4.  Ontogeny of shoot buds

Under the optimal conditions, meristems formed from the callus are random and scattered. On transferring to the medium supporting organized growth promotes first the appearance of localized clusters of cambium like cells (Figure 5.4 A). These meristemoids (or nodules or growing centers), which may become vascularized due to the appearance of tracheidal cells in the centre, are the site for organ formation in the callus, as seen in above figure 5.4 B-C. Initially, the meristemoids exhibits plasticity and can form shoots and roots.

5.  Factors affecting shoot-bud differentiation

The genotype and plant growth regulators are well known to affect regeneration frequency. Plant growth regulators play a major role in the regeneration which mainly depends upon the concentration and type of growth regulators used. For in vitro differentiation genotype plays equally, if not more critical role as the growth regulator. Besides, there are certain other factors which play a critical role in regeneration are:

5.1. Explant

Regenerability of an explant is influenced by several factors such as, the organ from which it is derived, the physiological state of the explant like age of the explant, young vs. mature, position of the explant on the plant and the explant size. Orientation of the explant on the medium and the inoculation density may also affect shoot-bud differentiation. There may be a decline in the number of shoots per culture and the percent cultures showing regeneration with increasing age of the seedlings.

5.2.  Preparation of explant

Sharma et al (1990) studied that in cotyledon cultures of Brassica juncea , shoot buds or roots are formed at the cut end of the petiole, depending on the culture medium. Lamina lacks this potential. However, the presence of laminar tissue is essential for the petiolar cells to exhibit totipotency. Therefore, the ideal explant to achieve regeneration is the lamina together with a short (1mm) petiole.

5.3.  Orientation of the explant

Orientation of explant is proved to be critical for organogenic differentiation in cotyledon cultures of B. juncea. Inoculating the cotyledons with their abaxial surface (lower surface away from the stem) in contact with the medium and the petiolar cut end embedded in the medium gave best response. The explants in which due to expansion and curling of the lamina, the petiole lost contact with the medium within 3-5 days after culture, failed to form roots or shoots. Generally, the explants inoculated horizontally on the medium produced three times more shoots than those planted vertically.

5.4.  Physical Factors

i	Explants grown on liquid or semi-solid medium give different degree of organogenesis. In few species, like tobacco, the medium with 1% agar showed only flower formation. With lowering the agar concentration the frequency of flower formation dropped and vegetative bud differentiation occurred. In liquid medium, the tissue exhibited callusing and vegetative bud formation.
ii.	The quality of light also influences organogenic differentiation. Alternating light and dark period (diffused light, 15-16 hrs) proved best. Callus maintained under continuous light remained whitish and may not exhibit organogenesis. Blue light promote shoot-bud differentiation whereas red light stimulated rooting in tobacco. Calli of Brassica oleracea grown in dark for 20 days formed shoot-buds 12 days after transfer to light while those shifted to light after 12 days of growth in dark differentiated shoots within 9 days.
iii.	Skoog (1944) studied the effect of a range of temperature on tobacco callus growth and differentiation. Growth of callus increased with rise in temperature up to 33°C, but for shoot-bud differentiation 18°C was optimum.