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Growth Regulators and Other Factors for Shoot Regeneration - In general, shoot regeneration is promoted by cytokinins, while auxins seem to have an inhibitory effect. The classical studies of Skoog and coworkers on shoot regeneration from tobacco pith culminated in the well known postulate that organ formation depended on the auxin/cytokinin ratio and not on their absolute concentrations. In tobacco a higher ratio of cytokinin led to shoot regeneration, while a higher ratio of auxin promoted root regeneration.The auxin/cytokinin ratio concept does not seem to apply equally well to other species.

(1) In some species, e.g., Convolvulus, Bacopa, Citrus, shoot regeneration occurs on GR-free media, but is promoted by a cytokinin.
(2) In some other cases, e.g., chickpea immature cotyledons, shoot regeneration occurs only when cytokinin is provided; presence of a low concentration of auxin may have a promotory effect.
(3) In plants like Cyclamen, auxin concentration alone determined whether a shoot or root would regenerate; in contrast, adenine (a cytokinin) affects only the frequency of their regeneration.
(4) Alfalfa presents a peculiar case: callus is first initiated on a 2, 4-0 and kinetin containing medium; shoot or root regeneration occurs when it is subcultured on a GR-free medium.
The ratio of auxin to kinetin in the first medium determines the type of organ formed: a high 2, 4-0 concentration results in shoot regeneration, while a high kinetin level supports root regeneration.

In general, GA3 inhibits shoot bud regeneration. But in some species, e.g., Chrysanthemum, Arabidopsis, GA3 promotes shoot regeneration. In contrast, a large number of species show enhanced shoot regeneration due to ABA (abscisic acid), which counteracts many effects of GA3.
The variable responses of different plant species to the exogenous GRs may be explained as follows. Shoot and root regeneration require specific levels of the different growth hormones, viz., auxin, cytokinin, and gibberellin. However, the endogenous levels of these hormones may vary considerably among different plant species so that a hormone may be either suboptimal, optimal or supraoptimal for shoot (or root) regeneration. The response of a plant species to an exogenous GR would, therefore, depend mainly on the endogenous level of that GR (and of other GRs as well) in that species.

Thus it has been postulated that in species like Chrysanthemum, GA3 occurs in suboptimal concentrations so that exogenous GA3 has a promotory effect. But in those species where its endogenous concentration is supraoptimal, ABA promotes shoot regeneration. It may be added that in some species auxin inhibitors enhance shoot bud differentiation.
Other Factors. Shoot regeneration is markedly affected by the genotype of explant in that different varieties of a given species show quite different frequencies of shoot regeneration. In alfalfa, breeding and selection drastically increased regeneration ability. In wheat, callus growth and regeneration ability are governed by genes called, tissue culture response (TCR) genes, which have been mapped on specific chromosomes.In addition, physical condition of medium (liquid or agar) has a marked influence on shoot regeneration; in some cases liquid medium was superior, while in others it was drastically inferior to agar medium. Light seems to have an  inhibitory effect, and even the quality of light may be important. The optimum temperature for shoot regeneration may vary with the plant species.
 

 
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