Definition: The term plant tissue culture is described in vitro and aseptic cultivation of any plant part on nutrient medium. OR the culture of plant seeds, organs, tissues, cells, or protoplasts on nutrient media under sterile conditions.

Factors Affecting Plant Tissue Culture Growth Media Minerals, Growth factors, Carbon source, Hormones Environmental Factors Light, Temperature, Photoperiod, Sterility, Media Explant Source Usually, the younger, less differentiated the explant, the better for tissue culture Genetics Different species show differences in amenability to tissue culture In many cases, different genotypes within a species will have variable responses to tissue culture; response to somatic embryogenesis has been transferred between melon cultivars through sexual hybridization Emphasize the implications for genetic involvement: Could there be undesirable genes linked to genes influencing tissue culture response?

Three Fundamental Abilities of Plants Totipotency the potential or inherent capacity of a plant cell to develop into an entire plant if suitably stimulated. It implies that all the information necessary for growth and reproduction of the organism is contained in the cell Dedifferentiation Capacity of mature cells to return to meristematic condition and development of a new growing point, follow by redifferentiation which is the ability to reorganise into new organ Competency the endogenous potential of a given cells or tissue to develop in a particular way

Types of In Vitro Culture Culture of intact plants (seed and seedling culture) Embryo culture (immature embryo culture) Organ culture 1. shoot tip culture 2. root culture 3. leaf culture 4. anther culture Callus culture Cell suspension culture Protoplast culture

Engraving of Henri-Louis Duhamel du Monceau by François-Hubert Drouais Engraving of Henri-Louis Duhamel du Monceau by François-Hubert Drouais. Duhamel du Monceau was the first to describe callus formation that he observed growing over the wound of an elm tree.

Callus formation

Theodor Schwann Theodor Schwann (7 December 1810 – 11 January 1882) was a German physiologist. His many contributions to biology include the development of cell theory.

Performed experiment on callus formation by decorticated trees such as Robinia, Pawlonia and Ulmus. Trecul (1853) Obtained very flourishing callus from Brassica rapa and proposed the polarity in development of buds from the upper portion and roots or callus and from the lower portion of a stem piece Vochting (1878) Described callus formation on isolated stem fragments and root slices. Rechinger (1893) Concept of in vitro Cell Culture Gottlieb Haberlandt (1902) (Father of Tissue Culture) Culture of Embryogenic tissue Hannig (1904)

Gottlieb Haberlandt Haberlandt first pointed out the possibilities of the culture of isolated tissues, plant tissue culture. His original idea presented in 1902 was called totipotentiality: “Theoretically all plant cells are able to give rise to a complete plant.”

*All these discoveries contributed to the establishment of totipotency of somatic cells under experimental conditions, thereby accomplishing the goals set by Haberlandt.

History In 1902 Haberlandt proposed that single plant cells could be cultured

Haberlandt did not culture them himself

1930’s White worked on T.C. discovery of plant growth regulators

1930’s importance of vitamins was determined for shoot and root culturing A,D,E,K,C, B complex

1930’s Indole-Acetic Acid IAA discovered in 1937

IAA 2,4-D Dicamba NAA IBA all synthetic hormones

1957-58 Miller and Skoog University of Wisconsin - Madison discovered Kinetin

Kinetin a cytokinin plays active role in organogenesis

1958 Steward developed somatic embryo from carrot cells

1958-60 Morel cultured orchids and dahlias freed them from a viral disease

1962 Murashige and Skoog published recipe for MS Medium

60’s & 70’s Murashige cloned plants in vitro promoted development of commercial plant T.C. labs

1966 raised haploid plants from pollen grains

1972 used protoplast fusion to hybridize 2 species of tobacco into one plant contained 4N

4N all chromosomes of both plants

70’s &80’s develop techniques to introduce foreign DNA into plant cells beginning of genetic engineering

