Morphological and molecular characterization of fertile tetraploid somatic hybrids produced by protoplast electrofusion and PEG-induced fusion between Lycopersicon esculentum Mill. and Lycopersicon peruvianum Mill.

Summary Mesophyl protoplasts of two genotypes of cultivated tomato (Lycopersicon esculentum Mill.) and one of its wild relative species (Lycopersicon peruvianum Mill.) were fused by using electrofusion and polyethyleneglycol-induced fusion. Forty-three fertile tetraploid somatic hybrid plants, each deriving from separate calli, were recovered from both fusion procedures. Electrofusion appeared more efficient than chemical fusion for the production of somatic hybrids. These plants appeared morphologically similar, whatever the fusion procedure and tomato genotype. They had intermediate leaf, inflorescence, and flower morphology. After self-pollination, the hybrids set fruit of intermediate size and color. The hybrid nature of these plants was confirmed by isoelectric focusing of the Rubisco small subunits used as nuclear markers. L. esculentum and L. peruvianum were distinguished by means of two chloroplast markers: CF1-ATPase subunit as analyzed by isoelectro-focusing and ct DNA restriction patterns. All hybrids displayed both ct markers of only one parent with no biased transmission. Mitochondrial (mt) DNAs were prepared from flower buds by using miniaturized CsCl gradients. Preliminary analysis indicated that mt genomes from the hybrids all differed from those of both parents. mt DNA Sall restriction enzyme analysis revealed that all but two hybrids contained one novel fragment of 13.5 kb. Gene mapping experiments showed that the mt apocytochrome b and ATPase subunit 9 homologies in the somatic hybrid mt DNA resembled L. esculentum and L. peruvianum, respectively; the mt nad5 probe distinguished at least four distinct patterns in the hybrids. These results indicated that mt DNA rearrangements involving intergenomic recombinations occurred through protoplast fusion. A greater mt DNA polymorphism was induced with chemical fusion than with electrofusion.     

Production of a triple mutant,chlorophyll-deficient,streptomycin-, and kanamycin-resistant Nicotiana tabacum,and its use in intergeneric somatic hybrid formation with Solanum melongena

Summary In order to produce a triple mutant, sexual crosses between a chlorophyll-deficient, streptomycin-resistant mutant of Nicotiana tabacum (SA) and a kanamycin-resistant transformant of N. tabacum (KR.) were carried out. From the offspring of this cross, a triple mutant (KR-SA) was selected. In N. tabacum KR-SA, chlorophyll deficiency is due to recessive mutation in the nuclear genome, streptomycin resistance is due to a dominant mutation in the chloroplast genome, and kanamycin resistance is shown to be a dominant nuclear marker. Cell suspension protoplasts of N. tabacum KRSA were fused with callus protoplasts of Solanum melongena by dextran treatment. Somatic hybrid plants were selected for streptomycin resistance and the ability to produce clorophyll in regenerated plants. By using this selection system, green plants were recovered from two colonies. When these green plants were then tested for kanamycin resistance, all analyzed plants carried this trait. In addition, the hybrid nature of these plants was confirmed by investigation of the peroxidase isozyme. The present results show that the use of N. tabacum KR-SA in studies of somatic hybridization makes it possible to select somatic hybrid plants easily and provides information of the N. tabacum genome.Chemical Regulation of Biomechanism, The Institute of Physical and Chemical Research, Wako 351-01, Japan     

以异源四倍体体细胞杂种为父本杂交培育三倍体柑桔植株的研究

以 3个柑桔原生质体融合而来的四倍体体细胞杂种为父本 ,与二倍体单胚性种柚子 (Citrusgrandis)以及单多胚混合型品种“华农本地早”桔 (C.reticulata)有性杂交 ,授粉后 90 d,发现种子干瘪 ,大部分种子的胚败育。将干瘪种子在 MT附加 1mg/L GA3 或 50 0 mg/L麦芽浸出物的培养基中 ,经培养抢救 ,有 2 5.6%的种子萌发成苗或继续进行胚的生长 ,后者进一步诱导能形成丛芽 ,经试管嫁接或诱导生根形成完整植株。共获得 6个组合 73棵完整植株 ,染色体数检查表明 ,2 0株为三倍体 (2 n=3x=2 7) ,32株为二倍体 (2 n=2 x=18) ,8株为非整倍体 ,其它 13株还有待于进一步检查。     

Somatic embryogenesis from immature leaves of in vitro grown tea shoots

Immature leaves of in vitro grown shoots of tea were cultured on various levels of 2,4-D. Somatic embryos were induced directly on leaves or via embryogenic callus produced at the basal regions of the leaves. Induction of embryogenesis appeared to be correlated with the maturity of the leaf explants, with younger leaves responding better. The embryogenic response of leaf explants also was correlated with the period of culture in 2,4-D containing liquid medium. Embryogenic calli or repetitive somatic embryos maintained their regeneration capacity for more than 3 years. Histological observation revealed somatic embryos were formed on various regions of the leaf midrib. Somatic embryos germinated and developed into plantlets on agar medium containing BA and IBA.Abbreviations BA 6-benzylaminopurine - IBA indole-3-butyricacid - 2,4-D 2,4-dichloro phenoxyacetic acid     

The growth pattern of chimpanzees: Somatic growth and reproductive maturation inPan troglodytes

