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Journal of Plant Physiology 168 (2011) 20282034

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Journal of Plant Physiology
" o u r n a l h o # e $ a g e % & & & ' e l s e v i e r ' ( e ) " $ l $ h
*ater relations in ulture #e(ia influene #aturation of avoa(o so#ati
e#bryos
+el,n -.r/ue01-art2n
a
3 4afael Ses#ero
a
3 -iguel 5' 6uesa(a
b
3
7ernan(o Pliego15lfaro
3

3 Carolina S.nhe014o#ero
b
a
IFAPA, Centro de Churriana, Cortijo de la Cruz s/n, 29140 Churriana, Mlaga, Sain
b
!earta"ento de #iolog$a %egetal, &ni'ersidad de Mlaga, Ca"us de (eatinos s/n, 290)1 Mlaga, Sain

Instituto de *orto+ruti,ultura Su-troi,al . Mediterrnea /0a Ma.ora1 2I*SM3&MA3CSIC4, !earta"ento de #iolog$a %egetal, Fa,ultad de Cien,ias, &ni'ersidad de
Mlaga, Ca"us de (eatinos s/n, 53290)1 Mlaga, Sain
a r t i l e i n f o
Arti,le histor.6
4eeive( 23 -arh 2011
4eeive( in revise( for# 6 June 2011 5e$te( 8 June 2011
7e.8ords6
8el strength
9s#oti $otential
Persea a"eri,ana
So#ati e#bryogenesis
*ater $otential
a b s t r a
t
5$$liation of
transfor#atio
n an( other
biotehnologi
al tools in
avoa(o
(Persea
a"eri,ana
-ill') is
ha#$ere( by
(iffiulties in
obtaining
#ature
so#ati
e#bryos
a$able of
ger#ination
at an
ae$table
rate' :n this
∨3 &e evaluate( the effet of (ifferent o#$oun(s
affeting #e(iu# &ater relations on #aturation of
avoa(o so#ati e#bryos' Culture #e(ia &ere
harateri0e( &ith res$et to gel strength3 &ater
$otential an( os#oti $otential' :#$rove( $ro(ution
of #ature so#ati e#bryos &as ahieve( &ith
gelling agent onentrations higher than those
onsi(ere( stan(ar(' <he os#oti agents suh as
sorbitol an( P=8 (i( not have $ositive effets on
e#bryo #aturation' <he nu#ber of &1o #ature
so#ati e#bryos $er ulture &as $ositively
orrelate( &ith #e(iu# gel strength' 8el strength
&as signifiantly affete( by gelling agent ty$e as
&ell as by gelling agent an( P=8 onentration'
-e(iu# &ater $otential &as influene( by sorbitol
onentration> inor$oration of P=8 to a ulture
#e(iu# (i( not affet #e(iu# &ater $otential' <he
highest #aturation results &ere ahieve( on a
#e(iu# gelle( &ith 10 g l
?1
agar' -oreover3 these
so#ati e#bryos ha( i#$rove( ger#ination rates'
<hese results orroborate the role of &ater restrition
as a ;ey fator ontrolling #aturation of so#ati
e#bryos'
@ 2011 =lsevier 8#bA' 5ll rights reserve('
Introdu,tion
5voa(o (Persea a"eri,ana -ill') is
ultivate( in #ost tro$ial an( subtro$ial
regions of the &orl( for its fruits3 &hih are
a$$rei1ate( for their high oil ontent an(
onstitute a &ell balane( su$$ly of $roteins3
arb
ohy(
rates
3
#ine
rals
an(
vita#
ins
(4ay3
2002)' 5vo1a(o $ro(ution
is li#ite( by several fungal
(iseases3 suh as the
avoa(o root rot an( the
&hite root rot3 %erti,illiu"
