Growth & Diffferentiation in Tissue Culture

7/22/2019 Growth & Diffferentiation in Tissue Culture

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Differentiation & morphogenesis in

plant tissue culture


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Growth & Development

Cell Theory1938

Totipotency

Plant v/s animals Differentiation

Dedifferentiation

Redifferentiation Positional v/s lineage

based differentiation


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Differentiation of embryo


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Trachery element differentiation in

Zinniaelegansmesophyll cells

Fukuda & Komamine Plant Physiol 1980, 65:61-64


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Pathways of plant regeneration


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Explants for regeneration


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Tran Thanh Vans experiments with

Thin cell layer explants


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Different programmed states within a leaf


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IAA mg/l

Kinetinmg/l

Skoo g and Miller 1957 . Symp. Soc. Exp. Biol. 11: 118- 131


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Factors influencing organogenesis

Genetic: Knotted 1 maize

Koshihikari

low regeneration v/sKasalath (high)

Nishimura et al PNAS 2005


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Factors influencing organogenesis

Physical conditions

Light: Blue - shoot , Redroot (tobacco)

Photoperiod: Pelargonium

Temperature

Solid v/s liquid: tobacco thin layers N. glauca x N. lagnsdorfii

Solid med - callus

Liquid med

shoot buds(oxygen tension)

Gaseous environment

Other aspects

Season Age

Size of explant


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Auxin-cytokinin interactions

Synergistic : cell division

Antagonistic: rt & sht organogenesis

Auxin resistant mutants (aux1, axr1) also confer

cytokinin resistance w.r.t. root growth inhibibiton External application of cytokinin leads to

increased IAA

Auxin influences cytokinin: Auxin at apical

meristem checks axillary bud growth Decapitationsurge of cytokinin in xylem

Apply NAA at cut tip - no surge


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Formation of other organs

Formation of Storage organs: Tubers, bulbs etc.

Sugar GA

In vitro flower formationon Arabidopsis root with

p35S:LFY

(Wagner et al Plant J 2004,

39:273-282)


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SAM organization

Colchicine-inducedchimeras


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ATH1 (A. thaliana Homeobox 1)

Pennywise (PNY) and

Poundfoolish (PNF) interact with

STM to control shoot

organogenesis


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Clavata and WUS interaction

Sharma et al. PNAS 2003, 100:11823


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Atta et al. Plant J 2009, 57:626-644

Callus proliferation from the xylem pericycle

cells in root and hypocotyl explants of

Arabidopsis


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Attaetal.PlantJ200

9,

57:626-644

Expression of various marker genes in shoot inducingmedium from callus induced from root explants


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Sugimoto K, Jiao Y, Meyerowitz EM Dev. Cell 2010, 18:463-471.

Tracking in vitro regeneration through

Molecular Markers


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No callus formation occurs in alf4 (aberant lateral root formation 4) mutant

supporting the requiremnt of lateral root initiation programme for callus induction.

Similarly, ABA which inhibits lateral root emergence does not affect callus formation

suggesting that further root development programme is not necessary for

continued growth of callus


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Lateral Root formation


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Cytokinins modulate auxin efflux to

induce root organogenesis

Pernisova et al PNAS 2009, 106:3609-3614

PIN1:GFP

expression in

roots originating

on hypocotylexplants at

different con. of

cytokinins


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Somatic embryogenesis

Discovery: Steward (1958); Reinert (1958) Occurrence:

Natural polyembryony: Citrus, mango, Malus

Experimental: > 100 species Somatic embryognesis stages

Induction

Maturation Conversion

> 3500 genes involved in embryo development

>40 genes for embryo body pattern


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Features of SE

Unequal division

Isolation from neighbouring cells

no plasmodesmatal contacts

Cutinisation

Callose

Highly cytoplasmic

Accumulation of starch Calcium oxalate crystals


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Differentiation of embryo


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A. thaliana embryo development


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WOX genes

Hecker et al. Development 2004, 131: 657-668


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WOX genes


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Factors influencing SE

Nuclear genes:

Wheat4B, 2DL, 2AL, 2BL

Maize A188

Cytoplasmic genes:

Chinese Spring (Embryogenic) but after mt-

recombination non-embryogenic


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Growth regulators

Auxin: Essential for induction; absence or low

levels for maturation

2,4-D: choice auxin; dicamba for grasses and

banana, picloram for pulses

Cytokinin: Not essential

ABA: For maturation

Ethylene: Inhibitory

Brassinosteroids: ?

GA: dormancy


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Media supplements etc.

Nitrogen: Ammonical form for induction

Carrot cells on 55mM KNO3No SE

55mM KNO3 + 5mM NH4ClSE

NH4 alone can support provided pH is kept at 5.4

Organic nitrogen (aa or amides) can support SE

Glutamine: Soybean, Brassica, wheat

Asparagine: Norway spruce

Sugar: Maltose highly promotive Higher concn. better quality (Osmotic effect?)

Osmotictreatment


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Applications

SE and somaclonal variation

Synthetic seed technology

Haploids and doubled haploids

Transformation

Selection for embryo specific proteins and

fatty acid patterns


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AtSERK expression

Seedling, SE-callus, Non-SE callus

Hetch et al. 2001. Pl Physiol 127:803-816

SERK first found in

carrot SE Also found in cells

undergoing SE in rice,

citrus, Arabidopsis,

Dactylus, maize, cocoa

etc. Also upregulated during

apomixis in Heiracium


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SERK

A family in Arabidopsis (Five SERK genes identified)

AtSERK1 also expressed in male & female tissue, in the

embryo up to torpedo stage

OsSERK induced by blast fungus, jasmonic acid, salicylic acid

and ABA

At SERK2 & 3 expressed in the same tissues and act

redundantly during anther development, loss of both gene

activity leads to male sterility

AtSERK1 and 3 : components of Brassinosteroid receptor


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MtSERK

Induced by auxin

Furtherupregulated by

cytokinin

Response of near isogenic lines to somatic embryogenesis

(Nolan et al., 2006)


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Stress and growth regulator response pathwaysinteract and integrate in somatic embryogenesis

Initiation of culture

Stress response pathway

Hormone response pathway

Embryo differentiation


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LEC1 & SE

Lotan et al. 1998, Cell, 93:1195-1205

LEC1 can induce SE on hormone

free medium and also without

tissue culture


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LEC & FUS

LEC1: CCAAT binding TF (CBF)

LEC2: B3DNA binding motif; required for suspensordev. cotyledon identity, progression through maturation

LEC genes act at both embryogenesis and maturation

phase

FUS3: B 3 domain TF; can upregulate ABA and downregulate GA

ABI3 is also a B3 domain TF

LEC1 induces FUS3 and ABI3


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PICKLE

Abnormal root phenotype

pkl mutant roots grow on hormone-free med

and produce SE

pkl phenotype suppressed by GA

Chromatin architecture; may be a repressor

of LEC1


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BABY BOOM

Boutilier et al. 2002 Plant Cell, 14 : 1737-1749

AP2/ERF transcription factor Related to Ethylene

response factor binding TF


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BBM

MADS box protein

Expressed in the embryo,helps maintain SE capacity forlong

Expressed during apomixisTaraxacum

During microspore SE inBrassica, SE alfalfa

lec1 mutant shows altered

AGL15 expression AGL15 controls GA

metabolism in Arabidopsisbinds to GA2oxidase gene

Regeneration of 35S::BBM on hormone-free medium

A & B control on hormone, C & D transgenics on

Hormon-free medium

AGL15

Growth & Diffferentiation in Tissue Culture

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