Tissue Culture of Anthurium andreanum: A Significant Review and Future Prospective
Anthurium, with over 108 described genera and 3750 species, is one of the most popular tropicals, highly appreciated for its brightly coloured and long lasting flowers. The exotic Anthurium industry plays a significant role in the global floriculture trade. To overcome the demerits of conventional propagation of Anthurium, plant tissue culture proved to be an influential tool that can complement conventional breeding and accelerate Anthurium development. This review presents a consolidated explanation of in vitro propagation and focuses upon contemporary information on biotechnological advances made in Anthurium.
May 27, 2010; Accepted: June 09, 2010;
Published: September 02, 2010
Floriculture puts its best foot forward to shift a considerable amount of capital
in productive chain every year. Anthurium, for its exquisiteness, durability
and by the terms of long vase-life stands out among most of the tropical cultivated
flowers. As a member of the Araceae family, Anthurium, the native to
the tropics of Central and South America (Gantait et
al., 2008), consists of 108 genera and approximately 3750 monocotyledonous
species (both herbaceous and creepers) (Viegas et al.,
2007). Principally, the arums are a tropical family, occurring naturally
on all continents, with an exception of Antarctica. The genus having highly
ornamental inflorescence and/or foliage are Anthurium, Caladium,
Dieffenbachia, Monstera, Philidendron and Spathyphyllum
(Gryum, 1990). A vast number of Anthurium species
are duly produced and traded internationally as cut-flowers, flowering potted
plants and landscape plants among which most of the cut-flower Anthuriums
are believed to be hybrids of Anthurium andraeanum Linden ex André
with several closely related species in the section Calomystrium (Croat
and Sheffer, 1983) and have been referred to as Anthurium andraeanum
Hort. (Kamemoto and Kuehnle, 1996). According to the
report of Kuehnle et al. (1992), the breeding
of two species, A. andreanum Linden ex André and A. scherzerianum
Schott., has resulted in many cultivars which are used as potted plants
as well as cut flowers, grown commercially all over the world. Until now, the
identification of the cultivars has mainly been based on the spathe colour (Kobayashi
et al., 1987). A large number of Anthurium cultivars displaying
an array of spathe colours (ranging from red, orange, pink, coral and white)
and belonging to the three spathe categories- namely, standard (single-coloured
heart-shaped), obaki (bicolours of green with another major anthocyanin colour)
and tulip-type are cultivated in order to meet the demand of the various market
preferences in terms of colours, shades, floral sizes and shapes. The production
of a single flower from each leaf axil takes place during the sympodial phase.
In fact, the commercial flower is an inflorescence, composed of the spadix (a
spike of minute flowers closely arranged round a fleshy axis) and the spathe
(a large sheathing bract enclosing the flower cluster) (Higaki
et al., 1984). The Anthurium gains its fame and respected
status by exhibiting its striking ensemble, which is created unitedly by its
spadix and its spathe, within the economically essential ornamentals, allowing
its use in interior and exterior decoration and also to its use as a cut flower
(Lopes and Mantovani, 1980; Castro
et al., 1986). In the worldwide market, the Anthuriums are
flowers with intense importance. The commercial value of Orchids and Anthuriums
among the tropical cut flowers are first and second, respectively (Dofour
and Guerin, 2003; Chen et al., 2003). Due to
the increasing demand for cut flowers in the global market, large numbers of
novel Anthurium cultivars are continually being imported from the Netherlands
and Hawaii for commercial cultivation (Nowbuth et al.,
PROBLEMS ASSOCIATED WITH CONVENTIONAL PROPAGATION
Propagation of Anthurium can be possible by both asexual and sexual
methods. Conventional vegetative or asexual propagation of Anthurium
is performed through terminal and single node cuttings along with the division
of newly emerging suckers or offshoots, which can also be induced by topping
(Mahanta and Paswan, 2001). In spite of producing homogenious
materials, vegetative propagation of Anthurium is a very measured process,
taking it years to develop commercial quantities of elite clones (De
Lima et al., 2006) and thus not proving very advantageous (Pierik
et al., 1974; Higaki and Ramussen, 1979;
Geier, 1990). The traditional propagation of hybrid is
challenging due to the growth of a low number of new plants from the base (Martin
et al., 2003). In other way Anthurium is sexually propagated
by seed (Dofour and Guerin, 2003), although, some taxa
fail to produce viable seeds due to incompatibilities (Sheffer
and Kamemoto, 1976). Moreover, the seed propagation leads to a very heterogeneous
progeny due to cross pollination (Bejoy et al., 2008).
A notable variation in the form of colour, quality, yield and the time of first
flowering can be observed in the plants which are derived from seeds (Jahan
et al., 2009), where the seeds cannot be conserved and must be collected
immediately after fruit maturation. Besides, the phase from pollination to seed
maturity (approximately 6 to 7 months) and the ensuring development of the plants,
which takes a further three years, is too lengthy (Hamidah
et al., 1997; Viegas et al., 2007).
