In vitro Propagation of Anthurium andreanum cv. Temptation

Seeds of Anthurium andreanum cv. Temptation collected from the flower spadices were germinated on MS medium supplemented with 1.0 mg l -1 BAP. After two weeks, 100 % of the seeds germinated. Four weeks later, micro-cuttings from the in vitro germinated seedlings were subcultured on Murashige and Skoog's medium containing 0.5 mg l -1 BAP and 1.0 mg l - 1 GA 3 . On an average, 5.9 shoots per explant were obtained. The highest number of roots per shoot and the longest roots were obtained on media containing Nitsch's basal salts with 1.0 mgl -1 IBA. Anthurium andreanum plants regenerated by organogenesis were transferred to pots and 80 per cent of the plants acclimatized successfully.

The Anthurium genus comprises about 1500 tropical species which are important ornamental plants and are normally propagated by seed (Dufour and Guerin, 2003).The traditional techniques of vegetative propagation such as the use of stem cuttings and suckers exist; they are tedious and not practical when carried out on a large scale.Vegetative propagation methods applied to these plants have not shown good results and tissue culture techniques appear as an alternative to increase the production (Pierik et al., 1974, Chen et al. 1997).Tissue culture can also provide a source of clean material which has become increasingly important due to outbreak of bacterial and other diseases.Anthuriums are highly amenable for in vitro propagation using different parts as explants.Plant regeneration of Anthurium andreanum has been achieved through adventitious shoot formation from callus (Pierik et al. 1974;Pierik and Steegmans, 1976) and direct shoot regeneration from lamina explants (Martin et al. 2003).The production of in vitro plants directly from proliferating axillary buds (Kunisaki, 1980), adventitious buds (Cen et al., 1993), leaf or petiole organogenic callus culture (Kuehnle and Sugii, 1991) and from somatic embryos derived from in vitro grown leaf blade explants (Kuehnle et al., 1992) has been reported.All workers found that there was great variation in the requirements of different genotypes.One such protocol for large-scale micropropagation of Anthurium andreanum is reported here.

Materials and Methods
Explants were obtained from plants of A.andreanum cv.Temptation germinated from seeds.The fruits were separated from spadices and sterilized for 15 minutes in 0.1 % mercuric chloride and then rinsed three times with sterile water for 10 minutes.Eighty seeds were collected and sterilized for 5 minutes in 0.1% mercuric chloride, and afterwards they were washed two times with sterile water for 10 minutes.After washing 4-5 times with sterile distilled water, the seeds were inoculated on basal medium consisting of Murashige and Skoog's (1962) salts and vitamins, 3% sucrose and 0.2% Gelrite.The media was buffered to a pH of 5.8 and dispensed in culture bottles before autoclaving at 121°C for 15 minutes.Basal MS medium was supplemented with various concentrations of BAP (0.5, 1.0, 1.5, 2.0 and 2.5 mg l - 1 ) for seed germination.One week later the radicle emerged and shoot developed under continuous light.After four weeks, micro-cuttings were subcultured on MS medium supplemented with BAP (0.5, 1.0, 1.5, 2.0 and 2.5 mg l -1 ) and GA3 (1.0 mg l -1 ) and incubated under continuous fluorescent light (50 µEM -2 S -1 ) at 25ºC.This process of subculturing was continued repeatedly every 30 days for three cycles.After three months, the micro-shoots were transferred to rooting medium consisting of Nitsch's and Nitsch's (1969) medium containing various concentration of IBA (0.25, 0.50, 0.75, 1.00 and 1.25 mg l -1 ).After three months, the seven months old rooted plantlets were removed from the culture bottles.
The roots were washed in tap water and the plants were transferred to pots containing a mixture of Cocopeat and Perlite (1:1).The plants were kept 119 in hardening chambers with high relative humidity (70-80%) and low light intensity.One month later, when the plants had acclimatized well, they were transferred to the greenhouse.

Results and Discussion
Among the different concentrations of BAP used for in vitro seed germination, best response of radicle emergence was obtained on MS + 1.0 mg l - 1 BAP (Table 1).Puchooa and Sookun (2003) had also reported that BAP is of essential significance for the development of shoots in Anthurium.However, high level of BAP (2.5 mg l -1 ) suppressed the growth.Teresa Vargas et al., (2004) had established an efficient regeneration system for Anthurium andreanum cv.Rubrun, seeds from in vitro seed germination on a medium supplemented with 2.2 mM BAP. Formation of microshoot was noticed after 4 weeks of inoculation.A two-fold increase in multiplication was observed in 8 to 10 weeks.A similar observation was made earlier by George and Ravishankar (1996) in Vanilla planifolia.Further, transfer to the MS media supplemented with BAP (1.0 mgl -1 ) and GA3 (1.0 mg l - 1 ) resulted in a two-fold proliferation of shoots at every subculture (Table 2).
The elongated shoots (4 to 5 cm long) were excised and cultured separately in NN medium containing various concentration of IBA to induce roots.The highest number of roots per shoot and the longest roots were obtained on the media containing NN+1.0 mgl -1 IBA (Table 3).Earlier, Somaya et al.(1998) observed high rooting percentage of Anthurium andreanum cultured in NN medium supplemented with IBA (1.0 mg l -1 ).
From the present study, an effective plant multiplication protocol has been standardized which can be exploited on the commercial scale.