Diplotaxis catholica + Brassica juncea somatic hybrids: molecular and cytogenetic characterization

Summary Intergeneric somatic hybrids Diplotaxis catholica (2n=18) + Brassica juncea (2n=36) were produced by fusing mesophyll protoplasts of the former and hypocotyl protoplasts of the latter using polyethylene glycol. Out of 52 somatic embryos, 24 produced plants of intermediate morphology. Cytological analysis of 16 plants indicated that 15 were symmetric hybrids carrying 54 chromosomes, the sum of the parental chromosome numbers. One hybrid was asymmetric with 45 chromosomes. Nuclear hybridity of five putative hybrids was confirmed by the Southern hybridization pattern of full length 18s-25s wheat nuclear rDNA probe which revealed the presence of Hind III fragments characteristic of both the parental species. The hybridization pattern of mitochondria specific gene probe cox I indicated that three of the hybrids carried B. juncea mitochondria and one carried mitochondria of D. catholica. Presence of novel 3.5 kb Hind III and 4.8 kb Bgl II fragments suggested the occurrence of mtDNA recombination in one of the hybrids. The hybrids were pollen sterile. However, seeds were obtained from most of the hybrids by back crossing with B. juncea.     

Production of wide hybrids and backcross progenies between Diplotaxis erucoides and crop brassicas

Intergeneric hybrids were produced between D. erucoides (), a wild species, and four cultivated species of Brassica, B. campestris, B. juncea, B. napus and B. oleracea, through embryo rescue. The hybrid nature of these plants was confirmed through morphological and cytological studies. Backcross pollinations with the pollen of the respective cultivars yielded BC progenies in the hybrids D. erucoides x B. juncea and D. erucoides x B. napus but not in D. erucoides x B. campestris and D. erucoides x B. oleracea. The hybrid D. erucoides x B. campestris was also used as a bridge species and crossed with B. juncea to raise the hybrid and backcross progenies. F₂ progenies were more amenable than f₁ hybrids for raising backcross progenies. Although D. erucoides is considered to be a close relative of B. campestris and B. oleracea, incompatibility barriers of this species with different cultivars do not reflect this relationship.     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

Microspore embryogenesis in anther cultures of two Indian cultivars of Brassica juncea (L.) Czern.

Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained. Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis. Embryogenesis was also stimulated by an initial culture at 5°C for 3 days. Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos. All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets. The androgenic plants were haploid (2n=18).     

Somatic hybrids between Brassica oleracea and B. campestris: selection by the use of iodoacetamide inactivation and regeneration ability

Summary An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed. Hypocotyl protoplasts of B. oleracea were fused with mesophyll protoplasts from three different varieties of B. campestris by the polyethylene glycoldimethylsulfoxide method. The selection of somatic hybrids utilized the inactivation of B. oleracea protoplasts by iodoacetamide (IOA) and the low regeneration ability of B. campestris. The efficiency of recovery of somatic hybrids depended upon the IOA concentration, and when 15 mM IOA was used, 90% of the regenerated plants were found to be hybrid. The somatic hybrids were examined for i) leaf morphology, ii) leucine aminopeptidase (LAP) isozyme and iii) chromosome number. All the hybrids had intermediate leaf morphology and possessed LAP isozymes of both parental species. The chromosome analysis revealed a considerable variation in chromosome number of somatic hybrids, showing the occurrence of multiple fusion and chromosome loss during the culture. Some of the hybrids flowered and set seeds.     

Influence of paclobutrazol on growth and development of fruit in Brassica juncea (L.) Czern and Coss

Foliar spraying of paclobutrazol(5, 10 and 20 g mL^–1) on Brassica juncea (cv. RLM 514) plants, at the green floral bud stage of the terminal inflorescence, exerted a profound effect on growth and metabolism of fruits (siliquae). Paclobutrazol caused a decrease in siliqua length but enhanced significantly siliqua width, seed size (diameter) and seed number per siliqua. Paclobutrazol increased dry matter production and also hastened the relative growth rate of the siliqua wall and seed. In paclobutrazol-treated plants the siliquae were darker green, and during active phases of growth both siliqua wall and seed exhibited higher total chlorophyll (Chl) content, decreased chl a/b ratios, greater hill reaction activity of chloroplasts and increased activities of -amylase, -amylase and invertase than controls. Paclobutrazol enhanced the levels of total soluble sugars, starch, protein and free amino acids in mature seeds. The seed oil content increased by 2 to 3 per cent with a treatment of 10 g mL^–1 pacolobutrazol.     