Seed viability, contamination and germination often creates difficulty and to
record high level of germination, seed requires continuous light condition which
is hardly available in natural field condition (Gantait
et al., 2008), apart from that the seeds are viable only for two
to three days and germination is very low (20 to 30%) (Jahan
et al., 2009).
PLANT REGENERATION SYSTEMS IN VITRO
Plant regeneration in vitro is an alluring alternative for mass multiplication
of outstanding cultivars at faster rates than conventional methods. A unique
concept proposed by Haberlandt (1902) and logically
proved for the first time by Steward et al. (1958),
states that, in vitro culture is one of the key tools of plant biotechnology
that exploits the totipotent nature of plant cells. In vitro culture
promotes accelerated multiplication of superior clones and is a pre-requisite
for the improvement of plants via genetic engineering technique. Tissue culture
has been exploited to generate genetic variability by producing haploids, somaclonal
and gametoclonal variants from which crop plants can be improved. It is also
utilized for enriching the health status of the plant material and to amplify
the number of desirable germplasm available to the plant breeder. Tissue culture,
in union with molecular tools and techniques, has been successfully used to
integrate specific traits through genetic transformation. The culture of single
cells and meristems can be effectively used to clean out the pathogens from
planting material and thereby dramatically improve the produce of established
cultivars. By using certain laboratory materials, typically taking one or two
decades to attain the trade market all the way through plant breeding, this
technology can be expected to have an ever accelarating impact on crop improvement
as we pass through the new millennium (Kothari et al.,
Anthurium is an ornamental plant used for its cut flower significance from commercial to household decoration. Commercial production of the Anthurium plants is growing globally to meet the increasingly high demand of its flowers. Its economic value has extensively increased over the last two decades and there is a growing prospective for continued further growth in both domestic and international markets. To accomplish this purpose and satisfy the increasing demand of innumerable quality propagules, a collection of research work has been carried out to explore morphogenic potential and attain regeneration of Anthurium and constant efforts are still in advancement to achieve further improvement in the biotechnological aspects. Anthurium plant regeneration has been achieved chiefly through direct or indirect organogenesis, but the other modes of regeneration such as somatic and zygotic or somatic embryogenesis have also been explored.
Organogenesis in vitro: Organogenesis in vitro is a versatile inclination concerning the de novo formation of organs (shoots or roots). Shoots can be derived either all the way through differentiation of non-meristematic tissues known as adventitious shoot formation or through pre-existing meristematic tissues known as axillary shoot formation. Both these approaches bring about a synergistic interaction of physical and chemical factors. A triumphant plant regeneration protocol requires an apt choice of explant, age of the explant, specific media formulations, definite growth regulators, genotype, carbon source, gelling agent and other physical factors which includes light regime, temperature and humidity. An array of research work has been performed to explore the morphogenic potential and achieve regeneration of A. andreanum (Table 1).
|| Organogenesis in vitro from Anthurium andreanum
|Mult Sht: Multiple shoot; Rt: Root; CW: Coconut water; AC:
Activated charcoal; Rt: Root; Ca: Callus; PLB: Protocorm-like bodies; Sht
Reg: Adventitious shoot regeneration; CW: Coconut water; AC: Activated charcoal;
Nitsch: Nitschs basal medium (Nitsch, 1969),
Pierik: Pierik medium (Pierik, 1987)
Effect of cultivars in regeneration: Cultivars are one of the key attributes that have left their impact on the organogenic response of the cultures in different plant species. The specificity of the cultivars in terms of their individual regeneration capacity stands out as the limiting factor resulting in a condition which demands for specific protocols for each cultivar. The fact concerning the reliability on cultivar specificity for in vitro regeneration acts as one of the most important factors for Anthurium tissue culture.
The outcome of cultivar difference on in vitro morohogenesis has been
reported as an obvious result among the Anthurium andreanum cultivars
(Pierik et al., 1974). Pierik
(1976) examined 38 cultivars of Anthurium andreanum and observed
moderate to strong callus formation from leaf explants in 31 responsive cultivars,
where callus induction was very meager in four with no response from rest of
the three. Performance variation of different cultivars on callus induction
and regeneration of Anthurium was studied comprehensively in total 15
different cultivars (Chen et al., 1997; Malhotra
et al., 1998; Puchooa, 2005; Duong
et al., 2007; Atak and Celik, 2009) using
altered explant sources and culture media compositions. In the study of Duong
et al. (2007), 10 cultivars namely, Neon, Choco, Sonate, Midori,
Pistache, Tropical, Safari, Arizona and Cancan were evaluated on the basis of
their performance over callus induction and adventitious shoot formation. In
terms of callus induction and proliferation, cultivar Pistache proved as the
best resultant, whereas, Carnaval and Cancan didnt reveal any response.
The potentiality of shoot regeneration was also varying from cultivar to cultivar,
where, the Tropical type resulted maximum number of shoots than the others.