HISTORY OF PLANT TISSUE CULTURE 1838-39 cellular theory (Cell is autonom and totipotent) Schleiden-Schwann 1902 First attempt of plant tissue culture Harberlandt 1939 Continuously growing callus culture White 1946 Whole plant developed from shoot tip Ball 1950 Organs regenerated on callus 1954 Plant from single cell Muir 1960 Protoplast isolation Cocking

HISTORY OF PLANT TISSUE CULTURE 1962 MS media Murashige - Skoog 1964 Clonal propagation of orchids Morel Haploids from pollen Guha 1970 Fusion of protoplasts Power 1971 Plants from protoplasts Takebe 1981 Somaclonal variation Larkin

Development of Tissue Culture Root-tip Culture White (1934, 1937) Embryo Culture (Use of coconut milk i.e. embryo sac fluid) Van Overbeek et al. (1941) Stem-tip Culture Loo (1945) Ball (1946) Role of Auxin Cambium cells of some tree species Gautheret (1939) Tumor tissues of hybrid White (1939) Carrot slices Nobecourt (1939) Discovery of Cytokinin Skoog (1955) Miller et al. (1955)

Hormonal Control of Organ Formation Skoog and Miller (1957) Improvement of Media Murashige and Skoog (1962) Preparation and Cloning of Single Cell Cultures Bergmann (1960) Regeneration of Single Cell to Whole Plant Regenerated plantlets Vasil (1965) Somatic embryogenesis Steward (1966) Kohlenbach (1966)

T.C. Media functions provide H2O provide mineral nutritional needs

T.C. Media provide growth regulators Provide vitamins provide organic compounds

T.C. Media provide access to atmosphere for gas exchange serve as a dumping ground for plant metabolites

T.C. Media H2O is usually distilled minerals must provide 17 essential elements energy source and carbon skeletons - sucrose is preferred

Vitamins thiamine pyridoxin nicotinic acid biotin

Vitamins citric acid ascorbic acid inositol

Growth Regulators auxins and cytokinins gibberellic acid abscissic acid

Basis for Plant Tissue Culture Two Hormones Affect Plant Differentiation: Auxin: Stimulates Root Development Cytokinin: Stimulates Shoot Development Generally, the ratio of these two hormones can determine plant development:  Auxin ↓Cytokinin = Root Development  Cytokinin ↓Auxin = Shoot Development Auxin = Cytokinin = Callus Development

Control of in vitro culture Cytokinin Leaf strip Adventitious Shoot Root Callus Auxin

pH of media usually 5.0-5.7

Media must be sterile autoclave at 250 F at 15 psi for 15 minutes

T.C. Stages Explanting- Stage I get plant material in sterile culture so it survives provide with nutritional and light needs for growth

Stage II rapid multiplication stabilized culture goal for a commercial lab difficult and time consuming to maintain

Stage II occurs in different pathways in different plants

Rooting - Stage III may occur in Stage II usually induced by changes in hormonal environment lower cytokinin concentration and increase auxin

Rooting may skip stage III and root in a greenhouse

Stage IV transplantation and aftercare usually done in greenhouse keep RH high (relative humidity)

Stage IV gradually increase light intensity and lower RH after rooting occurs allows plants to harden and helps plants form cuticle

Cuticle waxy substance promotes development of stomates plants in T.C. don’t have cuticle

Explant portion of plant removed and used for T.C. Important features size source - some tissues are better than others

Explant species dependent physiological age - young portions of plant are most successful

Explant degree of contamination external infestation - soak plant in sodium hypochlorite solution

Explant internal infection - isolate cell that is not infected roots - especially difficult because of soil contact

Explant herbaceous plants soft stem easier to culture than woody plants

Patterns of multiplication stage II - light 100-300 foot candles callus - shoots - roots stage III - rooting - light intensity 1000-3000 foot candles

Genetic transformation permanent incorporation of new or foreigh DNA into genome of cell

Transformation methods protoplast fusion cell wall is enzymatically removed from cell

Protoplasts naked plant cells from 2 different plants can be mixed together and forced to fuse

Protoplast fusion results in heterokaryon cell containing two or more nuclei from different cells homokaryon - from same cell

