Application of micro-propagation

APPLICATION OF MICRO-PROPAGATION

Sushil NyaupaneDepartment of Natural Resources and Environmental DesignNorth Carolina A&T State University

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.

Plant Tissue CulturePlant tissue culture refers to the techniques of growing plant cells, tissues, organs, seeds or other plant parts in a sterile environment on a nutrient medium.

Who they are ?

Haberlandt T. Murashige F. Skoog

4

A glance in History

In 1902, Haberlandt; father of plant tissue culture ( He proposed that plant cells could be cultured.

In 1930- White cultured tomato root tip and subculutred to fresh medium containing salts, yeast extract and sucrose and vit B

During this period, some plant growth regulators, additives and vitamins was discovered for the plant micro- propagation.discovery of first PGR ---IndoleAcetic Acid , called IAA, in 1937

In 1962, Murashige and Skoog published a recipe for MS media.In 1972, protoplast fusion has been done in toabacco.

What is micro-propagation?

Micropropagation is rapid clonal in vitro propagation of plants from cells, tissues or organs cultured aseptically on defined media contained in culture vessels maintained under controlled conditions of light and temperature.

Briefly, it is the art and science of multiplying plants in- vitro.

7

ExplantsExplant is an excised piece of tissue or organ taken from the plant to initiate a culture.They can be : shoot meristem, tip, bud leaf or stem (internode) root anther / microspore ovule embryo associated seed parts

Shoot Explants

Meristem TipShoot Tip

Dormant Bud

Somatic Embryo Culture

Leaf Explants

Seed explants

Pollen culture

Plant tissue culture types for micro-propagation

Then can you tell me about this??

Yes, this is from lab.

Tissue cultured banana

20

Steps of micro-propagationStage 0- Selection and preparation of the mother plant(Sterilization of the explant tissues)

Stage I- Initiation of culture ( Explant placed into growth media)

Stage II- Multiplication ( Explant transferred to shoot media and shoot can be constantly divided)

Stage III- Rooting ( Explant transferred to rooting media)

Stage IV- Transfer to soil ( Hardening off)

21

Why do micro-propagationA single explant can be multiplied into several thousand plants in less than a year.

Once established, it can give a continuous supply of young plants throughout the year i.e. irrespective of season.

Taking an explant does not usually destroy the mother plant, so rare and endangered plants can be saved somehow.

Clones through micro-propagation are true to type as compared with seedlings, which show greater variability

Why do micro-propagation?This allows fast selection for crop improvement - explants are chosen from superior plants, then cloned.

Plant tissue banks can be frozen, then regenerated through micro-propagation.

Disease and virus free plants can be produced through this technique.

Greater credibility in international market as plantlets are produced through micro-propagation( Phyto-sanitary perspective)

How possible is this ??Micro-propagation of almost all vegetables and fruit crops is possible.Some examples,mustard, corn, soybean, azalea, dwarfing sweet cherry, strawberry, mango, banana, rose, orchid, nutraceutical plants, rhododendron, citrus, potato, tomato,legumes etc.

Applications of micro-propagationSomaclonal VariationGermplasm ConservationMutation BreedingInducing mutationEmbryo cultureHaploid and Dihaploid productionIn Vitro Hybridization- protoplast fusionProduction of Disease free plantsMolecular farmingGenetic engineeringProduction of secondary metabolites

Applications ..Somaclonal variationSomaclonal variation is a general phenomenon of all plant regeneration systems that involve a callus phase.

There are two types of Somaclonal variation:Heritable genetic variation

Non- Heritable genetic variation

Applications..Advantages of somaclonal variationIt helps in crop improvementCreates additional genetic variantsPlants with resistant and tolerant to toxins, herbicides, high salt and even mineral toxicitySuitable for breeding purposesIncreased and improved production of secondary metabolites

Applications..Germplasm conservationMicropropgation is utilized in conserving genetic resources.

Depending upon the crop species and method of preservation, tissue culture can help in the preservation of genetic resources from 1 to 15 years.

Cyopreservation the preservation of germplasm in a dormant state at ultra-low temperatures, usually in liquid nitrogen (-196 C) is a type of tissue culture which can be used to preserve seeds.

Applications..Mutation Breeding 1927: Muller produced mutations in fruit flies using x-rays1928: Stadler produced mutations in barleyBasically, there are three groups of breedersMutation breeding is useless, we can accomplish the same thing with conventional methods Mutation breeding will produce a breakthrough given enough effort Mutation breeding is a tool, useful to meet specific objectives

Applications..Types of mutationSpontaneous (natural mutation)Some type of spontaneous mutation have played an outstanding role in development of valuable crop cultivars and hybrids.But limitation of this is that, it can not form the basis of modern plant breeding

Induced mutationAny mutation found in nature can be induced by mutation breeding.

