In Vitro Propagation of Citrus Rootstocks

Abstract

Present investigation was conducted to standardize a protocol for in-vitro propagation of citrus rootstocks viz. Rough lemon, Cleopatra mandarin Pectinifera and Troyer citrange. The shoot tip explant was found better for callus induction of these rootstocks than the nodal segment. Maximum callus formation (40.0% and 23.3%) of shoot tip explants was obtained in Cleopatra mandarin, Pectinifera, and Rough lemon and Troyer citrange, respectively in treatment MS basal media + 0.5mg/l Kin, 2.0mg/l NAA, and 2.0mg/l 2, 4-D. Furthermore, the maximum number of shoots per explant was obtained through the callus in Pectinifera, Rough lemon and Cleopatra mandarin in MS basal media + BAP 1mg/l. Maximum rooting of shoots (1.11%) was noted in rootstock Rough lemon followed by Cleopatra mandarin for the ½ MS media supplemented with 10mg/l IBA. Although the callus development and bud proliferation was recorded in rootstock Troyer citrange however, shoot and root formation did not occur. The potting media consisting of soil, sand and FYM in the ratio of 1:1:1 by volume was better with maximum survival rate of hardened plants six weeks after transferring to the pots under greenhouse for Rough lemon followed by Pectinifera and Cleopatra mandarin rootstock.

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Available online at www.notulaebotanicae.ro
Not. Bot. Hort. Agrobot. Cluj 37 (1) 2009, 84-88
Print ISSN 0255-965X; Electronic ISSN 1842-4309
Notulae Botanicae Horti Agrobotanici
Cluj-Napoca
In Vitro Propagation of Citrus Rootstocks
Suneel SHARMA, Atam PRAKASH, Ajinath TELE
1) Department of Horticulture, CCS Haryana Agricultural University, Hisar125004, Haryana, India; sharma.suneel@yahoo.com
Abstract
Present investigation was conducted to standardize a protocol for in-vitro propagation of citrus rootstocks viz. Rough lemon,
Cleopatra mandarin Pectinifera and Troyer citrange. The shoot tip explant was found better for callus induction of these rootstocks than
the nodal segment. Maximum callus formation (40.0% and 23.3%) of shoot tip explants was obtained in Cleopatra mandarin, Pectinifera,
and Rough lemon and Troyer citrange, respectively in treatment MS basal media + 0.5mg/l Kin, 2.0mg/l NAA, and 2.0mg/l 2, 4-D.
Furthermore, the maximum number of shoots per explant was obtained through the callus in Pectinifera, Rough lemon and Cleopatra
mandarin in MS basal media + BAP 1mg/l. Maximum rooting of shoots (1.11%) was noted in rootstock Rough lemon followed by
Cleopatra mandarin for the ½ MS media supplemented with 10mg/l IBA. Although the callus development and bud proliferation was
recorded in rootstock Troyer citrange however, shoot and root formation did not occur. The potting media consisting of soil, sand and
FYM in the ratio of 1:1:1 by volume was better with maximum survival rate of hardened plants six weeks after transferring to the pots
under greenhouse for Rough lemon followed by Pectinifera and Cleopatra mandarin rootstock.
Keywords: In-vitro multiplication, citrus rootstocks, pectinifera, troyer citrange, rough lemon, Cleopatra mandarin
Introduction
Usefulness of citrus rootstocks for the improvement
of canopy architecture, fruit production, quality and tol-
erance to biotic and a-biotic stresses of citrus crops is well
known. However these rootstocks, propagated by growing
open-pollinated seeds, are highly nucellar and produce true
to type plants. Depending upon the rootstocks, generally
1 to 40% zygotic seedlings are produced, which must be
culled from seed beds to maintain clonal uniformity. All
citrus cultivar selections are usually grafted to selected
rootstock seedlings. Some potentially valuable rootstocks
produce few or no seed and thus seed shortage of such pop-
ular rootstock occurs periodically. further, the demand of
quality planting material of important rootstocks in ample
number necessitates for their in vitro propagation.
