Chapter 2 Review - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/11365/10/10... · 2015. 12....
Transcript of Chapter 2 Review - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/11365/10/10... · 2015. 12....
Chapter 2
Review Of
Literature
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Chapter 2
REVIEW OF LITERATURE
The science of plant tissue culture takes its roots from path breaking research
in botany like discovery of cell followed by propounding of cell theory by Schleiden
(1838) and Schwann (1839). They proposed that a cell is the basic unit of an organism
and capable to regenerate into whole plant if an appropriate environment is given. But
Schleiden and Schwann had no experimental evidence to prove it. The Cell Theory
received much impetus from the famous aphorism of Virchow (1858), ‘‘Omnis cellula
e cellula’’ (All new cells arise from pre-existing cells) and by the very prescient
observation of Vötching (1878) that the whole plant body can be built up from ever so
small fragments of plant organs.
An important approach of tissue culture was discovered by Rechinger (1893)
who tried to determine experimentally the limit of plant divisibility permitting tissue
proliferation. He used isolated buds, slices of roots, stems and other materials. The
explants were placed on sand moistened with tap water. But he did not use nutrients
or aseptic conditions; his culture could scarcely be called tissue culture. However,
Rechinger’s experiments were suggested by a concept related to the tissue culture
principles; thus he was recognized as a true pioneer in this field (Gautheret 1983).
In 1902 Haberlandt, a German physiologist was the first to conduct
experiments designed to demonstrate totipotency of plant cells by culturing isolated
leaf mesophyll cells of Lamium purpureum, glandular hairs of Pulmonaria and cells
from petioles of Eicchornia crassipes on diluted Knop’s (1865) salt solution enriched
with glucose. Unfortunately, he failed largely because of the poor choice of
experimental materials, inadequate nutrients and infection (Vasil and Vasil 1972).
But, he boldly predicted that it should be possible to generate artificial embryos
(somatic embryos) from vegetative cells which encouraged subsequent attempts to
regenerate whole plants from cultured cells. This potential of a cell is known as
‘totipotency’, a term coined by Steward in 1968. Despite lack of success, Haberlandt
made several predictions about the nutrients’ requirement in experimental conditions
which could possibly induce cell division, proliferation and embryo induction.
Haberlandt is thus regarded as ‘father of tissue culture’.
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Initial progress in plant tissue culture came from the work of Molliard (1921)
in France, Kotte (1922) in Germany, Robbins (1922) in the United States, who
successfully cultured the fragments of embryos and roots. Unfortunately, the growth
of those cultured tissues could not be sustained for long even if they were transferred
to fresh medium. Innovative plant tissue culture techniques progressed rapidly during
the 1930s due to the discovery of natural auxin and vitamin B5
The breakthrough progress came from White (1934) who was the first to
demonstrate continuous culture of excised tomato root-tips on a medium containing
inorganic salts, sucrose and yeast extract (YE). Later, he (1937) replaced YE by
vitamin B namely pyridoxine, thiamine and proved their growth promoting effect.
One of the main thrust in the history of tissue culture is the induction of callus.
Gautheret (1934) is credited with the first successful attempt of callus induction from
cambial cells of some tree species. This was followed by the formation of continuous
callus cultures in carrot and tobacco independently endorsed by Gautheret, White and
Nobécourt in 1939.
which were necessary
for the growth of isolated tissues containing meristem. In 1926, Went discovered the
first plant growth regulator, indole-3-acteic acid (IAA) which is a naturally occurring
member of a class of plant growth regulator (PGR) termed as ‘auxin’.
Adding to the ongoing improvements in the culture media, van Overbeek
(1941) used coconut milk besides usual salts, vitamins and other nutrients for embryo
culture. After 1950, there was an immense advancement in the area of PGR. Skoog
and Tsui (1951) demonstrated continued induction of cell division and bud formation
in tobacco by adenine and high levels of phosphate. This led to further investigations
by Miller et al. (1955) who isolated ‘kinetin’ (Kn), a derivative of adenine (6-furyl
amino purine). They worked further and proposed the concept of hormonal control for
organ formation in 1957. Their experiment on tobacco pith culture showed that the
high concentration of auxin promoted rooting; whereas high kinetin induced bud
formation. Later studies led to the isolation of other naturally occurring as well as
synthetic cytokinins, elucidation of their role in cell division and bud development
and their extensive use in the micropropagation industry related to their suppression
of apical dominance resulting in the development of many axillary shoots.
In early 1960s, the most significant breakthrough in the field of plant tissue
culture was the development of a defined culture medium by Murashige and Skoog
(1962), prepared by increasing the concentration of salts twenty-five times higher than
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Knop’s solution. Today MS medium has been proved as the most effective and widely
used culture medium for various plant species. The role of tissue culture in plant
genetic engineering was first exemplified by Kanta et al. (1962). They developed a
technique of test tube fertilization which involved growing of excised ovules and
pollen grains in the medium thus overcoming the incompatibility barriers at sexual
level. In 1966, Guha and Maheshwari cultured anthers of Datura and raised embryos
which developed into haploid plants initiating androgenesis.
Recently, tissue culture technology gained unbeatable recognition in plant
science for successful micropropagation and improvement of plant species, leading to
its commercial application. A number of plant species have been micropropagted
around the globe, out of which the review of some important medicinal plants has
been categorized below;
2.1 Organogenesis
Organogenesis is a complex phenomenon involving the de novo formation of
organs (shoots or roots). Shoots can be derived either through pre-existing
meristematic tissues known as ‘axillary shoot formation’ or through differentiation of
non-meristematic tissues known as ‘adventitious shoot formation’. Both these
approaches require synergistic interaction of physical and chemical factors. A
successful plant regeneration protocol requires appropriate choice of explant, age of
the explant, definite media formulation, specific growth regulators, genotypes, energy
source, gelling agent and other physical factors including light regime, temperature
and humidity (Bhojwani and Razdan 1983).
2.1.1 Meristem, shoot tip and nodal segment culture
Meristem culture is based on suppressing the shoot apical dominance by
addition of cytokinins to the growth medium followed by axillary bud sprouting in
multiple shoots. As the cells of apical and axillary bud are uniformly diploid and least
susceptible to genotypic changes, they produce a large number of genetically stable
plants in a short span of time, thus it is regarded as the most common technique for
mass production of useful plant species.
The history of meristem culture began with the first successful shoot tip
culture of Nasturtium (Tropaeolium majus) by Ball in 1946. Since then meristem
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culture has attracted much attention of the plant scientists. However, demonstration of
practical utilisation of this important technique must be credited to Morel and Martin
(1952) who for the first time produced virus-free Dahlia plants from infected
individual by excising and culturing their shoot tips in vitro. Later, Morel extended
this approach for the production of virus free plants of orchids (Morel 1960). That
was the beginning of tissue culture. Thereafter, in the 1970s developed countries
began commercial exploitation of this technology.
The meristem tip must be small enough to eradicate viruses and other
pathogens, yet large enough to develop into a shoot. In case of meristematic
propagation, elimination of virus particles in explant cells is reached within a short
time. In many cases meristematic cells do not contain virus particles due to absence of
vascular connection with other plant parts. Now, meristem culture is considered as a
unique technique to produce pathogen-free (bacteria, fungi, viruses, viroides and
mycoplasma) plants of many species (Morel and Martin 1955, Walkey 1978,
Bhojwani and Razdan 1983, Biswas et al. 2007). Recently, Banerjee et al. (2010)
used apical meristem for reducing phytoplasma infection in Artemisia roxburghiana.
Plant tissue culture entered in the developing world during the 1980s. It was
earlier used to develop ornamental plants for export. With tree species, the technique
of tissue culture remained confined for many years to laboratory stage and had
generally invited only academic interest. But in most developing countries, the
shortage of biomass and the ever-increasing energy requirements created the need to
explore possibilities of mass propagation of trees by tissue culture. Using this
approach of micropropagation, significant achievements in in vitro cloning has been
made for various herbaceous and woody plants of medicinal, horticultural and
ornamental values (Sharma et al. 2002, Tripathi and Tripathi 2003, da Silva 2003,
Zhou and Wu 2006, Rout et al. 2006, Chaturvedi et al. 2007).
2.1.1.1 Effect of adenine-based cytokinins on shoot regeneration
Cytokinins are plant hormones promoting cell division and differentiation.
