ANNOUNCEMENTS
The summary of results obtained in the present study has been categorised and presented under the five sub-headings as mentioned below:
• In vitro regeneration
• Agrobacterium mediated transformation
• Microprojectile bombardment mediated transformation
• Molecular characterization of putative transformants
•Isolation of coat protein gene
1. In vitro regeneration by somatic embryogenesis:
• Seed sterilization protocol described in the present study could yield approximately 84 % aseptic cultures. Germination of seeds was observed between 10-20 days of incubation.
• Of the various explants tested viz. hypocotyl segments, leaf petiole, leaf lamina and root segments; the root explants were found to be the most responsive for induction of embryogenic callus.
• The combination of 2.22 μM BAP and 4.90 μM IBA proved to be the best for both induction and multiplication of somatic embryos from root explants.
• Of the various parameters tested for improving the induction of somatic embryogenesis, MS ½ as basal medium, sucrose (6%) as carbohydrate source, agar as gelling agent (0.8%) and culture of embryos in dark incubation were proved to have a positive effect on increase in number of somatic embryos.
• Although the multiplication rate of somatic embryo was significantly higher in liquid media in comparison to semi-solid medium, the embryos multiplied on liquid medium failed to germinate.
• Germination of somatic embryos started within 3 weeks of incubation and maximum germination frequency (76%) was observed on MS medium supplemented with 0.9 μM Kinetin. Glutamine incorporation (at 400 mg/l) was useful in prevention of leaf and healthy shoot formation.
• Though the germinated embryos formed tiny roots, the quality of roots was not suitable for transplantation. Consequently a separate rooting stage was necessary. Optimum rooting was observed when shoots were given a pulse treatment on medium supplemented with 8.57 μM IBA for 4 days in dark followed by transfer to growth regulator-free medium containing 500-mg/l activated charcoal and 400-mg/l glutamine. The hardening survival of in vitro raised plants was more than 85 % in the mixture of soil, agropeat and FYM
• This is the first report on somatic embryogenesis in Indian gynodioecious cultivar CO 7 using vegetative explants (root).
2. Agrobacterium mediated transformation
• In the present study, the somatic embryos were used as the target tissue for conducting genetic transformation experiments. This is because, somatic embryos offer considerable advantage over other plant tissues as they originate from a single cell.
• The transformation frequency of papaya cultivar “CO7” by Agrobacterium tumifaciens is largely influenced by the type of pre-culture technique and the duration, bacterial strain harboring plasmid pC1301 and the time period required for co-cultivation.
• In the present study we employed three disarmed strains of Agrobacterium tumefaciens namely LBA4404, EHA105 and A208 to evaluate their efficiency in transforming papaya somatic embryos. The results revealed that an LBA4404 strain was far more superior to the other two strains.
• Similarly, if liquid pre-culturing was performed 10 days prior to transformation, there was a considerable increase in the transformation efficiency. The mid-log phase inoculum of Agrobacterium (O.D ~0.7) was found to be optimal for infection with a concomitant increase in number of GUS positive cells.
• Another advantage of using liquid pre-culture was found to be in the filtration process wherein, the embryos were filtered using a Millipore vacuum filtration unit, following the pre-culture period. This process apart from allowing ease to handle somatic embryos, also provided the growth medium to get into superficial cell layers.
• The phytotoxic levels of hygromycin for papaya somatic embryos was found to be 250 mg/l.
• Amongst the two bactericidal antibiotics tested, Carbenicillin at 500 mg/l was most efficient, both in terms of organogenic response and bactericidal effect.
• Treating explants in Agrobacterium suspension for 30 minutes followed by incubation in dark and culturing in semi-solid medium was found to be the ideal condition of treatment for affecting transformation in the somatic embryos. In this study, co-cultivation of the treated explants in dark condition for 3 days resulted in high frequency of putative transformants.
• In the present study, an efficient transformation protocol for somatic embryos of Indian cultivar of papaya was developed using Agrobacterium tumefaciens.
3. Microprojectile bombardment mediated transformation
• In this study, papaya somatic embryos were used as explants for genetic transformation by microprojectile bombardment. Use of somatic embryo as explant offered considerable advantage of uniform geometry and rapid cell division.
• Osmotic conditioning by subjecting the culture plates to drying in a laminar hood for 1 to 1.5 hours was found to be beneficial for transformation.
• Subjecting the somatic embryos to physical injury during the pre-culture period enhanced the transformation frequency. The optimum time period following pre-culture treatment was found to be 10 days prior to bombardment. This procedure enhanced the regenerative capacity of somatic embryos.
