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Mudasir Iqbal Et Al
Mudasir Iqbal Et Al
net/publication/336890900
Article in International Journal of Current Microbiology and Applied Sciences · October 2019
DOI: 10.20546/ijcmas.2019.810.265
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1
Division of Fruit Science, 2Division of Plant Pathology, 3Division of Agroforestry, Faculty of
Agriculture, Main Campus, Chatha, Sher-e-Kashmir University of Agricultural Sciences &
Technology of Jammu, J&K-180009, India
*Corresponding author
ABSTRACT
Citrus, one of the most important group of fruit crops around the world, are propagated at
large scale with many difficulties. Propagation through seeds is challenging because of
Phytophthora foot rot together with recalcitrance of citrus seeds. Vegetative propagation of
Citrus species is mainly performed now-a-days by budding on seedling rootstocks. As
heavy losses are experienced among the susceptible seedlings due to Phytophthora and
Keywords Citrus tristeza virus (CTV), the interest in resistant rootstocks has greatly increased. The
Tissue culture, potential of conventional methods of citrus plant breeding of rootstocks are limited by
Micropropagation, physiological factors such as heterozygosity, inbreeding depression, nucellar
Callus, polyembryony and juvenility. Under such conditions advanced tissue culture techniques
Regeneration, provide best possible alternative for producing large number of resistant progenies from
Rooting, elite citrus genotypes. Plant tissue culture provides reliable and economical method of
Acclimatization maintaining pathogen free plants that allows rapid multiplication and international
Article Info exchange of germplasm. Generally, when in vitro propagation protocols are developed for
any specific plant species, specialized conditions for individual genotypes, elite species
Accepted: and even various developmental stages of the explants plants are selected via error-and-
17 September 2019 trial experiments. Because large diversity is observed in Citrus plant family, it takes many
Available Online: months to develop protocols for most suitable culture medium, best concentrations and
10 October 2019 combinations of plant growth regulators and other supplements for better development of
explant cultures. Therefore, in this review, we tried to put together results from difficult-
to-find literatures and listed all the identified findings, in which callus induction or somatic
organogenesis was used to develop citrus plants. Successful protocols of surface
sterilization method, culture establishment, shoot regeneration, in vitro rooting and
acclimatization are presented systematically.
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Mosambi). In addition Taye et al., 2018 used However, the disadvantages are very known,
fungicides like Kocide, Bayleton and Redimol including insufficient knowledge regarding
each with the concentration of 0.25 g/100 ml their genetic background. Das et al., (2000) in
of water for 15 and 20 minutes. The surface their study developed a protocol for
sterilization of explants with 70 % aqueous micropropagation of elite plants of sweet
solution of ethanol for 30 seconds followed by orange (Citrus sinensis) through nucellar
0.1 % mercuric chloride for 8-10 minutes and embryo culture and found that MS medium
then thoroughly washing with sterile distilled supplemented with NAA (1.0 mg/l) or 2, 4-D
water in citrus was reported by (Sharma et al., (1.0 mg/l) encouraged callus development in
2009). Pre-sterilization of excised explants both nucellar and zygotic embryos. Al-Khayri
with Benomyl (0.2%) can improve a cleanness and Al-Bhrany (2001) in their study on
and aliveness of all types of explants, micropropagation in lime Citrus aurantifolia
especially when followed through surface using nodal explants of mature tree nodes
sterilization done by mercury chloride (HgCl2) found best multiple shoot formation, i.e. 8.0
(Nurul, et al., 2012). Kour and Singh 2012 shoots per node on MS medium supplemented
removed expanded leaves of Rough lemon as with 1.0 mg/l BAP and 0.5 mg/l kinetin.