Since the discovery of first cytokinin i.e., Kn, a number of chemicals suited to the
definition of cytokinin has grown to include a large array of natural and synthetic
compounds, adenine and phenylurea derivatives. The natural cytokinins are adenine
derivatives and can be classified by their configuration of N6-side chain as isoprenoid
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or aromatic cytokinins (Fig. 7). Cytokinins with an unsaturated isoprenoid side chain
are the most prevalent, in particular those with a trans-hydroxylated N6-side chain i.e.,
trans-zeatin. However, cis-zeatin and N6-(∆2-isopentenyl) adenine (2-iP) are generally
minor components although exceptions exist (Durand et al. 1994, Emery et al. 1998).
Kn and N6-benzyladenine (BA) are the best known cytokinins with ring substitutions
at N6
Three adenine-based cytokinins viz., BA, Kn and 2-iP have been commonly
used for different approaches of micropropagation; however, zeatin rarely screened
for shoot multiplication. It is now well established that cytokinins are effective only at
optimum concentrations. Higher concentrations are not recommended for shoot
proliferation as callus production interferes with subsequent rooting and
acclimatization. Among all the adenine-based cytokinins tested, BA has been found
the most effective for axillary shoot proliferation, but it is recommended that a range
of concentrations should be tested to optimize shoot production. Ault (2002) reported
that in Hymenoxys acaulis var. glabra, BA at 20 µM induced significantly more
axillary shoots (10.3 shoots per explant) than did other cytokinins. In comparison,
23.3 shoots per node at 5.0 µM BA in Mucuna pruriens (Faisal et al. 2006d), 6.3
shoots per node at 8.87 µM BA in Tinospora cordifolia (Raghu et al. 2006), 12.9
shoots per node at 5.0 µM BA in Ocimum basilicum (Siddique and Anis 2008), 8.6
shoots per shoot tip in Spilanthes mauritiana (Sharma et al. 2009b), 20.7 shoots per
node in Veronica anagallis-aquatica (Shahzad et al. 2011), 14.37 shoots per node at
2.22 µM BA in Ceropegia spiralis (Murthy et al. 2010) have been reported.
-position. In the early years of cytokinin research, only cytokinins with an
isoprenoid side chain were thought to be endogenous compounds; however in the mid
1970s BA derivatives were also identified as natural cytokinins (Horgan et al. 1973 &
1975).
The important role of BA in shoot proliferation has been reported for various
taxa of Asteraceae family, such as Wedelia calendulacea (Emmanuel et al. 2000),
Echinacea purpurea (Koroch et al. 2002), Eclipta alba (Dhaka and Kothari 2005,
Husain and Anis 2006), Stevia rebaundiana (Debnath 2008, Sharma and Shahzad
2011), Centaurea ultreiae (Mallóon et al. 2011) etc. Similarly, the superiority of BA
over other cytokinins has also been reported for various Asclepiads like Gymnema
sylvestre (Komavalli and Rao 2000), Hemidesmus indicus (Sreekumar et al. 2000),
Decalepis hamiltonii (Anitha and Pullaiah 2002), Holostemma ada-kodien (Martin
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Figure 7. Structural modifications in some adenine and urea-based cytokinins
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2002), Ceropegia spp. (Beena et al. 2003, Nikam et al. 2008, Murthy et al. 2010,
Chavan et al. 2011), Marsdenia brunoniana (Ugraiah et al. 2010).
Borthakur et al. (2000) reported Kn as the best cytokinin for shoot
proliferation in Eclipta alba and Eupatorium adenophorum. Similarly, Özel et al.
(2006) found Kn as more effective cytokinin for regeneration in Centaurea
tchihatcheffii on optimal concentration of 4.5 mg l-1. While, on 2.5 mg l-1 Kn growth
in regenerants was very slow which improved considerably when the regenerants
were transferred to BA (1.0 mg l-1), NAA (2.0 mg l-1) and glutamic acid (50 mg l-1
In several studies, combination of two cytokinins proved to be advantageous
over single cytokinins treatment for apical and axillary bud sprouting. High frequency
shoot multiplication of Eclipta alba was obtained with MS medium containing BA
combined with Kn or 2-iP. The shoots regenerated on a combination of BA (4.4 µM)
and 2-iP (14.7 µM) grew faster than those initiated in BA and Kn combination
(Baskaran and Jayabalan 2007). In Decalepis hamiltonii, the MS medium composed
of 9.1 µM zeatin, 4.7 µM Kn and 0.6 µM IAA proved to optimal for maximum shoot
regeneration and multiplication (5.4 shoots per shoot tip). Further multiplication and
elongation was achieved on medium containing 2.5 µM 2-iP and 0.3 µM GA
(Giridhar et al. 2005). Whereas, Rani and Raja (2010) reported callus-free multiple
shoot formation in Tylophora indica as a function of BA activity alone, but internode
elongation was dependent on the synergistic effect of GA. Similarly, the shoot buds of
Eclipta alba were multiplied and maintained on BA and GA containing MS medium
(Dhaka and Kothari 2005).
)
combination. While, the promotive role of 2-iP on shoot regeneration has been
reported by Cellarova and Hocariv (2004) in Digitalis purpurea and Sujatha and
Ranjitha Kumari (2007 & 2008) in Artemisia vulgaris.
Generally, inclusion of low concentration of auxin to the cytokinin containing
medium results in high frequency shoot production through axillary or apical buds
and indicates the synergistic effect of a cytokinin and an auxin. The presence of NAA
and BA has increased shoot multiplication in Gymnema sylvestre (Reddy et al. 1998),
Mentha arvensis (Shahzad et al. 1999), Spilanthes mauritiana (Bais et al. 2002),
Santolina canescens (Casado et al. 2002), Clitoria ternatea (Shahzad et al. 2007),
Tylophora indica (Faisal et al. 2007), Gynura procumbens (Keng et al. 2009), Carlina
acaulis (Trejgell et al. 2009) etc. Corral et al. (2011) also investigated that the
addition of NAA with BA significantly improved the shoot proliferation efficiency in
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Crepis novena as compared to the combination of NAA and Kn. They reported that an
average of 49.77 shoots per axillary bud were produced in 100% of cultures on MS
medium comprised of 0.54 µM NAA and 4.44 µM BA. In contrast, Banerjee et al.
(2010) reported maximum shoot regeneration (38.0 shoots per explant) on the
combination of 13.95 µM Kn and 0.27 µM NAA in Artemisia roxburghiana. They
have advocated that the combination of 8.88 µM BA and 0.27 µM NAA was found to
be more stimulative for further multiplication and elongation during sub-culturing.
For Gymnema sylvestre, Komalavalli and Rao (2000) reported a maximum
number of shoots (57.2) induced from axillary node explants on MS medium
containing BA (1 mg l-1), Kn (0.5 mg l-1), NAA (0.1 mg l-1), malt extract (100 mg l-1)
and citric acid (100 mg l-1). Maximum shoot proliferation on BA and IAA
combination has been reported by Sivaram and Mukundan (2003), Debnath (2008)
and Sharma et al. (2009b). The combination of BA and IAA in addition to additives
like adenine sulphate (ADS), arginine, citric acid and ascorbic acid used to establish
the aseptic cultures of Leptadenia reticulata (Arya et al. 2003). Whereas, Devi and
Srinivasan (2008) found optimal response for micropropagation of Gymnema
sylvestre on MS medium containing 1 mg l-1 BA, 0.5 mg l-1 IAA, 100 mg l-1 vitamins
B2 and 100 mg l-1
Gantait et al. (2010) reported an elite protocol for accelerated quality-cloning
in Gerbera jamesonii using shoot tips in which MS medium supplemented with 0.5
mg l
citric acid using nodal explants. In contrast, Beena et al. (2003)
established a protocol for in vitro propagation of Ceropegia candelabrum through
axillary bud multiplication using 8.87 µM BA in combination with 2.46 µM IBA.
-1 NAA and 1.5 mg l-1 BA promoted earliest axillary bud initiation within 5 day in
91.6% of the inoculants. They achieved very high rate of shoot multiplication (14
shoots per explant) when MS medium was fortified with a relatively higher level of
BA (2 mg l-1) and 60 mg l-1
Many researchers studied the comparative efficiency of different explants for
maximum shoot production. Sivanesan and Jeong (2007) achieved more number of
shoots from nodal segments as compared to shoot tips in Pentanema indicum. They
also revealed the additive effect of ADS when added to the BA and IAA containing
MS medium. Trejgell et al. (2009 & 2010) reported maximum shoot regeneration
form the seedling derived shoot tips in comparison to the hypocotyl, cotyledon and
ADS within 27 day of incubation. According to Gantait
and Mandal (2010), ADS acts as an elicitor or enhancer of growth in synergism with
endogenous and exogenously supplemented PGRs in Anthurium anderanum.
