Development of transgenic banana cv Rasbale (syn Rasthali AAB) resistant to Fusarium wilt

In an attempt to genetically modify Banana cv Rasbale (syn Rasthali, AAB, Silk ), one of the favored cultivars of Southern India which is reaching extinction due to  infection by Fusarium oxysporum f.sp. cubense Race1, embryogenic cell suspensions of the cultivar were transformed with antimicrobial peptide gene cloned in-house from Allium cepa seeds and the construct developed in pCAMBIA 2301.  Around 62 original transformants were produced.  First batch of transformants (28 nos in total) which  tested positive for integration of the gene by PCR, RT-PCR and  Southern Blot, were further multiplied by micro propagation.  Level of expression of the transgene Ace-AMP1 was tested by ELISA using gene-specific antibody developed in Guinea pigs.  Plants with levels higher than 30 ng g-¹ leaf were selected for root challenging with Fusarium oxysporum f. sp cubense (FOC) in pots. The inoculum was prepared on sterilized sorghum grains for two weeks at 28°C.  Plants were root challenged with FOC inoculum (150 g plant^-1  containing 10⁸ CFU g^-1soil) in two replicates along with nontransgenic controls.  The scoring of disease was according to the extent of leaf yellowing, wilting and stem splitting  (0 = no symptoms, 5 = stem base splitting) (Nasir et. al (2003). The scores were converted into percent disease index (PDI).  Of the 21 root challenged  transgenic plants,  7 plants  showed 0-20  PDI, 3 plants 20-40 PDI,3 plants 40-60,4 plants 60-80 and the rest 80-100 PDI.  Except the 7 plants with a PDI of 0-20 all the rest progressively succumbed to the disease over a seven  months observation period.   Non-transgenic plants succumbed to the disease within a month.    Seven  plants showing 0-20 PDI were  multliplied for further field testing.  Second batch of thirty four remaining original transformants are being  evaluated similarly.