Page 1
REVIEW
The role of thin cell layers in regeneration and transformationin orchids
Jaime A. Teixeira da Silva
Received: 2 October 2012 / Accepted: 3 December 2012 / Published online: 21 December 2012
� Springer Science+Business Media Dordrecht 2012
Abstract Thin cell layers (TCLs) offer a simple yet
effective protocol that has contributed to major advances in
clonal micropropagation of orchids. TLCs have been suc-
cessfully used for protocorm-like body (PLB) and callus
induction in Aranda, Coelogyne cristata, Cymbidium spp.,
Dendrobium spp., Doritaenopsis, Paphiopedilum, Renan-
thera, Rhynchostylis, Spathoglottis, and Xenikophyton.
TCLs have also been a bulwark for genetic transformation
studies of select genera. This review takes an in-depth look
at how TCLs have been employed in orchid biotechnology
and provides in-depth protocols that will allow for the
generation of PLBs using TCLs. As PLBs in orchids are
deemed somatic embryos, these will be useful for large-
scale mass propagation in bioreactors or for long-term
storage as synthetic seeds.
Keywords Cymbidium � Orchids � Protocorm-like bodies �Thin cell layer
Introduction: what is a TCL?
The concept of thin cell layers (TCLs) was proposed
almost 40 years ago with ground-breaking work that
demonstrated that by excising thin and transverse slices of
tissues from pedicels of flowering Nicotiana tabacum
(tobacco), various tissues such as flowers, vegetative buds,
and roots can be induced via manipulation of the in vitro
milieu (Tranh Than Van 1973). By then, a substantial
amount of work had been done on the in vitro culture of
tobacco, including the fundamental study by Murashige
and Skoog (1962) that eventually led to the establishment
of a basal medium that would prove to be the most com-
monly used medium in plant tissue culture, although not
for orchids. In Tranh Than Van’s study, a 1 mm-thick layer
of cells epidermal peels (with variable area dimensions)
should be defined as a longitudinal TCL or lTCL, while a
transverse slice, a few mm thick, should be termed a
transverse TCL or tTCL. Certainly it was not the capacity
to culture tobacco tissue under sterile conditions, nor was it
the possibility to culture plant cells in vitro to generate a
whole plant—the original concept of totipotentiality (toti-
potency) which Haberlandt himself conceptualized almost
75 years earlier—that was revolutionary about TCLs.
Rather, it was the capacity to more strictly control the
outcome of an organogenic ‘‘programme’’, not so much by
the contents and additives of the medium or the sur-
rounding environment, but rather by the size of the explant
itself, that captivated the minds and attention of plant tissue
culture scientists since 1973. In the 40 years that have
ensued, TCLs have shown to be veritable tools in the
controlled organogenic potential of almost every group of
plants with now at least 130 papers having successfully
been published using TCL technology (Nhut et al. 2003a,
b; Teixeira da Silva et al. 2007a, b).
This mini-review will focus on the use of TCLs for
in vitro culture and micropropagation of orchids (Table 1).
Orchids were once considered to be particularly difficult-
to-propagate plants in vitro, but TCL technology has been
one method that has advanced their tissue culture, making
mass clonal propagation easier and more reproducible.
Moreover, the micropropagation of these valuable orna-
mental and cut-flower pot plants is possible without the
need for expensive labour and technology such as
J. A. Teixeira da Silva (&)
Faculty of Agriculture and Graduate School of Agriculture,
Kagawa University, Miki-cho, Ikenobe 2393,
Kagawa-ken 761-0795, Japan
e-mail: [email protected]
123
Plant Cell Tiss Organ Cult (2013) 113:149–161
DOI 10.1007/s11240-012-0274-y
Page 2
Ta
ble
1T
hin
cell
lay
ers
(TC
Ls)
inin
du
cin
go
rgan
og
enes
iso
rem
bry
og
enes
isin
orc
hid
s
Spec
ies
Org
anfr
om
whic
hT
CL
was
der
ived
Cult
ure
med
ium
,P
GR
san
dad
dit
ives
Type
of
resp
onse
Rem
arks
on
org
anogen
icre
sponse
invit
roR
efer
ence
s
Ara
nda
Deb
ora
hS
hoot
tips
MS
,K
C,
VW
wit
hB
A(0
–8.8
lM
),N
AA
(0.0
–5.5
lM
),A
C
(0.5
g/l
),C
W(5
–25
%).
Em
bry
ogen
esis
Max
imum
num
ber
of
PL
Bs
(13.6
/expla
nt)
pro
duce
din
VW
wit
h
20
%(v
/v)
CW
.P
LB
spro
fuse
lypro
life
rate
din
2.7
5N
AA
wit
h20
%(v
/v)
CW
inV
W.
PL
Bs
dev
eloped
into
pla
ntl
ets
on
soli
dV
Wco
nta
inin
g10
%(v
/v)
CW
and
0.5
g/l
AC
.
Lak
shm
anan
etal
.(1
995)
Spath
oglo
ttis
pli
cata
Nodes
and
leav
es
MS
wit
h5.3
7m
MN
AA
and
0.4
4m
MB
A(f
or
PL
Bs)
;
2.6
9–10.7
4m
MN
AA
and
8.8
8m
MB
A(f
or
pla
ntl
et
dev
elopm
ent)
Org
anogen
esis
PL
Bs
dev
eloped
pla
ntl
ets
on
MS
wit
h0.4
4–8.8
8m
MN
AA
and
0.5
4–10.7
4m
MB
A.
How
ever
,th
epro
cess
was
slow
erw
hen
PL
Bs
wer
em
ainta
ined
inm
ediu
mth
atfa
vore
dP
LB
regen
erat
ion.
The
opti
mum
com
bin
atio
nfo
rP
LB
induct
ion,
NA
A/B
A5.3
7/0
.44
mM
,del
ayed
pla
ntl
etdev
elopm
ent
for
1–2
month
s.P
LB
squic
kly
dev
eloped
pla
ntl
ets
when
tran
sfer
red
tom
ediu
mco
nta
inin
g2.6
9–10.7
4m
MN
AA
and
8.8
8m
MB
A.
Up
to145
pla
ntl
ets
could
be
dev
eloped
from
0.2
gP
LB
sin
1m
onth
and
appro
xim
atel
y15–20
pla
ntl
ets
could
be
dev
eloped
from
PL
Bs
ina
single
nodal
TC
Lse
ctio
n.
PL
Bs
could
also
regen
erat
efr
om
root
sect
ions.
Ten
get
al.
(1996
)
Rhyn
chost
ylis
gig
ante
aS
tem
and
shoot
tips
MS
wit
h3
lM
BA
and
3l
MT
DZ
Org
anogen
esis
The
opti
mal
com
bin
atio
nfo
rm
axim
um
bud
regen
erat
ion
was
3
lM
BA
and
3l
MT
DZ
,giv
ing
rise
to11.7
buds/
tTC
L.
Roots
wer
eobta
ined
wit
h10
lM
forc
hlo
fenuro
n(C
PP
U)
and
1%
sucr
ose
.T
he
invit
ropla
nts
([3
cmlo
ng)
obta
ined
4–6
wee
ks
afte
rth
ecu
lture
of
tTC
Ls
wer
etr
ansf
erre
dto
the
gre
enhouse
;
thei
rm
orp
holo
gy
was
norm
al.
Le
etal
.(1
999)
Cym
bid
ium
alo
ifoli
um
(L.)