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root explants of Carlina acaulis and Senecio macrophyllus. Similarly, in Stevia
rebaudiana shoot tips were proved to be the most effective explants for shoot
regeneration over nodal segments and leaf explants when cultured on MS medium
supplemented with 1.0 mg l-1 BA and 0.5 mg l-1
2.1.1.2 Effect of urea-based cytokinins on shoot regeneration
IAA (Anbazhagan et al. 2010).
However, Gonçlaves et al. (2010) reported maximum shoot proliferation from nodal
segments than apical shoot tips in Tuberaria major. The proliferation frequency was
not differed by cytokinin type when nodal segments were used. More than 6 shoots
were obtained on BA and zeatin supplemented MS media. However, a differential
proliferation was noticed for shoot tips depending upon the cytokinins. The difference
in shoot multiplication among different explants in response to exogenous PGR could
be a reflection of probable variation of endogenous PGR level (Yucesan et al. 2007)
or different tissue sensitivity to PGR (Lisowska and Wysokinska 2000).
Diphenylurea (DPU) was the first cytokinin-active phenyl urea identified
(Shantz and Steward 1955). Although, this discovery was linked to the detection of a
compound in liquid coconut endosperm, it was later found to be a contaminant from
prior chemical analysis of DPU. This fortuitous discovery however led to the
synthesis of a number of potent analogues such as forochlorfenuron [1-(2-chloro-4-
pyridyl)-3-phenylurea, CPPU] and thidiazuron [N-phenyl-(1, 2, 3- thidiazol)-5-ylurea
TDZ] (Fig. 7) with cytokinin activity exceeding that of natural cytokinins (Takahashi
et al. 1978, Mok et al. 1982). Synthetic phenylureas are less susceptible to the plants’
degrading enzymes than endogenous cytokinins and can persist in the plant tissues for
long periods of time (Mok and Mok 1985, Mok et al. 1987). Besides, there is no
evidence that any phenylurea cytokinin occurs naturally in plant tissues.
TDZ and CPPU have proven advantageous for micropropagation of a wide
range of recalcitrant plant species that do not respond well to amino puirne because of
their tremendous ability to stimulate shoot proliferation (Huetteman and Preece 1993).
The mode of action of these urea derivatives is still unclear even though it appears
quite sure that they inhibit cytokinin oxidase (CKOx) activity (Hare and van Staden
1994) and thus induce cytokinins accumulation within the cells (Victor et al. 1999).
TDZ mediated response has been reported to be influenced by Ca2++ (Yip and Yang
1986, Hosseini and Rashid 2000). Mundhara and Rashid (2006) and Sharma et al.
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(2011) reported an enhancement in number of responding explants when transient
Ca2++
Application of TDZ induces a diverse array of culture response in plant
tissues. These range from induction of callus to the formation of somatic embryos.
The activity of TDZ varies widely depending on its concentration, exposure time,
cultured explant and species (Murthy et al. 1998). The concentration at which TDZ is
most effective is 10-1000 times lower than the other PGRs (Huetteman and Preece
1993). Therefore, direct comparison between TDZ and purine-based cytokinins at
equimolar concentrations or at similar durations of the treatment is complicated. Short
duration exposure to TDZ has been proved very effective for morphogenesis (Tulać et
al. 2002). Higher levels, on the other hand, promote callus and somatic embryo
fromation (Huetteaman and Preece 1993, Rida et al. 2001, Fengyen and Han
2002). In most of the studies, continuous or more than critical exposure with TDZ
resulted in stunted or abnormal shoot development. Its deleterious effect has also been
well documented in several plant species (Huetteman and Preece 1993, Faisal et al.
2005, Khurana et al. 2005, Ahmad and Anis 2007).
was provided.
Primarily TDZ was used as a cotton defoliant (Arndt et al. 1976) and later
found to mimic cytokinin like activity that was 20 times more effective in dormancy
breaking (Wang et al. 1986). However, extended research showed that TDZ, unlike
traditional cytokinins is capable of fulfilling both the cytokinin and auxin requirement
of various regenerative responses (Mok et al. 1982, Murthy et al. 1998). The list of
plant species exhibiting morphogenesis in the presence of individual TDZ has
continued to increase over the years, facilitating the improvement of tissue culture
technology (Murthy et al. 1998). For some species, the combination of TDZ and
purine-based cytokinins (usually BA) has been found more effective to induce
morphogenetic response than either TDZ or BA alone as reported by Mohamed-
Yasseen (2002) in Hylocereus undatus.
The exploitation of TDZ for regeneration has been reported vastly superior
over adenine-based cytokinin for a number of woody plant species such as Hydrangea
quercifolia (Ledbetter and Preece 2004), Cassia angustifolia (Siddique and Anis
2007), Pterocarpus marsupium (Husain et al. 2007a) and Vitex negundo (Ahmad and
Anis 2007). Apart from woody plant species, TDZ has also shown a promise role for
regeneration in many other plants (including herbs and shrubs) belonging to diverse
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groups of families such as Hypericum perforatum (Murch et al. 2006), Bacopa
monniera (Tiwari et al. 2001), Artemisia judaica (Liu et al. 2003), Hordeum vulgare
(Ganeshan et al. 2003), Cineraria maritime (Banerjee et al. 2004), Oryza sativa (Gairi
and Rashid 2004), Hyoscyamus niger (Uranbey 2005), Psoralea corylifolia (Faisal
and Anis 2006).
2.1.2 Leaf culture
Direct regeneration from leaf is another alternative step for clonal propagation
and germplasm conservation. Direct de novo or adventitious shoot regeneration is
most preferred if Agrobacterium-mediated gene transfer is to be achieved and leaf
explants are the best suited for both adventitious shoot formation and Agrobacterium-
mediated gene transfer experiments. Hildebrandt et al. (1946) and Hildebrandt and
Ricker (1947) were the first who cultured the excised leaf tissues of tobacco and
sunflower under aseptic conditions. In a large number of studies, leaf has been proved
as one of the most potent explant for a number of plant species (Misra and Datta
2001, Beegum et al. 2007, Saritha and Naidu 2008, Zheng et al. 2009, Sahai and
Shazad 2010).
Leaf explants have been found to be the most regenerative at their proximal or
petiolar end as compared to leaf margin and mid rib portion. In view of this, high
frequency shoot induction at proximal region may be due to higher accumulation of
PGRs (Rajasekharan et al. 1987). There is a physiological gradient in the leaf explant
from proximal to distal end for de novo regeneration of shoot buds. In some reports,
excised petioles have been found to be more effective than leaf segments to exert
shoot organogenesis. The leaf vein is an extension of xylem and phloem of the stem
through the petiole which is surrounded by one or more layers of parenchymatous
cells, but not well specialized for a particular function yet having the capacity for cell
division. These parenchymatous cells are very sensitive to different growth stimuli,
such as PGRs and environmental conditions; therefore they are easier to be
differentiated to new organs than other cells in tissue culture (Gahan and George
2008).
Multiple shoot regeneration from proximal part of the leaves than distal ends
has been reported in a number of plant species including Tagetes erecta (Misra and
Datta 2001), Anthurium andraeanum (Martin et al. 2003), Euphorbia nivulia (Martin
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et al. 2005), Spilanthes mauritiana (Sharma et al. 2009b), Lysimachia species (Zheng
et al. 2009). However, Sreedhar et al. (2008) reported direct regeneration of shoot
buds on both side of midrib and rarely from regions of smaller veins, but never from
lamina indicating the presence of vascular cells appear to be crucial for de novo
organogenesis from immature leaf explants of Stevia rebaudiana.
2.1.2.1 Effect of adenine-based cytokinins on shoot regeneration
Among the adenine-based cytokinins, BA was found to be the most effective
to induce direct shoot bud regeneration from the leaf explants in a variety of plant
species. BA alone at 8.87 µM induced 38.0 shoot per leaf explant in Ophiorrhiza
prostrata (Beegum et al. 2007). For Chicorium intybus, BA was found nearly twice
more successful than Kn (Yucesan et al. 2007). Sreedhar et al. (2008) reported that a
combination of BA and Kn was found to be an ideal combination for de novo shoot
regeneration from leaf explants of Stevia rebaudiana. The MS medium containing
8.88 µM BA and 4.65 µM Kn resulted in the formation of highest number of shoots
per explant at the end of 7 week of incubation. Hedayat et al. (2009) evaluated direct
organogenesis from leaf and petiole segments of Tanacetum cinerariifolium and
reported that the leaf segments were highly responsive than petiole cuttings and
produced a maximum shoot regeneration (70%) on MS medium supplemented with
4.0 mg l-1 BA and 0.2 mg l-1 2, 4-D. The highest proliferation rate was observed on
MS medium supplemented with 1.5 mg l-1 BA and 2.0 mg l-1
For Coleus forskohlii, Sahai and Shahzad (2010) evaluated leaf size, position,
orientation and season of collection to select the most regenerative explant condition.
