PhD

Investigations on genetic transformation in black pepper (Piper nigrum
L.), variety Panniyur-l , using Agrobacterium tumefaciens strain AGL-1; 1303
harbouring antibiotic resistant selectable marker genes (npt II and hpt IV) and GUS
and GFP reporter genes were carried out in Plant Tissue Culture Laboratory,
College of Forestry, Kerala Agricultural University, Vellanikkara, Thrissur, during
the period from November, 1997 to August, 2000.
For the collection of explants for transformation work and related
studies, axenic seedlings of black pepper were raised. Of the different media used
for this, double sterilised moist sand was found to be the best.
Callus induction on cotyledonary leaf explants and callus growth was
found to be maximum in half MS medium supplemented with 1.0 mg }"1 each of
IAA and BA compared to other combinations ofIAA, BA and kinetin tried.
Of the different growth regulator combinations tried in half MS medium
for regeneration, only when callus cultured in half MS supplemented with 1.5
mg r' IAA + 1.0 mg rl BA were transferred to half MS supplemented with 1.0
mg rl each ofIAA and BA produced regeneration to the extent of three per cent.
In order to produce embryogenic calli for somatic embryogenesis,
zygotic embryos were cultured in liquid basal SH medium under different cultural
conditions. Somatic embryos were suspected to be produced when embryo with
endosperm were cultured in liquid basal SH medium on rotary shaker. However,
embryogenic calli were not generated.
Black pepper tissues, both leaf and callus, were found to be susceptible
to kanamycin and hygromycin when freshly prepared antibiotic stocks were used
and tissues subcultured at two week interval. Callus induction on leaf was
completely inhibited at 50 mg rl of kanamycin and callus growth at 100 mg r'.

Hygromycin at 10 mg rI completely suppressed callus induction on leaf and callus
growth at 30 mg r'.
Cefotaxime at a strength of 500 mg r' effectively killed the
Agrobacterium tumefaciens strain AGL-l; 1303 in pure bacterial suspension
cultures with an O.D.S6Omn = 1.0.
Proline was found to Increase the rate of multiplication of
A. tumefaciens strain AGL-l; 1303.
Leaf transformation was carried out by varying different factors
affecting transformation. None of the leaf explants showed callus induction in
screening media. Cefotaxime at strengths of 500 and 1000 mg rI could not
eliminate Agrobacterium effectively from the leaf tissue. Prolonged survival of the
Agrobacterium in the tissues could be the reason for non-multiplication of
transformed cells.
Callus transformation was carried out varying different factors affecting
transformation. Excessive exudation of phenols was noticed in all the treatments
compared to the controls. Heavy overgrowth of Agrobacterium was seen on
withdrawal of cefotaxime from the screening media. Cefotaxime at 500, 1000 and
1500 mg r' could not eliminate the Agrobacterium effectively from callus tissues.
Ineffective elimination of Agrobacterium, the super virulent character of the strain
and the hypersensitive response of the callus to bacterial infection and wounding
could be the reasons for failure of transformed cells to multiply.