Plant tissue culture offers important role in genetic improvement

tissue culture offers important role in genetic improvement of any plant
species and gained importance for its application in mass multiplication of
elite genotype, secondary metabolite production and in in vitro cloning of
plants. Among these, Micropropagation is a powerful tool for mass
multiplication of selected genotypes at faster rates (Bajaj, 1986). To overcome
many of the problems associated with conventional breeding programs,
micropropagation technique have gained greater momentum on commercial
application in the field of plant propagation (Paranjothy et al., 1990; Machey
et al., 1995). A considerable account of in
vitro studies had been undertaken both nationally and internationally in
agricultural and horticultural fields. Using different culture techniques it
has been possible to regenerate propagules with better qualities, greater
vigor, higher yield and disease resistance.

clonal propagation is possible through bud or shoot proliferation (Pierik,
1990), induction of bulbs or corms (Ziv, 1990), or somatic embryogenesis
(Ammirato, 1989). Tissue culture was utilized for eradication of viruses
through meristem culture (Morel and Martin, 1952), culture of single cells and
suspension cultures (Muir et al .,1954), establishment of auxin-cytokinin basis
of organogenesis (Skoog and Miller, 1957), somatic embryogenesis (Reinert,
1958) and large-scale culture of cells (Tulecke and Nickell, 1959).

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allows the production of large numbers of identical plants from small pieces of
stock plant in a relatively short period of time. In most of these cases, the
original piece of tissue is taken either from the leaf, shoot-tip, axillary
bud, stem or root tissue. This methodology is a true means of accelerated
asexual propagation and the plants produced by these techniques respond
similarly to any own-rooted plant (Akin-Idowu et al., 2009).

most important technique in micropropagation is meristem proliferation, wherein
shoot meristems i.e., shoot tips or apical meristems or axillary buds are
cultured to regenerate multiple shoots without any intervening callus phase. In
vitro tissue culture propagation systems are very efficient in Musa. These can
give high-quality, uniform plants free of disease and nematodes, and much of
the planting material used in commercial plantations, and increasingly in
smallholder production, comes from mass micropropagation. Shoot tip cultures
have been most widely used (Strosse et al., 2004), but suspension cultures are
also being developed (Roux et al ., 2001).


protocol for the micropropagation of banana was first standardized by Cronauer
and Krikorian (1984). Strosse et al., 2004 reported that shoot meristem can be
used as an explant and later on the protocol was  revised and modified by several researchers
for shoot tip culture of different varieties of banana (Jarret et al , 1985;
Wong, 1986; Khatri et al , 1997; Ganapathi et al , 1998; Malik et al , 2000;
Abeyaratne and Lathiff, 2002; Gubbuk and Pekmezci, 2004; Strosse et al , 2004;
Titov et al , 2006; Dangi et al , 2009; Karim et al , 2009 and Shirani et al ,

shoot-tip explants are isolated from underground suckers and rhizomes of
banana. These are most commonly infested by soil-borne bacteria and fungi,
which cause contamination during the initiation stage, resulting in loss of
inoculum. Over 60% contaminants in Musa shoot
tip cultures are the species of Bacillus (Van den houwe et al., 1998; Vora et
al., 2009). The procedures for various sterilization have been proposed by
several researchers.

hypochlorite is the most commonly used disinfectant for surface sterilization
of banana explants (Sandra and Krikorian 1984; Mendes et al., 1996; Muhammad et
al., 2004). For the explant disinfected after excision, a shorter treatment time
and a lower hypochlorite concentration (0.0525 percent) is also effective
(Vessey and Rivera, 1981; Krikorian and Cronauer, 1984).

hypochlorite is considered as one of the commonly used surface sterilant for
banana explants (Mendes et al., 1996; Muhammad et al., 2004). The disinfection
of explant with lower concentration of sodium hypochlorite is also effective (Vessey
and Rivera, 1981; Krikorian and Cronauer, 1984). Several authors reported the use of low concentration of
mercuric chloride for surface sterilization of explants (Banerjee and Sharma,
1988; Habiba et al., 2002; Molla et al., 2004; Titov et al., 2006). Double
disinfection method has also been followed by some researchers, where more than
one chemical sterilant has been used (Nandwani et al., 2000; Rahman et al.,
2002; Madhulatha et al., 2004).

avoid fungal and bacterial contaminations in in vitro cultures sometimes explants are treated with fungicides
and antibiotics (Van den Houwe 1998; Nadwani et al., 2000). Ethanol has been
used by a number of research workers for disinfection purposes (Silva et al.,
1998; Rahman et al ., 2002; Jalil et al ., 2003). For in vitro culture, Asif et al .(2001) reported that banana seeds
were soaked in 1.4 percent (v/v) sodium hypochlorite solution for 10 minutes
followed by a quick wash with 70 percent(v/v) ethanol before inoculation.

