APPLICATIONS OF PLANT TISSUE CULTURE SMG

A brief account of Applications of Plant tissue culture - Micropropagation, Meristem culture, Synthetic seeds, Embryo culture, In vitro mutagenesis, In vitro production of secondary metabolites

1. Applications of Plant Tissue Culture Dr.Saji Mariam George Associate Professor Assumption College Autonomous Changanacherry

2. Applications of Plant Tissue Culture Micropropagation A large number of plantlets can be produced from a small part of plant by in vitro tissue culture in a suitable nutrient medium. This method can be used for the production of large number of plantlets in rare, endangered and threatened species ; commercially important ornamental plants like orchids, Anthurium , Heliconia etc. ; Banana; plantation crops like Cardamom etc.

3. ii) Production of Virus free (disease free) plants by Meristem culture (Morel and Martin 1952) In vitro culture of shoot apical meristem Apical or lateral shoot tips are used – contains shoot apex and several leaf primordia – at the axils of which axillary shoots could potentially develop. Excised shoot tips are cultured on solid medium with high concentration of a cytokinin hormone – development of axillary shoots – separate each shoot and repeat the process to rapidly multiply the shoots. These shoots are planted into a solid medium containing auxin (root inducing hormone) – formation of plant let.

4. iii) Production of Artificial seeds (Synthetic seeds/ Synseeds ( Toshio Murashiege 1978 ) –) Uploaded by: Dr. Somdeep Ghosh , Oct 18, 2018

5. Somatic embryos of a well known strain can be produced by tissue culture and they can be encapsulated in a protective gel containing nutrients, growth regulators etc. to produce artificial or synthetic seeds. Bead of gel contains Somatic embryo Nutrients (serves as artificial endosperm) Growth regulators Pesticides Antibiotics

6. Synthetic Seeds contain identical somatic embryos of a well known strain Small in size since they contain only minimum essential amount of nutrients. Encapsulating agents Sodium alginate Polyacrylamide Nitrocellulose Agarose Polyoxyethylene

7. Synthetic Seed Production 1.) Desiccated systems – Somatic embryos are first hardened to withstand desiccation and then are encapsulated in a suitable coating material. Steps: Production of somatic embryos by tissue culture Hardening of somatic embryos to withstand desiccation Treating or coating mature somatic embryos with a suitable polymer and drying Treating somatic embryos with Abscisic acid (ABA) during maturation phase.

8. Encapsulation in a suitable encapsulating agent (Protective gel/ chemical membrane) eg . Synthetic seed production in Carrot (Kim and Janick 1989) Mixed equal volumes of carrot somatic embryo suspension and 5% solution of poly ethylene oxide ( a water soluble resin) and dried to form polyembryonic desiccated seeds.

9. 2.) Hydrated System Somatic embryos are enclosed in gels which remain hydrated. Somatic embryos are prepared by tissue culture 2% solution of sodium alginate filled in a burette is allowed to form a drop at the tip. Somatic embryo is inserted into the drop with a spatula.

10. The drop with somatic embryo is allowed to fall into 100m M Calcium chloride solution. The bead becomes hardened as Calcium alginate is formed After 20 – 30 minutes , the artificial seeds are removed, washed with water. A waxy coating can be provided over the seeds to prevent the sticky nature (difficult to handle on a large scale and dry rapidly in air)

11. Advantages of Hydrated Artificial seeds Reduced cost of vegetatively propagated elite lines Direct transplantation to field Genetic uniformity of plants

12. Significance / Advantages of Synthetic Seeds A potential technology for the use of somatic embryogenesis for large scale propagation of plants through automation Somatic embryos of suitable age can be produced in a bioreactor.

13. Disadvantages / Limitations of Synthetic seeds Quality of somatic embryos Low gas exchange in coated somatic embryos

14. iv) Embryo Culture ( Hanning , 1904 ) In vitro culture of embryo in a suitable medium to obtain seedlings. Hanning tested a variety of nutrient media containing sugars, mineral salts, plant decoctions, certain amino acids and gelatin.

15. Chemical composition of culture media for Embryo culture Carbon source – Sucrose/Glucose Mineral salts Amino acids Vitamins Growth regulators Gelatin/Agar Organic supplements Plant decoctions Coconut milk Casein hydrolysate Yeast extract pH: 5 – 7.5

16. Young embryos – need elaborate culture medium containing many nutrients Mature differentiated embryos – need only a medium with minimum nutrients – a few mineral salts and sucrose.

17. A medium containing Rich source of sucrose/glucose Ammonium nitrate Vitamins Casein hydrolysate Plant extracts with growth regulators pH 5 – 7.5 – show good response to embryo culture

18. Procedure Surface sterilization of seeds (5-10% Chlorox or 0.45% Sodium hypochlorite for 5 – 10 minutes) Dissect out the embryo Direct inoculation on to the medium or after surface sterilization with 70% ethanol (30-60 seconds) Incubation at 25  2 O C  Callus  Regeneration by organogenesis or embryoids (somatic embryos)  Plants

19. Other methods In cereals, transfer of embryos to the endosperm of other genera ( Stingl , 1907) Culture in Knop’s solution with 2.5 – 5% cane sugar and 1.5% Agar (Dietrich, 1924) Embryo placed on moist filter paper containing sucrose/glucose ( Laibach 1925, 1929) Embryo rescue in interspecific cross, Linum perenne x L. austriacum )

20. Types of Embryo culture Embryo culture involves the culture of Proembryos (immature embryos) Culture of heart shaped, globular proembryo in a suitable medium. Helps to understand – the differentiation process – Nutritional requirements of developing embryo 2. Intact seed containing undifferentiated embryo. (lacking radicle and plumule , eg : Orchids)

21. 3. Mature and intact seed embryo – helps to determine diverse parameters of embryonic growth 4. Dissected embryo-helps to analyze the interrelationship of different parts of the embryo. 5. Inviable or abortive embryos. (Embryo rescue) – In interspecific or intergeneric crosses.

