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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