An interspecific somatic hybrid between Actinidia chinensis and Actinidia kolomikta and its chilling tolerance

Protoplasts isolated from cotyledon-derived callus of Actinidia chinensisPlanch. var. chinensis (2N=2x=58) were fused with mesophyll protoplasts of Actiniadia kolomikta(Maxim. et Rupr.) Maxim (2N=2x=58) using a PEG method. Plantlets were regenerated from the fusion product clone 11. RAPD analyses, chromosome numbers of root tip cells and fluorescence peak position of leaf nuclei confirmed that clone 11 was an interspecific somatic hybrid (2N=4x=116) between A. chinensis and A. kolomikta. The chilling tolerance of the somatic hybrid was tested with in vitro leaves at low temperatures. Based on data of leaf thickness, electroconductivity, proline levels, malondialdehyde content and activity of superoxide dismutase, dendrogram cluster analysis suggested that the interspecific somatic hybrid was similar to A. kolomikta, and might have a higher capacity of cold resistance than A. chinensis.     

Production of male sterile interspecific somatic hybrids between transgenic N. tabacum (Bar) and N. rotundifolia (NPT II) and their identification by aflp analysis

Summary A protoplast fusion experiment was carried out aiming to obtain somatic hybrid plants of transgenic Nicotiana tabacum (bar) (+) N. rotundifolia (npt II). The bialaphos resistance marker (bar) was introduced into N. tabacum via Agrobacterium tumefaciens using vector pGV1500 carrying the bar gene phosphinothricin acetyltransferase. N. rotundifolia (npt II) was recovered after direct gene transformation of protoplasts by the pGP6 plasmid carrying the npt II gene for neomycin phosphotransferase. Both plasmids possessed 35S CaMV promotors. Hybrid selection was based on dual bialaphos— kanamycin resistance. Amplified fragment length polymorphism (AFLP) analysis of regenerated plants showed the presence of species-specific bands for both parents, which confirmed their hybrid nature. N. tabacum (bar) (+) N. rotundifolia (npt II) hybrids exhibited a great diversity in morphology. Fertile hybrids which possessed N. tabacum or N. rotundifolia morphology were recovered. Flow cytometric analysis revealed that the N. tabacum- and N. rotundifolio-like hybrids had nuclear DNA contents near that of N. tabacum (9.40±0.24pg) or N. rotundifolia (5.29±0.36 pg), respectively, and were highly asymmetric. Other hybrids combined traits from the two species at various levels—N. tabacum habit or branched, similar to N. rotundifolia. Their leaves varied in shape. The flowers of the hybrid plants were of N. tabacum or N. rotundifolia type, or had N. rotundifolia dimensions, pink with N. tabacum corolla or white with curly fused petals. All were self-sterile or male sterile. The nuclear DNA content varied from 8.90±0.30 to 19.57±0.33 pg. The data from the morphological and cytological analysis provided vidence that parental chromosome elimination in the hybrid clones was spontaneous and not species-specific and that diploidization of the tobacco genome might have occurred in some clones during in vitro culture. This reflects the genomic incompatibility between the two species.     

Selection and characterization of somatic hybrids between Lycopersicon esculentum and Lycopersicon peruvianum

Somatic hybrids of the cultivated tomato, Lycopersicon esculentum, and a wild species, L. peruvianum, were obtained by fusion of leaf protoplasts from both species in the presence of poly-ethylene-glycol (PEG) or in an electric field. The somatic hybrids were selected on the basis of kanamycin resistance of L. esculentum and the plant regeneration capacity of L. peruvainum. Chromosome counts in root tips and the determination of the number of chloroplasts in guard cell pairs revealed that the majority of these hybrids was tetraploid (2n = 4x = 48). The remaining hybrids were at the hexaploid level with chromosome numbers between 64 and 72. The hybrid nature of the regenerated plants was confirmed by analysis of isozyme markers and by their morphology. Most hybrids did flower and set fruits and seeds after selfing. According to RFLP analysis 6 out of the 10 hexaploid hybrids contained two genomes of L. esculentum and four genomes of L. peruvianum. One of these hexaploids had genomes of two different L. peruvianum genotypes and was therefore considered to be derived from a triple protoplast fusion. The hexaploid plants were less fertile than the tetraploids and more resembled L. peruvianum.     

