Plant tissue Culture pharmacognosy and phytochemistry I Unit 3

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. Plant Tissue Culture: Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues, or organs under sterile conditions on a nutrient culture medium of known composition. It is widely used to produce clones of a plant in a method known as micropropagation. Explant: An explant is the part of a plant which has got the regeneration potential and is capable to give rise to the whole plant. Explants types

3. Applications M icropropagation using meristem and shoot culture to produce large numbers of identical individuals. L arge-scale growth of plant cells in liquid culture as a source of secondary products. C rossing 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.

9. Organ Culture : In this, any organ of the plant such as shoot, leaf, can be used as an explant. A number of methods can be used for the organ culture, such as plasma clot method, raft method, grid method, and agar gel method. This method is used to preserve the structure and functions of an organism. Protoplast Culture: It is a cell without a cell wall. A protoplast can be cultured using the hanging-drop method, or micro-culture chambers. In protoplast culture, a number of phases can be observed: development of cell wall, cell division, regeneration of a whole plant. Shoot tip culture: The shoot tips are the tissues that are originally able to regenerate the plant. Shoot-tip culture isolated from plants can multiply in the form of a protocorm-like body, auxiliary bud, or shoot primordium and regenerate clone plants. Root tip culture: In culture, root tips are induced to grow like that of root system of an intact plant. A clone of excised roots can also be established from a single root culture by repeatedly cutting and transferring of the main root tips or of lat­eral tips into fresh medium in every subculture at the interval of definite period . Anther culture means plant regeneration from the haploid microspore cells with the aim of haploid and dihaploid plant production.

10. Hairy root culture: Hairy roots (HRs) are differentiated cultures of transformed roots generated by the infection of wounded higher plants with Agrobacterium rhizogenes. Bud Culture: Buds contain active meristems in the leaf axils which are capable of growing into a shoot. Single node culture is where each node of the stem is cut and allowed to grow on a nutrient medium to be developed into a shoot tip from the axil which ultimately develops into a new plantlet. Callus Culture: Callus is a more or less unorganized de-differentiated mass of cells arising from any kind of explant under in vitro cultural conditions. The cells in the callus are parenchymatous but may or may not be a homogenous mass of cells. Cell Suspension Culture: The growth of individual cells that have been obtained from any kind of explant tis­sues or callus refers to as the cell suspension culture. These are initiated by transferring pieces of tissue explant/callus into a liquid medium (without agar) and then placing them on a gyratory shaker to provide both aeration and dispersion of cells . Flower culture can be defined as the aseptic culture of excised floral bud on a chemically defined nutrient medium where they continue their development to produce a full bloom in a culture vessel.

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 .

14. 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.

15. 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

16. 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.

17. 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 /.