Sw
.an
dD
endro
biu
mnobil
eL
indl
PL
Bs
MS
med
ium
wit
hZ
R(1
4.0
lM
),B
A(1
1.0
lM
)an
dIB
A
(9.8
lM
)
Em
bry
ogen
esis
PL
Bs
wer
ein
duce
don
MS
med
ium
supple
men
ted
wit
hZ
Rat
14.0
mM
BA
for
C.
alo
ifoli
um
but
at11.0
mM
for
D.
nobil
e.T
he
aver
age
num
ber
of
PL
Bs/
TC
Sw
ashig
hin
both
case
s
(28.2
inC
.alo
ifoli
um
,34.0
inD
.nobil
e).C
yto
kin
ins
above
the
opti
mal
level
inhib
ited
PL
Bfo
rmat
ion.
PL
Bdev
elopm
ent
in
both
the
spec
ies
was
enhan
ced
by
the
use
of
susp
ensi
on
cult
ure
.A
hig
hfr
equen
cyof
shoots
(85
%in
C.
alo
ifoli
um
and
80
%in
D.
nobil
e)re
gen
erat
edfr
om
PL
Bs
could
root
on
MS
med
ium
conta
inin
g9.8
lM
IBA
.R
egen
erat
edpla
nts
wer
e
succ
essf
ull
yac
clim
atiz
edth
entr
ansf
erre
dto
the
fiel
d.
Nay
aket
al.
(2002
)
Hybri
dD
ori
taen
opsi
s(D
ori
tis
9P
hala
enopsi
s)N
ewca
ndy
9D
.(M
ary
Anes
9E
ver
spri
ng)
Lea
ves
MS
wit
h9.0
lM
TD
ZO
rgan
ogen
esis
TD
Zin
duce
dP
LB
sm
ore
effe
ctiv
ely
than
BA
or
Zea
.T
he
hig
hes
tper
centa
ge
of
PL
Bfo
rmat
ion
(72.3
%)
and
hig
hes
t
num
ber
of
PL
Bs
form
ed(1
8/e
xpla
nt)
wer
eobse
rved
on
thin
leaf
sect
ions
(1m
mth
ick),
whil
eonly
20
%(4
.3/e
xpla
nt)
of
larg
ele
afse
gm
ents
(5m
mth
ick)
wer
eab
leto
pro
duce
PL
Bs
under
the
sam
ecu
lture
condit
ions.
Inth
e2006
pap
er,
expla
nts
cult
ure
din
close
dves
sels
pro
duce
dm
ore
som
atic
embry
os
than
those
rear
edin
ven
tila
ted
ves
sels
.T
his
enhan
ced
form
atio
nco
nfi
rmed
the
gre
ater
involv
emen
tof
accu
mula
ted
ethyle
ne
under
non-v
enti
late
dco
ndit
ions,
bec
ause
wound-
induce
dti
ssues
from
thin
leaf
sect
ions
norm
ally
rele
ase
hig
h
level
of
ethyle
ne.
When
expla
nts
wer
ew
ashed
inli
quid
med
ium
and
inocu
late
don
the
sam
eso
lid
med
ium
,so
mat
ic
embry
opro
duct
ion
was
1.7
and
18.5
tim
eshig
her
than
inth
e
thin
sect
ion
cult
ure
san
dth
ick
segm
ent
cult
ure
s,re
spec
tivel
y.
Red
uci
ng
the
level
of
phen
oli
csin
expla
nts
atth
ein
itia
lst
age
of
cult
ure
appar
entl
yst
imula
ted
this
embry
o(i
.e.
PL
B)
regen
erat
ion.
Par
ket
al.
(2002,
2006
)
150 Plant Cell Tiss Organ Cult (2013) 113:149–161
123
Page 3
Ta
ble
1co
nti
nu
ed
Spec
ies
Org
anfr
om
whic
hT
CL
was
der
ived
Cult
ure
med
ium
,P
GR
san
dad
dit
ives
Type
of
resp
onse
Rem
arks
on
org
anogen
icre
sponse
invit
roR
efer
ence
s
Hybri
dC
ymbid
ium
cv.
Tw
ilig
ht
Mon
‘Day
light’
PL
Bs
VW
?var
ious
conce
ntr
atio
ns
of
NA
A,
KIN
,B
A,
and
mult
iple
med
iaad
dit
ives
,in
cludin
gC
W,
AC
Org
anogen
esis
/
Em
bry
ogen
esis
Lar
ge
var
iabil
ity
inre
sult
sdep
endin
gon
the
med
ium
and
gro
wth
condit
ions
and
expla
nt
use
d.
Lig
ht
mic
rosc
opy
and
scan
nin
g
elec
tron
mic
rosc
opy
use
dto
confi
rmdev
elopm
enta
lpro
cess
es
over
tim
ean
dth
atP
LB
sar
ein
fact
som
atic
embry
os
in
orc
hid
s,an
dfl
ow
cyto
met
ryto
confi
rmplo
idy
level
sover
the
dev
elopm
enta
lper
iod
and
RA
PD
anal
yse
sto
confi
rmgen
etic
stab
ilit
y.
Tei
xei
rada
Sil
va
ori
gin
ally
coin
edth
ete
rms
pri
mar
y
(1�)
and
seco
ndar
y(2
�)P
LB
s.
Tei
xei
rada
Sil
va
etal
.(2
005
,
2006a,
b,
2007a,
b),
Tei
xei
ra
da
Sil
va
and
Tan
aka
(2006),
Tei
xei
rada
Sil
va
(2012a,
b)
Den
dro
biu
mca
ndid
um
Wal
l
exL
indl.
PL
Bs
MS
wit
hdif
fere
nt
level
sof
NA
A,
BA
and
Kin
alone
or
in
com
bin
atio
n.
Org
anogen
esis
Hig
h-f
requen
cysh
oot
regen
erat
ion
poss
ible
wit
h1.2
mg/l
NA
A
and
1.2
mg/l
BA
.E
ffici
ency
of
shoot
regen
erat
ion
was
rela
ted
toth
eori
enta
tion
and
posi
tion
of
expla
nts
.W
ith
anupri
ght
ori
enta
tion
on
med
ium
,sh
oots
wer
ein
duce
dm
ore
effi
cien
tly.
Zhao
etal
.(2
007
)
Cym
bid
ium
bic
olo
rS
hoot
tips
Mit
raet
al.
(1976
)bas
alm
ediu
msu
pple
men
ted
wit
h3
lM
24-e
piB
L
Org
anogen
esis
86
%of
expla
nts
form
edP
LB
s,ea
chw
ith
65.0
PL
Bs/
TC
L.
Shoots
that
form
edw
ithin
12
wee
ks
dev
eloped
bes
ton
Mit
ra
etal
.(1
976
)bas
alm
ediu
mw
ith
2l
MT
RIA
.
Mal
abad
iet
al.
(2008a)
Eri
adalz
elli
Shoot
tips
Mit
raet
al.
(1976
)bas
alm
ediu
msu
pple
men
ted
wit
h
9.0
8l
MT
DZ
Org
anogen
esis
The
hig
hes
tper
centa
ge
of
PL
Bsu
rviv
alw
as96
%,
pro
duci
ng
hea
lthy
shoots
wit
h2–3
leav
es.
Shoots
form
edro
ots
when
cult
ure
don
the
sam
ebas
alm
ediu
msu
pple
men
ted
wit
h
11.4
2l
MIA
A.