The most recent report by Atak and Celik (2009) in Anthurium
andreanum cv. Arizona and Sumi, describes the significantly different impact
of cultivars on indirect organogenesis. The callus induction rate of Arizona
was significantly higher than that of the Sumi. The average number of adventitious
shoots regenerated from callus for Arizona cultivar also increased significantly
as compared to Sumi.
Direct regeneration of multiple shoots and roots was notably influenced by the
cultivars Midori, Kalapana, Tinora Red, Senator, Sonate and Lambada, as revealed
by the literatures (Lee-Espinosa et al., 2003;
Joseph et al., 2003; Martin et al., 2003;
Rivero-Bautista et al., 2005). In the study of
Martin et al. (2003) two commercially elegant cultivars
of Anthurium Tinora Red and Senator revealed their differential performance
in direct organogenesis from lamina explants. Cultivar Tinora Red proved to be
more regenerative than Senator in term of number and growth of meristemoids and
multiple shoots per explant though there was no significant difference recorded
in rooting in vitro.
Kuehnle et al. (1992) were the first to report
the influence of Anthurium hybrid cultivars UH780, UH965 and UH1060 on
somatic embryogenesis. UH780 and UH956, the two intraspecific hybrids including
the tulip-type hybrid UH1060, were cultured on MS medium with varied concentration
ratio of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylaminopurine (KIN).
Impact of different cultivars in production of somatic embryos was quite prominent
as UH1060 was the best performer in comparison to the other two.
Effect of explants in regeneration: Direct or indirect in vitro
organogenesis of the plant tissue is mostly governed by the suitable selection
of the explant. In support to this statement, Anthurium regeneration
is also reliant on the age and type of the explant concerned. Different explants
including explants such as in vitro seedling, axillary bud, shoot tip,
node, leaf, lamina, petiole, spathe and spadix (Pierik et
al., 1974; Leffringen and Soede, 1979; Kunisaki,
1980; Kuehnle and Sugii, 1991; Teng,
1997; Joseph et al., 2003; Vargas
et al., 2004; De Lima et al., 2006)
initiate the purpose efficiently in Anthurium tissue culture.
For direct organogenesis into multiple shoot and root regeneration, shoot tip
explants were successfully used (Somaya et al., 1998;
Lara et al., 2004; Dhananjaya
and Sulladhmath, 2006). The pioneering effort of successful shoot multiplication
of Cancan cultivar of Anthurium using apical shoot bud was carried out
by Gantait et al. (2008) (Fig.
1a, b). Nodal explant was recognized to be the other most responsive explant
to regenerate in vitro roots (Rivero-Bautista et
al., 2005). Shoot multiplication was witnessed in three cultivars Agnihotri,
Midori and Kalapana, initiating the culture with axillary buds (Mahanta
and Paswan, 2001; Lee-Espinosa et al., 2003).
Comparable results have been demonstrated by other authors using micro-cuttings
of in vitro seedlings explants (Vargas et al.,
2004; Maira et al., 2009). Yang
et al. (2002) and Martin et al. (2003)
fascinatingly studied the efficiency of direct shoot regeneration from lamina
Adventitious shoot and root regeneration subsequent to successful callus induction
were studied in detail using leaf explants (Pierik et
al., 1974; Lightbourn and Deviprasad, 1990;
Malhotra et al., 1998; Trujillo-Sanchez
et al., 2000; Zhang et al., 2001;
Zhao et al., 2004; Montes
et al., 2004; Puchooa, 2005; Nitayadatpat
and Te-Chato, 2005; Duong et al., 2007;
Viegas et al., 2007; Yang
et al., 2008; Beyramizade et al., 2008).
||In vitro propagation of Anthurium andreanum
(a) Initiation of shoot bud (b) Multiple shoot regeneration (c) Root induction
and elongation (d) Ex vitro acclimation of micropropagated plantlets
(e) Micropropagated plants at bloom stage and (f) Gel electrophoresis using
ISSR marker [(GT)8A] showing the monomorphic bands ensures clonal
fidelity (Lane M- 50 bp ladder, C1 to C7- in vitro regenerated clones
and P- mother plant)
Pierik et al. (1974) led the way out in this
aspect. Later, Teng (1997) in a novel approach employed
homogenised leaf inoculums to regenerate induced callus and then adventitious
shoots along with roots using liquid or raft culture. As stated in the study
of Bejoy et al. (2008), callus development was
restricted along the cut margins and mid rib zone of leaf lamina explants in
the cultivar Agnihotri. Jahan et al. (2009) reported
the advantage of leaf explants over spadix both in terms of callus induction
and development of multiple shoots. Very recently, efficiency of leaf explants
in indirect morphogenesis was assessed by Atak and Celik
(2009) in two cultivars of Anthurium andreanum Arizona and Sumi.