explants and then treated them with 10 % Srivastava et al., (2001) in their study on vitro
solution of teepol detergent for 10 minutes plant regeneration of Citrus aurantifolia
followed by thorough washing with distilled through callus culture, shoot tip, epicotyls and
water. They further preferred treatment of hypocotyl segments reported callusing on MS
explants with 70 % ethanol for 30 seconds medium enriched with BAP (5.0 mg/l) and
followed by 0.1 % mercuric chloride treatment observed highest per cent of callus and shoot
for 8 minutes and then rinsing with autoclaved regeneration with 5.0 mg/l BAP. Kamble et
distilled water three times. al., (2002) in their study on in vitro
micropropagation and callus induction in acid
Culture establishment lime (Citrus aurantifolia) cv. Sai Sarbati
observed the highest callus induction with
Callus formation is controlled by the level of epicotyl cultured on half-strength of MS
plant growth regulators (auxin and cytokinins) medium supplemented with NAA (10.0 mg/l)
in the culture media. Concentrations of plant and BAP (0.5 mg/l). Karwa Alka (2003)
growth regulators can vary for each plant carried research on in vitro propagation of
species and can even depend on the sources of Citrus reticulata (Nagpur mandarin) through
explants or individual plant. Culture mature seeds and found the highest (80%) of
conditions (temperature, light) are also shoot induction and multiple shoots per
important. Protocols developed in previous explants when cultured on MS medium
studies have shown that plant growth regulator supplemented with BA (8.80 μM), NAA (2.69
concentration and selection are vital for citrus μM) and kinetin (2.32 μM). Singh et al.,
callus induction. (2004) obtained multiple shoots on shoot tips
(2.0 to 3.0 mm) derived from mature plants (5
Explant type, media composition and callus to 6-year-old) of Citrus reticulata Blanco cv.
induction Khasi mandarin and C. limon Burm.f. cv.
Assam lemon, when cultured on Murashige
The major advantages of using seedlings and Skoog (MS) medium, supplemented with
explants over explants taken from field-grown 1.0 mg/l BAP, 0.5 mg/l kinetin, and 0.5 mg/l
mature plants are their high multiplication NAA. Mukhtar et al., (2005) reported that
rates and high regeneration potentials callus induction was greatest when shoot
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segments of lime were cultured on MS 4-D (1.0 mg/l) in combination with BAP (1.0
medium containing 2, 4-D and coconut milk. mg/l) produced early and highest percentage
Further, embryo proliferation was greatest on of callus with formation of somatic embryos
MS medium supplemented with kinetin (1.5 (Kaur, 2018). Taye et al., (2018), in their
mg/l). In addition, shoot induction was highest study on optimization of an in vitro
on MS medium along with BAP (2.0 mg/l). regeneration protocol for Rough lemon
Ali and Mirza (2006) observed optimal callus rootstock (Citrus jambhiri Lush.) via direct
induction response on MS medium, organogenesis reported that almost all IBA
supplemented with 2,4-D 1.5 mg/l from all and BA treatments resulted in almost cent
types of explants, with highest response (92%) percent shoot induction except IBA (0.1 mg/l),
and maximum shoot regeneration response (70 BA (1.5 and 2.0 mg/l). Further, it was reported
%) from callus on MS medium supplemented that among the explant sources, nodal
with BA 3 mg/l. Saini et al., (2010 observed segments induced a higher percentage of
maximum bud induction frequency of 83.97 % longer shoots in a shorter period of time than
on MS medium supplemented with BA (0.5 shoot tips.
mg/l) with an average of 8.6 buds per explant.
Kumar et al., (2011) obtained maximum Shoot regeneration and multiplication
callusing in epicotyl segments on MS medium
supplemented with NAA (10.0 mg/l) in The inherent capacity of plant cells to give rise
combination with BA (1.0 mg/l), KN (0.5 to complete plant is described as „Cellular
mg/l), sucrose (6%) and galactose (3%). totipotency‟. For a differentiated cell to
Savita et al., (2010) reported that the express its totiotency it first reverts to
maximum callus induction (98.66 %) were meristimatic stage and forms undifferentiated
found from leaf segments on MS medium callus tissue (dedifferentiation) followed by
supplemented with 2, 4-D (4.0 mg/l). Further forming whole plant or plant organ
in nodal segments, maximum callus induction (redifferentiation). Al-Khayri and Al-Bahrany
(96.00 %) was observed with 2, 4-D (1.0 mg/l) (2001) reported that multiple shoots from
and in root segments; it was 48.66 % on MS nodal segment of lime (Citrus aurantifolia
medium supplemented with 2, 4-D (2.0 mg/l). (Christm.) on MS medium supplemented with
BAP, kinetin and NAA. Ali and Mirza (2006)
Savita et al., (2011b) found maximum callus reported maximum shoot regeneration
induction of 91.66 % on MS medium response (70 %) from callus on MS medium
supplemented with 2, 4-D (2.0 mg/l) in supplemented with BA (3.0 mg/l). Perez-
combination with ME (500 mg/l). Further, Tornero and Tallo´nI.Porra (2008) tried
maximum shoot regeneration of 87.50 % was several combinations of BAP and Gibberellic
