Received: 14 October, 2011. Accepted: 6 December, 2011. Original Research Paper Floriculture and Ornamental Biotechnology ©2011 Global Science Books In Vitro Flowering Studies with Nine Cultivars of Perennial Ryegrass (Lolium perenne L.) Puthiyaparambil Josekutty • Shobha Devi Potlakayala • Rebekah Templin • Sairam Rudrabhatla * School of Science Engineering and Technology, Pennsylvania State University, 777W Harrisburg Pike, Middletown, PA 17057, USA Corresponding author: * svr11@psu.edu ABSTRACT We report here in vitro flowering in six cultivars namely; ‘Banquet’, ‘Gulf annual’, ‘Linn Perennial’, ‘Meridian’, ‘Quartet’ and ‘Tolosa’ of perennial ryegrass (Lolium perenne L.). The frequency of in vitro flowering varied from 2.5% for ‘Tolosa’ to 90% for ‘Gulf annual’. Out of the five media (RM1-RM5) tested to induce flowering in vitro, only RM5 medium (MS salt + vitamin + 0.50 mg L -1 6-benzyladenine (BA) + 2.0 mg L -1 thidiazuron (TDZ) + 38.0 mg L -1 CuSO 4 .·H 2 O) produced in vitro flowering. Vernalization (one week at 4°C) of the in vitro shoots improved the flowering efficiency by 35% compared to the non-vernalized control. Callus was induced from mature seeds on medium containing MS salts and vitamins with 4.0 mg L -1 2,4-dichlorophenoxyacetic acid (2,4-D) and 30 g L -1 sucrose. Unlike previously published reports of callus-based regeneration that requires several months in culture and associated somaclonal variation and albino shoot formation, we obtained normal shoots from mature seed-derived callus in 10-12 weeks. _____________________________________________________________________________________________________________ Keywords: in vitro breeding, pasture grass, tissue culture, turf grass Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; BA, 6-benzyladenine; CIM, callus induction medium; GM, genetically modified; Kn, kinetin; PBZ, paclobutrazol; RM, regeneration medium; TDZ, thidiazuron INTRODUCTION Ryegrass is the most widely cultivated forage grass in the world. Major species and hybrids of this genus include Lolium perenne L., L. multiflorum Lam., and L. x bouche- anum Kunth. It is a nutritious and palatable forage grass with good adaptability and growth habits. Therefore, it is a preferred pastoral grass in North America, Europe, Austra- lia, New Zealand and other temperate regions of the world (Jauhar1993; Harper et al. 2011; Sampoux et al. 2011). Traditional breeding and biotechnology procedures have been applied to achieve genetic improvement of rye- grass, resulting in the release of improved cultivars (Yamada et al. 2005; Wang and Yamada 2008; Ghesquiere et al. 2010; Harper et al. 2011). Resistance to rusts, was im- proved at a rate of +11.39% per decade in the past few decades and dry matter yield was improved at a rate of 3.18% per decade (Sampoux et al. 2011). Traditional breed- ing in ryegrass takes a long time due to the fact that it flowers in 12-18 months and typically sets seeds after a brief vernalization treatment (Jauhar 1993; Reheul and Ghesquiere 1996). Another major challenge with improving L. perenne through conventional breeding is its self-incom- patibility (Yamada et al. 2005; Yang et al. 2009). Addition- ally, the susceptibility of L. perenne cultivars to biotic stress (diseases) and abiotic stress such as cold, drought and salinity in the face of global climate change is a major chal- lenge for ryegrass breeders developing novel cultivars (Yamada et al. 2005; Kosmala et al. 2007). Indirect regene- ration of shoots through a callus and cell culture phases after prolonged periods of in vitro culture of ryegrass has been reported (Creemers-Molenaar et al. 1989, 1992; Dal- ton et al. 1988; Altpeter and Posselt 2000; Bradley et al. 2001; Newell and Gray 2005, Liu et al. 2006). Regenera- tion from cell culture derived protoplast cultures of ryegrass using conditioned medium is also reported (Folling et al. 1995). Polyploids, mixoploids and chimeras were among a group of in vitro regenerated rye grass plants after several months in culture (Creemers-Molenaar et al. 1992). Newell and Gray (2005) reported indirect regeneration of ryegrass plants from leaf base explants. Long period of incubation under in vitro ( 6 months) conditions resulted in somaclo- nal variation and albino shoot formation (Altpeter and Posselt 2000). Likewise, the effect of genotype on callus and shoot regeneration, rapid loss of regenerability from callus are some of the challenges for rapid cloning of rye- grass through tissue culture (Liu et al. 2006). Therefore, developing a protocol that allows rapid regeneration of callus and shoots in vitro with little or no genotype effect on regeneration and greater fidelity is highly desirable. Such an efficient and rapid regeneration protocol will improve the recovery of genetically stable transgenic ryegrass plants with greater efficiency. Genetic transformation of ryegrass with reporter genes using microprojectile bombardment (Spangenberg et al. 1995, 1998) and Agrobacterium-medi- ated genetic trans-formation (Bhalla et al. 1999; Altpeter et al. 2000; Altpeter 2006; Bajaj et al. 2006) have been repor- ted. More recently, value added genes have been incorpo- rated to develop salt resistant (Wu et al. 2005) and drought resistant (Zhao et al. 2007) transgenic ryegrass plants In vitro flowering and seed set (in vitro breeding) can complement and advance the gains made through conven- tional and biotechnology mediated genetic improvements of ryegrass. Many plants flower faster in vitro than ex vitro thus the life cycle of the plant and generation time could be cut down with in vitro breeding strategy as demonstrated by Ochatt and Sangwan (2008) in Arabidopsis thaliana. In vitro flowering of bamboo, a monocarpic species with a long pre-bearing period was developed to reduce its breed- ing cycle (John and Nadgauda 1998). Sairam and Goldman (2009) also have discussed possible applications of in vitro flowering technology such as; reducing the breeding cycle, eliminating seasonality of breeding during adverse climate, rapidly advancing traits through breeding and/or transgenic seed production. Therefore, the current study was directed at developing an in vitro flowering system for ryegrass cul- ®