&ilt3 anthra1nose an(
Cer,osora s$ot'
Conventional bree(ing in
avoa(o is ha#$ere( by its
long "uve1nile $erio(3
an( biotehnologial
a$$roahes oul(
therefore be useful
for this s$eies' 5n
effiient regeneration
$rotool fro# sin1gle
ells is a re/uisite for
a$$liation of
biotehnologial
#etho(s' :n avoa(o3
so#ati
e#bryogenesis (S=)
is the #etho( utili0e(
for a(ventitious $lant
regeneration (Bit0 et
al'3 200C)'
=#bryogeni
A--re'iations6 +C#3 #aturation #e(iu# onsisting of
the -S for#ulation &ith the #aronutrients of 8a#borg et
al' (1D68)> -SP #e(iu#3 -S #e(iu# (-urashige an(
S;oog3 1D62) su$$le#ente( &ith 0'1 #g l
?1
$ilora#> P=83
$olyethylene glyol> s'e'3 so#ati e#bryo> S=3 so#ati
e#bryogenesis> &1o3 &hite1o$a/ue'
1 Corres$on(ing author' <el'% E34
DC2131D4F> faG% E34 DC2131D44' 53"ail
address6 fer$liegoHu#a'es (7' Pliego1
5lfaro)'
01F61161F)I see front #atter @ 2011 =lsevier
8#bA' 5ll rights reserve(' (oi%
10'1016)"'"$l$h'2011'06'008
ultur
es
(eriv
e(
fro#
i##a
ture
avo
a(o
0ygot
i
e#br
yos
an
be
routi
nely
obtai
ne(
(Plie
go1
5lfaro an( -urashige3 1D88>
*it"a;sono an( Bit03
1DDDa)' Ao&ever3 the lo&
onversion rate of so#ati
e#bryos into $lants is the
#ain bottlene; in $lant
regeneration' 5s a
onse/uene3 although the
intro(ution of s$eifi
genes an( &i(ening the
geneti base follo&ing
generation of so#alonal
vari1ants are no& feasible
a$$roahes for avoa(o
bree(ing (Bit0 et al'3 200C)3
the lo& onversion rate of
the so#ati e#bryos into
$lants is li#iting the
a$$liability of these
tehni/ues'
=#bryo #aturation is a
;ey fator affeting $lant
onversion in so#ati
e#bryogenesis
syste#s (5##irato3
1D8F)' !uring the
#aturation stage3 the
so#ati e#bryos
un(ergo
#or$hologial an(
biohe#ial hanges3
suh as storage
$ro(ut (e$osition3
&hih are essential
for subse/uent $lant
(evelo$#ent'
8enerally3 au#u1
lation of storage
$ro(uts auses the
hange fro#
transluent to &hite1
o$a/ue olor
e#bryos (CaillouG et
al'3 1DD6)' *hite1
o$a/ue a$$earane
has been use( as
#aturity riterion in
(ifferent s$eies
(CaillouG et al'3 1DD6>
8arin et al'3 2000>
JKrgaar(3 1DDF)3
inlu(ing avoa(o
(Per.n16uesa(a et
al'3 2004> *it"a;sono
an( Bit03 1DDDb)'
*ater relations
bet&een the e#bryo
an( its environ#ent
$lay regulatory role in
e#bryo (evelo$#ent3
$artiularly (uring
#at1uration (5(a#s
an( 4inne3 1D80>
+ra(for(3 1DD4)' :n
onifer S=3 it has
been $ro$ose( that
re(ue( &ater
availability oul(
sti#ulate
#9 Mr:uez3Mart$n et al9 / ;ournal o+ Plant Ph.siolog. 168 (2011) 20282034 202D
a shift in the (evelo$#ental $rogra# of the ulture3 fro#
$rolif1eration of ells an( early so#ati e#bryos to the
$ro(ution of e#bryos at a(vane( stages ( Lli#as0e&s;a
et al'3 2000)'
*ater restrition has been a &i(ely use( strategy for
i#$rov1ing so#ati e#bryo #aturation both in gy#nos$er#s
