1. Plant Tissue Culture & Applications

2. Learning objective Define tissue culture
To get familiar with medium preparation and terminology of plant tissue culture
To practice aseptic culture of plant tissues.
What is tissue culture and why is it important?
What process is used for tissue culture propagation?

3. What is tissue culture?
Tissue culture is the term used for “the process of growing cells artificially in the laboratory”
Tissue culture involves both plant and animal cells
Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture)

4. Definition:
Plant tissue culture is the science of growing plant cells, tissues or organs isolated from the Mother plant, on artificial media in vitro under controlled conditions.

5. Why does tissue culture work?
There are numerous methods to propagate plants in tissue culture.
But the one principle that is constant is totipotency – all plants and plant parts have this potential.

6. Why does tissue culture work?
Totipotency:
The ability of a cell to differentiate and develop into a whole plant when given the correct conditions. This is because every cell has the genetic potential of the parent plant.

7. What’s the Background?
Tissue culture had its origins at the beginning of the 20th century with the work of Haberlandt (plants) and Alexis Carrel (animals)
The first plant growth hormone, indole acetic acid (IAA) – F.Kogl (1930
Haberlandt
Carrel

8. History
HABERLANDT is considered to be the father of plant tissue culture who conceived the concept of totipotency & cell culture in 1902.
Totipotency is the ability of a single cell to divide and produce all of the differentiated cells in an organism.
He was the first to consider culturing cells aseptically in a nutrient solution.
However, he was unsuccessful in his culture as he failed to recognize that cell differentiation requires plant growth regulators

9. The Background, II
The first commercial use of plant clonal propagation on artificial media was in the germination and growth of orchid plants, in the 1920’s
In the 1950’s and 60’s there was a great deal of research, but it was only after the development of a reliable artificial medium (Murashige & Skoog, 1962) that plant tissue culture really ‘took off’ commercially
Young cymbidium orchids

10. The Background, III
A more recent advance is the use of plant and animal tissue culture along with genetic modification using viral and bacterial vectors and gene guns to create genetically engineered organisms

11. What is needed? Tissue culture, both plant and animal has several critical requirements:
Appropriate tissue (some tissues culture better than others)
A suitable growth medium containing energy sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid
Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can over run a culture

12. What is Needed, II
Growth regulators - in plants, both auxins & cytokinins. In animals, this is not as well defined and the growth substances are provided in serum from the cell types of interest
Frequent sub culturing to ensure adequate nutrition and to avoid the build up of waste metabolites

13. Steps involved in the in vitro micropropagation
Cleaning of glassware
Preparation of nutrient medium
Selection and sterilization of explant.
Inoculation of aseptic explant in to nutrient medium.
Proliferation of shoots on a multiplication medium.
Transfer of shoots for sub-culturing.
Rooting and hardening of plantlets
Field trials.

14. Culturing (micropropagating) Plant Tissue - the steps
Selection of the plant tissue (explant) from a healthy vigorous ‘mother plant’ - this is often the apical bud, but can be other tissue
This tissue must be sterilized to remove microbial contaminants

15. The Steps, II
Establishment of the explant in a culture medium. The medium sustains the plant cells and encourages cell division. It can be solid or liquid
Each plant species (and sometimes the variety within a species) has particular medium requirements that must be established by trial and error

16. The Steps, III
Multiplication- The explant gives rise to a callus (a mass of loosely arranged cells) which is manipulated by varying sugar concentrations and the auxin (low): cytokinin (high) ratios to form multiple shoots
The callus may be subdivided a number of times
Dividing shoots
Warmth and good light are essential

17. The Steps, IV
Root formation - The shoots are transferred to a growth medium with relatively higher auxin: cytokinin ratios
The bottles on these racks are young banana plants and are
growing roots

18. The Steps, V
The rooted shoots are potted up (deflasked) and ‘hardened off’ by gradually decreasing the humidity
This is necessary as many young tissue culture plants have no waxy cuticle to prevent water loss
Tissue culture plants sold to
a nursery & then potted up

20. Why do Plant Tissue Culture?
A single explant can be multiplied into several thousand plants in less than a year - this allows fast commercial propagation of new cultivars
Taking an explant does not usually destroy the mother plant, so rare and endangered plants can be cloned safely
Once established, a plant tissue culture line can give a continuous supply of young plants throughout the year

21. Why do Plant Tissue Culture, II
In plants prone to virus diseases, virus free explants (new meristem tissue is usually virus free) can be cultivated to provide virus free plants
Plant ‘tissue banks’ can be frozen, then regenerated through tissue culture
Plant cultures in approved media are easier to export than are soil-grown plants, as they are pathogen free and take up little space (most current plant export is now done in this manner)

22. Why do Plant Tissue Culture, III
Tissue culture allows fast selection for crop improvement - explants are chosen from superior plants, then cloned
Tissue culture clones are ‘true to type’ as compared with seedlings, which show greater variability
To create exact copies of plants that produces particularly good flowers or fruits.
To quickly produce mature plants.
To produce multiple of plants in the absence of seeds or necessary pollination to produce seeds.
Used to regenerate the whole plants from plant cells that have been genetically modified.

23. 1. The technique of micro-propagation is an alternative approach to conventional methods of vegetative propagation, which has the enhanced rate of multiplication.
2. A million of shoot tips can be obtained from a small, microscopic piece of plant tissue within a short period of time and space.
3. The advantage in this type of propagation is that as shoot multiplication usually has a short cycle (2-6 weeks) and each cycle results in logarithmic increase in the number of shoots.
4. This method of propagation is more advantageous in case of bulb or corm pro­ducing plants, because mini-tubers or mini-corms for plant multiplication are available throughout the year irrespective of season.

