Domestication of Plant (Part - 4) - Notes, Botany, Class 12 PDF Download

Domestication of Plant

PLANT TISSUE CULTURE

•  This is one of the latest and most promising methods of crop improvement in such plant, where all other conventional methods of breeding fail.
•  Tissue culture technique is based on totipotent nature of plant cell.
•  Plant tissue culture is the technique of maintaining and growing plant cells, tissues and organs in sterilized culture medium, under controlled aseptic conditions (in-vitro).

Explant –Plant part that is excised from its original location and used for initiating a culture. it may be root tip, shoot bud, anther, embryo, ovule etc. Normally undetermind cells of plant are used as explant.

Surface Sterilization –The process of treatment of explant with specific antimicrobial chemicals like sodium hypochlorite, H₂O₂, Bromine water, C₂H₅OH etc.

•  The vessels, media and instruments are also sterilized by treated them with steam, dry heat or alcohol.

Culture medium or nutrient medium – Medium, which provides nutrition to explants & which is required for normal growth and development of explants.

•  Standard culture medium contains inorganic Salts, Vitamins, Sucrose (as a source of energy and carbon), growth regulators (2,4–D, Cytokinins, BAP–benzylaminopurine)
• Growth regulators are required for cell division and organogenesis in explant.
• Murashigice and Skoog's culture medium (Most commonly used culture medium.) Callus – By culturing explant in culture medium an undifferentiated mass of cells is obtained which is known as‘callus’.

Types of Cultures – 

(1) Callus culture & Suspension culture.

(2) Meristem culture

(3) Embryo culture

(4) Anther culture

(5) Protoplast culture

(1) Callus & Suspension culture – Callus culture – In callus culture when an explant is placed on a medium gel with agar many of the cells become meristematic and begin to divide and giving rise to callus in 2–3 weeks. The agar medium contains both type of growth regulators auxin like 2,4–D and cytokinin like BAP.

Suspension culture – In case of suspension culture a single cell or small group of cells is placed on liquid medium. The medium normally contains the auxin 2,4-D . These cells devide and form small groups of cells.

The suspension cultures are continuously agitated to break the cell mass in to smaller clumps and single cells and also maintain uniform distribution of cells and cell clumps in the medium. It also allows gaseous exchange.

Suspension cultures grow much faster than callus culture. Suspension culture can be maintained in either of the following two forms –

(i) Batch culture ⇒ are initiate as single cells in a flask and are propagated by transferring  regularly small groups of suspensions to a fresh medium.

(ii) Continuous culture ⇒ are maintained in a steady state for long period by draining out the used medium and adding from new medium.

With passage of time in a culture :-

(a) Cell tissue dry matter (biomass) ­

(b) The level of nutrients in the medium ¯

(c) The medium volume declines due to evaporation.

•  The process of transferring the cell culture into a fresh culture medium is called subculturing. It is nromally done after 4-6 week when callus develops to its maximum. During subculture only a part of the culture from a vessel is transferred into the new culture vessel.

Formation of plantlets :-

•  Gottlied Haberlandt was first one who grow isolated leaf cells by plant tissue culture.
•  Totipotency:- The ability of a plant cell to regenerate into a complete plant let.
•  Regeneration can be described as the development of specific structure like root, shoot or somatic embryo from cultured cells.
•  The concept of totipotency was given by "Haberlandt" and practical applications of totipotency was shows by "Steward."
•  Steward developed a complete carrot plant from a single cell obtained from root of wild carrot.

Shoot and root formation :-

•  The two important components of a plant are root and shoot.
•  The regeneration of root and shoot is controlled by two types of growth regulators.
•  The auxin like NAA (Naphthaline Acetic Acid) promotes root regeneration whereas cytokinins like BAP promotes shoot regeneration.
•  Callus cultures first kept on medium containing BAP, which initiates shoot formation from the callus.
•  When shoots become 2-3 cm. long, the culture is transferred to a medium containing auxin. Roots develop from the lower ends of these shoots and develop into young plant called plantlet.

Somatic embryo regeneration :-

•  Somatic embryo or embryoids are non-zygotic embryo like structures that develop in vitro cultures from somatic cells.
•  The medium having ammonium nitrate and auxin (2,4-D) favour induces their differentiation.
•  These young embryos develop into mature embryos either on the same medium or on another medium.
•  Mature somatic embryos germinate to yield complete plantlets.
•  The plantlets can be shifted from culture medium to green house for undergoing hardening process. 
•  In this process plantlets are exposed to reduced light and high humidity for a suitable period of time. The process of hardening is essential for plantlets as they adjust themselves and become capable to face unfavourable environmental conditions.
•  Now these plantlets are finally shifted to the normal fields.