Enhanced shoot production and proliferation has been achieved on medium
containing 2.0 μM BA and 0.1 μM NAA wherein, a highest number of 35.0 shoots per
explant were produced. In the same year, Krishna et al. (2010) provided a rapid in
vitro regeneration protocol using leaf explant of C. forskohlii. They used three
different segments of leaf i.e. proximal, middle and distal and cultured on MS basal
medium supplemented with different cytokinins. Comparison of shoot regeneration
response from different leaf segments at 5.0 mg l
NAA.
-1 BAP showed that the distal end
was comparatively most regenerative as induced 45.0 shoots followed by 23.0 and
21.0 shoots from middle and proximal ends. Further elongation was achieved on MS
medium augmented with 0.1 mg l-1 BA and 0.1 mg l-1 IAA combination. For Populus
33
tremula, Huang and Dai (2011) reported a high frequency of shoot regeneration on
10-20 µM zeatin as compared to other cytokinins. A maximum of 92.6 shoots were
induced from each petiole explant when culture on 20 µM zeatin added to MS
medium, while an average of 60.9 shoots were induced from leaf explants on similar
concentration of zeatin.
The existence of synergistic and additive interaction of auxin and cytokinins
combination involves a complex web of signal interactions such as increased
sensitization, receptivity, feedback inhibition and modulation of gene expression
resulting in variable translation of mRNA population (Cline 1991, Eklof et al. 1997,
Schmulling et al. 1997, Armstrong et al. 2004). In most of the studies, combination of
BA and NAA was found to be most effective for regeneration through leaf explants
such as in Chaememelum species (Echeverrigaray et al. 2000), Phellodendron species
(Azad et al. 2005), Saussurea species (Dhar and Joshi 2005). Through leaf disc
cultures of Sansevieria cylindrica, highest shoot regeneration frequency (80%) and
mean number of shoots per explant (13.5) were obtained in MS medium
supplemented with 10.0 µM BA and 0.1 µM NAA (Anis and Shahzad 2005).
Mohapatra et al. (2008) reported a maximum shoot (8.3 shoots per leaf) and shoot
length (2.1 cm) in 81.6% of cultures of Centella asiatica on MS medium
supplemented with 3.0 mg dm-3 BA and 0.05 mg dm-3 NAA. In Lysimachia
nummularia a maximum of 12.73 shoots per leaf explant were induced in 100% of
cultures on MS medium supplemented with 1.0 mg l-1 BA and 0.1 mg l-1
In contrast to BA and NAA combination, in Tagetes erecta leaf culture highest
shoot regeneration established on BA (13.3 μM) and IAA (17.1 μM) combination
(Vanegas et al. 2002). While in Chicorium intybus, 0.5 mg l
NAA (Zheng
et al. 2009). Similarly, Corral et al. (2011) reported mean number of 2.48 shoots per
explant on 2.22 µM BA and 2.69 µM NAA combination from leaf explant of Crepis
novoana. Superiority of NAA with BA over other auxins might be due to the fact that
NAA has more affinity for easy penetration through plasma membrane even without
active uptake as suggested by Nordstrom et al. (2004).
-1 Kn combined with 0.3
mg l-1 IAA gave optimum response with a mean of 19.7 shoots per lamina explant
(Yucesan et al. 2007) and in Ophiorrhiza prostrata combination of 8.87 µM BA and
2.46 µM IBA yielded maximum number of shoots per leaf explant (76.0) (Beegum et
al. 2007). Whereas, Saritha and Naidu (2008) reported direct organogenesis from
juvenile leaf explants of Spilanthes acmella on the medium augmented with BA and
34
IAA. Similarly, the combination of BA (2.0 mg l-1) and IAA (0.5 mg l-1) produced
maximum number of shoots (32.8) from leaf explants of field grown plants of
Solanum nigrum, whereas from in vitro derived leaf explants maximum number of
shoots (38.0) were obtained on BA (3.0 mg l-1) and IAA (0.5 mg l-1
As an additive, GA also plays a very significant role for the induction of shoot
buds from leaf explants. In this context, Sekioka and Tanaka (1981) were of the
opinion that GA can act as a replacement for auxin in shoot induction and thus a ratio
of cytokinins-GA may be decisive for differentiation in certain plant tissues. GA has
also found conducive for promotion of biomass production and enhanced xylem fibre
length in transgenic aspen (Eriksson et al. 2000). A combination of 14.43 µM GA and
4.44 µM BA in the absence of any auxin induced multiple shoot bud differentiation
from the leaf segments of Tagetes erecta (Misra and Datta 2001).
) combination
(Sridhar and Naidu 2011).
Pre-treatment with low temperature improved the regeneration potential of
plant tissues. The enhancement of plant regeneration by low temperature treatment
was related to the alternation of endogenous auxin-cytokinin balance and redox-state
which played a key role in the plant growth and development (Hou et al. 1997, Merce
et al. 2003, Andersone and Levinah 2005). Guo et al. (2007) reported an efficient
micropropagation system for Saussurea involucrata, an endangered Chinese
medicinal plant through leaf explants. A maximum of 66.0% of shoot regeneration
frequency and 5.2 shoots per explant were achieved when explants cultured on a
medium containing 10.0 µM BA and 2.5 µM NAA. Shoot organogenesis was
improved further when the leaf explants were pre-incubated at low temperature and
80.6% of shoot regeneration frequency was recorded with 9.3 shoots per leaf explant
at 4 °C by 5-day pre-treatment period.
2.1.2.2 Effect of urea-based cytokinins on shoot regeneration
Similar to the meristem culture, TDZ has been successfully exploited for
direct regeneration of shoot buds from leaf explant in a number of plant species
(Feyissa et al. 2005). Like adenine-based cytokinins, TDZ in combination with a
suitable auxin also favoured high frequency of shoot regeneration from leaf explants
(Orlikowska and Dyer 1993). Radhika et al. (2006) reported high frequency of shoot
regeneration and high number of shoots per regenerating leaf explant on a wide range
35
of TDZ and NAA combinations in Carthamus tinctorius. Later, Sujatha and Dinesh
Kumar (2007) compared the efficacy of TDZ plus NAA and BA plus NAA
combination for the leaf explants of eleven Carthamus species. They observed highly
prolific adventitious shoot regeneration on MS medium supplemented with 0.2 mg
dm-3 TDZ and 0.2 mg dm-3 NAA in C. tinctorius whereas 0.2 mg dm-3 TDZ plus 1.0
mg dm-3 NAA was found effective for shoot regeneration in C. arborescens. Zeng et
al. (2008) reported an efficient micropropagation system using leaves as explants for
Tigridiopalma magnifica. Up to 7.6 adventitious buds formed per leaf explant after a
40-day culture on MS medium containing 2.0 mg l-1 BA and 0.1 mg l-1 TDZ. During
30-day subculture, the proliferation rate of adventitious bud in cluster was 5.7 on MS
medium supplemented with 2.0 mg l-1 BA and 0.5 mg l-1
Ma et al. (2011) established an efficient propagation and regeneration system
via direct shoot organogenesis for an endangered species, Metabriggsia ovalifolia.
Among various PGRs tested, 2.5 µM TDZ was found to be the most effective to
induce a maximum of 36.7 shoots per leaf explant. Shoot regeneration capacity was
further enhanced when auxin was added to TDZ. Among a wide range of cytokinin
(Kn, BA and TDZ) and auxin combinations, 5.0 µM TDZ along with 0.5 µM NAA
induced a maximum of 79.1 adventitious shoots from each leaf explant.
NAA.
2.1.3 Cotyledon culture
Cotyledons are a potential source of regeneration because of their year-round
availability, ease of culture initiation and applicability to a number of genotypes
(Burger and Hackett 1982, Baker et al. 1999) and represent a good source not only for
micropropagation studies but also serve as a target tissue for transformation studies
(Franklin et al. 2004). Organogenesis from cotyledons was successfully obtained in
Citrullus lanatus (Chaturvedi and Bhatnagar 2001), Dalbergia sissoo (Singh et al.
2002a), Glycine max (Sairam 2003), Capsicum annuum (Joshi and Kothari 2007),
Pongamia pinnata (Sujatha et al. 2008).
