Shoot-Tip and Meristem Culture
Most of the horticultural and forest crops are infected by systemic disease caused by fungi, viruses, bacteria, Mycoplasma and nematode. While plant infected with bacteria and fungi may respond to treatments with bactericidal and fungicidal compounds, there is no commercially available treatment to cure virus infected plants. It is possible to produce disease free plants through tissue culture. Apical meristems in the infected plants are generally either free or carry a very low concentration of the viruses. The various reasons attributed to the escape of the meristems by virus invasion are:
a) Viruses move readily in a plant body through the vascular system which in meristems is absent,
b) A high metabolite activity in the actively dividing meristematic cells does not allow virus replication and
c) A high endogenous auxin level in shoot apices may inhibit virus multiplication. Meristem –tip cultures has also enabled plants to be freed from other pathogens including Viroids, mycoplasmas, bacteria and fungi. Therefore, main objective of shoot-tip and meristem –tip culture is the production of disease free plants through micro propagation.
It may be described as the culture of terminal (0.1-1.0mm) portion of a shoot comprising the meristem (0.05 -0.1) together with primordial and developing leaves and adjacent stem tissue.
Meristem cultures is the in vitro culture of a generally shiny special dome like structure measuring less than 0.1mm in length and only one or two pairs of youngest leaf primordia, most excised from the shoot apex.
The excised shoot tip and meristem can be cultured aseptically on agar solidified simple nutrient medium or on paper bridges dipping into liquid medium and under appropriate conditions will grow out directly into a small leafy shoot or multiple shoots. Alternatively, the meristem may form a small callus at its cut base on which a large number of shoot primordia will develop. These shoot primordia grow out into multiple shoots. Once the shoot have been grown directly from the excised shoot tip or meristem, they can be propagated further by nodal cuttings. This process involves separating the shoot into small segment each containing one mode. The axillary bud on each segment will grow out in culture to form a yet another shoot. The excised stem tips of orchids in culture proliferate to form callus from which some organised juvenile structures known as protocorm develop. When the protocorm are separated and cultured on fresh medium, they develop into normal plants. The stem tips of Cuscuta reflexa in culture can be induced to flower when they are maintained in the dark.
Exogenously supplied cytokinins in the nutrient medium plays a major role for the development of a leaf shoot or multiple shoots from the meristem or shoot tip. Generally high cytokinins and low auxin are used in combination for the culture of shoot tip of meristem. Addition of adenine suifate in the nutrient medium also induces shoot tip multiplication in some areas. BAP is the most effective cytokinins commonly used in shoot tip or meristem culture. Similarly, NAA is most effective auxins used in shoot tip culture. Coconut milk and gibberlic acid are also equally effective for the growth of shoot apices in some cases.
1. Remove the young twings from the healthy plant. Cut the tip portion of the twig.
2. Surface sterilize the shoot apices by incubation in a sodium hypochlorite solution ( 1% available chlorine) for 10 minutes. The explants are thoroughly rinsed 4 times in sterile distilled water.
3. Transfer each explant to a sterilize petridish.
4. Remove the outer leaves from each shoot apices with pair of jweller’s forceps. This lessens the possibility of cutting into the softer underlying tissues.
5. After the removal of all the outer leaves, the apex is exposed. Cut off the ultimate apex with the help of scalpel and transfer only those less than 1 mm in length to the surface of the agar medium or to the surface of Filter Paper Bridge. Flame the neck of culture tube before and after the transfer of excised tips. Binocular dissecting microscope can be used for cutting the true meristem or shoot tip perfectly.
6. Incubate the culture under 16 hrs light at 25 0C.
7. As soon as the growing single leafy shoot or multiple shoots obtained from single shoot tip or meristem, transfer them to hormone free medium to develop roots.
8. The plants form by this way are later transferred to pots containing compost and kept under green house condition for hardening.