1. Genetic Transformation of Guava (Psidium guajava L.)
Maneesh Mishra and Uzma Jaleel
Biotechnology Laboratory, Central Institute for Subtropical Horticulture, Lucknow
INTRODUCTION
Guava (Psidium guajava L.) belonging to family Myrtaceae is an important commercial fruit crop.
Guava production is impeded by a fungal disease -guava wilt.
Conventional methods to control this disease has not been successful.
Transgenic technology can be gainfully utilized for development of wilt resistant guava.
We report for the first time a repeatable shoot tip transformation protocol in guava using Agrobacterium tumefaciens harbouring endochitinase gene.
MATERIALS AND METHODS
Agrobacterium tumefaciens strain LBA4404 harbouring the endochitinase gene from Trichopderma harzianum in pBINAR vector under control of 35S promoter and NOS terminator (Supplied by IIHR, Bangaluru) was used for transformation.
Overnight grown LBA-4404 (Agrobacterium tumefaciens) cells were harvested by centrifugation and resuspended in 10mM MgSO4 soluion to maintain optimum OD at 600 nm.
0.3 cm long shoot tips excised from in vitro grown guava seedlings were used as explant for transformation.
A total of 5000 explants were micro-wounded by tungsten particles (06-1 µm) using Gene Pro-HE-2000 ballistic gun apparatus at 10, 11 and 12 Kg/cm2 pressure and 9, 10 and 11 cm distance.
Wounded explants were infected with Agrobacterium for 45 minutes followed by co-cultivation on MS media for 48, 72 and 96 hours.
Various antibiotics (Cefotaxime 250 mg/l, Streptomycin 100 mg/l, Carbenicillin 100 mg/l, and Rifampicin 100 mg/l) in different combinations were used to control the infection of Agrobacterium.
Agrobacterium treated shoot tips were proliferated and selected on MS medium supplemented with BAP (2 mg/l) and Kanamycin (300 mg/l) for 24 weeks.
Selected shoots were rooted on MS media supplemented with IBA (100 ppm) and activated charcoal (200 mg/l).
Putative transformants were PCR analyzed for endochitinase and npt-II genes using gene specific primers.
1. In vitro grown Seedlings
2. Explants for infection
3. Agro-infection
4. Control of Agro-infection, induction and multiplication on selection media
5. Selection on selection media
6. Rooting of selected shoot
7. Rooted putative
transformants
Table 1: Gene specific primers used for PCR diagnostics of putative transformants
Primer pair
Sequence
Offset
NptII Forward
5’-TCTCACCTTGCTCCTGCC-3’
480 bp
NptII Reverse
5’-AGGCGATAGAAGGCGATG-3’
Chitinase Forward
5’-TTAATTTGTTACGGAATCATAGA-3’
1200bp
Chitinase Reverse
5’-TTGAGACCGTTTCGGATGTT-3’
Table 2: PCR conditions used for gene specific primers of nptII and endochitinase genes
PCR steps I
(1cycle) II
(30 cycles)
III
(1 cycle)
Name of gene
Initial denaturation
Denaturation
Annealing
Extension
Final extension
Npt II
94 oC for 3 min.
94 oC for 1 min.
54.4 oC for 1 min.
72 oC for 1 min.
72oC for 10 min.
Chitinase
53 oC for 1 min.
72 oC for 2 min.
Results
0.3 cm long shoot tips excised from in vitro grown seedling were found most responsive to transformation.
Highest transformation efficiency was achieved when explants were microwounded at 11 kg/cm2 pressure using ballistic gun.
72 hours co- cultivation was found ideal for integration of Agrobacterium under dark conditions at 25 oC temperature and 55% RH.
Cefotaxime and Streptomycin (250 mg/l each) solution was found effective in controlling Agrobacterioum.
Kanamycin selection pressure of 300 mg/l was best.
21 putative transformants were found to be PCR positive out of 5000 explants.
DNA samples of the possible transformants are being subjected to Southern hybridization for furthur confirmation of gene transfer and integration.
Conclusion
Out of 5000 shoot-tip explants targetted for individual transformation events, 21 possible transformants were obtained showing transformation efficiency of 0.42 %.