Liquid Culture Systems for in vitro Plant Propagation

374 Pratap Kumar Pati et al.
Roses are generally propagated by vegetative methods such as cuttings,
layering, budding and grafting. Seeds are also used for propagation of
species, new cultivars and for production of rootstocks (Horn, 1992).
However, in vitro propagation methods have attracted attention of many rose
growers for having enormous potential for mass multiplication of elite
clones and production of healthy, and disease-free planting material,
especially in ornamental roses, namely Rosa hybrida (Short and Roberts,
1991; Horn, 1992; Taslim and Patel, 1995; Rout et al., 1999). Among oilbearing
roses there is no report on in vitro propagation in R. bourboniana
and only fragmentary information is available for R. damascena (Khosh-
Khui and Sink, 1982; Ishioka and Tanimoto, 1990; Koronova and
Michailova, 1994; Kumar et al., 2001).
In rose micropropagation, agar-gelled medium is most frequently used.
However, the use of liquid media during different stages of in vitro
propagation and improvement within the existing tissue culture protocols has
been successfully demonstrated for multiplication of R. damascena and R.
bourboniana. The different stages, namely, initiation of aseptic culture,
shoot proliferation, rooting of microshoots, hardening and transfer to soil
have been standardized for production of tissue culture raised plants in a cost
effective manner. The present work also highlights the significance of
osmotic potential for enhanced performance of rooting or microshoots in
liquid cultures compared to agar-gelled cultures.
2. Materials and methods
2.1 Initiation of aseptic cultures and shoot proliferation on agargelled
and liquid media
Nodal explants (2.5-3.0 cm), taken from field-grown plants of R.
damascena and R. bourboniana were thoroughly washed with Tween-80 for
20 minutes, followed by surface sterilization with an aqueous solution of
0.04% (w/v) mercuric chloride for 7-8 minutes and subsequent rinsing (3-4
times) with sterilized distilled water. These were inoculated and maintained
for 3-4 weeks on Murashige and Skoog (1962) medium (MS) containing
3.0% (w/v) sucrose and 0.8% (w/v) agar for initial screening. The pH of the
medium was adjusted to 5.8 before autoclaving. Cultures were incubated at a
photosynthetic photon flux density (PPFD) of 20 �mol m -2 s -1 from cool
white fluorescent lamps at 25�2 o C. Daylength was 14h in a 24h light/dark
cycle.