Protocols for Micropropagation of Woody Trees and Fruits

MICROGRAFTING GRAPEVINE FOR VIRUS INDEXING 261
Once the shoots are induced, the pots can be transferred to the greenhouse and
allowed to grow under optimal conditions. Green shoots for tissue culture establishment
can be selected from the resulting plants (Figure 1). Field-grown plants may
also be used to establish tissue cultures but they usually carry more contamination,
therefore surface sterilization must be more thorough when using them as source
material.
The indicator plants used for viral indexing must be maintained separately from
plants that have unknown virus status. A regular spray programme to control the
insect vectors of viruses, such as mealy bugs, is also important.
Figure 1. Vitis vinifera growing in the greenhouse ready for initiating tissue cultures.
2.1.2. Explant Excision and Sterilization
Green shoots harvested from the greenhouse-grown plants (0.5 cm diameter) should
be cut into pieces, each having a single bud. They can be surface sterilized by
dipping in 70% ethyl alcohol for 30 s followed by 1% sodium hypochlorite solution
containing 0.2% Tween® 80 for 20 min. For better sterilization, the container with
the shoots immersed in the sterilant should be shaken on a rotary shaker (50 rpm).
The explants are rinsed three times for 1 min in sterile water. Aseptically cut the
bleached ends of stem pieces before they are placed into tissue culture. Usually
whole shoot pieces do not result in active shoot production as readily as when young
shoots are split in half aseptically and placed horizontally on the media with the cut
surface touching the medium and the bud facing up.