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New Phytol.

1997), 137,

1-8

Genetic approaches in plant physiology

BY

M. KO OR NN EE F* , C. AL ON SO - BL AN CO AND

A. J. M. PEETERS

Department ofGenetics, Wageningen Agricultural University, Dreijenlaan2 6703HA

ageningen

The Netherlands

{Received3April 1997;accepted 24 June 1997

SUMMARY

The use ofgeneticsin plant biology aimsat thephysiologicalandmolecular genetical characterizationof the

phenotypic

variation for the trait under s tudy. Efficient mutant and gene isolation procedures have been developed

for

a

number

of

plant models such

asArabidopsis thaliana.

For

this,

the map position

of

the genes and insertion

mutagenesisare used. The latter also allows the characterization

of

genes that are not easily recognized in mutant

approaches,by using enhancerorgene-trapping procedures and reverse genetics.Inaddition to mutants, natural

variationpresent among wild and cultivated varieties ofa species provides an important source ofgenetic variation.

The

use of molecular markers, advanced mapping populations and specific cytogenetic stocks in case of polyploids,

enablesa detailed characterization

of

such natural variation even when

it

is

of

a quantitative and polygenic nature.

Examplesof the various genetic approachesare given.

Key words: Arabidopsis thaliana, abscisic acid ABA), gene isolationandmutagenesis, photomorphogenesis,

quantitative trait locus QTL).

INTRODUCTION

The use of genetics as a tool to dissect complex

biological processesinplants hasalong history.For

instance, a tobacco mutant called MarylandMam-

moth and different soybean varieties led to the

discover} of photoperiodism by Garner Allard

1920). Late r, during the fifties, genetic dwarfs in

peaandmaize convinced manyofthe importanceof

gibberellins asplant growth hormones. Why there-

after geneticshas notbeen used very muchinplant

physiology is less clear, despite that mutants were

shown to be crucial toolsfor theunderstandingof

biosynthetic pathways inmicro-organisms and de-

velopmental processes, e.g. inDrosophila. Towards

the end of the seventies, students of

E.

coliand

Drosophila

genetics such asSomerville andMeyer-

owitz became convinced that genetics was the wayto

go ahead in plant science. These authors,aswellas

Laibach, Redei and Feenstra before them, had

realized that

Arabidopsis thaliana

L.) Heynh. wasa

model speciesfor plant genetics, because this small

self-fertilizing crucifer has avery short generation

time. Genetics becomes even more powerful whenit

can

be

combined with molecular genetics , which

links DNAwith thephenotype. Its small genome

and ease of transformat ion were additional factors.

which established Arabidopsis as the general model

organism

in

higher plant molecular genetics.

Cur-

rently, the whole genome of this plant is being

sequenced Bevanetal.. 1997), and the impact of this

information is rapidly increasingas can beseen,for

example, from thefrequent use thatisalready made

of the partly sequenced cDNAs called expressed

sequence tags ESTs).

Besides Arabidopsis, other plant species suchas

petunia.

Antirrhinum,

maize, pea, tomato, barley and

rice have been studied for a long time, resultingin

large collectionsofgenetic stocksandgenetic maps.

They became very interesting genetic models for

specific developmental processes because of the

particular characteristics of these species, whichled

to the characterizationofsomeoftheir mutants.For

instance, for

Petunia

and

Antirrhinum,

mutations

affecting flower colour and morphology were

analysed in detail. With the advent of molecular

biology. Antirrhinum, maize and, somewhat later.

Petunia could beused for gene cloning, since well

characterized transposable elements were available.

Unfortunately, these species, except Petunia, are

difficult orimpossibleto transform, which limitsthe

complementation proof

of

having cloned

the

gene

and the further characterizat ion of those genes.

Tomato and, even more so, rice, have relatively

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M. Koornneef C. Alonso-Blanco and A.J. M. Peeters

resulting in the isolation of several disease-resistance

genes (Jones Jones , 1997) . T he la rger genom es of

barley and pea make these classic model species less

amenabletosome molecular approach es . H owev er ,

the va lue of these species sho uld not be u n d e r -

estimated, given their suitability not only for genetics

bu t a lso for plant physiology and plant biochem is try.