Callus induction, proliferation and regeneration of shoots were consistently
excellent. Efficacy of leaf and petiole over callus-mediated prorocorm-like
body (PLB) induction supported by histological details was reported by Yu
et al. (2009) in cultivar Valentino. Totipotent callus induction
on ½ MS supplemented with 2,4-D and BA was achieved following growth
and differentiation of PLBs. In vitro roots also proved to be an attractive
explants source for Anthurium micropropagation as they are readily obtainable.
Recovery of adventitious plantlets from in vitro root cuttings was efficiently
achieved by Chen et al. (1997) in UH1060, Alii
and Anuenue cultivars of Anthurium under weak light condition and modified
MS with BA supplementation. Apart from substantial achievement of leaf as explants,
successful appliance of shoot tip, micro-cuttings, petiole and node as explants,
for indirect organogenesis was also reported by several authors (Kuehnle
and Sugii, 1991; Somaya et al., 1998; Dhananjaya
and Sulladmath, 2003; Han and Goo, 2003; Vargas
et al., 2004; Yu and Paek, 1995). Prakash
et al. (2001) studied on the regeneration efficiency of petiole explants
of A. andreanum cv. Mauritius Orange. Callus was produced initially at
the cut ends and later on the entire surface of petiole explants in almost six
to eight weeks of culture. Subsequent development of shoots from shoot primordia
and later regeneration of roots under in vitro shoots was achieved with
For somatic embryo development and plant regeneration in A. andreanum,
leaf and calli were used as explants to initiate the process (Kuehnle
et al., 1992; Prakash et al., 2002;
Xin et al., 2006; Sontikul
and Te-chato, 2007; Beyramizade et al., 2008).
Earlier Kuehnle et al. (1992) developed a successful
in vitro protocol of production of somatic embryos and subsequent regeneration
of plants from Anthurium hybrids using whole leaf blade. Later on, Xin
et al. (2006) have also postulated the superiority of leaf over the
other explants like petioles and roots in terms of embryogenic potentiality.
Further, this report on efficiency of leaf to develop somatic embryo has been
supported by the study of Beyramizade et al. (2008)
in an elegant Anthurium cultivar Tera.
It is significant to evaluate key events in somatic embryogenesis with zygotic
embryogenesis to establish whether similar developmental events take place in
somatic embryos and to consider their quality (Flinn et
al., 1991). At present, there are insufficient information on zygotic
embryogenesis of Anthurium to make such comparisons and Anthurium
genetic improvement. Matsumoto et al. (1998) reported
the first work on the ovule or zygotic embryo culture from Anthurium andreanum
cv. Kalapana using floral parts as explants. They also analyzed an array
of morohological, anatomical and histochemical aspects of zygotic embryogenesis.
Scanning the literature we can conclude that leaf explants have been found to be the most effective for indirect organogenesis (adventitious shoot formation) and plant regeneration in Anthurium, whereas apical or axillary meristems have been most frequently used for clonal multiplication. In addition to this, other explants have also proved their potentiality for regeneration depending upon the parameters like cultivar, plant growth regulator regime and others.
Impact of culture conditions: Kuehnle et al.
(1992) explored the importance of light regime on somatic embryogenesis
of Anthurium andreanum. They observed that the initiation of somatic
embryo requires typical dark period with 23°C temperature, whereas, conservation
and maturation of embryos preferred a 16 h photoperiod of 54 μmol/m2/sec
and 25±2°C temperature.
Effect of low light intensity in direct regeneration from in vitro root
was assessed by Chen et al. (1997), where as
low as 4 μmol/m2/ sec illumination successfully incubated the
root explants. Martin et al. (2003) observed that
the effectiveness of direct plant regeneration was significantly influenced
by the pH of the medium. The effect of pH was studied by culturing young brown-coloured
lamina explants on the optimal medium. Of the different regimes of pH (5.0 to
6.0) tested in their study, pH at 5.5 developed a higher number of shoots directly
from the lamina explant. Explants cultured on medium with lower or higher regimes
of the optimal pH exhibited a higher percentage of yellowing and browning.
Duong et al. (2007) observed the effect of pH
on different phases of organogenesis, where pH 6.0 was optimum for all stages
except in vitro rooting in pH 6.2. In their study, both callus multiplication
and plantlet regeneration were achieved at 10 h photoperiod with 45 μmol/m2/sec
illumination at 25±2°C and 70-75% RH.
In contrast to that Bejoy et al. (2008) reported
the effectiveness of continuous dark period over alternative light and dark
period in callus induction of Anthurium. Callus induction frequency was
quite high in dark regime, while the explants kept in light didnt develop
callus and turned brown rapidly. In accordance with the above finding, Atak
and Celik (2009) also preferred the role of continuous dark phase of one
month for callus initiation.
