Factor Affecting the Organogenesis
In vitro organogenesis is controlled by a number of factors other than phytohomes such factors are discussed below:
1. Size of Explant:
Organogenesis is generally dependent upon size of explant. The large explant consisting parenchyma, vascular tissues and cambium have greater regenerative abilitythan the smaller explant. Small group of homogenous tissues taken from epidermal or subepidermal layer could directly give rise to complex organs like flower or bud or roots.
2. Source of Explant:
The most suitable part of the plant for starting culture will depend on species. Leaves and leaf fragment of many plant species like Begonia, Solanum, Nicotina, Crepis , etc have shown capacity to regenerate shoot buds. Bulb scale of Hillium, sps, regenerate adventitious bulbelts, flower stem explant of Tulip asps. Regenerate shoot bud , inflorescence axis of Haorthia sp. Also forms shoots and root section of Convovulus sp. Produce shoot bud in culture.
3. Age of the Explant:
Physiological age of explant is important for in vitro organogenesis. In Nicotiana species, regeneration of adventitious shoot is only noted if the leaf explant is collected from vegetative stage i.e. before flowering. Leaf explants of Echeveria sp. That are collected from young leaves only produce roots, whereas older leaves initiate only shoot buds and leaves of medium age produce both shoots and roots.
4. Seasonal Variation:
Bulb scales of Lilium speciosum regenerate bulblets freely in vitro when explant is taken during spring and autuma period of growth but same explant collected from summer or winter season does not produce any bulblets.
5. Oxygen Gradient:
In some cultures, shoot bud formation takes place when the gradient of available oxygen inside the culture vessel is reduce. But rooting requires a high oxygen gradient.
6. Quality and Intensity of Light:
The blue region of spectrum promotes shoot formation and red light induce rooting. The treatment of blue light followed by treatment of red light also stimulates the organogenesis phenomenon. In some cultures artificial fluorescence light favours rooting and inhibits in others. In case of Pisum sativum shoot bud initiation takes place in dark followed by sudden treatment of lights.
Normally, organogeneses in culture take place with an illumination of 2000- 3000 lux. However, the callus tissue of Nicotina tabacum also produces shoot bud or embryo when tissue is exposed to high intensity of light of 1000-15000 lux.
Most tissue culture are grown successfully at twp. Around 25 0C. In number of bulbous species optimum temperature may be much lower of about 15-18 0C. Increase in temp upto upto 330C may be associated with rise in growth of tobacco callus but for shoot bud initiation a lower temp of about 18 0C may be optimum.
8. Culture Medium:
Medium solidified with agar favours bud formation although there are some reports about the development of leaf shoot buds on culture grown in a liquid medium.
9. PH of the Medium:
The PH of the culture medium is generally adjusted between 5.6 and 5.8 before sterilization. The pH may have a determining role in organogenesis.
10. Ploidy Level:
Variation in chromosome number i.e anuploidy, polyploidy, etc of plant cell in culture has been well documented. With the increase in chromosome instability there is a general decline in morphogenetic potentiality of callus tissue. So the most important factor in maintaining organogenic potential of callus tissue is the maintenance of chromosome stability. Frequency of subculture can affect the chromosome stability of cell culture. So in order to maintain chromosome stability, cultures are subcultured frequently and regularly.
11. Age of Culture:
A young culture frequently produces organs. But the organogenic potential may decrease and ultimately disappear in old culture. In certain cultures of some plants, the plant regeneration capacity may retain indefinitely for many years.