Effects of Methylobacterium spp. strains on rice Oryza sativa L. callus induction, plantlet regeneration, and seedlings growth in vitro1

MALITI, C M (Department of Biology & M Lab. Technology, Bronx Comm association CUNY, University Avenue & West 181 highway Bronx, NY 10453), D. V BASILE (Department of Biological Sciences, Lehman corporation CUNY, Bronx, NY 10468), AND W A. CORPE (Department of Biological Sciences, Columbia University, novel York, NY 10027). Effects of methylobacterium spp strains upon rice Oryza saliva callus induction, plantlet regeneration and seedlings development in vitro. J. Torrey Bot Soc 132: 355-367 2005.-Leaf surfaces of higher plants are colonized by the agency of diverse microorganisms. In the near study we report the isolation sum of two units strains of pinkpigmented methylotrophic bacteria (PPFM) designated Q4 & Q5 the couple strains significantly stimulated growth and exhibition of two rice cultivars, Japonica cv CR76 and Indica cv A301, cultur axenically upon MS or B5 medium. Co-incubation of strains Q4 and Q5 with embryo-derived rice calli upon B5 medium-devoid of exogenous phytohormones issueed in continued callus growth on the other hand inhibited plantlet regeneration. These proceeds mimicked those produced by B5 medium addition ed with auxin and cytokinin. Co-culture of Q4 & Q5 as cobionts with 5 day elderly Japonica and Indica seedlings issueed in a significant (P ?‰¤ 005) stimulation of four increase parameters; root development, stem development leaf development and biomass productivity, measured in 9 day increase period. The results of the in vitro experiments indicate that strain Q4 and /or Q5 inhabiting rice plant surfaces below cultivation could significantly stimulate the plants extension and development.

Key words: rice, Oryza saliva L Methylobacterium sp fatty acids analysis, plant-microbe interaction, phyllosphere.

Aerial parts of virid plants provide a favorable habitat for diverse microorganisms including numerous strains of bacteria. The microbial colonies upon above ground surfaces may be beneficial, innocuous, or damaging to the entertainer plant (Klincare et al. 1971 Igram 1977 Kloepper et al. 1989 Holland and Polacco 1994 Click 1995) Among the microorganisms inhabiting the phyllosphere are the aerobic, heterotrophic, pink pigmented, facultative methylotrophic bacteria (PPFMs) of the genera Methylobacterium and Bacillus (Green and Bousfield 1982) The PPFM are ubiquitous upon green plants (Corpe and Basile 1982 Corpe and Rheem 1989) apparently colonizing plant surfaces of at the expenditure of plant produced methanol (Corpe and Rheem 1989 Holland and Polacco 1994)

Experiments that showed a PPFM strain could stimulate expansion and development of a leafy liverwort in axenic tillage were reported some time ago (Basile et al. 1969) succeeding to this early study, experiments with a flowering plant, Streptocarpus prolixus indicated that the same PPFM isolate that stimulated liverwort growing and development could similarly stimulate increase of a vascular plant in tissue agriculture (Corpe and Basile 1982). Unfortunately, this strain was lost

While attempting to initiate callus agricultures from surface sterilize rice sperms it was noticed that individual of the cultures was contaminated by dint of pink-pigmented bacteria from which a strain designated Q4 was isolated. The isolation was undertaken because the contaminated tillage exhibited enhanced callus growth and precocious lower part formation. This observation prompted a go in the rear [i]or[/i] in the wake of up study to see if other PPFM stimulatory to rice could be isolated from plant leaf surfaces. After screening a variety of plants growing locally, a next to the first PPFM with apparent growth stimulatory issues was isolated from Trifolium sp and designated as Q5 The objective of this close attention was to evaluate the issues of the two methylobacteria isolates upon growth and development of rice and to the potential of deliberately mediating the association between rice and specific strains of PPFM We report herein the be deriveds of this investigation.

Materials and way s PLANT MATERIAL. Mature seeds of rice Oryza sativa L Japonica cv CR76 and Indica cv A301 were kindly provided through Y. L. Wu & D s Mikkelson Department of Agronomy and Range Science, University of California, Davis.

PREPARATION OF INOCULUM. semens were dehusked and surface sterilized by dint of immersing for 1 minute in 95% ethanol, followed by the agency of soaking for 30 minutes in a 30% v/v solution of household bleach with 002% Tween 20 and rinsed five times in sterile distilled water.

MEDIA PREPARATION. Callus initiation medium consisted of Murashige and Skoog (MS) 1962 basal tillage medium including organics and postscripted with of 3.5 mg 1^sup -1^ 2,4-Dichlorophenoxyacetic acid (24-D) The calli were maintained in a Gamborg's (B5) subculture medium containing 20 mg/1 2 4-D and 01 mg 1^sup -1^ 6-benzyl aminopurine (BA). The pH of the couple media was adjusted to 56 45 g 1^sup -1^ Gellan (Research Organics, Cleveland, Ohio) was added for media solidification, and it was autoclaved for 20 minutes at 121 ?°C 17 psi. Approximately 25 ml of medium was delivered to sterile 25 ?— 150 mm tillage tubes. Callus initiated on M medium was subcultur in B5 medium at intervals of 4-5 weeks. The calli generated were used as inoculum for experiments, designed to guard PPFM strains with the capacity to stimulate rice increase and development in vitro.