Linked epistasis for six quantitative traits in Indian mustard (Brassica juncea (L.) Czern & Coss)

Summary Gene effects, and interactions, and associations between days-to-flower initiation and maturity, number of secondary branches and siliquae per plant, and 1,000-seed weight and yield per plant were studied in a cross of Indian mustard (Brassica juncea (L.) Czern & Coss) using the parents and F₁, F₂, F₃, B₁, B₂, B₁₁, B₁₂, B₂₁, B₂₂, B_1S, B_2S, B₁F₁, B₂F₁, B_1bip, B_2bip, F₂P₁, F₂F₁, and F_2bip generations. A linked digenic model was adequate for all characters studied. According to this model, the main effects, additive and interactions between linked pairs of genes, were present in varying proportions for days-to-flower initiation and maturity and number of siliquae per plant. The contribution of linked epistatic effects, however, was much greater than that of additive effects. Dominance effects contributed significantly to the inheritance of days-to-flower initiation. Duplicate epistasis was observed for all traits except 1,000-seed weight where epistasis was of the complementary type. A complete association among the genes of similar effect (increasing or decreasing) was observed for number of secondary branches and siliquae, and yield per plant. Coupling phase linkage was observed for days-to-flower initiation whereas repulsion phase linkage was observed for daysto-maturity and 1,000-seed weight.     

Transformation of Brassica juncea (L.) Czern with bacterial codA gene enhances its tolerance to salt stress

The codA gene for biosynthesis of glycinebetaine from Arthrobacter globiformis was used for transforming Brassica juncea cv. Pusa Jaikisan (which lack any means to synthesize glycinebetaine) through Agrobacterium mediated transformation. The stable insertion of the codA gene in the shoots obtained on medium with kanamycin and hygromycin was confirmed by PCR analysis of the nptII gene. Southern hybridization with a codA probe further demonstrated its successful integration. Immunoblot analysis revealed the presence of choline oxidase demonstrating that the bacterial codA gene had been successfully transcribed and translated. The seeds of transgenic lines showed enhanced capacity to germinate under salt stress as compared to that of the wild type. Further, the seedlings of transgenic plants that expressed codA gene showed significantly higher growth than that of the wild type under salt stress conditions. These results demonstrated that the introduction of a biosynthetic pathway for glycinebetaine into Brassica juncea significantly enhanced their salt tolerance. Hence, homozygous genotypes of selected transformed lines can be exploited for improving the salt tolerance of the desirable cultivars of Brassica juncea through breeding programmes.     

The role of cotyledonary tissue in the differentiation of shoots and roots from cotyledon explants of Brassica juncea (L.) Czern.

In cotyledon cultures of Brassica juncea, shoots and roots invariable differentiate at the cut end of the petiole. Organogenesis occurred only if the proximal cut end of the petiole was in contact with the medium. In the absence of the petiole, differentiation from the lamina was rare. Hence investigations were carried out to study the influence of the cotyledonary lamina on regeneration of shoots and roots from the petiolar cut end. The lamina tissue was surgically removed from the cotyledon explants at different durations (0–10 days) after culturing them on either root-forming (basal medium) or shoot-forming (basal medium containing 5.0 M N⁶-benzyladenine) media. The differentiation of roots or shoots from the petioles was dependent on the presence of the lamina for at least 7 days of culture. Quantitative removal of the laminar tissue had a corresponding negative effect on shoot bud differentiation from the petiole. The nature of the lamina factor was found to be auxin-like.     

Production and characterization of intergeneric somatic hybrids of Trachystoma ballii and Brassica juncea

Intergeneric somatic hybrids, Trachystoma ballii (2n=16)+B. juncea (2n=38), were obtained by fusing mesophyll protoplasts of T. ballii and hypocotyl protoplasts of B. juncea using polyethylene glycol. The heterokaryotic fusion frequency was around 23%. Plants were regenerated from 10 out of 2×10⁴ calli of which four were hybrids. Hybrids were intermediate between the parents in general morphology. However, in some characters one of the parents dominated. All the plants were symmetric in their chromosomal constitution as revealed by the formation of 26 bivalents at metaphase-I of meiosis. Two trivalents and 2 univalents were also observed in some pollen mother cells. Hybrid nature was also confirmed by Southern hybridization of DNA of one regenerated plant restricted with Hind III and probed with the nick translated plasmid pTA71 carrying a wheat nuclear r-DNA sequence. Hybrid plant RT 1 showed bands characteristic of both parents. All the plants were absolutely pollen sterile. However, on backcrosses to B. juncea seeds were obtained.     

