Micropropagation is a method of propagating plants that uses very small parts of plants that are grown in sterile culture. There are generally four stages involve in Micropropagation.
Stages of Micropropagation
STAGE 1 - Establishment of explant in culture: We can take virtually any part of the plant.
Vegetative portions: Shoot tip, meristem, leaves, stems, roots.
Reproductive portions: Anthers, pollen, ovules, embryo, seed, spores.
Considerations for selecting plant parts:
1. Part of plant used
2. Disinfestation or surface sterilization: Must clean plant tissues of all contaminating microorganisms
3. Medium: Must contain all components necessary to nourish explant
a. Inorganics: Macronutrients(N,P,K,Ca,Mg) & Micronutrients(B,Co,Cu,Mn,I,Fe,Zn)
b. Organics - carbon source - needed since plants do not seem to photosynthesize well in culture
c. Vitamins: Thiamine (essential), Myoinisitol, B vitamins, folic acid, & biotin
d. Growth regulators: Cytokinins, Auxins, GA, ABA rarely used
e. Complex organics – natural orange juice, coconut milk, bananas
f. Inert supports: Agar, foam rubber, filter paper bridge, liquid
4. Growth regulators: Basic research findings of Miller and Skoog have been born out by many
In general: Cytokinins induce shoot bud formation and Auxins induce root formation
5. Environmental conditions:
Light- light intensity , photoperiod, quality important.
Temperature - there are usually high and low cutoffs. In some cases a specific temperature is needed. But of all environment, specific temperatures are generally not required beyond the all purpose.
STAGE 2 – Multiplication Propagules to be multiplied.
Propagules: organs that are multiplied up. Can be shoot, embryos, meristematic nodules, leafy callus.
During this stage selections of amounts and kinds of hormones are critical. Often the concentrations of hormones determine the morphology of propagules.
Methods of multiplication
1. Axillary shoot formation: Axillary meristem located in the axil of a leaf and giving rise to an axillary bud. Shoot tips and meristems. Already have organization of shoot. Can use lower amounts of hormones than for other structures where shoot morphology is not present.
2. Adventitious shoot formation:
a. callus – organogenesis: With most structures other than shoots usually must go through callus
first. Get dedifferentiation and then redifferentiation. The process is similar to that seen with root primordia formation.
b. Organ formation directly without callus: Occurs but is not as common. eg. African violet, peperomia.
Structures arising not at their usual sites.
3. Callus embryogenesis - callus in first stage. Callus then is placed in liquid culture. For callus and liquid culture - use 2,4-D, NAA. Then plate it out without auxin. Then get embryos to form.
STAGE 3 - Pretreatment for transfer to soil: Need root formation and adjustment to greenhouse conditions. For root initiation growth regulators are important. Generally aux/cyt>1. NAA used for herbaceous plants. IBA - for woody plants. Reduce sugar to encourage autotrophic growth. Light - reduce or increase depending on specific plant. Lower or in darkness for 1 week for root formation. Higher - 10,000 lux to adapt it to higher light intensities in GH or field.
STAGE 4 - Transfer to soil: Must maintain high RH or plants will wilt and desiccate very rapidly. Seems to be less wax on most plants. Stomates on some do not seem to function normally. Either remain open or close too slowly. Disease - succumb to fungus - have not been exposed to any in vitro. Dormancy - often trees and bulb crops need chilling in order to grow properly once in greenhouse.