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PLANT & TISSUE CULTURE - C2 (Regeneration Pathway) Cheat Sheet (DRAFT) by

Brief summary of Chapter 2 (Regeneration Pathway) of Plant and Tissue Culture Subject

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Regene­ration Ability of an Explant

depends on:
1. Organ from which it is derived
2. The physio­logical state of explant/ Differ­ences in the stage of the cells in the cell cycle
3. Size of the explant
4. Orient­ation of the explant on the medium
5. Inocul­ation density
6. Availa­bility or ability to transport endogenous growth regulators
7. Metabolic capabi­lities of the cells

Plant Regene­ration Pathways

1. Histog­enesis (Micro­pro­pag­ation; Pre-ex­isting Meristems)
Uses merist­ematic cells to regenerate whole plant (shoot cultur­e/nodal culture)
2. Organo­genesis
Relies on the production of organs either directly from an explant or callus structure
3. Somatic Embryo­genesis
Embryo­-like structures which can develop into whole plants in a way that is similar to zygotic embryos are formed from somatic cells

1. Histog­enesis (Micro­pro­pag­ation)

-most commonly used tissue explants are the merist­ematic ends of the plants like the stem tip, auxillary bud tip & root tip
-these tissues have high rates of cell division & produce required growth regulating substances including auxins & cytokinin
Stage 0: Prepar­ation of donor plant
-if possible, mother plant should be ex vitro cultivated under optimal conditions to minimize contam­ination in the in vitro culture
 
-explant should be selected from young and healthy part that actively grow
 
-colle­ction prior to flowering
Stage I: Initiation stage
1. Explant isolated is surface sterilized and transf­erred into nutrient medium
 
2. Combined applic­ation of bacter­icide and fungicide (gener­ally)
 
3. Cultures are incubated in growth chamber either under light or dark conditions according to the method of propag­ation
 
*disin­fec­tants: sodium hypoch­lorite, calcium hypoch­lorite, ethanol, mercuric chloride (HgCl2)
Stage II: Multip­lic­ation stage
-aim: increase the number of propagules
 
-number of propagules is multiplied by repeated subcul­tures until the desired (or planned) number of plants is attained
 
-repeated enhanced formation of axillary shoots from shoot tips or lateral buds
 
-4-8 weeks subcul­turing intervals (1 cycle)
 
-multi­pli­cation is very labor-­int­ensive
 
1. Higher concen­tration of cytokinin provided
 
2. Lower concen­tration of auxin provided
 
3. Gibber­ellins (GA’s) may be added to promote etiola­tion, especially in species that form rosettes.
Stage III: Rooting stage
1. Plants must be rooted by using media containing auxin or by dipping explant bases in auxin solutions.
 
a) may use the same culture media used in multip­lic­ation stage
 
b) sometimes, it is necessary to change media, including nutrit­ional modifi­cation and growth regulator compos­ition to induce rooting and the develo­pment of strong root growth
 
2. Higher concen­tration of auxin provided
 
3. Lower concen­tration of cytokinin provided
Stage IV: Acclim­ati­zation Stage
-aim: in vitro plants need to be weaned and hardened by undergoing acclim­ati­zation
 
1. Micros­hoots are moved from sucrose in jar (heter­otr­ophic stage) to photos­ynt­hesis (photo­aut­otr­ophic stage)
 
2. Increasing the light intensity (to harden the plants)
 
3. reducing sugar, inorganic salts and humidity (to harden the plants)
 
4. The plants are then transf­erred to an approp­riate substrate (sand, peat, compost etc.) and gradually hardened under greenhouse
 
*Medium must be removed prior to transp­lan­tation to prevent contam­ina­tion.

