P l u n t C e l l . ' l - i s s u au n d O r e u n ( ' u l t u r e 3 5 : 1 3 1 - 1 3 9 . 1 9 9 3 .
) 1993 Klun,cr At'udanic Publislrrs. I'rinted in the lletlrrltuttls.
Effect of copper on shoot and root regenerationin wheat, triticale, rape
and tobacco tissue cultures
L a rszl oP ur nhaus c r ' & G a b o r Gv u l a i r
-Department
' ('crc,tl
of Genetics and Plant
. Hungary;
R< , s eur ghI ns ti tttta
. H -6 7 0 1 Sz t, gt,d
. PO B -? 9 1
Breeding. University of Agricultural Sciences,H-2103 Godolld, Hungary
R e c e i v c d 2 i J u l v 1 9 9 2 ; a c c e p t e c il n r e v i s e c lf o r m 6 M a v 1 9 9 3
Ke y w or ds : c allus . pla n t re g e n c ra ti o n ,s i l v e r
Abstract
C u SO* ( 0. 1- 1( X )pM ) s i g n i fi c a n tl yc n h a n c e ds h o o t regenerati onfrom cal l i of w heat and tri ti cal e and of
to b a cco leaf dis c c ultu rc s . In c u l tu rc s o f w h e a t a n d tri ti cal e, C uS O* al so sti mul ated root formati on.
Wh e n equal c onc en tra ti o n so f C u SO* w e rc a p pl i ed i n di fferent medi a. i t w as found that the
co mp onc nt s of t he b a s a l me d i a h a d o n l y mo d i f yi ng effects. C uS Or pretreatment promoted pl ant
survival when regcncrated wheat plants were transfcrred directly tc-rptttting soil. [n contrast with
Cu SOr , A gNO , , which a l s o s ti m u l a tc ds h o o t re g enerati on.i nhi bi tcd rooti ng i n w hcat and tri ti cal e. In
Bra ssi c a nopLt sc allus c u l tu re s , Ag N O . s tro n g l y i ncreasedmorphogcnesi s.w hereas C uS Ot had no
significant effect.
c d, K N - ki neti n, N A A Ab b re vic t t ions : B A - b e n z y l a d e n i n,e 2 .1 -rl- 2 .z l - di chl orophcnoxyacetiaci
n a p h th alc neac et icac i d
Introduction
Sh o o t r egener at ion is o f c ru c i a l i m p o rta n c e i n
the application of in vitro methods for plant
i mp ro vem ent . I t has b c c n s h o w n th a t th e fre q u cn cy of s hoot r ege n e ra ti o nd c p c n d s o n g e n o typ c. c. g. in whc at ( Tri ti c u m a e s ti v u mL .) (Se a rs
& De ck ar d 1982) . T h c i m p o rta n c e o f g ro w th
re su l a tor s , c s pc c ially a u x i n s a n d c y to k i n i n s , i n
ca l l u s i nduc t ion and p l a n t re g e n c ra ti o nh a s a l s o
b ce n s hown. T he et h y l c n c i n h i b i to r Ag N O . h a s
si g n i frcant ly im pr ov e d . s h o o t re g e n c ra ti o n i n
speciessuch as whcat, lYicotianaplumbaginifolia
Vi v. (P ur nhaus eret a l . 1 9 8 7 )a n d ra p e (B ra s s i c a
n u p u s L . ) ( D e B l o c k c t a l . 1 9 8 9 ) .A m o n g e t h y l ene inhibitors, other heavy mctals, e.g. Co
( R o u s t a ne t a l . 1 9 8 9 ;S e t h ie t a l . 1 9 9 0 ;C h r a i b i e t
al. 1991) and Ni (Roustan et al. 1989). also
proved to be effectivc in stimulating mor-
phogenesi si n cal l us cul tures. A l though somc
heavy metal s arc w i del y used as mi croel ements.
little is known about thcir effects on the morphogenesi si n ti ssuc cul tures.
In our previ ous experi ments. C u at concentrati ons 5 to 1000 ti mes hi gher than i n thc
ori gi nal Murashi ge& S koog (1962) mcdi um (0.1
pM C uS O_,)stri ki ngl y enhancedshoot and root
regenerati oni n w heat cal l us cul tures, and 1 pM
Cu resulted in a significantly higher increase in
shoot regeneration than trcatment with KN or
B A at concentrati onsrangi ng from 0.5 to 4 mg
'
(P urnhauser 1991). H i gh concentrati onsof
I
Cu also increascd shoot formation in callus
cul turesof C ucumi s mel o L. (Garci a-S ogoet al .
1991).
The present paper reports thc effects of
C uS Or on regenerati on from somati c or androgenic calli or leaf disc culturcs of several
r32
species [whcat. triticalc (X TriticosecaleWittma ck) , r ape and to b a c c o ]. a n d c o mp a rc s th e
effccts of Cu with those of Ag and also thc
effects of Cu added to four basal media formulac
wi d e l y us ed in plan t ti s s u ec u l tu rc .