During the past years, micro-propagation techniques
have been widely used for several plant species. Also, the
plant regeneration in citrus species has been reported by
various workers (Singh et al., 1994; Parthasarathy et al,
2001). Tissue culture technique could be used for propa-
gation of citrus rootstocks and thus, the number of plants
produced would not be limited by their seed supply, rather
more uniform disease free and quality plant populations
might be produced. In spite of micro-propagation of citrus
genotypes reported by several workers, a very few reports
of in vitro multiplication of citrus rootstocks Pectinifera,
Troyer citrange, Cleopatra mandarin and Rough lemon are
available in the literature (Chaturvedi and Mitra, 1974).
In view of this, the present study was undertaken with the
objectives to develop protocol for callus induction and
regeneration in the above mentioned rootstocks, and to
make their utility more effective in orchard establishment
of citrus crop.
Materials and methods
The explants viz. shoot tips and nodal segments were
collected from Pectinifera (Citrus depressa Hayata), Troyer
citrange (Poncirus trifoliata (L.) Raf. x Citrus sinensis Os-
beck), Rough lemon (Citrus jambhiri Lush.) and Cleo-
patra mandarin (Citrus reshni Hort. ex. Tan.) rootstocks
growing at Experimental Orchard of the Department of
Horticulture, CCS HAU, Hisar. The surface sterilization
of explants was done with 70% aqueous solution of etha-
nol (v/v) for 30 seconds followed by 0.1% HgCl2 (w/v)
for 8-10 minutes and then washed properly with sterile dis-
tilled water under aseptic condition.
In order to induct callus as well as for the regeneration
of plants, the explants were cultured on MS basal medium
(1962) supplemented with different concentrations of
phyto-hormones viz. BAP, kinetin, NAA, and 2, 4-D. Sur-
face sterilized and thoroughly washed explants viz. shoot
tips and nodal segments were cultured on MS medium
containing different concentrations of Kinetin, NAA and
2, 4–D.
Explants were aseptically inoculated on culture me-
dium under white light. five explants were inoculated per
flask and about 150 explants were kept in every set of ex-
periment. All cultures were incubated at 25±20C in cul-
ture room under 16/8hrS cycles of light (200 Lux) and
darkness. The proliferated shoots (3-4cm) were inoculated
individually on MS, half MS media alone or supplemented
with various concentration of IBA. The rooted plantlets

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Sharma, S. et al./ Not. Bot. Hort. Agrobot. Cluj 37 (1) 2009, 84-88
85
higher than the nodal segment explants for all the root-
stocks. The interaction between media and explants was
not significant.
This study revealed that among various explants used,
the per cent callus formation was found satisfactory only
in shoot tip explant in all the rootstocks. The difference
in per cent callus formation between rootstocks of citrus
might be due to the plant genotype. The results are in con-
formity with various earlier authors (Chaturvedi and Mi-
tra, 1974; Goyal and Arya, 1981). The maximum per cent
callus formation in shoot tip explants was obtained in MS
basal + 0.5mg/l Kin, 2.0mg/l NAA and 2.0mg/l 2, 4-D
treatment in all the rootstocks. The ratio of auxin and cy-
tokinin concentrations in culture media is known to play
a major role in establishment of callus cultures in several
citrus species (Chaturvedi and Mitra, 1974).
It can be inferred from the data in Table-2 that shoot
tip and nodal explants of rootstocks Pectinifera, Cleopatra
mandarin and Rough lemon of citrus produced more (2.61,
2.11 and 1.92, respectively) number of shoots through cal-
lus in 1.0mg/l BAP treatment, better than all other treat-
were washed thoroughly and transferred into small pots
containing potting media prepared of soil, sand and fYM
in the ratio of 1:1:1 by volume, and kept at 25±10C All the
experiments were carried out following the factorial com-
pletely randomized design (Gomez and Gomez, 1984).
