Molecular investigations, cryopreservation and genetic transformation studies in papaya (Carica papaya L.) for cold hardiness

Papaya is one of the major fruit crops of the tropical regions of the world. The cold sensitivity of this crop has limited its cultivation to tropical and warm subtropical regions. The Caricaceae family consists of genera with stress tolerance traits which can be potentially transferred to papaya using a combination of biotechnology and breeding.; Carica and Vasconcella genomes were probed for the presence of cold inducible sequences similar to those found in the Arabidopsis genome using the polymerase chain reaction. These studies indicated the presence of possible cold inducible sequences in the Vasconcella genome but which were absent in the Carica genome.; A genetic transformation approach was used to transfer CBF transgenes into papaya. The CBF (C repeat Binding Factor) gene family is known to induce the cold acclimation pathway in Arabidopsis. Embryogenic cultures were transformed using the Agrobacterium tumefaciens mediated protocol. Transformed cultures harboring CBF: NPT II gene sequences were selected on maintenance medium containing 300mg L-1 kanamycin sulfate. Further selection was accomplished on somatic embryo development medium containing 150 mg L-1 kanamycin sulfate. Plantlets were regenerated and the presence of the transgene was confirmed by Southern blot hybridization.; To address the problems of loss of embryogenic potential and somatic embryo development over time, a cryopreservation protocol was developed. Slow and fast cooling methods in combination with different cryoprotectants and tissue stages were studied to determine the optimum time, cryoprotectants and method of cooling. Proembryonic masses (PEMs) used in combination with vitrification and fast cooling resulted in excellent regrowth and regeneration of plantlets following cryopreservation.; These studies should be important for research conducted for stress tolerance of papaya. The cryopreservation protocol developed for papaya should help in the long term storage and maintenance of germplasm and experimental material.