A favourable effect of controlled culture condition during Anthurium
organogenesis in vitro (pH 5.8), with a temperature range of 25±2°C,
16 h photoperiod, 60% RH and 1500-3000 lux light intensity was studied (Mahanta
and Paswan, 2001; Gantait et al., 2008; Jahan
et al., 2009). For PLB induction and regeneration, a specific condition
at 25±2°C with 12 h photoperiod at 25-30 μmol/m2/sec
proved to be favourable according to the report of Yu et
Initiation of aseptic cultures: Sterilization: For commencement of aseptic
cultures, meticulous information on the physiological status and the susceptibility
of the plant species to diverse pathological contaminants are obligatory. In
favour of most of the explants, the universally adopted system involves surface
disinfection of initial explants with 70% (v/v) ethanol for 1 min followed by
0.1% mercuric chloride (HgCl2) for 2 min (Mahanta
and Paswan, 2001) or 0.1% HgCl2 alone for 7-12 min and rinsing
in sterile distilled water (Martin et al., 2003;
Duong et al., 2007; Bejoy
et al., 2008) or treating with 1-3% sodium hypochlorite (NaOCl) for
15-20 min (Teng, 1997; Vargas et
However, Gantait et al. (2008) sterilized the
shoot tips using antifungal solution cetrimide for 5 min followed by NaOCl and
0.1% HgCl2. Later, Jahan et al. (2009)
effectively used 70% (v/v) ethanol for 1 min, 1.5% NaOCl for 8 min as disinfectant
and added 0.01% Tween-20 as surfactant.
Most recently, to diminish the contamination caused by fungus, endogenous and
exogenous bacteria, Atak and Celik (2009) surface sterilized
the explants for 1 min in 70% (v/v) ethanol, soaked in gentamicin solution for
30 min and then again soaked in 20% (v/v) commercial bleach [containing 5% (v/v)
NaOCl] for 12 min. However, soaking in solution of gentamycin after surface
sterilization resulted in the maximum percentage of disinfected explants. Such
discrepancy could be clarified as all through surface sterilization, the bare
ends of the explants are separated leaving the fresh conducting tissue, which
allows the antibiotic solutions to infiltrate deep within the tissue ensuing
in superior rates of disinfection.
Experiment with plant growth regulators: Plant Growth Regulators (PGR)
signify a crucial role to pave the way in which plants grow and develop. They
control the pace of growth of the individual organs and integrate these organs
to generate the plants. There is a synergistic requirement of both auxins and
cytokinins to induce cell division and growth in plant tissue cultures. Experiments
on whole plants and excised tissues have largely established the existence of
antagonistic and additive interactions linking these two types of plant growth
regulators (Kothari et al., 2010). Shoot regeneration
in A. andreanum is also highly influenced by the media formulations containing
plant hormones and other growth regulators.
Pierik et al. (1974) for the first time tested
different hormonal regimes for indirect organogenesis in Anthurium. Leffringen
and Soede (1979) were the first authors to report the efficiency of cytokinin
alone to induce multiple shoots from a single shoot tip explant. MS medium was
supplemented with either 4.4-13.3 μM benzyleadenine (BA) or KIN. Soon after,
Kunisaki (1980) used Coconut Water (CW) as a growth
enhancer successfully to induce multiple shoot buds. Axillary buds were cultured
on MS basal with 15% CW and 0.2 mg L-1 BA and a high frequency multiple
shoot induction and proliferation were recorded. In accordance with the above
findings, later, the report of Mahanta and Paswan (2001)
also favoured the role of CW (150 mg L-1) in a combination with vitamin
B5 (0.5 mg L-1), Poly Vinyl Pyroledon (PVP) (200 mg L-1)
and indole-3-acetic acid (IAA) (0.1 mg L-1) in direct shoot multiplication
from axillary bud explants of A. andreanum cv. Agnihotri.
Lightbourn and Deviprasad (1990) introduced Nitsch
basal medium (Nitsch, 1969) replacing MS for indirect
organogenesis of A. andreanum. A low level of 2,4-D (0.5 mg L-1)
alone efficiently induced in vitro callus and its subsequent proliferation,
whereas, exactly similar concentration of BA was supplemented alone for successful
regeneration of adventitious shoots. With the advancement of the previous idea
Kuehnle and Sugii (1991) employed a relatively high level
of cytokinin source in combination with low level of auxin to produce more number
of calli with a higher proliferation rate in Anthurium hybrids. Pierik
basal medium (Pierik, 1987) was fortified with 0.36 μM
2,4-D + 4.4 μM BA when petiole explants were inoculated for dedifferentiation
of tissue. For regeneration of shoots from calli, their study supports the reports
of their predecessors, BA at 2.2 μM proved to be the best for single cytokinin
source. Yu and Paek (1995) established two different
protocols in other way to specify cytokinin sources in callus culture for two
different cultivars. For Hazrija, MS supplemented with 0.5-2 mg L-1
BA proved most efficient in callus induction as well as growth. On the other
hand, MS plus 0.5-2 mg L-1 iP was the best combination for Ingrid
cultivar. The effectiveness of cytokinin alone in both callus induction as well
as adventitious shoot regeneration was once again proved by the study of Chen
et al. (1997) from root explants of Alii, Anuenue and UH1060 cultivars
of Anthurium. BA at the rate of 2.2 μM supplementation to MS proved
to be the ideal PGR, although response was varied from cultivar to cultivar.