In pea , thesystem of nitro gen fixation c ouldbe

s tudied genet ica l ly, and wel l charac ter ized mutants ,

e.g. inthe ph ytoc hrom e, f lowering and gibbere l l in

pa thways , resul ted

in

excellent research, c om ple-

m e n t a r ytotha tofthe 'g ene ra l ' m ode ls .Inbarley,

s imilar approaches provided important molecular

da ta on seed germinat ion, and the recent map-based

cloning of the mildew resistance gene

Mlo

(Blischges

etal 1997) indicates that these limitations, because

of the large genome, canbeovercome . T hu s ,it is

clear that genetics nowadaysis abasic toolinplant

science. In orde r to apply this genet ic approach ,

genetic variation, which takes

a

range

of

forms,

is

essential. Thegenera t ion of mu tant va r iat ion is

genera l ly notapro blem because efficient mu tag ens

and mutagenes is procedures are available . M ore

difficult

is

the identification

of

m u t a n t p h e n o t y p e s

tha t arerelevant to theresearch topic . Form o r -

phological processes the phenot>^pe itself allows the

detec t ion of mu tan ts ; however , for some biochem ica l

and physiological traits , either thep h e n o t y p e is

relatively subtle, or the phenotype is too general, e .g.

genera l reduct ion of plan t size or vigour .An

important l imita t ion infinding mu tan ts istha tfor

many genes redundancy is present , which means tha t

muta t ions in such genes do not resul t in an obvious

vis ible phenotype s ince the redundant counterpar t

produces enough produc t to atleast partly) su b-

s t i tute for thefunction of themuta ted gene .In

addi t ion

to

induced m utan ts , another sou rce

of

genet ic var ia t ion is provided by the na tura l var iants

present withinaspecies andbycytog enetic stocks

such as chromoso mal su bs t i tut io ns . Once genet ic

variation has been identified and characterized, the

appl ica t ion of molecular genet ic techn iques to amen -

able species allows the cloning of the mutated genes.

Cloning and charac ter iza t ion

of

genes have some -

t imes given c lues about the na tureofthe observed

defec ts in the mutants . An important recent example

is the identification ofArabidopsis and tomato genes

produc ing dwar f i sm when muta ted , in whichen-

coded prote ins arerelated to steroid biosynthes is

pro te ins in mam mals (L ie t

al

1996). This prom pted

severa l authorstotes t t he effectof brass inos teroids

on these dwarf mutants , ofwhich many couldbe

rever ted to the phenotype of the wild type , whereas

others were classified as insensitive to this group of

c o m p o u n d s ( K a u s c h m a n n

et

al,\996 . Brass inos-

te roid mutants have common charac ter is t ics , such as

be ing dw arf with small round leaves and having a de-

e t io la ted phen otype when grown in da rkness. T hese

brass inos teroidsas na tura l p lan t hormones . On

othe r hand ,

it is

very im po rtan t to find an effect

the phenotypebygenes tha t h ave been c loned

sequenced but forwhich nom u t a n t p h e n o t y p

known, s ince such a phenoty pe provides key

formation about the funct ion

of

those genes . N

approaches such as reverse genetics are be

developed to genera te mutants , s ta r t ing with a DN

sequence of unknown funct ion.

THE USE OF MUTANTS TO CLONE GENES AND

THE USE OF GENES TO ISOLATE MUTANTS

A number of procedures are now available to isol

the cor re sponding DNA

of

genes only know n

the i r mutant phenotype . Themos t impor tan t

the se me thods are gene tagging and map-ba

cloning.

1. Gene tagging

Inse r t ion of D N A in a coding sequence in m

cases dis rupts the gene, resul t ing in a m u t

phenotype . Since the sequence of the inser ted DN

is known,

it is

possible

to

clone the genom ic pl

DNA f lanking the inser t ion and thereby par t of t

d i s rupted gene . In p lan t s , T-DNA and t r ansposab

elements a re most f requent ly used fortagging.

Arabidopsis, several large collections of T - D

inser t ions have been genera ted (Fe ldmann, 199

and, more recent ly, the int roduc t ion of ma

transposa ble e lements such as the A c/ D s (Bancro

et al.,1992 and the En/ I (Aa r t set al 1995) tw

element systems have been used successfully.

