Plant Tissue Culture Methods for Growth and Reproduction Study
2.
a)
De
p
ending
on the part used
for
cultu
r
e
Organ
C
u
l
t
u
r
e
i.
Root
tip
cultu
r
e
(Me
r
istem –
r
oot
tip
cultu
r
e)
Root
a
pic
a
l
m
e
ris
t
em
i
s
m
a
inly
re
s
p
ons
i
ble
for
t
he
g
ro
wth
of
roots
b
y
ce
l
l d
i
v
is
i
on,
ce
l
l
en
l
arge
m
ent
an
d
ce
l
l
u
l
a
r
d
i
f
fer
e
n
t
iati
o
n.
T
i
ps
o
f
t
h
e
l
a
t
e
r
al
r
o
ots
a
r
e
s
t
erilis
e
d,
e
x
ci
s
ed
and
t
r
a
ns
f
e
rre
d
t
o
fresh
mediu
m
.
T
h
e
l
a
t
e
r
al
r
o
ots
co
nt
inu
e
t
o
g
ro
w
a
nd
p
ro
v
i
d
e
s
e
v
e
r
al
ro
ots,
w
h
ich
after
se
v
en
d
a
ys,
are
used
t
o
e
x
per
i
men
t
al
cu
l
ture
s
.
By
this
cu
l
t
u
r
e
m
eth
o
d
i
t
i
s
possible
to
st
u
d
y
the
n
ut
r
itio
n
al
r
eq
u
i
rem
en
t
s
o
f
r
o
ots,
sh
oot
an
d
ro
ot
g
r
o
w
t
h,
co
nditi
o
ns
req
u
i
re
d
for
t
h
e
d
e
v
el
o
p
me
nt
o
f
seco
n
dary
v
ascu
l
a
r
t
i
ss
u
es,
l
a
t
e
r
al
r
oot
an
d
bu
d
formation,
no
d
u
l
a
ti
o
n
etc.
43
ii.
Shoot-tip
cultu
r
e
(M
e
r
i
stem – shoot
tip
cu
l
tu
r
e)
The
shoot
a
p
e
x
o
r
shoot
-
tip
(
10
0-100
0
µm
lon
g
)
con
s
i
s
t
s
of
the a
p
i
c
al
meris
t
em
an
d
on
e
t
o thr
e
e
a
d
j
acent leaf p
r
imordi
a
.
A
c
tively
g
r
o
wing
sh
o
ot
-
tip
is
surface
s
t
e
r
i
l
i
z
ed
a
n
d
is
p
lac
e
d
o
n a d
e
fined
c
ulture
me
d
ium
unde
r s
t
e
r
ile
con
d
ition
s
.
Sho
o
t
Node
c
ulture
i
s
s
i
mplifi
e
d
form
o
f
shoot
c
u
l
ture,
w
h
i
c
h
is
y
e
t
a
n
o
t
h
e
r
method
for
p
r
o
d
u
c
tion
from
p
r
e
-
e
x
i
s
ting
meris
t
em.
I
t
i
s
the
simpl
e
st
met
h
od
b
u
t
is
a
s
sociat
e
d
with
t
he
least g
e
n
e
tic
va
r
iat
i
o
n
.
S
o
me
of
t
h
e
crop
s
p
ecies
that
have
b
e
e
n
fre
e
d
of
viruses
by
this
techniq
u
e,
th
e
y
i
n
c
l
u
d
e
soyabe
a
n,
s
w
e
e
t
p
o
tato, su
g
ar
ca
n
e
an
d rhub
a
rb.
Th
i
s
me
t
hod
is
used
w
i
th
both
monocot
and
d
ic
o
t
spec
i
es.
44
iii.
Le
a
ves
or
leaf
pr
i
mord
i
a
cu
l
tu
r
e
T
h
e
g
r
o
w
t
h
o
f
l
e
a
v
es
o
n
t
he
c
u
lt
ur
e
m
e
di
u
m
de
pe
n
ds
upo
n
the
stage
o
f
the
l
e
a
v
es
dur
i
ng e
x
cis
i
o
n
.
It
is
o
b
s
e
r
v
ed
t
h
at
e
x
plan
t
s
f
r
o
m
i
m
m
a
t
u
r
e
y
o
u
n
g
l
e
a
v
es
grow
better
than
e
x
p
l
ants
from
o
l
der
l
e
aves.
L
eaves
(80
0
µ
m
lo
n
g) m
a
y
b
e
d
e
t
a
c
hed
f
r
o
m shoo
t
s,
sur
f
a
c
e
s
t
e
ri
l
i
z
e
d
a
nd pla
c
ed
o
n
a
s
o
l
i
d
i
f
ied
m
e
di
u
m
w
h
e
r
e
t
h
ey
wi
l
l
r
e
m
ain
i
n
a
h
e
a
lthy
c
o
n
d
it
io
ns
f
or
a
l
o
ng
per
i
o
d
.
It
is
bel
i
e
ve
d
t
h
at
leaf
c
u
lt
u
re
d
ep
e
n
ds
up
on
t
h
e
phys
i
o
l
og
ical
state
an
d
the
ag
e
o
f the
l
e
af.
