MSc

An experiment on “Validation of tests for sex determination in papaya (Carica papaya L.)” was carried out at the Department of Pomology and Floriculture and Department of Plant Biotechnology, College of Agriculture, Vellayani during 2011-13 to compare the efficiency of chemical tests developed for identification of sex forms in papaya in order to find a reliable test for sex determination of papaya plants in the pre-flowering stage. Attempts were also made to identify the sex forms in papaya through molecular characterization using Random Amplified Polymorphic DNA (RAPD) technique. The colorimetric tests along with molecular analysis can be an efficient tool for more accurate prediction of sex forms in papaya.
Papaya varieties selected for the present study were Pusa Dwarf, Pusa Nanha and Washington. Two months old seedlings raised in polythene bags were serially numbered and transplanted into the main field. Two to three newly emerged leaves were collected from one and half months old nursery seedlings and the filtered aqueous extract was prepared. Chemical tests such as Almen reagent test, Ammonium molybdate test, Ferric chloride test and Titanous chloride test were carried out at pre-flowering stage and the results were compared with that from the field established plants after flowering. Almen reagent test was found to be the best in determining the male and female sex forms in papaya varieties. The accuracy of prediction of sex form was 67 per cent for males and 71 per cent for females.
For molecular analysis, genomic DNA was isolated from young leaves of all the varieties using Cetyl trimethyl ammonium bromide (CTAB) method. A total of 10 random primers (OPA3, OPA13, OPB1, OPB4, OPB17, SO1, SO7, RY01, RPI01, and RPI02) were used for study.Out of these, six primers (OPA13, OPB1, OPB17, SO1, SO7 and RY01) yielded amplification products with the DNA from both male and female sex forms of all the three varieties. Among these six primers, four primers (OPA13, OPB1, OPB17 and SO1) gave polymorphic banding pattern.
The primer SO1 (5’CCACCACGAC 3’) proved to be highly effective for the discrimination of male papaya plants of all the three varieties tested. RAPD profiles with SO1 primer showed two distinct bands, one with 500 bp and another with approximately 400 bp in male plants of all the three varieties. Primer OPB1 could differentiate sex forms of two varieties, viz. Pusa Dwarf and Pusa Nanha, while male and female plants of variety Washington could not be differentiated. With OPB1, one male specific band of approximately 1250 bp was present in Pusa Dwarf and Pusa Nanha while in Washington variety that band was present in both the sex forms. OPA13 helped to distinguish Pusa Nanha female plants with the presence of two bands at approximately 400 bp and more than 1.2 kb size. With OPB 17, an amplicon of approximately 300 bp was found distinctly in the variety Washington. But it was unsuccessful in differentiating sex forms in other two varieties.
The present investigation showed that the Almen reagent test and RAPD profiling with the decamer primer SO1 (5’CCACCACGAC 3’) can be used for the determination of the sex forms of papaya in the pre-flowering stage.