Materials and methods
Spring wheat (Triticum aestivum L.) line
CIMM Y T No. 215w a l sg ro w n i n th e g re e n h o u s e
(d a y/ night t em per a tu re s b e i n g a p p ro x i ma te l y
2 0 'C/ 15" C) . T his li n e o ri g i n a tc d fro m th c m a tcri a l of t he 7t h I n te rn a ti o n a l W h c a t D ro u g h t
Stre s sNur s er y . 198 8 -1 9 8 9 ,a n d p ro v e d to b e a
g o o d r egener at ingg c n o ty p e (F e l fo l d i & Pu rn hauser 1992). Calli were initiated from irnmaturc
cmbryos (ca. 1-1.5mm long) on medium consi sti ng of M ur as hig c a n d Sk o o g ' s (\9 6 2 ) i n o rganic nutrients (MS), I mg I ' thiamine-HCl
I
( F l u k a C h e m i e A G . , B u c h . S w i t z . ) . 1 ( Xm
) gI
m e s o - i n o s i t o l( R c a n a l . B u d a p c s t , H u n g . ) . 2 %
su cros e ( Reanal) , 0 .1 o /c Ox o i d -a g a r. a n d
'
I mg I
2, 4- o, Hig h l y p u ri fi e d w a te r o b ta i n e d
fro m a B ar ns t ead ( D u b u q u c , IA. U SA ) N a v o Puse Il cartridge purification systemwas used for
p re p ar at ion of t he m e d i u m. In o rg a n i c c h e m i c a ls
a n d g r owt h r egulat o rsu s e d i n e x p e ri m e n tsw e re
Sigma (St. Louis. MO.. USA) and Serva
(Hci delber g/ New Y o rk ) p ro d u c ts . re s p e c ti v c l y .
Fo u r week s af t er ini ti a ti o n . c a l l i w e re tra n s fe rre d
t
t c rt h e s a m c m e d i u m w i t h 0 . 5 m g l
2 , 4 - r r .a n d - l
w e e k s lat er c alli were s u b c u l tu re da g a i n o n to th e
sa me m c dium . E x p e ri m e n ts o n re g e n e ra ti o n
w cre t hen per f or me d . u s i n g c a l l u s p i e c e s c o n ta i n i ng c om pac t an d y e l l o w i s h -g re e np a rts a s
d e scr ibedpr c v ious l y (Pu rn h a u s c rc t a l . 1 9 tt7 ).In
th e fi r s t ex per im en t. c a l l i w e re tra n s fe rre do n to
MS m c dia f r ee of 2 ,4 -t>a n d s u p p l e me n te dw i th
d i ffe rent c onc c nt r a ti o n so f C u S O ., (0 -1 0 0 p M )
o r A g N O . ( 0 - l 0 0 p M ) . I n t h e s e c o n de x p e r i me n t, CuS O * at d i ffe re n t c o n c e n tra ti o n sw a s
a p p l i e dt o t h e m e d i a M S , N 6 ( C h u e t a l . 1 9 7 5 ) .
85 (Gamborg ct al. 1968)and SH (Schenk&
Hildebrandt 1972). CuSOr-free versionsof these
b a sal m edia wer e u s e d a s c o n tro l s . F o r e a c h
su b cult ur c . c alli t o b e tra n s fe rre dw e re c u t i n to
p i e cc s about 5 m m i n d i a me te r.
Hexaploid triticale (X TriticosecaleWittmack)
cal l i w crc i nduced from i mmature embryos of
the N B 3T12 l i nc provi ded by D r. S . B aenzi nger
( U n i v e r s i t y o f N e b r a s k a ,L i n c o l n , N E ) . I n o u r
earl i er tests. thi s l i ne proved to be a good
regenerati nggenotype (Fel fol di & P urnhauser
1992).C ondi ti ons of pl ant grow th. cal l us i nducti on and mai ntenance w cre the same as for
w heat except that tri ti cal c cal l i w ere mai ntai ned
t
2.4-o.
o n l y o n c e o n M S m e c l i u mw i t h 0 . 5 * g I
For thc experi ment on regenerati on. cal l us
pi ecescontai ni ngw hi te cmbryoi ds w ere sel ectcd
and transferredto the same mcdi um as used i n
the fi rst experi ment on w heat (ot descri bed
abovc).
Brassicanapus L. cv. Olga callus cultures were
i nduced from hypocotyl segmcnts(5-7 mm l ong)
of seedl i ngsgermi nated at 30' C i n darknesson
N 6 mcdi um for onc w cek. The cul ture medi um
contai ncd MS i norgani c nutri cnts, organi cs and
I
agar as l i sted above. suppl ementedw i th 1 mg I
I
BA (Scthi et al. 1990), and
NAA. I mg I
C u S O r ( 0 . 1 - 1 0 0p M ) o r A g N O . ( 1 0 - 1 ( Xp) M )
l\,licotianatubacunt leaf disc culturc (Marcc-rtri gi ano 1986)w as i ni ti atcd from a stcri l e shoot
culturc of the SRI line (Maliga et al. I9l3)
mai ntai ncd on hormone-free MS medi um at
24" C , at 2000l ux under a 16-h photoperi od.
Leaf di scs(B mm i n di amcter) w ere cut from the
fi rst ful l y devel oped l eaves and pl accd w i th
abaxial surface downwards onto a shoot inducti on mcdi um contai ni ns + strcnsth
- m c sMS
o - i nsal
o s ts.
itol,
I
1mg I
t h i a m i n e - H C l i. O r i r n gl '
'
BA and CuSO",
27c,sucrosc.0.8% agar, 2 -g I
(0
at di ffercnt concentrati ons 1-1000 pM). Leaf
di scsw ere i ncubatedi n thc dark for 4 w eeks at
24 " C .
A nthcr cul turc of w hcat w as i ni ti ated from
H ungari an cul ti vars Mv 15 and Mv 16. For
i nducti on of androgeni c cal l i , C -17 mcdi um
(H ui j un X . i n P auk et al . 1991) w as used w i th
somc modification (replacement of C-17 microel ementsw i th those of MS ). P rctreatment of
ti l l ers. i sol ati on of anthers and the cul ture condi ti ons w ere si mi l ar to those descri bedby P auk
et al. (1991). Calli 1mm in diameter were
harvested and transferred to + strength MS
medi um w i th or w i thout hormones (0.5 +
'
N A A and K N ) and contai ni ng di ffer0.5 mg I
ent concentrati onsof C uS O., (0, 0.1 and 2 frM).
