Production and genetic characterization of somatic hybrids between leaf mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) and broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

Inter-specific somatic hybrids of leaf mustard (Brassica juncea) and broccoli (Brassica oleracea) were established to introduce cytoplasmic male sterility (CMS) and Verticillium dahliae Kleb. resistance from broccoli to leaf mustard. Protoplasts isolated from hypocotyls and cotyledons of inbred lines of leaf mustard and broccoli were fused using 40% (w/v) polyethelene glycol and then cultured in modified k8p medium supplemented with 0.2 mg/l 2,4-dichlorophenoxi-acetic acid, 0.5 mg/l 6-benzyladenine (BA), 0.1 mg/l naphthaleneacetic acid (NAA), and 0.1 mg/l kinetin (Kin), and 0.4 M mannitol as osmoticum. At the eight- to ten-cell stage, divided cells were transferred to Kao’s basal medium supplemented with 0.3 M sucrose as carbon source and 0.1% agarose, 2 mg/l BA, 2 mg/l zeatin (ZEA), 1 mg/l NAA, and 0.5 mg/l Kin for callus proliferation. After 35 d, when small calli reached 2–3 mm in diameter, calli were transferred to regeneration medium containing 5 mg/l ZEA and 2 mg/l indole-3-acetic acid. The chromosome numbers of putative somatic hybrids varied from 46 to 54. A total of ten plants showed a 0.5-kb, CMS-specific band, while two regenerated plants showed a missing band similar to that of leaf mustard by polymerase chain reaction amplification using Ogura CMS-specific primers. Hybrid state was confirmed by random amplified polymorphic DNA analysis. Regenerated plants had normal petals and stamens, but only two plants produced pollen and set seed.     

Production and characterization of intergeneric somatic hybrids between <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> and their backcrossing progenies

Alien chromosome addition lines have been widely used for identifying gene linkage groups, assigning species-specific characters to a particular chromosome and comparing gene synteny between related species. In plant breeding, their utilization lies in introgressing characters of agronomic value. The present investigation reports the production of intergeneric somatic hybrids Brassica napus (2n = 38) + Orychophragmus violaceus (2n = 24) through asymmetric fusions of mesophyll protoplasts and subsequent development of B. napus-O. violaceous chromosome addition lines. Somatic hybrids showed variations in morphology and fertility and were mixoploids (2n = 51–67) with a range of 19–28 O. violaceus chromosomes identified by genomic in situ hybridization (GISH). After pollinated with B. napus parent and following embryo rescue, 20 BC₁ plants were obtained from one hybrid. These exhibited typical serrated leaves of O. violaceus or B. napus-type leaves. All BC₁ plants were partially male fertile but female sterile because of abnormal ovules. These were mixoploids (2n = 41–54) with 9–16 chromosomes from O. violaceus. BC₂ plants showed segregations for female fertility, leaf shape and still some chromosome variation (2n = 39–43) with 2–5 O. violaceus chromosomes, but mainly containing the whole complement from B. napus. Among the selfed progenies of BC₂ plants, monosomic addition lines (2n = 39, AACC + 1O) with or without the serrated leaves of O. violaceus or female sterility were established. The complete set of additions is expected from this investigation. In addition, O. violaceus plants at diploid and tetraploid levels with some variations in morphology and chromosome numbers were regenerated from the pretreated protoplasts by iodoacetate and UV-irradiation. Z. Zhao and T. Hu make equal contributions to this work.     

Production and characterization of somatic hybrids between upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) and wild cotton (<Emphasis Type="Italic">G. klotzschianum</Emphasis> Anderss) via electrofusion

Symmetric somatic hybrid plants between Gossypium hirsutum Coker 201 and G. klotzschianum were obtained through electrofusion. The fusion products were cultured in KM₈P medium supplemented with 2.685 M -naphthaleneacetic acid and 0.465 M kinetin, and the regenerated plants were morphologically, genetically, and cytologically characterized. Nuclear-DNA flow cytometric analysis revealed that the plants tested (31 of 40) had a relative DNA content close to the total DNA contents of the two parents. Subsequent genome DNA analysis using random amplified polymorphic DNA markers revealed 16 of 18 plants were true somatic hybrids. Cytological investigation of the metaphase root-tip cells of seven hybrids revealed there were 72–81 chromosomes in the hybrids, a value close to the expected 78 chromosomes. The morphology of the hybrids was distinct from that of the parents and from that of the regenerants from protoplasts of Coker 201. Somatic hybridization represents a potent and novel tool for transferring genomes of wild cottons containing economically important traits to cultivars in breeding programs. This is the first report on the regeneration of somatic hybrids via protoplast fusion in cotton.     

