Plant tissue culture, basic requirements for plant tissue culture, culture media for plant tissue culture, explants, types of plant tissue culture, Applications of plant tissue culture, edible vaccine, Applications of edible vaccines.

1. Plant tissue culture

2. Explants types

3. Applications
• Micropropagation using meristem and shoot culture to produce large numbers
of identical individuals.
• Large-scale growth of plant cells in liquid culture as a source of secondary
products.
• Crossing distantly related species by protoplast fusion and regeneration of the
novel hybrid
• The new plantlets can be grown in a short amount of time.
• The new plantlets and plants are more likely to be free of viruses and diseases.
• The process is not dependent on the seasons and can be done throughout the
year.
• On a larger scale, the tissue culture process helps to supply the consumer
market with new subspecies and variety.

4. History
• 1902 Gottlieb Haberlandt proposed that plant cells can be cultured on artificial
media and developed the concept of in vitro cell culture.
• In 1904 Hannig initiated work on excised embryo culture and later cultured embryos
from several cruciferous species.
• In 1922 Kotte and Robbins suggested root and stem tips as possible explants to
initiate in vitro tissue culture.
• In 1926 Went discovered the first plant growth hormone i.e., Indole Acetic Acid
(IAA).
• In 1934 White reported vit. B as growth supplement in plant tissue culture was
reported by White.
• In 1937 White formulated the first synthetic plant tissue culture medium.
• In 1941 Van Overbeek introduced coconut water as a media component and
demonstrated its beneficial effects on in vitro tissue culture.
• In 1946 Ball raised whole plants from shoot tips of Lupinus.
• In 1954 Muir successfully induced cell division in mechanically isolated single cells.

5. • In 1955 Skoog and Miller reported the discovery of Kinetin, which is a type of
cytokinin and promotes cell division.
• In 1957 Skoog and Miller described chemical control hypothesis of root and
shoot differentiation by manipulating the ratio of concentrations of auxin and
kinetin.
• In 1962 Murashige and Skoog formulated MS medium with higher salt
concentrations.
• In 1964 Guha and Maheshwari produced the first androgenic haploid Datura
planty by anther culture.
• In 1971 Protoplasts were subcultured in vitro, and plants were regenerated from
their culture.
• In 1993 Kranz and Lorz produced fertile maize plants through in vitro
fertilisation.
• In 2000 Transgenic rice engineered for the production of provitamin A (beta-
carotene) in rice endosperm was developed and is called ‘Golden Rice’.
• In 2016 Somatic embryogenesis was introduced in plant transformation through
the embryonic genes.

6. Basic requirements
• Washing area: The place to wash glassware, plastic ware and other lab ware
used in PTC. Washed lab ware need to be stored at a clean and dry place.
• Various media components for the preparation of Nutrient Media.
• Facility to sterilize nutrient media and store media at low temperatures.
• Facilities for maintenance of cultured tissues in aseptic conditions under
controlled environment i.e., light, temperature and humidity.
Culture vessels Glassware Incubator

7. Scissors, scalpels, forceps Spirit burner Autoclave
Spirit burner pH meter Laminar air flow cabinet

8. Types of Plant tissue culture
• Seed Culture: In this culture, the explants are obtained from an in-vitro derived
plant and introduced into a laboratory where they proliferate. The explant
should be sterilized to prevent it from tissue damage.
• Embryo Culture: This involves the in-vitro development of an embryo. For this,
an embryo is isolated from a living organism. Both, a mature or an immature
embryo can be used in the process. Mature embryos can be obtained from ripe
seeds. The immature embryos are obtained from the seeds that failed to
germinate. The ovule, seed or fruit is already sterilized, therefore, it does not
need to be sterilized again.
• Callus Culture: A callus is an unorganized, dividing mass of cells. When the
explants are cultured in a proper medium, the callus is obtained. The growth of
callus is followed by organ differentiation. The culture is grown on a gel-like
medium composed of agar and specific nutrients required for the growth of the
cells.
• Pollen culture is a technique of production of haploid plants by the culture of
pollen grains isolated from an anther of a plant under the aseptic condition on
artificial media of known composition.

11. • Single cell culture: An explant is the part of a plant which has got the
regeneration potential and is capable to give rise to the whole plant.
• Leaf culture: a new method in the propagation of plants using leaves of the
plants.