Protoplast fusion allowed to regenerate cell wall and then grow into callus callus turns to shoots

Shotgun approach DNA coated micro bullets of gold or tungston shot into growing cells DuPont holds the patent

Shotgun approach injures cells random success rate

PEG Polyethylene glycol pores open similar to electroporation

Ti Plasmids Tumor inducing Agrobacterium temefasciens infect cells with agrobacterium which contains desired DNA

Ti Plasmids monocots resist agrobacterium infection researchers are working to overcome this

Luciferase an enzyme put into tobacco using Ti plasmid

Luciferase when transformed tobacco plants are watered with solution containing Luciferin they break it down and emit light

Luciferase glowing in the dark like a fire fly

Screening techniques used to identify if culture has taken on desired new trait

Examples sensitivity to antibiotics color sensitivity to excess deficiencies of substances in growth media

Conventional plant breeding egg cell gives half the chromosomes and almost all of the cytoplasm male only gives its chromosomes

Cont……. This condition is called maternal cytoplasmic inheritance

Microinjection single cells from culture are held stationary with gentle suction injected with a tiny syringe loaded with DNA

Microinjection done under electron microscope

Electroporation desired DNA in solution outside cell high energy pulses - 50,000 volts for a millisecond

Electroporation cause tiny pores to open allows DNA to enter the cell

Fundamental principles: There are three fundamental principles (1) The plant part must be isolated from the rest of plant body. (2) The plant part (ex plant; isolate cell that is not infected) must be in a controlled , preferably defined , nutrient medium. (3) Aseptic condition must be maintained.

Types of plant cultures: (1) Callus Culture. (2) Cell Suspension Culture. (3) Anther Culture. (4) Ovule Culture. (5) Embryo Culture. (6) Protoplast Culture. (7) Micro Propagation.

History: (1) HABERLANDT. (2) WHITE. (3) NOBECOURT. (4) VAN OVERBEEK. (5) SKOOG. (6) BRAUN. (7) STEWARD. (8) MURI.

(1) Callus Culture technique When an excised and isolated piece of tissue is cultured on a nutrient medium, an unorganized mass of cell appears, is called callus. This callus is transferred on to different media to regenerate plants. this technique is called tissue culture.

Plant tissue culture

(2) Cell Suspension Culture It is the culture of isolated cells or very small cell aggregates dispersed in liquid medium. The cell suspension is obtained by agitating pieces of callus in liquid medium on gyrating shaker.

(3) Anther Culture Anther culture is a mean to produce plants with a gametic number of chromosomes by aseptic culture of anther. The technique give rise to haploid plants either directly or through formation of a haploid callus.

(4) Ovule Culture Ovule culture techniques is an important technique in modern plant breeding. it is much easier to culture whole ovule than to isolate a single embryo, especially in small seeded plants. E.g., tobacco

(5) Embryo Culture The embryos are isolated from young seeds and placed on a solid medium containing nutrients and vitamins. Embryos are cultured at 25°c,first in dark until seedlings are about 2 cm long and root formation has started, and than in light until the seedlings can be planted in soil.

(6) Protoplast Culture It is one of the most significant and recent developments in the field of plant tissue culture. the protoplast are usually isolated from cultured cell or leaf mesophyll cell by treating them with enzyme solutions. the isolated protoplast may be used to regenerate the plants directly, or for the production of somatic hybrids through fusion.

(7) Micro Propagation It is in vitro asexual propagation of crop plants. This technique is advantages over the conventional practice of asexual propagation as only a small amount of plant is needed, species highly resistant to conventional bulk propagation can be propagated by this method and it is non season dependent. Micro propagation is used for rapid multiplication of stocks, elimination of diseases, germ plasm preservation, and induction of mutation.

Applications: Tissue culture technique has helped in studying various biochemical and physiological processes in pure cultures without complications of other factors. The effect of various hormones are differentiation and growth processes can be studied by using tissue culture technique. Organogenesis has been studied by inducing formation of roots , shoot tips etc from callus on nutrient medium.