T
h
e
s
ho
ot fo
r
m
in
g
p
ot
e
nt
i
als
d
i
f
f
er
i
n
t
h
e
leaf
c
ul
t
u
r
es
a
s
p
er
t
h
e
d
er
i
v
at
io
n
o
f
t
h
e
expla
n
t
a
nd
t
h
e
h
o
rm
on
a
l
factors
i
n
v
o
l
v
e
d
.
45
iv.
F
l
o
w
er
cu
l
tu
r
e
(M
e
r
i
stem - floral
cu
l
tu
r
e)
Fl
o
w
ers
t
w
o
d
a
y
s
a
f
ter
pol
l
i
na
t
ion
a
r
e
ex
cis
e
d,
ste
r
i
l
i
z
e
d
b
y
imme
r
s
i
o
n
i
n
5
%
c
a
lcium
h
y
po
chlo
r
ite,
r
ep
ea
t
e
dly
tr
a
ns
f
e
r
r
e
d
med
i
u
m
.
wa
she
d
t
o
cu
l
t
u
r
e
with
s
t
e
r
i
l
i
ze
d
w
at
e
r
and
a
gar
t
u
b
es
c
o
ntain
in
g
an
Wh
e
n
fruits
c
u
l
t
u
r
e
d,
s
u
c
h
f
low
e
rs
p
rod
uce
f
r
u
i
ts.
L
a
rg
er
a
re
o
bta
ine
d
on
m
e
di
u
m
s
u
p
p
le
m
e
nt
e
d
wi
t
h
g
r
o
w
t
h
ho
r
m
on
e
s
.
F
lo
wers
excis
e
d
b
e
f
o
r
e
d
o
no
t
pro
d
uce
fruits.
p
o
l
l
i
n
a
t
ion
Th
i
s
cul
t
u
re
sys
t
e
m
is
u
s
ef
u
l
in
st
u
dy
i
n
g
veg
etati
v
e
micro
c
lima
t
es
o
r
nu
t
riti
o
nal
e
f
f
e
c
ts
o
n
t
he
an
d
rep
r
od
u
cti
v
e
proc
e
ss
e
s
o
f the
p
l
an
t
.
46
v.
An
t
her
and
po
l
l
e
ns
cultu
r
e
Y
o
u
ng
flo
w
er
buds
ar
e
r
e
mo
v
ed
from
the
pla
n
t
and
s
u
rface
s
t
e
r
ili
z
ed.
I
m
mature
sta
g
e
o
f
ant
h
e
r
o
r
late
st
a
ge
of
fi
l
l
ed
p
o
l
len
u
sua
l
ly
grow
a
b
n
o
rmally
or
the
a
n
ther
cont
a
ining
s
t
a
r
ch
d
e
v
e
lo
p
ment
is
g
e
n
e
r
a
lly
in
e
f
f
e
c
t
ive
a
nd
h
e
nce
f
or
b
e
t
t
er
r
e
spo
n
s
e
al
w
a
y
s
s
e
l
ect
matu
r
e
a
n
ther
o
r p
o
lle
n
.
The
a
n
the
r
s
are
then
carefu
l
ly
e
x
c
i
s
e
d
and
t
r
a
ns
f
e
r
r
e
d
to
an
appropria
t
e
n
u
trient
mediu
m
.
The
a
n
the
r
s
ar
e
g
e
n
e
r
a
l
l
y
cul
t
u
r
ed
on
a
solid
a
g
ar
medium
where
they
d
e
v
e
lop
into
emb
r
y
o
ids
for
a
n
th
e
r
cul
t
u
r
e
u
n
d
er alternate
l
ig
h
t
an
d
dar
k p
e
rio
d
.
Anther
cul
t
u
r
e
Pol
l
en
o
r
p
o
llen
g
r
ai
n
s
o
f
di
f
fer
e
nt
s
p
eci
e
s
h
a
ve
bee
n
succe
s
sfully
t
o o
b
tain
lar
g
e
n
u
mber
o
f
h
a
ploid
pla
n
t
s
.
g
r
a
i
n
s
r
e
mo
v
ed
f
r
o
m
the
a
nt
h
e
r
eith
e
r
m
ech
a
n
i
c
a
lly
o
r
by
n
a
tur
a
lly
d
e
h
i
s
c
e
nce.
A
n
the
r
s
plac
e
d
in
5
m
l
o
f
liquid
medi
u
m
in
a
p
e
tri
dish
c
o
ntaini
n
g
p
o
ll
en
g
r
ains
with
p
a
r
a
film
an
d
in
c
uba
t
e
d
.
A
f
ter
d
e
v
e
lop
e
d.
in
the
cul
t
u
r
e
media
ar
e
s
e
aled
in
c
u
b
ation
h
a
p
l
oid
pla
nt
lets
are
47
v
i
.
Ovule
a
n
d
e
m
bryo
cultu
r
e
Mat
u
re
e
m
b
ryos
a
re
e
x
c
i
s
ed
from
ri
p
en
e
d
o
v
u
l
e
/
s
ee
d
s
and
c
u
lt
u
red
m
a
i
n
l
y
to
a
void
i
n
h
i
b
i
ti
o
n
i
n
the
s
e
ed
for
ge
r
mina
t
i
o
n
.
V
ery
s
mall
g
l
ob
u
l
a
r
e
m
bryos
re
q
u
i
r
e
a
de
l
i
c
ate
b
a
l
a
n
c
e
o
f
the
ho
r
mone
s
.