an( angios$er#s' *ater availability #ani$ulation has been
a((resse( through (ifferent #eans% a((ition of lo& #oleular
&eight o#1$oun(s ($las#olysing agents) suh as inorgani
salts3 a#inoai(s an( sugars> use of high #oleular &eight
o#$oun(s (non1$las#olysing agents) suh as $olyethylene
glyols (P=8) an( (eGtrans (Stasolla et al'3 2002) an( (rying
$roess (=tienne et al'3 1DD3)' 5lthough gelling agents are
#ainly use( to reate se#isoli( #e(ia3 they an also
influene the availability of &ater to tissues (Miv3 1DD1)'
!es$ite the &i(e a$$liation of various gelling agents3 a
fe& stu(ies have been arrie( out to un(erstan( the effet on
&ater availability of su$$le#enting the ulture #e(iu# &ith
o#$oun(s of (ifferent ty$e ( Lli#as0e&s;a and S#ith3
1DDF> Lli#as0e&s;a et al'3 2000)'
<he ai# of this stu(y &as to o$ti#i0e #aturation
on(itions of avoa(o so#ati e#bryos by #ani$ulating
&ater relations through the a((ition of (ifferent o#$oun(s to
a ulture #e(iu#' :n $aral1lel3 the ulture #e(iu#
$hysiohe#ial $ara#eters suh as &ater $otential3
os#oti $otential an( gel strength &ere (eter#ine( &ith the
ob"etive of eG$laining the behavior of e#bryogeni tissues
ulture( un(er the (ifferent #aturation on(itions'
Materials and "ethods
Initiation and "aintenan,e o+ e"-r.ogeni, ,ultures
5voa(o (Persea a"eri,ana -ill') e#bryogeni ultures
&ere initiate( fro# i##ature 0ygoti e#bryos3 ultivar N!u;e1
FO3 aor(1ing to Pliego15lfaro an( -urashige (1D88)' 9ne to
t&o ## 0ygoti e#bryos &ere ulture( on -S #e(iu#
(-urashige an( S;oog3 1D62) su$$le#ente( &ith 0'1 #g l
?1
$ilora# (-SP) an( 6 g l
?1
agar (5112D63 Sig#a5l(rih)'
Cultures &ere inubate( in (ar;ness at 2C P 1
Q
C'
-aintenane of establishe( e#bryogeni ultures &as
arrie( out on the sa#e ulture on(itions use( for initiation'
Subultures &ere arrie( out at #onthly intervals'
So"ati, e"-r.o "aturation
7or #aturation3 e#bryogeni sus$ensions &ere initiate(
fro# ultures in #aintenane on(itions' 7ollo&ing the
$rotool of *it"a;sono an( Bit0 (1DDDa)3 0'4 g of friable allus
&as inoulate( in 40 #l li/ui( -SP #e(iu# in 100 #l
=rlen#eyer flas;' Sus$en1sions &ere inubate( on an orbital
sha;er (120 r$#) un(er lo& light intensity (C #ol #
?2
s
?1
)3
$rovi(e( by 8roluG la#$s (Syl1vania3 =rlangen3 8er#any)3
an( a 16 h $hoto$erio( at 2C P 1
Q
C' 5fter D (ays they &ere
sieve( se/uentially through 2 an( 1 ## $ore #esh' 9ne
hun(re( #g of the fration that $asse( the 2 ## $ore #esh
an( &as retaine( in the 1 ## $ore #esh &ere inou1late( in
2C ## R 1C0 ## test tubes ontaining 2C #l of #aturation
#e(iu#'
-aturation #e(iu# (+C#) onsiste( of the -S
for#ulation &ith the #aronutrients of 8a#borg et al' (1D68)
(Per.n16uesa(a et al'3 2004)' 7our e#bryogeni lines &ere
use( for #aturation eG$eri#ents'