tissue cultures often suffer from excessive blackening caused by phenolic
exudation released from wounded tissues. These will form a barrier round the
tissue, preventing nutrient uptake and hinders the growth. Therefore, during
the first 4-6 weeks, fresh shoot-tips are transferred to new medium at every
1-2 weeks of interval. Alternatively, the in
vitro cultures can be kept in complete darkness for one week. Jarret et al
., (1985) suggested that addition of ascorbic acid or citric acid in
concentrations ranging from 10-150 mg/l, to the growth medium will minimize the
blackening of the tissue, or the explants can be  dipped in antioxidant solution (cysteine 50
mg/l) prior to their transfer to culture medium. The similar effect of ascorbic
acid in controlling the phenolic oxidation has been reported by several investigators
((Chawla, 2002; Abeyaratneb and Lathiff, 2002).

charcoal is used in control of lethal browning. Lu et al., (1990) first
reported its use in micro propagation and it was suggested that charcoal was
responsible for adsorption and desorption which controlled the release of
nutrients in the production of synthetic seeds (Ganapathi and Higgs et al.,
1999). Similar effect was observed by several investigators ((Fridborg et al.,
1978; Thomas, 2008).

micro propagate different types of plant tissues and organs various kinds of
nutrient media are required as per the explant genotype. For banana
micropropagation, MS-based media are widely adopted. Generally, they are
supplemented with sucrose as a carbon source at a concentration of 30-40 g/l.
Several media formulations have been reported for banana shoot tip culture with
slight modifications of MS media (Browning et al., 1987). Other popular media
include B5 (Gamborg et al ., 1968), SH (Schenk and Hildebrant (Schenk and Hildebrant,
1972), N6 (Chu et al ., 1975) and Linsmaier and Skoog (LS) (Lismaier and Skoog,
1975) media (Hussein, 2012; Saad and Elshahed, 2012). Some investigators
initiate the cultures on the same media and later used for multiplication while
other used low concentrations of hormones for culture initiation. These authors
reported different media compositions, especially various plant growth
regulators, optimizing their concentration that provided maximum number of

investigators used only single cytokinin for culture initiation (Cronauer and
Krikorian, 1984; Thomas et al., 1995; Silva et al., 1998; Rahman et al., 2004;
Molla et al ., Roels et al ., 2005), while others used mixture of cytokinins
(Nandwani et al ., 2000; Rahman et al ., 2002). A combination of cytokinin and
auxin was also used for banana culture initiation by a number of researchers
(Hwang et al., 1984; Drew et al., 1989; Zaffari et al., 2000; Muhammad et al.,
2004).The rate of shoot proliferation is the most important factor for
micropropagation, which was found maximum using 5 mg/l of BA for Grand naine
variety of banana (Cronauer and Krikorian, 1984; Bairu et al., 2008). Sandra
and Krikorian (1984) achieved 9.1 shoots/explants during in
vitromultiplaication of ‘phillipine Lacatan’ and ‘Grand Naine’ on a modified
Murashige and Skoog (1962) medium supplemented with 5.0 mg/l BAP
(6-benzylaminopurine). On the other hand Rahman et al ., (2002) achieved 4.52
shoots/explants on the same concentration of BAP on MS medium during in vitro
multiplication of cv. Bari-1, indicating the genotypic response towards

was found to be the most effective over rest of the adenine-based cytokinins
including zeatin, kinetin and 2-ip (Cronauer and Krikorian, 1984; Vuylsteke and
De Langhe, 1985; Swamy et al , 1983; Wong, 1986). Similarly Nor-Aziah and
Khalid (2002) used higher concentration of BAP during regeneration of in vitro
banana plants from scalps and whole meristems. scalps were induced on MS
supplemented with coconut water and high concentration of BAP (75µM). The
average number of regenerated shoots produced from scalp was six-fold higher
than that generated from single meristems. Venkatachalam et al .,(2006)
achieved direct shoot regeneration from leaf sheaths of silk banana (AAB) when
cultured on medium containing 22.4 µM BAP. Diphenyl urea derived cyokinin, TDZ
was found even better and more economical than BA for in vitro shoot
proliferation responses of Musa (Arinaitwe et al , 2000; Gubbuk and Pekmezce,
2004). Some researchers have incorporated coconut water (5-15%) in MS media as
a supplementary growth regulator (rich in natural cytokinin) for in vitro
propagation of different varieties of Musa (Abeyaratne and Lathiff, 2002;
Srangsam and Kanchanapoom, 2003; Titov et al , 2006).