12. Nutritional requirements for plant tissue culture
• Some of the important media are: White’s ’s Medium, MS (Murashige and
Skoog) Medium, B5 (Gamborg’s) Medium, LS (Linsmaier and Skoog) Medium
• Organic Nutritional: Vitamins like thiamine (B1), Pyridoxin (B6), Nicotinic Acid
(B3). Antibiotics like Streptomycin, Kanamycin. Amino Acids like Arginine,
Asparagine
• Inorganic Nutrients: Micronutrients: Manganese (Mn), Iron (Fe), Molybdenum
(Mo), Zinc (Zn), Copper (Cu), Boron (B). Macronutrients: Nitrogen (N), Sulphur
(S), Phosphorus (P), Potassium (K), Magnesium (Mg), Calcium (Ca).
• Carbon and Energy Sources: Lactose, Maltose, Galactose, Raffinose, Cellobiose.
• Growth hormones: Auxin, cytokinins, Gibberllins, absisic acid.

13. Plant Tissue Culture Steps
Selection and Sterilization of Explant, Preparation and Sterilization of the
Culture Media, Inoculation, Incubation, Sub-Culturing, Transfer of Plantlets.

15. Edible Vaccines
• Edible vaccines are subunit vaccines where the selected genes are introduced
into the plants and the transgenic plant is then induced to manufacture the
encoded protein. Foods under such application include potato, banana, lettuce,
corn, soybean, rice, and legumes.
• An edible vaccine is a food, typically plants, that contain vitamins, proteins or
other nourishment that act as a vaccine against a certain disease. Once the
plant, fruit, or plant derived product is ingested orally, it stimulates the immune
system. Specifically, it stimulates both the mucosal and humoral immune
systems.Edible vaccines are genetically modified crops that contain antigens for
specific diseases. Edible vaccines offer many benefits over traditional vaccines,
due to their lower manufacturing cost and a lack of negative side effects.
However, there are limitations as edible vaccines are still new and developing.
Further research will need to be done before they are ready for widespread
human consumption. Edible vaccines are currently being developed for measles,
cholera, foot and mouth disease, Hepatitis B and Hepatitis C.

16. Applications
• edible vaccines are easy to produce, purify, sterilize, and distribute.
• they do not require more expensive manufacturing equipment, only rich soil,
the cost to grow the vaccines is significantly lowered.
• edible vaccines do not require sterilized production facilities or the biosafety
standards required to cultivate certain pathogenic agents for traditional
vaccines which are expensive to implement and maintain.
• They are also easier and less expensive to store since they do not require strict
refrigerated storage.
• The seeds from an edible vaccine plant can also be easily dehydrated and
preserved for cheap and quick distribution.
• Eating a vaccine is a simpler means of administration compared to injection,
making them extremely economical. This reduces the need for medical
personnel and sterile injection conditions that are not always achievable in
developing countries.
• Edible vaccines are considered a “pharmafood” which is a food source that
increases health while also fighting diseases

17. Production
• Edible vaccines are subunit vaccines; they contain a antigen proteins for a
pathogen but lack the genes for the full pathogen to form. The first steps in
making an edible vaccine is the identification, isolation, and characterization of
a pathogenic antigen. In order to be effective, the antigen needs to elicit a
strong and specific immune response. Once the antigen is identified and
isolated, the gene is cloned into a transfer vector. One of the most common
transfer vectors for DNA being used for edible vaccines is Agrobacterium
tumefaciens. The pathogen sequence is inserted into the transfer DNA (T-DNA)
to produce the antigenic protein. It is then inserted into the genome,
expressed, and inherited in a mendelian fashion, which results in the antigen
being expressed in the fruit or plant.
• Techniques for production of Edible vaccines: Stable transformation, Transient
transformation, Bombardment method.

19. References
• https://en.wikipedia.org/wiki/Plant_tissue_culture.
• https://microbenotes.com/edible-vaccines/.
• https://www.plantcelltechnology.com/pctblog/different-types-of-tissue-
culture-processes/.
• Wijerathna-Yapa, A, Hiti-Bandaralage, J. Tissue Culture—A Sustainable
Approach to Explore Plant Stresses. Life 2023, 13, 780.
• Hesami M et. al.Advances and Perspectives in Tissue Culture and Genetic
Engineering of Cannabis. International Journal of Molecular Sciences
22(11):5671.
• https://biologyease.com/types-of-plant-tissue-culture/.