observed with BA (3.0 mg/l). In vitro acid (GA3) to optimize the proliferation phase
multiplication of C. jambhiri through the and found that the numbers of shoots were
nodal explant on MS medium supplemented dependent on the BA and GA concentrations
with BAP (1.5 mg/l) and malt extract (500 and the best results were observed with 2.0
mg/l) established highest number of shoots per mg/l BAP and 1.0 or 2.0 mg/l GA. Sharma et
explant in minimum time (Kour and Singh al., (2009) obtained maximum number of
2012). Kasprzyk-Pawelec et al., (2015) shoots per plant through the callus in
observed best shoot induction when the leaf Pectinifera, rough lemon and Cleopatra
explants were cultured on Murashige and mandarin on MS basal medium with 1.0 mg/l
Tucker media (MT) supplemented with BAP BAP. Saini et al., (2010) reported higher
(3.5 mg/l). MS medium supplemented with 2, number of elongated shoots on MS medium
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having BA 0.5 mg/l and GA3 1.0 mg/l, while micro propagation technique for Sour Orange
studying direct shoot organogenesis and plant (Citrus aurantium L.) using nodal explants of
regeneration in rough lemon. Upadhyay et al., mature trees, and reported that best shoot
(2010) found MS medium supplemented with formation of 7.4 shoots per node on MS
BAP (2.0 mg/l) in combination with KN (1.0 medium containing BAP (1.0 mg/l) combined
mg/l) and NAA (0.1 mg/l) as the best with Kinetin (0.5 mg/l). Kaur (2016) during in
treatment multiplication medium with vitro plant regeneration in Rough lemon
maximum shoot length and highest number of (Citrus jambhiri Lush.) through epicotyl
leaves. Kumar et al., (2011) concluded that the segments by direct shoot organogenesis
maximum shoot regeneration of 76.09 % was obtained maximum number of elongated
achieved on MS medium supplemented with shoots (8.50) on MS medium having BAP (0.5
NAA (0.5 mg/l) in combination with BA (3.0 mg/l) combined with Gibberellic Acid (GA3)
mg/l) and KN (0.5 mg/l) and highest (1.0 mg/l). Taye et al., (2018) observed longer
regeneration potential on medium shoots with 0.1 mg/l GA3 than culture medium
supplemented with sucrose (6.0 %) and without this plant growth regulator. Kaur
maltose (2.0 %) and it decreased ever more (2018) developed an efficient protocol for in
with increase in the age of callus from 40 to vitro embryogenic callus induction and
120 days. Savita et al., (2010) established a regeneration of Rough lemon (Citrus jambhiri
protocol for micropropagation of C. jambhiri Lush.). It was reported that MS medium
via callus induction and regeneration and fortified with NAA (0.5mg/l) combined with
reported that callus raised from leaf segments BAP (3.0 mg/l) and kinetin (1.0 mg/l) had
showed maximum regeneration of 57 % on good regeneration potential, highest number of
MS medium supplemented with NAA (0.5 shoots and shoot length and took minimum
mg/l) and BA (1.0 mg/l), where as nodal number of days for regeneration.
segments showed better regeneration of 71.89
% on MS medium augmented with NAA (0.5 In vitro rooting
mg/l) and BA (3.0 mg/l. Savita et al., (2011b)
further developed an efficient In vitro good quality of root induction is a
micropropagation protocol for Citrus jambhiri known phenomenon due to plant growth
Lush. using cotyledons as explants and regulators (auxins). The plant growth
reported maximum shoot regeneration (87.50 regulators (IAA, IBA and NAA) have been
%) on MS medium supplemented with BA popularly considered as rooting hormones in
(3.0 mg/l). It was also reported that the callus plant tissue culture. Paudyal and Haq (2000)
retained regeneration capacity (58.33 %) even found that NAA was superior to IBA for in
after 420 days of culture. Kasprzyk-Pawelec et vitro root induction (75%) in Pummelo when
al., (2015) in in vitro organogenesis using shoots were transferred into half strength MS
Citrus limon L. Burm cv. „Primofiore‟ leaf medium supplemented with 1.3, 2.7 and 5.4
explants reported the best shoot induction μM of NAA. Krishan et al., (2001) has found
when the leaf explants were cultured on good response for in vitro rooting in Mosambi
Murashige and Tucker media supplemented (Jaffa). Further recorded longest regenerated
with 3.5 mg/l BAP. Sarker et al., (2015) found roots of 5.33 cm on half strength MS medium
that semi solid MS medium having BAP (1.5 supplemented with NAA (0.5 mg/l) combined
mg/l) in combination with GA3 (0.5 mg/l) with IBA (0.5 mg/l). Singh et al., (2001)
established as best medium formulation for observed paclobutrazol showing significant
proper shoot regeneration and elongation. effect on rooting in citrus. Further, they
Kanwar et al., (2016) conducted a study on recorded that root length reduction was more
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pronounced in Assam lemon than Sweet lime, medium half-strength was supplemented with
may be due to reduced the biosynthesis of NAA (1.0 mg/l). Also concluded that half
gibberellins as a result of paclobutrazol strength of MT medium without auxin resulted
addition. Al-Khayri and Al-Bhrany (2001) in the maximum rooting of regenerated shoots.
