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.     

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.     

Creating favourable morphological and yield variations for rapeseed by interspecific crosses between Brassica rapa and Brassica oleracea

Brassica napus is a most important oilseed grown worldwide with a limited genetic background, due to the short history of speciation, domestication and cultivation. To create novel germplasm for rapeseed breeding, we made interspecific crosses followed with chromosome doubling between B. rapa and B. oleracea to generate novel B. napus with favourable agronomic traits. The resynthesized (S0) hybrids were confirmed by SSR and cytogenetic analysis, and the fertility was increased from 32.7% in S0 generation to ~97.31% in S1 generation. The plant shapes of the progeny were dramatically improved compared to the diploid parents and B. napus cv. ‘Yangyou 6’, especially for the branch initiation height, branch number and pod number. The single‐plant yield was significantly improved in S1 progeny for the variations in branching sites and number. Significant improvement in plant shape and yield was observed on S2 generation compared to the local elite commercial open‐pollinated cultivar, which would be further fixed by intensive selection and pyramiding breeding. Such variation is of great value for breeding rapeseed with improved plant architecture and harvest index.     

Cytoplasmic male sterility with self-incompatibility,a novel approach to utilizing heterosis in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

The Polima cytoplasmic male sterility (CMS) system has been successfully used in three/two-line hybrid production in rapeseed (Brassica napus L.). However, the sterility of the Polima (pol) CMS lines is sensitive to temperature fluctuations. Also, traces of pollen can cause self-pollination within the CMS lines, which results in reduced levels of F₁ hybrid seed purity and leads to a significant yield loss. Self-incompatibility (SI) is another important approach for hybrid seed production in rapeseed. Despite having a wide range of restorers and being easily selected in a breeding program, SI system has some drawbacks. In this study, SI genes from a self-incompatible line of Brassica napus were transferred to a pol CMS line and S372A, a novel line of combined cytoplasmic male sterility with self-incompatibility was bred. Due to the SI genes, this line produced very few seeds when it was selfed at low temperature and no seeds at high temperature. This suggested that the line with CMS + SI had combined the advantages and overcome the disadvantages of both the pol CMS and SI systems. Furthermore, our results showed that most of the maintainers and all the restorers of the pol CMS system were also maintainers and restorers of the CMS + SI line, respectively. This indicates that the CMS + SI system can be easily used to establish three-line hybrids of rapeseed, and we believe this novel system could be extended to other species of Brassica.     

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%.     

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.     

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.     

Genotypic Variation for Tolerance to Transient Drought During the Reproductive Phase of Brassica rapa

Brassica rapa L. is a genetically diverse parent species of the allotetraploid species, oilseed rape (B. napus) and a potential source of drought tolerance for B. napus. We examined the effect of a 13‐day drought stress period during the early reproductive phase, relative to a well‐watered (WW) control, on subsequent growth and development in nine accessions of B. rapa and one accession of Brassica juncea selected for their wide morphological and genetic diversity. We measured leaf water potential, stomatal conductance, water use, and leaf and bud temperatures during the stress period and aboveground dry weight of total biomass at maturity. Dry weight of seeds and reproductive tissue were not useful measures of drought tolerance due to self‐incompatibility in B. rapa. The relative total biomass (used as the measure of drought tolerance in this study) of the 10 accessions exposed to drought stress ranged from 47 % to 117 % of the WW treatment and was negatively correlated with leaf‐to‐air and bud‐to‐air temperature difference when averaged across the 13‐day stress period. Two wild‐type (B. rapa ssp. sylvestris) accessions had higher relative total and non‐reproductive biomass at maturity and cooler leaves and buds than other types. We conclude that considerable genotypic variation for drought tolerance exists in B. rapa and cooler leaves and buds during a transient drought stress in the early reproductive phase may be a useful screening tool for drought tolerance.     

Interspecific amphiploid‐derived alloplasmic male sterility with defective anthers,narrow disc florets and small ray flowers in sunflower

The cytoplasmic male‐sterility (CMS)/fertility‐restoration system is important for hybrid sunflower (Helianthus annuus L.) seed production. The objective of this study was to characterize two novel alloplasmic CMSs, designated CMS GRO1 and CMS MAX3, with defective anthers, narrow disc florets with no swollen corolla, and short, narrow ray flowers derived from two tetraploid amphiploids (AMPs). Among 26 tested lines, only AMP Helianthus cusickii/P 21 and HA 410 failed to restore male‐fertility. Segregation of CMS, male‐fertile plants and plants with reduced male‐fertility was observed both in the testcross progeny of a six line half‐diallel cross of F₁s with CMS MAX3 and in an F₂ population of CMS GRO1 × RHA 274. Male‐fertility restoration was controlled by at least two dominant genes. Detailed analysis of the mitochondrial genes may provide insight into the differences between these CMSs and other CMS lines. The new CMSs will facilitate the studies of the incompatibility between cytoplasmic and nuclear genes, especially for the alloplasmic CMS involving perennial species, and also provide unique ornamental flower types and CMS sources for hybrid sunflower breeding.     