Results and discussion
Plant regeneration through indirect organogenesis
Maximum callus formation (40.0% and 23.3%, re-
spectively) in treatment 0.5mg/l Kin, 2.0mg/l NAA and
2.0mg/l 2, 4-D was observed in all the rootstocks, which
was significantly better than all other media treatments,
followed by 26.67% in treatment 0.5mg/l Kin, 5.0mg/l
NAA and 5.0mg/l 2, 4-D in Cleopatra mandarin and
Pectinifera, and 16.7% and 23.3% in treatment 0.5mg/l
Kin and 5.0mg/l NAA in Rough lemon and Troyer cit-
range, and Cleopatra mandarin and Pectinifera, respec-
tively (Tab. 1). The maximum per cent callus formation
was obtained in shoot tip explants, which was significantly
Tab. 1. Effect of phytohormones on per cent callus formation in explants of citrus rootstocks
Treatment
(mg/l)
NAA
-
2.0
5.0
2.0
5.0
-
-
-
-
2.0
5.0
Rough
lemon
B
0.0
0.0
6.67
0.0
13.3
0.0
6.7
0.0
0.0
20.0
13.3
Cleopatra
mandarin
B
0.0
0.0
13.3
0.0
13.3
0.0
13.3
0.0
0.0
33.3
20.0
Pectinifera
Troyer
citrange
B
0.0
0.0
6.7
0.0
13.3
0.0
6.7
0.0
0.0
20.0
6.7
Kin
-
-
-
0.5
0.5
-
-
0.5
0.5
0.5
0.5
2,4-D
-
-
-
-
-
2.0
5.0
2.0
5.0
2.0
5.0
A
0.0
0.0
13.3
0.0
20.0
0.0
6.7
0.0
0.0
26.6
20.0
Mean
0.0
0.0
10.0
0.0
16.7
0.0
6.7
0.0
0.0
23.3
16.6
A
0.0
0.0
20.0
0.0
33.3
0.0
20.0
0.0
0.0
46.5
33.3
Mean
0.0
0.0
16.6
0.0
23.3
0.0
16.7
0.0
0.0
40.0
26.6
A
0.0
0.0
20.0
0.0
26.7
0.0
13.3
0.0
0.0
46.5
26.7
B
0.0
0.0
13.3
0.0
20.0
0.0
6.7
0.0
0.0
33.3
26.7
Mean
0.0
0.0
16.7
0.0
23.3
0.0
10.0
0.0
0.0
40.0
26.7
A
0.0
0.0
13.3
0.0
20.0
0.0
6.7
0.0
0.0
26.7
13.3
Mean
0.0
0.0
10.0
0.0
16.7
0.0
6.7
0.0
0.0
23.4
10.0
C.D (5%) Media: 2.41, Explants: 1.45, Media x Explants: NS
A – Shoot tip, B – Nodal segment, Kin – Kinetin, NS – Non significant
Tab. 2. Effect of phytohormones on average number of shoots per explant obtained through the callus in shoot tip and nodal segment
explants of Citrus rootstocks
Treatment
(mg/l)
BAP
0.0
0.5
1.0
1.5
2.0
0.0
0.0
0.0
0.0
Rough
lemon
B
0.00
0.67
1.67
0.67
0.33
1.67
1.00
0.33
0.33
Cleopatra
mandarin
B
0.00
1.17
1.83
0.67
0.33
1.67
0.83
0.50
0.33
Pectinifera
Kin
0.0
0.0
0.0
0.0
0.0
0.5
1.0
1.5
2.0
AMean
0.00
0.92
1.92
0.58
0.42
1.75
1.00
0.42
0.33
AMean
0.00
1.19
2.11
0.69
0.30
1.89
0.95
0.45
0.28
ABMean
0.00
1.56
2.61
1.00
0.50
2.36
1.00
0.45
0.30
0.00
1.17
2.17
0.83
0.50
1.83
1.00
0.50
0.33
0.00
1.22
2.39
0.72
0.27
2.11
1.06
0.39
0.22
0.00
1.94
2.72
1.50
0.67
2.39
1.17
0.56
0.28
0.00
1.17
2.50
0.50
0.33
2.33
0.83
0.33
0.33
C.D (5%) Media: 0.24, Explants: , Media : x Explants: NS
* No shoot formation was observed in rootstock Troyer citrange in all the treatments.