researchers have incorporated coconut water (5-15%) in MS media as a
supplementary growth regulator (rich in natural cytokinin) for in vitro
propagation of different varieties of Musa (Abeyaratne and Lathiff, 2002;
Srangsam and Kanchanapoom, 2003; Titov et al , 2006). In vitro multiplication
of banana is normally carried in the presence of high cytokinin levels which
inhibit root formation and elongation.

during in vitro multiplication shoots may lack roots and are growing in the
form of bunches which cannot be transferred directly to field conditions. Prior
to transfer in free living conditions, individual shoots are separated from
cluster and grown on root induction media. The concentration of cytokinin in
the rooting medium should be lower than auxins in the multiplication medium, so
that cytokinin/auxin ratio becomes low which is favorable for root induction as
reported by Gupta (1986) and Wong (1986). However most of the investigators
omit cytokinins entirely from the rooting medium.

most frequently incorporated auxins in rooting medium were NAA, IAAand IBA.
Indole butyric acid (IBA) was effective for root induction of in vitro raised
banana plants and frequently used for this purpose. Muhammad et al., (2000,
2004) and Habiba et al., (2002) regenerated roots on half strength MS medium
having 1 and 2 mg/l IBA respectively.                              Molla et al., (2004)
reported that a good number of healthy roots were produced on half MS media containing
0.4, 0.5 or 0.6 mg/l IBA. Nandwani et al., (2000) also found 1.0mg/l IBA
suitable in MS medium during mass propagation of Basrai. Madhulatha et al.,
(2004, 2006) used IBA and NAA in combination during optimization of liquid
pulse treatment for production of in
vitro rooted plants of cv. Nendran (Musa
spp. AAA).

acetic acid (NAA) was another auxin used frequently at lower concentration for
root induction of in vitro raised banana plants. Arinaitwe et al., (2000)
achieved rooting on MS medium containing 1.2µM NAA during the study of
proliferation rate effects of cytokinins on Kibuzi, Bwara and Ndizwemiti banana
cultivars. Rahman et al., (2004) used different concentrations of NAA for root
induction of Musa sapientum and found
that 2mg/l was better.

plants were delicate plants because they were produced in a closed, sterile
environment and grown on nutrient-rich artificial media under controlled
conditions with high humidity and low light intensity. The transfer of rooted
plantlets from aseptic culture conditions direct to the external environment
can result in significant loses of plants.

removed from the tissue culture environment, micropropagated plants must be
allowed to adjust to the outside environment with its varying light levels,
changing temperature, reduced humidity, lower nutrient availability, and
pathogen presence. Tissue-cultured plants are generally poor in cuticle,
therefore lose water rapidly upon transfer to natural conditions.

plantlets, placed in portrays containing cocopeat, were allowed to harden in
green house for 45 days, optimized at 27°C, 70% RH and 15,000 lux2 (Shailesh et
al ., 2010).

and development of in vitro raised
plants of cultivar Pioneira (Musa sp. AAAB) during hardening was studied by
Silva et al., (1998). In vitro rooted
plantlets were transferred to plastic bags containing organic substrate. Jasrai
et al., (1999) developed protocols for hardening of in vitro derived banana plants without greenhouse facilities. In vitro raised plants were transferred
in polythene bags which were perforated six cm from the base. The bags
containing the plants were placed inside a plastic tray. High humidity was
maintained by spraying water after every two hours. On an average 92 percent of
the plantlets survived.

performance of these micropropagated plants was reported by a number of
authors. Nokoe and Ortiz (1998) suggested the optimum plot size for banana
field trails.

was observed that 16 ± 3 plants per plot were needed to evaluate the growth
characteristics and yield potential of the cultivars. The recommended optimum
plot size considered on average, 13 ± 3 plants per plot for the plant crop, and
15 ± 2 plants per plot for the ratoon crop. Hwang et al., (1984); Smith and
Drew (1990) reported that tissue culture plants had bigger pseudostem and
retained more healthy leaves than those originating from suckers.

et al., (1993) achieved 20.4 percent higher yield than conventional plants, due
to larger bunches and a shorter cycle to harvest. On the other hand in vitro derived plants of plantain (Musa spp. AAB) did not show any higher
yield (Vuylsteke and Ortiz, 1996) and more phenotypic variation observed in
tissue culture plants.

plantlet size for tissue culture banana plants was studied by Fraser and
Eckstein (1998). They reported that small plants of 100 mm took three weeks
longer to harvest and six percent lower yields as compared with 300 mm plants.
It was advisable that 200 mm plants should be planted at least 100 mm below
soil surface, preferably in a furrow.