Nuclear genetic control of variation in simazine tolerance in oilseed brassicas

Summary Brassica napus is a natural allotetraploid derived from the diploid species B. rapa L. (syn. campestris L.) and B. oleracea L. Somatic hybrids synthesized from highly heterozygous lines of these two diploid species were evaluated for fertility. The hybrids were obtained from two fusion experiments which differed in the B. rapa full-sibling parent used as the source of protoplasts. Both B. rapa siblings were lelf-incompatible (SI) yet contained different S-alleles; the B. oleracea species parent was self-compatible (SC). Eight tetraploid hybrids examined had very high female and male fertility; eight hybrids with higher ploidy had low fertility. Hybrids derived from one B. rapa sibling were self-incompatible, whereas those derived from the other B. rapa sibling were fully self-compatible. These data suggest that the different S-alleles of each B. rapa sibling displayed varying penetrance relative to the SC of the B. oleracea parent when combined in B. napus.Abbreviations SC self-compatibility - SI self-incompatibility     

Genetic and histological characterization of a novel recessive genic male sterile line of <Emphasis Type="Italic">Brassica napus</Emphasis> derived from a cross with <Emphasis Type="Italic">Capsella bursa</Emphasis>-<Emphasis Type="Italic">pastoris</Emphasis>

In a previously made cross Brassica napus cv. Oro (2n = 38) × Capsella bursa-pastoris (2n = 4x = 32), one F₁ hybrid with 2n = 38 was totally male sterile. The hybrid contained no complete chromosomes from C. bursa-pastoris, but some specific AFLP (amplified fragment length polymorphism) bands of C. bursa-pastoris were detected. The hybrid was morphologically quite similar to ‘Oro’ except for smaller flowers with rudimentary stamens but normal pistils, and showed good seed-set after pollination by ‘Oro’ and other B. napus cultivars. The fertility segregation ratios (3:1, 1:1) in its progenies indicated that the male sterility was controlled by a single recessive gene. In the pollen mother cells of the male sterile hybrid, chromosome pairing and segregation were normal. Histological sectioning of its anthers showed that the tapetum was multiple layers and was hypertrophic from the stage of sporogenic cells, and that the tetrads were compressed by the vacuolated and disaggregated tapetum and no mature pollen grains were formed in anther sacs, thus resulting in male sterility. The possible mechanisms for the production of the male sterile hybrid and its potential in breeding are discussed.     

Microspore mutagenesis of Brassica species for fatty acid modifications: a preliminary evaluation

A microspore mutagenesis protocol was developed for Brassica rapa, Brassica napus and Brassica juncea for the production of double haploid lines with novel fatty acid profiles in the seed oil. Freshly isolated Brassica microspores were first cultured with ethyl methane sulphonate (EMS) for 1.5 h. The EMS was removed and the microspores were then cultured according to the standard Brassica microspore culture protocol. This protocol was used to generate over 80 000 Brassica haploid/double haploid plants. Field evaluation of B. napus and B. juncea double haploids was conducted between 2000 and 2003. Fatty acid analysis of the B. napus double haploid lines showed that saturated fatty acid proportions ranged from 5.0% to 7.7%. For B. juncea, saturate proportions ranged from 5.4% to 9.5%. Of the 7000 B. rapa lines that were analysed, 197 lines had elevated oleic acid (>55%), 69 lines had reduced α‐linolenic acid (<8%) and 157 lines had low saturated fatty acid proportions (<5%), when compared with the parental lines.     

Phenotypic and seed structural comparison in hybrids of different Brassica rapa and B. oleracea

Brassica napus is an important oil species with short history and narrow genetic background. Interspecific hybrids from crosses between B. oleracea and different B. rapa were obtained. We found the hybrids with white petal resembling B. oleracea, the flavonoid and phenolic content decreased in hybrids, agreeing with the expressional changes of flavonoid biosynthesis genes. Seed coat of hybrids resembled diploid parents, or partly resembled to each parent with a clear outline. The palisade layer in hybrids was thicker than parents, with similar pigment accumulation as B. oleracea but more than B. rapa. Differentially sized protein bodies (PBs) were found in hybrids. The radical and inner cotyledon of all hybrids were identified with larger but less PBs than parents. The average size of PBs in outer cotyledon of resynthesized B. napus was also larger than parents, but the number of PBs was not significantly reduced. The phenotypic and seed structural variations after polyploidization of B. napus would be interesting for genetic broadening and breeding of rapeseed.     

