Towards Agrobacterium-mediated transformation of Capsicum annuum L. var. 'Sweet banana'

Abstract

The aim of this research project was to develop an Agrobacterium-mediated transformation system and a regeneration protocol for Capsicum annuum L. ('Sweet banana'). The upper hypocotyl of an 11-day old seedling of this variety formed a rosette of shoot buds when cultured in liquid shoot inductive medium supplemented with 3%(w/v) sucrose and 5mgL-l benzylaminopurine for a period of 20 days. Shoot formation was inhibited by the antibiotic kanamycin at 50mgL-¹ or higher. Fresh weight change of the upper hypocotyl decreased drastically at this concentration of kanamycin. A minimum of 4 days culture from the outset in the medium containing 50 mgL-¹ kanamycin was required for the inhibition of shoot formation. Once shoot induction had occurred transfer to medium having kanamycin did not affect the development of shoot buds. Explants were inoculated with rapidly growing cultures of three Agrobacterium tumefaciens strains and then selection of transformants was carried out by culturing the explants in shoot inductive medium with 50mgL-¹ kanamycin for 20 days. Attempts to transform the upper hypocotyl explant with A. tumefaciens having the binary plasmid pBI 121 (with kanamycin-resistance selectable gene and β-glucuronidase marker gene) were unsuccessful. Transformation of mature plant tissue with A. tumefaciens containing pIG 121 (P-glucuronidase-intron) showed that the variety of Capsicum was susceptible to the strains C58::pIG 121 and LBA4404::pIG 121 but not to A4T::pIG 121. Leaf, stem, petal and anther explants expressed GUS activity after 48 hours co-cultivation. Having shown that C.annuum L. (‘Sweet banana') could express introduced genes, further co-cultivation experiments were carried out with the upper hypocotyl explant. The upper hypocotyls explant may be recalcitrant as shown by the lack of GUS expression after several trials. Further studies have to be carried out to determine the culture conditions that will render the explant competent. The shoot bud rosette induced on the upper hypocotyl developed leaf-like structures when cultured on Murashige and Skoog basal medium. Supplementing the basal medium with 0.05mgL-¹ napthaleneacetic acid and 0.02 or 0.5mgL-¹ indolebutyric acid produced roots on the excised shoot buds from the rosette but the shoots did not develop further. The optimization for the whole plant regeneration protocol is another area for further research.