Reg
ener
ated
pla
ntl
ets
gre
wnorm
ally
wit
ha
90
%su
rviv
alra
te.
Mal
abad
iet
al.
(2008b
)
Aer
ides
macu
losu
mS
hoot
tips
Mit
raet
al.
(1976
)bas
alm
ediu
msu
pple
men
ted
wit
h
13.6
2l
MT
DZ
Org
anogen
esis
Ahig
hper
centa
ge
(81
%)
of
PL
Bs
surv
ived
and
ult
imat
ely
pro
duce
dhea
lthy
shoots
wit
h2–3
leav
es.
Shoots
roote
dw
hen
cult
ure
don
the
sam
ebas
alm
ediu
msu
pple
men
ted
wit
h
12.2
5l
MIB
A.
Reg
ener
ated
pla
ntl
ets
gre
wnorm
ally
wit
ha
90
%su
rviv
alra
te.
Mal
abad
iet
al.
(2009a)
Lip
ari
sel
lipti
caS
hoot
tips
Mit
raet
al.
(1976
)bas
alm
ediu
msu
pple
men
ted
wit
h4
lM
24-e
piB
L
Org
anogen
esis
93
%of
expla
nts
form
edP
LB
s,ea
chw
ith
71.0
PL
Bs/
TC
L.
Shoots
form
edin
12
wee
ks
and
roote
dbes
ton
Mit
raet
al.
(1976)
bas
alm
ediu
mw
ith
10.7
4l
MN
AA
.
Mal
abad
iet
al.
(2009b
)
Den
dro
biu
mdra
conis
Young
stem
s
MS
wit
hdif
fere
nt
level
sof
NA
A,
BA
and
Kin
alone
or
in
com
bin
atio
n.
Org
anogen
esis
PL
Bs
form
edsh
oots
wit
hin
6–7
wee
ks.
The
opti
mal
PG
R
com
bin
atio
nfo
rm
axim
um
PL
Bdev
elopm
ent
was
2m
g/l
BA
and
1.0
mg/l
NA
A,
68
%of
expla
nts
form
ing
anav
erag
eof
11
PL
Bs/
expla
nt.
Shoot
dev
elopm
ent
was
bes
ton
MS
med
ium
conta
inin
gsu
crose
and
CW
.P
lantl
ets
6–8
cmin
hei
ght
wer
e
tran
spla
nte
din
toco
conut
husk
pea
tw
ith
92
%su
rviv
alin
a
nurs
ery.
Ran
gsa
yat
orn
(2009)
Den
dro
biu
mgra
tiosi
ssim
um
Pro
toco
rms,
shoots
,
shoot
tips
MS
?2
mg/L
Kin
(pro
toco
rms)
;M
S?
5m
g/L
Kn
and
1.0
mg/L
NA
A(s
tem
TC
Ls)
Org
anogen
esis
PG
R-f
ree
MS
med
ium
fail
edto
induce
PL
Bs
from
stem
expla
nts
.
How
ever
,pro
toco
rm-
and
stem
-der
ived
TC
Ls
dev
eloped
PL
Bs
wit
hin
3–4
wee
ks
on
MS
conta
inin
gP
GR
s.H
ighes
t
per
centa
ge
of
PL
Bfo
rmat
ion
(83
%)
and
hig
hes
tnum
ber
of
PL
Bs
(18/e
xpla
nt)
was
poss
ible
on
pro
toco
rmT
CL
s,but
only
66
%of
stem
TC
Ls
pro
duce
dP
LB
sw
ith
anav
erag
eof
9
PL
Bs/
expla
nt.
Pla
ntl
etco
nver
sion
from
PL
Bs
was
succ
essf
ull
y
achie
ved
on
PG
R-f
ree
med
ium
.
Jaip
het
and
Ran
gsa
yat
orn
(2010
)
Hybri
dC
ymbid
ium
cv.
Sle
epin
gN
ym
ph
PL
Bs
KC
?5
%(v
/v)
CW
Org
anogen
esis
5P
LB
s/tT
CL
form
edin
30-d
ay-o
ldP
LB
sw
ith
83
%of
tTC
Ls
resp
ondin
gbut
am
uch
low
erper
centa
ge
in60-d
ay-o
ldP
LB
s.
Vyas
etal
.(2
010)
Plant Cell Tiss Organ Cult (2013) 113:149–161 151
123
Page 4
Ta
ble
1co
nti
nu
ed
Spec
ies
Org
anfr
om
whic
hT
CL
was
der
ived
Cult
ure
med
ium
,P
GR
san
dad
dit
ives
Type
of
resp
onse
Rem
arks
on
org
anogen
icre
sponse
invit
roR
efer
ence
s
Coel
ogyn
ecr
ista
taL
indl.
PL
Bs
MS
med
ium
wit
hT
DZ
(0.5
,1.0
,2.0
and
3.0
mg/l
),N
AA
(0.5
and
1.0
mg/l
)an
dB
A(0
.5,
1.0
,1.5
and
2.0
mg/l
)
eith
erin
div
idual
lyor
inco
mbin
atio
ns
or
wit
hban
ana
pow
der
(30
g/l
)an
dco
conut
pow
der
(30
g/l
)
Org
anogen
esis
BA
inm
ediu
mw
asdis
tinct
lybet
ter
for
shoot
mult
ipli
cati
on.
The
hig
hes
tper
centa
ge
of
expla
nts
pro
duci
ng
shoots
,w
ith
a
max
imum
aver
age
of
8.1
/expla
nt,
was
achie
ved
on
1.0
mg/l
NA
Aan
d0.5
mg/l
BA
wit
hC
P.
Shoots
pro
duce
dan
aver
age
of
15
roots
/expla
nt
on
hal
f-st
rength
MS
wit
h2.0
mg/l
IBA
and
2.0
mg/l
BA
.P
loid
yan
alysi
sof
regen
erat
edpla
nts
usi
ng
flow
cyto
met
ryre
vea
led
the
sam
eplo
idy
level
(dip
loid
).
Nai
ng
etal
.(2
011)
Paphio
ped
ilum
Dep
erle
and
P.
Arm
eni
Whit
e
FB
sM
Sw
ith
4.4
3l
MB
Aan
d4.5
2l
M2,4
-Dfo
rth
ecu
lture
of
Paphio
ped
ilum
Arm
eni
Whit
ean
dw
ith
44.3
9l
MB
Aan
d
26.8
5l
MN
AA
for
the
cult
ure
of
Paphio
ped
ilum
Dep
erle
.
Org
anogen
esis
Inboth
spec
ies,
only
sect
ions
that
conta
ined
the
bas
eti
ssue
of
FB
sw
ere
able
topro
duce
shoots
and
pla
nts
.F
Bse
ctio
ns
bet
wee
n1.5
and
3.0
cmfr
om
Paphio
ped
ilum
Dep
erle
wer
e
able
topro
duce
shoots
,but
only
sect
ions
of
FB
s[
2.5
cmfr
om
Paphio
ped
ilum
Arm
eni
Whit
ew
ere
regen
erab
le.
The
smal
l
bra
ctat
the
FB
bas
ehar
bore
da
new
min
iatu
reF
B,
whic
h
furt
her
har
bore
da
pri
mit
ive
FB
wit
hdom
e-sh
aped
mer
iste
m-
like
tiss
ues
that
pre
sum
ably
led
topla
nt
induct
ion.
No
call
us
form
ed.
Lia
oet
al.