Identification of AFLP and SCAR markers linked to the male fertility restorer gene of <Emphasis Type="Italic">pol</Emphasis> CMS (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

The pol cytoplasmic male-sterility system has been widely used as a component for utilization of heterosis in Brassica napus and offers an attractive system for study on nuclear–mitochondrial interactions in plants. Genetic analyses have indicated that one dominant gene, Rfp, was required to achieve complete fertility restoration. As a first step toward cloning of this restorer gene, we attempted molecular mapping of the Rfp locus using the amplified fragment length polymorphism (AFLP) technique combined with bulked segregant analysis (BSA) method. A BC₁ population segregating for Rfp gene was used for tagging. From the survey of 1,024 AFLP primer combinations, 13 linked AFLP markers were obtained and five of them were successfully converted into sequence characterized amplified region (SCAR) markers. A population of 193 plants was screened using these markers and the closest AFLP markers flanking Rfp were at the distances of 2.0 and 5.3 cM away, respectively. Further the AFLP or SCAR markers linked to the Rfp gene were integrated to one doubled-haploid (DH) population derived from the cross Quantum × No.2127-17 available in our laboratory, and Rfp gene was mapped on N18, which was the same as the previous report. These molecular markers will facilitate the marker-assisted selection (MAS) of pol CMS restorer lines.     

Generation of tribenuron‐methyl herbicide‐resistant OsCYP81A6‐expressing rapeseed (Brassica napus L.) plants for hybrid seed production using chemical‐induced male sterility

Chemical‐induced male sterility (CIMS) is a method for hybrid rapeseed (Brassica napus L.) production. Some sulphonylurea herbicides such as tribenuron‐methyl (TBM) are used as chemical hybridization agents (CHAs) in CIMS systems. However, the male parents must be protected from herbicide injury with a shield during spraying of the female parents with CHAs to induce male sterility. Thus, using herbicide‐resistant rapeseed lines as the male parents can significantly simplify the seed production procedure and reduce the cost in hybrid seed production. A rice cytochrome P450 hydroxylase, OsCYP81A6, has been previously characterized to confer resistance to bentazon and sulphonylurea herbicides. We demonstrate here that the introduction of OsCYP81A6 renders rapeseed plants resistant to TBM. Compared with wild‐type plants, the transgenic plants displayed normal stamen development and male fertility when treated with 0.05 mg/l of TBM, the dose used for inducing male sterility in hybrid seed production. These results indicate that the OsCYP81A6‐expressing rapeseed plants can be used as the male parents for hybrid rapeseed production using CIMS.     

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.     

Independent Inheritance of Flower Colour and Male-Fertility Restorer Characters in Brassica napus L.

Available material of oilseed (Brassica napus L., AACC) comprises two yellow-flowered breeding lines and a white/pale-flowered line of resynthesized rape. The flower colour white/pale is dominant over yellow, and is controlled by a gene located in the C-genome. The yellow-flowered genotypes acted as restorer lines and the white/pale-flowered genotype as a maintainer line in a cytoplasmic male sterility system. The segregation pattern of flower colour and male fertility restorer characters were studied in F₂ generations of crosses between these lines, also in a three-way cross additionally including a yellow flowered B. campestris (AA) line. Evidense was obtained in support of the conclusion that the flower colour and male fertility restorer characters are monogenically controlled and independently inherited. Whether the male fertility restorer gene is located in the A or C genome remains to be determined.     

Development of self-incompatible Brassica napus: (III) B. napus genotype effects on S-allele expression

Use of self‐incompatibility (SI) as a pollination control method for Brassica napus hybrid production requires the development of a sufficient number of S‐alleles that are expressed consistently in a range of B. napus lines. Self‐incompatibility (SI) alleles have been transferred from Brassica oleracea and Brassica rapa into B. napus var. oleifera. An understanding of expression of these alleles in B. napus is essential for their commercial use. Four SI B. napus doubled haploids containing the B. oleracea S‐alleles S2, S5, S13 and S24 were crossed to three B. napus cultivars to measure the B. napus genetic background effect on S‐allele expression. A line x tester analysis indicated that the largest source of variation in the expression rate of SI was the S‐allele itself. The B. napus genotypes tested contained modifier gene(s), some that enhanced SI expression and others that inhibited SI expression. The B. napus Canadian cultivar ‘Westar’ generally had a negative effect on SI expression while the European cultivar ‘Topas’ had a positive effect on the B. oleracea S‐allele expression. The B. oleracea S‐allele S24 was very similar in expression to the B. rapa allele W1. The application of these results for the use of B. oleracea S‐alleles for hybrid production in B. napus is discussed.     

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.