Differentiation & morphogenesis in plant tissue culture

Growth & Development

Cell Theory 1938 Totipotency Plant v/s animals Differentiation Dedifferentiation Redifferentiation Positional v/s lineage based differentiation

Differentiation of embryo

Trachery element differentiation in Zinnia elegans mesophyll cells

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

Pathways of plant regeneration

Explants for regeneration

Tran Thanh Vans experiments with Thin cell layer explants

Different programmed states within a leaf

IAA mg/l

Kinetin mg/l

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

Factors influencing organogenesis

Genetic: Knotted 1 maize Koshihikari low regeneration v/s Kasalath (high) Nishimura et al PNAS 2005

Factors influencing organogenesis

Physical conditions Light: Blue - shoot , Red root (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)
Other aspects Season Age Size of explant

Gaseous environment

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
Decapitation surge of cytokinin in xylem Apply NAA at cut tip - no surge

Formation of other organs

In vitro flower formation on Arabidopsis root with p35S:LFY (Wagner et al Plant J 2004, 39:273-282)

Formation of Storage organs: Tubers, bulbs etc. Sugar GA

SAM organization

Colchicine-induced chimeras

ATH1 (A. thaliana Homeobox 1) Pennywise (PNY) and Poundfoolish (PNF) interact with STM to control shoot organogenesis

Clavata and WUS interaction

Sharma et al. PNAS 2003, 100:11823

Callus proliferation from the xylem pericycle cells in root and hypocotyl explants of Arabidopsis
Atta et al. Plant J 2009, 57:626-644

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

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

Tracking in vitro regeneration through Molecular Markers

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

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

Lateral Root formation

Cytokinins modulate auxin efflux to induce root organogenesis

PIN1:GFP expression in roots originating on hypocotyl explants at different con. of cytokinins

Pernisova et al PNAS 2009, 106:3609-3614

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

Features of SE
Unequal division Isolation from neighbouring cells
no plasmodesmatal contacts Cutinisation Callose

Highly cytoplasmic Accumulation of starch Calcium oxalate crystals

Differentiation of embryo

A. thaliana embryo development

WOX genes

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

WOX genes

Factors influencing SE
Nuclear genes:
Wheat 4B, 2DL, 2AL, 2BL Maize A188

Cytoplasmic genes:
Chinese Spring (Embryogenic) but after mtrecombination non-embryogenic

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

Media supplements etc.

Nitrogen: Ammonical form for induction
Carrot cells on 55mM KNO3 No SE 55mM KNO3 + 5mM NH4Cl SE 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?)

Osmotic treatment

Applications
SE and somaclonal variation Synthetic seed technology Haploids and doubled haploids Transformation Selection for embryo specific proteins and fatty acid patterns

AtSERK expression

SERK first found in carrot SE Also found in cells undergoing SE in rice, Seedling, SE-callus, Non-SE callus citrus, Arabidopsis, Hetch et al. 2001. Pl Physiol 127:803-816 Dactylus, maize, cocoa etc. Also upregulated during apomixis in Heiracium

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

MtSERK
Induced by auxin Further upregulated by cytokinin

Response of near isogenic lines to somatic embryogenesis (Nolan et al., 2006)

Stress response pathway Initiation of culture Hormone response pathway Embryo differentiation

Stress and growth regulator response pathways interact and integrate in somatic embryogenesis

LEC1 & SE
LEC1 can induce SE on hormone free medium and also without tissue culture

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

LEC & FUS

LEC1: CCAAT binding TF (CBF) LEC2: B3 DNA binding motif; required for suspensor dev. cotyledon identity, progression through maturation LEC genes act at both embryogenesis and maturation phase FUS3: B 3 domain TF; can upregulate ABA and down regulate GA ABI3 is also a B3 domain TF LEC1 induces FUS3 and ABI3

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

BABY BOOM

AP2/ERF transcription factor Related to Ethylene response factor binding TF

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

BBM

AGL15
MADS box protein Expressed in the embryo, helps maintain SE capacity for long Expressed during apomixis Taraxacum During microspore SE in Brassica, SE alfalfa lec1 mutant shows altered AGL15 expression AGL15 controls GA metabolism in Arabidopsis binds to GA2oxidase gene

Regeneration of 35S::BBM on hormone-free medium A & B control on hormone, C & D transgenics on Hormon-free medium