Intergeneric somatic hybrid production through protoplast fusion between Brassica juncea and Diplotaxis muralis

Summary The need to transfer genetic traits from Diplotaxis muralis (2n=42) to Brassica juncea (2n=36), a major oil seed crop of the tropical world, was realised. Since the two plant types are sexually incompatible, attempts were made to evolve parasexual hybrids as the result of protoplast fusion. Protoplasts of hypocotyl-derived calli of two cultivars of B. juncea were fused with normal and -irradiated mesophyll protoplasts of Diplotaxis muralis. Regeneration of 110 plants from the fused products was successfully achieved. Upon analysis of some of them, we realised that true somatic hybrids and partial somatic hybrids had been generated. Thus the primary goal of evolving intergeneric hybridisation products between these two plant types was fulfilled.     

Random chloroplast segregation and mitochondrial genome recombination in somatic hybrid plants of Diplotaxis catholica+Brassica juncea

Detailed molecular analysis of the somatic hybrid plants of Diplotaxis catholica+B. juncea indicated random chloroplast segregation. One of the five hybrid plants analyzed derived its chloroplasts from D. catholica and two hybrids had chloroplasts of B. juncea origin. Two hybrid plants maintained mixed population of chloroplasts. The mitochondrial (mt) genomes of the fusion partners had undergone recombinations. Occurrence of fragments specific to both the parents in HindIII digestion followed by atp 9 probing, as in hybrid DJ5, provided evidence for intergenomic mitochondrial recombination between D. catholica and B. juncea. Similar mt genome organization in two hybrids (DJ3 and DJ6) suggested that intergenomic recombination may be preferred at specific sites. Hybrid DJ1 had about 70% similarity to D. catholica in mt genome organization. mt genomes of hybrids DJ2, 3, 5, and 6 differed from B. juncea by 14.3–28%. The significance of these novel mt genome organizations in developing novel male sterility systems is discussed. Received: 4 April 1997 / Revision received: 19 December 1997 / Accepted: 28 March 1998     

Somatic hybrids between unilateral cross-incompatible Petunia species

Summary Somatic hybrid plants regenerated following the fusion of leaf mesophyll protoplasts of Petunia parodii with those isolated from a cell suspension of albino P. inflata. These two species exhibit a unilateral cross-incompatability with a pre-zygotic mode of reproductive isolation preventing hybridizations with P. inflata as the maternal parent. Selection of somatic hybrids relied on the fact that unfused or homokaryon protoplasts of P. parodii did not develop beyond the cell colony stage while those of the putative somatic hybrids and albino P. inflata parent produced callus. Green somatic hybrid calluses were readily identified against the white background of P. inflata following complementation to chlorophyll synthesis proficiency and continued growth in hybrid cells. Shoots, and ultimately flowering plants, were identified as somatic hybrids based on their floral morphology and colour, chromosome number and the fact that they segregated for parental characters. The frequency of somatic hybrid production was comparable to that previously established for two sexually compatible Petunia species.     

Isozyme studies in Indian mustard (Brassica juncea L.)

Summary A comparative study of peroxidase and esterase isozymes was carried out at five developmental stages of siliqua in order to characterize twelve genotypes of Indian mustard. The studies showed nearly the same number of isozyme bands at every stage for peroxidase and a varying number of isozyme bands for esterase. The appearance and disappearance of bands, along with their intensity scores, indicated the role of different isozymes at different stages of siliqua development. It has been ascertained that these patterns, especially the intensity scores, can be successfully used to characterize different Indian mustard genotypes.     

Alternaria incidence in some alloplasmic lines of Indian mustard (Brassica juncea (L.) Coss.)

Summary The cytoplasmic substitution lines of Brassica juncea (L.) Coss were evaluated for their field resistance to Alternaria blight (Alternaria brassicae). The euplasmic B. juncea cv. RLM 198 had a mesothetic reaction while alloplasmic B. juncea lines with cytoplasms of B. campestris, B. chinensis, and B. japonica were highly susceptible. B. nigra cytoplasm did not have any effect on the disease reaction of the B. juncea genome. However, the alloplasmic lines with the cytoplasm of B. napus and B. carinata revealed a comparatively higher degree of resistance. The study underlined the utility of cytoplasmic manipulations in modifying the phenotypic expression of nuclear genes.     