Fine-scale genetic mapping of a hybrid sterility factor between <Emphasis Type="Italic">Drosophila simulans</Emphasis> and <Emphasis Type="Italic">D. mauritiana</Emphasis>: the varied and elusive functions of "speciation genes"

Background  

Hybrid male sterility (HMS) is a usual outcome of hybridization between closely related animal species. It arises because interactions between alleles that are functional within one species may be disrupted in hybrids. The identification of genes leading to hybrid sterility is of great interest for understanding the evolutionary process of speciation. In the current work we used marked P-element insertions as dominant markers to efficiently locate one genetic factor causing a severe reduction in fertility in hybrid males of Drosophila simulans and D. mauritiana.     

Ectopic expression of <Emphasis Type="Italic">MNX</Emphasis> gene from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> involved in auxin biosynthesis confers male sterility in transgenic cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) plants

A transgenic male sterile line of upland cotton was generated by the ectopic expression of the monooxygenase (MNX) gene from Arabidopsis thaliana via Agrobacterium-mediated transformation. The bacterium harbored a plasmid pBinplus carrying a 1.25-kb MNX coding sequence together with a GUS reporter gene; the former was driven by the MS2 promoter of a male sterility gene in Arabidopsis, and the latter was under the control of CaMV 35S promoter. Twenty-seven putative transgenic plants (T₀) were obtained, all of which showed GUS activity and positive signals of NPTII and MNX genes by PCR analysis, and also showed male sterility to some extent. It was further confirmed by Southern blotting that one copy of the NPTII and MNX gene was integrated in the genome of the plants which expressed male sterility to a higher degree. Northern blotting assay also demonstrated that the transgenes stably transcribed in the genome of the transgenic plants in F₄ generation. The male sterile plants usually display lower plant height, shortened internodes, shrunken anthers without pollen grains or with some abortive pollen grains, and unusual leaves with deeper multi-lobes. Microscope observations on the meiosis processes of pollen mother cells (PMCs) showed that the abortion of pollen grains mainly resulted from abnormalities of meiosis such as direct degeneration of PMCs, degenerations of dyad and tetrads, amitosis, lagging chromosomes, and the multi-polar segregations of chromosomes and so on. This study indicates a method of developing novel cotton male sterile materials for potential application in agriculture and for engineering of male sterility in other important crops.     

In-vitro development of ovules obtained after pollination of cotton (<Emphasis Type="Italic">Gossypium</Emphasis> spp) flowers with pollen from okra (<Emphasis Type="Italic">Abelmoschus esculentus L. Moench)</Emphasis>

The in vitro response of ovules obtained after pollination of cotton flowers with pollen from Abelmoschus esculentus was studied. For this, 492 cotton flowers from five G. hirsutum varieties, four G. barbadense varieties and 10 F1 interspecific hybrids, were pollinated with pollen from A. esculentus and 5,069 ovules were cultured in vitro. From the cultured ovules, 69 embryos were isolated and 16 of them grew into plants. However, only three of them survived after transplantation. Finally, one plant which originated from the interspecific cross (B403 × Acala Sindos) × A. esculentus reached maturity. The mature plant (Pa0) had no morphological traits from A. esculentus. On the contrary, traits from both cotton species were observed. The flowcytometric analysis of the Pa0 plant indicated that it was hypoaneuploid. Root tip chromosome counts of its offsprings revealed a progressive chromosome increase from the Pa1 to Pa4 generation. Plants with 52 chromosomes or hypoaneuploids with a lower level of chromosomes (46–51) could be isolated from the Pa4 generation. These plants exhibited morphological traits from both cotton species and they were fertile. No signs of A. esculentus morphological characteristics were observed in these plants. It was concluded that aneuploid partial interspecific cotton plants could be produced after pollination of cotton interspecific hybrids with pollen from A. esculentus and application of an in-ovule embryo rescue technique.     

Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey     

Production of interspecific somatic hybrids between tuber mustard (<Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">juncea</Emphasis>) and red cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

In the present investigation, the interspecific somatic hybridization between tuber mustard and red cabbage was established in order to introduce valuable genes from red cabbage (Brassica oleracea) into Brassica juncea. Prior to fusion treatment, protoplasts of red cabbage were inactivated with 2 mM iodoacetamide to inhibit cell division. Micro-calluses were obtained at a frequency of 10.3% after approximately 5 weeks culture following protoplast fusion. Some of the fusion-derived calluses possessed red pigmented cells after being transferred to proliferation medium, and they were presumably considered to be somatic hybrid cell lines. Plantlets were regenerated from 12 cell lines, of which nine plantlets exhibited characteristics intermediate of both parents in terms of plant morphology. With the exception of common protein bands featured by two parents, there were unique banding patterns produced in the hybrids by using SDS-PAGE analysis. By chromosome countings, it was showed that they ranged approximately from 2n=30 to 42 in chromosome numbers. Their hybridity were further confirmed by RAPD analysis revealing that genes of both parents were partially incorporated into the hybrids. Positively, all these hybrids were capable of seed-setting. The pod-setting was 4.2 in somatic hybrid H₇ when backcrossed with tuber mustard.     

Different fertility and meiotic regularity in allohexaploids derived from trigenomic hybrids between three cultivated <Emphasis Type="Italic">Brassica</Emphasis> allotetraploids and <Emphasis Type="Italic">B</Emphasis>. <Emphasis Type="Italic">maurorum</Emphasis>

The wild species Brassica maurorum Durieu (MM, 2n = 16) is useful for the improvement of Brassica crops. Herein, interspecific reciprocal crosses between B. maurorum and three cultivated Brassica allotetraploids were carried out with the aid of embryo rescue. Trigenomic hybrids with Brassica napus (AACC, 2n = 38) and Brassica carinata (BBCC, 2n = 34) were produced from reciprocal crosses, but the hybrids with Brassica juncea (AABB, 2n = 36) were obtained only when B. maurorum was used as female. All the hybrids were morphologically intermediate between their parents, and were male and female sterile. By in vitro chromosome doubling of the trigenomic hybrids, the allohexaploids (AACC.MM/MM.AACC, 2n = 54; BBCC.MM, 2n = 50; MM.AABB, 2n = 52) were established and characterized for their phenotype and cytology. The fertilities of three allohexaploids were different, for AACC.MM and MM.AACC failed to produce seeds by selfing, but BBCC.MM showed low seed-set and MM.AABB had good seed-set. They also expressed variable extents of male meiotic regularity as to chromosome pairing and segregation, with MM.AABB > BBCC.MM > AACC.MM/MM.AACC, the same order as their fertility. So their meiotic behavior contributed to the fertility. Finally, the potential of these allohexaploids as a bridge for genetic improvement of Brassica crops was discussed.     

Efficient production of genetically engineered,male-sterile <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> using anther-specific promoters and genes derived from <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">B. rapa</Emphasis>

Prevention of transgene flow from genetically modified crops to food crops and wild relatives is of concern in agricultural biotechnology. We used genes derived from food crops to produce complete male sterility as a strategy for gene confinement as well as to reduce the food purity concerns of consumers. Anther-specific promoters (A3, A6, A9, MS2, and MS5) were isolated from Brassica oleracea and B. rapa and fused to the β-glucuronidase (GUS) reporter gene and candidate genes for male sterility, including the cysteine proteases BoCysP1 and BoCP3, and negative regulatory components of phytohormonal responses involved in male development. These constructs were then introduced into Arabidopsis thaliana. GUS analyses revealed that A3, A6, and A9 had tapetum-specific promoter activity from the anther meiocyte stage. Male sterility was confirmed in tested constructs with protease or gibberellin insensitive (gai) genes. In particular, constructs with BoCysP1 driven by the A3 or A9 promoter most efficiently produced plants with complete male sterility. The tapetum and middle layer cells of anthers expressing BoCysP1 were swollen and excessively vacuolated when observed in transverse section. This suggests that the ectopic expression of cysteine protease in the meiocyte stage may inhibit programmed cell death. The gai gene also induced male sterility, although at a low frequency. This is the first report to show that plant cysteine proteases and gai from food crops are available as a novel tool for the development of genetically engineered male-sterile plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Marker-assisted breeding of a photoperiod-sensitive male sterile <Emphasis Type="Italic">japonica</Emphasis> rice with high cross-compatibility with <Emphasis Type="Italic">indica</Emphasis> rice