Growth of chimpanzees reared at the Kumamoto Primates Park of Sanwa Kagaku Kenkyusho Co. Ltd. was studied cross-sectionally from the viewpoints of somatic growth and reproductive maturation. Distance and velocity curves were expressed using spline function method. Males showed adolescent growth acceleration in body weight, with a peak at 7.86 yrs of age, but not in trunk length. Females showed continuous rapid growth from mid-juvenile to adolescent phase in both body weight and trunk length, but no isolated adolescent spurt. The Sanwa chimpanzees matured at about 12.5 yrs of age for females and 15.0 yrs for males. The mean adult weights and trunk lengths were 53.2 kg and 507.8 mm for males and 42.7 kg and 481.6 mm for females. The Sanwa chimpanzees had similar growth patterns to those of the Yerkes chimpanzees, although they showed a slight delay in infancy, and a higher growth rate from the early juvenile phase onwards. Growth patterns in these two laboratories may be regarded as “normative” for laboratory-reared chimpanzees. They matured earlier than wild chimpanzees by more than two years. The major reason for the retarded maturation in wild chimpanzees is the delay of growth from infant to the early juvenile phases (0–4 yrs of age), probably owing to a limited nutritional supply from the mother. Development of the testes comprised three phases: slow growth from infant to juvenile (until 6.4 yrs); rapid growth around adolescence (until 9.2 yrs); and adult (mean testicular volume, 187 cm³). Setting the nutritional standard at 2,000–2,600 Cal/day (= Kcal/day) per adult, calories were considered for captive chimpanzees in each age class.     

Production and identification of somatic hybrids between Solanum tuberosum and S. papita by using the rolC gene as a morphological selectable marker

A successful hybridization of a diploid clone of Solanum tuberosum with a rolC-transgenic, diploid S. papita clone is reported. By using leaf expiants of this S. papita clone, which after transformation expressed kanamycin resistance, intact protoplasts were obtained, but these protoplasts did not develop to microcalli or regenerate to mature plants. However, protoplasts of the S. tuberosum clone showed a high capacity to regenerate plants from isolated protoplasts. On a medium containing Kanamycin only calli regenerated to plants, which revealed a rolC phenotype (reduced apical dominance with a large number of adventitious shoots and a pale green color of leaves) and later on turned out to be true hybrids. Self fusions of S. papita never developed to microcalli and those of S. tuberosum ceased to develop on the kanamycin-containing medium. Identification of somatic hybrids was done by RFLP and RAPD analysis. In the greenhouse, out of four selected hybrids only FK3.1 was successfully crossed with two standard S. tuberosum varieties (Datura, Desirée). Out of all the seeds germinated, only rolC-negative F¹ seedlings were further characterized. Within the seedling population obvious differences were evident in respect of the S. papita and S. tuberosum characteristics.     

Synthesis of hexaploid (AABBCC) somatic hybrids: a bridging material for transfer of ‘tour’ cytoplasmic male sterility to different Brassica species

Most of the alloplasmic cytoplasmic male sterility (CMS) systems are known to be associated with a number of floral abnormalities that result from nuclear-cytoplasmic incompatibilities. One such system, tour, which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp. While the restorer for this CMS has been reported to be present in B. napus, in B. juncea, where the abnormalities are more pronounced, no restorer has yet been identified. Rectification of these floral abnormalities through mitochondrial recombinations and chloroplast replacement might result in the improvement of this CMS system. As organelle recombinations can possibly be achieved only by somatic cell hybridization, fusion experiments were carried out between hygromycin-resistant B. juncea AABB carrying tour cytoplasm and phosphinotricin-resistant, normal B. oleracea CC to generate AABBCC hexaploid somatic hybrids. The presence of selectable marker genes facilitated the selection of hybrids in large numbers. The resulting hybrids showed wide variation in floral morphology and organelle composition. Regenerants with normal, male-sterile flowers having recombinant tour-or oleracea-type mitochondria and oleracea-type chloroplasts were obtained. Hybrids with male-fertile flowers were also obtained that had recombined tour mitochondria. The AABBCC hexaploid hybrids synthesized in the present study were successfully utilized as a bridging material for transferring variability in the organelle genome simultaneously to all the digenomic Brassica species, and all of these hybrids are now being stabilized through repeated backcrosses to the allopolyploid crop brassicas.     

Transient reduction in secreted 32 kD chitinase prevents somatic embryogenesis in the carrot (Daucus carota L.) variant ts11

At the nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot (Daucus carota L.) cell variant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type cells. The causative component in the conditioned medium has been identified as an acidic 32 kD endochitinase. An antiserum raised against the 32 kD chitinase detected this protein in culture medium from ts11 embryo cultures grown at the permissive temperature as well as at the nonpermissive temperature. No difference in biochemical characteristics or in effect on ts11 embryo development could be detected between the 32 kD chitinase purified from wild-type cultures and the chitinase from ts11 cultures grown at the permissive or at the nonpermissive temperature. Compared to the amount present in a ts11 embryo culture at the permissive temperature, a reduction in the amount of 32 kD chitinase was observed during the temperature-sensitive period at the nonpermissive temperature. These results imply that the arrested embryo phenotype of ts11 is not the result of a structural difference in its 32 kD chitinase, but is the result of a transient decrease in the amount of 32 kD chitinase present. Morphological observations indicate that the ts11 phenotype is pleiotropic and also affects the cell wall of nonembryogenic cells. © 1995 Wiley-Liss, Inc.