Somatic hybridization between birdsfoot trefoil (Lotus corniculatus L.) and L. conimbricensis Willd

Summary Somatic hybrid plants were produced by fusion of birdsfoot trefoil (Lotus corniculatus) cv Leo and L. conimbricensis Willd. protoplasts. Birdsfoot trefoil etiolated hypocotyl protoplasts were inactivated with iodoacetate to inhibit cell division prior to fusion with L. conimbricensis suspension culture protoplasts. L. conimbricensis protoplasts divided to form callus which did not regenerate plants. Thus, plant regeneration from protoplast-derived callus was used to tentatively identify somatic hybrid cell lines. Plants regenerated from three cell lines exhibited additive combinations of parental isozymes of phosphoglucomutase, and L. conimbricensis-specific esterases indicating that they were somatic hybrids. The somatic chromosome number of one somatic hybrid was 36. The other somatic hybrid exhibited variable chromosome numbers ranging from 33 to 40. These observations approximate the expected combination of the birdsfoot trefoil (2n=4x=24) and L. conimbricensis (2n=2x=12) genomes. Somatic hybrid flowers were less yellow than birdsfoot trefoil flowers and had purple keel tips, a trait inherited from the white flowered L. conimbricensis. Somatic hybrids also had inflorescence structure that was intermediate to the parents. Fifteen somatic hybrid plants regenerated from the three callus lines were male sterile. Successul fertilization in backcrosses with birdsfoot trefoil pollen has not yet been obtained suggesting that the hybrids are also female sterile. This is the first example of somatic hybridization between these two sexually incompatible Lotus species.Formerly USDA-ARS, St. Paul, Minn, USA     

Effect of nitrogen and chlormequat chloride on the seed yield and oil content of mustard (Brassica juncea L. Czern & Coss)

The growth of mustard was increased significantly when treated with up to 80 kg N ha^–1 (N₈₀). Spraying with (2-chloroethyl) trimethylammoniumchloride (chlormequat chloride) increased seed yield and seed protein content. Spraying nitrogen fertilized plots with chlormequat chloride, increased leaf area, leaf area ratio, leaf area duration, number of siliquae plant^–1, number of seeds siliqua^–1 and length of siliqua. Reducing, non-reducing and total sugars in the leaves at 80 days after sowing were also affected significantly. Chlorophyll a, b and total chlorophyll were little affected. The number of siliquae plant^–1 was highly correlated with seed yield in both the seasons of experimentation. The correlation coefficient value () was 0.586 in 1982/83 and 0.912 in 1983/84.The total accumulation of nutrients, i.e. nitrogen, phosphorus and potassium in seed and straw was significantly affected by N₈₀ × chlormequat chloride interaction.     

Generation mean analyses for flowering and maturity in Indian mustard (Brassica junces (L.) Czern & Coss)

Summary Components of generation means were partitioned for days to flower initiation and maturity in three crosses of Indian mustard (Brassica juncea (L.) Czern and Coss) cultivars. A linked digenics model was adequate for flowering in cross II and maturity in Cross I. All three types of digenic interactions among the linked pairs of genes, additive X additive (i), additive X dominance (j) and dominance X dominance (1), contributed significantly in the inheritance of flowering in cross II and maturity in cross I. A complete association among the genes of greater effects in higher mean parent was detected for flowering in cross II and maturity in Cross I. Duplicate epistasis was evident for flowering in Crosses I and II and maturity in Crosses I and III.Inadequacy of all the fitted models for days to flowering in Cross III and maturity in Cross II indicated the presence of higher order interactions.Part of PhD (plant breeding) Thesis, submitted by senior author to GBPUAT, Pantnagar (Nainital) U.P., India (unpublished). Research paper No. 4262     

Interspecific somatic hybridization between Brassica napus and Brassica hirta (Sinapis alba L.)

Summary Somatic hybridization between Brassica napus and B. hirta (or Sinapis alba) is described. No cybrid plant with B. napus nucleus exhibiting cytoplasmic male sterility was recovered. Somatic hybrids were identified morphologically and, for some of them, by cytological observations. They were also characterised by Southern hybridization of nuclear rDNA. Chloroplast and mitochondrial DNA restriction analysis showed that 2 plants out of 14 have B. hirta ctDNA, one the B. napus mtDNA and the other a hybrid. Nine possess B. napus ctDNA with a hybrid mtDNA. For six of them, mtDNA patterns present novel bands, suggesting intergenomic recombination during fusion. These hybrids will be included in the breeding program.