PhD

A study on ‘Agrobacterium mediated genetic transformation’ in Dendrobium, Sonia 17 was conducted in the Plant Molecular Biology and Biotechnology Centre, College of Agriculture, Vellayani during 2002 – 2004.

Protocorms were obtained from the in vitro germination of Dendrobium seeds on half – strength MS medium supplemented with coconut water 150 ml l-1, sucrose 30gl-1 and agar 6 gl-1. Protocorm like bodies were produced from the in vitro culture of the shoot apices on half-strength MS medium with coconut water 150mgl-1, activated charcoal, 0.5 gl-1, sucrose 30gl-1, agar 6gl-1 and BA 0.2 gl-1.

Experiments were conducted to evaluate the sensitivity of both protocorms and PLBs to different doses of antibiotics viz., ampicillin, rifampicin, cefotaxime, carbenicillin, kanamycin and hygromycin. It was observed that ampicillin 400 and 450 mgl-1 induced complete bleaching of the protocorms and PLBs, respectively. In the seventh and eight week, bleaching was observed in protocorms with rifampicin 400 mgl-1, and browning and death with rifampicin 450 mgl-1. With PLBs none of the treatments showed browning and death. Even increased doses of 450 mgl-1 induced only bleaching. Increased doses of cefotaxime 450 mgl-1 induced browning of the PLBs and cefotaxime 350 mgl-1 caused the browning of the protocorms in the eighth week. Bleaching was observed with carbenicillin 350 mgl-1. Carbenicillin 450 mgl-1 induced browning and subsequent death of PLBs. Bleaching of protocorms and PLBs was noticed with kanamycin 100 mgl-1 from the sixth week onwards. Total inhibition of the growth of the protocorms and PLBs was observed with hygromycin 50 mgl-1. Out of the six antibiotics used the orchid tissues were highly sensitive to kanamycin 100 mgl-1 and hygromycin 50 mgl-1.

Effect of cefotaxime for the elimination of Agrobacterium was studied. It was observed that Agrobacterium was effectively killed by cefotaxime 200 mgl-1.

Two strains of Agrobacterium viz., LBA 4404 and EHA 105 were used for the experiments. The strain LBA 4404 habouring PBI 121, pCAMBIA 1301 and pCAMBIA 2301 took 38.0, 35.0 and 30.8 hours respectively for growth. The strain EHA 105 harbouring the binary plasmids PBI 121, pCAMBIA 1301 and pCAMBIA 2301 showed growth in 36.5, 30.0 and 29.9 hours respectively.

When the protocorms were used as explants, none of the transformants were obtained in all the binary plasmids PBI 121, pCAMBIA 1301 and pCAMBIA 2301. The maximum of 2.0 percentage transformants were obtained when the PLBs of 0.2 cm were co-cultivated. When the PLBs of 0.4 and 0.5 cm size were co-cultivated. 0.5 per cent transformants were obtained. The per cent explants (98.2) retained after co-cultivation was maximum when the explants were infected by bacterial suspension. But the transformation efficiency was the highest 1.0 in the pelleting method. Overcrowding occured when 50 explants were kept in a petriplate during co-cultivation which led to the overgrowth of the bacteria. (With 20, 30, and 40 explants the transformation efficiency of 0.5, 0.5 and 1 per cent was obtained.)

In a study conducted to optimize the time required for infection, transformants (1.0 per cent) were obtained with the infection process for 15 minutes using the bacterial pellets. The infection done by diluting the bacterial suspension to 1:5 (v/v) produced 0.5 per cent transformants. Wounding was found beneficial and 18.9 kanamycin and hygromycin resistant PLbs were obtained when compared to unwounded explants (1.1), when the infection was done with bacterial suspension diluted to 1:5 v/v. With the infection using the bacterial pellets the wounded PLBs showed best response and maximum of 20.1 antibiotic resistant PLBs were obtained where as only 5.3 PLBs were recovered with the unwounded explants.

Transformants were recovered only with the wounded PLBs of 0.2 cm size co-cultivated in the dark for 2.0 and 3.0 days. None of the transformants were produced from the explants co-cultivated for more than three days. The maximum transformation efficiency of 2.0 per cent was obtained when LBA 4404 (pCAMBIA 1301) and EHA 105 (pCAMBIA 2301) were co-cultivated for three days and the infection was carried out with the bacterial pellets. The loss of explants due to fungal contamination was avoided by blotting the explants in a blotting paper before inoculation. This practice helped in the retention of 80.3 per cent explants whereas only 30.1 per cent explants were retained after co-cultivation when the explants were not blotted. The transformation efficiency was increased to 3.0 per cent when acetosyringone 100 M was added.

Out of the total kanamycin resistant PLBs 69.23 per cent were positive for GUS activity. The GUS expression was observed as blue spots and blue patches on the surface of the PLBs. The antibiotic sensitive PLBs did not stain. Amplification of DNA of the four plants which stayed green and grew in the presence of kanamycin revealed the expected 0.7 kb fragment. Genomic DNA from those plants gave good PCR amplification. No amplification of the expected band was observed in the control. The height of the transformed plants ranged from 0.8 to 1.8 cm. They produced 1-3 leaves in one year growth in vitro under stringent antibiotic selection conditions. The root length ranged between 0.5 to 1.5 cm and the leaf length from 0.5 to 1.1 cm.