Teng (1997) carried out the pioneer work in Anthurium
introducing PGRs in liquid or raft culture to improve indirect organogenesis.
MS with 0.9 μM 2,4-D + 2.2-4.4 μM BA influenced the inoculums size
and regeneration frequency to a great extent. Larger (1000 μm) inoculums
produced 1.8-3.3 times higher number of shoots than the smaller ones (500-1000
Young leaf explants of three A. andreanum cultivars (Nitta, Osaki and
Anouchka) were cultured on MS medium with the auxin 2,4-D, indole-3-butyric
acid (IBA) and the cytokinin (BA), with an additional supplementation of ammonium
nitrate (NH4NO3) and Activated Charcoal (AC). In this
unique study of Malhotra et al. (1998) effect
of NH4NO3 and AC apart from the regular PGRs was significantly
observed particularly for callus induction, adventitious shoot regeneration
and in vitro rooting respectively. 200 mg L-1 NH4NO3
in combination with BA and 2,4-D in Nitsch successfully enhanced callus
growth and relatively higher level of NH4NO3 (730 mg L-1)
induced adventitious shoots from callus. On the other hand, 0.04% AC stimulated
a huge success in rhizogenesis in vitro when combined with 1 mg L-1
IBA. The similar study on the same cultivars was carried out by Puchooa
(2005) and the details absolutely support the findings of the previous report
of Malhotra et al. (1998) regarding the usefulness
of 2,4-D, NH4NO3 and AC, except in the recent study it
was found that the relatively low level of NH4NO3 (720
mg L-1) was sufficient for shoot regeneration. In a more advanced
report of Yang et al. (2008), the noteworthy
impact of NH4NO3 on callus induction and proliferation
has been observed when supplemented in permutation with CaCl2. In
accordance to these reports successive studies of Duong
et al. (2007), Gantait et al. (2008)
and Atak and Celik (2009), treatment with AC was necessary
for root induction and elongation occurring on medium. Combinations of AC and
auxin (IAA or IBA) have been very often used as root elongation promoters. However,
the addition of AC seems to provide additional beneficial advantage. It is reported
that AC eliminates light and provides a reasonable physical environment for
the rhizosphere and helps rooting in A. andreanum (Gantait
et al., 2008) (Fig. 1c). This statement was further
supported by Gantait et al. (2009a,
b) in Dendrobium and vanilla respectively, where they observed the
enhanced effect of AC in rooting in vitro.
Employment of Adenine Sulphate (ADS), an additive to PGRs, has been shown to
promote induction and elongation of shoot buds in A. andreanum (Nitayadatpat
and Te-Chato, 2005). MS medium plus ADS devoid of any PGR fruitfully stimulated
shoot regeneration. It seems that ADS acts indirectly on bud induction as an
elicitor or enhancer of growth in combination or synergism with endogenous or
exogenously added growth regulators. Addition of ADS to the medium significantly
improved the multiple shoot elongation. Recently, Gantait
et al. (2008) emphasized the importance of ADS in the medium for
enhanced multiple shoot bud induction and elongation from the cultured shoot
tip of A. andreanum cv. Cancan (Fig. 1a, b). The highest
number of shoot buds per explant was obtained when MS medium was fortified with
0.5 mg L-1 BAP + 60 mg L-1 ADS.
Yu et al. (2009) reported the upshot of low
auxin and high cytokinin levels on callus induction to subsequent PLB production.
A half strength of MS with 0.9 μM 2,4-D plus 8.88 μM BA successfully
induced callus growth. When the level of BA was decreased to 4.44 μM, it
hastened the formation of PLB.
Bud regeneration from somatic embryos cultured in vitro of three hybrid
cultivars (UH965, UH780 and UH1060) of Anthurium was studied in detail
by Kuehnle et al. (1992). They reported 1-4 mg
L-1 2,4-D compiled with 0.33-1 mg L-1 KIN to be the best
for somatic embryo development and 0.2 mg L-1 BA to be ideal for
adventitious shoot bud regeneration. In support to this, Xin
et al. (2006) and recently Beyramizade et
al. (2008) reported the similar significance of 2,4-D and KIN combination
in embryogenesis and BA alone in shoot regeneration.
