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).
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.
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.