Microp­rop­agation Flow Chart

Conven­tional propag­ation vs Microp­rop­agation

**2. Organo­genesis

-refers to the production of advent­itious plant organs i.e. roots, shoots and leaves that may arise directly from the meristem or indirectly from the undiff­ere­ntiated cell masses (callus)
-ability of non-me­ris­tematic plant tissues to form various organs
-produ­ction of roots, shoots or leaves
-organs may arise out of pre-ex­isting meristems or out of differ­ent­iated cells
-may involve a callus interm­ediate but often occurs without callus
-involves the callus production and differ­ent­iation of advent­itious meristems into organs by altering the concen­tration of plant growth hormones in nutrient medium
 
Type of Organo­genesis
1. Direct Organo­genesis
a) Directly from an explant
b) Axillary bud formation and growth
2. Indirect organo­genesis
a) Callus culture
1) Dediff­ere­nti­ation - less committed, more plastic develo­pmental state
 
2) Induction - Cells become organo­gen­ically competent and fully determined for primordia production
 
3) Differ­ent­iation
 
Charac­ter­istics
-relies on the inherent plasticity of plant tissues, and is regulated by altering the components of the medium
 
-auxin to cytokinin ratio determines which develo­pmental pathway
 
-induce shoot formation by increasing the cytokinin to auxin ratio of the culture medium.
 
-these shoots can then be rooted relatively simply
 
Control of Organo­genesis
1. Auxin: Stimulates Root Develo­pment
-↑ Auxin ↓Cytokinin = Root Develo­pment
2. Cytokinin: Stimulates Shoot Develo­pment
-↑ Cytokinin ↓Auxin = Shoot Develo­pment
 
- Auxin = Cytokinin = Callus Develo­pment
 
Advantage
1. Mass multip­lic­ation of elite germplasm.
2. Source material for protoplast work or genetic transf­orm­ation
3. Conser­vation of endangered genotypes
*Organ­oge­nesis may not produce clones!

Organo­genesis Flow Chart

Organo­genesis Process

3. Somatic Embryo­genesis

-in vitro method of plant regene­ration widely used as an important biotec­hno­logical tool for sustained clonal propag­ation
-process by which somatic cells or tissues develop into differ­ent­iated embryos, then develop into whole plants without undergoing the process of sexual fertil­ization
-Plant growth regulators play an important role in the regene­ration and prolif­eration of somatic embryos
-usually involves a callus interm­ediate stage which can result in variation among seedlings
A) Plant regene­ration via somatic embryo­genesis occurs by the induction of embryo­genic cultures from zygotic seed, leaf or stem segment and further multip­lic­ation of embryos
B) Mature embryos are then cultured for germin­ation and plantlet develo­pment, and finally transf­erred to soil
 
1. Direct Somatic Embryo­genesis
-Embryos initiate directly from explant in the absence of callus formation.
 
-Though common from some tissues (usually reprod­uctive tissues such as the nucellus, styles or pollen), direct somatic embryo­genesis is generally rare
2. Indirect Somatic Embryo­genesis
-Embryos initiate from callus developed from explant
 
Explant → Callus induction → Callus Embryo­genic develo­pment → Maturation → Germin­ation
 
1) Initial stage (embryo initia­tion)
high concen­tration of 2,4-Di­chl­oro­phe­nox­yacetic acid (selective herbicide) is used
 
2) Second stage (embryo produc­tion)
embryos are produced in a medium with no or very low levels of 2,4-D
   
*supplying a source of reduced nitrogen (specific amino acids/­casein hydrol­ysate) can also improve
 
- also regarded as a valuable tool for genetic manipu­lation
-The process can also be used to develop the plants that are resistant to various kinds of stresses and to introduce the genes by genetic transf­orm­ation. advent­itious
 
Various terms for non-zy­gotic embryos
1. Advent­itious embryos
Somatic embryos arising directly from other organs or embryos.
2. Parthe­nog­enetic embryos
Somatic embryos are formed by the unfert­ilized egg.
3. Androg­enetic embryos
Somatic embryos are formed by the male gameto­phyte.
 
