Articles
PLANT RECOVERY FOLLOWING TRANSFORMATION OF AVOCADO WITH ANTI-FUNGAL PROTEIN AND SAM HYDROLASE GENES
Article number
738_55
Pages
447 – 450
Language
English
Abstract
In our studies to enhance fruit shelf life and resistance to root diseases, embryogenic Suardia and Hass avocado cultures were transformed with SAM hydrolase and anti-fungal protein (AFP) genes, respectively.
Embryogenic cultures were induced and maintained according to previously developed procedures (Witjaksono and Litz, 1999). Embryogenic Suardia suspension cultures were cocultivated with an acetosyringone-activated Agrobacterium tumefaciens strain EHA101 harboring binary vector pAG-4092 which contained the nptII gene and the samK gene driven by an avocado fruit ripening-specific cellulase promoter.
Transformed embryogenic tissues were selected in liquid cultures with 50 mg L-1 kanamycin.
Embryogenic Hass suspension cultures were cocultivated with an acetosyringone-activated A. tumefaciens strain EHA105 harboring binary vector pGPTV-BAFP which contained the bar selectable marker gene, uidA (GUS) reporter gene and AFP gene driven by the 35S promoter.
Transformed embryogenic cultures were selected in liquid medium with 3 mg L-1 phosphinothricin, and monitored by the X-Gluc assay.
Enlarged and opaque somatic embryos were obtained from the transformed cultures by plating onto semisolid MS medium with 20% coconut water.
The rate of normal germination was low and most somatic embryos produced only small shoots.
Therefore, shoots from somatic embryos were rescued by micrografting onto in vitro seedlings.
To obtain plants ex vitro, in vitro-derived shoots were also grafted onto three-week-old seedlings.
Confirmation of transformation has been conducted by PCR. Transgenic plants are growing vigorously in the greenhouse.
Embryogenic cultures were induced and maintained according to previously developed procedures (Witjaksono and Litz, 1999). Embryogenic Suardia suspension cultures were cocultivated with an acetosyringone-activated Agrobacterium tumefaciens strain EHA101 harboring binary vector pAG-4092 which contained the nptII gene and the samK gene driven by an avocado fruit ripening-specific cellulase promoter.
Transformed embryogenic tissues were selected in liquid cultures with 50 mg L-1 kanamycin.
Embryogenic Hass suspension cultures were cocultivated with an acetosyringone-activated A. tumefaciens strain EHA105 harboring binary vector pGPTV-BAFP which contained the bar selectable marker gene, uidA (GUS) reporter gene and AFP gene driven by the 35S promoter.
Transformed embryogenic cultures were selected in liquid medium with 3 mg L-1 phosphinothricin, and monitored by the X-Gluc assay.
Enlarged and opaque somatic embryos were obtained from the transformed cultures by plating onto semisolid MS medium with 20% coconut water.
The rate of normal germination was low and most somatic embryos produced only small shoots.
Therefore, shoots from somatic embryos were rescued by micrografting onto in vitro seedlings.
To obtain plants ex vitro, in vitro-derived shoots were also grafted onto three-week-old seedlings.
Confirmation of transformation has been conducted by PCR. Transgenic plants are growing vigorously in the greenhouse.
Authors
R.E. Litz, S.H.T. Raharjo, D. Efendi, Witjaksono, M.A. Gómez-Lim
Keywords
Persea americana, somatic embryo, micrografting, air layering
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