-aturation #e(ia &ere $re$are( by
a((ing the a$$ro$riate o#$onents an(
a("usting the $A to C'F43 $rior to a((ing
the gelling agent' -e(ia &ere heate( in
an autolave (121
Q
C an( 0'1 -Pa) for
F #in to #elt the gelling agent' <&enty1
five #l of the #olten #e(iu# &ere
(is$ense( in 2C ## R 1C0 ## test
tubes &hih &ere
overe( &ith $oly$ro$ylene losures
La$1Sts' -e(ia &ere steril1i0e( by
autolaving for 1C #in at 121
Q
C an(
0'1 -Pa'
-aturation eG$eri#ents &ere arrie(
out (uring three sub1ultures &ith
suessive transfers to fresh #e(iu# of
the sa#e o#$osition at CF &ee;
intervals' :nubation &as in (ar;ness at
2C P 1
Q
C'
<&enty ultures (test tubes) &ere
use( $er #aturation treat1#ent' 7resh
&eight inrease an( nu#ber of &1o
so#ati e#bryos $ro(ue( $er ulture
&ere reor(e( at the en( of eah
subulture' 5s in $revious stu(ies
(CaillouG et al'3 1DD6> Per.n16uesa(a et
al'3 2004> *it"a;sono an( Bit03 1DDDb)3
&hite1o$a/ue a$$earane of so#ati
e#bryos &as use( as a riterion of
#aturity'
5++e,t o+ gelling agent t.e and
,on,entration
:n this eG$eri#ent3 the effet of three
gelling agents at (iffer1ent
onentrations &as stu(ie( on
#aturation of avoa(o so#ati
e#bryos' +C# #e(iu# su$$le#ente(
&ith 30 g l
?1
surose &as soli(ifie( &ith
eah of the follo&ing gelling agents%
agar (5112D63 Sig#a5l(rih) at 63 83
10 an( 12 g l
?1
3 agargel (5133013
Sig#a5l(rih) at 3'C3 C3 F'C an( 10 g
l
?1
an( gellan gu# (8elrite) (811D103
Sig#a5l(rih) at 1'F3 3'43 C'1 an( 6'8
g l
?1
'
5++e,t o+ os"oti, agent t.e and
,on,entration
<he effets of t&o os#oti agents of
(ifferent nature ($enetrat1ing an( non1
$enetrating) &ere stu(ie( in the seon(
eG$eri#ent' -aturation #e(iu#
ontaining 30 g l
?1
surose &as
su$$le#ente( &ith (ifferent
onentrations of sorbitol (S160213
Sig#a5l(rih) (2'C3 C an( F'CT (&)v))
or $olyethylene glyol (P=8)3 -
r
80003
(P1213D3 Sig#a5l(rih) (2'C3 C an(
F'CT (&)v))' 5ll #e(ia &ere gelle( &ith
6 g l
?1
agar'
<er"ination o+ so"ati, e"-r.os
*hite1o$a/ue so#ati e#bryos U4
## (evelo$e( in +C# gelle( &ith 10 g
l
?1
agar &ere ulture( on -1 #e(iu#
(S;ene an( +arlass3 1D83) gelle( &ith
1'F g l
?1
gellan gu# after $artial
eGision of otyle(ons3 e'g'3 longitu(inal
uts &ith re#oval of about one thir( of
the otyle(ons' Cultures &ere inubate(
un(er a 16 h light $ho1to$erio( $rovi(e(
by 8roluG la#$s (Sylvania3 =rlangen3
8er#any) (40 #ol #
?2
s
?1
) at 2C P 1
Q
C' 8er#ination &as arrie( out (uring
three subultures of C &ee;s eah'
<he ger#ination eG$eri#ent &as re$eate( t&ie' *ithin
eah eG$eri#ent3 C0 so#ati e#bryos &ere use(' =#bryos
&ere onsi(1ere( ger#inate( &hen shoot an()or root
elongation &as U2 ##'
Mediu" gel strength
-e(ia &ere $re$are( as $reviously in(iate( an(
(is$ense( in 2C ## R 1C0 ## ulture
tubes until the li/ui( al#ost reahe( the