Em
b
ryo
i
s
d
i
s
s
e
c
t
ed
from
the
ovul
e
/
s
eed
an
d
pu
t
i
n
to
c
u
lt
u
re
medi
a
.
Th
i
s
type
o
f
c
u
lt
u
re
i
s
re
l
at
i
vely
e
a
s
y
a
s
the
e
m
b
r
yos
re
q
u
i
re
a
s
i
mple
nu
t
r
i
ent
medi
u
m
c
o
nt
a
i
n
i
n
g miner
a
l
s
a
lt
s
,
s
u
gar
a
nd
agar
for growth
and
developmen
t
.
Al
s
o,
mult
i
cell
u
l
a
r
imm
a
ture
c
u
lt
u
red
a
s
ep
t
i
c
a
l
ly to
ob
t
a
i
n
i
s
re
s
c
u
ed,
two
ge
n
o
m
e
s
are
to
pr
o
du
c
e
a
fer
t
i
l
e
p
l
an
t
.
e
mb
r
y
o
s
are
d
i
sse
c
ted
ou
t
a
nd
viab
l
e
hybri
d
s
.
O
n
ce
the
e
mb
r
yo
ne
e
d
e
d
to
b
e
c
o
m
b
i
ned
to
g
et
h
er
By
th
i
s
me
t
hod
do
r
ma
n
cy
pe
r
i
o
d
o
f
s
e
eds
c
a
n
b
e
s
h
ort
e
n
e
d,
a
s
well
a
s
ha
p
l
o
i
d
s
c
a
n
b
e
pr
o
du
c
e
d
.
By
ovule
c
u
lt
u
re,
i
t
i
s
po
s
s
i
b
l
e
to
gro
w
,
s
t
udy
v
a
r
i
ous
nu
t
ri
t
i
o
nal
re
q
u
i
rements
an
d
s
t
ag
e
s
young
em
b
ryos
o
r
zygo
t
e
.
48
v
i
i.
Ovari
e
s
cultu
r
e
Ova
r
i
e
s
excised
af
t
er
p
o
ll
i
nat
i
on
can
pro
d
u
c
e
f
r
u
i
ts
on
a
sim
p
le
med
i
um
contain
i
ng
mi
n
eral
s
a
lts,
sugar
and
vitam
i
n
s
.
Ova
r
i
e
s
t
a
ken
f
r
om
u
n
-
p
ol
linat
e
d
flow
e
rs
f
a
i
l
t
o
produce
f
r
u
i
ts
on
s
uch
a
simple
med
iu
m
but
can
deve
l
op
i
nto
seed
l
ess
f
r
u
i
ts
h
o
rmone
s
.
on
a
med
i
um
supp
l
emented
with
B
y
this
cul
t
uri
n
g
me
t
hod
can be studie
d
.
phy
s
io
l
ogy
of
f
r
u
i
t
deve
l
op
m
ent
H
a
p
l
o
i
ds
can
be produce
d
.
R
a
re
hy
b
rids
can a
l
so
b
e prod
u
ced
b
y ov
a
ry
cultur
e
.
D
o
r
m
ancy
peri
o
d
of seeds
can be reduce
d
.
49
viii
.
Nuc
e
llus
cultu
r
e
Nucellar
tissu
e
s
e
x
cised
fr
o
m
u
n
fe
r
tilised
ov
u
l
e
s
h
a
ve
no
a
d
ve
n
titive
cont
a
in
i
ng
embryoi
ds
.
If
cult
u
red
on
a
medium
malt
e
x
t
ract
a
n
d
a
r
e
aden
i
n
e,
embryoi
d
s
are
g
e
rm
i
n
a
t
e
.
fo
r
med
but
th
e
y
u
n
a
b
l
e
to
When
such
em
b
ryoids
are
e
x
c
ised
and
cultured
o
n
a
medium
c
o
n
taining
gibberelli
n
,
pla
n
tlets
may
be
formed
which
can
th
e
n
be
tra
n
s
p
la
n
t
e
d
to
the
fiel
d
.
Diseas
e
-
f
r
ee
cult
u
r
e
.
cl
o
n
e
s
can
be
o
b
tained
by
n
u
ce
l
l
u
s
50
i
x
.
Seed
cu
l
tu
r
e
T
h
e
see
ds
a
r
e
t
r
ea
te
d
w
ith
70
%
a
lco
h
ol
for
abo
ut
two
minu
t
es,
w
a
s
hed
w
i
t
h
sterile
d
i
s
tilled
w
a
t
e
r
,
trea
t
e
d
w
i
th
surface
steri
l
iz
i
ng
agent
On
c
e
a
g
ain
r
i
n
s
ed
wi
t
h
for
spec
i
fic
per
i
o
d
.
ste
r
i
l
i
ze
d
d
istill
e
d
w
a
t
e
r
an
d
k
e
pt
for
g
e
r
mina
t
ion
by
p
lac
in
g
t
h
e
m
on
dou
ble
l
a
y
ers
of
p
r
e
-
w
i
t
h
ste
r
i
l
i
z
e
d
fi
l
ter
p
ape
r
,
pla
c
ed
i
n
p
etr
i
-
d
i
s
h
m
o
ist
ene
d
ste
r
i
l
i
z
e
d
dis
t
i
l
l
ed
w
a
t
e
r
o
r
p
l
aced
o
n
m
o
ist
ene
d
cott
o
n
s
wab
i
n
petr
i
-dish.