(2) Meristem culture :– 

•  Use of an explant that contains pre-existing shoot meristems and produce shoot from them
•  Explant (shoot tips or nodal segments) are cultured on a medium containing cytokinin (usually BAP)
•  Cytokinin promotes axillary branching by overcoming apical dominance.
•  Now multiple shoot produced from each explant. Each shoot are cultured, but when axillary branching does not take place the single shoot is cut into nodal segments, which are then cultured.
•  When the shoots becomes 2–3 cm. long they are excised and rooted on a suitable medium.
•  When the  plantlet has grown a few leaves it is transferred to soil after hardening.
•  Meristem culture can be used for :–

1.  Rapid clonal multiplication.
2.  Production of virus free plants.
3.  Conservation of germplasm.
4.  Production of transgenic plants.

(3) Embryo culture :–

• Application of embryo culture method to prevent the abortion of the interspecific hybrid embryo is called Embryo rescue.
• Excision of young embryos from developing seeds and their cultivation on a nutrient medium is called embryo culture.
• Aim :– Aim of embryo culture is to allow to young embryos to develop into complete seedlings & overcoming hybridization barriers..

Applications :– (I) In some interspecific crosses the endosperm of developing hybrid seeds degenerate very early so young hybrid embryo which gets devoid of nutrition also dies in such cases the young hybrid embryo is excised and cultured in vitro to obtain hybrid seedling.

(II) Seeds of some plants like orchid lack stored food. In such cases embryo culture allows seedling development from the embryos. This method is also used for rapid clonal propagation in orchid.

(III) In some species seeds may remains dormant due to inhibitors present in the endosperms/seed coat.

• Embryo culture in such cases allows embryo development by eleminating the inhibitors responsible for dormancy.

(4) Anther culture & haploid production :–

• Anther culture is also known as pollen grain culture or androgenic haploid culture.
• When anthers of a plant species are cultured on a suitable medium, then haploid plants are produced, this method is called anther culture.
• This technique was first used in India to produce haploid plants of Datura innoxia by Guha and Maheshwari (1964).
• Sometime diploid plants are also formed among haploid plants. Source of these diploid plants is anther wall (which is diploid)
• In anther culture there can be two situation :– (i) Nucleus within a pollen grain may continue to divide and give rise to a pollen embryo or (ii) Continued division in a pollen produces a callus which then regenerate shoots.
• Haploid can also be produced by culturing – unfertilised ovules.

Androgenic haploid plants have some variations which are called gametoclonal variation.
Haploid plants are very useful in plant breeding because,

(i) They have single set of chromosome, so even a very small change or mutation can be detected in haploids.

(ii) These haploids are used to produce homozygous diploids (by colchicine treatment) and these homozygous diploids are used as parents in crossing.

(iii) Use of haploids in producing pure lines has reduced the period required for  developing new varieties from 10 years to 5 years.

(5) Protoplast culture :–

• Somatic hybrid : A hybrid produced by fusion of somatic cells of two species or  varieties.
• The process of production of somatic hybrid is somatic hybridisation.
• Protoplast : Cell wall less plant cell is called protoplast.

Steps of somatic hybridisation :– (A) Removal of cell wall → 2 method (i) Mechanical method → Old method

Plant tissue Plasmolysing solution                                                                Deplasmolysing Cut from sharp knife     Solution       Protoplast

(ii) Enzymic method → New method .

Discovered by – Cocking . In this method cell wall is digested by using pectinase & cellulase enzyme.

(B) Fusion between protoplast → 2 methods

(i) Spontaneous fusion :– During enzymetic treatment some protoplast fused together and form multinucleated structure which is called homokaryons or homokaryocytes. . This is a intraspecific fusion. . Not very useful in study.

(ii) Induced fusion :– . Protoplast of two different species are fused together by induced fusion. . Substance which induces the fusion of protoplast is called fusogen or fusogenic agent. .

Fusogenic substance and condition :– . By treatment of NaNO₃ . By treatment of Ca⁺² ions at high pH . By treatment of  polyethylene glycol [PEG] . By high voltage electric shock

(C) Culture of the fused protoplast :–

• Product of fused protoplast of two different species is called heterokaryon.
• Heterokaryons are mainly used in tissue culture.
• When the fused protoplasts are cultured on a suitable medium they regenerate cell walls and begin to divide to ultimately produce plantlets.

Importance of somatic hybridisation :– (i) It allows the production of hybrids between different lines and species that can not be produced normally by sexual reproduction. eg. Rice & Carrot

• Pomato is a somatic hybrid between potato and tomato.
• Bromato – Brinjal & tomato (ii) Use of somatic hybrid . For gene transfer . Transfer of cytoplasm . Production of useful allopolyploids.

Sp. Points :–

• Somatic hybridisation is also called parasexual hybridisation.
• First somatic hybrids were obtained between two species of tobacco Nicotiana gluca and N. langsdorfit by Carlson et. al.