(2011
)
Xen
ikophyt
on
smee
anum
Shoot
tips
Mit
raet
al.
(1976
)bas
alm
ediu
msu
pple
men
ted
wit
h
11.3
5l
MT
DZ
(for
PL
Bin
duct
ion)
Org
anogen
esis
Shoots
could
root
on
Mit
raet
al.
(1976
)m
ediu
m?
8.5
6l
M
IAA
Mulg
und
etal
.(2
011)
Ren
anth
era
Tom
Thum
b
‘Qil
in’
Lea
fbas
eV
W?
1.0
mg/l
TD
Z,
1.0
g/l
pep
tone,
and
10
%C
WO
rgan
ogen
esis
Only
38.3
3%
of
cult
ure
sfo
rmed
PL
Bs.
PL
Bs,
when
sub-
cult
ure
d12
tim
eson
VW
med
ium
wit
h0.5
mg/l
BA
,1.0
g/l
pep
tone,
100
ml/
lC
Wan
d0.5
g/l
AC
over
a2-y
ear
per
iod
show
eda
2.7
7-
to3.2
0-f
old
incr
ease
per
subcu
lture
.
VW
?1.0
g/l
pep
tone,
1.0
mg/l
NA
Aan
d1.0
g/l
AC
was
suit
able
for
pla
ntl
etfo
rmat
ion;
85.3
3%
of
pla
ntl
ets
dev
eloped
from
PL
Bs
wit
hin
60
day
scu
lture
.V
Wm
ediu
mco
nta
inin
g
100
g/l
BH
,1.0
g/l
pep
tone,
1.0
mg/l
NA
A,30
g/l
sucr
ose
and
1.5
g/l
AC
was
suit
able
for
pla
ntl
etgro
wth
.99.6
7%
of
accl
imat
ized
pla
ntl
ets
surv
ived
.A
bout
50,0
00
pla
ntl
ets
could
be
pro
duce
dsu
cces
sfull
yw
ithin
3yea
rs,
show
ing
no
phen
oty
pic
var
iati
on.
Wu
etal
.(2
012)
2,4
-D,
2,4
-dic
hlo
rophen
oxyac
etic
acid
;24-e
piB
L,
24-e
pib
rass
inoli
de;
AC
,ac
tivat
edch
arco
al;
BA
,N
6-b
enzy
laden
ine
(use
dunif
orm
lyev
enw
hen
BA
P,
i.e.
ben
zyla
min
opuri
ne,
has
bee
nin
dic
ated
inth
eori
gin
al;
Tei
xei
rada
Sil
va
2012b
);B
H,ban
ana
hom
ogen
ate;
CP
,co
conut
pow
der
;C
W,co
conut
wat
er;
FB
,fl
ow
erbud;
IAA
,in
dole
-3-a
ceti
cac
id;
IBA
,in
dole
-3-b
uty
ric
acid
;K
C,K
nudso
nm
ediu
m;
Kin
kin
etin
;M
S,M
ura
shig
ean
dS
koog
(1962)
med
ium
;N
AA
,
a-n
aphth
alen
eace
tic
acid
;P
GR
,pla
nt
gro
wth
regula
tor;
PL
B,
pro
toco
rm-l
ike
body;
TC
L,
thin
cell
layer
,T
CS
,th
incr
oss
sect
ion;
TD
Z,
thid
iazu
ron
(N-p
hen
yl-
N’-
1,2
,3-t
hia
dia
zol-
5-y
lure
a);
TR
IA,
tria
conta
nol;
VW
,V
acin
and
Wen
t
(1949)
med
ium
;Z
ea,
zeat
in;
ZR
,ze
atin
-rib
osi
de
152 Plant Cell Tiss Organ Cult (2013) 113:149–161
123
Page 5
bioreactors. Despite several decades of orchid tissue cul-
ture research, no in-depth assessment of the use of TCLs
exists, even though their importance was recently high-
lighted by Hossain et al. (2012). This review covers
exclusively those studies in which the term TCL or thin
section culture (TSC) were strictly used, even though a new
term, thin tissue layer or TTL, was suggested (Teixeira da
Silva 2008). Indeed, by definition of explant size and
proportion, there may be other studies in orchids that in
fact used TCLs, but the authors chose not to use this term.
How have TCLs been applied to orchid tissue culture?
The basic, fundamental and common vision of plant tissue
culture has always been how to perfect a protocol such that
a desired organ or plant of interest can be generated,
inexpensively, reproducibly and in large numbers and
remains one of the most fundamental techniques in plant
biotechnology, including in orchid transformation studies
(Teixeira da Silva et al. 2011; Table 2), germplasm con-
servation and eco-rehabilitation (Wu et al. 2012), long-
term storage through synthetic seeds (Sharma et al. 2012),
and in vitro flowering (Teixeira da Silva unpublished data),
and promises to remain so for the long-term future.
Thin cell layers have been used to culture several orchid
species in vitro: Aranda Deborah (Lakshmanan et al.
1995), Coelogyne cristata (Naing et al. 2011), Cymbidium
spp. (Begum et al. 1994; Nayak et al. 2002; Teixeira da
Silva and Tanaka 2006; Teixeira da Silva et al. 2005,
2006a, b, 2007a, b; Teixeira da Silva and Tanaka 2009a;
Malabadi et al. 2008a, b; Hossain et al. 2008, 2009, 2010),
Dendrobium sp. (Nayak et al. 2002), Dendrobium candi-
dum (Zhao et al. 2007), Dendrobium draconis (Rangsayatorn
2009), Dendrobium gratiosissimum (Jaiphet and Rang-
sayatorn 2010), Doritaenopsis (Park et al. 2002), Paphio-
pedilum Deperle and P. Armeni White (Liao et al. 2011),
Renanthera Tom Thumb ‘Qilin’ (Wu et al. 2012),
Rhynchostylis (Le et al. 1999), Spathoglottis (Teng et al.
1996), and Xenikophyton smeeanum (Mulgund et al.
2011) (Table 1). Table 1 only indicates how TCLs were
effectively used by different authors for very specific
hybrids or species, and the reader is cautioned into
extrapolating the positive results for one genotype for
another genotype. For example, it is easy to understand
how the TCL technique can be used effectively for some
(the majority) hybrid Cymbidium cultivars, but not for all
(Table 3). The advantage of the TCL system is to produce
high-frequency organ regeneration and to reduce the time
interval required to generate a desired organ. In was esti-
mated that more than 80,000 plantlets could be produced
from a single transverse TCL in a year compared to 11,000
plantlets produced by a conventional shoot tip method
(Teixeira da Silva 2003). In general, cells from 6 to
7 week-old PLBs are highly meristematic and thus TCL
tissue should be derived from these PLBs (Prakash et al.
1996). Using the plant Growth Correction Factor (GCF), it
will now be easier to make direct comparisons between
protocols that involve different sized explants, including
TCLs (Teixeira da Silva and Dobranszki 2011). To better
understand this concept, an example is given using Cym-
bidium hybrid Twilight Moon ‘Daylight’. For this hybrid, a
single PLB can yield an average of 8.4 PLBs per whole
PLB when cultured on Teixeira Cymbidum or TC medium
(Teixeira da Silva 2012a), but only 2.6 PLBs per lTCL or
1.4 PLBs per tTCL. However, it is evident (see Teixeira da
Silva and Tanaka 2006) that one whole PLB can yield
several lTCLs or tTCLs, 6 or 8, respectively for this cul-
tivar. In all cases, new PLBs can only form from the sur-
face area of a PLB and never from the internal tissue.