The incomplete fertility of japonica × indica rice hybrids has inhibited breeders’ access to the substantial heterotic potential of these hybrids. As hybrid sterility is caused by an allelic interaction at a small number of loci, it is possible to overcome it by simple introgression at the major sterility loci. Here we report the use of marker-assisted backcrossing to transfer into the elite japonica cv. Zhendao88 a photoperiod-sensitive male sterility gene from cv. Lunhui422S (indica) and the yellow leaf gene from line Yellow249 (indica). The microsatellite markers RM276, RM455, RM141 and RM185 were used to tag the fertility genes S5, S8, S7 and S9, respectively. Line 509S is a true-breeding photoperiod-sensitive male sterile plant, which morphologically closely resembles the japonica type. Genotypic analysis showed that the genome of line 509S comprises about 92% japonica DNA. Nevertheless, hybrids between line 509S and japonica varieties suffer from a level of hybrid sterility, although the line is highly cross-compatible with indica types, with the resulting hybrids expressing a significant degree of heterosis. Together, these results suggest that segment substitution on fertility loci based on known information and marker-assisted selection are an effective approach for utilizing the heterosis of rice inter-subspecies.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Biolistic transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) with the <Emphasis Type="Italic">phyA</Emphasis> gene from <Emphasis Type="Italic">Aspergillus ficuum</Emphasis>

Transgenic cotton with an increased level of phytase activity was generated from cotton (Gossypium hirsutum L.) cv. ND94-7 by subjecting shoot-apex explants to particle bombardment. These tissues were transformed with plasmid pC-KSA2300 carrying a selectable marker (for kanamycin) and a target gene (phytase, or phyA, from Aspergillus ficuum). Primary plants were regenerated in a medium containing 75 mg l⁻¹ kanamycin. Of 1,534 shoot apices, 52 (3.4%) survived on this selection medium. Southern and Northern blot analyses confirmed that phyA was stably integrated and expressed in those primary transgenics. The progenies of the primary transgenic plants were found to have a 3.1- to 3.2-fold increase in root extracellular phytase activity, resulting in improved phosphorus (P) nutrition. Growth also was enhanced when they were supplied with phytate, and their P content was equivalent to that of wildtype plants supplied with inorganic phosphate. These results demonstrate that the expression of phyA in cotton plants improves their ability to utilize organic P in response to a deficiency.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated co-transformation of multiple genes in upland cotton

Two cotton genotypes, Simian 3 (SM 3) and WC, were co-transformed using a mixture of four Agrobacterium tumefaciens cultures of strain LBA4404, each carrying a plasmid harboring the following genes, Bt + sck (for Bacillus thuringenesis protein and modified Cowpea trypsin inhibitor), bar (for glufosinate), keratin, and fibroin. The frequency of callus induction, embryogenesis, and plant regeneration were notably different between the two genotypes. However, there were no differences between the two genotypes for number of plantlets carrying multiple gene copies of different gene combinations as well as transformation frequency for different gene combinations. PCR analysis indicated that more than 80% of plantlets carried the nptII gene for kanamycin resistance. Overall, the co-transformation frequency of two or more genes was about 35%. Southern blot analysis confirmed integration of target genes into the cotton genome, and the number of copies of the transgene(s) varied from one to four. Multiple transgene expression was confirmed by RT-PCR analysis in some transgenic lines. Further analysis of T₁ plants demonstrated that multiple transgenes were inherited and expressed in progenies. Fei-Fei Li and Shen-Jie Wu are joint first authors.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis> Wright,an important pharmaceutical crop

Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated from initial infected callus explants.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> -mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) using a heterologous bean chitinase gene

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l⁻¹ kanamycin and 200 mg l⁻¹ cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and βglucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T₁ plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. ‘Western’ immunoblot analysis of leaves isolated from T₀ transformants and progeny plants (T₁) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae.Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.     