Grow th regul atorsand C uS O* w ere added to
133
th e me dium pr ior t c -ra u to c l a v i n g .T h e p H w a s
a d j u ste d t o 5. 8 bc f or e a u to c l a v i n g .F i l te r-s tc ri l i ze d AgNO . was add e d to th c a u to c l a v e dme d i a
a t 4 5 "C. G las s P et r i d i s h e s (1 0 0 x 2 0 m m ) c o n ta i n i n g 25 m l m edium a n d 9 (w h e a t. tri ti c a l e a n d
Bra .ss ic as om at ic c u l tu rc s ), l 0 - 1 5 (w h e a t a n drogenic cultures) or l0 (l,,lic'otianu)pieces of
i n o cu l a on it wer c s e a l e dw i th c o mme rc i a lp o l y e th yl e n c c ling f ilm . E a c h e x p c ri me n t r,v z rs
re p l i ca te cl 3 t o 13 t ime s . T h e c u l tu re s . e x c c p t
l Vi co ti ana( s ee abov e ), w e re i n c u b a te d a t 2 5 ' C
( l (X)0l x i 16 h/ day ) an d w e re c v a l u a te dfo r s h o o r
re g cn e r at ion af t er 5 w e e k s fo r w h e a t a n d tri ti ca l e . and af t c r 1 w e e k s fo r B . n o p u s . T h e
ro o ti n g r es pons e of w h e a t a n d tri ti c a l e w a s
d e tcrm inc d in t er m s o f d ry ro o t w c i g h t. T h i s w a s
a cco mplis hedby r em o v i n g c a l l i a n d s h o o tsfro m
th e medium . m c lt ing th e a g a r i n a m i c ro w a v e
o ve n , a nd was hing t h e ro o ts i n ru n n i n g w i l rm
w a te r p r ior t o dr y ing a t 7 0 ' C fo r 5 h .
Th c v iabilit y of r e g c n e ra te d w h c a t p l a n tl e ts
wzrs te st ed in t he gre e n h o u s eb y tra n s p l a n ti n g
th cm wit h wholc s ho o ts a n d ro o ts d i re c tl y fro m
th c Pctr i dis hc st o gr e e n h o u s ep o tti n g s o i l a t th e
e n d o f t he c ult ur c peri o d .
1 -h e c x per im ent alu n i t w a s c o n s i d e re dto b e a
Pe tri dis h. T o im pr o v c th e n o rma l i ty o f d a ta ,
tra n sfo r m at ionswer e u s e d a s s u g g e s te db y L i ttl e
& H i l l s ( 1972) . A ll t h e p e rc e n ta g ev a l u e s a n d
sh o o t num ber dat a pe r d i s h e sw e re tra n s fo rme d
b y a rcs in. t he dr y r oo t w e i g h t d a ta w e re tra n s fo rmccl logar it hm ic al l y ,e x c e p t th o s e i n T a b l e I
w h cre s quar e r oot t r a n s fo rm a ti o nw a s a p p l i e d .
A nal ysi s of vari arncc and D uncan' s mul ti pl e
rangc test (Little & Hills 1972) were performed
(at p :5V c, l evcl of si gni fi cance)for the experi ments. w hi ch w ere dcsi gnedas randomi zedcompl ete bl ocks.
Results
The effec'tof Cu on wheat and triticale somatic
cullus cultures
In w heat somati c cal l i . C uS O. dramati cal l y i ncreasedshoot rcgenerati onover a w i de rangc of
c o n c c n t r a t i o n( T a b l c 1 ) . O n M S m e d i u m l a c k i n g
C uS O* . the rcgenerati on frequcncy w as onl y
13% , w hcrcerson MS mcdi um contai ni ng the
ori gi nal concentrati onof C uS O, (0.1 pM) 35tl a
of thc cal l i produced shoots. A more than tw ofol d further i ncreaseoccurred at hi gher C uS O.
c o n c c n t r a t i o n (s1 - 1 0 0 p M ) A t t h e o p t i m a l c o n centrati on of l 0 pM C uS O.,. B 6V c of the cal l i
produced shoots. Therc w as al so a l arge change
i n thc number of shoots/regenerati ngcal l us.
Thus. a thrcc-fol d further i ncreasew as achi cvcd
by usi ng C u. On C uS O* -free medi um, the mean
shoot number/regenerati ngcal l us w as onl y 1.1.
w hi l e at thc opti mal C uS O., conccntrati on
( 10 pM) 3.6 shoots/regenerati ngcal l us w ere
produced. C uS O* al so sti mul atedroot formati on
from w heat cal l us. The root l cngth, number of
roots per cal l us, and the total dry root mass
T u b l c l - E f f e c t o f C u S O , a n d A g N O . o n t h c r e g e n c r a t i o no f w h c a t ( C I M M Y T N o . . 1 - 5a) n c l t r i t i c a l c ( N S 3 T l 2 ) s o m a r i cc a l l u s
c u l t u r e s . C l u S O . ,a n d A g N O . w e r c a p p l i c c l i n h u r m o n c - a n d C u S O . , - f r c cM S m e d i u m .
('or.rccntration
(pM )
(% ) Cialli
producing shoots
Mean shoot no. pcr
r c g e n e r a t i n gc a l l u s
Drv root weight
pcr dish (mg)'
CUSO.
