Agric. Rev., 23 (1) : 1 - 13, 2002
MICROPROPAGATION IN CITRUS - A REVIEWI.P. Singh
National Research Centre for Citrus,Amravati Road, Shankar Nagar, Nagpur - 440 010, India
ABSTRACTCitrus trees are propagated both by seed and by vegetative means. Vegetative propagation
is preferred because it ensures true to type plants, uniform quality, regular bearing etc. Tissueculture technique particularly micropropagation, is now gaining popularity due to many reasons.Micropropagation has many advantages over conventional methods of vegetative propagationand its application in citrus is currently.expanding world wide. The goal of micropropagation is toobtain a large number of genetically identical, physiologically uniform and developmentally normal plantlets preferably with a high photosynthetic potential to survive the harsh ex vitro conditions, in a reduced time period and at a lowered cost. The performance of micropropagatedplantlets should be at par in comparison to plants raised by conven,~nal method.
Citrus is the most important fruit crop and yield due to infection and preservation ofin the world and is produced in over 100 coun- germplasm. Hence micropropagation is a verytries in all six continents, and it· is often re- useful tool for a production of large number ofgarded as golden fruit or queen of aH fruits (Nita, planting materials. Besides, this technique is1996). In India, citrus fruits rank third in area also useful for saving the citrus species whichand production after mango and banana with are facing extinction.an estimated production of 37.0 Lakh to~nes Tissue culture in citrus ,and an area of 4.4 Lakh hectares (Chadha, The importance of tissue culture in cit1997) and contributes 4.78% towards world's rus research was recognised long back, andtotal citrus. Total cultivation worldwide exceeds amply emphasized by Bitters and Murashige2x106 hectares and production at over 63 mil- (1967) and Kochba and Spiegel-Roy (1976).lion metric tonnes, far surpasses that of all de- The far reaching significance of tissue cultureciduous fruit crops. in citrus breeding for improvement and aug-
The important commercial citrus fruits ,menting production was discussed by Kochbain India are the mandarin orange (Citrus and Spiegel-Roy (1977) and various other asreticulata Blanco), followed by sweet orange pects of citrus tissue culture by Button and(C. sinensis Osbeck) and acid., lime Kochba, (1977) and Spiegel-Roy and Kochba(C.aurantifolia Swingle) and lemon (C./imon (1980). .Burm). In India, mandarins constitute about Micropropagation410/0,. sweet orange 23% and limes and lem- . Regeneration of plan'tlets can beons about 23% of total citrus produced (Ghosh, achieved from a wide range of explants such1997). CitnJstrees are propagated both byseed as stem sections (Grinblat 1972,. Chaturvediand by vegetative means. There is huge de- and Mitra 1974, Raj Bhansali and Arya 1978,mand of planting material. Non availability of 1979; Barlass and Skene 1982), root secscientifically propagated planting material from tions (Raj Bhansali and Arya 1978, Simelite clones for plantation are the maincon- etal,1989 and Duran-villa eta/" 1989), root.straints in citrus cultivation. In recent years, meristems (Sauton. et al., 1982), shoot·andtissue culture techniques (micropropagation) are shoot tips (Sim etal, 1989, Singh etaJ., 1994,increasingly used for rapid donal propagation Kitto and Young 1981 & Barlass and Skeneof several economic plants, restoration of vigour 1982) and leaf sections (Chaturvedi and Mitra
~,~
2 AGRICULTURAl.:. REVIEWS
1974, Yelenosky 1987). Protocols have been slow increase in clonal multiplication.standardised for mass multiplication of differ- A summary of results obtained froment citrus species of NEH Region in lCAR R~. micropropagation of citrus are given in Table'Complex for NEH Region, Meghalaya 1.