to$ of the tube' 5fter 24 h e/uilibration
at 2C P 1
Q
C3 an( aor(ing to
Lli#as0e&s;a and S#ith (1DDF)3 the
fir#ness of the #e(iu# gel &as
#easure( using the V<1$lus <eGture
5naly0er (Stable -iro Syste#s Bt('3
Surrey3 SL) &ith a 4 ##1(ia#eter
stainless steel ylin(er $robe' <he tests
&ere al&ays $erfor#e( in the enter of
the tube3 in or(er to avoi( e(ge effets'
<he trigger fore &as fiGe( at 3 g3 the
test (istane (e$th at 2 ##3 $re1test
an( test s$ee( at 1 ## s
?1
an( $ost1
test s$ee( at C ## s
?1
' !ata of gel
strength &ere obtaine( using the
<eGture 5naly0er soft&are (<==323
v'3'0'1'0) as the #aGi#u# $ea; fore'
7or eah #aturation #e(iu#3 at least
three re$liates fro# (ifferent tubes
&ere #easure('
2030 #9 Mr:uez3Mart$n et al9 / ;ournal o+ Plant Ph.siolog. 168 (2011) 20282034
Fig9 19 =ffet of gelling agent ty$e an( onentration on #aturation of avoa(o so#ati e#bryos' !ata re$resent the #eans of three subultures' !ifferent letters in(iate signifiant
(ifferenes at P W 0'0C'
=ater and os"oti, otential o+ the "ediu"
-e(ia &ere $re$are( as in(iate( an( 20 #l &ere (is$ense( in D0
## Petri (ishes' Petri (ishes &ere seale( &ith $arafil# an( store( for 24
h in a ulture ha#ber at 2C P 1
Q
C' 5fter e/uilibration3 a ylin(er &as
sa#$le( fro# the entral $art of eah Petri (ish using an 8 ##1(ia#eter
or; bearer' <he &i(th of the slies &as virtually onstant3 sine in all
ases eGatly the sa#e volu#e of #e(iu# &as $oure( into the Petri
(ishes' *ater $otential &as #easure( &ith a $syhro#eter (+eruto et al'3
1DDD)' 8el slies &ere autiously $ut in a her#eti C1C2 Sa#$le
Cha#ber onnete( to a A433 < (e& $oint #irovolt#eter (*esor :n'3
Bogan3 SS5)' Sa#$les &ere allo&e( to e/uilibrate for 2 #in an( the
&ater $otential &as #easure( 30 s after the ooling stage'
7or eah #aturation #e(iu#3 at least three #easure#ents &ere
arrie( out in (ifferent Petri
(ishes'
9s#oti $otential &as also
(eter#ine( using a $syhro#eter
by a((ing C0 B of the #e(iu#
&ithout the gelling agent to 8 ##
$a$er (iss' 5s $reviously
in(iate(3 they &ere e/uilibrate(
for 2 #in an( then #easure( 30 s
after the ooling stage' <hree
#ea1sure#ents of eah sa#$le
&ere ta;en
Statisti,al anal.sis
:n #aturation eG$eri#ents3 the
(ata re$resent the average of
three subultures' !ata &ere
analy0e( by 5J9X5 follo&e( by
o#1$arison of #eans using the
least signifiant (ifferene test
(BS!)' <he (egree of orrelation
bet&een variables &as esti#ate(
by using the PearsonOs oeffiient'
<he signifiane level use( &as
0'0C in all ases'
>esults
5++e,t o+ gelling agent t.e and
,on,entration
<he gelling agent ty$e ha( a signifiant influene on allus $ro1
liferation rate &ith values ranging fro# 0'6F g in agargel to 0'80 g in agar1
gelle( #e(ia ((ata not sho&n)'
Ju#ber of &1o so#ati e#bryos $er ulture &as signifiantly affete(