Specifically, the surface area of a whole PLB, a lTCL or a
tTCL is 8, 1 and 1 mm2, respectively. Therefore, using the
GCF, the true productivity of lTCLs and tTCLs can be
calculated from a single, whole PLB, main it thus possible
to compare productivity data across experiments of labo-
ratories. Thus, if a whole PLB were to be converted to
lTCLs or tTCLs, hypothetically one would be able to
derive 20.8 and 11.2 new PLBs, respectively, far more than
a single, intact PLB, demonstrating thus the power of the
TCL in orchid organogenesis. Consequently, the actual and
hypothetical values for several cultivars can be calculated
(Table 3).
Table 2 Use of thin cell layers (TCLs) for orchid transformation
Species Gene(s) Explant Method References
Hybrid Cymbidium Twilight Moon
‘Day Light’
gusA, nptII, bar callus, PLB TCLs PB Teixeira da Silva and Tanaka
(2009b, 2011)
Dendrobium ‘Madame Thong-In’ nptII, DOH1 antisense PLBs TCLs At Yu et al. (2001)
Dendrobium sp. gusA, nptII, DOMADS1 PLBs TCLs PB Yu et al. (2002)
Dendrobium sp. nptII, DSCKX1 PLBs TCLs At Yang et al. (2003)
At = Agrobacterium tumefaciens; bar = phosphinothricin acetyltransferase; DOH1 = a Class 1 knox gene (Dendrobium orchid homeobox);
DOMADS1 = Dendrobium orchid MADS-box gene; DSCKX1 = Dendrobium Sonia cytokinin oxidase; gusA = b-glucuronidase (also known as
uidA); nptII = neomycin phosphotransferase II; PB = particle bombardment
Plant Cell Tiss Organ Cult (2013) 113:149–161 153
123
Page 6
Cymbidium hybrid as a model orchid for TCL studies
Cymbidium hybrid orchids will be the main focus of this
review for three main reasons. Firstly, until recently, only
terrestrial cymbidiums had been successfully propagated
in vitro (Hasegawa 1987), mainly through the culture of
shoot tips (Morel 1960), whereas Cymbidium hybrids were
much more difficult to propagate. Secondly, precisely
because it is a difficult plant to propagate efficiently, by
being able to manipulate organogenesis precisely in vitro
would make it a suitable model plant and this has already
been made possible (Teixeira da Silva and Tanaka 2006).
Thirdly, by showing that the TCL technique is applicable
to an expensive ornamental market commodity, hope is
created for use of the technique in both developing and
developed countries for the mass propagation of conven-
tional cash crops as well as difficult-to-propagate species,
for research and for business.
Cymbidium tissue culture has been reviewed elsewhere
(Nayak et al. 2006), so only most important and fundamental
concepts will be defined here although three detailed proto-
cols will be presented. The first concept that requires defi-
nition is a protocorm-like body or PLB. Simply, a PLB is an
organ that looks like a protocorm, but is not one, since a
protocorm is derived from a seed. A PLB is not derived from
a seed, although a PLB may derive from a protocorm. Where
then does the original PLB derive from? This basic important
fact is always overlooked in almost every single tissue cul-
ture and micropropagation protocol available for almost
every orchid. In the case of hybrid Cymbidium, where
plantlets—originally derived from the tissue culture of
sterilized shoot tips—are cultured on a highly organic sub-
strate (e.g. one supplemented with banana), from a flask of
about 100 rooted shoots, about 1 % of plants spontaneously
form a PLB at the base of the leaf sheath. The capacity for
PLBs as suitable explants for callus formation and somatic
embryogenesis was later demonstrated (Huan et al. 2004).
The term somatic embryogenesis can be interchangeably
used with PLBs (Teixeira da Silva and Tanaka 2006) since in
fact PLBs are somatic embryos (Fig. 1a) that develop a shoot
and root system from each PLB. PLBs and embryogenic
callus (Fig. 1b) can be induced under a wide range of med-
ium variations (Teixeira da Silva et al. 2005), biotic (Teixeira
da Silva et al. 2006b) and abiotic factors (Teixeira da Silva
et al. 2006a), in the light (Fig. 1b) and dark (Fig. 1c). PLBs,
PLB segments or PLB TCLs can be used in synseeds
(Fig. 1d).
The most basic protocol for PLB proliferation in orchids is
exemplified in Fig. 2. A spontaneously formed PLB or pri-
mary (1�) PLB, once cultured on appropriate medium, can
then form secondary (2�) PLBs (Begum et al. 1994), albeit at a
low multiplication rate. Every time a PLB is used (whole or in
part) for a sub-culture, it is considered a 1� PLB and any PLB
that is derived from a 1� PLB is a 2� PLB (Fig. 2). A terciary
(3�) is essentially the same as a 2� PLB (in terms of its origin),
although it is strictly clonal, i.e. of the same size, shape and
dimensions, and would be used in a commercial, microprop-
agation setting. The terms primary (1�), secondary (2�), and
tertiary (3�) PLBs were originally coined by this author
(Teixeira da Silva) in Teixeira da Silva and Tanaka (2006).
The methodology described in this review does not
include any process related to the tissue culture protocol that
goes beyond the plantlet stage in vitro, since this would be
beyond the scope of TCL technology, and thus unrelated to
the focus of this review, although the interchange between
the in vitro and ex vitro environment must always be kept in
Table 3 Inter-cultivar variation in Cymbidium hybrid PLB formation based on explant size
Cymbidium cultivar Parentage BioU
cultivar
code
No. PLBs/explant1 Ratio of No.
PLBs/explant
Half-PLB2 tTCL2 lTCL2 (Half-PLB:
tTCL:lTCL)
Aroma Candle ‘Hot Heart’ Jenteel ‘Pair Look’ 9 Seaside ‘Crown Princess’ 91-8 2.6 0.4 0 0.87:0.13:0
Pretty Poetry ‘Malachite’ Mini Sarah ‘Artisan’ 9 Eastern Star ‘Green Fields’ 167-1 3.2 1.1 0.4 0.68:0.23:0.09
Alice Beauty ‘No. 1’ Alice Luna 9 Sleeping Beauty ‘Mistuko’ 204-1 1.8 0.2 0 0.90:0.10:0
Twilight Moon ‘Day Light’
(TMDL)
Lovely Bunny ‘Romeo’ 9 Hiroshima Golden Cup
‘Sunny Moon’
246-2 8.3 6.4 3.6 0.45:0.35:0.2
Spring Night ‘No. 12’ Tiny Sour 9 TMDL 485-12 4.6 2.3 1.2 0.57:0.28:0.15
Dream City ‘No. 1’ Great Katy ‘Tender’ 9 Lucky Flower ‘Anmitsuhime’ 536-1 2.2 0.8 0.3 0.67:0.24:0.09
Call Me Love ‘Snow
Princess’
Jenteel ‘Pair Look’ 9 Great Katy ‘Tender’ 553-1 3.1 2.6 0.8 0.48:0.40:0.12
Energy Star ‘No. 4’ Morning Moon ‘Great Tiger’ 9 TMDL 649-4 6.4 3.4 1.3 0.58:0.31:0.12
Sweet Moon ‘No. 2’ Yellow Candy ‘Lemon Fresh’ 9 TMDL 653-2 7.2 2.8 1.1 0.65:0.25:0.10
1 n = 30 (10 9 3)2 Prepared according to Teixeira da Silva and Tanaka (2006) on Teixeira Cymbidium (TC) medium (Teixeira da Silva (2012a)
154 Plant Cell Tiss Organ Cult (2013) 113:149–161
123
Page 7
mind when establishing an in vitro protocol (Teixeira da
Silva 2004; Teixeira da Silva et al. 2007a).