(Parthasarathy et al 1996, Parthasarathy, Bud establishment and shoot proliferation1993, Parthasarathy and Parthasarathy, 1993, S ful shoot proliferation has beenparthasarathy and Nagaraju, 1992, 1994a, achieved ~cc~ citrus types belonging to 191994b,1996a 1996b).. species by Parthasarathy etal (1998). Among
In the field of micropropagation in cit- cytokinins they found BAP is the most suitablerus, it was the efforts of Murashige, etaI. (1972) cytokinin for proliferation of shoots. Morethat triggered the advent of shoot tip culture in number of shoot, leaves and nodes were foundcitrus varieties. They developed the technique with BAP than kinetin. BAP at 0.75 mg/!mainly for obtaining virus-free propagative produced maximum number of shoots, theybudwood in order to lower the economic losses were shorter than the shoots produced by lowercaused. by citrus virus and virus-like diseases. concentration of BAP (0.1 mg/l). One cm longMicropropagation has been the most success- in vitro shoot tips of 33 citrus types/cultivarsful out come of plant tissue culture research belonging to 19species of citrus when culturedwith profound commercial appUcation mostly on MS media. supplemented with 0.75 mg/!in herbaceous plants (Murashige, 1974, 1978; BAPrevealed that proliferating ability of enVasil and Vasil, 1980). The spectacular suc- demic species like C indica, C assamensisandcess achieved have been mostly in ornamental C latipes was low (Table 2).and fruit crops which have revolution~ed the The shoot meristem culture of citrus'tissue culture (~ey, 1997; Monaco.et al provides an important means of virus elimina1977; Chaturvedl 1979; Chaturv~dl and tion (Chaturvedi and Sharma, 1987). They alsoSharma, 1979; George and Shernngton, suggested an alternate procedure for meristem1984). culture by culturing single node segments ob-
Micropropagation has many advan- tained from field grown plants. Chaturvedi andtages over conventional methods of vegetative Mitra (1974) established from callus culture ofpropagation (cutting or seed) and its ~pplica- Citrusgmndismaximum number of shoot budstion in Horticulture, Agriculture and Forestry using 0.25mg/! of BAP with O.lmg of NAA.is currently expanding world wide (Jeong This was probably one of the first cases of sucet al, 1995). The goal of micropropagation cess with citrus. Barlass and Skene (1982)is to obtain a large number of genetically iden- produced successfully micropropagated plant- '.tical, physiologically uniform and developmen- lets using 10mg/! of BAP for shoot proliferatally normal plantlets preferably with a high tion and same concentration of NAA for rootphotosynthetic potential to survive the harsh lng in case of Carrizo citrange, trifoliate orex vitro conditions, in a reduced time period ange, Cleopatra mandarin, Rangpur lime andand at a lowered cost. sweet orange. Starrantino and Caruso (1988)
In recent years micropropagation is obtained success in citrange with SAP (0.5m~increasingly used for rapid donal multiplication, 1) with lBA supplementation ~0.5 rng/l). Ed~~srestoration of vigor, yield and conservation of and Burger (1984) also obtamed success wnhgermplasms. Tissue culture method of plant epicotyle segment using BAP (0.~5 mg/ll a~dpropagation is useful alternative in plant propa- NAA (1 mg/l) in case of Troyer cltrange..Slffigation when conventional methods permit only et al. (1989) presented protocol for
Tab
le1.
Sum
mar
yof
-lite
rat6
reonmicroprop~gation
ofc
itrus
.
Spe
cies
Co
mm
on
Age
of
Exp
lant
Sho
otre
gene
ratio
nm
ediu
mR
oot
indu
ctio
nm
ediu
mR
efer
ence
sn
am
em
ater
ial
sour
ceB
asal
med
ium
Gro
wth
Bas
alm
ediu
mG
row
th(a
nd%
sour
ce)
com
pone
nt(a
nd%
sour
ce)
com
pone
nt(m
gII)
(mgI
I)
1---_._-~
69
23
45
78
---_
._--_
..
Cit
rus
Wes
tIn
dian
SS
tem
,ro
ot
Mod
.M
S(5
%)
BA
(0.5
%)+
Mod
.M
S(2
%)
NA
A(1
-2)
Raj
Bha
nsal
iau
rant
jfoIi
aac
idlim
eM
E(1
000)
(C/M
)IA
A(2
),011
and
Ary
a(C
IM)
IBA
(1-2
)(1
978a
,b)
PK
M.1
SS
tem
MS
(3%
)B
A(0
.25)
+M
S3
/4(3
%)
NA
A(3
)T
hiru
mal
ai&
NA
A(O
.l)
Tha
mbu
raj,
19
97
Kag
zilim
eS
Ste
mM
SB
AP
(0.7
5)
Par
thas
arat
hy&
Nag
araj
u,1
99
6C
.ja
mbh
iri
Soh
myn
dong
SS
tem
MS
BA
P(.
75
)P
arth
asar
athy
&N
agar
aju
19
96
~C
.au
rant
ium
Sou
ror
ange
S,M
Ste
mM
S(3
%)
K(l
)+N
AA
(l)
No
Cha
nge
No
chan
geB
ouzi
d(1
975)
CIJ
.B
rezi
liaS
Ste
mM
S(3
%)
BA
P(2
)M
S(3
%)
IBA
(l)+
NA
AC
anet
ai.
,1
99
2r..:
> S'"P
trifo
liate
xC
itrem
on1
45
2M
Ste
mM
S(5
%)
BA
MS
l/2
(2.5
%)
Thi
amin
e(0
.2)
Mas
etai
.,·1
994
zC
.{e
mai
l(0
.5)+
NA
A(0
.5)
&N
AA
(0.1
5)
<:>
c.gr
andi
sS
hadd
ock
SS
tem
,le
afM
od.
MS
(5%
)B
A(0
.25
),N
och
ange
NA
A(0
.1-0
.5)
Cha
tUlv
edia
nd....
