The following points highlight the thirteen factors influencing organogenesis in plant tissue culture.

The thirteen factors are: (1) Size of Explant (2) Source of Explant (3) Age of the Explant (4) Seasonal Variation (5) Oxygen Gradient (6) Quality and Intensity of Light (7) Temperature (8) Plant Hormones (9) Culture Medium (10) Agar-Agar (11) pH of the Medium (12) Ploidy Level and (13) Age of Culture.

Factor # 1. Size of Explant:

Organogenesis is generally dependent upon the size of the explant. The large explant con­sisting of parenchyma, vascular tissue and cam­bium have greater regenerative ability than the smaller explant. Small groups of homogeneous tissue taken from the epidermal and sub-epider­mal layer could directly give rise to complex or­gans such as flower or buds or roots. A remark­able capacity to regenerate shoot buds in vitro is displayed by certain ferns such as Davallia Platycerium. The tissue pieces obtained by aseptically homogenizing the plants in a blender produce numerous new plants.

Factor # 2. Source of Explant:

The source of explant cultured is important in determining the potential of organogenesis. The most suitable part of the plant for starting culture will depend on the species. Leaves and leaf fragments of many plant species like Bego­nia, Solanum, Nicotiana, Crepisetc. have shown the capacity to regenerate shoot buds. Many of the monocotyledonous species with special­ized storage organs possess a profound capac­ity to produce buds.

The bulb scale of Hilium sp regenerates adventitious bulblets in culture. Flower stem explant of Tuhpa sp. regenerates shoots in vitro. Inflorescence axis of Haworthia sp.; also forms shoots in culture. Root sections of Convolvulus sp. Ipomea sp. etc. produce shoot buds in culture. In conifers, the rate of adventi­tious shoot formation on cotyledons is correlated with the growth rate of the parent tree and with the size of the seed.

Factor # 3. Age of the Explant:

The physiological age of the explant is an­other factor which often plays an important role in organogenetic phenomenon. Regeneration of adventitious shoot bud is only noted in case of Nicotiana sp. if the leaf explants are collected from the vegetative phase i.e. prior to flowering. Leaf explants of Echeveria sp. that are collected from young leaves produce only root, whereas older leaf initiates only shoot buds and leaves of medium age produce both shoots and roots.

Factor # 4. Seasonal Variation:

The effect of seasonal variation on plant is another factor which exercises an influence on or­gan formation. Bulb scales of Lihum speciosum regenerates bulblets freely in vitro when the ex- plant is taken during spring and autumn period of growth. But the same explant collected from summer or winter season does not produce any bulblet.

Factor # 5. Oxygen Gradient:

Oxygen gradient in a tissue culture often exercises an influence on organ formation. In some cultures, shoot bud formation takes place when the gradient of available oxygen inside the culture vessel is reduced. But rooting requires a high oxygen gradient.

Factor # 6. Quality and Intensity of Light:

The quality and intensity of incident light on culture may play an effective role in the pro­motion of organogenesis. Studies on spectral light on organogenesis reveals that the blue re­gion of the spectrum promotes shoot formation and red light induces rooting.

The treatment of blue light followed the treatment of red light also stimulates the organogenetic phenomenon. Hence the nature of organogenesis can be regu­lated by exposure to light of different wave length. This sort of action of light on organogen­esis will help us in understanding the action of auxin and cytokinin on organogenesis. In some culture, artificial fluorescent light favours root­ing and inhibits in others. In case of Pisum sativum, shoot bud initiation takes place in dark followed by the sudden treatment of light.

Normally, organogenesis in culture takes place with an illumination of about 2,000-3,000 lux (16 hrs. photoperiod) The callus tissue of Nicotiana tabacum also produces shoot bud or embryo when the tissue is exposed to high in­tensity of light—10,000-15,000 lux.

Factor # 7. Temperature:

Most tissue cultures are grown successfully at temperatures around 25° C, but the usual en­vironmental temperatures of the species concern­ed should be taken into account. In a number of bulbous species, the optimum temperature may be much lower— 15°C in case of Galanthus and 18°C for some cultivars of Narcissus and Allium. Tropical species require higher temperatures, the optimum for date palm being 27°C and for Monstera deliciosa 30°C.

Factor # 8. Plant Hormones:

Of the many factors that influence Organo­genesis in vitro, the most important single factor seems to be the phytohormones. In their classi­cal experiments with cultured stem pith tissue of tobacco, Skoog and Miller (1957) demonstrated that different types of organogenesis (Fig 7.1) can be achieved by varying the concentrations of auxins and cytokinin in the culture medium— when the concentrations of cytokinin are high relative to auxin, shoots are induced; when the concentrations of cytokinin are low relative to auxins, roots are reduced; and at intermediate concentrations the tissues grow as unorganized callus (Fig 7.2).

