Tissue Culture

Plant Tissue Culture and its Application By; Nasir Hussain

Tissue Culture • Production of microbe-free plant material in an in vitro conditions from any part of the plant in an aseptic (sterile) environment, such as sterilized nutrient medium. • It is non conventional method of vegetative propagation for any crop specie. • Conventionally vegetative propagation take place through cuttings, budding, grafting and layering etc.

Tissue Culture Techniques • • • • • • • • •

Micropropagation . Production of pathogen free plants. Meristem Culture. Somatic embryogenesis. Organ Culture. Callus Culture. Embryo culture. Anther and pollen grain Culture Protoplast culture etc.

Advantages of Tissue Culture • Elimination of viruses from infected plants • Rapid multiplication of clones (elite) • Vegetative propagation of difficult to propagate species • All the year round propagation of clones • True to type plantlet production • Rapid multiplication of seedlings (in cases where seed is hard to get)

Applications of Tissue Culture • Organ, meristem, shoot. • Undifferentiated (callus suspension). • Embryo rescue. • Somatic embryogenesis.

• Micropropagation, disease elimination, germplasm preservation. • In vitro selection, secondary metabolites production • Wide hybridization • Artificial seeds, genetic engineering.

Applications of Tissue Culture • Anther, microspore culture. • Protoplast

• Homozygous line production. • Gene pool diversification

Tissue culture methods for virus eradication Thermotherapy, chemotherapy and meristem tip culture. • Incubation of plants or of cultures at relatively high temperatures (32° to 40°C) either alone or • in combination with the excision and culture of very small stem tips is the only method of ridding infected cultivars of their viruses. • Antivirus (Ribavirin).

Tissue culture methods for virus eradication • Excise and culture the apical meristem of an infected plant because this region is most likely to be free of viruses. • It involves a very delicate dissection and such small explants are difficult to grow in culture. • A very small stem tip which has, in addition to the apical meristem, one or two leaf primordia. It is much easier to grow in culture. • High magnification stereomicroscope is also needed to excise the meristems (0.1-0.5 mm).

Tissue culture methods for virus eradication • However, some viruses invade the apical meristem and, in these cases, it is necessary to apply heat treatments to the plant. In the past, this has meant heat treatment of the whole plant followed by stem tip culture. An easier method involves the application of the heat treatment to aseptic cultures.

MERISTEM TIP CULTURE • Meristem tip refers to the region of shoot apex lying distal to the youngest leaf primordial, whereas shoot apex includes Meristem tips plus a few sub adjacent leaf primordial.

MERISTEM TIP CULTURE • Meristem tip culture is a method in which shoot meristem (Meristem tip) is cultured in vitro with a few leaf primordial. • ELISA (Enzyme-Linked Immunosorbent Assay ) protein based. • PCR nucleic acid base. • Clonal multiplication of virus free stock

MERISTEM TIP CULTURE

Micropropagation • Micropropagation is the production of whole plants from small sections of plant such as a shoot tip, node, meristem, embryo, or even a seed • Plant tissue culture is basically the same thing, except that it implies the use of callus tissue generated from plant cells cultured in-vitro.

Micropropagation • Why does micropropagation work? – Plant cells have the ability to reproduce the whole plant from single cells. This is called totipotency. – Totipotency is the ability of a single cell to express the full genome in the cells to which it gives rise by cell division.

Micropropagation • What part of the plant can be used for micropropagation? – Any part of a plant can be used. – Meristems, shoot and root tips, leaf tissue, anthers, embryos, flowers, virtually all parts of a plant can be used.

Micropropagation • The only limitation is that each plant is propagated differently and not every plant will respond the same way. • Each genus, species and variety may require a different tissue which will obtain the best results.

Tip bud

Leaf

Axillary bud

Internode

Root

Starting material for micropropagation

Micropropagation Methods • Micro cuttings • Small sections of plant tissue are cut from the mother plant • Placed into media • Grown out or subdivided again to produce more plants

Micropropagation Methods • Shoot Tip Culture (less 10 mm) • Meristem culture (0.1-0.5 mm) Surface sterilize, wash & culture. Higher cytokinins to over come apical dominance. Adventitious bud production. sub-culture frequently.

Basic in vitro propagation ...

Micropropagation Stages • The Basics of Micropropagation • The Four stages – Stage one – Explant establishment or initiation – Stage two – Multiplication – Stage three – Rooting – Stage four – Acclimatization or hardening off

Benefits for Micropropagation ... ♣ Rapid multiplication of clones ♣ Difficult species ? ♣ Genetic uniformity ? ♣ Aseptic conditions ♣ Micro- stock plants ♣ Controlled environment

Other applications ... ♣ in vitro micro-grafting ♣ Genetic conservation ♣ Plant improvement ♣ Experimental system