A – Shoot tip, B – Nodal segment, Kin – Kinetin, NS – Non significant

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86
In citrus rootstocks Rough lemon, Cleopatra manda-
rin and Pectinifera the maximum bud proliferation (76.7
and 90.0%, respectively) was recorded in treatment BAP
1.0 mg/l and rated best among all the media treatments
(Tab. 3). It was followed by rootstock Cleopatra mandarin
(86.3%) in treatment BAP 0.5 mg/l, Pectinifera (80.0%) in
treatment BAP 1.5 mg/l, and Rough lemon (70.0%) with
Kin 0.5mg/l. The highest (86.67 and 93.33%, respectively)
bud proliferation was obtained in nodal segment explants
in Cleopatra mandarin and Pectinifera followed by Rough
lemon (86.7%) and Troyer citrange (73.3%), which was
better than explant shoot tip in all the rootstocks. The dif-
ferences in per cent bud proliferation might be attributed
to the genetic behavior of the rootstocks. Shoot regenera-
tion (66-100%) from shoot tip and nodal stem segment
of mature plants was reported in C. mitis Blanco (Sim et
al., 1989).
It is clear from the data given in Tab. 4 that maximum
number of multiple shoots per explant (2.10, 2.00 and
1.87, respectively) was recorded in rootstocks in the treat-
ment MS +BAP 1.0mg/l in Pectinifera, Cleopatra manda-
rin and Rough lemon. This treatment was found superior
ments and it was followed by2.36,1.89 and 1.75, respec-
tively in treatment Kin 0.5mg/l. No shoot formation was
observed in rootstock Troyer citrange in all the treatments.
The minimum average number of shoots (0.28 in Cleo-
patra mandarin, 0.30 in Pectinifera and 0.33 in Rough
lemon) was obtained through callus in treatment 2.0mg/l
Kin. There was no culture response in MS basal media
treatment without supplementation of growth hormones.
furthermore, significantly higher number of shoots was
obtained through callus in rootstock Cleopatra mandarin.
Thus it can be inferred that significantly higher number
of shoots obtained through callus resulted in Pectinifera
was superior to the rootstocks Rough lemon and Cleopa-
tra mandarin. The difference in the number of shoots ob-
tained through callus among the rootstocks might be due
to the differences in their genetic makeup. Chaturvedi and
Mitra, (1974) reported that isolated stem callus of Citrus
sinensis also regenerated plantlets.
Plant regeneration through direct organogenesis
Tab. 3: Effect of phytohormones on per cent bud proliferation in explants of citrus rootstocks
Treatment
(mg/l)
BAP
0.0
0.5
1.0
1.5
2.0
0.0
0.0
0.0
0.0
Rough lemonCleopatra mandarin PectiniferaTroyer citrange
Kin
0.0
0.0
0.0
0.0
0.0
0.5
1.0
1.5
2.0
ABMean
56.7
66.7
76.7
63.3
56.7
70.0
70.0
46.7
40.0
C.D (5%) Media: 3.59, Explant:2.40, Media x Explant:7.19
ABMean
66.7
83.4
90.0
70.0
60.0
66.7
73.3
53.3
46.7
ABMean
60.0
76.7
90.0
80.0
70.0
66.7
76.7
56.7
50.0
AB Mean
53.3
66.7
70.0
60.0
43.3
50.0
53.3
38.3
30.0
53.3
60.0
66.7
53.3
53.3
60.0
60.0
40.0
40.0
60.0
73.3
86.7
73.3
60.0
80.0
80.0
53.3
40.0
66.7
80.0
86.7
66.7
60.0
66.7
73.3
53.3
46.7
66.7
86.7
93.3
73.3
60.0
66.7
73.3
53.3
46.7
53.3
73.3
86.7
73.3
66.7
66.7
73.3
53.3
46.7
66.7
80.0
93.3
86.7
73.3
66.7
80.0
60.0
53.3
46.7
66.7
66.7
60.0
40.0