Somatic Embryo Develo­pment
-Auxin must be removed for embryo develo­pment
Continued use of auxin inhibits embryo­genesis
-Polarity is establ­ished early in embryo develo­pment.
-Signs of tissue differ­ent­iation become apparent at the globular stage and apical meristems are apparent in heart-­stage embryos.
Develo­pment Stages
1. Zygote
4. Torpedo
2. Globular
5. Cotyle­donary
3. Heart
6. Germin­ation
 
Charac­ter­istics
1. Bipolar structure – shoot and root pole
2. Source of protop­lasts and suspension cultures.
3. Clonal propag­ation

Somatic Embryo­genesis Process

Somatic Embryo Develo­pment Stages

Embryo­gen­esis, Organo­gen­esis, Microp­rop­agation

-Both of these techno­logies can be used as methods of microp­rop­aga­tion.
-It is not always desirable because both of them may not always result in popula­tions of identical plants which is needed for microp­rop­agtion.
-The most beneficial use of somatic embryo­genesis and organo­genesis is in the production of whole plants from a single cell (or a few cells).
a) High probab­ility of mutations
b) The method is usually rather difficult.
c) Losing regene­rative capacity become greater with repeated subculture
d) Induction of embryo­genesis is very difficult with many plant species
e) A deep dormancy often occurs with somatic embryo­genesis

Difference of S. Embryo­genesis and Organo­genesis

Organo­genesis
Somatic Embryo­genesis
-monopolar structure
-bipolar structure with a closed radicular end
-has vascular connection with the mother tissue
-has no vascular connection with the mother tissue

Compare Organo­genesis and Embryo­genesis

Organo­genesis
Embryo­genesis
-Explant or callus is subcul­tured on shooting medium to induce shoot formation
-Explant or callus is subcul­tured on embryo­genesis medium to induce formation of pro-em­bry­ogenic cell masses (PEMs)
-Group of cells differ­entiate to form shoots (5,000­-10­,000)
-PEMs are form from single cells and subcul­tured into the same medium for PEM prolif­eration (hundred thousands to million)
-Each shoot of approp­riate size is identified and excised indivi­dually and subculture on rooting medium to induce rooting (labour intensive)
-PEMs are split and subcul­tured onto medium with less auxin in batches (less labour) to grow and differ­entiate further
 
-Somatic embryos are subcul­tured on medium without hormone for germin­ation

Different problems in Plant tissue culture

Problem
Descri­ption
Way to Overcome
1. Recalc­itrance
-inability of plant tissue culture to respond to culture manipu­lation
-Antio­xidant Protec­tion: Antiox­idants are special compounds that have the capability of neutra­lizing reactive molecules and particles - so called free radicals
 
-loss of morpho­genetic competence and totipo­tency capacity
-Juvenile tissue can be selected as explant
 
-Free radica­l-m­ediated stress has a role in tissue culture recalc­itr­ance.
-Parts of the desired plant rejuve­nated by treatments like cytokinin spray on selected branches
 
-Free radicals and their reaction products react with macrom­ole­cules such as DNA, proteins and enzymes, causing cellular dysfun­ction and, as a result, the cultures become recalc­itrant
-
 
-All aerobic organisms are totally dependent upon redox reactions and the transfer of single electrons and many life processes involve free radical interm­edi­ates.
-
2. Contam­ination
-source: a) carry over of microo­rga­nisms on the surface or in the tissues of explants; b) faulty procedures in the laboratory
-Wear gloves and a lab coat and keep long hair tied back.
 
-Bacteria, fungi, mould and yeasts are common contam­inating microo­rga­nisms in tissue culture.
-Work in a laminar flow hood when passaging cells.
 
-Many of the microo­rga­nisms that are likely to be present interc­ell­ular, in plant tissues will be capable of growth on the plant tissue culture medium, although some may be inhibited by the high salt or sucrose concen­tration and the pH
-Wipe down working surfaces with ethanol.
   
-Use sterile equipment.
   
-Inspect all equipment and media for visible contam­ination before use.
   
