Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF CRESCENTIA CUJETE L. 435
and then transplant into 4-inch plastic pots containing a soil-less mix (Promix
BX, Premier Horticulture Ltd, Quebec, Canada).
3. After potting, water the plants with an N-P-K (20-20-20) fertilizer solution
(Plant Products Co. Ltd, Brampton, On, Canada) weekly.
4. Place the pots in a growth chamber set for 16 h light at 26°C, 8 h dark at
24°C, and 95% relative humidity. Reduce the relative humidity by 5% every
week for 5 weeks and then keep it constant at 70%.
5. At the end of acclimatization period, transfer the plants to 1-gallon pots and
place the potted plants in the greenhouse. Most of the transplants survive
and grow to the size of juvenile trees, which can be planted outdoors, 1 month
after transplanting (Figure 2H).
3. CONCLUSION
C. cujete L. is a widely distributed medicinal tree commonly known as the
Calabash Tree. The current study was undertaken to establish protocols for in vitro
maintenance and multiplication of axenic cultures of C. cujete and to develop a
large-scale production system for biochemical characterization and phytomedicine
production. The results of this study provide an optimized system for mass multiplication,
and production of sterile, consistent plant material of C. cujete.
The culture system significantly influenced the micropropagation of C. cujete
shoot cultures. Differences were observed between solid and liquid culture as well
as between the two liquid culture systems, the flask and temporary immersion
bioreactors. Liquid culture proved to be more effective in promoting shoot growth
than solid medium and no hyperhydricity of in vitro grown plantlets was observed
with liquid culture. Plantlets obtained from the temporary immersion liquid culture
were longer and of better quality than those obtained from both solid medium
culture and flask-liquid culture. Temporary immersion culture systems for micropropagation
have been shown to be beneficial for many plant species, and offer
significant advantages including: 1) efficient supply of liquid nutrition and oxygen
transfer, 2) less shear and hydrodynamic forces, 3) minimal hyperhydricity of
regenerants in the liquid medium, 4) improvement of plant quality and survival
during acclimation, and 5) automation at a relatively low cost (Etienne & Berthouly,
2002). The results with the temporary immersion bioreactor culture of C. cujete
shoots provide further evidence that enhanced atmospheric gas exchange and
reduced stress and hydrodynamic forces can improve shoot proliferation and quality
in a scaled-up micropropagation culture system with the liquid medium.
The principle prerequisite for the development of high-quality phytopharmaceutical
products is a consistent source of high-quality plant material. The development
of a regeneration protocol and scaled-up micropropagation system for C. cujete
represents a significant advancement for phytopharmaceutical production of this
medicinal species. The bioreactor multiplication can provide mass qualities of sterile,
consistent, standardized and optimized plant material which can be utilized for the
biochemical characterization of medicinally active constituents. Finally, the de novo
regeneration protocol may be useful for genetic improvement using Agrobacterium
and other gene transfer methods.