24. 5. Smaller size of propagules are advantageous for storing and transporting as it takes lesser space.
6. The propagules can be maintained in soil-free environment which facilitates their storage on a large scale.
7. Stocks of germplasm can be maintained for many years using this method of propagation.
8. This method is more applicable where disease free propagules are wanted. This in vitro technique helps to raise pathogen free plant and to maintain them.

25. 9. Micro-propagation is very useful in case of dioecious plants, because there the seed progeny yield is 50% male and 50% female, but this technique helps to get the progeny according to the desired sex.
10. A major advantage of micro-propagation happens to be the minimum growing space required in commercial nurseries. Thousands to millions of plantlets can be maintained within the culture vials. This is specially useful for maintaining the horticultural species.
11. This method is more helpful in case of slow growing plants where the seeds are produced after a long term and the seeds are the only propagule. This method can overcome the difficulty to obtain the propagules.
12. Through seed production genetically uniform progeny is not possible always. But the micro-propagation method will help to maintain the genetic uniformity in the propagules.

26. Disadvantages of Micro propagation
Contamination of Cultures:
During micro propagation, several slow-growing microorganisms (e.g. Bacillus sp) contaminate and grow in cultures. The microbial infection can be controlled by addition of antibiotics or fungicides. However, this will adversely influence propagation of plants.
Contamination risks
Maintenance of aseptic (sterile) environment difficult.
Rapid spread of contaminants = widespread loss.

27. Disadvantages of Micro propagation
Brewing of Medium: (Browning)
Micro propagation of certain plants (e.g. woody perennials) is often associated with accumulation of growth inhibitory substances in the medium. Chemically, these substances are phenolic compounds, which can turn the medium into dark colour. Phenolic compounds are toxic and can inhibit the growth of tissues. Brewing of the medium can be prevented by the addition of ascorbic acid or citric acid or polyvinyl pyrrolidone to the medium.

28. Genetic Variability:
When micro propagation is carried out through shoot tip cultures, genetic variability is very low. However, use of adventitious shoots is often associated with pronounced genetic variability.
Risk of mutation arising
Artificial environment induces mutations.

29. Vitrification:
During the course of repeated in vitro shoot multiplication, the cultures exhibit water soaked or almost translucent leaves. Such shoots cannot grow and even may die. This phenomenon is referred to as vitrification. Vitrification may be prevented by increasing the agar concentration (from 0.6 to 1%) in the medium. However, increased agar concentration reduces the growth rate of tissues.

30. Cost Factor:
For some micro propagation techniques, expensive equipment, sophisticated facilities and trained manpower are needed. This limits its use.

31. Production of Disease-Free Plants:
Many plant species are infected with pathogens — viruses, bacteria, fungi, mycoplasma and nematodes that cause systemic diseases. Although these diseases do not always result in the death of plants, they reduce the quality and yield of plants. The plants infected with bacteria and fungi frequently respond to chemical treatment by bactericides and fungicides.
However, it is very difficult to cure the virus-infected plants. Further, viral disease are easily transferred in seed- propagated as well as vegetatively propagated plant species. Plant breeders are always interested to develop disease-free plants, particularly viral disease-free plants. This have become a reality through tissue cultures.

32. Disadvantages Specialized equipment required Laminar flow cabinets
Autoclave
Water purification systems
Glassware etc…
High labor cost is the most limiting factor
Skilled labor required
Responses to tissue culture conditions varies
Trial and error to determine optimum media or conditions。

33. 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.

34. 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, O2 and CO2 concentrations.

35. 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.

36. 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.

37. Factors affecting tissue culture
Growth medium (Artificial)
Nutrients
Plant hormone
Vitamins
Environmental factors (Controlled)
Light intensity
Photoperiod
Temperature
Sterility

38. Terminology Explant Living tissue transferred
from a plant to an artificial
medium for culture.
It can be any portion of
the shoot, leaves, roots,
flower or cells from a plant.

39. Terminology In vitro culture
From Latin- “within the glass” performing an experiment in a test tube.
All types of culture including animal cells, in vitro fertilization, etc.
Tissue culture
Inclusive term for growth of cells and tissues in a sterile environment
undifferentiated plant cells
plant callus
plant tissue

40. Terminology Micropropagation
The production of whole plants from small sections of a plant, called an “explant”.
Apical bud
Axillary bud
Meristem
Usually the method used by commercial tissue culture laboratories is micropropagation, since a whole plant (including shoots and roots) is produced, which is genetically identical to the mother plant.
Nowadays, tissue culture, in vitro culture and micropropagation are sometimes used interchangeably.

41. Subculture
After a period of time, it becomes necessary, due to nutrient depletion and medium drying, to transfer organs and tissues to fresh media.
In general, callus cultures are subcultured every 4-6 weeks. Theoretically plant cell and tissue cultures may be maintained indefinitely by serial subculturing.

42. Types of Growth:  Organized Growth:
It occurs when plant cell or tissue (explant) are transferred to culture media and continue to grow with their structure preserved.
 Unorganized Growth:
It occurs when pieces of whole plants are cultured in vitro and the cells aggregate and typically lack any recognizable structure. e.g. Callus and Cell Suspension cultures.