As far as the literature is concerned, single cotyledon explant can produce
multiple shoot buds from the proximal cut ends due to the presence of highly
meristematic cells (Guerra and Handro 1988). Similar results were also reported by
Hisajima (1982) who found that up to 10 million shoots of almond species could be
obtained from a single seed explant within a year after several subcultures. This type
36
of response has been initiated from the seeds of many species, particularly legumes
(Vasanth et al. 2004, Maina et al. 2010).
2.1.3.1 Effect of adenine-based cytokinins on shoot regeneration
Webb et al. (1984) showed that cotyledon age can influence the regeneration
response; with older cotyledons has less ability for direct shoot regeneration than
younger ones. According to Hunter and Burritt (2002), cotyledon age influences the
shoot-forming ability of cotyledon explants. Zhang and Cui (2001) studied the
stimulatory effect of different cytokinins on direct plant regeneration from 5-day old
cotyledon explants in Cucumis sativus. 1.0 mg l-1
Singh et al. (2002a) compared the regeneration potential of semi-mature and
mature cotyledons lacking embryonic axes in Dalbergia sissoo. Shoot buds were
induced from the proximal region of semi-mature cotyledons on MS medium
supplemented with 4.44 μM BA and 0.26 μM NAA. Adventitious shoot bud
formation was also noticed from the mature cotyledons. However, unlike the semi-
mature explants, the mature cotyledons exhibited shoot bud differentiation on MS
medium containing 22.20 μM BA without NAA. Pre-culture of mature cotyledons in
liquid MS medium containing 8.88 μM BA for 48 h improved shoot bud regeneration
up to six-fold.
zeatin had a highest efficiency
(85%) over BA, Kn, TDZ.
Vega et al. (2006) examined regeneration efficacy from three different regions
(proximal, middle and distal) of cotyledon explants in six sun flower inbred lines. A
decreasing regeneration was observed from proximal to distal sections for all inbred
lines. Shoot differentiation depends upon the presence of proximal region of explant
regardless of the genotype. Maximum regeneration frequency (87.1%) was noticed for
N 834 genotype. This was in accordance with other studies in which regenerated
plants were obtained from cotyledons at high level of cytokinins (Joshi and Kothari
2007).
Rashid et al. (2010) assessed the in vitro response of two genotypes of tomato
(Lycopersicon esculentum) viz., Punjab Upma and IPA-3 for direct regeneration from
cotyledon explants. They noticed that direct regeneration was significantly influenced
by the genotype. The MS medium supplemented with Kn (0.5 mg l-1) and BA (0.5 mg
l-1) was found optimum for inducing direct shoot regeneration. At this combination of
37
BA and Kn Punjab Upma exhibited a better response in terms of shoot regeneration
per cent (92.49) and average number of shoots per explant (4.78) in when compared
to IPA-3.
Chaturvedi et al. (2010) compared the regenerative potentiality of cotyledon
explants of some Indigenous varieties of Cucurbits. The 7 day-old seedlings were
used as explant source. They reported direct shoot regeneration for the first time from
cotyledons of Cucurbita pepo under the influence of BA (5.0, 10.0 μM) and 3.0 μM
each of BA and 2-iP. Indirect as well as direct regeneration was observed in Cucumis
melo var. utilissimus; BA alone (5.0, 10.0 μM) supported shoot-bud differentiation
indirectly via callusing while in combination with 2-iP at 1.0 μM each promoted
direct regeneration of shoot-buds in cultures.
2.1.3.2 Effect of urea-based cytokinins on shoot regeneration
Fragmentary reports are available on TDZ mediated direct organogenesis from
cotyledonary leaves. In most of the cases TDZ has been found to induce indirect
organogenesis (Radhika et al. 2006). Murthy et al. (1996) achieved stimulation of
direct organogenesis and somatic embryogenesis from cotyledons of Cicer artietinum
when implanted on BA and TDZ amended MS medium. Multiple shoots formed de
novo without an intermediary callus phase at the cotyledonary notch of the seedlings
within 2 to 3 weeks of culture initiation. TDZ was found to be more effective as
compared to BAP as an inductive signal of regeneration. The TDZ induced multiple
shoot formation at all the concentrations tested (1.0 µM to 10.0 µM), although
maximum morphogenic response was observed at 10.0 µM TDZ. Addition of NAA
alone or in combination with BAP to the MS medium failed to invoke similar
response. When the TDZ supplemented medium was amended with L-proline, the
resultant regenerants were mostly somatic embryos. Histological investigations
confirmed the switch in the regeneration pathway from directly formed adventitious
shoots to embryogenesis (Murthy et al. 1996). High frequency of adventitious shoot
regeneration (33.33%) and the highest number of shoots per explant (6.5) from
cotyledons of Carthamus tinctorius was optimized at 0.5 mg l-1 TDZ and 0.25 mg l-1
IBA containing MS medium (Başalma et al. 2008).
38
2.2 Indirect organogenesis
The undifferentiated mass of profusely dividing cells known as callus and
callus mediated regeneration is termed as indirect organogenesis. In vitro callus can
be induced from various parts of the plants like shoot tip, node, inetrnode, hypocotyl,
cotyledon, root, leaf or floral organs and has multiple uses (Pande et al. 2002,
Khurana et al. 2005, Sahai et al. 2010, Parveen and Shahzad 2011). The induction of
callus growth and subsequent differentiation and organogenesis is accomplished with
the differential application of growth regulators and the controlled environmental
conditions in the culture medium (Tripathi and Tripathi 2003). Explants when
cultured on the appropriate medium, usually with both an auxin and cytokinin, gave
rise to an organized, growing and dividing mass of cells. In culture, callus
proliferation can be maintained more or less indefinitely, provided that the callus is
sub-cultured on the fresh medium periodically. During long-term culture, the culture
may lose the requirement for auxin and/or cytokinins. This process is known as
‘habituation’.
Gao and Bjork (2000) reported callus induction and plant regeneration in
shoot tip explant of Valeriana officinalis with the manipulation of various
combination and concentrations of auxins (IAA, IBA and NAA) and cytokinins (BA
and Kn). Influence of different PGRs on high frequency plant regeneration via leaf
callus was also reported in Coleus forskholii (Reddy et al. 2001). Rehman et al.
(2003) reported regeneration via leaf derived callus of Cichorium intybus on modified
MS medium containing 2.0 mM IAA, 5.0 mM Kn and 1000 mg l-1
Faisal and Anis (2003) reported callus formation from leaf explants of
Tylophora indica with the application of dichlorophenoxy acetic acid (2,4-D) or
trichlorophenoxy acetic acid (2,4,5-T) in which 100% cultures showed callus
induction on MS medium supplemented with 2,4,5-T at high level of 10.0 µM. The
characteristics of calli were also greatly influenced by the concentration of auxins and
casein hydrolysate
(CH) with the production of at least five or more shoots from each callus. Efficient
regeneration system has also been achieved from leaf derived callus in Solanum
laciniatum (Okslar et al. 2002). Koroch et al. (2003) established a protocol for the
induction of adventitious shoots from leaf calli of E. pallida. They reported optimum
shoot regeneration frequency (63%) and number of shoots per explants (2.3 shoots per
explants) on 26.6 µM BA and 0.11 µM NAA containing MS medium.
39
cytokinins. In general, maximum callus induction frequency was observed on a high
level of auxin with low cytokinin level. Nodular callus was initiated from young leaf
segments of Pluchea lanceolata, when cultures on Wood and Braun medium (1961)
containing 2.0% sucrose and 5.0 mg l-1 Kn (Kumar et al. 2004). However,
considerable amount of callus formation was observed with the combination NAA
and BA in Leucaena leucocephala (Maity et al. 2005). The maximum callus induction
frequency from stem callus of Ruta graveolens was observed in a combination of 2, 4-
D and BA (Faisal et al. 2006b). Explants cultured on control medium (PGR-free MS
medium) became necrotic and showed no sign of callus formation. Nandagopal and
Ranjitha Kumari (2006) used ADS for high frequency shoot organogenesis from leaf-
derived callus of Cichorium intybus. They observed highest percentage of callus
induction and multiple shoots proliferation was on MS plus B5
Auxins generally stimulate callus formation, but in some cases phenyl urea
derivative i.e., TDZ was also found to possess callus induction properties. Phippen
and Simon (2000) reported callus and shoot induction in Ocimum basilicum when leaf
explants were placed on MS medium supplemented with 16.8 μM TDZ alone. The
combination of TDZ with an auxin (NAA) greatly influences the callus formation
frequency in leaf explants of Cimicifuga racemosa (Lata et al. 2002). Shahzad et al.
