TITLE:
Agrobacterium Tumefaciens Based Transformation of Pelargonium x Hortrum cv. ‘Samba’ with Anti-1-aminocyclopropane-1-carboxylate Synthase cDNA to Regulate Ethylene Biosynthesis
AUTHORS:
Rajinder S. Ranu, Jianguo Fan, Sarada Krishnan, Pradeep Agarwal., Amitva Mitra
KEYWORDS:
ACC Synthase; Ethylene Biosynthesis; Transformation; Geranium Regeneration; Agrobacterium Tumefaciens
JOURNAL NAME:
Engineering,
Vol.4 No.10B,
January
16,
2013
ABSTRACT: Phytohormone, ethylene plays an important role in
plant growth and development including fruit ripening and flower senescence.
The synthesis of 1-aminocylo-propane-1- carboxylate (ACC), the immediate
precursor of ethylene, from S-adenosyl-methionine is catalyzed by ACC synthase;
and which is also a rate limiting step in the ethylene biosynthetic pathway. Therefore,
it plays a key role in ethylene biosynthesis and the genes that code for ACC synthase
are of special interest. Moreover, in zonal geraniums, ethylene bursts released
from cuttings can have profound impact on the viability of explants for plant
propagation. Biotechnological approach involving genetic modification that may
reduce ethylene levels has potential for increasing the shelf-life of cuttings
for plant propagation. These considerations have led us to clone several cDNA
of ACC synthase genes from Pelargonium x
hortorum cv. ‘Sincerity’. To transform geranium cells with Agrobacterium tumefaciens an in vitro regeneration system was developed using very young petiole
explants. An Antisense construct of ACC synthase cDNA (PHSacc41) ligated into
binary vector pAM696 was introduced into A.
tumefaciens EHA 105 cells. Petiole explants were incubated with the Agrobacterium for 15 min and then
co-cultivated for several days on MS medium containing 5 mM BAP and 1 mM IAA in
the dark without the antibiotics. Selection for transformants was
carried out in the presence of kanamycin and timentin. Transgenic plantlets generated
were examined for inserted gene cassette by PCR and Southern blotting. Recovery
of positive transformants that survived selection suggested that it is possible
to transform and introduce genes via transformation in hybrid geraniums for
genetic modification.