31

Applications..Inducing Mutations Physical mutagens (Irradiation)These mutagens helps in the chromosome aberrations and point mutations.Neutrons, Alpha raysGamma, Beta, X-raysChemical mutagens By using different carcinogenic chemicals which are highly toxic in nature and eventually result in point mutations.

Applications..Embryo Culture Embryo culture is usually done from the need to rescue embryo from wide crosses where fertilization occurred, but not the embryo development.Objectives:Rescue F1 Hybrid from wide crossesOvercome seed dormancy by addition of hormone to media. For ex. GATo overcome immaturity in seed To rescue valuable genotype from dead or dying plant To speed up the generations in a breeding program

Applications..Embryo culture as a source of genetic variation Hybridization Can introduce new genetic combinations through inter-specific crosses Can transfer mutant alleles between species Polyploidy It combines embryo culture with chromosome doubling to create new polyploid species

ApplicationHaploid and dihaploid productionPlants produced through the anther culture are the haploids. Doubling the chromosomes without going into series of backcrossing produce homozygous plants. This technique shortens the time of breeding by half.

Application In Vitro Hybridization( Protoplast fusion)Created by degrading the cell wall using enzymes.Very fragile in nature.Protoplasts can be induced to fuse with one another.Methods:ElectrofusionPoly Ethylene Glycol(PEG)Addition of calcium ions at high PH Values

Applications..Production of Disease free plant Heat treatment Plants grow faster than viruses at higher temperature.Meristemming Viruses are transported from cell to cell through plasmodesmata and vascular tissue.Apical meristem are virus free in nature. So, micro-propagation of these cells gives virus free plantlets.Even, not all the cells in the plant are infected. For example, adventitious shoots formed from single cells can give virus free shoots.

Applications.. Molecular farmingWhere plants are treated as bioreactors for the production of specific compounds. Range from simple peptides to a thermoplastic. Two types of products: 1)High value compounds with small scale production requirements such as pharmaceutical products. For ex. Malaria epitope. 2)Compounds needed on a bulk scale with low production costs (plant biotechnology has greatest potential in this area)For ex. -Amylase (food + detergent industries)-starch manipulation

ApplicationsGenetic Engineering

Genetic engineering would not be possible without the development of plant tissue culture.

Genetic engineering requires the regeneration of whole plants from single cells.

Efficient regeneration systems are required for commercial success of genetically engineered products.

Application..Protoplast fusion between male sterile cabbage and normal cabbage was done and cybrids were selected that contained the radish mitochondria and cabbage chloroplast.

Applications.. Production of secondary metabolitesSecondary metabolites are those cell constituents which are not essential for survival.For example, alkaloids, glycosides, terpenoids,latex,tannins etc. In vitro production of secondary metabolites is much higher from differentiated tissues compared to non-differentiated tissues. Azadirachtin from Azadirachta indica Insecticidal Berberine from Coptis japonica Antibacterial, anti inflammatory Capsaicin from Capsicum annum Cures Rheumatic pain

Problems of micro-propagation Expensive laboratory equipment and service No possibility of using mechanization Plants are not autotrophic Poor Acclimatization to the field is a common problem (hyperhydricity) Risk of genetic changes if 'de novo' regeneration is used Mass propagation cannot be done with all crops to date. In cereals much less success is achieved Regeneration is often not possible, especially with adult woody plant material.

Do you have any idea about this orchid?

Micro-propagation a tool for commercialization (Taiwan story)According to Taiwan Today, Jan 1, 2016The nations orchids are exported to 36 countries in Northern America, Northern Europe and South Africa.

This country began the export with US $ 23 million in 2004 but in 2015 they have exported with worth US $ 130 million.They are selling a Phalaenopsis or moth orchid in Dubai at the price of US $ 1,000.

Won the bid to host the 23rd World Orchid Conference in 2020.

44

What may be the reason for this achievement ?According to John Feng, CEO of SOGO TEAM CO LTD., they are exploiting micro-propagation as a technique in mass production as well as in variety improvement.

In I-Hsin Biotechnology Corps Tissue Culture Lab only, they have conducted 12,000 breeding experiments which have yielded 2,300 Phalaenopsis varieties.This all is being possible through tissue culture.

ALBIFLORA ORCHID OF NEPALIn Nepal a total of 377 species of orchids belonging to 100 genera reported.

46

Future of micro-propagation

Perhaps some of the greatest discoveries of plant tissue culture are yet to come.

SO BE READY !

47

THANK YOU !