蒲氏钩蝠蛾幼虫血淋巴物质与其耐寒性

作为冬虫夏草寄主,蒲氏钩蝠蛾具重要的经济研究价值,主要分布于青藏高原色季拉山,具有较强的耐寒性,其耐寒性机理尚不明了。通过调查蒲氏钩蝠蛾生境土壤温度与血淋巴物质组成周年变化,以期探讨其耐寒性机理。实验结果表明:土壤温度呈规律性升降趋势,1、2月最低,8月逐步上升至最高,而后逐渐下降;血淋巴中主要物质按含量从高到低排列依次为:蛋白质(8.37—74.2 mg/m L)、总糖(12.65—36.12 mg/m L)、脂肪(7.7—12.32 mg/m L)以及甘油(1.66—3.91 mg/m L);在温度较高的夏季,蛋白质、总糖以及脂肪等物质含量处于较低水平,相反,在寒冷的冬季,此三者的含量均居较高水平;此3类能源物质含量与野外土壤温度均呈极显著的负相关关系,因而血淋巴总热量值在夏季较低而冬季较高,与野外土壤温度亦呈极显著负相关关系;与之相反的是,血淋巴中甘油含量的周年变化规律与野外土壤温度无明显的相关关系。因此,血淋巴中蛋白质、总糖以及脂肪等物质对于蒲氏钩蝠蛾耐寒性有着非常重要的作用,幼虫通过积累3类物质,增加血淋巴能量值,抵御青藏高原的高寒气候;可能由于生境的不同,甘油在蒲氏钩蝠蛾中的作用与其它昆虫有差异,在耐寒性提高中的功能尚需进一步研究。实验结果为了解蒲氏钩蝠蛾的强耐寒性以及适应高寒环境的生理生化机制提供理论依据。     

Morphological and molecular characterization of somatic hybrid plants between Lycopersicon esculentum and Solanum nigrum

Summary Mesophyll protoplasts of tomato (Lycopersicon esculentum Mill. var. cerasiforme) and of an atrazine-resistant biotype of black nightshade, (Solanum nigrum L.), were fused by using polyethylene glycol/dimethyl sulfoxide (PEG/DMSO) solution and three somatic hybrid plants, each derived from a separate callus, were recovered. A twostep selection system was used: (1) protoplast culture medium (modified 8E) in which only tomato protoplasts formed calluses; and (2) regeneration medium (MS2Z) on which only S. nigrum calluses produced shoots. These selective steps were augmented by early isozyme analysis of putative hybrid shoots still in vitro. Phosphoglucoisomerase (PGI) and glutamate oxaloacetate transaminase (GOT) mapped to five loci on four chromosomes in tomato confirmed the hybrid nature of the nuclei of regenerated shoots. The somatic hybrid plants had simple leaves, and intermediate flower and bud morphology, but anthesis was reduced to 5% due to premature bud abscission and the pollen grains were non-viable. Southern DNA blot hybridization using a pea 45 S ribosomal RNA gene probe reconfirmed the hybrid nature of the nuclear genome of the three plants. A ³²P-labeled probe of Oenothera chloroplast DNA (cpDNA) hybridized to cpDNA restricted with EcoRI or EcoRV indicated the presence of the tomato cpDNA pattern in all three hybrids. Likewise, the plants were all found to be atrazine sensitive. Analysis with two mitochondrial (mt)DNA-specific probes, maize cytochrome oxidase subunit II and PmtSylSa8 from Nicotiana sylvestris, showed that, in addition to typical mitochondrial rearrangements, specific bands of both parents were present or missing in each somatic hybrid plant.Michigan Agricultural Experiment Station Journal Article No. 12433     

Selection of a Nicotiana plumbaginifolia universal hybridizer and its use in intergeneric somatic hybrid formation

Summary A Nicotiana plumbaginifolia cell strain carrying a positive (dominant) trait, resistance to azetidine-2-carboxylate (A2C), was selected in strain NX1 which lacked nitrate reductase activity (a negative or recessive trait). This universal hybridizer strain, denoted NXA^r, was fused with dextran to a Daucus carota strain, PR, which carried glyphosate (GLP) resistance. A large number of hybrids were selected in a medium with NO ₃ ^- as the sole nitrogen source and A2C as inhibitor, conditions which prevent the growth of both parents. When the selected colonies were then tested for GLP resistance, 93% carried this trait. In addition the hybrid nature was indicated by additive chromosome numbers, both A2C and GLP resistance in suspension cultures, intermediate nitrate reductase activity and the presence of banding patterns for three isozymes which match those of the parents. Southern hybridization analysis using an enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) probe, pMON 6145, also showed the presence of the gene from both parents in the hybrid strains based on restriction length polymorphisms. The PR strain contains increased levels of EPSPS which confers GLP^r due to gene amplification. Since the universal hybridizer can be used as a fusion partner with any wild-type line many protoplast fusion studies can be carried out easily.Abbreviations A2C azetidine-2-carboxylate - 2,4-D 2,4-dichlorophenoxyacetic acid - EPSPS 5-enolpyruvylshikimic acid-3-phosphate synthase - GLP glyphosate - HAT hypoxanthine, aminopterin, glycine and thymidine medium - IDH isocitrate dehydrogenase - MDH malate dehydrogenase - 5MT 5-methyltryptophan - NBT nitroblue tetrazolium - PGI phosphoglucoisomerase - SDS sodium dodecylsulfate     

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.