NA
A(O
.I),
Mitr
a(1
974)
r..:>
0+
ME
(500
)0 r..:
>P
umm
elo
SS
tem
MS
BA
P(0
.75
)P
arth
asar
athy
&N
agar
aju,
19
96
C.
volk
amer
iand
Vol
kam
erle
mon
SS
tem
MS
BA
PO
.75
,P
arth
asar
athy
&N
agar
aju
19
96
C.
lim
etti
oide
sS
wee
tlim
eS
Ste
mM
od.
MS
(3%
)B
A(0
.5),
Mod
.M
S(2
%)
NA
A(1
-2-5
)+R
ajB
hans
ali
(C/M
)K
(0.2
5),
NA
A(C
/M)
K(O
.l)an
dA
rya
(197
9)(0
.2)+
ME
(500
)C
./im
on
Le
mo
nS
,MS
tem
.M
S(3
%)
K(l
)+N
AA
(l)
No
chan
geN
och
ange
Bou
zid
(197
5)S
Roo
tM
od.
MS
(3%
)B
A(I
)+2
,4-
MS
(3%
)S
auto
net
al.
(0(0
.1)
(198
2)A
ssam
lem
onM
Sho
ottip
sM
S(3
%)
BA
(l)+
Kin
eti
nM
S(3
%)·
BA
P(0
.25
)+N
AS
ingh
etal
.,(0
.5)+
NA
A(0
.5)
A(0
.5)+
IBA
(0.5
)1
99
4C
.m
adur
ensi
sC
alam
ondi
nS
Ste
mM
S(5
%)
BA
(0.1
-10)
MS
(5%
)N
AA
(O.I
)G
rinbl
at(1
972)
NA
A(O
.Il,
+M
E(5
00
)+
ME
(50
0)
(Con
td.
---_
.__._
---------
...1
23
45
67
89
SS
tem
Whi
te1
94
3B
arbi
tone
(5)
or
No
chan
geN
och
ange
Ran
gasw
amy
(2%
)T
ukey
,M
E(5
0)(1
975)
19
38
(0.5
%gl
ucos
e)S
Sho
otti
pW
hite
19
43
Ade
nine
(40)
or
No
chan
geN
och
ange
Ran
gasw
amy
(2%
)C
CM
(40
%v/
v)(1
975)
C.
para
disi
Gra
pefr
uit
SS
tem
,le
afM
od.
MS
(5%
)B
A(0
.5),
No
chan
geN
och
ange
Raj
Bha
nsal
i&(M
rnN
AA
(0.1
5),
Ary
a(1
978c
)+
ME
(lO
OO
)C
.re
ticul
ata
Man
dari
nS
,MS
tem
MS
(3%
)K
(1)+
NA
A(I
)N
och
ange
No
chan
geB
ouzi
d(1
975)
Cle
opat
raS
,MS
tem
MS
(3%
)B
A(2
)N
och
ange
No
chan
geB
arla
ssan
dM
anda
rin
Ske
ne(1
982)
MS
hoot
apex
1/2
MS
(1.5
%)
or
BA
(0.5
)o
rB
AN
och
ange
No
chan
geB
arla
ssan
dM
S(3
%)
(2)
Ske
ne(1
982)
»K
hasi
man
darin
MS
hoot
tips
MS
(3%
)B
AP
(I)+
MS
(3%
)B
AP
O.2
5+
Sin
ghet
a1.,
C)
Kin
etin
--N
AA
(0.5
)+1
99
422 ("
)(0
.5)+
NA
(0.5
)IB
A(0
.5)
c::C
.Iim
onia
Ran
gpur
lime
S,M
Ste
mM
S(3
%)
BA
(2)
Whi
te1
94
3N
AA
(2-5
)B
arla
ssan
dCj c::
(2%
)S
kene
(198
2)~
C.
sine
sis
Sw
eetO
rang
eS
Ste
m,
leaf
Mod
.M
S(5
%)
BA
(0.2
5)N
AA
No
chan
geN
AA
(0.1
-0.5
)C
hatu
rved
iand
r-(O
.I)+
ME
Mitr
a(1
974)
i(5
00)
MG
ima
ryB
ud
Mod
.M
S(5
%)
BA
(0.2
-2)
No
chan
geN
och
ange
Altm
an
and
Gor
e(1
974)
(J1
S,M
Ste
mM
S+
Hel
les,
BA
(0.5
)+IB
AN
och
ange
No
chan
geB
ouzi
d(1
975)
19
53
(3%
)(1
)S
Ste
mM
od.
MS
(5%
)B
A(0
.5)+
ME
Mod
.M
S(2
%)
NA
A(1
-2)1
MR
ajB
hans
ali
(CIM
)(5
00)
(2),
+IB
A(1
-2)
and
Ary
a(1
978b
)S
Ro
ot
Mod
.M
S(3
%)
BA
(1)+
2,4-
MS
(3%
)S
auto
net
a1.