T
h
e
se
eds
a
r
e
g
e
r
mi
n
at
e
d
i
n
da
rk
a
t
2
5-
28
°
C
and
s
m
all
p
art
o
f
the
see
d
l
i
ng
i
s uti
l
iz
e
d for
the
i
n
iti
a
tion
o
f
ca
l
l
u
s
.
x.
Cotyledon
cultu
r
e
Im
m
a
t
u
r
e
c
oty
l
e
d
o
n
d
e
v
elo
p
s
i
n
t
o
s
o
m
atic
e
mb
r
y
os,
s
h
oot
b
uds
a
nd
c
o
m
ple
t
e
pl
a
nts
i
f
c
u
l
t
ur
e
d
o
n
a
s
u
it
a
ble
n
utr
ien
t
med
i
u
m
.
51
xi.
Endos
p
e
r
m
cultu
r
e
If
e
n
d
o
sperm
is
cult
u
red
o
n
a
pro
p
er
n
u
trient
medium
co
n
tinued
prolifer
a
t
i
on
of
cal
l
us
mass
tak
e
s
place
an
d
subse
q
ue
n
tly
plant
i
s
rege
n
erate
d
.
It
is
us
e
ful
for
pla
n
t
bre
e
ding
an
d
h
o
rticulture
s
.
It
also
us
e
ful
in
the
pro
d
uction
of
pla
n
t
l
ets
as
an
alt
e
rnative
to
the
c
o
nv
e
ntional
meth
o
ds
of
crossing
ind
u
cti
o
n
i.
e
.
te
t
raploi
d
s
with
dipl
o
ids
or
t
riploids
which
is
a
p
plica
b
le
to
fruit
tree
s
.
52
x
i
i.
Fruit cultu
r
e
The
c
u
lture
o
f
fru
i
t
t
issues
a
s
w
h
ole
organ
or
i
s
o
lated
tissue
se
c
t
ion
su
c
h
a
s
o
v
a
r
y
h
a
s
bee
n
s
u
c
ces
s
f
u
lly
t
o mature
fruits
e
.
g
. s
t
r
a
w
b
e
r
r
y
.
c
u
lt
u
r
e
d
t
o
gi
v
e
r
i
se
Usua
l
ly
w
h
e
n
a
n
i
s
o
lat
e
d
p
o
rtion
o
f
the
into
a s
t
e
r
ile e
n
vi
r
o
n
ment.
I
t
im
m
e
d
iat
e
ly
lo
s
e
s
s
t
r
u
c
t
u
r
al
i
ntegr
i
ty
r
a
pidly
di
v
iding
c
allus
mas
s
.
L
o
ss
o
f
s
t
r
u
c
t
u
r
al
i
n
teg
r
i
ty
is
ass
o
ci
a
ted
fruit
tiss
u
e
i
s
intro
d
u
ced
and
degenera
t
e
s
into a
corr
e
sp
o
n
d
ingly
with
an
alter
a
tion
o
f
p
h
ysiol
o
gy
that
is
s
u
bseq
u
e
n
t
ly
r
e
fle
c
ted
in
the
p
r
o
d
uc
t
ion
o
f
a
n alter
e
d
metab
o
li
s
m
.
So
it
is
n
o
t
p
o
ssi
b
le
to
ma
k
e
a
mea
n
ingf
u
l
s
t
u
d
y
o
f
f
r
ui
t
s
d
e
v
e
lopi
n
g
using
callus
d
e
ri
v
ed cul
t
u
r
e.
The
use
o
f
f
r
u
it
cul
t
u
r
e
i
s
t
o
ser
v
e
a
s
a
bio
a
s
s
ay
s
ys
t
em
t
o
stu
d
y
fruits matur
a
tion e
v
e
n
ts
within
a controlled
e
n
vi
r
o
n
ment.
x
i
i
i
.
Pl
a
nt
cell
cultu
r
e
C
u
lture
o
f
i
solat
e
d
c
e
l
l
s
or
v
e
ry
s
m
a
l
l
a
g
g
r
e
g
a
tes
r
e
m
a
i
ning
dispersw
e
d
in liquid
mediu
m
.
53
b)
HAI
R
Y
/
TRAN
S
F
O
RMED
RO
O
TS
(TRAN
S
INFE
C
TION)
Cer
t
a
i
n
soil
bac
t
eria
of
the
genus
A
g
r
obac
t
erium
(
G
ram
nega
t
ive
bac
t
eri
a
)
in
f
ec
t
s
a
wide
range
of
pl
a
nt
sp
e
c
i
es
and
c
auses
the
inf
e
c
t
ion
i
n pl
a
nt t
e
r
m
ed
a
s
“
Hairy
roo
t
”
dis
e
as
e
.
The
disease
i
s
trans
f
o
r
m
ed
b
y
e
t
h
e
i
r
geno
m
e
t-
D
NA fr
o
m
a
bac
t
eri
a
l
pl
a
s
m
id
t
o
pl
a
nt
ha
i
ry
root ce
l
ls.
A l
a
r
ge
nu
m
ber
of
s
m
a
l
l
fine
ha
i
r
y
r
oots
cover
e
d
with
root
h
a
i
rs
orig
i
na
t
ed
dire
c
t
l
y
f
rom the
exp
l
ant
i
n
response
t
o
Ag
r
obac
t
erium
r
hizogenes
or
Ag
r
obac
t
erium
tume
f
ac
i
ens
.
