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.     

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.     

Cytological observation of anther structure and genetic investigation of a new type of cytoplasmic male sterile 0A193‐CMS in Brassica rapa L.

Cytoplasmic male sterility (CMS), a maternally transmitted failure in pollen formation, is an effective pollination control system in hybrid rapeseed (Brassica napus) breeding. However, CMS is not widely used in the related oilseed species Brassica rapa. In the past years, several male sterile plants have been isolated from the B. rapa landrace ‘0A193’, collected in Shaanxi, China, in 2011. It is noteworthy that the fertility expression of 0A193‐CMS was affected by temperature. In contrast to pol CMS, fertility tests with 18 B. rapa and 9 B. napus accessions suggest that a different system of maintaining and restoring is responsible for the observed phenotype. Further on, genetic investigation evidenced that fertility of 0A193‐CMS is controlled by both cytoplasmic and one pair of nuclear recessive genes. Interestingly, plants of the 0A193‐CMS type possess a highly specific fragment of the mitochondrial gene orf222, a crucial regulator of male sterility in nap CMS. Our study broadens the CMS resources in B. rapa and provides a highly applicable alternative to pol CMS and ogu CMS for hybrid breeding production.     

Production and characterization of an alloplasmic and monosomic addition line of Brassica rapa carrying the cytoplasm and one chromosome of Moricandia arvensis

Intergeneric hybridization was performed between Moricandia arvensis and four inbred lines of Brassica rapa following embryo rescue. Three F₁ hybrid plants were developed from three cross combinations of M. arvensis × B. rapa, and amphidiploids were synthesized by colchicine treatment. Six BC₁ plants were generated from a single cross combination of amphidipolid × B. rapa ‘Ko1-303’ through embryo rescue. One BC₂ and three BC₃ plants were obtained from successive backcrossing with B. rapa ‘Ko1-303’ employing embryo rescue. Alloplasmic and monosomic addition lines of B. rapa (Allo-MALs, 2n = 21) were obtained from backcrossed progeny of three BC₃ plants (2n = 21, 22 and 23) without embryo rescue. An alloplasmic line of B. rapa (2n = 20) degenerated before floliation on 1/2 MS medium due to severe chlorosis. Allo-MALs of B. rapa (2n = 21) showed stable male sterility without any abnormal traits in vegetative growth and female fertility. Molecular analyses revealed that the same chromosome and cytoplasm of M. arvensis had been added to each Allo-MAL of B. rapa. This Allo-MAL of B. rapa may be useful material for producing cytoplasmic male sterile lines of B. rapa.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Genetic Variation for Heat Tolerance During the Reproductive Phase in Brassica rapa

We investigated heat tolerance at the reproductive stage in six spring‐type B. rapa accessions and one B. juncea accession as a control. Plants were subjected to two temperature treatments for seven days in controlled environmental rooms, beginning one day before the first open flower on the main stem inflorescence. The high‐temperature treatment ranged from 25 °C to 35 °C during 16 h light and 25 °C during 8 h dark. The control temperature treatment was set at 23 °C during 16 h light and 15 °C during 8 h dark. Soil moisture was maintained at close to field capacity to avoid drought stress. Main stem buds that emerged during the treatment period were tagged, and pod and seed production was recorded at each reproductive node. Leaf temperature depression and leaf conductance increased in the high‐temperature treatment which indicated that plants were not drought stressed. A leafy vegetable type of B. rapa from Indonesia was the most tolerant to high temperature, as defined by its ability to set seed equally well in the control and high‐temperature treatments, followed by an oilseed type from Pakistan. Pollen viability remained above 87 % in all accessions and treatments. We conclude that bud number and length, and pod number produced under high temperatures, might provide a useful preliminary screen for high‐temperature tolerance and that B. rapa may be a valuable source of heat tolerance in canola (B. napus).     

Tetralocular mustard, Brassica juncea: New promising variability through interspecific hybridization

Lines of Brassica juncea with tetralocular (four-valved) pods were produced by interspecific hybridization between dilocular (two-valved) B. juncea var. Pusa Barani and tetralocular yellow-seeded turnip rape (Brassica rapa ssp. trilocularis) var. YID-1. Some selected lines were significantly superior to their parents for some agronomic traits, including seed yield and harvest index. Line 11 was the best among these and recorded 70% yield advantage in plot yield.     

Separation and identification of Brassica rapa BrAFP and its gene cloning and expression under freezing stress

Antifreeze proteins (AFPs) are proteins that inhibit ice nucleation by non‐colligative combined with ice, to inhibit its growth and to modify ice crystal morphology. In this study, 'Longyou 6' (Brassica rapa) with the cold resistance was used as a model. A 38 kDa band of protein extracted from leaves of plants exposed to low temperature was enhanced on the electropherogram. The 38 kDa protein can significantly inhibit growth of ice crystals and modify its morphology, thus suggesting this protein has an AFP in winter turnip rape. The protein was identified as beta‐1,3‐glucanase by mass spectrometry, named BrAFP1. Its corresponding gene contains 1,032 bp bases, coding 343 amino acids with a molecular weight of 38,102 kDa. The BrAFP1 is a predicted hydrophobic protein with a signal peptide. Under cold stress, the plants showed morphological characteristics of cold resistance, and BrAFP expression was significantly up‐regulated at ?4°C from 0.5 to 4 days compared with the control (at room temperature), suggesting that the BrAFP gene played an important role in cold acclimation in B. rapa.     

Development of molecular markers linked to the Leptosphaeria maculans resistance gene Rlm6 and inheritance of SCAR and CAPS markers in Brassica napus × Brassica juncea interspecific hybrids

Leptosphaeria maculans causes blackleg disease on Brassica napus, an economically important oilseed crop. Brassica juncea has high resistance to blackleg and is a source for the development of resistant B. napus varieties. To transfer the Rlm6 resistance gene from B. juncea into B. napus, an interspecific cross between B. napus “Topas DH16516” and B. juncea “Forge” was produced, followed by the development of F₂ and F₃ generations. Sequence characterized amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers linked to the L. maculans resistance gene Rlm6 were developed. Segregation of SCAR and CAPS markers linked to Rlm6 were confirmed by genotyping of F₂ and F₃ progeny. Segregation of CAPS markers and phenotypes for blackleg disease severity in F₂ plants had a Mendelian ratio of 3:1 in resistant vs. susceptible plants, respectively, supporting the assumption that genetic control of resistance was by a single dominant gene. The molecular markers developed in this study, which show linkage with the L. maculans resistance gene Rlm6, would facilitate marker‐assisted backcross breeding in a variety development programme.     

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.     

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.