0(0
.1)
(198
2)S
,MS
tem
MS
(3%
)B
A(2
)W
hite
,1
94
3N
AA
(2-5
)B
arla
ssan
d(2
%)
Ske
ne(1
982)
SS
hoot
apex
1/2
MS
(1.5
%)
or
BA
(0.5
)o
r(2
)W
hite
19
43
NA
A(2
-5)
Bar
lass
and
MS
(3%
)(2
%)
Ske
ne(1
982)
cv.
Mos
ambi
SN
odal
segm
entM
SB
AP
(0.2
5)+
MS
(3%
)N
AA
(I)+
IBA
Moh
anty
eta1
.,1
M(1
.00)
(2)
19
78
(Con
td.
12
34
5·
67
89
e.si
nesi
sx
Citr
ange
SS
tem
Mst
Nits
chan
dB
A(l
O)+
No
chan
geN
AA
(l)
Pri
mo
Mill
oan
dP.
trifo
liate
Nit
sch
,19
65
NA
A(1
0)H
arad
a(1
976)
(5%
)
SR
oot
Mod
.MS
(3%
)B
A(1
)+2
,4-0
MS
(3%
)S
auto
net
al.
(0.1
)(1
982)
S,M
Ste
mM
S(3
%)
BA
(2)
Whi
te,
19
43
NA
A(1
-2)
Bar
lass
and
(2°1c
))S
kene
(198
2)S
,MS
hoot
apex
l/2
MS
(1-5
%)
or
BA
(0.5
)o
rW
hite
,1
94
3N
AA
(1-2
)B
arla
ssan
dM
S(3
%)
BA
(2)
(2%
)S
kene
(198
2)C
arriz
oci
tran
geS
Ste
mM
S(5
%)
BA
(22
urn)
MS
l/2
(2.5
%)
NA
A(5
.41l
ffi)
Moo
re,
19
86
P.tr
ifolia
tex
.Tro
yer
citr
ange
SS
tem
MS
(3%
)B
AP
(0.0
8)
--
Lukm
aneta
l.e.
sine
nsis
(199
0)T
roye
rci
tran
geS
Epi
coty
lM
S(5
%)
BA
P(l
)+M
SN
AA
(2)
Edr
iss
and
~S
egm
ents
NA
A(l
)B
urge
r,(1
984)
Pon
ciru
sT
rifo
liate
oran
geS
Roo
tM
od.M
S(3
%)
BA
(1)+
2,4
-0M
S(3
%)
Sau
ton
etal
.I\
:) wtr
ifolia
te0.
1)(1
982)
- zS
Ste
mM
S(3
%)
BA
(2)
Whi
te,
19
43
NA
A(2
)B
arla
ssan
d9
(2%
)S
kene
(198
2)!""
'e.
indi
caIn
dian
wild
SS
tem
MS
(3%
)B
AP
0.7
5P
arth
asar
athy
I\:) 0
Ora
nge
eta
l.1
99
70 I\
:)
SS
tem
MS
(3%
)B
AP
O.5
Soi
lrite
Bar
uah
etal
.,1
99
6e.
lati
pes
Kha
sipa
peda
SS
tem
MS
(3%
)B
AP
O.7
5P
arth
asar
athy
eta/
.1
99
7K
hasi
Pap
eda
SS
tem
MS
(3%
)B
AP
(0.5
)S
oil r
it!"
Bar
uah
etaI
.,1
99
7C
.ass
amen
sis
Ada
Jam
irS
Ste
mM
S(3
%)
BA
p(0.
5)-
Soi
lrite
Bar
uah
etal
.,1
99
7.
e.un
shiu
cv.
Aos
him
aM
Sho
ottip
sM
S(3
%)
GA
3(50
1lffi
)+M
SN
AA
(0.1
1lffi
)O
mur
aan
dU
nshi
u1
J.lm
BA
+o
rIB
A(1
01lff
i)H
idak
a1
99
2.