observed the highest rooting on medium Ali and Mirza (2006) reported that MS
containing either NAA (1.5 mg/l) alone or medium supplemented with NAA (0.5 mg/l)
NAA (0.5 mgl/1) combined with (IBA 2.0 provided 70 % of rooting response in Citrus
mg/l). Further they observed that the highest jambhiri. EI-Sawy et al., (2006) found that
number of roots were produced on a treatment rooting in citrus was best using micro shoots
containing both NAA (2.0 mg/l) and IBA (2.0 regenerated from nodal explants. Treatments
mg/l) whereas, most of the elongated roots including MS medium with IBA at 0.0, 0.5
were found in the treatment containing 0.5 and 1.0 mg/l and NAA at 0.0, 0.5 and 1.0 mg/l
mg/l of either NAA or IBA. Kaya and Gubbuk were evaluated for rooting and NAA at 0.5
(2001) conducted a study on in vitro mg/l resulted in best rooting response among
propagation and rooting in some citrus all the treatments. Pe´rez-Tornero et al.,
rootstock through tissue culture in Troyer (2008) obtained highest rooting percentages
citrange and Carrizo on MS medium on media containing IBA (3.0 mg/l) alone or
supplemented with BAP (1.0 mg/l), NAA (1.0 in combination with) IAA (1.0 mg/l. The
mg/l) and GA3 (1.0 mg/l). They observed that average root length was affected significantly
they had optimum growth and development by the IBA and IAA concentrations. Root
other than MS supplemented with BA (1.0 length was greater when only 3.0 mg/l IBA
mg/l) and NAA (1.0 mg/l) in Sour orange cv. was used, also explants had a better
Trunk. Wang et al., (2002) achieved 87 % appearance, with greener and larger leaves.
rooting frequency, when in vitro raised shoots While studying in vitro propagation of citrus
were cultured into MT medium supplemented rootstocks viz. Rough lemon, Cleopatra
with NAA at 0.5 mg/l in Citrus reticulata var. mandarin Pectinifera and Troyer citrange
tankan hayata. Singh et al., (2003) has studied Sharma et al., 2009 reported maximum
the effect of bio-regulators on rooting of in rooting of shoots (1.11 %) in rootstock Rough
vitro raised micro shoots in two Citrus lemon followed by Cleopatra mandarin for the
species, namely, Khasi mandarin and Sweet MS media (half strength) supplemented with
lime and recorded that medium having NAA IBA (10 mg/l). Saini et al., (2010) reported
at 0.1 mg/l resulted in the maximum rooting highest rooting percentage of 77 % on MS
(87.71 %) and longer root length of 46.79 mm. medium containing NAA (1.0 mg/l) combined
Paclobutrazol increased root diameter but with IBA (1.0 mg/l) in Citrus jambhiri. Savita
reduced root length. The growth regulators in et al., (2010) found best rooting response (71
Sweet lime registered a lower rooting %) with NAA (0.5 mg/l) and reported that
percentage (6.83 %) than mandarin (51.75 %). callus from root segments did not regenerate
Karwa and Chikhale (2004) studied that the in Citrus jambhiri. While studying, an
effect of various growth hormones on in vitro efficient plant regeneration protocol from
clonal propagation of Citrus sinensis and callus cultures of Citrus jambhiri Lush.
found that IBA (2.64 μM/l) as best treatment (Savita et al., 2011) reported maximum
with 100 % of the explants producing roots rooting response (91.67 %) on half strength
among different concentration of IBA (0.98 to MS medium supplemented with NAA (0.5
4.9 μM/l). Silva et al., (2005) found that mg/l). Kasprzyk-Pawelec et al., (2015)
rooting in Citrus reshni mandarin was best reported best rooting response of 82 % using
achieved, when in vitro raised shoot on MS the MS medium with NAA (1.0 mg/l). Kaur
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(2016) obtained highest rooting percentage of and coco peat mixture placed in the shade with
96 % and root number on MS medium low light intensity, succeeded in showing
containing IBA (0.1 mg/l) combined with IAA normal growth and functioning of the plants.
