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Plant transformation mediated by Agrobacterium rhizogenes: optimization of the infection process

Luciano Cônsoli; Salete Aparecida Gaziola; Maria Lúcia Carneiro Vieira

Departamento de Genética, ESALQ-USP, Caixa Postal 83, 13418-900 Piracicaba, SP, Brasil. Send correspondence to M.L.C.V.

ABSTRACT

Because of its ability to transfer part of its Ri plasmid to plant genomes, Agrobacterium rhizogenes can be used as a vector for the transfer of genes of interest. The objective of this study was to determine factors for the optimization of the process of plant tissue infection in two species, Carica papaya L. and Centrosema pubescens Benth. Liquid cultures of the wild A. rhizogenes strains LBA 9402, A₄T and 8196 were used to inoculate the explants, obtained 20 days after germination. Three methods were used for explant inoculation: a scapel, a hypodermic needle, and a platinum loop. Strain LBA 9402 was highly efficient in infecting papaya and C. pubescens explants, whereas strains A₄T and 8196 only infected C. pubescens explants. For both cultures, inoculation methods that promoted wounds in the plant tissue (scalpel and needle) were more efficient than the platinum loop method. Papaya hypocotyl segments were more responsive to infection than leaf and cotyledon explants, with 61% of explants presenting on average 3.9 roots/explant. C. pubescens leaf and epicotyledon explants gave similar responses (4.33 and 3.35 roots/explant, with a 54% and 77% rate of explant infection, respectively). When a growth curve was constructed for strain LBA 9402, plotting culture growth phase against infecting ability of the bacteria, the bacteria was found to present a greater ability to infect hypocotyledon and leaf explants of C. papaya and C. pubescens, respectively, at the end of the log phase (T24-T30).

Keywords: Plant transformation; Agrobacterium rhizogenes; infection.

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