AgNO,
Whcat
Triticllc
Wheat
Triticale
Wheat
Triticale
0
0.1
I
t0
100
0
o
U
U
0
0
o
I
I0
1(X)
l3Dr
3-5C
U;lA
86A
83A
7 3A B
71AB
fi) BC
-52D
7 9A B C
9 0A B
92A
79 AI]C
6tt c
7I]BC
70c
l.1c
2.,1B
3 . 3A
3 . 6A
3 . 1A
2.28
1 . 8B
1 . 9B
1 . 1C
2 . 7A
2 . . sA
2 . 3A B
2 . 08 C
2 . 0B C I
1 . 8c
1 . 7c l
30c
628
t 2 1A
l3t3A
1.12A
2tc
23C
l.l D
3.1C
62.B
8.1A
86A
9.1A
33C
33 Cl
12D
t)
F-tcst
:i
'All
i n o c u l i r e g c n e r a t c crlo o t s
tMcans
w i t h i n c o l u m n sf o l l o w c c lb v t h c s a m e c a p i t t t l l c t t e r a r c n o t s i g n i f i c a n t l vc l i f f c r c n t a c c o r d i n st o D u n c a n ' s m u l t i p l e r a n g c t c s t
" ' S i g n i f i c a natt l e a s ta t p : 5 1 1
134
i n cre a s ed wit h in c re a s i n g c o n c e n tra ti o n o f
Cu SO *. A s c om par c d w i th th e C u S O * -fre c c o n t r o l , t h e 0 . 1 p M t r e a t m e n td o u b l e d a n d t h e 1 1 0 0 p M CuS O * c onc e n tra ti o n sre s u l te di n a fo u rto five-fold increase in dry root weight.
Strong enhanc ing c ffe c ts o f C u S O * o n s h o o t
and root production were also observed in other
w h e at c ult iv ar s ( F ig . 1 ). S u p p l e me n ti n gC u SO* frcc M S m edium wi th 1 to 1 0 0p M Ag N O ., a l s o
significantly improved shoot regeneration in
wh e at and t r it ic ale (T a b l e 1 ) b u t to a l e s s e r
cxtcnt t han CuS O * . T h e ma i n d i ffe re n c e b c tw e e n t he ef f ec t s o f C u SO* a n d A g N O. w a s
th a t, w her easCuS O . s tro n g l y i n c re a s e dth e ro o t
p ro d u c t ion at all i n v e s ti g a te d c o n c e n tra ti o n s,
AgNO. significantly reduced rooting.
Fo r y ounger whe a t c a l l i w h i c h w e rc m a i n I
2 ,4 -u -c o n ta i n i ng
ta i n e c l only onc e on 0 .5 m g I
me d i um , t he r egen e ra ti o no n h o rm o n e -fre eMS
mcd i u m wit h t he o ri g i n a l C u S O * c o n c e n tra ti o n
wa s h igher ( in t he C IMMY T N o . 4 5 l i n e g e n e ra l ly 50-60% of these calli produced shoots) than
for cal l i mai ntai nedtw i ce on thi s medi um. In thi s
caseA gN O., i ncreasedthe shoot number si mi l arl y to C uS O* (data not show n).
C u S O , ( 0 . 1 - 1 0 0p M ) a l s oe n h a n c e ds h o o t a n d
root regenerati oni n the tri ti cal e cal l us cul tures
(Tabl c l ). H ow ever. i n contrast w i th w heat,
tri ti cal e cal l i (previ ousl y mai ntai ned onl y once
'
2,4-o) show cd a
on MS medi um w i th 0.5 mg I
much hi gher regcnerati on frequency (52o/o)on
hormone-freeMS medi um l acki ng C uS O* . The
number of shoots per regenerati ngcal l us w as
l ow er for tri ti cal e than for w heat at hi gh (l 100pM) C uS O, concentrati ons.In addi ti on, thc
effect of 1 pM CuSO., did not differ significantly
(p:5% lcvel) from that of 0.1 pM. ln triticalc,
C uS O* agai n sti mul ated root formati on, al though the differcnccs between the effects of
C uS O* i n the 0-1 pM concentrati onrangc w ere
not so largc as for wheat. The cffects of AgNO.
on shoot and root regcncrati onw ere qui te si mi l ar for tri ti cal e to those for w heat (Tabl e 1).
C uS O" (0.1-10 pM) enhanced the shoot arrd
F . i g . 1 . E f f e c t o f C u S O , o n t h e shoot (A) and root (B) regeneration of wheat ( S i e t e C e r r o s c u l t i v a r ) c a l l i . C u S O , w a s a p p l i e d i n
hormone- and CuSO,-free MS medium.
135
root regeneration of wheat in all media tested
(MS , N 6, 85 and S H ). A t equal concentrati ons
of C uS O* , thc components of the basal medi a
had onl y modi fyi ng effects (Fi g. 2). For shoor
production, thc analysis of variance (data not
shown) showed significant differences among
C uS Or treatments(p :0.5o/o l evel ). A nal ysi s of
variance for dry root weight showed that the
CuSO* x media interaction was significant (data
not show n). In addi ti on, among the medi a tested, the N6 saltsappearedto be the best cofactors
of copper for both shooting and rooting. In this
experi ment, smal l cr C uS O+ concentrati onsteps
wcre used and it seemed that the optimal concentration for shooting was between 1 and
10 pM. The hi ghest shoot producti on w as obt a i n c d a t 2 p M ( 0 . 5m g l r . ; i n e a c h c a s e . b u t
hi gher C uS O* concentrati on(10 pM) gave hi gher increase in root formation. The original
CuSOr concentrations in the different basal
media (MS and 85: 0.1pM; N6: 0 pM; SH:
t).8 pM), exceptthat i n the S H medi um, w ere far
bel ow the opti ma w hi ch w ere determi ned i n our
e x p e r i m e n t s( 2 - 1 0 p M ) .
!0
.-{
-l
,. 20
0.)
H
tr
r.J
x
-1
c
rr 10
.)
0.1 0.8
1
2
CUSO4 (uM)
l
Ai
l.+u
Fn
120
ul
lr\r\
The effect of Cu on wheat androgenic callus
cultures
| \./\J
rr1
!
