Preparation of the Explants
The preferred tissue culture explants for stevia are, shoot tips, nodal segments and leaf discs. Shoot tips and nodal segments are mostly sued for direct shoot generation, which is followed by root formation by transferring the growing shoots in another medium containing appropriate root formation hormones.
Shoot Tips :
For shoot tip culture, relatively large explants (sometimes up to 1.0 cm in length), consisting of the shoot apical meristem and a number of leaf primordial and small un-expanded leaves are used. The dominance of apical meristem is reduced and development of precocious axillary shoots is stimulated by incorporation of appropriate growth regulators in the growth medium.
Use of nodal segment is most prevalent in the published works on stevia tissue culture. An axillary meristem is located in the nodal zone, which gives rise to axillary buds, which when treated with cytokinin in in-vitro conditions can lead to production of multiple shoots. The length of the nodal segments should be in the range of 0.5 to 1.0 cm.
Leaf Discs :
Leaf discs are used for formation of undifferentiated callus first. Then the callus tissue is transferred in a medium containing hormones for shoot initiation. After shoot are formed, they are transferred in another medium containing specific hormones for root formation. The lamina between the midrib and the margin is used as the source of explants. Generally 5 to 10 mm2 area of the leaf is cut with sharp instruments either as circular discs or a rectangular pieces and use as explants.
Surface Sterilization of the Explants :
The first important condition for the successful tissue culture procedures is the maintenance of aseptic condition. Sterilization eliminates contamination by microorganisms – which can grow in the tissue culture medium and decompose the plant tissue. To maintain an aseptic culture condition, all culture vessels, media and instruments used in handling tissue, as well as the plant explants should be surface sterilized. Plant material can be surface sterilized by variety of methods. The most common method for surface sterilization of explants from stevia is depicted in the flow diagram at the right hand side.
Preparation of Nodal Segments
Leaf Disc Explants
Transfer of the explant on the media:
Control of contamination largely depends upon the skill of the technician while transferring the sterilised explant/sub-culturing into the sterile culture vial containing nutrient media under aseptic condition. Dust from the surface, hair, hands and clothes are the potential sources of contamination. Before starting the transfer procedure the surface of transfer area and hands should wiped with 95% ethanol; sterile clothes (aprons) should be used. All the metallic equipment’s used for transfer (inoculating needle, forceps, and scalpel) should be dipped into 95% ethanol and then flamed and cooled. The tissue material should not touch the edge of culture vessel during transfer. The entire process of the transfer is to be done in a laminar flow hood. 30 minutes before the actual transfer, the UV lamps in the laminar air flow cabinet is to be turned on and it is to be turned off before the transfer process. All the transfer process is to be done within 15 cm radius of the flame of the Bunsen burner in the laminar flow hood table.