A specific application of tagging is the use

enhance rorpro mo tor t raps (Sun daresan, 1996) .

this system

the

T - D N A

or

transposab le e lem

carriesareporter gene witho ut a promo tor or wit

min imal prom otor . W hen such a constru c t is inser t

near to a gene the express ion pa t te rn of the repor t

gene might reflect theexpress ion pa t te rn of t

endogenous gene , whose promotor

or

enhancer w

used todrive the express ion ofthe rep or ter ge

Because of r e d u n d a n c y , andalso beca use cert

funct ions might not be essent ia l , such inser t ionsd

not a lways resul t in physiologica l and morp hologic

mutant phenotypes . Very sophis t ica ted sys temso

this type , which inc lude gene t raps , have bee

developed recent ly (Sundaresan

et al

1995) . Su

trap l ines have been shown to bevery usefulf

marking specific tissues (Scheres et al 1994)o

processes . An important novel appl ica t ion of taggin

is in reverse genetics (Koes etal 1995). Plants wi

inser t ions in c loned genes , for which some sequen

information

is

available,

can be

identified

by t

ability to amplify D N A fragm ents m polymer

chain reac t ion (PCR). One PCR pr imer is based o

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Genetics in plant physiology

sought. Again a mutant phenotype wil l not always be

observed when therespective gene is disrupted ,

becauseofredundancy .

2. Map-based cloning

Map-based c loning requi resadetailed genetic m ap -

posit ion ofthe locus a nd the availability of easily

screenable l ibrar ies harbour ing la rge p lant DNA

fragments , e.g. in yeast and bac teria l artificial

chrom osom es (YA Cs and BAC s) . Th e avai labi li ty of

complete physical maps, where such clones have

been grouped and sorted into contigs that are related

to the genetic map are now available for most of the

rabidopsis genome (Schmid t et al., 1995, Zachgo et

al., 1996). This enormously facil i tates map-based

loning since it rest r ic t s chrom osom e walkingto

M ar t in ,

1995). Final proof that theright gene hasbeen

the

complementa t ion

of

by D N A

. O ther gene-cloning strategies based on mutants

Ausubel ,

is

performed compa r ing wi ld type

and

et al.,1990).

SE OF MUTANTS TO DISSECT TRAITS

for

of the m uta ted genes and pro-

Clon ing of the corre spon ding genes has often

provided impor tant ,

and

somet imes uniqu e ,

in-

formation on the mode of action of these genes and,

fur thermore , it offers the possibility of modifying

these processes,

e.g. by

reint roducing

the

cloned

genes into plants, thereby over-expressing or under-

expressing the genes.

Three different pathways, namely those affecting

abscisic acid (ABA), phytochrome and the transi t ion

to fiowering can serve as illustrations for the genetic

dissection of physiological pathways.

1. Abscisic acid

T he p lant ho rm one abscisic acid (ABA) affects man y

processes in plants . Its role in contro lling seed

dormancy

and

stomatal closure, toge ther with

its

germination and growth-inhibit ing effect ledtothe

isolation ofm ut an ts affected inABA biosynthesis

and action (Table 1) . The aba mutants , which are

defective in epoxy -carotenoid levels and ABA

provided conclusive proof for the carotenoid path-

way of ABA biosyn thesis in higher plants (Du ckha m,

Linfo rth Tay lor, 199 1; Rock Zeevaart , 1991).

M ar inet al.(1996) cloned this gene using transp oson

tagging. The ABA mutants showed the impor tance

of ABA in stress resistance (reviewed by Giraudat et

al., 1994). Three different genes of ABA-insensit ive

{abi)mu tants have been c loned. ABIl (Leung et al.,

1994; Meyer , Leube Grill, 1994) and ABI2

(Leu ng, M erlot Gira uda t , 1997) were both shown

to encode protein phosphatase 2Cs, which apparently

play

a

role

in

ABA s ignal t ransduct ion . T he ABI3

gene encodes a seed-specific transcription factor that

t ransmi ts

the

ABA signal,

as

well

as

u n k n o w n

developmental s ignals, to a number of seed-develop-

ment-specif ic genes (Parcy Gira uda t , 1997). T he

relevanceofABAinseed germ inationisshownby

the lack of seed dormancy, characteristic of almost all

ABA related mutants identif ied sofar. Recip rocal

Loci involved in the biosynthesis or mode of action of ABA , and phenotype of the mutants at these loci