The
ha
i
ry
ro
ots
are
produced
by
in
o
cu
l
a
t
ing
the
host
pl
a
nt
when
grown
i
n
a
hor
m
one-f
r
ee
m
e
dium
give
rise
to
cop
i
ous
roots
referred
t
o
a
s
'
transfor
m
ed
roots'
or
'
ha
i
ry
roots
'
.
Th
e
se
are
fast
growi
n
g,
hi
g
hly
bran
c
hed
adv
e
nt
i
tious
roo
t
s
a
t
the
site of
infe
c
t
i
on.
They
are
gene
t
ical
l
y
stab
l
e
and
a
f
fect
a
wide
range
of
di
c
ot
y
l
e
donous
plants and have sa
m
e
m
e
t
abo
l
ic
fea
t
ure
s
.
54
Esta
b
lishment/Meth
o
d
o
l
o
gy
o
f
Hairy
Ro
o
t
Cult
u
r
e
T
h
e
ex
p
lant
m
a
ter
i
al
is i
n
oc
u
lated
with
a
sus
p
ens
i
on
of
A
g
r
o
b
acterium
r
h
i
z
o
ge
n
es
ge
n
erat
e
d
b
y
g
r
owi
n
g
b
a
cter
i
a
in
YMB
m
e
d
i
u
m f
o
r
two
d
a
y
s
a
t
2
5
°C
with
g
y
r
atory
(r
o
u
n
d
circl
e
)
sha
k
ing,
pelleti
n
g
b
y
centrif
u
gati
o
n (
5×
1
0
r
p
m;
20
m
i
n
)
and
sus
p
e
n
d
i
ng
t
h
e
bact
e
ria
in
YMB
m
edi
u
m
to
form
a t
h
ick
suspensio
n
.
T
ransf
o
r
m
a
t
i
on
m
ay
b
e
ind
u
ced
a
s
as
c
ep
t
ic
p
l
ants
grown
fr
o
m
see
d
s
,
o
f
o
n
detac
h
ed
leaves,
leaf
d
i
sc
s
,
peti
o
les
o
f
stem
seg
m
ents
fr
om
g
r
een
h
o
use
p
l
ants
f
o
ll
o
w
i
n
g
sterilization
o
f
excised tiss
u
e
with
1
0
%
(
v
/
v
)
d
o
m
estos
f
o
r
2
0
m
i
n
u
t
es.
Scrat
c
hi
n
g
the
m
idrib
o
f
a
le
a
f
o
r
the
stem
o
f
a
plan
t
let,
with
the
need
l
e
o
f
a
h
y
poder
m
i
c
s
y
ri
n
ge
c
ontain
i
ng
t
he
t
h
ick
bacterial
sus
p
ens
i
on
allows
i
n
oc
u
lati
o
n
with
s
m
all
(ab
o
ut
5
-
10
µl)
d
r
o
p
lets
co
n
tain
i
ng
A
g
r
o
b
acteri
u
m
r
h
i
zo
g
ene
s
.
In
some
spec
i
es
a
p
r
o
f
usi
o
n
o
f
r
o
o
t
s
m
a
y
a
p
pear
d
i
r
e
ctly
a
t
t
h
e
site
of
i
n
oc
u
lati
o
n, but
in
o
t
her
a
callus
will
f
o
r
m
i
n
itially
and
r
o
o
t
s
e
m
e
r
ge
su
b
seq
u
ently
fr
o
m
it.
In
either
c
ase
hair
r
o
t
n
or
m
a
lly
ap
p
ear
with
in
1
-4
we
e
ks
alth
o
u
g
h
t
h
e
suscepti
b
ility
o
f
species
to
i
n
fection
is
varia
b
le.
55
A
d
vantages
of
Hairy Root
C
u
ltu
r
e
Gene
t
ic
and
gr
o
wth
ki
n
e
t
ic
sta
b
i
l
ity
over
p
rol
o
nged
per
i
od
of
growth
i
n
v
i
t
r
o.
Ease
of
c
ul
t
ure
i
n
v
i
t
r
o
using
si
m
p
l
e
m
ed
i
a
lac
k
ing
ph
y
tohor
m
one
s
.
Many
pl
a
nt c
e
ll
cu
l
ture
sy
ste
m
s,
which did not produ
c
e
ad
e
q
u
a
t
e
a
m
ount of
d
esired
co
m
p
ounds
i
s
being
reinv
e
sti
g
a
t
ed
using
h
a
i
ry
root
cu
l
ture
m
e
t
hod
s
.
A
div
e
rsified
range
of
p
l
a
nt
species
has
been
tran
s
for
m
ed
using
various
bac
t
eri
a
l
stra
i
n
s
.
Pl
a
nt
reg
e
ne
r
a
t
io
n
,
pl
a
nt
i
m
prov
e
m
e
nt
and
gen
e
t
i
c
m
anipulat
i
on
in
pl
a
nt
ca
n
be done.
Reproduc
i
ble
& predictab
l
e
l
e
ve
l
s
of
product s
y
nth
e
si
s
.