O.l
J.lm
NA
A
Sou
rce:
(But
ton
etal
.,1
97
7an
dS
ingh
,2
00
0)
(J1
6 AGRICULTURAL REVIEWS
Table 2. Proliferating ability of shoot tips of certain citrus species in vitro
Spp. Species/Cultivars Mean Mean No. ofNo. shoot No. shoot length nodes
(em)
1. C reticulata Blanco Cv. Khasi mandarin 5.9 7.7 32. C reticulata Blanco Cv. Shuntala 9.5 1.1 43. C sinensisOsbeck Cv. Malta 7.5 6.0 3.14. C sinensisOsbeck Cv. Soh bitara 2.2 7.4 2.75. C limon Burn Cv. Assam lemon 2.9 19.5 4.11
·6. C Dmon Burn Cv. Jaintia lemon 5.09 5.97 2.457. • C UmonBurn Cv. Gol Neembu 3.0 6.3 3.28. C UmmettiodesTanaka Cv. Sweet lime 2.7 3.9 2.179. C UmmettiodesTanaka Cv. Sweet lime sour 2.7 5.1 4.0010. C medica Unn Cv. Citron Seeded 3.2 13.7 38.711. C medica Unn Cv. Citron Gandharaj 2.2 11.4 2.512. C medica Unn Cv. Soh mad 3.7 12.6 3.113. C aurantiloUa Swingle Cv. Kagzi lime 5.5 8.8 2.614. C meyeriiY. Tim Cv. Lemon major 4.0 1.1 3.51-5. C. jambhiri Lush Cv. Soh myndong 4.0 6.3 2.216. C aurantium Linn Cv. Karun jamlr' 2.9 7.8 2.617. CparadisiMacfCv.Grapefruit 2.7 12.1 2.618. C macroptera Mont Cv. Satkara 3.7 4.3 4.419. CkarnaRPCV.SohSarkar 2.7 13.1 3.120. C vo/kameriana Pasq Cv. Volkameriana 3.8 6.5 3.021. C grandis Osbeck Cv. Pummelo 3.5 10.9 3.222. C teshniTanaka Cv. Cleopatra mandarin 6.6 9:1 3.223. C taiwanicaTanaka Cv. Taiwanica 4.7 11.2 3.124. C madurensis Lour Cv. Calamondin 8.3 12.1 3.225. C /atipesTanaka Cv. Khasi Papeda 3 1.8 626. C assamensis Dutt & Bhattacharya Cv. Ada jamir 3.5 1.2 627. C indica Tanaka Cv. Indian wild orange 2 1.0 4.528. C hybridCv. Kara mandarin 3.2 4.1 2.629. C hybridCv. Kinnow (C nobiUsx C deJiciosa) 2.3 2.0 5.230. Carrizocitrange (P trilo/iatax C. sinensiS) 7.5 10.1 4.031. Troyer citrange(P trilo/iatax C sinensiS} 13.0 2.1 13.532. Tangelo Dancy(C reticulata xC paradis1J 6.7 7.1 4.533. Citremon(P triloliatax C lemon, 5.7 13.3 4(,
(Source, Parthasarathy, eta/,1998)
Fresh cultureweight (gm)
0.10.1440.160.10.120.10.070.090.1550.1650.070.160.070.1670.140.1550.170.090.1670.120.110.120.130.20.2970.2970.110.0760.130.560.6830.260.22
mlcropropagation of calamondin. Duran Villa etal. (1992) regenerated plants from long termet aJ (1989) defined tissue culture techniques root culture of lime, Citrus aurantifoliaand cultures conditions for morphogenesis, (Christm.) Swing. Can et al (1992) standardcallus culture and plantlet of sweetorange (Cit- ized the in vitro clonal propagation of sourrus sinensi$Osb.), citron (Citrus medica I.) and orange (C aurantium var. Brezilia) by usinglime (C aurantilolia Christm.Swing.). A me- epicotyl segments. MS medium containing'dium containing 22 IlM BA with or without 2mg/l BAP with or without 4mg/l GA3 was5.4 IlM NAP was optimum for shoots initia- optimum for shoot initiation. The optimumtion in case of Carrizo citrange, Cleopatra rooting of in vitro regenerated shoots was obmandarin and sour orange seedling explants, served with Img/l of each IBA & NAA. Inbut the 3 genotypes varied greatly in number Poncirus tri/oJiata and Carrizo citrange, theof shoots p:oduced(Moore, 1986). Bhatt highest rate of induction of adventitious shoots
Vcl.23,No.1,2002 7
and globular embryoids was obtained on a cul- role for root induction. Murashige (1977)ture medium supplemented with 5mg/1 BAP emphasised that high light intensity also induces(Beloualy, 1991). Malt extract has been found better rooting and causes hardening of plantsto be an ideal supplementaion for induction of which renders them more tolerant to moistureadventive embryogenesis in citrus stress and diseases. One fourth salt concen(Parthasarathy and Nagaraju, 1994). tration as MS medium help in induction ohootsParthasarathy (1993) found that MS medium (Skirvin and Chu, 1979). James and co-workwas the best for inducing and increasing the ers have found the importance of phlorogluciweight of embryoides without any supplemen- nol (PG), a phenolic compound found in xytation. Lukman et a!' (1990) produced mul- lem sap orapple for rooting of number of rotiple shoots of Troyer citrange in vitro from saceous fruit cultivars. A low salt medium isshoot apices. BAP at 0.08mg/1 associated with found satisfactory for rooting of shoots in a1.0mg /1 of GA3 induced the proliferation of large number of plant species, Often wherethe apices. Multiple shoots were obtained from shoot multiplication induced on full, strengthshoot tip (2,3mm) derived from mature plants MS medium, the salt concentration was reduced(5 to 6 years old) of Citrus reticulata Blanco to half (Garland and Stoltz, 1981) for bettercv. Khasi mandarin and C limon Burm. cv. rooting. Addition of activated 'charcol in rootAssam lemon when cultured on MS medium expression medium improved the overall rootsupplemented with 1.0 BAP, 0.5 kinetin and ing capacity in Pinus panaster (Elizabeth and0.5 NAA (mg/1) (Singh eta!., 1994). Grosser Oliver, 1995). Elizabeth and Oliver (1995)and Chandler (1986), obtained multiple shoots further reported that 98%' of Juvenile explantof Swingle citrumelo rootstock with cumarin to Pinus pinasterrooted easily while only 49%at 90-150 ~lM. mature explant rooted.