(0.5 mg/l). Sarker et al., (2015) found best Kumar and Rao (2012), by using lower
root induction (100 %) on MS medium having relative humidity, higher light intensity and
NAA (0.5 mg/l) in Citrus aurantifolia. Kaur septic environmental condition, reported good
(2018) observed that rooting of regenerated amount of success as regards hardening.
shoots was highest in MS supplemented with Normah et al., (1997) reported 83.33 %
NAA (1.0 mg/l) and IBA (1.0 mg/l) and took survival of regenerated plantlets of Citrus
minimum number to rooting in Citrus halimii under ex-vitro conditions. Al-Khayari
jambhiri. Taye et al., (2018) reported longest and Al-Baharany (2001) reported 90 %
roots with MS medium (half strength) survival of regenerated plantlets of Citrus
supplemented with GA3 (0.1 mg/l). aurantifolia. Rani et al., (2004) reported 67%
survival rate of rooted plantlets of Kinnow.
Hardening and planting out Altaf et al., (2008) reported 76 % survival of
regenerated plantlets of Citrus jambhiri.
In vitro propagation technique has been Citrus is vast genera comprising of many
widely used for development of disease free economically important species and varieties
plants, their improvement and rapid across the world. Citrus species are infected
multiplication in many crop plants. However, by several microorganisms like bacteria,
its wider use often gets restricted by high fungi, viruses and mycoplasma causing severe
percentage of plant loss or death whenever economic losses. Microorganism infestation is
transferred to natural environmental easily transferred through seed as well as
conditions. The acclimatization and survival vegetative means of propagation. The demand
of in vitro hardened plantlets in natural field and need of citrus industry is to develop high
conditions is the ultimate and important step. yielding progenies as well as to get biotic and
Eden and Cerruti (2008) successfully abiotic stress resistant root stock as a planting
acclimatized 7-8 cm heighted and well rooted material. Therefore, like majority of vast
shoots in partial shading that can initially genera and plant species, Citrus also needs
reduce light by 50 percent. Anita et al., (2000) improvement to develop resistant genotypes.
found that bacterial inoculum enhanced the The conventional citrus breeding methods are
survival rate of in vitro hardened plantlets and limited due to difficulties such as
there was increase (30-50%) in survival rate. heterozygosity, inbreeding depression,
Pospisilova et al., (1999) indicated different nucellar polyembryony and juvenility. Under
abnormalities from in vitro acclimatized plants such conditions in vitro standardized protocol
due to the suddenly changed environmental of citrus micropropagation would prove useful
conditions. Hazarika and Parthasarathy (2002) for rapid multiplication of plants. It can be
have also reported the beneficial effects of concluded that the citrus species can be
reduced humidity and antitranspirants use for successfully be micropropagated employing
successful in vitro hardening and ex vitro seedling explants like leaf, epicotyl and nodal
survival of citrus plantlets. Darwesh and segments though callus induction with good
Rasmia (2015) studied the in vitro isolated multiplication rates and regeneration potential
plantlets transferred to acclimatize in on different media composition with different
greenhouse in peat of moss and perlite (2:1) combinations and concentrations of plant
kept in plastic cover with inside 100% growth regulators cited within the manuscript
humidity and noted their better normal (Fig. 1).
growth. During the present work, the fine sand
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Fig.1 Typical events during propagation of Citrus spp. through callus induction as exemplified
by Citrus jambhiri. A. Inoculation of leaf explants B. Callus induction from leaf segments C.
Callus regeneration D. Shoot regeneration after subculturing E. Rooting of regenerated shoots F.
Planting out after acclimatization
B C
A
D E F
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Mudasir Iqbal, V. K. Wali, Parshant Bakshi, Kiran Kour, Vijay K. Razdan, B. K. Sinha and
Sood, K. K. 2019. In vitro Propagation of Citrus Species through Callus Induction and
Regeneration: A Review. Int.J.Curr.Microbiol.App.Sci. 8(10): 2282-2295.
doi: https://doi.org/10.20546/ijcmas.2019.810.265
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