Seed*

dormancy

Stomatal

closure*

ABA sensitivity

growth inhibition*

Gene function

References

BB

-t

t

Zeaxanthin epoxydase

Conversion of xanthoxin to ABA

aldehyde

Addition of sulphur to Moco of

ABA aldehyde oxidase

Protein phosphatase 2C

Protein phosphatase 2C

Seed-specific transcription factor

Mar in et al. (1996)

Schu^artz et al. (1997)

Schwartz et al. (1997)

Leung et al. (1994);

Meyer et al. (1994)

Leung et al. (1997)

Giraudat et al. (1992)

Finkelstein (1994)

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M.

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C. Alonso-Blanco and A.J. M. Peeters

Table 2 Arabidops i s

mutan ts affecting photorecep tive pigmen ts

Sensitivity to *

Gene

Bf

R

F R

Gene function

References

HYl

HY2

HY3 PHYB)

HY4

HY5

PHYA +

Heme oxygenase J

synthase I

Phytochrome B

Cryptochrome

Transcription factor

Phytochrome A

Parks Quail (1991); Ter ry

(1997)

Parks Quail (1991); Terry

(1997)

Reed

et al.

(1993)

Ahmad Cashmore (1993)

Oyama

et al.

(1996)

Whitelam

et al.

(1993)

* The sensitivity of mutants at these loci to light of specific wavelengths is less than ( ), slightly less than (+

the same as

(

+

)

wild type.

t B, Blue light; R, Red light; FR , Far Red light.

X

Heme oxygenase and phytochrom obilin synthase control the two last steps of phytochrom e chromo ph

biosynthesis.

c rosses revea led tha t ABA produced by the embryo

controls germinat ion (Karssen

et al.,

1983) . Wi thout

ABA, seeds do not require gibbere l l in (GA) for

germinat ion as shown by the ir res is tance to the

gibbere l l in biosynthes is inhibi tors te tcyc lac is and

paclobutrazol (Leon-Kloosterz ie l

et al., 1996a, b).

In addi t ion, the insens i t ivi ty to such inhibi tors of

seed germination led to the isolation of

aba2

and

aba3

mutants and to the isolation of mutants affected

specifically in seed dormancy, which probably rep-

resent genes tha t control one of the downstream

processes affected by ABA.

phytochrome-B-de f ic ien t

hy3

mutant sugges ted

several authors that screening for insensitivity to F

might yie ld phytochrome-A-def ic ient mutants ,

indeed was la te r proven. Since

phyA

mu tants h

no obvious phenotype in white light, this specif

screen was required to f ind them. From the mome

these well defined mutants were available they ha

been used to specify the modes of action of the

dif ferent phytochromes , e .g. in seed germinat i

(Botto et al., 1995) , anthocy anin format

(Kerckhoffs

et al.,

1997) and fiowering (Bagnall

al.,

1995).

2.