(2006) documented maximum callus formation from mature green cotyledons on 0.6
µM TDZ supplemented MS medium in Acacia sinuata. In Hydrastis canadensis, high
frequency of indirect organogenesis was achieved on 2.5 μM TDZ and 5.0 μM NAA,
however sub-culturing of the parent tissue on BA (5.0 μM) containing medium
maximized the production of shoots (He et al. 2007). Faisal et al. (2005) developed a
protocol for high-frequency shoot regeneration and plant establishment from petiole-
derived callus of Tylophora indica. In this plant, optimal callus was developed from
petiole explants on MS medium supplemented with 10.0 µM 2, 4-D and 2.5 µM TDZ.
Adventitious shoot induction was achieved from the surface of the callus after
transferring onto shoot induction medium. The highest rate (90%) of shoot
multiplication was achieved on MS medium containing 2.5 µM TDZ. For Phyllanthus
amarus, Nitnaware et al. (2011) reported maximum callus induction from leaf explant
on 2.26 µM 2, 4-D and 2.32 µM Kn that exhibited higher shoot regeneration (32.4
shoots per culture) after transfer to MS medium containing TDZ.
medium containing
6.66 μM BA, 2.85 μM IAA and 1.36 μM ADS.
40
2.3 Other factors influencing regeneration
Several factors influence the efficiency of in vitro regeneration such as basal
medium, growth regulators, types of additives, age of explants, age of culture,
photoperiod which have been time-to-time reviewed by various workers (Batra 2001).
2.3.1 Effect of different culture media on shoot regeneration
One of the most important factors governing in vitro growth and
morphogenesis of plant tissues is the composition of the culture medium. The basic
nutrient requirements of cultured plant cells are very similar to those of whole plants.
Several media formulations are used for the majority of all cell and tissue culture
work. These media formulation include those described by Murashige and Skoog (MS
1962), White (1963), Linsmaier and Skoog (1965), Gamborg et al. (B5
The development of culture medium formulations was a result of systematic
trials and experimentations. The MS medium was developed for tobacco, based
primarily on the mineral analysis of tobacco tissue. Previously, it was referred as a
‘high salt’ medium due to its high content of potassium and nitrogen salts. The LS
medium is basically MS medium with respect to its inorganic portion, but only
inostiol and thiamine HCl are retained among the organic components. The B
1968), Nitsch
and Nitsch (NN 1969), Schenk and Hilderbrandt (SH 1972) and Lloyd and McCown
(WPM 1980).
5
medium was devised for soyabean callus culture and has lesser amounts of nitrates
and especially NH4+ than MS. Although, B5
For most of the plant species MS medium was proved to be the best for
micropropagation studies. Whereas, Faisal et al. (2007) examined the effect of
different strengths of MS medium (¼ MS, 1/3 MS, ½ MS and MS), each comprising
2.5 µM BA, 0.5 µM NAA and 100 mg l
was originally developed for the purpose
of obtaining callus or for use with suspension culture but it also works well as a basal
medium for whole plant regeneration. The SH medium was also formulated for callus
culture of monocots and dicots while the White’s medium was designed for tissue
culture of tomato roots. Whereas, the NN medium came in to existence for anther
culture and contains a salt concentration intermediate to that of MS and White’s
media (Beyl 1999).
-1 ascorbic acid for axillary shoot proliferation
41
in Tylophora indica. A sharp decline in shoot proliferation efficiency of the explant
was noticed on gradual reduction in salts’ concentration.
However, Chuenboonngarm et al. (2001) successfully used B5 medium for
micropropagation of Gardenia jasminoides through shoo tip cultures. Similarly,
Nandagopal and Ranjitha Kumari (2006) reported the highest percentage of callus
induction and multiplication shoot proliferation on MS and B5
The comparative influence of different culture media formulations on in vitro
response has been assessed by various groups. In Gymnema sylvestre, Komalavalli
and Rao (2000) found MS medium as the best basal medium for shoot sprouting
(62%), number (3.2) and length (2.2) with little callus formation followed by B
medium supplemented
with 6.66 µM BA and 2.852 µM IAA and 1.360 µM ADS. While, in nodal culture of
Terminalia bellerica a maximum number of shoots per explant (10.6) was obtained on
SH medium, but shoots were stunted and exhibiting yellow leaves which intensified
on subsequent subcultures on the same fresh medium. However, growth of shoots was
better on MS medium (Rathor et al. 2008). Woody plant medium (Lloyd and
McCown 1980) has been reported to be more suitable medium for in vitro
regeneration study of woody tree species. The pivot role of WPM for shoot
proliferation has been reported in Cornus florida (Kaveriappa et al. 1997).
5
Husain et al. (2008) and Husain and Anis (2009) achieved the highest shoot
multiplication as well as shoot length on MS basal medium over half-strength MS,
WPM and B
, SH,
WPM and white’s medium. They noticed that shoot buds sprouted on White’s
medium showed only limited development even if they were maintained for longer
period. Similarly, in vitro propagation of various plants belonging to Asclepiadaceae
has also been shown to have optimum growth in MS medium (Chi Won and John
1985, Pattnaik and Debata 1996, Komalavalli and Rao 1997).
5 basal media in Pterocarpus marsupium and Melia azedarach
respectively. Similar results have also been reported on many woody plant species
including Swartzia madagascariensis, Lagerstromia parviflora and Smilax china
(Berger and Schaffner 1995, Tiwari et al. 2002, Song et al. 2010). Song et al. (2010)
provided a comparative analysis of different culture media formulations (½ MS, MS,
2MS, WPM, B5 and SH) to achieve an efficient micropropagation protocol for Smilax
china. As reported in most of the studied the MS medium exhibited higher growth
than those of others. When three different strengths of MS medium were considered,
½ MS resulted in the highest shoot regeneration over MS and 2MS basal media.
42
On the other hand, Warakagoda and Subasinghe (2009) compared these media
(MS, WPM and B5) for in vitro seed germination of Jatropha curcas. In their study
among three media tested, B5 was the best for seed germination of J. curcus.
Therefore, for culture establishment only B5
Mhatre et al. (2000) used four different nutrient media with various
modifications to determine their role in shoot regeneration in Vitis vinifera. The
following media compositions were chosen for the micropropagation protocol;
was selected.
G16, initiation medium, comprised of NN major and minor salts, LS vitamins,
Fe EDTA, 2% (w/v) sucrose, 10 mg l-1 thiamine HCl, 40.53 mg l-1 ADS, 218.4
mg l-1 monobasic sodium phosphate, 2.25 mg l-1 BAP and 0.09 mg l-1
GM2, multiplication medium, comprised of WPM major and minor salts, B
NAA.
5
vitamins, Fe EDTA, 3% (w/v) sucrose, 2 mg l-1 calcium pantothenate, 168 mg
l-1 monobasic sodium phosphate 0.5 mg l-1 IBA and 2.2 mg l-1
MS2, shoot elongation medium, comprised of MS major and minor salts, MS
vitamins, Fe EDTA, 2% (w/v) sucrose, 0.5 mg l
BAP
-1 Bap and 0.2 mg l-1
GR1, rooting medium (liquid), comprised of half-strength MS major and
minor salts, full strength MS vitamins, Fe EDTA, 1% (w/v) sucrose and 0.1
mg l
IAA
-1
They reported that these modified media (G16 and GM2) resulted in healthy
proliferation, none of the culture exhibited hyperhydricity. For this they suggested
that both G16 and GM2 media contain monobasic sodium phosphate in addition to
BAP and this could be responsible for a synergistic effect of cytokinins and NH
IAA.
4+
In a report of 2004, Nas and Read hypothesized that the composition of
minerals and organic substances in proportions similar to those found in the seed
composition could provide an optimum tissue culture medium for micropropagation
of higher plants. Their hypothesis would help to avoid factorial treatments, labour and
explant requirement for obtaining defined tissue culture medium with optimum
response. Using their hypothesis, they first developed a new tissue culture medium
(Nas Medium, NM 2004) for hybrid hazelnuts (Corylus avellana). Threefold higher
shoot length was observed on NM medium than any other media. Moreover, potential
multiplication rates observed on NM (up to 107%) and WPM (up to 85%) were higher
than those on other media. When the composition of NM was further improved (Nas
ions.
43
and Read medium, NRM) in accordance with their hypothesis, shoot length (up to
three fold) and potential multiplication rate (up to 93%) were further enhanced.