Three model protocols for orchid (hybrid Cymbidium)
in vitro culture
General methodological requirements for all three
protocols
In order to conduct PLB regeneration studies from regular
explants or from TCLs for several Cymbidium cultivars
(Table 3), the following are required: Petri dishes (100 mm
diameter, 15 mm high) (Falcon, Osaka, Japan); Kinetin
(Kin); a-naphthaleneacetic acid (NAA); Tryptone; Bacto
agar (Difco Labs); surgical blades (Hi stainless platinum or
carbon steel; Feather Safety Razor Co., Ltd., Osaka,
Japan); Whatman No. 1 filter paper (9 cm diameter). All
plant growth regulators (PGRs) and tryptone are of tissue
culture grade (Sigma-Aldrich, St. Louis, USA).
In order to establish initial PLBs, young shoots of any
Cymbidium hybrid should be excised from 3-year-old
matured plants growing in a greenhouse without any visi-
ble symptoms of bacterial, fungal or viral infection. Shoots
Fig. 1 The induction of hybrid Cymbidium (Twilight Moon ‘Day Light’) PLBs (a) on TC medium, friable embryogenic callus in the light (b) or
dark (c) using thin cell layers, which can also be used as synseed (d)
1 PLB
2 PLB
A
B
C
Fig. 2 Protocol 1 for spontaneous orchid PLB induction. Culture of a
whole 1� PLB (a) results in the formation of a plantlet (shoot and
adventitious root formation; b). 2� PLBs, whose formation is erratic
after 30–45 days (c), and whose rate of formation is low (and
uncontrollable), can be harvested and employed as 1� PLBs (a) in a
second round of 2� PLB formation. This method is not recommended
for micropropagation (i.e. 3� PLB formation) due to great differences
in size, shape and developmental stage. Dashed line indicates the
medium line. Figure not to scale
Plant Cell Tiss Organ Cult (2013) 113:149–161 155
123
Page 8
are placed under running tap water for 30 min, surface
sterilized in 1.5 % (v/v) sodium hypochloride for 15 min,
transferred to fresh sterilization solution for another
15 min. After rinsing shoots three times with sterile dis-
tilled water (SDW; *5 min each time), apical meristems
(*5–10 mm terminal tips) should be isolated in a sterile
Petri dish. These apical meristems serve to induce 1� PLBs
on PGR-free half-strength Murashige and Skoog (MS)
basal salt medium (Murashige and Skoog 1962). After
*6 months, 1� PLBs will appear spontaneously at the base
of rooted shoots. These PLBs will then be used in Protocols
1–3. A ‘‘universal’’ medium for 2� PLB formation, inde-
pendent of the protocol, is suitable for plating 10 1� PLBs
on 40 ml medium/100-ml Erlenmeyer flask of PLB-
induction medium, which consists of Vacin and Went basal
medium (Vacin and Went 1949) supplemented with Nitsch
micro elements (Nitsch and Nitsch 1967), 2 mg l-1 tryp-
tone, NAA and Kin at 0.1 mg/l each, 2 % sucrose (w/w),
pH = 5.8 ± 0.1 and 8 g/l Bacto agar. The medium is
autoclaved at 121 psi for 21 min. 1� or 2� PLBs should be
cultured at 25 ± 0.5 �C in a 16-h photoperiod provided by
fluorescent tubes with a low photon flux density of
30–40 lmol m-2 s-1.
Experimental pitfalls or general notes of caution for all
three protocols
The sharpness of the blade is one of the most important
factors that determines the success of Protocols 2 and 3, in
particular Protocol 3, which requires thin explants, the
TCLs. Feather blades made in Japan are highly recom-
mended since they can be autoclaved, sterilized, boiled,
sterilized with 98 % ethanol and still remain sharp for
explant preparation several times. Several other makers
from brands around the world do not given the same
‘‘perfect slice’’. In general, to avoid explant damage, even
with feather blades, blades should be replaced after pre-
paring 40–50 explants (conventional half-moon explants or
TCLs). 1� PLBs are not guaranteed to form on MS med-
ium, although personal experience has shown that the
inclusion of 1 g/l of activated charcoal (AC; acid washed,
Sigma-Aldrich) and culture in the dark can make MS a
suitable basal medium. Ideally a ripe banana-based med-
ium, rich in carbohydrates, sometimes, supplemented with
coconut milk, will yield more 1� PLBs. Once initial shoots
begin to elongate (before roots elongate, or cut off roots),
transfer PLBs to 0.5 % (w/v) Gelrite supplemented with
2 % (w/v) ripe banana (mashed by hand) and 10 % (v/v)
coconut water; this results in strong growth (shoot and
root) of plantlets. Green, unripe banana should be avoided.
A high level of irradiation ([80 lmol m-2 s-1) can inhibit
2� PLB formation, sometimes completely. Darkness, on the
other hand, with this PGR combination, is also not that
effective, and it is better to substitute 0.1 mg/l Kin with
1 mg/l 6-benzyladenine (BA; Sigma-Aldrich). In this case,
2� PLBs form, but these are white (Fig. 1c) and not as
numerous, but once transferred to light, regain their pho-
tosynthetic capacity. Another alternative is to supplement
the Kin/BA medium with 1 % (w/v) AC, and place the
cultures in the light; it is possible that the AC mirrors a
darkened natural environment of Cymbidium in tree tops.
Most protocols in the literature on ‘‘Cymbidium’’ are
mainly on terrestrial cymbidiums, which, like Dendrobium
spp., are much easier to propagate in vitro.
Protocol 1
This protocol is used for inducing ‘‘conventional’’ (such as
2�) PLBs from whole 1� PLBs. As 1� PLBs develop, 2�PLBs are formed, and these may then be separated out and
re-plated on the same medium to induce tertiary 3� PLBs
(Fig. 2). This protocol results in very few (average = 1.68,
n = 40) 2� PLBs per 1� PLB. Hypothetically, sub-culture–
to–sub-culture would yield a 13.49 multiplication rate
after 5 consecutive sub-cultures (2 months each). In other
words, with a single initial 1� PLB, a total of 535 3� PLBs
can be obtained after a 10-month period, assuming that
every single 1� and 2� PLB is used, that every single 1� and
2� PLB survives and that every single 1� and 2� PLB is
able to differentiate. A typical sub-culture should be made
once every 2 months before the apical meristems have time
to develop into shoots, and before roots can emerge from
the base of the 1� PLB. Oxidation and browning of 1� PLBs
can take place rather quickly (within 10 min); therefore, 1�PLBs should be plated immediately following dissection.
Naturally, depending on the cultivar, the size of the 1� PLB
will differ. However, for hybrid Cymbidium cultivars
(Table 3), 1� PLBs of standard diameter (4–6 mm) should
be used. Larger 1� PLBs may already be in too an advanced
developmental stage, and may have already started to form
a shoot and adventitious roots, which tends to reduce the
PLB-inducing potential of the 1� PLB. Too small a 1� PLB
will also result in poor 2� PLB formation because of too
much tissue damage and reduced surface area.
