Plant tissue culture (1)

Plant Tissue Culture Types and Techniques

1. Techniques of Tissue Culture

2. Tissue Culture The process of growing cells artificially in the laboratory Involves both plant and animal cells Produces clones , in which all product cells have the same genotype (unless affected by mutation during culture)

3. Requirements Tissue culture has several critical requirements: Appropriate tissue A suitable growth medium Aseptic (sterile) conditions Growth regulators Frequent subculturing

4. What is plant tissue culture ? Plant tissue culture is a technique of growing plant cells, tissues, organs, seeds or other plant parts in a sterile environment on a nutrient medium

5. How? Adult plant cells are totipotent , meaning they have the ability to give rise to a fully differentiated plant. Because of this, it is possible to collect cells from a mature plant and use those cells to produce clones of that plant.

6. Basic Tissue Culture Procedures

7. Micropropagation The art and science of plant multiplication in vitro Usually derived from meristems (or vegetative buds) without a callus stage Tends to reduce or eliminate somaclonal variation, resulting in true clones Can be derived from other explant or callus (but these are often problematic)

8. Steps of Micropropagation Stage 0 – Selection & preparation of the mother plant sterilization of the plant tissue takes place Stage I  - Initiation of culture explant placed into growth media Stage II - Multiplication explant transferred to shoot media; shoots can be constantly divided Stage III - Rooting explant transferred to root media Stage IV - Transfer to soil explant returned to soil; hardened off

9. An unspecialized and unorganized, growing and dividing mass of cells, produced when explants are cultured on the appropriate solid medium (with both an auxin and a cytokinin and correct conditions) During callus formation there is some degree of dedifferentiation both in morphology and metabolism, resulting in the lose the ability to photosynthesis Callus Culture

10. Callus cultures may be compact or friable Compact callus shows densely aggregated cells Friable callus shows loosely associated cells and the callus becomes soft and breaks apart easily Habituation: it is the lose of the requirement for auxin and/or cytokinin by the culture during long-term culture Callus

11. Cell-suspension cultures When friable callus is placed into the appropriate liquid medium and agitated, single cells and/or small clumps of cells are released into the medium and continue to grow and divide, producing a cell-suspension culture The inoculum used to initiate cell suspension culture should neither be too small to affect cells numbers nor too large too allow the build up of toxic products or stressed cells to lethal levels Cell suspension culture techniques are very important for plant biotransformation and plant genetic engineering

12. Embryo Culture Embryos are allowed to grow for some time in an artificial medium Embryo culture developed from the need to rescue embryos from wide crosses where fertilization occurred, but embryo development did not occur Artificial embryo culture media basically contain glucose, pyruvate , and energy-providing components, but the addition of amino acids, nucleotides, vitamins, and cholesterol improve the performance of embryonic growth and development

13. Somatic Embryogenesis When embryos regenerate from somatic cells or tissues (which are haploid,diploid etc.) it is termed as Somatic Embryogenesis. Somatic embryogenesis is a process by which the somatic cells or tissues develops into differentiated embryos.

14. Somatic Embryogenesis Types: Direct somatic embryogenesis: The embryo is formed directly from a cell or small group of cells such styles or pollen without the production of an intervening callus. Direct somatic embryogenesis is generally rare Indirect somatic embryogenesis Callus is first produced from the explant and then embryos are produced from the callus tissue or from a cell suspension cultures.

15. Organogenesis Definition : The production of organs, either directly from an explant or from a callus culture. Organogenesis depend on adventitious organs arising either from a callus culture or directly from an explant or on the formation of axillary bud to regenerate whole plants from some types of tissue culture.

16. Shoot and Root Culture Shoot culture is promoted by cytokinins and auxins like NAA (naphthalene acetic acid) The shoot and root cultures are generally controlled by auxin- cytokinins balance Usually an excess of auxin promotes root culture, whereas that of cytokinin promotes shoot culture

17. Meristem Culture Cultivation of axillary or apical meristems is called " Meristem culture". Meristem culture involves development of already existing shoot meristem . Technique is used for quick vegetative propagation of a large number of plant species in a short period. Meristem culture produces virus free callus and eventually plantlet.