Photo receptive pigmen ts

The control of growth and development by the

qual i ty , qua nt i ty and dura t ion of l ight is descr ibed as

photomorphogenes is . Plants perce ive information

from l ight through pigment sys tems such as phyto-

chrome and c ryptochrome . The complexi ty of the

regula t ion of photomorphogenes i s by phytochrome

comes from the fact that different types of phyto-

chr om e enco ded by at least 45 different genes exist

(Pra t t , 1995) . Th ese phy toch rom es differ in the ir

photo-s tabi l i ty and the ir tempora l and develop-

mental expression. For some processes these dif-

fe rent types of phytochrome might have dif fe rent

modes of ac t ion. Mutants a t the

hyl-hy5

loci, wh ich

are defective in specific aspects of photomorpho-

genes is and which are recognized by the ir e longated

hypocotyls in white light, were first described by

Koornneef, Rolff Sprui t (1980) . Subsequent ly , the

molecular nature of all f ive mutants was elucidated

(Table 2) . The ident i f ica t ion of the blue l ight

receptor depended ful ly on the c loning of the

HY4

gene (Ahm ad Cash mo re , 1993) , and the sub -

sequent charac ter iza t ion of the c loned gene (Lin

et

al.,

1995) . The compar i son of the phytochrome

3. Floral initiation

The t rans i t ion f rom the vegeta t ive to the repr

duct ive meris tem which produces f iowers , is poor

unders tood a t the molecular leve l . To increase o

unders tanding of this important process in high

plants a genet ic approach has been sugges te

Genet ic var ia t ion for this t ra i t i s abundant . F

ins tance , dozens of mutants have been found

Arabidopsis

that either delay or advance the tr

s i t ion to f lowering (Hau ghn , Schul tz M art ine

Zapater , 1995

;

Peete rs Ko orn neef 1996), as wel

in other spec ies , but in none of these mutants h

flowering been completely abolished. In contrast

mutant s w i thout a r eproduc t ive phase , em f m u t a

( S u n g

et al.,

1992) lacking the vegetative phase h

been isolated. The hypothesis that fiowering is t

default state in

A rabidopsis,

which is repressed by

E M F produ c t s , was then e s tab l i shed (Sung

et a

1992;

M a r t i n e z - Z a p a t er et al., 1994; Weigel , 199

The effect of these product(s) can be modified b

var ious processes control led by the f lowering- t im

genes and also by environmental factors such as lig

and tempera ture . A number of the f lowering t im

genes ,

inc luding LD (Lee et al., 1994a ) , C

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Genetics in plant physiology

{G Oand

LD

and a

{FGA . Using

a

system with

GO gene productisswitched on,a careful

of

the floral initiation proce ss

in

relat ion

to

by the develop men tal switch

be performed (Simon, Igeno Coup land ,

The

fact tha t

the

genetics

of the

floral

a comp lex regulat ion

com-

hard ly accessible for experim ental analysis .

URAL GENETIC VARIATION

ofa species prese nt in natu re contains

at

m any different g enes.

In

contras t

onedoes not expect variants

are

stron gly affected

in

vigo ur, since selection

of

variat ion

for

basic research, al though

is thetype that has been,and

is,

exploited

for

plant breeding. Fu r therm ore ,

ofna tur al se lection has ledtogenotypes

to specific enviro nm ents. Th is a daptat ion

in

ecological/

the

species, w hich

is

very obvious

in

of cult ivated pla nts . Th is source of

to

physiological

has not bee n v ery accessible for genetic

and

even less

so for

mo lecular genetic

by quanti tat ive

on theexpression of the

A

n u m b e r

of

developments

in

gene tics, such

as

in m arker technology (Rafalski

and in

the improvemen t

of

statistical

the

genetic detection

of

single

the genetic variation for such

are

identified,

be

similar

to

The analys is

of

Q T L s

is

based

on

the association

at

specific positions

on the

thema p posit ion ofthe

The

availability

of

efficient m ark er

in a

s laborious way as com pared with other m arker

such as R FL Ps and i sozymes . Th e problem

can be

solved

by

using

zygous mapping populat ions such as setsofdoubled

haploids (DHs), recombinant backcross l ines

(RBLs), also called backcross inbred l ines (BILs)

(Ramsey et

al,

1996), introgression lines (Us) (Fshed

Zam ir, 1995) or substi tut ion l ines. M ore advanced

material

of

this kind are near-isogenic l ines ( N IL s) ,

differing in a small introg ression from a corre-

sponding genotype .

The

mult ip le

use of

these

populat ions without having to genotype the material

again with molecular markers, makes these genetic

stocks extremely valuable. Th is can be d emo nstrated

by the analysis of traits as different as flowering

(Jansen

et al,

1995)