Experiments with carbon sources: Plant regeneration in vitro
requires the carbon source for induction and proliferation of differentiated
or undifferentiated organs. 3% sucrose is the frequent carbohydrate supply in
Anthurium in vitro culture (Leffringen and Soede,
1979; Somaya et al., 1998; Yang
et al., 2002; Viegas et al., 2007;
Gantait et al., 2008; Maira
et al., 2009). Muraghige and Skoog (1962)
have postulated that, generally the supplementation of 3% sucrose is better
than 2 or 4% for the purpose of tissue culture. Nevertheless, there are many
accounts on the use of other carbohydrate sources and concentrations for direct
or indirect organogenesis in Anthurium. During somatic embryo development
of Anthurium Kuehnle et al. (1992) used
supplementation of both 1% glucose with 2% sucrose or 3% sucrose, or 4% sucrose
and 2% glucose as carbon sources. In this study it was found that a combination
of glucose and sucrose in place of sucrose alone significantly improved the
somatic embryogenesis of Anthurium, which was entirely supported by
the advanced report of Xin et al. (2006) in the
similar morphogenetic (somatic embryogenesis) study.
Somatic embryogenesis: Somatic embryogenesis acts as a key component
of in vitro propagation when conjugated with a traditional breeding approach
and molecular techniques. According to Stasolla and Yeung
(2003), somatic embryogenesis provides a precious implement to boost the
pace of genetic enhancement of commercial crop species.
Somatic embryos morphologically resemble zygotic embryo but regenerates from
somatic cells through a systematic sequence of distinctive morphological stages
and is considered profitable over other in vitro propagation systems
as it curtails the multiplication time span and proves to be potential as an
efficient regeneration system with comparatively high genetic integrity (Kothari
et al., 2010). The aforesaid statement certainly is a significant
issue in plant tissue culture, which is still progressive in case of A. andreanum
In Anthurium micropropagation, using bulking up via a callus stage,
followed by adventitious bud formation, has been proposed as an interesting
possibility for large scale production by Pierik et al.
(1974) but according to Geier (1990) this method
results in formation of off-types. For many applications, somatic embryos have
powerful advantages for mass propagation; that is, the high multiplication rate,
the ease of use of liquid medium, the handling of enormous numbers of embryos
at one time and the possible use of bioreactors (Merkle et
al., 1990). As an alternative, most commercial tissue culture laboratories
now try to avoid adventitious propagation and favour axillary bud proliferation
techniques for Anthurium (Hamidah et al.,
1997). However, there are still grave problems with weaning of this material
and moreover, the rate of the propagation is time-consuming. As a remedy to
the aforesaid problems with conventional in vitro and in vivo propagation
techniques, an embryogenic-like callus of A. andraeanum, cultured on
medium containing 2,4-D and BA was developed by Kuehnle and
Sugii (1991). Plants were readily obtained from that callus but regeneration
from somatic embryos was not demonstrated. Afterwards, Kuehnle
et al. (1992) reported somatic embryogenesis and plant regeneration
in A. andraeanum hybrids using an induction medium containing 2,4-D and
Direct or indirect somatic embryogenesis and plant regeneration from leaf or
in vitro calli using as explants have been achieved by several authors
(Prakash et al., 2002; Xin
et al., 2006; Sontikul and Te-chato, 2007;
Beyramizade et al., 2008). Nitschs basal
medium (Nitsch, 1969) supplemented with natural additives
like coconut water, banana pulp, wheat malt and ragi malt has been observed
to promote somatic embryos formation and subsequent shoot regeneration in Liver
Red cultivar of Anthurium from leaf explants (Prakash
et al., 2002). A higher level of auxin (2,4-D) with comparatively
lower level of cytokinin (BA) supplementation in MS have significant effect
or even they accelerate the pace of somatic embryogenesis in A. andreanum
(Xin et al., 2006). In accordance to the previous
report, germination of somatic embryos has been induced by higher 2,4-D and
lower KIN level with modified MS, as revealed by the study of Sontikul
and Te-chato (2007) and Beyramizade et al. (2008).
As understood by all these statements, a very low concentration of BA alone
as cytokinin source is established to be potent for shoot regeneration.
|| Somatic embryogenesis in Anthurium andreanum
|Rt: Root; Em: Somatic embryo; Sht Reg: Adventitious shoot
regeneration; CW: Coconut water; AC: Activated charcoal; WM: Wheat malt;
BP: Banana pulp; RM: Ragi (Eleusine coracana) malt; Nitsch: Nitschs
basal medium (Nitsch, 1969)
The in vitro plant regeneration system, an integral part of plant biotechnology,
still carries the difficulty of ex vitro acclimatization of plants to
triumph over. Acclimatization virtually denotes the climatic or environmental
adjustments of an organism, particularly a plant that has been transferred to
a new atmosphere. Ex vitro adaptation of a micropropagated plant to a
greenhouse or a field setting is indispensable because there is, in general,
a noteworthy discrepancy between the in vitro environment and the greenhouse
or field condition. Unbeaten acclimatization measures offer most advantageous
circumstances for a high proportion of endurance of plants; they diminish the
percentage of dead and injured plants in the in vitro process and boost
the plant growth and establishment. Ultimately, an efficient acclimatization
procedure saves the resources of time, labour, money and cuts off the cost of
production of competent and deliverable produce (Gantait,
Substantial efforts have been directed to optimize the conditions for in
vitro stages of micropropagation, but the process of acclimatization of
micropropagated plants to the soil environment has not been studied exhaustively
in A, andreanum. Accordingly, the transplantation stage continues to
be a major bottleneck in the micropropagation of Anthurium. Plantlets
regenerated in vitro have been incessantly exposed to a unique microenvironment
that has been standardised to offer nominal stress and best possible conditions
for plant multiplication. Hazarika (2003) proposed the
gradual systematic acclimatization of in vitro generated plantlets in
accordance to their physiological and anatomical characteristics.
