Callus Induction and Organogenesis from Pueraria tuberosa (Roxb. ex Willd.) DC

Pueraria tuberosa (Roxb. ex Willd.) DC. is a perennial woody climber, commonly known as Indian Kudzu in English, Vidarikand in Hindi and Vidari in Sanskrit. The tubers are used in different systems of medicine viz. Ayurveda, Folk, Homoeopathy, Siddha, Tibetan and Unani. The present study aims to develop an effective protocol for optimum callus induction and organogenesis in Pueraria tuberosa. Callus cultures were first established by inoculating tender leaf explants in Murashige and Skoog's (MS) medium supplemented with different concentrations of 6-Benzylaminopurine (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg.L-1) with 1-Naphthaleneacetic acid (0.5 mg.L-1). Maximum callus induction and shoot organogenesis was observed in MS medium containing 1.5 mg.L-1 BAP with NAA 0.5mg.L-1. Fresh weight of the organogenic greenish granular hard callus was 4.70±0.10 gm. Shoot organogenesis was observed after 65 days of culture. Maximum shoot buds developed from callus on MS media augmented with 1.5 mg.L-1 BAP with 0.5 mg.L-1 NAA.


INTRODUCTION
Pueraria tuberosa belonging to family Fabaceae is distributed throughout tropical parts of India. It is a large climbing shrub with trifoliate leaves and bluish-purple flowers. Ayurvedic physicians use the tubers for the management of general weakness, fertility disorders and also as anti-ageing, Pandey et al., (1998). The tuberous roots act as a galactagogue, stimulant and emollient, Warrier et al., (1995). Its tubers are rich in isoflavonoids and terpenes with puetubersoanol, and tuberosin , Khan et al., (1996). Puerarin, diadzein, genistein and genistin are the isoflavonoids present in callus cultures of P.tuberosa, Kamlesh et al., (2006). Increased isoflavonoid production was reported by elicitation effect in cell cultures of P.tuberosa, Shaily and Ramawat, (2008

Plant sample and experiment designing
Young, tender leaves collected from three months old vegetatively propagated plants were first washed in running tap water for 10 minutes and then soaked in soap water for 3 minutes. The leaves were cut into small fragments and then immersed in cefotaxime (200 mg.L-1), tetracycline (200 mg.L-1) and bavistin (15 g.L-1) for 5 minutes. The bavistin treated explants were then washed with double distilled sterile water for five times. These explants were then sterilized by mercuric chloride solution for 3 minutes. After several distilled water wash, 4-5 explants were inoculated into MS (Murashige and Skoog, 1962) media supplemented with 3% (w/v) sucrose, 0.2 % (w/v) clerigel and1ml lactic acid taken in petridish. The pH of the media was adjusted to 5.8 after the addition of various concentrations of BAP and NAA. The culture medium was autoclaved at 121°C, 15 psi pressure for 20 minutes. The media were incubated at 25 ± 2 °C under 16 hour photoperiod at a relative humidity of 55 percent with a light intensity of 3000 lux.
Leaf explants were inoculated into media supplemented with various combinations of BAP (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg.L-1) with NAA (0.5 mg.L-1). Percentage of callus induction, fresh weight and dry weight of callus, colour and texture of callus and the number of days for callus induction were the different parameters observed and recorded. Subcultures were routinely carried out every 10 days interval into fresh media. Growth of callus on different media compositions after 50 days of inoculation were measured in terms of fresh weight and dry weight. Dry weight of the callus was measured by drying at 60 o C in a hot air oven to a constant weight.

Statistical Analysis
Experiments were conducted with three replications, having 30 samples each. The effect of various treatments on different growth parameters was measured quantitatively and statistically tested using analysis of variance (ANOVA) using "R-statistics package" version 11.0. The significance of the mean values of various treatments was assessed by Duncan's New Multiple Range Test (DMRT) at p < 0.05.

III. RESULTS
The combined effect of BAP and NAA on callus induction was studied by culturing leaf explants on MS medium supplemented with various concentrations of BAP (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg.L-1) with 0.5 mg.L-1 NAA (Table 1). MS basal media without plant growth regulators were taken as control. The explants failed to establish callus on control media. Various types of callus such as compact, friable and granular were observed in different colours as green, creamy yellow and white. Decrease in percent of response (60%) was observed at lower concentration of BAP (0.5 mg.L-1) with 0.5 mg.L-1NAA. Organogenesis was not recorded from the white friable callus of this media. The fresh weight and dry weight of the callus of this particular media composition were found to be the lowest among other treatments (Table 2). Creamy yellow friable callus (Fig 1A) initiated within 12 days on media with 1 mg.L-1BAP and 0.5 mg.L-1 NAA and the fresh weight of callus harvested from this media after fifty days was 1.60±0.15 gm. Only 2 shoots were differentiated from this callus after 65 days of culture. It was observed that the callus growth was best in MS media augmented with 1.5 mg.L-1 BAP with 0.5 mg.L-1 NAA. This particular media composition resulted in 84% callus induction within 7days of inoculation (Table 1). After 50 days of culture, the fresh weight and dry weight of callus at this optimum treatment was 4.70±0.10 and 2.43±0.15 gm respectively. The green granular hard callus (Fig 1 C) resulted after 50 days of culture showed initiation of buds.
These buds developed into 5-6 shoots after 65 days of culture. At higher concentrations of 2 mg.L-1 BAP with NAA 0.5 mg.L-1, the percentage of callus induction reduced (80.3%) and the number of days for callus induction increased to14 days. The green buds which originated from the dark compact callus (Fig 1 E) of this medium developed into 3-4 shoots after 65 days of culture. During subculture after 50 days, the dark brown regions of this callus were removed to reduce contamination. The shoots elongated to a height of 3-4 cm after 85 days of culture (Fig 1F). Frequent sub culturing was done to enhance the survival rate of the callus.

IV.
DISCUSSIONS Auxin and cytokinin balance is an important factor in the control of cell division in tissue culture. In the present study, different concentrations of auxin and cytokinin influenced callus production from leaf explants. Highest percent of organogenic green granular hard callus was obtained on leaf explants grown on the medium containing 1.5 mg.L-1BAP and 0.5 mg.L-1NAA (Table 1 and