In lh"tro Rooting In the micropropagation practice, usu-Number of factors have been observed ally natural auxin IAA and synthetic auxins NAA
to be associated with rooting of microshoot. and IBA are used for rooting (Nemeth~ 1986).That includes nature of cuttings, rooting co- Jones et al. (1977) used filter sterilized IBA infactor, synergistic role of exogenously applied their experiments. Auxins alone or with cytogrowth hormone and endogeneously present kinins, GA3, ABA and phenolics exert theirco factors in the rooting, the relative efficiency effect mainly during the root induction and iniof different auxins, their combination and meth- tiation phase. Crabapple (Malus spp.) rootingods of application (Audus, 1972; Weaver, of in vitro produced shoots was obtained on1972; Haissig, 1974). Existence of certain basal medium containing 5 or 10mg/1 IBAco-factors for rooting or auxin synergist in tis- (NQrton and Noe, 1982). Excellent rooting ofsues of cutting have been demonstrated by Van in Wtro derived shoots was obtained onOverbeek et al. (1946). Certain easy to root 1/2 strength MS medium containing 0.1 Of
varieties can be rooted each in absence of leaves 0.2Mg/1 NAA. An increasing percentage ofcutting in a combination of IBA with Arginine root~d shoots and more roots/shoots were obHel, NH4SO4 and Sucrose which is cofactor tain~ with a concentration range of NAA fromfor rooting in addition to IAA and is reviewed 0 to:1mg/1 in Quince (Cydonia oblonga Mill).(Hackett, 1970, Haissig, 1974). Role of dif- (AU rttarri etal., 1986). At the optimal NAAferent nitrogen source have been reported to concfntration of 0.5mg/1, the shoots reachedbe important in combination with auxin(s). 90%pf rooting. The average root length was
Many factorsdo play very important maxiIttum in control medium without auxin and
A corr~ination of 0.5mg NAA, 0.5mgIBA and 0.25mg BAP/liter was found best forroot development in microshoot of Assamlemon (Citrus limon) and Khasi mandarin (Citrus reticu/ata)cv. of citrus (Singh et ai, 1994).Duran-Villa (1989) tested different concentration (0-50mg/l) of NAA for rooting ofmicroshoots of 3 Citrus species. The optimalconcentrations of NAA to induce root formation on stem segments were 10mg/l for sweetorange and lime and 3mg/l for citron. Beloualy(1991), tried NAA and GA
3for rooting in
Poncirus trifo/iata, Carrizo citrange and Citrusaurflntium microshoots developed throughembyrogenesis. Rooting was promoted by asupplement of 1mg/l GA3 or 1mg/l NAA. GA3enhanced stem elongation and rooting in embryoids and NAA stimulated adventitious rootformation. .
Can et a/., (1992) obtained best rooting of microshoots of sour orange (Citrusaurantium var. Brezilia) in the medium contained 1mg/l IBA and 1mg/l NAA. NAA alonewas also effective on the rooting but to a lesserextent than the combination with IBA. In another experiment Lukman et a! (1990) reported NAA at various levels (0,0.1,0.3,0.5,0.7 and 1.Omg/l) did not promote rooting inTroyer citrange microcutting. On the otherhand transferred to vermiculite provided approximately 17% rooting. Juvenile shoots oftrifoliate orange rooted readily in media containing 5 f..l.M NAA (60%) and 10 f..l.M NAA(90%) (Barlass and Skene, 1982). In this case,roots were produced after 3 weeks exposureto 10 f..l.M NAA (as for both Juvenile and mature citrange shoots at all NAA concentration~
applied). 2mg NAA/l found best for rootingof Troyer citrange microshoots (Edriss &Burger, 1984). Similarly Parthasarathy andNagaraju (1996 a) found NAA supplementa-
8 AGRICULTURAL REVIEWS
decreased with increasing NAA concentrations. tion at the rate of 0.05 mg/l induced goodOn the contrary, the root number per shoot rooting of microcuttings on three citrus speincreased with the auxin concentration. cies while for Csinesis cv. Musambi NAA at
0.2 mg/l was best.