2.3.2 Effect of different carbon sources on shoot regeneration
Carbohydrate compounds, normally found in the sieve-tube exudates of plants
have been positively related with a suitable carbon source used in plant tissue culture
medium (Welander et al. 1989). It is well documented that a specific carbohydrate
may have different effects on morphogenesis in vitro. A carbohydrate, generally
sucrose, is an indispensible ingredient of all culture media, as the photosynthetic
ability of cultured tissue is limited because of low irradiance and limited gas exchange
(Kozai 1991). It is also required as an osmotic agent (Thorpe 1985). Being easily
translocable and resistant to enzymatic degradation due to non-reducing nature,
sucrose is the most effective of choice among various carbohydrates for of plant tissue
culture studies (Pontis 1978). But, now it is well established that carbohydrate
requirements may show differences according to the species (Thompson and Thorpe
1987).
A concentration of 20 to 40 g l-1
Although sucrose is the most widely used carbohydrate in tissue culture, some
reports indicate that it may cause hypoxia and ethanol accumulation due to fast
metabolism and result in a significant decrease in osmotic potential of the medium
(Neto and Otoni 2003). These conditions could in turn interfere with the nutrient
uptake process. This interference would most likely result in the failure of absorption
of diffusion or diffusion of some important elements. In such a critical situation, some
reducing sugar like mono- or disaccharides and sugar alcohols such as glucose,
sucrose (a disaccharide made up of glucose
and fructose) is the most often used carbon or energy source, since this sugar is also
synthesized and transported naturally by the plant. Whereas, Murashige and Skoog
(1962) stated that the use of 3% sucrose is better than 2 or 4%. The sugar
concentration chosen is dependent on the type and age of the explant in culture. To
justify this fact, Gürel and Gülsen (1998) investigated the requirement of sucrose
concentration during three successive stages, namely initiation, transplantation and
multiplication for Amygdalus communis cultures. Comparatively higher concentration
of sucrose (5 and 6%) was required during initiation and transplantation stages as
compared to multiplication phase (3 and 4%).
44
fructose, sorbitol and maltose may also be used to find an alternative carbon source
(Nicoloso et al. 2003, Mosaleeyanon et al. 2004, Skrebsky et al. 2004, Pati et al.
2006, Rodrigues et al. 2006, Bandeira et al. 2007, Luo et al. 2009, Dobránszki and da
Silva 2010, Mohamed and Alsadon 2010).
The response of in vitro cultures to different carbon sources added to the
medium has been compared for a number of plant species. Among three carbon
sources (glucose, fructose and sucrose), sucrose proved to be the best for shoot
regeneration in Pentanema indicum (Sivanesan and Jeong 2007). Similar result was
obtained in Artemisia vulgaris (Sujatha and Ranjitha Kumari 2008). In fact, sucrose
has been commonly used as a carbon source in tissue culture media (Fuentes et al.
2005). This is due to its efficient uptake across the plasma membrane (Borkowska and
Szezebra 1991).
Both sucrose and glucose gave a similar rate of proliferation in sour cherry
(Borkowska and Szezebra 1991), Bixa orellana (Neto and Otoni 2003). While,
Debnath (2005) reported the best response at 20 g l-1
Sorbitol, a polyol that occurs abundantly in plant species, is a good carbon
source for Malus species and Prunus persica tissue culture (Chong and Taper 1972,
Coffin et al. 1976). The promotive effect of sorbitol on shoot multiplication rather
than sucrose has also been reported for some plant species of Rosaceae (Pua and
Chong 1984, Kadota et al. 2001, Ahmad et al. 2007).
sucrose in terms of explant
response and shoot developing potential, although glucose supported shoot growth
equally well or better than sucrose depending upon cultivar type of Vaccinium vitis-
idaea. But, carbohydrate concentration had a little effect on shoot vigour. Abou-
Rayya et al. (2010) found glucose as the most effective carbon source for stimulating
the production of shoots, fresh weight and shoot length followed by sucrose and
fructose. In Solanum nigrum, Siridhar and Naidu (2011) also reported the highest
number of shoots (24.0) on 4% fructose, but maximum shoot length (11.0 cm) was
observed on 4% sucrose. The results obtained are in line with the earlier observation
in Mulbery (Vijaya Chitra and Padmaja 2001), where addition of fructose instead of
sucrose in the multiplication medium increases the shoot number and also growth of
shoots.
Energy source has also found to enhance the alkaloid content along with
optimum morphogenesis. In Nepeta rtanjensis, Misic et al. (2005) noticed significant
enhancement in shoot growth and nepetalactone accumulation on glucose. Besides,
45
they also determined the effect of different concentrations of carbohydrates in culture
media on the internal carbohydrate status for the same plant species.
2.3.3 Effect of different pH on shoot regeneration
Scragg (1993) reported that at low pH, cells release H+ to the extracellular
environment affecting the absorption of nutrients, especially the NH4+ while at high
pH, cells release OH- ions thus the absorption of NO3- is adversely affected. Based on
the current report, it is hypothesized that the inhibitory effect of pH on shoot height is
likely to be due to reduced uptake of NH4+ and NO3
-
Martins et al. (2011) studied the influence of low pH (4.5, 5.0 and 5.75) on in
vitro growth and biochemical parameters ((lipid peroxidation, proline and
carbohydrate content, antioxidant enzymes activities and total soluble protein) of
Plantago almogravensis and P. algarbiensis. It was observed that medium pH did not
affect in vitro proliferation and rooting. Interestingly, cultures of both species modify
the initial pH value to the same final value. Results have shown that the lowest pH
tested induced an increase in the level of lipid peroxidation in roots of both species
and in shoots of P. algarbiensis, indicating plasma membrane damage. An
accumulation of carbohydrates was observed in roots of P. almogravensis cultured at
pH 4.5 and 5.0. Based on the results obtained it was concluded that Plantago species
are apt to grow in vitro in medium with pH values much lower than the usually used
in tissue culture, which is in agreement with the fact that both species colonize acid
soils.
at low and high pH respectively.
Every species requires an optimum pH for shoot regeneration and their subsequent
proliferation. In most of the studies optimum regeneration was achieved at 5.8 pH
(Sahai et al. 2010, Shahzad et al. 2011). However, Bhatia and Ashwath (2005) and
Naik et al. (2010) suggested the requirement of acidic pH for maximum biomass
production of Lycopersicon esculentum (5.5 pH) and Bacopa monniera (4.5 pH)
respectively.
2.4 In vitro rooting of microshoots
Rooting is an important step in micropropagation studies (Moncousin 1991).
Although, a number of plants root spontaneously in culture (some monocotyledons
and other herbaceous species), shoots of most species multiplied in vitro lack a root
46
system (Yeoman 1987). Rooting can be achieved either by transferring the
microshoots to medium lacking cytokinins with or without a rooting hormone or by
treating the shoots as conventional cutting after removal from sterile culture medium.
There is a great variation between species in the ease with which cultured shoots can
be rooted and systematic trials are often needed to find the most effective conditions
for rooting. All cytokinins inhibit rooting and BA (which is widely used for shoot
multiplication) does so strongly, even after transfer to cytokinins-free medium
(George and Sherrington 1984). The use of Kn or 2-iP in place of BA in the final
stage of multiplication often improves subsequent rooting (e.g. in Pinus as reported by
Webb and Street 1977). On the other hand, MS basal medium devoid of PGR has
been found to induce in vitro rooting in some plant species (Cristina et al. 1990,
Saxena et al. 1998, Faisal and Anis 2003, Ray and Bhattacharya 2008). The ease of
root formation on auxin free medium may be due to the availability of endogenous
auxin in the regenerated shoots (Minocha 1987).
The concentration of rooting hormone (generally auxin) is often required to
provide sufficient stimulus to initiate roots while preventing the excessive formation
of callus. Root elongation may be inhibited by the levels of auxin required to initiate
roots and the use of IAA which rapidly breaks down in cultured tissues is a useful
way of overcoming this problem without having to provide a second rooting medium.
The requirement of IAA for best rooting has also been reported in Artemisia vulgaris
(Sujatha and Ranjitha Kumari 2007), Stevia rebaudiana (Ahmed et al. 2007,
Anbazhagan et al. 2010), Gerbera jamesonii (Gantait et al. 2010) etc.
Many species require the stronger axuin i.e., IBA or NAA to stimulate root
fromation. Among the auxins tested, IBA was found to be the most suitable for in
vitro rooting (Sreekumar et al. 2000, Faisal and Anis 2002, Liu et al. 2003, Sivaram
and Mukundan 2003, Anis and Shahzad 2005, Feyissa et al. 2005, Faisal et al. 2005a,
Faisal et al. 2006c, Faisal et al. 2007). Half-strength MS with IBA was proved to the
best for in vitro root induction in Saussurea obvallata (Dhar and Joshi 2005), Centella
asiatica (Mohapatra et al. 2008) and Spilanthes acmella (Saritha and Naidu 2008).
