Received: 1 May, 2008. Accepted: 21 September, 2008. Original Research Paper Floriculture and Ornamental Biotechnology ©2008 Global Science Books In Vitro Mutation in Rosa hybrida cv. ‘Pusa Gaurav’ and Selection through RAPD and ISSR Markers Sunil Kumar Senapati • Anuradha Mahapatra • Gyana Ranjan Rout * Plant Biotechnology Division, Regional Plant Resource Centre, Bhubaneswar, 751 015, Orissa, India Corresponding author: * grrout@rediffmail.com ABSTRACT Apical and axillary meristems of Rosa hybrida cv. ‘Pusa Gaurav’ were pretreated with various concentrations (0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0 μM) of oryzalin (C 12 H 18 N 4 O 6 ) to induce variation in vitro. Depending on the concentration of oryzalin used, the mean survival rate of meristem culture decreased from 98.9% (5.0 μM, 0 h) to 9.85% (30 μM, 36 h), but the lethal dose (LD 50 ) was 20 μM oryzalin pretreated for 24 h. Oryzalin-treated microshoots were used to multiply shoots. The maximum rate of shoot multiplication occurred on Murashige and Skoog (MS) medium supplemented with 2 mg/l BAP (6-benzylaminopurine), 0.25 mg/l IAA (indole-3-acetic acid), and 50 mg/l Ads (adenine sulfate) and 20 μM oryzalin. The elongated shoots were rooted in half-strength MS medium supplemented with 0.25 mg/l IBA (indole-3-butyric acid) and about 65% rooted plants survived in the greenhouse. Molecular [Randomly amplified polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR)] analyses were used to determine the genetic variability of the meristems of oryzalin-treated plants. Six out of 20 RAPD primers and five out of 12 ISSR primers revealed polymorphisms (30.5 and 61.8%, respectively) among the in vitro plants indicating the efficiency of oryzalin to induce in vitro variability in hybrid rose and to detect variation through molecular markers. A comparative morphological analysis between the control and the mutant was done, which showed minor variations in some morphological characters (plant height, number of branches, foliage size, thorn density, flower diameter, flower depth, number of petals. Our findings will provide fundamental insight into an improved rose breeding program. _____________________________________________________________________________________________________________ Keywords: hybrid rose, molecular marker, mutant, oryzalin Abbreviations: Ads, adenine sulfate; BAP, 6-benzylaminopurine; IAA, indole-3-acetic acid; ISSR, Inter simple sequence repeat; Kn, kinetin; LD 50 , lethal dose; NAA, 1-napthalene acetic acid; RAPD; randomly amplified polymorphic DNA INTRODUCTION Over the past few decades, breeders have faced many prob- lems in rose improvement due to low sexual reproduction and poor germination of seed because of embryo abortion. Since the first report by Elliott in 1970 on shoot multiplica- tion and rooting of rose many studies have been conducted using different concentrations of hormones for the initiation of callus, maintenance and regeneration of shoots and roots from the callus and directly from nodal segments, lateral and axillary buds and shoot tips (Rout et al. 1991; Vijya and Satyanarayan 1991; Rout et al. 1992; Chu et al. 1993; Syamal and Singh 1994; Kintzois et al. 1999; Dobois et al. 2000; Mohapatra and Rout 2005; reviewed in Khosh-Khui and Teixeira da Silva 2006). The application of tissue cul- ture techniques for the regulation and commercial propaga- tion of rose has been developed more recently. Among the rose cultivars, about 20% were developed on the basis of spontaneously derived sports (Krussmann 1974). This rela- tively high percentage may provide an opportunity to apply mutation induction techniques to rose breeding programs. Thus, induced mutation (both physical and chemical muta- gens) in combination with in vitro culture could provide an opportunity to increase variability of an economically im- portant cultivar and to obtain information about the poten- tial of spontaneously arising sports in new variety. Induced mutation has been used to generate a wide range of mutants for enhanced response to abiotic and biotic stress (Jain 2005; Kumar et al. 2006). Oryzalin (C 12 H 18 N 4 O 6 ) is a selec- tive surface applied herbicide used as a chemical mutagen, inducing spindle inhibition. It has much more affinity for plant microtubules and is less hazardous to human health than other chemical mutagens like colchicine (Hugdahl and Morejohn 1993). Molecular marker techniques such as Ran- domly Amplified Polymorphic DNA (RAPD) and Inter Sim- ple Sequence Repeat (ISSR) have proven to be a powerful tool for estimating genetic variation in various plant groups (Williams et al. 1990; Pharmawati et al. 2004; Mohapatra et al. 2005). To our knowledge this is the first study on in- ducing mutations in meristem cultures of rose which was carried out to develop improved varieties through in vitro mutagenesis by using oryzalin as a chemical mutagen in hybrid rose and to evaluate genetic variability among the mutants by using molecular markers. MATERIALS AND METHODS Material and in vitro culture Cuttings (about 15 cm long from the apical end) of Rosa hybrida cv. ‘Pusa Gaurav’ were collected from the rose garden of the Re- gional Plant Resource Center, Bhubaneswar, India and brought to the laboratory in a beaker with distilled water. Nodal segments with internodes (~ 0.25 cm long) were used as the explant source. About 0.5-1.0 cm of internodes containing nodes were cut and washed thoroughly under tap with 2% (v/v) detergent (Labolene, Qualigen, India) for 15 min. This was followed by washing in run- ning tap water for 15 min. Surface sterilization was made with 0.1% (w/v) mercuric chloride (Qualigen, India) for 15 min and subsequently washed with sterile distilled water three times. Twenty nodal explants were cultured in the test tubes (25 x 150 mm) containing MS (Murashige and Skoog 1962) medium sup- plemented with 6-benzylaminopurine (BAP), kinetin (Kn), ade- nine sulfate (Ads) (Sigma, USA) in different concentrations and combination with indole-3-acetic acid (IAA) and 1-naphthalene acetic acid (NAA) (Sigma). Each tube having one nodal explant ®