Protocol 2
This is a protocol for proliferation of 2� PLBs from 1�PLBs in consecutive sub-cultures (Teixeira da Silva et al.
2005, 2006a, b; Teixeira da Silva and Tanaka 2006). When
the 1� PLB grows, 2� PLBs form on the 1� PLB, usually at
the base. These are separated out, placed in an autoclaved
glass Petri dish with a double sheet of Whatman No. 1 filter
paper laid at the base. Using a feather blade, the top 1 mm
156 Plant Cell Tiss Organ Cult (2013) 113:149–161
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of the 1� PLB, which contains the apical meristem, is cut.
The bottom, brown part of the 1� PLB, if existent, should
also be sliced off (Fig. 3). It is always useful to have one
Petri dish prepared for every 10–20 1� PLBs that need to be
prepared. For a total of 1,000 1� PLBs, 1,000 ml of sterile,
double-distilled water (SDDW) is sufficient. Into each Petri
dish, 10–20 ml of SDDW is injected so that the filter paper
is always soaked with a thin layer of SDDW at the base.
The ‘‘trimmed’’ 1� PLB (i.e. without a shoot apical meri-
stem and base) is now sliced symmetrically to yield two
half-moon explants (often referred in the literature as half-
moon PLBs). The PLBs should never be allowed to dry-out
by always almost completely covering the Petri dish so that
the air flow from the clean bench does not desiccate them.
Caution should be taken also not to submerge the PLBs in
SDDW, as an apparent hyperhydric response occurs. In
general, PLBs are extremely sensitive to injury, water, light
or temperature stress, even if small deviations from the
ideal medium (TC). 1� PLBs that have been left standing
for more than 30 min should be discarded. In 1� ? 2� PLB
conversion, there is always inevitably a basal part of the
PLB that is white or opaque and callus-like in appearance,
or that has a hyperhydric appearance due to direct contact
with the medium. This tissue generally has poor PLB-
induction potential and should never be used for PLB
induction. Moreover, 1� PLBs should never be used for 3�PLB production, and only 2� PLBs that form on the outer
layer of 1� PLBs should be used. Usually the latter are
almost perfectly round, and do not have a morphologically
distorted base. These are placed cut-surface down on the
medium, embedded about 1 mm into the medium. Explants
(1� half-moon PLBs) should never be placed with the intact
surface down on the medium, simply placed on top of the
medium, nor should they be totally embedded into the
medium because in such cases PLBs will never form. After
about 45–60 days (the number depending on the treatment
tested, i.e. the actual experimental protocol), 2� PLBs form
on the outer, epidermal surface of the PLB. These should
be allowed to enlarge and only uniform sized (opti-
mum = 4–6 mm) 2� PLBs should be used for PLB pro-
duction, i.e. micropropagation. Each cultivar forms PLBs
of very different diameters, which is likely to influence the
organogenic outcome, too. Usually the ‘‘mother’’ PLB, i.e.
the 1� PLB, will gradually die away and turn brown. This
will take about 60 days to occur, at which time, ideal sized
2� PLBs will have formed, which can and should be used
for whatever experimental purpose they are required, or for
micropropagation. In principle, different sized 2� PLBs
should never be used for experiments since initial 2� PLB
size strongly affects the experimental outcome. Moreover,
any PLBs that have formed leaves, or where the leaf pri-
mordia have already emerged from the PLB should never
be used in PLB proliferation experiments. In this sense,
PLBs can and should be thought of as storage organs, and
the translocation of resources to the developing leaf
‘‘weakens’’ the sink strength of the PLB, thus making,
2 PLBs
1 PLB
A
B
C
D
Fig. 3 Protocol 2 for PLB proliferation. A whole 1� PLB (a) is not
cultured (unlike Protocol 1). Rather, the shoot apical meristem (SAM)
and basal part of the PLB that is in contact with the medium are
dissected or removed, yielding a ‘‘trimmed’’ (or top-less) 1� PLB (b).
Note: trimming should take place before the SAM begins to elongate
into a shoot. This ‘‘trimmed’’ 1� PLB is cut symmetrically length-wise
to yield two half-moon-shaped explants (c). When each half-moon-
shaped PLB explant is re-plated on the same medium, several 2�PLBs form near or at cut surfaces (primarily) and on the surface after
30–45 days (d); the rate of formation is higher than in Protocol 1, and
can be harvested and employed as 1� PLBs (a) in a second round of 2�PLB formation. This method is recommended for micropropagation
(i.e. 3� PLB formation) because of high levels of PLB formation, each
of more-or-less uniform size, shape and developmental stage. Dashedline indicates the medium line. Doted lines indicate lines of
sectioning. Tick symbol correct level (manages to remove the
SAM); wrong symbol incorrect level (does not manage to remove
the SAM). In the literature, the term ‘‘mericlone’’ is often mistakenly
used to indicate clonal plants derived from the zone that includes cells
exclusively from the SAM, although these studies often do not
provide histological proof of such cellular origin. Figure not to scale
Plant Cell Tiss Organ Cult (2013) 113:149–161 157
123
Page 10
apparently, the ability to form new PLBs lower. Protocol 2
results in a large number (average = 8.21, n = 40) of 2�PLBs per 1� half-moon PLB. Hypothetically, sub-culture–
to–sub-culture would yield a 4,0009 multiplication rate
after 4 consecutive sub-cultures (3 months each). In other
words, with an initial two 1� PLB half-moon explants, a
total of *36,350 3� PLBs can be obtained after a 12-month
period, assuming that every single 1� and 2� PLB is used,
that every single 1� and 2� PLB survives and that every
single 1� and 2� PLB is able to differentiate.
Protocol 3
This protocol is specifically targeted for TCL-induced (2�)
PLB formation from 1� PLB tTCLs (Teixeira da Silva and
Tanaka 2006; Teixeira da Silva et al. 2007a, b). When the
1� PLB grows, 2� PLBs form on the 1� PLB, usually at the
base. Following the general guidelines for Protocol 2, only
ideal size and shaped 2� PLBs are selected. Using a new
feather blade for every 6–8 PLBs, a 0.5–1 mm deep inci-
sion in the shape of a square, 3–5 9 3–5 mm in area, is
made. This area is sliced to separate the epidermal
0.5–1 mm in one continuous move, thus creating an lTCL
(Fig. 4b–d). It is imperative to prepare the lTCL in a single
stroke (e.g. as one would when slicing an envelope with a
brand new letter opener). If the explant is prepared in
several strokes (e.g. as slicing a hard baguette with a bread
knife), then the explant itself tends to get damaged, both on
the surface and on the sub-surface. Although the inner
tissue (sub-epidermal layers and below) of a PLB never
forms 2� PLBs, any damage to this tissue results in rapid
browning of all tissue (within 1 week) and eventual
necrosis (within 1–2 weeks) of the whole TCL, making
TCL a simple but challenging technique. It is thus imper-
ative to change the feather blade regularly and water the
cut lTCLs or tTCLs in SSDW. Using a new feather blade
for every 6-8 PLBs, and only using the central 5 mm girth
of the 1� PLB, a 0.5–1 mm transverse slice is made
throughout the whole PLB, thus creating a tTCL (Fig.