Somaclonal variations

variations were observed among in vitro regenerants due to pre-existing genetic
variation within the explant and by the influence of exogenous phytohormones (Lakin
and Scowcroft, 1981; Evans et al., 1984). The genetic variations in tissue
culture-raised plants has been reported at morphological, chromosomal,
biochemical and molecular levels in many plant species has been extensively
reviewed many by researchers (Brown, 1991; Karp, 1991, Geering, 2005). The
variations expressed in tissue cultured plants may be the result of oxidative
stress damage imposed upon explants due to culture initiation, subsequent
subculture, wounding, exposure to sterilants during sterilization, high
concentration of phytohormones and lighting conditions  (Krishna et al. 2008; Tanurdzic
et al. 2008; Smulders and de Klerk 2011; Nivas and DSouza 2014). Nwauzoma and
Jaja (2013) study confirms increasing the number of cell cycle in vitro induces somaclonal variations
and also decrease multiplication rate in micropropagated banana.

(1997) also reported that tissue such as leaves, stems and roots produce more
variations than the meristematic tissue such as axillary buds and shoot tips. Currais
et al. (2013) also reported that genetic variations generally occur in undifferentiated
cells, isolated protoplasts, calli, tissues and morphological traits of in vitro raised plants. Shchukin et al.,
(1997) observed 5.3% variations in shoot tip cultures of cv. Grandnaine in
comparison with plant lets derived through somatic embryogenesis.  Studies of Israeli et al., (1996) suggested
that in the Cavendish group of bananas, shoot tips might be chimeric and that
dissociates of these chimeras might possibly result in the recovery of dwarf
variants. With the extensive use of in
vitro techniques, somaclonal variation is commonly observed in Musa propagation (Vuylsteke et al., 1991)
and the genetic origin of the phenotypic variation is a subject of more

et al., (1996) reported a somaclonal variant with mosaic-streaked leaves
obtained from Grandnaine (AAA) cultivar. Trujillo and Garcia (1996) reported a
somaclonal variant (CIEN BTA-03) resistant to Yellow Sigatoka (YS;
Microphaerella musciola Lench) regenerated from adventitious shoots induced on
excised shoot apices of Williams cultivar (susceptible to the disease), grown
on MS medium (Murashige and Skoog, 1962) with 15 mg/l of N6-benzyladenine (BA).

variation has also been evaluated at the DNA level in Musa. Kaemmer et al., (1992) used random amplified polymorphic DNA
(RAPD) and microsatellite fingerprinting to construct phylogenetic dendograms
of Musa spp. and characterized a
somaclonal variant of Grandnaine (AAA) named Novaria. Bhat et al., (1995)
reported restriction fragment length polymorphism (RFLP) and RAPD studies in 57
Musa cultivars. Faure et al., (1993)
compared RFLP and RAPD markers as techniques for mapping analysis and concluded
that RAPD markers are highly polymorphic for making maps. Venkatachalam et al.,
(2007a) investigated the occurrence of somaclonal variations in Musa acuminata var. ‘Nanjanagudu
Rasabale’ cultured on media supplemented with high concentration of cytokinin
using RAPD and ISSR and reported the 
absence of genetic instability in the regenerants. The genetic
variability in micropropagated plants of ‘Robusta’ and ‘Giant Governor’ was
observed by utilizing the markers technique RAPD and ISSR by Ray et al., 2006.

 Polymorphism at DNA level among the somaclonal
families which were phenotypically normal was reported in strawberry (Damiano,
1997) in Triticum (Brown et al ., 1993), in rice (Godwin et al ., 1997), in
Populus deltoids (Vijay et al ., 1995) and in date palm (Saker et al ., 2000;
Saker et al ., 2005). Such modifications included gene methylation changes, DNA
rearrangements and alterations in copy number. 
Unambiguous identification is especially important in
clonally-propagated crops such as banana (Kestner, 1983). Among many
researchers, AFLP is the marker technology of choice since it combines the
reliability of classical restriction-based fingerprinting with the speed and
convenience of polymerase chain reaction (PCR)-based marker techniques (Vos et
al., 1995; Powell et al., 1996; Lu et al., 2002; Ude et al., 2002a, 2002b). The
AFLP technique rapidly generates hundreds of highly replicable DNA markers,
thus allowing high resolution genotyping (Loh et al., 2000). Several studies
suggested  that the AFLP markers is
effective for genetic diversity analysis in Musa
and the level of polymorphism comparatively higher than  other 
markers  used  in  the
diversity  analysis (Crouch et al., 1999;
Wong et al., 2001; Kour et al.,2011; Tripathy et al.,2016). In the present
study also a sincere attempt was made for the evaluation of morphometric
characters of the regenerants in the field condition and evaluation of genetic
variations using AFLP markers.