22. Applications of Embryo culture 1. Shortening the breeding cycle – develop into seedlings – avoid a long time of seed maturity. 2. Overcoming dormancy (in certain fruit trees) 3. Recovery of distant hybrids (Embryo rescue – in interspecific or intergeneric crosses) 4. Propagation of orchids – young or mature embryos are cultured. 5. Propagation of rare plants – Makapuno coconut (has soft, solid, fatty tissue in place of liquid endosperm).

23. v) In vitro Mutagenesis Used to improve cultivars of vegetatively propagated plants. Materials used: Highly regenerative cell lines. Protoplasts Shoot apical meristem

24. Use of Physical Mutagen – Gama Rays (from 60 Co) Irradiation of explant – shoot tips, in vitro plant parts. Culture of irradiated explant in a medium containing auxins and cytokinins (72 -96hrs- recover from radiation shock) Selection of desirable mutants – resistant to a pathogen, herbicide, heavy metal toxicity etc.

25. Selection of Fusarium wilt tolerant mutants Add the toxin produced by the pathogen Fusarium – Fusaric acid to the medium. Remove dead tissues at regular intervals to avoid harmful effects on rate of shoot regeneration. Subculture at regular intervals (3 -4 weeks) on a fresh culture medium containing the toxin Fusaric acid to dissociate chimeras ( two or more genetically distinct tissues) and maintain the stability of mutant traits.

26. Use of Chemical Mutagen – Ethyl Methane Sulphonate (EMS) Most common method. Immerse the explant in a solution of optimum dose of EMS for 30 minutes. Wash the explant repeatedly in sterile distilled water. Transfer the explant to liquid growth medium.

27. Culture on fresh media at least twice to remove residual mutagen. Transfer the explant to sterile Whatman filter paper to remove excess liquid growth medium. Transfer to a semi – solid M S basal growth medium containing growth regulators for shoot regeneration and finally plant regeneration.

28. A Scheme for producing induced mutants by in vitro mutagenesis In vitro Culture Mutagen treatment Regenerate induced mutants Screening of regenerated plants (Proto plast / cell cultures/ adventitious buds/ somatic embryos) (Physical or Chemical ) (By Somatic embryogenesis/ adventitious shoot formation/ axillary bud break) Uniformly mutated Plants ( Homohistonts ) Chimeric Plants Reinitiate in vitro culture ( Homohistonts ) Field Testing (confirm stability of mutants Release new variety

29. vi) In Vitro Secondary metabolite production In vitro plant tissue cultures can be used for the commercial production of secondary metabolites – recognized since early 1950’s. Use of Batch cultures – Continuous cultures – Techniques are available for the induction and selection of stable genetic variants. Secondary Metabolites – Chemical substances derived from primary metabolites – Alkaloids, Terpenoids , Flavonoids , Phenolics , oils, steroids etc.

30. Not directly involved in the primary metabolic processes. Potential sources of drugs, pigments, flavours High economical and pharmaceutical importance.

31. Alkaloids - Natural, nitrogenous compounds derived from aminoacids and heterocycles of pyrrole , pyrimidine etc. High medicinal value. e.g. Quinine from Cinchona officianalis _ Cure for malaria Vincristine and vinblastin – from Catharanthus roseus – Anticancerous potential Taxol from Taxus brevifolia ,, Digoxin from Digitalis purpurea - for cardiovascular disorders Reserpine from Rauvolfia serpentina - for hypertension

32. Terpenoids Isoprenoids – (Five Carbon Isoprene units) Monoterpenes , sesquiterpines , diterpines . Helps in plant defense – antimicrobial (bactericidal, fungicidal, antiviral) eg . Azadirachtin , Nimbin , Nimbidin etc. from Azadirachta indica ( Neem ); Menthol Camphor Carotenoid pigments Polyterpenes - Rubber etc. Other Effects Cytotoxic Spermicidal Anticancerous

33. Flavonoids – Red, blue and purple pigments of plant tissues Antimicrobial Insecticidal

34. Advantages of in vitro secondary metabolite production Plant cells produce secondary metabolites only in small amounts. Secondary metabolites have complex structure – chemical synthesis is economically unattractive Plant cells can be easily cultured under aseptic, controlled nutritional and environmental conditions - can avoid variations in climate and soil .

35. Easy to incorporate precursors in suspension cultures (Difficult to administer to plant growing in native). Commercial production by Batch cultures, continuous cultures etc. Refined culture systems to improve biochemical yields .

36. Examples Commercial Production of Shikonin ( Naphtoquinone – an antiseptic, a dye for silk & cosmetics), from cell cultures of Lithospermum erythrorhizon . Taxol – (Alkaloid – for breast and ovarian cancer treatment ) from Taxus brevifolia . .

37. Extraction of Secondary Metabolites Explant  Sterilization  Induction of Callus  Cell Suspension Culture  Cell Plating  Cell colonies  Testing for high production potential (for desirable product)  High yielding clones  Large scale culture of cells in bioreactor  Extraction of Desirable secondary metabolites

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