4e–h). Protocol 3 results in a very large number (aver-
age = 14.48, n = 40) of 2� PLBs per 1� PLB lTCL but in
much fewer (average = 6.08, n = 40) 2� PLBs per 1� PLB
tTCL (the reason is related to the total surface area of a
tTCL being much less than that of an lTCL: see Table 3 for
more data, discussion and rationale using the GCF). Note
that two lTCLs can be prepared from an ideal-sized 1�PLB, while 5 tTCLs can be prepared from the same mother
explant. Hypothetically, sub-culture–to–sub-culture would
yield a 24,2809 multiplication rate after 3 consecutive sub-
cultures (3 months each) for lTCLs. In other words, with
two initial 1� PLB lTCLs, a total of *351,700 3� PLBs
can be obtained after a 9-month period, assuming that
every single 1� and 2� PLB is used, that every single 1� and
2� PLB survives and that every single 1� and 2� PLB is
able to differentiate. For tTCLs, these values are lower, but
1 PLB A
B
X2 or 3
X3-7
2 PLBs
2 PLBs
2 PLBs
C
D
E
F
G H
Fig. 4 Protocol 3 for micropropagation of orchids using PLB TCLs.
A whole 1� PLB (a) is not cultured as in Protocol 2. Rather, the shoot
apical meristem (SAM) and basal part of the PLB that is in contact
with the medium are dissected or removed, yielding a ‘‘trimmed’’ (or
top-less) 1� PLB (b). Trimming should take place before the SAM
begins to elongate into a shoot. This ‘‘trimmed’’ 1� PLB now enters
the lTCL (b–d) or the tTCL (e–h) pathway. In the lTCL pathway, 2–3
lTCLs (0.5 mm thick, 3 9 3 mm) can be safely prepared from a
single ‘‘trimmed’’ 1� PLB (c). When each lTCL is re-plated on the
same medium, numerous 2� PLBs form over the entire surface after
20–25 days, and can be harvested at 30–45 days (d); the rate of
formation is higher than in Protocols 1 and 2, and can be harvested
and employed as 1� PLBs (a) in a second round of 2� PLB formation.
This method is recommended for micropropagation (i.e. 3� PLB
formation) because of high levels of PLB formation, each of more
uniform size, shape and developmental stage than those harvestable
from Protocol 2. In the tTCL route, a single ‘‘trimmed’’ 1� PLB can
yield between 3 and 7 (best = 5) ‘‘slices’’ or tTCLs (e, f). When each
tTCL is re-plated on the same medium, numerous 2� PLBs form only
on the surface containing PLB surface (internal tissue never forms
PLBs; g = side view, h = top view) after 20–25 days, and can be
harvested at 30–45 days (g, h); the rate of formation is higher than in
Protocol 1 but never more than Protocol 2 or the lTCL method, and
can be harvested and employed as 1� PLBs (a) in a second round of 2�PLB formation. This method is not recommended for micropropaga-
tion (i.e. 3� PLB formation) because of insufficiently high levels of
PLB formation, even though each is uniform in size, shape and
developmental stage (as for the lTCL route). Dashed line indicates the
medium line. Doted lines indicate lines of sectioning. Tick symbolcorrect level (manages to remove the SAM); wrong symbol incorrect
level (does not manage to remove the SAM). Figure not to scale
158 Plant Cell Tiss Organ Cult (2013) 113:149–161
123
Page 11
still significant, if considering a micropropagation plant
factory: Hypothetically, sub-culture–to–sub-culture would
yield a 4,6209 multiplication rate after 3 consecutive sub-
cultures (3 months each) for tTCLs. In other words, from 5
initial 1� PLB tTCLs, a total of *28,100 3� PLBs can be
obtained after a 9-month period, assuming that every single
1� and 2� PLB is used, that every single 1� and 2� PLB
survives and that every single 1� and 2� PLB is able to
differentiate. However, these values are absolute values,
and if we consider that 3–8 tTCLs can be prepared from a
single PLB or that 2–10 lTCLs can be prepared from a
PLB, depending on the genotype used (Table 3), the
hypothetical number of PLBs that can be generated from a
single PLB is enormous.
Debunking truths about TCLs and TCL protocols
1. TCL technology is based on a combination of scientific
principles and artistic skills, just as it is the case for other
in vitro culture protocols, but with one exception, Pro-
tocol 3, which is exclusively relevant to TCLs. This
protocol requires special attention to selecting appro-
priate sizes of PLB-derived explants, technical skills,
and careful handling of explants. Part of the success may
lie in the use of new Teixeira Cymbidium (TC) medium
(Teixeira da Silva 2012a), which has been found to be
more effective than VW- and MS-based basal media.
2. TCL technology is purely a tissue culture protocol, and
does not involve any high-tech histological, biochem-
ical, or genetic techniques, although the use of TCLs has
incredible potential, when used in conjunction with any
of these techniques, for assessing cellular and ultra-
structural processes, controlling developmental events,
assisting genetic transformation protocols, and improv-
ing regeneration and micropropagation of difficult-to-
propagate species (see several chapters in Nhut et al.
2003a). It is not uncommon to see the use of poorly
prepared PLBs which are either of non-standard size, or
from which leaves have already started to develop, even
within respected journals. Use of the TCL, together with
implementation of the GCF, would potentially eliminate
lab-to-lab, genotype-to-genotype and genus-to-genus
bias and error, and allow for more standardized clonal
material for synseed production and long-term preser-
vation through cryopreservation, bioreactor mass prop-
agation, or genetic transformation.
3. Flow cytometry or molecular markers, useful and
simple techniques, can be used to assess the ‘‘true-
ness-to-type’’ of an explant (e.g. Teixeira da Silva et al.
2007a, b). By understanding the ploidy level of explants
through a quick (\30-min analysis) assay, the origin can
be determined and hence the appropriateness for the
proposed study. Different tissues from orchid plants
contain variable levels of ploidy, or endopolyploidy
(Teixeira da Silva and Tanaka 2006), and thus selection
of genetically stable tissue is advised.
4. Protocol 1 frequently results in mixed organogenesis,
including PLBs, adventitious roots, shoots and callus.
Protocol 2 results primarily in PLBs, some callus and
occasionally shoots. Protocol 3 results exclusively in
PLB production, although the perceived number is
lower than ‘‘ideal’’ protocols in the literature that tend to
employ Protocol 1-like methodologies. Due to the
multiple-organogenic pathways that would result from
the use of different protocols, in particular from Protocol
1, the estimated output (total number of 3� PLBs) would
be extremely skewed, slightly skewed or almost not
skewed when referring to Protocols 1, 2 and 3,
respectively. Such imbalances caused as a result of
comparing protocols that use different sized explants
can be eliminated by implementing the GCF. To give the
reader a more realistic perspective, 3� PLBs, i.e. of
uniform size, shape, and developmental stage would/
could be the material used to generate clonal hybrid
Cymbidium in a commercial orchid micropropagation
unit since shoots would all emerge very much synchro-
nously and root and shoot development would result in
very little variation. Studies that have been exclusively
established from Protocol 1, as is found in[95 % of all
papers published for most orchid species in vitro (see
Hossain et al. 2013), result in some organogenic
outcome, although the programme is not ‘‘pure’’, and
thus not very prone to commercial exploration, or can
result in a large number of regenerants being discarded
due to the lack of uniformity in size, i.e. somaclonal
variation. Protocol 3 strengthens the importance of
TCLs as a tool for controlling organogenesis and for
creating clones that have similar size, appearance, and
developmental characteristics.
5. One of the strongest positive aspects of TCLs is the
inherent capacity to strictly control an organogenic
programme more than a conventional explant, which
has multiple advantages and applications in plant tissue
culture.
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