and

seed dorm ancy

(van der

Schaar et

al,

1997) in the sam e set of R IL s of the two

most widely used Arabidopsis ecotypes, Landsberg

erecta {her andColum bia (Col). This popula t ion

also serves

as the

standard map ping popula t ion

in

Arabidopsis

(Lister De an, 1993).Acareful choice

of pa ren ts e.g. by cho osing extrem es of the genot^^pic

variat ion within

a

species, extends these oppo r-

tuni t ies even more . Ex amplesof'imm or ta l ' m app ing

populat ions based

on

very different gen otyp es

are

t h e R I L s inrice deriv ed from acrossof anupland

japonica variety with anindicalowland variety (Wan g

etal, 1994);inbarleytheD H s have been der ived

from crosses between malt ing

and

fodder cultivars

(Kleinhofs et al, 1993),and in

Arabidopsis

crosses

between European andAfrican ecotypes (Alonso-

Blanco

et al,

unpubl ished) . Af ter

the

location,

quantification and analysisofthe interactionsofloci

controll ing

the

trait

has

been made ,

it

will

be

impor t an t to characterize the individual loci . In

order

to

obtain genotypes w ith only mon ogenic

differences, the further backcrossing with a recu rrent

parent (often one

of

the parents

of

the initial cross)

wil l be necessary, when working with RILsorD H s .

This ' Me ndel i s ing ' of aQ T Lcan befacilitatedby

markers l inked

to the

respective loci

and

also

by

selectionofthe phenot>-peinbackcross po pulat ion s.

W h e n N I L s

are

available, this process

of

' M e n d e l i s i n g ' Q T L s hasalready been perform ed.

An al ternative approach

to the

dissection

of

natura l

genetic variation is to perform a backcrosspro-

gramme with phenotype-based selection from

the

beginning (Fig. 1). After an u m b e r ofbackcrosses,

the analysis with molecular markers wil l indicate

what chromosomal regions

of

the donor parent

are

still present in theselected linesandthereby show

the

map

posit ion

of

puta tive Q TL s . O nce

a NIL

with monogenic segregation has been obtained,the

refinement

of

the m ap posit ion

can be

done

in the

progeny of thecross of such a NILcarryingthe

introgressed gene, with

the

recurrent parent .

The

selection of recombinants a round the locus of

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M.Koornneef C. Alonso-Blanco and A.J. M. Peeters

RP DP

I I

Parents

F I

o

c

x:

F2

i

3

D

o

o

c

o

CO

0

T D

X3

0

0

O

_

O5

C

F8

I^ L RIL pop ulatio n

Genotyping with molecular markers covering the genome

Phenotyping for the trait

o

interest and QTL mapping

1

1

Constructiono NILs BILs) con taining a single QTL

Physiological and genetical characterisation of the NILs

Figure 1 A

schematic o utline

of

the production and use

of

recom binan t inbred lines RI Ls) and near isogenic

lmes NIL s). RP, recurrent parent; DP , donor paren t;

S,

selected plan t; BI L, backcross inb red line.

cloning procedures. A potential problem is that it

will be difficult to distinguish if one gene or more

than one very closely linked genes determine the

traits that segregate monogenically. The determi-

nation of a very detailed map position will be

especially important in those species where the

complete physical map is available and for which in

the near future the complete sequence of the genome

will become available. When the biochemical func-

tions of the genes located in the region of the QTL

are known, one might 'guess' the candidate gene.

Knowledge of the position of open-reading frames

will also allow the selection of clones that can be used

for transformation, which will provide the proof of

the successful cloning by complementation. Al-

though in natural alleles it will not be clear whether

one is dealing with alleles that make a functional

To knock out the wild type allele, a mutatio

approach can be followed to find null mutants. T

detailed map position is also important when tra

posons are used for this, because this will permit

choice of a genotype with a transposable element

the vicinity of the target gene and thereby incre

the chance of finding insertions in the target ge

since transposable elements have a tendency to ins

predominantly to linked sites (Sundaresan, 199

Furthermore, when many ESTs are mapped in

region of interest, it will enable the use of DN

sequences in combination with transposons to p

form reverse genetics. Such EST probes can also

used for the detection of deletions caused

irradiation mutagenesis.

Examples of 'natural' monogenic traits that ha

been cloned are many disease resistance genes (Jon

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Genetics in plant physiology

and for which map-based cloning efforts have been

initiated are the flowering-time genes

FRI

(Clarke

Dean, 1993) andFLC (Lee et al 19946) in

Arabidopsis.

CONCLUDING REMARKS

The use of genetics has been successfully exploited

to dissect plant developmental processes.

In

par-

ticular, the combination of genetics with biochem-

istry and molecular biology allows the study of the

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