Organogenesis in tobacco

This basic concept has been used to regenerate a wide variety of dicotyle­donous plants. In general monocotyledonous plants do not show a pronounced response to cy­tokinins and need high concentration of auxins such as 2, 4-D to obtain changes in the develop­ment of cultured tissue. Other plant hormones, particularly abscisic acid and gibberellins have some dramatic action on in vitro organogene­sis.

Relationship of the auxin and cytokinin concentration of a culture medium to their effects on the growth and development of plant cells

Endogenous ethylene retards organ initia­tion during early stages of culture but in the later stages it helps shoot initiation. Very lit­tle is known about how phytohormones awake a particular pattern of organogenesis. Various hy­potheses have been put forward to explain the mode of action of phytohormones on organogen­esis.

Such hypotheses have been placed into two broad categories, depending upon how much em­phasis is given on the developmental state of the responding cell. In the first category of hypo­theses, phytohormones are regarded as primary morphogens.

According to these hypotheses, the responding cell or groups of cells are competent to react to the hormones but are not commit­ted to a particular development fate. When the cells are treated with hormones, the cells start to move a specific developmental pathway. The alternative view is that hormone responsive cells are already determined and that the hormones stimulate the expression of the committed state.

On the basis of large number of available evidences it can be suggested that the hormones act in both ways depending upon the particular experimental system. Histological studies of cul­ture of cereals reveal that organ primordia are ar­rested in their development by auxin in the cul­ture medium.

Again, in dicotyledonous plants, different types of development can be obtained by treating the same cloned line of cells with dif­ferent combinations of hormones. In some cases, the hormones show different actions at different stages in the same developmental pathway.

Factor # 9. Culture Medium:

The essential components of plant cell cul­ture medium are the macro or major salts and micro or minor salts. Besides these, vitamins, amino-acids, carbohydrates etc. are also requir­ed for in vitro growth and development of plant cells. Inorganic nitrogen’s most important role in the plant cells is its presence in the structure of the protein molecule. In addition, nitrogen is found in such important molecules as purines, pyrimidine’s and coenzymes.

Phosphate is found in plants as a constituent of nucleic acids, the co-enzymes NAD and NADP and most impor­tant as a constituent of ATP. Increase in phos­phate concentration induces shoot formation and suppresses root initiation. One well known role played by calcium in the plant cell is its function as a constituent of cell walls in the form of cal­cium pectate.

Similarly, iron, zinc and molybde­num are parts of certain enzymes. The organic supplements required in plant culture medium include a carbon source and vitamins; sucrose is used as a carbon source but may be substituted with glucose. Other sugars are used less often. Vitamins most often added to culture medium include inositol, nicotinic acid, pyridoxine, thi­amine, calcium pantothenate and biotin.

Thi­amine is required for plant growth, while others enhance growth in some system. Vitamin E has been shown to regulate cell aggregation and Vi­tamin D induce root formation. Various complex nutritive mixtures of undefined composition like casein hydrolysate, coconut milk, malt-extract etc. are sometimes added to the culture medium to increase cell growth.

Factor # 10. Agar-Agar:

Agar-agar is not an essential component of the culture medium. In plant tissue culture, the culture medium is gelled with agar. The quantity of agar is a factor that may have a de­termining role in organogenesis. Commercially available agar contains impurities.

So “Difco” or Bacto-agar containing less impurities are gen­erally used in plant tissue culture medium. With high concentration of agar, the nutrient medium become hard and does not allow the diffusion of nutrient to the growing tissue. So the concentra­tion of agar also plays a role in organogenesis.

Factor # 11. pH of the Medium:

The pH of the culture medium is generally adjusted between 5.6 to 5.8 before sterilization. The pH is another factor that may have a deter­mining role in organogenesis.

Factor # 12. Ploidy Level:

Variation in chromosome number i.e. aneuploidy, polyploidy etc. of plant cell in culture has been well-documented. It is generally observed that with the increase of chromosome instabil­ity there is a gradual decline in morphogenetic potentiality of the callus tissue. So the most important factor in maintaining organogenic po­tential of the callus tissue is the maintenance of chromosome stability. It has been suggested that the frequency of subculture can affect the chro­mosome stability of cell cultures. So, in order to maintain chromosome stability, cultures are sub-cultured frequently and regularly.

Factor # 13. Age of Culture:

Age of culture often exercises an influence on organ formation. 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.