In vitro clonal multiplication of safed musli (Chlorophytum borivilianum Sant. et. Fernand.), a rare Indian medicinal herb, has been achieved on Murashige and Skoog's (MS) medium supplemented with 22.2 ~ benzyladenine using young shoot bases as explants. Shoots multiplied at a rate of four-fold every 3 weeks. All shoots rooted when transferred to MS medium with 3/4-strength inorganic and organic constituents and 9.8 laM indolebutyric acid and 67% of the micropropagated plants were successfully established in pots. Such plants produced normal fasciculated storage roots as in wild plants.

Multiple shoot buds could be induced directly from internode explants of Celastrus paniculatus inoculated on Murashige and Skoog's (MS) medium containing different growth regulators. The best response was obtained when 4.44 µM 6-benzylaminopurine (BAP) was incorporated in the medium. Incorporation of indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA) did not improve response, rather promoted callusing. Adventitious shoot buds could be multiplied and elongated on MS medium containing 2.22 µM BAP. Rooting of shoots (80 %) was obtained when their bases were dipped in pre-autoclaved indole-3-butyric acid (IBA) solution (2.45 mM) for 10 min followed by their implantation on medium containing ¼ MS salts, 1.0 % sucrose and 0.6 % agar. Out of 500 plantlets subjected to hardening, 410 were successfully hardened under greenhouse conditions. Twenty plants were established in field while remaining of them were transferred to nursery conditions without any mortality.

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A deviation from usually found characteristics of stomata in Wrightia tomentosa was noted during in vitro propagation. Increase in stomatal frequency in leaves of plants grown in vitro was observed with 29.4 % malformed stomata. The stomata were spherical, wide open, did not close in detached leaves even after 3 h. The leaves exhibited 93.4 % total water loss during 3-h period. Stomatal frequency, percentage of malformed stomata and rate of water loss declined in subsequent rooting phase. Nevertheless, for high survival rate plantlets were hardened under gradually decreasing air humidity either in partially opened glass bottles containing Soilrite TM moistened with ¼ Murashige and Skoog nutrients or in pots covered with polyethylene bags. The stomatal characteristics of hardened plants were comparable to seedlings. Survival rate was more than 95 %.Additional key words: ex vitro transfer, hardening, in vitro culture, stomatal frequency, water relations. ⎯⎯⎯⎯The transfer of woody plant species from in vitro vessels to ex vitro conditions is considered to be a critical stage in successful micropropagation. The major reason for high mortality rate during ex vitro transfer lies in excessive water loss attributed to poor stomatal functioning (Blanke and Belcher 1989), reduced leaf epicuticular wax (Sutter and Langhans 1979) and high stomatal densities (Desjardins et al. 1988). Hronková et al. (2003) have reported that the acclimation of in vitro derived plants to ex vitro conditions affected the stomata on adaxial and abaxial sides differently. Failure of stomata to close in response to darkness, applied ABA, or high CO 2 concentrations has also been reported in apple and cauliflower plants propagated in vitro (Brainerd and Fuchigami 1982). The cause of failure of stomata to close could be due to guard cell wall properties (Ziv et al. 1987), the deformation of stomata (Blanke and Belcher 1989) or the K + transport across the guard cells (Assmann 1993). Manipulation of irradiance and relative humidity (RH) in Rosa multiflora induced changes in stomatal and epidermal cell anatomy (Capellades et al. 1990).Micropropagation of Wrightia tomentosa, a threatened tree species of Aravallis in Rajasthan, India has been reported (Purohit et al., 1996). Despite high production rate, in vitro plants exhibited severe water loss and thus very high mortality during weaning period. Present study investigates stomatal features, relative water loss during different phases of micropropagation and methods for successful hardening of the plantlets for their large-scale production.Shoot cultures were initiated from aseptically grown seedlings of W. tomentosa according to the method described by Purohit et al (1996). Shoots induced from cotyledonary nodes on Murashige and Skoog (1962; MS) medium containing 22.2 μM 6-benzylaminopurine (BAP) were repeatedly subcultured every 3 weeks on lower concentrations of BAP (8.9 μM) in the medium. Elongated shoots (2 -3 cm) were rooted in vitro by giving pulse treatment of pre-autoclaved indolebutyric acid...

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