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Presentation on Commercial Exploitation of Micro-propagation in fruit crops & its Techniques

presented by

Pawan Kumar Nagar M.Sc. (Horti) PomologyReg. 04-2690-2015BACA, AAU, Anand

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MICRO-PROPAGATION

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What is MicropropagationWhat is MicropropagationA whole plant can be regenerated from a small tissue or plant cells in a suitable culture medium under controlled environment. The plantlets so produced are called tissue-culture raised plants.

The advantage is that in a relatively short time and space a large number of plants are obtained.

Type of micropropagationType of micropropagation1.Direct micropropagation

2.Indirect micropropagation

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Why do MicropropagationWhy do Micropropagation A single explant can be multiplied into several thousand plants in less

than a year - this allows fast commercial propagation of new cultivars.

Once established, a plant tissue culture line can give a continuous supply of young plants throughout the year.

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).

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Advantages of micropropagationAdvantages of micropropagation From one to many propagules rapidly Multiplication in controlled lab conditions Continuous propagation year round Potential for disease-free propagules Inexpensive per plant once established Precise crop production scheduling Reduce stock plant space Long-term germplasm storage Production of difficult-to-propagate species

DisadvantagesDisadvantages

Specialized equipment/facilities required More technical expertise required Protocols not optimized for all species Plants produced may not fit industry standards Relatively expensive to set up

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genetically

modifiedplant breeding

does not produce seeds

does not respond well to vegetative

reproduction7

Steps of MicropropagationSteps of Micropropagation

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Micropropagation involved in 5 steps:

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Steps involved in the Steps involved in the in vitroin vitro MicropropagationMicropropagation

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. 11

12Source : Cadila pharmaceuticals limited

Stage 0 : Selection and maintenance of stock plants for culture initiation

EXPLANT

SHOOT TIPS FROM YOUNG SUCKER

APICAL MERISTEM (1-2 Cm3)

SURFACED STERILIZATION

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Explant

Cell, tissue or organ of a plant that is used to start in vitro cultures.

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Stage- I : Initiation and establishment of aseptic culture

16Source: Jain Irrigation Systems Ltd

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Initiation

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Stage –II : Multiplication of shoot

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Stage -III: Rooting

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Stage –IV: Acclimatization / Hardening

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Healthy/elite plantlets are exposed to the natural conditions in a step wise manner.

It is a gradual acclimatization of in vitro grown plants to in vivo condition. The plantlets are transferred to the pots/polyghene bag and immediately

irrigated with inorganic/nutrient solution. Plants are kept in the hardening room where controlled conditions of light,

humidity and temperature are maintained. Plants are maintained under high humidity for 10-20 days and subsequently

transferred in the field so as to grow under natural conditions. The success rate of micropropagation depends on the survival of the plantlets when transferred from culture to the soil (field).

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Secondary Hardening

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Plants transferred to nursery bags

Kept for 6 to 8 weeks under 50% shade

Regular foliar sprays

Variation if observed is discarded

Plant ready for sale (1 feet height)

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Factors Affecting Plant micro-propagation

1. Growth Media

2. Environmental Factors

3. Explants Source

4. Genetics

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Method of Micro-propagation

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Method of micro-propagationA. Meristem tip culture

B. Callus culture

C. Suspension culture

E. Protoplast culture

D. Embryo culture

F. Ovule culture

G. Anther culture

H. Cell culture 32

In addition to major use of tissue culture techniques for rapid clonal multiplication of plants, this technique is highly important for several purpose as under:1. Production and maintenance of pathogen free stock plants;2. Long term in vitro conservation of germplasm.3. Selection and regeneration of transgenic plants.4. Conservation of germplasm.

Other Implications of Micro-propagation

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Micro-propagation Techniques & ProcedureA. Micro-Propagation by NodesB. Micro-propagation by Node Cuttings in a Liquid

Medium Procedure 1. Sterilize Petri dishes and prepare the laminar flow chamber by disinfecting the internal surfaces with alcohol. Sterilize the tools with an instrument sterilizer and place them on a sterile dish.2. Open the tube, take off the plantlet and place it on a Petri dish with the help of forceps.3. Remove the leaves and cut the nodes.4. Open a tube containing fresh sterile medium and place a node inside, trying to plunge it slightly into the medium with the bud up. Close the tube.5. Seal the tube with a gas-permeable plastic tape and label it correctly.It is recommended to place two explants in 16 x 125 mm tubes, three in 18 x 150 mm tubes, five in 25 x 150 mm tubes, and 20-30 in magenta vessels.

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Applications of Tissue Culture

1. Embryo culture2. Meristem culture3. Micropropagation4. Somatic embryogenesis and Organogenesis5. Somaclonal variation and in vitro selection6. Anther culture Haploid & Dihaploid Production7. Protoplast culture (In vitro hybridization –

Protoplast Fusion)8. Germplasm preservation

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Micro-propagation technology developed for some Horticultural Crops in India

Sr. No.

Crop Institute

1. Annona IIHR, Banglore; BARC, Mumbai; NCL, Pune

2. Banana NCL, Pune; IIHR, Banglore; TNAU, Coimbatore

3. Citrus NBRI, Lucknow; NRC- Citrus, Nagpur4. Grape IARI, New Delhi5. Guava GBPUAT, Pantnagar6. Papaya IARI, New Delhi

7. Pineapple BARC, Mumbai

8. Strawberry TERI, New Delhi

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Tissue culture Banana

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Tissue culture Date palm

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Tissue culture Pomegranate Tissue culture Pomegranate

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Tissue Tissue cultureculture papaya papaya

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Tissue culture strawberry

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