OJ
p
BO
]J
u'
OU
qJ
+)
a)
40
.)
l-t
l-.1
20
o
0
0,1
1
C u S o 4( u M )
10
B
Fig.2. Effect of CuSO, on the shoot (,4) and root (B)
r e g e n c r a t i o n o f w h e a r ( C I M M Y T N o . 4 - 5 )c a l l u s c u l t u r e s i n
d i f f c r e n t b a s a lm e d i a M S ( U ) . N 6 ( I ) . B - s( ) a n d S H ( x )
f r e c o f h o r m o n e sa n d C u S O , .
In w heat embryoi ds i nduced i n anther cul turc,
CuSO* was also important for regeneration
(Tabl e 2). W hen hormone-free regenerati on
medi a w ere used, 0.1 pM C uS O* resul ted i n
significant increasesin shoot and root formation
as compared with the Cu-free control. Some
androgeni ccal l i of Mv 16 and Mv 15 cul ti vars,
I8c/o and 5ah on average, respectively, did not
form roots and shoot-forming calli showed a
strongerrooti ng acti vi ty (data not show n). In Mv
16 and Mv l 5 cul ti vars.the shoot formati on w as
l.l and L2 times higher, respectively,on
medi um contai ni ng 0.1 pM C uS O* than on a
CuSO-,-free one. The root formation was doubled for both cultivars. At a higher concentration
of C uS O* (2 pM), sl i ght further i ncreasesi n
shoot and root production were detected. With
h o r m o n e ( 0 . 5m g I I N A A + 0 . 5 m g l ' t c N ) contai ni ng I strength MS medi um (w i th 0.1 p" M
C uS O* ), w hi ch i s commonl y appl i ed for regeneration, the shoot and root formation was gener-
136
a l l y l owc r t han t ha t i n h o rmo n e -fre em e d i u m a t
the same (0.1pM) or higher (2 pM) CuSO*
co n tent . T he pr opo rti o n o f a l b i n o s h o o tsd i d n o t
ch a n gc s ignif ic ant l ya s a c o n s e q u e n c eo f C u o r
h o r m o n e t r e a t m e n t( T a b l e 2 ) .
The effect of Cu on the viability of regenerated
wheat plunts
Tra ns f er r ing t he re g e n e ra te dp l a n ts to p o tti n g
soil. significant differenccs wcrc observed in
p l a n t s ur v iv al as a c o n s e q u e n c eo f th e d i ffe re n t
Cu pr c t r eat m ent s( T a b l e 3 ). P l a n ts rc g c n c ra ted
fro m s om at ic c allu s c u l tu re s o f th c C IM M YT
No . 45 linc on C u S O * -fre e o r n o rm a l MS
me d ium did not s u rv i v c th e tra n s fe r.w h i l e th o s e
d e ve loped on M S m e d i u m c o n ta i n i n g 1 p M
C u SO * s ur v iv c d. Pl a n ts re g e n e ra te dfro m a nd ro g c nic em br y oid s o f th e M v 1 5 c u l ti v a r o n
h o rm one- f r ee . L M S me d i u m c o n ta i n i n g 2 p M
C u SO r wer c m os t v i g o ro u s a n d h a d th e h i g h e st
s u r v i v a l r a t e ( 1 0 0 c / c ) .C u S O , a t 0 . 1 p M a l s c - r
rcsult ed in a hig h s u rv i v a l ra te (7 3 % ). T h e
poorest survival rate (20c/r,
) was observed in
p l a nt s dc r iv ed f r om th e C u -frc c c o n tro l .
T a h l e - 1. E f f e c t o f C u S O . , p r c t r e a t m c n t s o f c u l t u r e s o n t h e
survival of rcgcnerated wheat plants aftr-r potting t() grccn
house.
CluSO.,
prctrcatmcnts
(pM)
Number of plants survived/transplantccl
R c g e n e r a t e df r o m
callus culturc of
CIMMYT No. .15
linc
Rcgcnerated from
anther culture of
Mv 15 cultivar
(J
0 /l 0
0,26
30/30
21l(',l
1 1 /1 - 5
0.1
I
)
15/ 1-5
The eJfect of Cu on Brassica callus cultures
In Brassica napus on MS mcdium with the
o r i g i n a l C u S O * c o n c e n t r a t i o n( U . 1 p M ) , s h o o t
rcgcnerati on w as l essthan l )c/c,.In contrastw i th
w heat and tri ti cal c, hi gher C uS O* conccntrati ons
di d not i ncrease shoot rcgenerati on. A t the
hi ghest concentrati on( 100pM) shoot regenerati on w as compl ctcl y i nhi bi ted and the percentagc
of root-produci ngcal l i w as decreased(Tabl e ,l ).
A gN O. si gni fi cantl y i ncreased thc number of
7 ' u b l e2 . E f f c c t o f C i u S C ) .o n t h c s h o o t a n d r o o t p r c d u c t i o n i n a n t h e r - d e r i v e dc a l l i o f w h c a t c u l t i v a r s M r , 1 - 5a n c l M v l 6 u s i n g
hormonc-free or hurmone-containing I strength M S m e d i u m ' .
Cultivar
Supplcmcntation
CuSO,
(pM)
Mv 16
Mv 15
0
0.I
2
0.t
2
F-tcst
0
0.1
2
0.I
2
F-tcst
NAA
( m gI ' )
KN
0
(,
(J
0
0.-5
0.-5
U
0
0..5
0.-5
0
(,
(,
(l
0.-5
0..5
0
0
0.5
0.-5
(?r) Calli
producn
i g
shoclts
Mean shoot
no. per
shoot
forming
calli
(?i)Albino
shootsl
( 1 7) C a l l i
producing
roots
Drv root
w ei g h t p e r
c l i s h( m g )
3 9c '
6.5Ats
7tA
.56B
6 1B
I . 6A B
1 . 7A
t . 7A
1 . 4B
1 . .B
+
20
8.5B
9 1A
92A.