18. Anther culture Anther culture is a technique by which the developing anthers at a precise and critical stage are excised aseptically from unopened flower bud Cultured on a nutrient medium where the microspores within the cultured anther develop into callus tissue or embryoids Give rise to haploid plantlets either though organogenesis or embryogenesis.

19. Pollen Culture Pollen or microspore culture is an in vitro technique by which the pollen grains preferably at the uninucleated stage ,are squeezed out aseptically from the intact anther then cultured on nutrient medium develop into haploid embryoids or callus tissue that give rise to haploid plantlets by embryogenesis or organogenesis

20. Protoplast The living material of a plant or bacterial cell, including the protoplasm and plasma membrane after the cell wall has been removed .

21. Ovule culture Utilized for raising hybrids which normally fail to develop due to abortion of the embryo in early stages Ovules can be easily excised from the ovary and cultured on the basal medium Loss of hybrid embryo due to premature abcission of fruits may be prevented by ovary culture Addition of fruit/vegetable juice increase the initial growth

22. Ovary culture Sucessfully employed to interspecific hybrids between sexually incompatible species Ovaries are excised from the flower and cultured at the zygote or two celled proembryo stage for obtaining for obtaining normal development on culture medium Coconut milk added as supplement to medium promote formation of fruits In anethum , addition of kinetin in the medium caused polyembryonony which gave rise to multiple shoots

23. Advantages Mass production of various plant cultivars 6 million plants per year from one explant. Much higher production rate than other asexual propagation methods. Especially beneficial for: Plants in high demand or valuable plants. Plants that are slow or difficult to propagate. Endangered species.

24. Advantages Production of pathogen-free plants M aintaining disease-free plants by micropropagation. Germplasm preservation Germplasm: the DNA of a species In the past: seeds limited shelf-life don’t preserve uniform characteristic (variability)

25. Advantages Continuous year round production Unaffected by climate Propagated in controlled lab conditions The ability to change specific conditions to meet the needs of a particular plant species. Mainly, nutrient, light and temperature requirements.

26. Advantages The original plant is not destroyed in the process - a factor of considerable importance to the owner of a rare or unusual plant.

27. Disadvantages Specialized equipment required Laminar flow cabinets Autoclave Water purification systems Glassware etc… High labor cost is the most limiting factor Skilled labor required

28. Disadvantages Contamination risks Maintenance of aseptic (sterile) environment difficult. Rapid spread of contaminants = widespread loss. Risk of mutation arising Artificial environment induces mutations . R esponses to tissue culture conditions varies Trial and error to determine optimum media or conditions 。

29. Factors affecting tissue culture The areas in which tissue culture techniques can be used are very wide. The choice of technique is dependent on what one wants to achieve. It may be mass production , breeding of new varieties , or producing virus-free plants . To be able to successfully propagate plants in vitro , understanding how and why these factors affect plant growth in an in vitro environment is crucial .

30. Factors affecting tissue culture The in vitro growth and development of a plant is determined by a number of factors: The genetic make-up of the plant Source of explants Nutrients Environmental factors: light, temperature, pH, O 2 and CO 2 concentrations.

31. Factors affecting tissue culture The genetic make-up of the plant. The genetic make-up is a decisive factor at every stage in the plant. It determines, for example, if a plant is a monocotyledon or dicotyledon, or which temperature is optimal for growth. The type of in vitro environment that must be created in the lab to ensure that growth and development of the explant takes place, is totally dependent on the genotype of the plant.

32. Factors affecting tissue culture Source of explant Young explant vs. old explant Usually the younger, less differentiated explant, the better for tissue culture Type of explant – leaf, stem, root, meristem, etc.

33. Factors Affecting Tissue Culture Growth medium (Artificial) Nutrients Plant hormone Vitamins Environmental factors (Controlled) Light intensity Photoperiod Temperature Sterility