Plant tissue-culture, anther and pollen culture, callus-culture

Plant tissue-culture:- Plant tissue culture is ideally done in 7 steps: -

1. Selection of Explant:-

•  Explant:- The part of the plant which is selected for culturing on the medium is called explant.

2. Sterilization of Explant:-

•  To destroy the spores of bacteria or fungi present on the surface of explant, the following sterilizing agents can be used:-

i. NaOCl = 1-1.4%

ii. Ca (OCl)2 = 9-10%

iii. H2O2 = 10-12%

iv. AgNO3 = 1%

v. Hg2Cl2 = 0.01 - 1%

3. Sterilization of Glasswares:-

•  Glass wares are sterilised by dry hot air in the oven.

4. Sterilization of Culture medium:-

•  The culture medium is sterilised at 121 ° C temperature and 15 p.s.i. pressure in autoclave for 30 minutes. Sterilization occurs by hot steam in the autoclave.

5. Pouring and Inoculation:-

•  The LAF cabinet is used for this step. [LAF = Laminar Air Flow]

•  In LAF first of all the internal environment is sterilised by UV light for 15 minutes.

•  The air in the LAF flows through the HEPA filter to the user.

    [HEPA = High Efficiency Particulate Air]

•  The pore size of HEPA filter is 0.3µm through which 99.97% of airborne particles cannot pass out.

•   Pouring:- The medium is removed from the autoclave and brought to the LAF cabinet. Now, we pour the medium in the culture vessel, in which the explant is to be cultured. This is called Pouring.

•   Inoculation:- When the medium cools and freezes, the explants are transferred on to the medium in the culture vessel. This is called Inoculation.

6. Incubation:-

•  Culture vessels are sealed with cotton plugs and placed in the Incubator.

•  A suitable amount of temperature, light and humidity is provided to the explant in the incubator.

•  In a few weeks, the seedlings are developed from explants.

7. Hardening and Acclimatization:-

•  The plant is removed from the adapted environment of the incubator and transferred to the green house. Where the plant has to face some adverse conditions.

•  Plants adapt to the green house environment in 15 - 20 days.

•  Now they are transferred to the soil of the field. Here the plants get adapted in a few days.

anther and pollen culture, callus-culture

Anther Culture:-

1. Introduction:-

·    Definition:- It is an artificial technique by which the developing anthers are collected from a closed floral bud at a fine and delicate stage and cultured on a nutrient medium. Where the microspores develop into callus tissue or embryo like structure, that develop haploid plants through organ formation or embryo formation.

·    So far anthers have been cultured in about 250 plant species. The three families are more common -

i. Solanaceae

ii. Criciferae

iii. Poaceae

2. History:-

·    W. Tulecke (1953):- He first observed that mature pollens of the gymnosperm plant Ginkgo biloba can be induced to form haploid callus in culture.

·    S. Guha and P. Maheshwari (1964):- They first observed that the embryos are developed from the microspores directly in anther culture of Datura innoxia.

·    J. P. Bourgin and J. P. Nitsch (1967):- They obtained fully haploid plants by the anther culture of tobacco (Nicotiana tabacum).

·    Niizeki and Oono (1968):- They were Japanese scientists. They developed haploid plants for plant breeding by anther culture in paddy.

3. Principle:-

·    The haploid plant is produced using the totipotency of the microspore.

·    Only one set of chromosomes is present in the microspore.

·    In the process of haploid plant development, the normal growth and functioning of the microspore of developing male gametes is halted. It is forced into a new metabolic pathway for somatic cell division.

·    Anther culture does not alter the natural habitat and environment of the microspores present inside the anther. In culturing conditions, the diploid tissue of the anther survives on the medium without cell division and induces somatic cell division of the microspore by providing nutrition.

Pollen Culture:-

1. Introduction:-

·    Definition:- Pollen grain or microspore culture is an artificial process in which pollens or microspores are extracted from the intact anther at its uni-nucleated state and cultured on the nutrient medium under sterilized conditions.

·    Androgenesis:- The process of artificial development of haploid plants by a series of cell division and differentiation from a totipotent pollen is called androgenesis. It is of two types -

i. Direct Androgenesis:- The microspore behaves like a zygote and by some changes the it forms a embryo like structure which further develops into haploid plants. This is called embryogenesis.

ii. Indirect Androgenesis:- The microspore divides repeatedly to form the callus tissue. Differentiation leads to development of haploid plant. This is called organogenesis.

2. Principle:-

·    The haploid plant is developed using the totipotency of the microspore.

·     Only one set of chromosomes is present in the microspore.

·     In the process of haploid plant development, the normal development and function of the microspore of the male gametes formation stops. It is forced into a new metabolic pathway for somatic cell division.

·    In pollen culture, diploid somatic cells of the anther also sometimes become active under culturing conditions and grow to form unwanted diploid callus or plantlets. Sometimes the chimera is formed. A callus or plantlet whose some cells are haploid and some cells are diploid, is called chimera. To avoid this problem, free pollens extracted from the anthers are cultured on the nutrient medium.

Callus Culture:-

1. Introduction:-

·    Callus:- The non-specific and unorganized group of cells growing by mitosis is called callus.

·    Callus is produced when the explant is cultured on a solid agar medium containing the suitable amount of auxin and cytokinin. 2,4 - D is commonly used as auxin.

·    There is some degree of dedifferentiation in morphology and metabolism during callus formation. Due to which they lose the ability of photosynthesis.

·    Callus is a blob of tissue in which most cells are undifferentiated.

·    Callus develops naturally when there is a wound in a plant.

·  Callus Culture:- When the callus is cultured on artificial solid agar medium in sterilized conditions, it is called callus culture.

2. History:-

·     R. J. Gautheret (1934-37):- He first succeeded in developing callus from the cambium of the Salix capraea plant.

·     P. Nobecourt (1939):- He first established the callus culture having unlimited growth potential on a solid agar medium. He started work on the tap root of Daucus carota.

·    J. Van Overbeck, M. E. Conklin and A. F. Blakeslee (1941):- He first discovered the importance of coconut milk in callus culture.

·    S. M. Caplin and F. C. Steward (1948):- They succeeded in achieving growth on a medium containing coconut milk by isolating differentiated non - cambial cells from carrot.

·    F. Skoog (1954-1955):- He succeeded in obtaining callus culture from a piece of tobacco stem cutting on a auxin containing medium. Callus remained active for some time and failed to grow.

·   F. Skoog and C. O. Miller (1957):- He first introduced the concept of hormonal control of organ formation from callus tissue. They reported that equal amounts of auxin and kinetin in the medium stimulate the continuous growth of callus tissue.

3. Principle:- Three important requirements must be met for a successful initiation of callus culture -

i. Preparation of plant material under sterilized conditions. Surface sterilization is done for this.

ii. Selecting the appropriate nutrient medium in which auxin and cytokinin are present in appropriate proportion. There should be a ratio of 1: 1 for callus growth.

iii. Incubation of culture under controlled physical conditions.

Ø  It is necessary to have controlled conditions of heat, light and humidity present for the correct initiation of callus tissue formation.

Ø  A temperature of 25 ± 2 ° C is considered appropriate.

Ø  In some plants, 24 hours of darkness is required for developing callus such as carrot. Other plants require 16 hours of light period and 8 hours of dark period. The intensity of artificial light should be 2000 to 3000 lux. Cold and white lamps are used to provide artificial light.

Ø  Generally 55 to 60% relative humidity is maintained in the culture chamber.