AcclimatizationThe survival and growth of in-vitro
propagated plants after removal from culturehas been a major problem in themicropropagation of citrus. Parthasarathyet al (1999 b) developed efficient and reproducible procedure for successful in-vitro rooting and acclimatization of micropropagatedshoots of important citrus species in a singlestep with very high establishment in the soil.The protocol involved direct planting of 6-8week old microshoots(2-2.5 cm long) in sterile soil rite topped over FYM Better rooting(80-90 %) and and very high ex-vitro survival(90-97%) was achieved using this protocol. Thepresent study describes an efficient and reproducible procedure for successful in-vitro rooting and acclimatization of micropropagatedshoots of important citrus species in a singlestep procedure with very high establishmentin the soil.Transfer of rooted plantlets from sterile to non-sterile conditions with reduced humidity lead to very high mortality (Brainerd andFuchigami, 1981; Sutter and Langhans, 1982)and p09r growth and delay in the attainmentof completely acclimatized plants (Wardleetal, 1983; Ziv eta/., 1983). In vitro rootedplants often lack root hairs and die shortly after transplanting (Donnelly et ai, 1985). Theprocess of acclimatizing in-vitro-rooted plantsinvolves various steps of acclimatization andextended periods (Cao, 1990). Debergh andMaene (1981) reported that the entire processof rooting in vitro and hardening has been estimated to account for approximately 35 to70% of the total cost of micropropagation.However, in citrus the success rate has beenreported to range from 22% (Fred etai, 1986)to 60% (Singh et ai, 1994). Hidaka andKajiura (1989) achieved 61.4-91.1% survival
Vol. 23, No.1, 2002 9
in a mist house but the cames used in their plant weight, shoot and root weight over mistudy are not commonly available. In addition, cro propagated plantlets during initial periodserveral months have to elapse before the plants (upto 2 months) (Singh et aI., 1997). Later oncan be transferred to the soil. a reverse growth trend was observed.Performance of micropropagated plantlets Micr~propagated.exhibited better gr~wth over
It is very important to evaluate s~edltngplantlets In terms of plant heIght, stemmicropropagated plantlets with conventionally gIrth, total canopy, numbers of branches andpropagated plants for recommending this plant- numbers of leaves. The reason may be attribing material to farmers. However, very little uted to root system. The micropropagatedwork have been done on this important aspect plantlets showed nlore fibrous roots, whichof micropropagation. Singh (2000) evaluated' helps in absorption of more nutrients from themicropropagated plant with conventionally soil than seedling. Palma et al. (1997) foundgrown seedling at Umaim Meghalaya. Studies better secondary roots in microcutting of Crevealed that seedlings showed comparatively macrophylla than seedlings.higher shoot and root length as well as higher
Table 3. Comparative growth of micropropagated and seedling plants of citrus sp. (1 year)
SI.No. Species/ Method of Plant Root No.of No. of Shoot Root Plantcultivar propagation height length leaves secondary wt. (g) wt. (g) wt. (g)
(em) (em) roots
1 SAT S 10.88 14.72 15.45 46.5 1.06 0.51 1.57M 16.53 18.72 20.27 64.75 1.36 0.64 1.99
2 LAT S 27.98 29.95 26.4 63.75 2.90 1.78 4.68M 28.78 30.72 27.42 75.55 2.94 1.88 4.82
3 SLS S 20.13 22.97 20.95 52.97 . 1.43 0.76 2.18M 32.05 26.07 33.32 66.30 2.36 1.41 3.77
4 SOB 5 22.83 23.40 25.95 62.00 2.70 1.72 4.25M 31.75 27.40 29.95 83.00 3.37 1.03 5.37
5 5 14.00 19.55 19.05 45.00 0.54 1.38 0.77M 17.58 20.75 21.22 67.00 0.62 2.30 0.95
6 ADA 5 19.38 22.67 21.2 73.25 3.78 2.34 5.66M 19.78 22.97 21.82 78.00 3.81 0:54 5.70
7 . KM 5 14.68 20.87 22.92 45.00 0.86 0.79 1.29M 17.80 21.70 24.02 60.00 0.93 0.96 1.43
8 SM 5 26.58 23.27 29.9 89.50 4.65 1.27 6.77M 34.73 24.9 37.32 102.75 4.98 1.99 7.45
9 JL 5 38.10 32.07 28.07 86.37 5.00 2.23 7.23tv! 41.90 31.82 29.37 89.82 5.24 2.33 7.57
10 P 5 25.78 24.27 22.8 86.92 2.71 2.30 5.01M 27.85 24.35 23.75 96.75 2.77 2.34 5.11
11 CV 5 24.98 27.32 2~.57 56.85 1.17 0.43 1.71M 29.85 27.00 2.47 72.87 1.39 0.5 2.18
12 AL 5 25.85 21.62 2a07 87.5 2.77 2.11 3.73M 34.95 26.62 2 35 97.0 3.14 2.47 4.41
S= Seedling M= MicropropagatedSatkara (Citrus macroptera Mont.) 5AT, Khasi papeda (Citr".~ latipes Tanaka) LAT, 5weet lime (Citrus limettioidesTanaka)5LS, Soh Bitara (Citrus sinensisosbeck) 50B, Indian Wild orange (Citrus indica Tanaka) I, Ada Jamit (CitrusassamensisDutta & Bhattacharya) ADA, Khasi mandarin (C{frt/S reticulata Blanco)KM, Soh myndong (CitrusJambhiriLush)5M, Jaintia lemon (Citrus limon Burm)JL, Pummelo (t:'itrus grandis Osbeck)P, Assam lemon (Citrus limonBurm)AL, Volkalller Lemon (Citrus volkameriana Pasq.) CV
(Source : Singh, 2000)
10 AGRICULTURAL REVIEWS
Micrografting (1999) standardize the STG technique of SweetThe development of shoot tip graft- orange at NRCC, Nagpur.
