Factors Affecting Shoot Multiplication
Morphogenesis and Proliferation rate of culture depend on various factors, influencing the relative incidence of organogenesis or embryogenesis.
i) Physiological Status of Plant Material:
Successful cultures are rarely obtained from senescing tissues. Explant isolated from more recently produced parts of the plant are more regenerative than those from older regions. The regenerative potential of tissue culture diminished with each year of maturation. Papaya tissue cultures can be established in hot summer months, whereas flower stem explants of Tulipa gives rise to shoots only when excised during the dry storage phase.
ii) Culture Media:
The standard tissue culture media are more suitable for achieving stage I and II of Micropropagation. Only stage III requires some modifications. As proposed by Skoog and Miller (1957), that organ differentiation in plant is regulated by an interplay of auxin and cytokinins should work as guide when developing a new medium for a new plant for micro propagation. To induce adventitious root formation, after axillary shoot propagation, cytokinins is usually omitted and auxin added. GA and ABA in a medium also reported to inhibit root formation. Activated charcoal may also induce the formation of adventitious rots in some species. Its presence in medium reduce the light supply to in vitro regenerated shoots and helps in removing inhibition by the absorption of all such compounds released in culture.
iii) Culture Environment:
Although in vitro regenerated shoots are heterotrophs, the light absorbed by the photosynthetic pigments in cultured tissue, plays an important role in inducing the morphogenesis of these tissues. The optimum light intensity found for shoot multiplication in most of the species is 100 lux. The quality of light also controls the organogenic differentiation and growth of shoot in cultures. For example, blue light (467 nm) induces bud formation in tobacco shoots and even doubled the number of lettuce shoots regenerated from callus cultures. Red and far-red light induce root formation. A diurnal illumination of 1 hr day and 8 hr night is generally found satisfactory for multiplication and proliferation of shoot. Most micro propagation culture are normally maintained at 25 0C.
The genotype differs for regeneration capabilities in culture. The microprogation system develops for one particular cultivar will not automatically be applicable to another even within the same species. It has been found in grape that genotype with vigorous germination and branching capacity propagated rapidly.
v) In Vitro Rooting:
Media having low concentration of salt have been satisfactory for rooting of shoots micropropagated at stage II. Roots are mostly induced in the presence of suitable auxin in the medium although the shoots of some plants. Narcissus and strawberry may readily root on a hormone free medium.
vi) Acclimatisation of Plants Transferred to Soil:
Micropropagation on a large scale can be successful only when plants after transfer from culture to the soil show high survival rates and cost involved in the process is low. Tissue cultured plants generally show some structural and physiological abnormalities which , include, a) Abnormal leaf morphology and anatomy, (b) Poor photosynthetic efficiency, (c) marked decrease in epicuticular wax and (d) malfunctioning of stomata. These characteristics as well as heterotrophic mode of nutrition and poor mechanism- for- water loos control further render micropropagated plants vulnerable to transplantation shock.
It is essential to wash thoroughly the lower part of tissue cultured plants/ shoots before their transfer to potting mix (pumice, peat, vermiculite, soil, sand or their mixtures in different proportions). Transplanted plantlets or shoots are immediately irrigated with an inorganic nutrient solution and maintained under high humidity for the initial 10-15 days.
Storage organs have been induced in cultured shoots of several species. These structure donot require hardening and can be directly transplanted in soil. a well known example chloramequat. Other example is dioscorea species. In vitro cormlet formation in gladiolus requires high concentration of sucrose.