Capab
i
l
i
ty
t
o
s
y
nth
e
size
(novel)
secondary
m
e
t
abolit
e
s
sp
e
c
i
fic
to
th
a
t
pl
a
nt
s
p
e
c
i
e
s
from
whi
c
h
th
e
y
have
dev
e
loped
i
n
equ
a
l
or
even high
e
r a
m
ount
co
m
pared
t
o fie
l
d grown
pl
a
nts.
More ac
c
u
m
ul
a
t
i
on
of
secondary
m
etabol
i
te
s
.
E
g.
Lev
e
ls
o
f
stero
i
dal
a
l
ka
l
oid
solasodine
i
s
sig
n
if
i
can
t
ly
hi
g
her
i
n ha
i
ry root
cu
l
tures th
a
n ca
l
lus
or
su
s
pension cu
l
tures.
56
P
r
obl
e
ms
associ
a
ted
w
ith H
a
i
r
y Ro
o
t
Cultu
r
e
Exc
e
ss
bac
t
eri
a
l
growt
h
.
Slow
regen
e
rat
i
o
n
.
Decreased
ce
l
l
div
i
sion and transfor
m
a
t
ion due
t
o
stres
s
.
N
o
transfor
m
a
t
ion.
Loss
of gene
e
f
fe
c
ts.
A
p
pli
c
ations
of
Hairy
Root
C
u
ltu
r
e
57
S
r
.
N
o
.
P
l
a
n
t
P
r
o
d
u
c
t
o
b
t
a
i
n
e
d
1
Art
e
m
is
i
a
annu
a
Artim
i
si
n
in
2
Hy
o
scya
m
us
m
utic
u
s
H
y
osc
y
amine
3
N
i
coti
a
na
ta
b
ac
u
m
N
i
coti
n
e,
A
n
at
a
b
i
ne
4
P
o
d
o
p
h
yl
l
um
sp
p
.
L
i
g
n
a
n
s
5
P
a
na
x
g
i
n
s
en
g
P
o
l
y
a
c
e
t
y
l
en
e
ana
l
o
gue
s
6
S
o
l
a
n
u
m
av
i
cu
l
are
S
o
l
a
so
d
i
n
e
7
W
ith
a
n
i
a
so
m
n
i
fera
Witha
n
o
l
i
d
es
8
V
a
l
er
i
a
n
a
w
a
l
l
i
chi
V
a
l
p
o
triat
e
s
c)
P
r
otoplast
cultu
r
e
Proto
p
l
a
st
are
p
l
a
n
t
cel
l
s
w
i
th
a
p
l
a
s
m
a
m
e
m
b
ra
ne
but
without
ce
l
l
wa
ll
,
b
ec
au
s
e
o
f
th
i
s
t
h
e
p
r
o
t
o
pla
s
t
p
ro
v
i
de
the
st
a
r
t
ing
p
oi
nt
f
o
r
many
of
t
h
e
t
ec
h
niq
u
e of
g
e
ne
t
ic
m
a
n
i
p
ul
a
tor
of
p
l
an
t
s,
i
n
part
i
cu
la
r
the
ind
u
c
t
i
on
of
s
o
m
a
clo
n
al
v
a
r
ia
tion,
so
m
atic
hybridization
and
gen
e
tic
transf
e
r
.
They
are
cultivated
in
liquid
a
s
well
a
s
on
solid
m
edi
a
.
Isolation
of
P
r
otoplasts
is
b
y
two
me
t
ho
ds
.
Proto
p
l
a
sts
can
be
i
s
ol
a
ted
f
r
o
m
a
l
m
o
st
nodu
l
es,
a
ll
plant
p
a
r
t
s
i.
e
.
,
roo
t
s,
l
e
a
ve
s,
f
r
u
its,
t
u
b
e
rs,
ro
o
t
e
n
dosp
e
r
m
,
p
ol
l
en
c
e
lls,
and
c
e
lls
of
callus
tissu
e
.
1.
2.
M
e
chanical
me
t
hod
En
z
ymatic
me
t
hod
58
1.
M
e
ch
a
ni
c
al
m
e
thod
The
ce
lls
were
k
ept
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oly
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lys
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brane)
and cut
with
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ine knif
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.
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cut
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t
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ll
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t
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m
echani
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edure
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yie
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d
of
pro
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lasts
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ized
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ac
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ce
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and
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ic
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m
ethod
i
s labo
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ious
and ted
i
ou
s
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59
1
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on
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assif
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o
two
gro
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p
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2.
1
Seq
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enti
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m
at
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is
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nvo
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s
two
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ps
w
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first
m
a
c
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ed
p
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a
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t
tissues
are
i
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with
p
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pr
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.
cell
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in
m
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re
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s a
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l
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l
as
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s
.
62
Plant tissue culture methods such as root tip culture, shoot-tip culture, leaf culture, flower culture, and anther and pollen culture allow for the study of growth, reproduction, and genetic variations in plants. These techniques involve culturing various plant parts under sterile conditions to investigate factors influencing plant development, nutritional requirements, and reproductive processes. Each method serves a specific purpose in understanding plant physiology and can help in producing virus-free plant materials.