69C
19D
3 6A B
.13A
24C
3 1B C
118
58B
72A'
60B
.56B
I . 8A B
2 . 1A
2 . 1A
t . 1B
t.] B
93
90
99
97
92
NS
:t-5D
82B
9-sA
70 I]C
68C
-)l
-) 1,
-)+
36
NS
12
t2
l1
tt
ll
NS
r'
'Exprcssed
as pcrccntagcof shoot (green and albino) number per treatment
tMeans
w i t h i n e a c h c o l u m n f o l l o w e d b y t h e s a m e c a p i t a l l e t t e r a r c n o t s i g n i f i c a n t l yd i f f e r e n t a c c o r c l i n gt o D u n c a n ' s m u l t i p l c r a n g e
tcst
or NS: not signihcant
" ' S i g n i f i c a nat t l c a s t a I p : 5 % .
r37
Toble 1. Effcct of CuSO., and A g N O . o n t h e s h o o t a n d r o o t
f t r r m r r l i t r n o l B r t t . s . s i t 'nua p t t s c v . O l g a c a l l u s c u l t u r e o n
htlrnrone(|-Imul
N A A * B A ) - c o n t a i n i n qM S m c d i u m .
C o n c c n t r a t i o n( p M )
CuSO,
0.1
I
I0
1(X)
0.1
0.I
F-tcst
AgNO.
(,
0
0
0
10
100
(%) Calli
producing
shoots
( ? Z) C a l l i
producing
r()ots
78,
3B
8B
0B
60A
68A
2 1B C
2 8B C
2:l BCI
13C
12A
,13B
80
70
(n
..t
lC
60
r.l-.{
d
qJ
D//
f'
0.)
o+o
f{
OJ
E30
-t
'Means
within each columns followcd b,v the same capital
letter are not signilicantlv different according to Duncan's
multiplc rangc test
* S i s n i f i c a n ta t p : 5 c 1 ,
!20
d
'.10
0.1
regcnerating calli on MS medium from 8o/oto a
ma xi mum of 68% ( a t 1 0 0p M Ag N O .). H o w e ve r. i n B . napus as c o mp a re d w i th w h e a t a n d
tri ti ca l e . only f ew c a l l i p ro d u c e d ro o ts . D u ri n g
treatments. the percentage of root-forming calli
in Brassica followed a trend similar to that of
sh o o t r egener at ion. O n me d i a c o n ta i n i n g
AgNO., more than 90a/oof B. napus calli were
g re e n , whilc in ot her tre a tme n tsth e p ro p o rti o n
wa rsb e low 70o/ c .
The effect of Cu on 1,,1.tabacum leaf disc
cultures
C u SOr t r eat m ent inc r e a s e dth e s h o o t n u m b e r i n
l V. ta b ac um leaf dis c c u l tu re s (F i g . 3 ). O w i n g to
the auxin-free and high cytokinin-containing
medium. there was neither callus nor root formati o n i n t his c ult ur e. I n th e I s tre n g thMS c o n tro l
(0.1 pM CuSO*), on uuerage+S shoots per
i n o cu l um wer e f or m e d . C u SO. fro m 1 0 to
100 pM significantly increased shoot regenerati o n , wher eashigher c o n c e n tra ti o n s(3 0 0 p M a n d
1000pM) significantly decreased it (F-test was
si g n i fi c antat p : 0. 1o /c ).A s c o mp a rc d w i th th a t
o f t h e I s t r e n g t hM S c o n t r o l ( 0 . 1 p M C u S O r ) ,
taken as I00o/a, the highest shoot regeneration
wa s 1 4 8% ( at 100pM C u S O * ) a n d th e l o w e s t
wa s 5 o /c( at 1000pM C u SO,,).
10
30
100 300
1000
C U S O o( u 1 " 1 )
Fig. 3. Effect of CuSO., on thc shoot rcgcncration of
'
llicotianu tabacunt lcaf clisc culture on hormone (2 mg I
B A ) - c o n t a i n i n g M S m e c l i u m ( t r c a t m c n t s f o l l o w c d b - v -t'h e
s a m e c a p i t a l l e t t e r a r e n o t s i g n i f i c a n t l vd i f f e r c n t a c c o r d i n g t o
Duncan's multiple rangc tcst).
Discussion
The present study demonstrates that Cu ions
effectively enhance shoot and root regeneration
i n w heat and tri ti cal c cal l us cul tures. and shoot
regeneration in l/. tabacum leaf disc culture.
Experiments with several other wheat cultivars
and with different basal media also revealed the
highly reproducible effect of CuSOr.
