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 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.     

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.     

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.     

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.     

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.     

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.     

Hybridization in Brassica juncea × Brassica campestris through ovary culture

Summary Ovary culture has been employed for the production of interspecific hybrids of a partially compatible cross of Brassica juncea (2n=36) × Brassica campestris (2n=20). Five to seven days old ovaries cultured on White's medium supplemented with casein hydrolysate (300 mg/l) and sucrose (5%) produced more seeds than any other media tried, but seed development was better on media fortified with plant hormones. The seed yield was better in B. juncea × B. campestris than their reciprocal cross. The plants obtained from ovary-derived seeds were transferred to the field; they were intermediate in some morphological characters and chromosome number (2n=28) as compared to their parents. The flower buds generally did not open and had poorly developed anthers with mostly sterile pollen. The pod size/setting was very much reduced, but healthy seeds were obtained.     

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.     

Synthesis of Alloplasmic Brassica campestris as a New Source of Cytoplasmic Male Sterility

Cytoplasmic male, steriles of Brassica campestris ssp. oleifera var. brown sarson were obtained m BO generation progenies Following the repeated back-crossing of the synthetic alloploid B. oxyrrhina (2n = 18, OO) ×B. campestris. (2n = 20, AA). Alloplasmic B. campestris plants resemble B. campestris in morphology and growth pattern and do not exhibit any trait of B. oxyrrhina, However, the leave; arc mildly chlorophyll deficient in the earlier stages and turn green at late stages In the development. The reproductive parts do not show and abnormalities except that the anthers are smaller and slenderer that the normal ones and are non dehiscent, containing only sterile pollen.     

Inheritance and mapping of a restorer gene for the rapeseed cytoplasmic male sterile line 681A

A novel cytoplasmic male sterility‐fertility restoration system has been developed in rapeseed (Brassica napus). The cytoplasmic male sterile line 681A was derived from a spontaneous male sterile mutant in a newly released double‐low rapeseed cultivar ‘Xiangyou 13′. The restorer line 714R was identified in the interspecific progeny from a B. napus×B. juncea‐cross. Genetic analysis showed that fertility restoration for 681A cytoplasmic male sterility was controlled by a single dominant nuclear gene which might originate from B. juncea. The RAPD marker S1039_‐520 was found to be linked to the restorer gene in F₂ progeny of 681A × 714R with a recombination frequency of 5.45%.     

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.     

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.     

Dissection of the Brassica nigra Genome by Monosomic Addition Lines

Hyperploid derivatives of Diplotaxis erucoides × Brassica nigra hybrids were used to extract seven out of the eight possible monosoinic addition lines for B. nigra (genome B). The fertility and transmission of the lines varied depending on the added chromosome. However, these parameters were high enough to assure the maintenance of the addition lines. Although no phenotypic changes were observed, the plants carrying extra chromosomes were slower in development than diploid D. emcoides. Each of the B. nigra chromosomes was recognizable cytologically by size and heterochromatin distribution. Seven of these were characterized by a series of isozymes and RFLP markers. Ribosotnal DNA sequences were detected in two independent B. nigra chromosomes. Two probes disclosed fragments dispersed in more than one chromosome.     

Identification of Maintainer Genes in Brassica napus L. for the Male-Sterility-Inducing Cytoplasm of Diplotaxis muralis L.

Male fertility of F¹ interspecific hybrid plants derived from crosses between cytoplasmic male-sterile Brassica campestris in Diplotaxis muralis cytoplasm and 147 B. napus cultivars was Investigated. F¹, plants obtained, from crosses with the B. napus cultivars‘Mangum’and‘Hinchu’were male-sterile while F¹ plants derived from all other crosses were male-fertile. This indicated that these two cultivars carried maintainer genes far the male-sterility-inducing cytoplasm of D. muralis. Sterility was stable In plants derived from backcrosses of male-sterile F; plants with‘Mangun and‘Hinchu’but the seed set of backcross plants was low. With restorer genes readily available in B. napus, these findings could lead to the development of a new cytoplasmic male sterility system for the breeding of B. napus hybrid cultivars.     

Identification of stable maintainer and fertility restorer lines for 'Polima' CMS in Brassica campestris

‘Polima’ cytoplasmic male sterility (CMS) was transferred from ‘Polima’ Brassica napus ‘ISN 706’to five different cultivars of Brassica campestris (‘Pusa kalyani’, ‘Pant toria’, ‘Candle’, ‘Tobin’ and ‘ATC 94211′) by repeated backcrossing. It was observed that, while ‘Polima’ CMS manifested complete and stable male sterility in the nuclear backgrounds of ‘Pusa kalyani’, ‘Pant toria’, and ‘Tobin’, the cultivars ‘Candle’ and ‘ATC 94211’possessed the restorer gene for this CMS in the heterozygous condition. An analysis of F₁ and F₂ generations of ‘Polima’‘Pusa kalyani’בCandle’ and ‘Polima’‘Pusa kalyani’בATC 94211’ revealed that restoration is controlled by a single dominant gene. Identification of stable maintainers and restorers of ‘Polima’ CMS could facilitate the development of hybrid varieties in B. campestris.     

Production of intergeneric hybrids between Brassica juncea and Diplotaxis virgata through ovary culture, and the cytology and crossability of their progenies

The cytogenetic study was investigated in the intergeneric F₁ hybrid, F₂and backcross progenies (BC₁). The plants used were Brassica juncea(2n=36) and Diplotaxis virgata(2n=18). Three intergeneric F₁ hybrids between two species were produced through ovary culture. They showed 36 chromosomes. It might consist one genome of B. juncea and two genomes of D. virgata. The morphology of the leaves resembled that of B. juncea. The color of the petals was yellow that was like in D. virgata. The size of the petal was similar to that of B. juncea. The mean pollen fertility was15.3% and the chromosome associations in the first meiotic division were(0–1)_IV+(0–2)_III+(8–12)_II+(12–20)_I. Many F₂ and BC₁seeds were harvested after open pollination and backcross of the F₁ hybrids withB. juncea, respectively. The F₂seedlings showed different chromosome constitutions and the range was from 28 to54 chromosomes. Most seedlings had 38chromosomes followed by 36, 40 and 54. The BC₁ seedlings also showed different chromosome constitutions and the range was from 29 to 62. Most seedlings had both 40and 54 chromosomes followed by 36, 46 and52. In the first meiotic division of F₂ and BC₁ plants, a high frequency of bivalent associations was observed in all the various kinds of somatic chromosomes. Many F₃ and BC₂ seeds were obtained by self-pollination and open pollination of both F₂ and BC₁ plants, and by backcrossing both F₂ and BC₁plants with B. juncea, respectively,especially, three type progeny with 36, 40or 54 chromosomes. The somatic chromosomes of the F₃ and BC₂ plants were further investigated. The bridge plants between B. juncea and D. virgata with 36 chromosomes may be utilized for breeding of other Brassica crops as well as B. juncea. This revised version was published online in July 2006 with corrections to the Cover Date.