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2. a) Depending on the part used for culture Organ Culture i. Root tip culture (Meristem root tip culture) Root apical meristem is mainly responsible for the growth of roots by cell division, cell enlargement and cellular differentiation. Tips of the lateral roots are sterilised, excised and transferred to fresh medium. The lateral roots continue to grow and provide several roots, which after seven days, are used to experimental cultures. By this culture method it is possible to study the nutritional requirements of roots, shoot and root growth, conditions required for the development of secondary vascular tissues, lateral root and bud formation, nodulation etc. 43
ii. Shoot-tip culture (Meristem shoot tip culture) The shoot apex or shoot-tip (100-1000 m long) consists of the apical meristem and one to three adjacent leaf primordia. Actively growing shoot-tip is surface sterilized and is placed on a defined culture medium under sterile conditions. Shoot Node culture is simplified form of shoot culture, which is yet another method for production from pre-existing meristem. It is the simplest method but is associated with the least genetic variation. Some of the crop species that have been freed of viruses by this technique, they include soyabean, sweet potato, sugar cane and rhubarb. This method is used with both monocot and dicot species. 44
iii. Leaves or leaf primordia culture The growth of leaves on the culture medium depends upon the stage of the leaves during excision. It is observed that explants from immature young leaves grow better than explants from older leaves. Leaves (800 m long) may be detached from shoots, surface sterilized and placed on a solidified medium where they will remain in a healthy conditions for a long period. It is believed that leaf culture depends upon the physiological state and the age of the leaf. The shoot forming potentials differ in the leaf cultures as per the derivation of the explant and the hormonal factors involved. 45
iv. Flower culture (Meristem - floral culture) Flowers two days after pollination are excised, sterilized by immersion in 5% calcium hypochlorite, repeatedly transferred medium. When fruits are obtained on medium supplemented with growth hormones. Flowers excised before do not produce fruits. washed to culture with tubes containing an sterilized water and agar cultured, such flowers produce fruits. Larger pollination This microclimates or nutritional effects on the and reproductive processes of the plant. culture system is useful in studying vegetative 46
v. Anther and pollens culture Young flower buds are removed from the plant and surface sterilized. Immature stage of anther or late stage of filled pollen usually grow abnormally or the ineffective and hence for better response always select mature anther or pollen. The anthers are then carefully excised and transferred to an appropriate nutrient medium. The anthers are generally cultured on a solid agar medium where they develop into embryoids for anther culture under alternate light and dark period. Anther culture Pollen grains removed from the anther either mechanically or by naturally dehiscence. Anthers placed in 5 ml of liquid medium in a petri dish containing pollen grains with parafilm and incubated. After developed. anther containing starch development is generally or pollen grains of different species have been successfully to obtain large number of haploid plants. in the culture media are sealed incubation haploid plantlets are 47
vi. Ovule and embryo culture Mature embryos are excised from ripened ovule/seeds and cultured mainly to avoid inhibition in the seed for germination. Very small globular embryos require a delicate balance of the hormones. Embryo is dissected from the ovule/seed and put into culture media. This type of culture is relatively easy as the embryos require a simple nutrient medium containing mineral salts, sugar and agar for growth and development. Also, multicellular immature cultured aseptically to obtain is rescued, two genomes are to produce a fertile plant. By this method dormancy period of seeds can be shortened, as well as haploids can be produced. By ovule culture, it is possible to grow, study various nutritional requirements and stages young embryos or zygote. embryos are dissected out and viable hybrids. Once the embryo needed to be combined together 48
vii. Ovaries culture Ovaries excised after pollination can produce fruits on a simple medium containing mineral salts, sugar and vitamins. Ovaries taken from un-pollinated flowers fail to produce fruits on such a simple medium but can develop into seedless fruits hormones. By this culturing method can be studied. Haploids can be produced. Rare hybrids can also be produced by ovary culture. Dormancy period of seeds can be reduced. on a medium supplemented with physiology of fruit development 49
viii.Nucellus culture Nucellar tissues excised from unfertilised ovules have no adventitive containing medium malt embryoids are germinate. embryoids. If cultured on a extract are formed but they and adenine, unable to When such embryoids are excised and cultured on a medium containing gibberellin, plantlets may be formed which can then be transplanted to the field. Disease-free culture. clones can be obtained by nucellus 50
ix. Seed culture The seeds are treated with 70% alcohol for about two minutes, washed with sterile distilled water, treated with surface sterilizing agent Once again rinsed with sterilized distilled water and kept for germination by placing them on double layers of pre- sterilized filter paper, placed in petri-dish moistened sterilized distilled water or placed on moistened cotton swab in petri-dish. The seeds are germinated in dark at 25-28 C and small part of the seedling is utilized for the initiation of callus. for specific period. with x. Cotyledon culture Immature cotyledon develops into somatic embryos, shoot buds and complete plants if cultured on a suitable nutrient medium. 51
xi. Endosperm culture If endosperm is cultured on a proper nutrient medium continued proliferation of callus mass takes place and subsequently plant is regenerated. It is useful for plant breeding and horticultures. It also useful in the production of plantlets as an alternative to the conventional methods of crossing i.e. tetraploids with diploids or triploids which is applicable to fruit trees. induction 52