The compositions of the macro and micro
el ements i n most of the standard medi a w ere
developed by evaluating the effects of different
mi neral s on the cal l us grow th of certai n model
speci es.U nti l now . i n most casesthe rol e of C u
has not been investigatedor it has not proved to
be significant. In Murashige and Skoog's experiment, for exampl e, C u addcd i n thc range from
0.03 to 30 pM was without effect on the growth
of tobacco tissue, but Cu was added (at random)
at a concentrati on of 0.1 pM because i t i s
"known to be an essential element for plant
grow th" . Later. thi s C u concentrati on w as
138
a d o pt c d f or s ev er a lo th e r c u l tu re m e d i a s u c h a s
B . ( G a m b o r g e t a l . 1 9 6 8 ) .A c c o r d i n gt o S c h c n k
& Hildc br andt ( 19 7 2 ). th e C u i o n a p p c a rc d
sti mulat or y at a hi g h e r l e v e l fo r c a l l u s g ro w th
a n d so t hc y us c d 0. 8 p M i n th e S H me d i u m . C h u
e t a l. ( 197- 5)did n o t m e n ti o n th e c ffe c t o f C u
a n d t hey did not u s e i t i n th e N 6 me d i u m
d e vc loped f or r ic e a n th e r c u l tu re . So me o th e r
m o r e r a r e l y u s e dm e d i a ( c . g . L i t v a y e t a l . 1 9 8 5 )
co n ta in a higher am o u n t o f C u b u t th c s p e c i fi c
e ffe c t of Cu on mo rp h o g e n e s i sh a s n o t b e e n
d cscr ibed. I n ou r e x p c ri me n ts , w e fo u n d
g cn o t y pic dif f c r c nc e s i n th e mo rp h o g e n i c re sp o n s e of t is s uc c u l tu rc s to C u tre a tmc n ts . In
wh e ar ts om at ic c ultu re . fo r e x a m p l e , th e u s e o f
Cu rc s ult ed in a s tro n g s ti m u l a ti o n i n rc g e n e ra t i o n . b u t i n B . n a p u st h e t r e a t m e n t( 0 . 1 - 1 ( X p
)M
Cu SO *) was inef f e c ti v co r i n h i b i to ry .
It has bec om c e v i d c n t th a t s e v e ra l h e a v y
mctals as m ic r o ele me n tsp l a y i mp o rta n t ro l c s i rr
th e re gener at ionof p l a n t ti s s u ec u l tu re s .Bc s i d e s
Cu a nd A g ot her he a v y m e ta l ss u c h a s C o a n c 'N
l i
(R o u s t an et al. 1 9 ti 9 ) a l s o s ti m u l a tc m o rp h o g c nes is .T hc r e s u l ts w i th Ag N O ., i n T . a e sti vu m and B . nopu s i n th e p rc s e n t s tu d v . a rc i n
a rccor danc ewit h th c p re v i o u s re p o rts . Si n c c
e t h y l e n ei n h i b i t o r so t h e r t h a n h e a v y m e t a l s .c . g .
a mi n oc t hox y v iny lg l y c i n e .a l s o p ro mo te re g e n e ra rti on ( S ongs t adet a l . l 9 B 9 ) a n d e rh y l c n e d e cre a s c sit ( P ur nhau s e re t a l . 1 9 8 7 ,So n g s ta de t a l .
l 9 BB ) . it is lik ely t h a t Ag c n h a n c e sre g e n c ra ri o n
th ro u gh c t hy lene in h i b i ti o n . H o w e v c r. C u i o n
h a s n ot been r egard e d a s a n e th y l e n e i n h i b i to r.
a n d thus it s ef f ec t si n w h e a t. tri ti c a l e a n d to b a cco c ould hav c an o th e r e x p l a n a ti o n . C u t' i s
kn o wn t o be a c omp o n c n t o r a c ti v a to r o f m a n y
i mp or t ant enz y m es i n v o l v e d i n c l e c tro n tra n s p o rt, pr ot c in and c a rb o h y d ra te b i o s y n th e s i s,
p o l yp henol m et abo l i s ma n d s o o n ; i t i s th e re fo re
p o ssi blc t hat s om c C u e n z y me s mi g h t p l a y a n
i mp or t ant r ole in pl a n t re g e n e ra ti o n .T h e i mp o rtance of enzymes is indicated by the fact that in
ti ssuec ult ur esc lf m a i z e (Ev e re tt e t a l . 1 9 8 5 )a n d
b a rl ey ( Coppens & D e w i tte 1 9 9 0 ) th e z y mo grams of certain enzymes are useful as markcrs
of morphogenesis.
To opt im iz e t he n u tri e n t re q u i re me n to f ti s s u e
cu l tur es in dif f er en t s p e c i e s ,i t s e e ms th a t th e
mi n e ral c om pos it i o n , e s p e c i a l l y o f m i c ro e l c -
ments. shoul d be cxami ned to achi eve maxi mal
regenerati on.
Acknowledgement
Thi s w ork w as supported i n part by the H ungari an N ati onal S ci enceFund (OTK A ) grant no.
6102.
References
Chraibi KM. Latchc A. Roustan JP & Fallot J (1991)
Stimulation of shoot rcgcncration frclm cotvledons of
H e l i u r t t l t u so n n t t s b v c t h v l c n c i n h i b i t c l r s .s i l v e r a n d c o b a l t .
Plant Ccll Rcp. l0: 201 207
C o p p e n s L & D e r . v i t t c D ( 1 9 9 0 ) E , s t e r a s ea n d p e r o x i d a s e
z v r " n o g r a m fsr o m b a r l c v ( H o r d c u n t v u l g u r e L . ) c a l l u s a s a
b i o c h e r n i c a l m a r k e r s y ' s t c m o f em b r v o g e n e s i s a n d o r { a n o g e n e s i sP
. lant Sci. 67: 97-10-5
I ) e B l o c k M . D e B n r u w e r D & T e n n i g P ( 1 9 8 9 )T r a n s f o r m a ticrn of Brussi<'unupus and l}rc.r.ricu olernceu using .4grobucteriunt tune.fhcien.rancl the expression of the bar and
n ( o g c n e s i n t h c t r a n s g e n i cp l a n t s . P l a n t P h y s i o l . 9 I :
69.1-701
E'n,eretN
t P . W a c h N I . l & A s h w o r t h D J ( 1 9 8 - 5 )B i o c h c m i c a r l
m a r k c r s o f c m b r v o q L - n c s i sn t i s s u c c u l t u r e s o f t h c m a i z e
i n b r c d B 7 3 . P l a n t S c i . , 1 1 :1 3 3 - 1 - 1 0
F e l f o l d i K & P L r r n h a u s eLr ( 1 9 9 2 ) I n d u c t i o n o f r e g e n e r e r t i n e
callus culturcs frorn immature embrvos of ,l,l wheat and 3
t r i t i c a l c c u l t i v a r s .C c r e : r l R e s . C o r n m . 2 0 : 2 7 3 - 2 1 1
Gambors OL. Miller RA & O jima K ( 1968) Nutrient
r e q u i r e m e n t so f s u s p e n s i o nc u l t u r c s o f s o y b e a n r o o t c c l l s .
