engleski

Spontaneus formation of somatic embryos in hairy-root lines of pumpkin (Cucurbita pepo L.)

INTRODUCTION Agrobacterium rhizogenes is a soil phytopathogen that causes the so-called hairy-root disease (Riker and Banfield 1932). The expression in plant cells of T-DNA sequences originally located on the Ri plasmid results in organized cell proliferation leading to abundant root proliferation. Hairy-root cultures are genetically stable, suitable for different manipulations and able to regenerate whole plant. In this paper we report our data on the virulence of two wild strains of A. rhizogenes (8196 and 15834) with regard to their ability to infect pumpkin cells and induce hairy-root proliferation, physiological and phenotypic characteristics of hairy-root lines cultivated on hormone-free nutrient medium and the role of external factors (pH and autoclaving duration of nutrient media) on embryogenic callus induction MATERIALS AND METHODS Agrobacterium rhizogenes wild strains: 8196 (mannopine-type) and 15834 (agropine-type) were used for plant inoculation. Excised cotyledons from 8-day-old pumpkin seedlings germinated in vitro were used for induction of transgenic roots. The primary hairy roots, which developed within 2-3 weeks of inoculation with A. rhizogenes strain 8196, were explanted and cultured on solid MS (Murashige and Skoog 1962) medium (0.9 % agar, 3 % glucose) without hormones, but with carbenicillin (500 mg/l) which was also added to the medium during the second and third subculture (3-4 weeks each). After that, they were subcultured monthly on a semi-solid or liquid MS medium. Root tips (1.5 cm) from established hairy-root lines were subcultured on solid MS medium (pH 4.5 and 5.7) and autoclaved 18 or 30 min. Four combinations of pH values and autoclaving duration were tested in order to establish conditions capable of evoking the development of somatic embryos in transgenic roots. Total genomic DNA was isolated from C. pepo tissue according to Edwards et al. (1991). Southern blotting was carried out using standard methods (Ausuber et al. 1994). For Southern hybridization, 10 mg of HindIII digested genomic DNA was subjected to electrophoresis in 0.7% agarose gel and transferred onto positively charged nylon membrane (Boehringer Manheim). DNA was probed with 32P-labeled HindIII fragment no. 19 from p218 (Koplow et al. 1984). The X-ray film was exposed at -70°C for three days. The influence of growth regulators on transgenic embryos was investigated by suplementing nutrient media with IAA and BAP in different combinations: MS + 4 mg/l BAP MS + 2 mg/l BAP + 0.5 mg/l IAA MS + 2 mg/l BAP + 2 mg/l IAA MS + 0.5 mg/l BAP + 2 mg/l IAA MS + 2 mg/l IAA RESULTS AND DICUSSION Cotyledons detached from the 8-day-old seedlings cultivated in vitro were susceptible to hairy-root transformation, but only with strain 8196. Cotyledons inoculated with strain 15834 failed to form hairy roots, which was previously reported (Katavic et al. 1991). Primary hairy -roots appeared within 5-7 days in the inoculated wound area. All the clones showed phenotypic characteristics of pRi-transformed culture: adaptation, plagiotropism and substantial increase of biomass. The callus induction in pumpkin hairy-root lines was the most frequent when the nutrient medium with pH 5.7 was autoclaved 30 minutes. Calli were small, green-grey coloured with many tiny roots. After a long period in culture three hairy-root lines (31Cp, Cp1 and Cp2) grown on semi-solid medium spontaneously generated friable callus with embryo-like structures. Subdivided embryogenic calli proliferated continually on the hormone-free medium. Strong phenotypic alternations among the embryos formed were observed. Embryos showed variable growth rates and sizes, and differed in morphology. The Southern analysis of embryos DNA showed the presence of T-DNA of Agrobacterium rhizogenes. Transgenic embryos retained abnormal morphology even after being transferred to media supplemented with different combinations of growth regulators (IAA ; BAP). The investigated external factors did not explain the ground of embryogenic callus induction. Our investigations showed that a few pumpkin 8196 hairy-root clones spontaneously generated an embryogenic callus after being a long period in culture.

Cucurbita pepo; somatic embryogenesis; hairy roots

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