-NO cross over - Do not pass your hands/arms over any open bottle, plate or tube.
   
-Use proper antibi­otics in your culture media.
   
-When finished, dispose of materials properly, wipe down working surfaces with ethanol, and turn on UV lamp within laminar flow hood for 10 minutes to sterilize the area.
3. Phenolic browning
-Many plants are naturally rich in polyph­enolic compounds that are commonly regarded as inhibitory agents.
-Culture bottles are kept in dark condition
 
-In most of the cases, when these plants are cultured in vitro, the culture medium turns brown.
-Addition of antiox­idants (Polyv­iny­lpy­rro­lidone, PVP-40) to medium was more effective to reduce the browning.
 
-Phenolic browning caused by the accumu­lation and oxidation of phenolic compounds.
-inhib­iting the activity of the phenyl­alanine ammonia lyase enzyme (PAL), thereby reducing the biosyn­thesis of phenolic compounds
4. Seasonal variation
-relative humidity, dry season affects the medium and nutrient medium evaporates rapidly when too dry
-Choose explant in its most responsive season
 
-extreme moist climate such as poor tropical region, fungi is effected on media
-Use in vitro plantlets as explant
 
-dust in air is also a major source of bacterial contam­inants
-Contr­olled enviro­nment
 
-germi­nation of shoots and roots also delayed due to the seasonal variation
-
5. Vitrif­ica­tion( hyperh­ydr­icity)
Hyperh­ydr­icity is the physio­logical malfor­mation due to excessive hydration, low lignif­ication and reduced mechanical strength of tissue culture generated plants.
-Culture are sub-cu­ltured frequently to overcome this vitrif­ication
 
Hyperh­ydr­icity in plant tissue cultures are those factors triggering oxidative stresses such as high salt concen­tra­tion, low calcium content in culture medium, gas built up within the container, high relative humidity, low light intensity, gas accumu­lation in the atmosphere of the jar, length of time intervals between subcul­tur­es.High ammonium concen­tra­tion, culture bottles kept in same container.
-Vitri­fic­ation can be lessen by raising the agar and/or sugar concen­tra­tion, addition of ethyle­ne-­inh­ibi­tors, amino acid, phenolic glycosides phlori­dzin, naringin or esculin hydate, using two-phase media, bottom cooling of the culture vessel­s,v­ent­ilation of the vessels, adding silver nitrate
6. Somaclonal Variation
-genetic variations along with phenotypic changes found in the in vitro cultured cells
-Avoiding long term cultures
 
-Somac­lonal variations occur as a result of genetic hetero­geneity (change in chromosome number and/or structure) in plant tissue cultures.
-Axillary shoot induction systems
 
-cause: a) Expression of chromo­somal mosaicism or genetic disorders; b) ii. Sponta­neous mutations due to culture conditions
-Regularly reinit­iating clones from new explants.
 
-factors: a) Genotype and explant source; b)Duration of cell culture; c) Growth hormone effects
-Prevent usage of 2,4-D IN media
 

Limita­tions of Somaclonal Variations

i. Most of the somaclonal variations may not be useful.
ii. The variations occur in an unpred­ictable and uncont­rolled manner.
iii. Many a times the genetic traits obtained by somaclonal variations are not stable and heritable.
iv. Somaclonal variations are cultiv­ar-­dep­endent which is frequently a time consuming process.
v. Somaclones can be produced in only those species which regenerate to complete plants.
vi. Many cell lines (calli) may not exhibit regene­ration potential.

Nodal Cutting

Function: Removes the inhibitory effect of the shoot apex on bud outgrowth (Apical dominance)

Nodal Cutting Image

Gibber­ellins

Growth hormones that stimulate cell elongation and cause plants to grow taller.

Rosette

Circular arrang­ement of leaves or of structures resembling leaves

Etiolation

Etiolation is a process in flowering plants grown in partial or complete absence of light.
It is charac­terized by long, weak stems; smaller leaves due to longer intern­odes; and a pale yellow color.