However, NAA usually give rise to short and thick roots which may have the
advantage of being better able to withstand accidental damage during planting out
(Lane 1979). The stimulatory effect of NAA on root formation has been reported in
many medicinal plants like Tagetes erecta (Misra and Datta 2001), Carthamus
47
tinctorious (Radhika et al. 2006), Trichosanthes dioica (Malek et al. 2007),
Lysimachia species (Zheng et al. 2009)
Sometimes combination of two different auxins or axuin with cytokinin found
to induce optimum rooting response. The combination of IBA and IAA was found to
induce best rooting in Ficus religiosa (Siwach and Gill 2011). Regenerated shoots of
Anthurium andraeanum were best rooted on half-strength MS medium with 0.54 µM
NAA and 0.93 µM Kn (Martin et al. 2003). Similarly, Beegum et al. (2007) reported
best rooting in Ophiorrhiza prostrata when shoots were cultured on 10.74 µM NAA
and 2.32 µM Kn containing half-strength MS medium.
Different phenolic compounds like, phloroglucinol (PG), chlorengenic acid
(CA) and salicyclic acid (SA) also facilitate in vitro rooting in recalcitrant species and
PG was found to be a promotive phenolic compound for root induction in
Pterocarpus marsupium (Anis et al. 2005, Husain et al. 2007a). However,
Sakhanokho and Kelley (2009) observed that SA in combination with NaCl had a
beneficial effect on root formation along with shoot multiplication and plant survival
in Hibiscus moscheutos.
Auxin promotes ethylene production that inhibits adventitious root formation
in some species like pea cuttings (Nordstrom and Eliasson 1993) and Prunus avium
shoot cultures (Biondi et al. 1990). Ma et al. (1998) demonstrated that the use of
ethylene inhibitors such as AgNO3 and CoCl2 may promote root formation in shoot
cultures of apple. Effect of AgNO3 on root fromation was also examined by Bais et
al. (2000) for Decalepis hamiltonii. They reported best rooting response with the
application of 40.0 µM AgNO3
2.5 Synseed production
. However, Reddy et al. (2002) used triacontanol
(TRIA) for in vitro rooting in D. hamiltonii.
Since the formulation of the concept of synseed by Murashige (1977), a
number of studies have been undertaken in this area of plant biotechnology. The first
report on synseed development was published by Kitto and Janick (1982) who
produced desiccated carrot synseeds by coating the multiple somatic embryos in a
water-soluble resin, polyoxyethyelene glycol (Polyox). Later on, Janick et al. (1993)
extended this technology for encapsulating a mixture of carrot somatic embryos and
embryogenic calli using Polyox. Redenbaugh et al. (1984) was the first to develop
48
hydrogel encapsulation technique for somatic embryos of alfalfa. Since then,
encapsulation in hydrogel remains to be the most studied strategy of synseed
production (Ara et al. 2000, Rai et al. 2009).
A number of coating agents such as agar, sodium alginate, potassium alginate,
sodium pectate carrageenan, sodium alginate with carboxymethyl cellulose, gelatin,
gelrite, guargum, tragacanth gum etc. have been tested as hydrogels (Ara et al. 2000,
Rai et al. 2009). Among these, sodium alginate has been frequently selected because
of its moderate viscosity, low spin ability of solution, low toxicity, quick gellation,
low cost and bio-compatibility characteristics (Swamy et al. 2009). Moreover, sodium
alginate and calcium salt has been reported as the best combination since the ions are
non-damaging, easy to use, have a low-price and provide an easy germination of
encapsulated propagules.
The encapsulation matrix composition is one of the important factors
significantly affecting the re-growth performance of encapsulated tissue. For effective
re-growth, the requirements of definite ingredients of hydrogel matrix (inorganic,
organic, PGRs, carbohydrate etc.) are species specific. Generally, the addition of
nutrients to the gel matrix results in improved re-growth performances (Chand and
Singh 2004, Sundararaj et al. 2010, Ahmad and Anis 2010).
Additionally, synseeds are reported to be highly susceptible to bacterial,
fungal and other infections in the greenhouse (Vij and Kaur 1994). To reduce
microbial contamination, various antimicrobial agents such as bavistin (Pattnaik et al.
1995), vitrofural G-1 (Nieves et al. 2003), plant preservative medium (PPM) (Micheli
2002) could be added to the gel matrix. However, such chemicals induce impairment
in convertibility that could be successfully alleviated by adding PGRs in gel matrix. In
agreement to this, additive effect of PPM and thidiazuron (TDZ) on overall plantlet
development was observed by Lata et al. (2009) in Cannabis sativa synseeds.
Previously, the synseed production was limited mostly to those plants in which
somatic embryogenesis had been reported, but embryogenesis was not well
documented for most of the plant species. In response to this, the possibility of using
non-embryogenic vegetative propagules such as shoot tips, nodal segments,
organogenic or embryogenic calli etc. has been explored as a suitable alternative to
somatic embryos (Ara et al. 2000, Danso and Ford-Llyod 2003, Rai et al. 2008, Faisal
and Anis 2007, Sharma et al. 2009a & b, West and Preece 2009, Ahmad and Anis
2010, Ozudogru et al. 2011).
49
With the use of such non-embryogenic plant propagules, synseed technology
has been widely exploited to a range of plant species such as Morus spp. (Pattnaik et
al. 1995, Pattnaik and Chand 2000), Eucalyptus grandis (Watt et al. 2000), Adhatoda
vasica (Anand and Bansal 2002), Dalbergia sissoo (Chand and Singh 2004), Ananas
comosus (Gangopadhyay et al. 2005), Chonemorpha grandiflora (Nishitha et al.
2006), Punica granatum (Naik and Chand 2006), Cannabis sativa (Lata et al. 2009),
Spilanthes mauritiana (Sharma et al. 2009b), Zingiber officinale (Sundararaj et al.
2010), Vitex negundo (Ahmad and Anis 2010) etc.
Amongst various vegetative propagules, nodal segments are most suitable for
encapsulation studies as they posses pre-existing axillary meristem, however in vitro
root induction is a major obstacle encountered in case of recalcitrant woody plant
species. It is assumed that encapsulation inhibits the oxygen supply to explants and
suppresses root induction (Piccioni 1997). Thus, to induce rooting different
approaches have been exploited for root induction in synseeds.
Piccioni (1997) suggested a method of incubating the explants in dark for root
primordia induction and thereafter addition of PGRs in the gel matrix for higher
conversion from synseeds. Pre-treatment of explants either with cytokinin or auxin
was also suggested for improved synseed conversion frequency (Pattnaik et al. 1995,
Soneji et al. 2002, Chand and Singh 2004, Germanà et al. 2011). Pinker and Abdel-
Rahman (2005) emphasized that the addition of IAA to the gel matrix (prepared in
modified MS) exhibited 100% root formation in encapsulated nodal segments of
Dendraanthema × grandiflora. Nishitha et al. (2006) suggested the addition of silver
nitrate (AgNO3
Addition of growth regulators to the germination medium eliminates the
requirement of an additional in vitro root induction experiment prior to
acclimatization. Ahmad and Anis (2010) found that the addition of Kn and NAA to
MS basal medium improved the germination frequency of synseeds and induced a
mean of 2.8 roots per synseed of Vitex negundo. Similar response has also been
recorded previously for Pimpinella pruatjan (Roostika et al. 2006) and Tylophora
indica (Faisal and Anis 2007). On the other hand, Gangopadhyay et al. (2005) devised
a two step method to achieve maximum synseed conversion into complete plantlets in
Ananus comosus; firstly, the microshoots were retrieved from synseeds and in the
) along with IBA to enhance the conversion frequency in synseeds of
Chonemorpha grandiflora.
50
second step, these microshoots were rooted in liquid medium (supplemented with IBA
and Kn) supported with Luffa-sponge.
Although a large number of plants can be produced in tissue culture through
direct or indirect embryogenesis and organogenesis, but their delivery is cumbersome.
Direct sowing of the synseeds in the soil or other type of substrates helps in escaping
acclimatization procedure. The technique provides an ideal delivery system enabling
easy flexibility in handling and transport of propagules as compared to large parcels
of seedlings. Thus, germination of synseeds on nutrient free substrate is a prerequisite
for sowing under non-sterile condition.
In this context, Mandal et al. (2000) suggested that the successful conversion
of synseeds into plantlets on simple planting substrate such as sand/soil/soilrite/vermi-
compost is necessary for their use in commercial-scale propagation. