INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY 1560–8530/2003/05–3–298–302 http://www.ijab.org In Vitro Induction of Polyploids in Watermelon and Estimation Based on DNA Content HASNAIN RAZA 1 , M. JAFAR JASKANI, M. MUMTAZ KHAN AND TANWIR A. MALIK† Institute of Horticultural Sciences and †Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad–38040, Pakistan 1 Corresponding author E-mail: mnhasnain19@yahoo.com ABSTRACT Polyploids are desired to produce seedless fruits in watermelon. In vitro techniques pave way towards production of tetraploids by culturing explants on media containing colchicine. Cotyledon, embryonic end of seed, epicotyl and hypocotyl explants were cultured on MS media supplemented with BA (1 mg L -1 ). Shoot proliferation response was maximum in cotyledonary explants cultured on 0.01% colchicine level for four days. The DNA content of regenerated plants was estimated using spectrophotometery. DNA content was found double in tetraploids (4.41μg mL –1 ) than diploids (2.18 μg mL –1 ) in unit gram of sample. Cotyledon explants cultured on colchicine supplemented medium (0.01%) for four days resulted in maximum tetraploids than plantlets regenerated from epicotyl and hypocotyl explants. Embryonic ends and cotyledon as explants produced aneuploids when cultured on the same media for seven days. Key Words: Polyploids; Colchicine; Watermelon; In vitro; Ploidy estimation INTRODUCTION Watermelon (Citrullus lanatus Thunb) is an important Cucurbetaceous vegetable. In Pakistan, it is grown over an area of 19 thousand hectares with a production of 420 thousand MT (FAO, 2002). Seedlessness is an important and desirable breeding objective in horticultural crops. In watermelon polyploidy is utilized to produce seedless fruits. Seedless watermelons are triploids (3x=33) and result from crossing a tetraploid (2n=4x=44) seed parent with a diploid (2n=22) pollen parent (Andrus et al., 1971; Kihara, 1951). Seedless (triploid) watermelons are preferred by consumers because of high fruit quality and absence of seeds; even consumers are willing to pay 50% more per pound (Marr & Gast, 1991). Polyploid watermelons are also found to be resistant to watermelon fruit blotch (Acidivorax avenae subsp. citrulli) and nematodes (Montalvo & Esnard, 1994; Garret et al., 1995). Moreover, in triploid watermelons orange flesh turns into deeper orange color as it ripens and flavor can even improve after harvesting. Also, their tough sunburn-resistant rind makes them excellent for long- distance shipping. However, production of seedless watermelons have been hampered by high seed cost ($150-$200/1000 seeds) and poor seed germination. High seed cost has generally attributed to difficulties in obtaining a sufficient number of tetraploid plants as they exhibit low fertility and generally require at least 8-10 years of self pollination before enough plants are obtained for commercial triploid seed production (Compton & Gray, 1992). Moreover, in vivo treatment of colchicine results in a mixed population of diploid, tetraploid, aneuploids and sectoral and periclinal chimeras. The induction of tetraploid in vitro offers an alternative method to obtain tetraploid plants because of reduction in number of aberrant plants produced and also reduce the time span required for triploid seed production from 10 years to approximately 1-2 years and also allows use of male sterile lines of tetraploids without cumbersome maintenance lines (Compton & Gray, 1992; Compton et al., 1993). The objective of the study was to produce watermelon polyploids in vitro for breeding seedless watermelons. As in advanced countries people are willing to pay 50% more for seedless watermelon than seeded watermelons, there is a potential for seed companies and growers to export seedless watermelon which in turn will be good source of foreign exchange earning. MATERIALS AND METHODS Seeds of diploid watermelon (Citrullus lanatus Thunb.) cultivar ‘Sugar Baby’ were sterilized in 2.5% NaOCl plus one drop 100 mL -1 Tween 20 for 30 min followed by three rinses with autoclaved double distilled water. The seeds were soaked for 5 h for ease in removing seed coat. The embryos were surface disinfected for 20 minutes in 1.25% NaOCl plus one drop 100 mL -1 of surfactant Tween 20 prior to three rinses with sterile double distilled water and were cultured on MS medium supplemented with Benzyl Adenine (BA; 0, 1 or 5 μM) to test the effect on seed germination and regeneration. Transversly cut epicotyl (E 1 ), transversly cut hypocotyl (E 2 ), cotyledons (E 3 ) and the embryonic end (E 4 ) of in vitro grown seedlings were used as explants. Later the best level of BA in the medium was used for explants culturing on following media formulations for 4 days (D 1 ) and 7 days (D 2 ).