ing (STG) as an in vitro multiplication technique Several factors influence the rate ofwas a conse~uenc~ of the hi9? eco.nom~c losses success of grafting or virus elimination. Succa~sed by CItruS VlruS an~, Vlrus hke dlseas~s, cessful grafts have been obtained using manywhich made the use of .VlruS free. propagative different scion cultivars of the commerciallybud .wood necessafoY. It IS a techmque t~at P?- grown citms species, and significant differencest.entJ,ally can. c~m~lne t~e adv~tage at rapId, in grafting success among three different typesIn ~t~o multJphcatJon Wlt~ t~e Increased pro- have not been noted when appropriateducti~tytha~ results from In vitro graft ~tw~en rootstocks were used. Both good and poorsupenor sCion and rootstock combmatton rootstocks scion combinations have been iden(Gebha~dt and Gol~bach, 1988). The ~et~od tified but it is not known if grafting success inmost ,WIdely used In the past t.o obtain VlruS vitro is related to graft compatibility in vivofree ~Itrus plants was the .select~on,of nucellar (Navarro 1981). The frequency of successfulseedlings of polyembryomc c~lt~va~s (Weather graft increases with the size of shoot tip, whileand Calavan, 1959). Th.e Itmlta~lon of thIS the percentage of virus free plants declines,method wa~ the long pen~ requl~ed fo.r nu- Murashige et a/. (1972) obtained new citrus,cellar seedhng? to proceea.from Juveml~ ~o plants by grafting shoot tip on rootstock seedadult p~ase b~fore becommg commerclahy lings grown in vitro. Navarro et aJ. (1975) de-'productive (ROlstacher, 1977). veloped a routine procedure of shoot tip graft-
The in vitro micrografting ,technique ing toobtain 30 to 50% successful grafts whichhas proved to be very useful in the regenera- were transplanted to soil with over 95% surtion of whole orchards of citrus infected by vi- vival. Most of the micrografted plants were freeruses (Jonard, 1986). This technique was first of the citrus virus and virus like diseases whichdeveloped by Murashige et a/. (1972) with a were present in the source plant and they didview to obtaining healthy citrus trees. Later not have juvenile characters (Roistacher et aJ.,Navarro 'ef al. (1975), Navarro and Juarez. 1976 and Murashige eta/., 1972). Edriss and(1977) , Roistacher eta/. (1976) and Roistacher Burger(1984), reported that the placement ofand Kitto (1977) extended micrografting tech- apical meristem on the seedling rootstock isnique to diverse species of citrus. The efficiency critical. They showed that placement of theof the technique in eradication of virus infected shoot tip in contact with the vascular ring or incitrus stocks has been well demonstrated (Russo the cortical surface in an inverted T incisionand Starrantino 1975 and Youtsy 1978), In have been the most successful treatment.India work on shoot tip grafting in citrus con- Navarro ef a/. (1,975) and Singh (1999), studtinuing at NRCC, Nagpur, Biotech lab ICAR ied the age effect of growing rootstock seedResearch Complex for NEH region, Assam Ag. lings. The greatest success was achieved usingUniversity,Jorhat and BCKV, West Bengal. seedlings two weeks after sowing. They alsoVijaya Kumari et a/. (1994) have followed a reported an inverse relationship between themodified procedure of Navarro for Nagpur size of shoot tip and the micrografting successmandarin and standardized the STG technique rate. Pretreatment of the shoot tip and/or seedfor Nagpur mandarin. Mukhopadhyay et a/. ling rootstcokhave been shown to increase the(1997) defined the STG technique for micrografting success rate. Jonard eta/. (1983)Darjeeling oranges. Parthasarathy eta/. (1996) treated peach shoot tip in 0.1 mg zeatin/l andand Singh eta/. (1997) have also standardize increased the success rate by 300();0.the STG technique for Khasi mandarin'. Singh Starrantino and Caruso (1988) observed that
Vcl.23,No.l,2002 11
by dipping the apex and decapited seedlings (1984) who used rectangular triangle hole infor ten minutes before micrografting in a solu- stead of inverted cut for insertion of scion.bon of BAP (0.5 ppm) the percentage of With the modified technique more than 60%sprouting increased from 73% to 91%. The successful grafts were obtained while the instandard procedure of micrografting developed verted T cut method yielded less than 20%.by Navarro was modified by Su and Chu
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