E x p . C - ' c l lR
. e s . - 5 0 :l - 5 1 - 1 - 5 8
G a r c i a - S o g oB . R o i g t - A & M o r c n o V ( 1 9 9 1 ) E , n h a n c e m c n t
o f m o r p h o e c n c t i c r e s p o n s e i n c o t y l e d c l n - d e r i v c dc x p l a n t s
t:t (lucuntis melo induced by copper ions. Acta Hortic.
2E9: 229-230
L i t t l e T M & H i l l s F J ( 1 9 7 2 ) :S t a t i s t i c am
l e t h o d si n a s r i c u l t u r a l r c s e a r c h .U n i v e r s i t v o f C a l i f o r n i a . D a v i s . C a l i f o r n i a .
212 p.
L i t v a y J D . V c r m a D C & J o h n s o n M A ( 1 9 8 5 ) I n f l u e n c eo f
loblolly pine (Pinu.stucleu L.) culturc mcdiums and its
c o m p o n e n t s o n g r o w t h a n d s o m a t i c e m b r y o g e n e s i so f w i l d
c a r r o t ( D u u c ' u sc u r o t t u L . ) . P l a n t C e l l R e p . , t : 3 2 , 5 - 3 2 8
Maliga P. Brcznovits A & Marton L (1973) Streptomicyn
resistant plants from callus culturc of haploid tobacco.
Naturc 221:29-30
M a r c o t r i g i a n o M ( 1 9 6 8 ) O r i - q i no f a d v c n t i t i o u s s h o o t s r e g e n e r a t c d f r o m c u l t u r e d t o b a c c o l e a f t i s s u e. A m c r . J . B o t . 7 3 :
1,15I - l.l-57
Murashise T & Skoog F (1962) A rcviscd medium for rapid
g r o r v t h a n d b i o a s s a y sw i t h t o b a c c o t i s s u e c u l t u r e s . P h v s i o l .
Plant. 15: 113-497
Pauk J. Manninen O. Mattila YS & Pulli S (1991) An-
139
droscnesis in hexaploid spring whcat F. populations and
thcir parcnts using a multiple-step regencratitln svstcm.
Plant Bree. 101:18-27
P u r n h a u s c rL ( 1 9 9 1 ) S t i m u l a t i o no f s h o o t a n d r o o t r e g e n e r a t i o n i n w h c a t ( ' l - r i t i c u mo e s t i v u n t )c a l l u s c u l t u r e sb y c o p p e r .
C l e r e a lR c s . C o m m . 1 9 : 4 1 9 4 2 3
PurnhauserL. Mcdgyesy P. Czako M. Dix PJ & Marton L
( 1 9 8 7 ) S t i r n u l a t i o n o f s h o o t r e g e n e r a t i o ni n ' l ' r i t i c u n t o e s t i v u m a , n d l l i < : o t i u t t up l u n f u a g i n i J ' o l i uY i t . t i s s u e c u l t u r e s
using the ethvleneinhibitor AeNO.. Plant Cell Rep. 6:
1.1
R o u s t a n J P . L a t c h c A & F a l l o t J ( 1 9 t 3 9 )S t i m u l a t i o n o f
Duuc'tts curote sclm:rtic embrvogenesis by' inhibitors of
c t h v l e n c s y n t h e s i s :c o b a l t a n d n i c k e l . P l a n t C e l l R e p . 8 :
182-I u.5
Schcnk RU & Hildcbrandt AC (1972) Medium and techn i q u e s f o r i n d u c t i o n a n d s r o w t h o f m o n o c o t v l e c l o n o u sa n c l
d i c o t y l e d o n o u sp l a n t c e l l c u l t u r e s .C a n . J . B o t . 5 0 : 1 9 9 201
S e t h i U . B a s u A & G u h a - M u k h e r j e eS ( 1 9 9 0 )C o n t r o l o f c el l
p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n b y m o c l u l a t o r so f e t h y l e n e
b i o s y n t h e s i s a n d a c t i o n i n f J r u s s i c uh y p o c o t v l e x p l a n t s .
Plant Sci. 69:225-229
S c a r s R G & D e c k a r c l L E ( 1 9 8 2 ) T i s s u c c u l t u r e v a r i a b i l i t , vi n
r v h e a t : C a l l u s i n d u c t i o n a n d p l a n t r c g e n e r a t i o n .C r o p S c i .
22: -5'16-5-50
S o n g s t a dD D . D u n c a n D R & W i l d h o l m J M ( l g t t l J ) E f f e c t o f
l - a m i n o c v c l o p r o p a n e - 1 - c a r b o r l 'al icci d . s i l v e r n i t r a t c . a n d
norbornadicnc on plant regcncrution frttm maize callus
cultures. Plant Clcll Rep. 7: 262-265
S o n g s t a dD D , P e t r a c e kP D . S a m s C E & C o n g e r I I V ( 1 9 8 9 )
E f f e c t o f 1 - a m i n o c v c l o p r o p a n-el - c a r b o x v l i c a c i d a n d
aminocthox,vvinylglycineon cthylenc cmanaticttt and
s o m a t i c e m b r v o g e n e s i sf r o m o r c h a r d g r a s s l e a f c u l t u r e s .
Plant Ccll Rcp. 8: 677-619