xii. Fruit culture The culture of fruit tissues as whole organ or isolated tissue section such as ovary has been successfully to mature fruits e.g. strawberry. Usually when an isolated portion of the into a sterile environment. It immediately loses structural integrity rapidly dividing callus mass. Loss of structural integrity is associated alteration of physiology that is subsequently reflected in the production of an altered metabolism. So it is not possible to make a meaningful study of fruits developing using callus derived culture. The use of fruit culture is to serve as a bioassay system to study fruits maturation events within a controlled environment. cultured to give rise fruit tissue is introduced and degenerates into a correspondingly with an xiii. Plant cell culture Culture of isolated cells or very small aggregates remaining disperswed in liquid medium. 53
b) HAIRY/ TRANSFORMED ROOTS (TRANSINFECTION) Certain soil bacteria of the genus Agrobacterium (Gram negative bacteria) infects a wide range of plant species and causes the infection in plant termed as Hairy root disease. The disease is transformed bye their genome t-DNA from a bacterial plasmid to plant hairy root cells. A large number of small fine hairy roots covered with root hairs originated directly from the explant in response to Agrobacterium rhizogenes or Agrobacterium tumefaciens. The hairy roots are produced by inoculating the host plant when grown in a hormone-free medium give rise to copious roots referred to as 'transformed roots' or 'hairy roots'. These are fast growing, highly branched adventitious roots at the site of infection. They are genetically stable and affect a wide range of dicotyledonous plants and have same metabolic features. 54
Establishment/Methodology of Hairy Root Culture The explant material is Agrobacterium rhizogenes generated by growing bacteria in YMB medium for two days at 25 C with gyratory (round circle) shaking, pelleting by centrifugation (5 10 rpm; 20min) and suspending the bacteria inYMB medium to form a thick suspension. Transformation may be induced as asceptic plants grown from seeds, of on detached leaves, leaf discs, petioles of stem segments from green house plants following sterilization of excised tissue with 10% (v/v) domestos for 20 minutes. Scratching the midrib of a leaf or the stem of a plantlet, with the needle of a hypodermic syringe containing the thick bacterial suspension allows inoculation with small (about 5-10 l) droplets containing Agrobacteriumrhizogenes. In some species a profusion of roots may appear directly at the site of inoculation, but in other a callus will form initially and roots emerge subsequentlyfrom it. In either case hair rot normally appear with in 1-4 weeks although the susceptibility of species to infection is variable. inoculated with a suspension of 55
Advantages of Hairy Root Culture Genetic and growth kinetic stability over prolonged period of growth in vitro. Ease of culture in vitro phytohormones. Many plant cell culture systems, which did not produce adequate amount of desired compounds is being reinvestigated using hairy root culture methods. A diversified range of plant species has been transformed using various bacterial strains. Plant regeneration, plant improvement and genetic manipulation in plant can be done. Reproducible & predictable levels of product synthesis. Capability to synthesize (novel) secondary metabolites specific to that plant species from which they have developed in equal or even higher amount compared to field grown plants. More accumulation of secondary metabolites. Eg. Levels of steroidal alkaloid solasodine is significantly higher in hairy root cultures than callus or suspension cultures. using simple media lacking 56
Problems associated with Hairy Root Culture Excess bacterial growth. Slow regeneration. Decreased cell division and transformation due to stress. No transformation. Loss of gene effects. Applications of Hairy Root Culture Sr. No. 1 Artemisia annua 2 Hyoscyamus muticus 3 Nicotiana tabacum 4 Podophyllum spp. 5 Panax ginseng 6 Solanum aviculare 7 Withania somnifera 8 Valeriana wallichi Plant Product obtained Artimisinin Hyoscyamine Nicotine,Anatabine Lignans Polyacetylene analogues Solasodine Withanolides Valpotriates 57
c) Protoplast culture Protoplast are plant cells with a plasma membrane but without cell wall, because of this the protoplast provide the starting point for many of the technique of genetic manipulator of plants, in particular the induction of somaclonal variation, somatic hybridization and genetic transfer. They are cultivated in liquid as well as on solid media. Isolation of Protoplasts is by two methods. Protoplasts can be isolated from almost roots, leaves, fruits, tubers, root cells, and cells of callus tissue. 1. 2. Enzymatic method all plant parts i.e., endosperm, pollen nodules, Mechanical method 58
1. Mechanical method The cells were kept in a suitable plasmolyticum (lysis of plasma membrane) and cut with a fine knife. Cells were cut only through the cell wall, releasing intact protoplast. This mechanical procedure gave low yield of protoplasts and could be utilized for only highly vacuolated cells. and non meristematic The method is laborious and tedious. 59
1. Mechanical Method Cells Plasmolysis Plant Tissue Microscope Observation of cells Release of protoplasm Cutting cell wall with knife Collection of protoplasm
2. Enzymatic Method Leaf sterilization, removal of epidermis Filtration through Nylon mesh Macroenzyme (Pectinase) in 13% Mannitol Isolated cells + 2% Cellulose 1 and a half hour Cellulose + Pectinase 2-3 hrs at 20-22 C Protoplast
2. Commercial preparations of purified cell wall degrading enzymes such as macroezyme, cellulase and hemicellulose became available that gave further progress to enzymatic isolation of protoplasts. By this method very large number of protoplast are obtained compared to mechanical method. Cells are not damaged or broken Osmotic shrinkage of protoplast is much less. Enzymatic method Enzymatic method of protoplast isolation can be classified into two groups. 2.1 Sequential enzymatic This involves two steps where first macerated plant tissues are incubated with pectinase to get single protoplast. cells followed by cellulase treatment to get 2.2Mixed enzymatic This involves simultaneous separation of cells and degradation of their walls to convert protoplast by immersing plant tissues in mixture of pectinases and cellulases. 62
