A × 'Zhongdou 5' cross was used for tagging the restorer genes for NJCMS1A with 664 pairs of simple sequence repeat primers selected randomly from the genetic linkage map of soybean published by Cregan et al. (1999) . Satt626 on linkage group M and Satt300 on linkage group A1 of the integrated linkage map by Song et al. (2004) were found to link to the two restorer genes of NJCMS1A. The maximum-likelihood estimates of the genetic distance between the two markers, Satt626 and Satt300, and the two restorer genes of 'Zhongdou 5' were 9.75 and 11.18 cM, respectively.     

Bud pollination and hybrid seed production in detergent-induced male sterile plants of Brassica juncea

The efficacy of a synthetic detergent (Surf Excel) as a potential chemical hybridizing agent in Brassica juncea was studied. Foliar sprays with various concentrations of the detergent caused reductions in plant height, number of branches and leaves per plant, size of leaves, anther size, pollen per flower, ovules per flower, pollen fertility, fruits per plant, fruit size, seeds per fruit, total yield per plant and 100 seed weight as compared with those of untreated plants. The style in the floral buds of plants sprayed with different concentrations of Surf Excel elongated and so the receptive stigma protruded from the buds which facilitated cross‐pollination by honey bees. The plants sprayed once with 2% Surf Excel exhibited an elongated style with a raised receptive stigma and 100% pollen sterility without causing a significant reduction in total yield.     

Genetic analysis of fertility restoration against photoperiod-sensitive cytoplasmic male sterility in Triticum aestivum cv. Norin 61

Triticum aestivum cv. Norin 26 with the Aegilops crassa cytoplasm becomes almost completely male sterile when grown under a long-day condition (15 h of light or longer), but is highly male fertile under a short-day condition (14.5h or less). This type of male sterility is called photoperiod-sensitive cytoplasmic male sterility (PCMS). Genetic analyses were made of the fertility-restoring (Rf) genes effective against PCMS that are present in T. aestivum cv. Norin 61. Conventional and monosomic studies indicated that restoration of fertility is controlled by multiple Rf genes located on at least four chromosomes: 4 A, 1D, 3D and 5D. The genetic mechanism of fertility restoration by the genes of‘Norin 61’differs from the mechanisms reported for‘Chinese Spring’and a‘Norin 26’mutant line.     

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.