• The frequency of transformation in papaya cultivar “CO 7” using microprojectile bombardment was largely influenced by the vacuum pressure, helium pressure, distance between rupture disk and macrocarrier, distance between macrocarrier and stopping screen and target distance. It was also observed that the vacuum of 28 inches of Hg, helium pressure of 1100 psi, ¼” gap distance (distance between the rupture disc and micro-carrier assembly), 10 mm distance between macrocarrier and stopping screen and 9 cm target distance from stopping screen were the most optimum parameters for an effective transformation of papaya somatic embryo.
• Whatman No.3 filter paper discs were used as support for embryos in culture plates. The filter paper discs had sufficient pore size to allow easy passage of nutrients from the tissue culture medium and they eased the process of microprojectile bombardment and successive sub-cultures.
4. Molecular characterization of putative transformants:
• The DNA isolation procedure employed in the present study yielded reproducible results of high quality DNA. The protocol was simple and inexpensive and was successfully demonstrated in papaya which contained large amount of secondary metabolites such as polyphenols, tannins and polysaccharides.
• In all, 1700 lines (1250 lines generated by microprojectile bombardment and 450 lines generated from Agrobacterium mediated transformation experiments) were analyzed for the presence of GUS gene by PCR.
• Of the 1700 lines analyzed, 280 lines (172 lines generated by microprojectile bombardment and 108 lines generated from Agrobacterium mediated transformation experiments) were PCR positive for the GUS gene.
• All the 280 PCR positive lines were analyzed by dot blot assay after 2-3 passages of multiplication to screen out the chimeras from proper transformation events. Of the 280 lines assayed, only 170 gave positive hybridization signals. The intensity of signal varied considerably despite using equal quantity of DNA.
• Of the 170 PCR positive lines obtained from microprojectile method, 102 were dot blot positive i.e. 60% of the PCR positive and 8.16% of the lines selected from transformed plates; while of the 108 PCR positive lines obtained by Agrobacterium methodology, 68 were dot blot positive i.e. 63% of PCR positive and 15.11% of the lines selected from transformed plates.
• The Southern analysis revealed that there were only 21 single copy insertions for GUS gene of all the lines tested and proven to be positive by dot blot analysis. The majority of lines were double or multiple copy insertions. In the present study, 9 single copy insertions were obtained using Agrobacterium mediated transformation and 12 single copy insertions using microprojectile mediated transformation.
• In the present study, of the total transgenic plants analysed, 86% of the transformants generated by Agrobacterium methodology possessed more than one copy of the transgene, while 88% of the transformants generated by microprojectile methodology had more than one copy of the transgene.
5. Isolation and cloning of PRSV coat protein gene
• In the present study, the viral coat protein gene was isolated using the viral RNA as a precursor.
• The sequence was characterized as coat protein gene sequence of PRSV-P isolate collected from Haryana region in India.
• The sequence was 852 bp long and had 42% GC content and 58% AT content. The average di-nucleotide content of the sequence was found to be 4.65 %. The thermodynamic parameters of the sequence based on Universal Genetic Code were deduced based on the sequence analysis for PCR amplification; 50 mM Sodium, 1.5 mM Magnesium and Probe concentration of 0.10 μM.
• The multiple sequence analysis of PRSV coat protein nucleotide sequences from India revealed that the Haryana sequence showed 94% similarity with the coat protein gene sequence from Ranchi and Delhi strains. The phylogenetic analysis revealed that the PRSV-P isolates from the Indian sub-continent are the most diverse of all the sequences tested in this study.
• The multiple sequence alignment of PRSV CP amino acid sequence from around the world revealed that Haryana isolate grouped together with the CP sequence from Bangladesh and formed a separate lineage, whereas the rest of the South East Asian isolates (Japan, Vietnam, Thailand, China, Sri Lanka, Philippines and Taiwan) formed another lineage.
• The nucleotide sequence for Haryana isolates codes for 284 amino acids in a single ORF. The amino acid composition analysis of the sequence revealed that the sequence coded for 116 hydrophobic residues (40.85%), 102 hydrophilic residues (35.92%) and 66 neutral residues (23.24%). The average molecular weight of the coded protein was calculated to be approximately 32480.03 whereas the mono-isotopic molecular weight was approximately 32459.98. The isoelectric point of the protein was calculated at pH=6.4.
• The amino acid sequence showed the presence of conserved sequences of the potyvirus group such as: the DAG motif, KE repeats, WCIEN motif and QMKAAA motif. Amino acid sequence comparison of the Haryana isolate with those of other PRSV-P isolates reported from different geographical locations revealed a highly variable N-terminal region. The CP gene of Haryana isolate had a deletion of 15 nucleotides, corresponding to five amino acids, in this region.
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