46.7
46.7
26.7
26.7
60.0
66.7
73.3
60.0
46.7
53.3
60.0
40.0
33.3
A – Shoot tip, B – Nodal segment, Kin – Kinetin
Tab. 4: Effect of phyto-hormones on number of multiple shoots per explant obtained through callus in shoot tip and nodal segment
explants of citrus rootstocks
Treatment
(mg/l)
BAP
0.0
0.5
1.0
1.5
2.0
0.0
0.0
0.0
0.0
Rough
lemon
Cleopatra
mandarin
B
0.87
2.00
2.40
2.00
1.00
1.20
0.00
0.00
0.00
Pectinifera
Kin
0.0
0.0
0.0
0.0
0.0
0.5
1.0
1.5
2.0
ABMean
0.3
1.1
1.87
1.24
0.67
0.60
0.00
0.00
0.00
AMean
0.44
1.40
2.00
1.50
0.80
0.60
0.00
0.00
0.00
ABMean
0.5
1.44
2.10
1.64
1.60
0.60
0.00
0.00
0.00
0.00
0.60
1.53
0.87
0.47
0.00
0.00
0.00
0.00
0.60
1.60
2.20
1.60
0.87
1.20
0.00
0.00
0.00
0.00
0.80
1.60
1.00
0.60
0.00
0.00
0.00
0.00
0.00
0.87
1.60
1.20
0.73
0.00
0.00
0.00
0.00
1.00
2.00
2.60
2.07
1.40
1.20
0.00
0.00
0.00
C.D (5%) Media: 0.12, Rootstock: 0.08, Media x Rootstock: 0.23
* No shoot formation was observed in rootstock Troyer citrange in all the treatments.
A – Shoot tip, B – Nodal segment, Kin – Kinetin

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87
shoot placed for rooting. Large sized shoots show higher
rooting percentage (Barua et al., 1996).
The per cent survival of in vitro derived plants in pot-
ting media was recorded six weeks after transferring to
the pots under greenhouse condition. The survival of
plants varied with potting media and rootstocks (Tab. 6).
Maximum survival was obtained in soil + sand + fYM
(1:1:1v/v) media, which was significantly better than
all other potting media and was followed by soil + river
sand (1:1v/v) and soil + sand (1:1v/v). The survival was
significantly higher (80.00 and 71.43%, respectively) in
soil + sand + fYM (1:1:1) media in Rough lemon and
Cleopatra mandarin. The higher plant survival in the soil
+ sand + fYM (1:1:1) potting mixture can be attributed
to the retention of optimum moisture with adequate aera-
tion. These results are in conformity with the observations
made by Goyal and Arya (1987).
The per cent survival of in vitro derived plants in pot-
ting media was recorded six weeks after transferring to
pots under greenhouse condition. The survival of plants
varied with potting media and rootstocks (Tab. 6). Maxi-
mum survival was obtained in soil + sand + fYM (1:1:1
v/v) media, which was significantly better than all other
potting media and was followed by soil + river sand (1:1
v/v) and soil + sand (1:1 v/v). The survival was signifi-
to all other treatments, and it was followed by (1.64, 1.50
and 1.24, respectively) in treatment MS + BAP 1.5mg/l
in these rootstocks of citrus. Troyer citrange did not show
multiple shoot formation. The maximum number of mul-
tiple shoots per explant (2.60, 2.40 and 2.20, respectively)
was found in nodal segments as compared to shoot tip ex-
plants in above rootstocks. The interaction between media
and rootstock was found significant. The difference in the
multiple shoots per explant between the two rootstocks
might be attributed to the difference in their genetic
makeup. Parthasarathy and Nagaraju (1996) recorded the
maximum number of shoots when MS basal media sup-
plemented with BAP (0.75mg/l) in C. limon.
It is clear that maximum number of roots per shoots
per explant (1.34) was obtained in rootstock Rough lemon
followed by Cleopatra mandarin and Pectinifera in treat-
ment ½ MS + IBA 10mg/l (Tab. 5). It was rated better
than all other rooting media. In rootstock Troyer citrange
there was no rooting occurred at all. No rooting was ob-
served in majority of the media treatments. However, the
treatments MS + IBA 15mg/l and MS + IBA 10mg/l were
also found effective for root formation in these rootstocks.
Interaction between media and rootstocks was found to
be significant. Rooting percentage relies on the size of the
Tab. 6: Effect of potting media on survival % of citrus rootstocks under greenhouse
Potting media mixture
Soil + Sand (1:1)
Soil + River sand (1:1)
Soil+ Sand + FYM (1:1:1)
Mean
Rough lemon
50.00
71.43
80.00
67.14
Cleopatra mandarin
45.45
69.23
71.43
63.04
Pectinifera
61.54
66.67
73.33
67.18
Mean
52.33
69.11
74.92
C.D (5%) Media: 3.41, Rootstock: 1.73, Media x Rootstock: 4.62
Tab. 5: Effect of media and IBA on number of roots/ shoot in citrus rootstocks
Media
(MS or ½ MS)
MS
MS
MS
MS
MS
MS
MS
MS
½ MS
½ MS
½ MS
½ MS
½ MS
½ MS
½ MS
½ MS
Treatments
IBA (mg/l)
0.0
1.0
2.0
4.0
5.0
10.0
15.0
20.0
0.0
1.0
2.0
4.0
5.0
10.0
15.0
20.0
C.D (5%) Media: 0.22, Rootstock: 0.07, Media x Rootstock: 0.32
Rough
lemon
0.00
0.00
0.00
0.00
0.00
0.64
0.78
0.00
0.00
0.00
0.00
0.00
0.00
1.34
0.63
0.00
Cleopatra mandarin PectiniferaMean
0.00
0.00
0.00
0.00
0.00
0.53
0.74
0.00
0.00
0.00
0.00
0.00
0.00
1.27
0.65
0.00
0.00
0.00
0.00
0.00
0.00
0.60
1.08
0.00
0.00
0.00
0.00
0.00
0.00
1.11
0.71
0.00
0.00
0.00
0.00
0.00
0.00
0.59
0.86
0.00
0.00
0.00
0.00
0.00
0.00
1.24
0.66
0.00
IBA – Indole butyric acid

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88
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Singh, S., B. K. Ray, S. Bhattacharya and P. C. Deka (1994). In
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cantly higher (73.33%) in soil + sand + fYM (1:1:1 v/v)
media in Pectinifera followed by Rough lemon and Cleo-
patra mandarin. There was no shoot formation in Troyer
citrange, hence, plants were not available for planting in
greenhouse. The higher plant survival in the soil + sand
+ fYM (1:1:1 v/v) potting mixture can be attributed to
the retention of optimum moisture with adequate aera-
tion. These results are in conformity with the observations
made by Goyal and Arya (1987).
In the present investigation efforts have been made to
standardize the protocol for in vitro multiplication of Cit-
rus rootstocks. It may be possible to propagate some Cit-
rus rootstocks viz. Rough lemon, Cleopatra mandarin and
Pectinifera in vitro if they are desirable enough to justify
the labour and expense that would be involved. However,
further studies are needed to be carried out to optimize the
procedures for the establishment of tissue culture derived
plants in soil, to examine genetic uniformity and evalu-
ate their performance in field in comparison with sexual
propagated plants.
Acknowledgement
The authors are thankful to Professor Dr. P.S. Partap,
Department of VegeTab. Science, CCS HAU, Hisar for
his kind help, valuable suggestions and critical reading of
this manuscript.