Professional Documents
Culture Documents
Application
Development of
superior cultivars
Germplasm storage
Somaclonal variation
Embryo rescue
Ovule and ovary cultures
Anther and pollen cultures
Callus and protoplast culture
Protoplasmic fusion
In vitro screening
Multiplication
Tissue Culture Applications
Micropropagation
Germplasm preservation
Somaclonal variation
Haploid & dihaploid production
In vitro hybridization – protoplast fusion
Micropropagation
Features of Micropropagation
• Clonal reproduction
– Way of maintaining heterozygozity
• Multiplication stage can be recycled many times to
produce an unlimited number of clones
– Routinely used commercially for many ornamental species,
some vegetatively propagated crops
• Easy to manipulate production cycles
– Not limited by field seasons/environmental influences
• Disease-free plants can be produced
– Has been used to eliminate viruses from donor plants
Microcutting propagation
• It involves the production of shoots from pre-existing
meristems only.
• Requires breaking apical dominance
• This is a specialized form of organogenesis
Steps of Micropropagation
• Stage 0 – Selection & preparation of the mother plant
– sterilization of the plant tissue takes place
• Stage I - Initiation of culture
– explant placed into growth media
• Stage II - Multiplication
– explant transferred to shoot media; shoots can be constantly
divided
• Stage III - Rooting
– explant transferred to root media
• Stage IV - Transfer to soil
– explant returned to soil; hardened off
COMPARISON OF CONVENTIONAL &
MICROPROPAGATION OF VIRUS
INDEXED REGISTERED RED
RASPBERRIES
Conventional Micropropagation
Space: More, but less expensive (field) Less, but more expensive
(laboratory)
Required to
prevent viral Screening, fumigation, spraying None
infection:
Ways to eliminate viruses
Heat treatment.
Plants grow faster than viruses at high temperatures.
Meristemming.
Viruses are transported from cell to cell through
plasmodesmata and through the vascular tissue. Apical
meristem often free of viruses. Trade off between infection
and survival.
Not all cells in the plant are infected.
Adventitious shoots formed from single cells can give virus-
free shoots.
Elimination of viruses
Plant from the field
Micropropagation cycle
Indirect Somatic Embryogenesis
Explant → Callus Embryogenic → Maturation → Germination
1.Callus induction
2. Embryogenic callus development
3.Maturation
4.Germination
Induction
• Auxins required for induction
– Proembryogenic masses form
– 2,4-D most used
– NAA, dicamba also used
Development
Auxin must be removed for embryo development
Continued use of auxin inhibits embryogenesis
Stages are similar to those of zygotic embryogenesis
– Globular
– Heart
– Torpedo
– Cotyledonary
– Germination (conversion)
Maturation
• Require complete maturation with apical
meristem, radicle, and cotyledons
• Often obtain repetitive embryony
• Storage protein production necessary
• Often require ABA for complete maturation
• ABA often required for normal embryo
morphology
– Fasciation
– Precocious germination
Germination
• May only obtain 3-5% germination
• Sucrose (10%), mannitol (4%) may be required
• Drying (desiccation)
– ABA levels decrease
– Woody plants
– Final moisture content 10-40%
• Chilling
– Decreases ABA levels
– Woody plants
Plant germplasm preservation
In situ : Conservation in ‘normal’ habitat
–rain forests, gardens, farms
Ex Situ :
–Field collection, Botanical gardens
–Seed collections
–In vitro collection: Extension of micropropagation techniques
•Normal growth (short term storage)
•Slow growth (medium term storage)
•Cryopreservation (long term storage
DNA Banks
In vitro Collection
Use :
Recalcitrant seeds
Vegetatively propagated
Large seeds
Concern:
Security
Availability
cost
Ways to achieve slow growth
• Preculturing
– Usually a rapid growth rate to create cells with small vacuoles
and low water content
• Cryoprotection
– Cryoprotectant (Glycerol, DMSO/dimetil sulfoksida, PEG)
to protect against ice damage and alter the form of ice crystals
• Freezing
– The most critical phase; one of two methods:
• Slow freezing allows for cytoplasmic dehydration
• Quick freezing results in fast intercellular freezing with little
dehydration
Cryopreservation Requirements
• Storage
– Usually in liquid nitrogen (-196oC) to avoid changes in ice
crystals that occur above -100oC
• Thawing
– Usually rapid thawing to avoid damage from ice crystal
growth
• Recovery
– Thawed cells must be washed of cryo-protectants and nursed
back to normal growth
– Avoid callus production to maintain genetic stability
Somaclonal Variation
Variation found in somatic cells dividing mitotically in culture
A general phenomenon of all plant regeneration systems that
involve a callus phase
Some mechanisms:
Karyotipic alteration
Sequence variation
Variation in DNA Methylation
Haploid Barley
2n = X = 7
H. Bulbosum
chromosomes
eliminated
Triticale
Haploid Plant Production
Embryo rescue of interspecific
crosses
– Creation of alloploids
Anther culture/Microspore
culture
– Culturing of Anthers or
Pollen grains (microspores)
– Derive a mature plant from a
single microspore
Ovule culture
– Culturing of unfertilized
ovules (macrospores)
Specific Examples of DH uses
• Evaluate fixed progeny from an F1
– Can evaluate for recessive & quantitative traits
– Requires very large dihaploid population, since no prior selection
– May be effective if you can screen some qualitative traits early
• For creating permanent F2 family for molecular marker
development
• For fixing inbred lines (novel use?)
– Create a few dihaploid plants from a new inbred prior to going to
Foundation Seed (allows you to uncover unseen off-types)
• For eliminating inbreeding depression (theoretical)
– If you can select against deleterious genes in culture, and screen
very large populations, you may be able to eliminate or reduce
inbreeding depression
– e.g.: inbreeding depression has been reduced to manageable level
in maize through about 50+ years of breeding; this may reduce
that time to a few years for a crop like onion or alfalfa
Somatic Hybridization
Development of hybrid plants through the fusion of somatic
protoplasts of two different plant species/varieties
Somatic hybridization technique
1. isolation of protoplast
Cells Plasmolysis
Plant Tissue
Release of protoplasm
Cutting cell wall with knife
Collection of protoplasm
Mechanical Method
Plasmolysed Plasmolysed
cells cells
Release of Protoplasm
Protoplasm released released
isolated cells
cellulase
Isolated
Protoplasm
Enzymatic Method
Chemofusion Mechanical
Intraspecific Intergeneric Electrofusion
Fusion
Uses for Protoplast Fusion
Combine two complete genomes
– Another way to create allopolyploids
In vitro fertilization
Partial genome transfer
– Exchange single or few traits between species
– May or may not require ionizing radiation
Genetic engineering
– Micro-injection, electroporation, Agrobacterium
Transfer of organelles
– Unique to protoplast fusion
– The transfer of mitochondria and/or chloroplasts between
species
Spontaneous Fusion
• Protoplast fuse spontaneously during isolation
process mainly due to physical contact
• Types of fusogens
• PEG
• NaNo3
• Ca 2+ ions
• Polyvinyl alcohol
Induced Fusion
= chloroplast
= mitochondria
Fusion
= nucleus
heterokaryon