Fine mapping of a male sterility gene <Emphasis Type="Italic">MS-cd1</Emphasis> in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

A dominant male sterility (DGMS) line 79-399-3, developed from a spontaneous mutation in Brassica oleracea var. capitata, has been widely used in production of hybrid cultivars in China. In this line, male sterility is controlled by a dominant gene Ms-cd1. In the present study, fine mapping of Ms-cd1 was conducted by screening a segregating population Ms79-07 with 2,028 individuals developed by four times backcrossing using a male sterile Brassica oleracea var. italica line harboring Ms-cd1 as donor and Brassica oleracea var. alboglabra as the recipient. Bulked segregation analysis (BSA) was performed for the BC₄ population Ms79-07 using 26,417 SRAP primer SRAPs and 1,300 SSRs regarding of male sterility and fertility. A high-resolution map surrounding Ms-cd1 was constructed with 14 SRAPs and one SSR. The SSR marker 8C0909 was closely linked to the MS-cd1 gene with a distance of 2.06 cM. Fourteen SRAPs closely linked to the target gene were identified; the closest ones on each side were 0.18 cM and 2.16 cM from Ms-cd1. Three of these SRAPs were successfully converted to dominant SCAR markers with a distance to the Ms-cd1 gene of 0.18, 0.39 and 4.23 cM, respectively. BLAST analysis with these SCAR marker sequences identified a collinear genomic region about 600 kb in scaffold 000010 on chromosomeA10 in B. rapa and on chromosome 5 in A. thaliana. These results provide additional information for map-based cloning of the Ms-cd1 gene and will be helpful for marker-assisted selection (MAS).     

Somatic hybrids obtained by asymmetric protoplast fusion between <Emphasis Type="Italic">Musa</Emphasis> Silk cv. Guoshanxiang (AAB) and <Emphasis Type="Italic">Musa acuminata</Emphasis> cv. Mas (AA)

To attempt to introduce genetic information of disease resistance from Musa acuminata cv. Mas (AA) to Musa silk cv. Guoshanxiang (AAB) and obtain somatic hybrids, we developed an asymmetric protoplast fusion with 20% (w/v) polyethylene glycol (PEG). The protoplasts derived from embryogenic suspension cultural cells of cv. Guoshanxiang (AAB) and cv. Mas (AA) were, respectively treated with 1.5 mM iodoacetamide (IOA) and with ultraviolet light (UV) at an intensity of 50 W/m² for 120 s. A total of 47 regenerated green plants were obtained and eight of which were survived in greenhouse. Six of the survived plants were identified as hybrids by RAPD analysis and only three hybrids were retained vigorously in field. The hybrid nature of the three plants was further confirmed according to their ISSR (inter-simple sequence repeat) patterns and the results indicated that they were true somatic hybrids. Chromosome analysis revealed that the three hybrids possessed an aneuploid chromosome number (2n = 34).     

Fine mapping of the recessive genic male sterility gene (<Emphasis Type="Italic">Bnms3</Emphasis>) in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

The Brassica napus oilseed rape line, 7-7365AB, is a recessive epistatic genic male sterile (RGMS) two-type line system. The sterility is controlled by two pairs of recessive duplicate genes (Bnms3 and Bnms4) and one pair of recessive epistatic inhibitor gene (Bnrf). Homozygosity at the Bnrf locus (Bnrfrf) inhibits the expression of the two recessive male sterility genes in homozygous Bnms3ms3ms4ms4 plants and produces a male fertile phenotype. This line has a good potential for heterosis utilization but it is difficult to breed heterotic hybrids without molecular markers. To develop markers linked to the BnMs3 gene, amplified fragment length polymorphism (AFLP) technology was applied to screen the bulks of sterile and fertile individuals selected randomly from a population of near-isogenic lines (NIL) consisting of 2,000 plants. From a survey of 1,024 primer combinations, we identified 17 AFLP markers linked to the BnMs3 gene. By integrating the previous markers linked to the BnMs3 gene into the genetic map of the NIL population, two markers, EA01MC12 and EA09P06, were located on either side of the BnMs3 gene at a distance of 0.1 and 0.3 cM, respectively. In order to use the markers for male sterile line breeding, five AFLP markers, P05MG05, P03MG04, P11MG02, P05MC11₂₅₀, and EA09P06, were successfully converted into sequence characterized amplified region (SCAR) markers. Two of these, P06MG04 and sR12384, were subsequently mapped on to linkage group N19 using two doubled-haploid mapping populations available at our laboratory derived from the crosses Tapidor × Ningyou7 and Quantum × No2127-17. The markers found in the present study should improve our knowledge of recessive genic male sterility (RGMS), and accelerate the development of male sterile line breeding and map-based cloning.