by the gelling agent ty$e (P W 0'0000) an( onentration (P W 0'0000)
attaining the highest nu#bers on 83 10 an( 12 g l
?1
agar
an( F'C g l
?1
agargel (7ig' 1)' :n
general3 better results &ere
obtaine( &ith agar' 5gargel3 a
blen( of agar an( gellan gu#3
gave rise to inter#e(iate values3
although #ore si#ilar to those
obtaine( &ith agar than &ith
gellan gu#' 8elling agent
onentration &as a rit1ial fator
in the $ro(ution of &1o so#ati
e#bryos' :n general3
onentrations higher than those
onsi(ere( stan(ar( enhane(
#aturation of so#ati e#bryos in
the three gelling agents teste('
Jevertheless3 so#ati e#bryos
obtaine( in a #e(iu# soli(ifie(
&ith 12 g l
?1
agar &ere s#aller
than those obtaine( in slightly
lo&er onentrations'
5++e,t o+ os"oti, agent t.e
and ,on,entration
9s#oti agent ty$e3
onentration an( the interation
ty$e R onentration3 signifiantly
affete( ulture gro&th (uring the
#aturation $hase ((ata not
sho&n)' 5 greater fresh &eight
inrease o#$are( &ith the
ontrol &as observe( in a
#e(iu# &ith CT P=8 (1'10 g vs'
0'D6 g in the ontrol)' *hen
sorbitol &as a((e( to ulture
#e(ia3 signifiantly lo&er gro&th
rates &ere obtaine( at all
onentrations teste( in(iating a
negative or1relation bet&een
sorbitol onentration an( the
ulture gro&th' 7resh &eight &as
only u$ to 0'32 g in the #aturation
#e(iu# su$$le#ente( &ith F'CT
sorbitol'
9s#oti agents in the
#aturation #e(iu# signifiantly
influ1ene( ulture a$$earane
(7ig' 2)' P=8 signifiantly
inrease( the $ro(ution of
otyle(onary e#bryos' <he higher
the onentration of P=8 in the
#aturation #e(iu#3 the larger the
so#ati e#bryos (evelo$e(' <he
so#ati e#bryos &ere
harateri0e( by a beige1to1yello&
olor an( hy$erhi(ri a$$earane'
*hite1o$a/ue so#ati e#bryos
&ere rarely observe( in P=81
su$$le#ente( #e(ia' Sor1bitol
ause( a (eterioration of ultures'
Calli bea#e (ar; in olor3 losing
the e#bryogeni harateristis
even at the lo&est on1entration
(2'CT sorbitol)' Ao&ever3 &1o
so#ati e#bryos &ere $ro(ue(
but e#bryo /uality &as
signifiantly affete(' 9ur results
reveale( a signifiant effet of the
os#oti agent ty$e (P W 0'0003)
an( onentration (P W 0'0000) on
the nu#ber of &1o so#ati
e#bryos $er ulture (7ig' 3)'
<here &as also an interation of
os#oti agent ty$e &ith os#oti
agent onentration3 &hih influ1
ene( this $ara#eter (P W
0'0003)'
:n aor(ane &ith the
$revious results3 +C# gelle( &ith
10 g l
?1
agar &as selete( as the
i#$rove( #e(iu# for #aturation
of avo1
(han? .ou +or e'aluating
#C0 eas.Con'erter !es?to
<his *or( (ou#ent &as onverte( fro# P!7 &ith an evaluation
version of +CB easyConverter !es;to$ soft&are that onl.
,on'erts the +irst @ ages of your P!7'
5tivate your soft&are for less than I20
htt$%))&&&'$(fonline'o#)easyonverter)
C<4BE Cli; on the lin; belo& to $urhase