Mahanta and Paswan (2001) successfully transferred in
vitro Anthurium plantlets in the plastic pots containing the growing medium
of soilrite-perlite at the ratio of 10:1 and reported 60% survival rate after
four weeks of transfer. Effect of vermicompost and sand mixture (1:3 v/v) in ex
vitro establishment under green house followed by net house condition was
reported by Martin et al. (2003), where 95% survival
rate was achieved. Maximum survival rate of 98% was reported by Han
and Goo (2003) in cultivar Atlanta using a combination of vermiculite and
perlite (1:1 v/v) as growth substrate. Vargas et al.
(2004) studied the acclimatization of cultivar Rubrun using soil and organic
humus (1:1 v/v). In their study micropropagated plantlets ensured 80% success
rate within four weeks. In an exceptional investigation, De
Lima et al. (2006) evaluated the associated impact of arbuscular mycorrhizal
fungi (AMF) on acclimatization of Anthurium. They reported the symbiotic
favour to the plantlets with higher water content and vigorous growth than the
control due to multiple inoculations of three AMF species (Glomus etunicatum,
Gigaspora albida and Acaulospora longula).
Bejoy et al. (2008) used coarse river sand and
charcoal (3:1 v/v) mixture for ex vitro transfer and acclimatization
of tissue cultured plantlets of Agnihotri which regained growth within four
to five weeks with 89% chance of survival. Accordingly, Gantait
et al. (2008) studied the effectiveness of organics like charcoal
and coconut fibre in a 1:1:1:1 (v/v) combination with sand and soil. The two-step
(with six week duration) process of acclimatization, with the success rate of
85% was standardised. The initial sand and soil mixture (1:1 v/v) ensured the
primary acclimatization and set the plants to be transferred further (Fig.
1d, e). They also reported the utility of intermittent
water spraying to ensure high humidity during this period. More recently, Jahan
et al. (2009) established the acclimatization procedure of callus,
derived from Anthurium plantlets, on sand, loamy soil and coco-peat (1:1:1
v/v) ensuring 85% survival rate.
Clonal fidelity assessment: There are three major modes of developing
an adventitious system suitable for mercantile scales, namely, -organ culture,
somatic embryogenesis and nodule culture. George (1996)
reviewed these and the other strategies in detail. A key contemplation in using
an adventitious system is the prospective of recuperating extraordinarily high
numbers of genetic variants (somaclonal variation). In an industrial scenario,
this risk is often serious enough to eradicate any further concern of micropropagation
as a cloning strategy. This is especially true for suspension/callus-based systems
which seed to generate the higher incidences of somaclonal variation in recovered
propagules. One of the foremost basis of such a genetic crisis, however, is
the unintended occurrence of adventitious bud formation within the shoot cultures
There are inadequate reports available on clonal fidelity assessment of
in vitro raised propagules of A. andreanum. Sontikul
and Te-chato (2007) reported the evaluation of genetic identity using isozyme
markers. In their study different banding patterns of esterase revealed the
somaclonal variation of plantlets regenerated through somatic embryogenesis.
In an exceptional study Gantait et al. (2008)
reported the appraisal of genetic clonality of micropropagated plantlets with
the aid of molecular markers for the first time in Anthurium. They used
ten inter simple sequence repeats (ISSR) markers to assess the uniformity of
in vitro generated clones along with their mother plant, where monomorphic
ISSR banding pattern revealed that all the micropropagated propagules are genetically
true to type (Fig. 1f). The direct organogenesis seems to
be the potent factor to restore the genetic integrity. In the recent most studies
of Gantait et al. (2010) the efficiency of ISSR
in clonal fidelity study on micropropagated Allium ampeloprasum was confirmed
CONCLUSION AND FUTURE PROSPECT
Noteworthy escalation intended towards in vitro improvement of A. andreanum propagation has been made, but still there is an extensive way to go in this track. Important advances have been materialized in regeneration systems in vitro. Significant efforts have been made to combat various types of hindrances with special emphasis on restoration of genetic integrity and in vitro conservation through long term shoot culture, but the results have not been quite satisfactory as more exhaustive study is expected in this aspect.
We believe that future investigations on the other modes of regeneration (e.g., haploid production, anther culture and protoplast culture etc.) and use of advanced biochemical and molecular markers to assess the differential stages will help to unknot novel mechanisms for the development of more proficient strategies with improved in vitro methods.
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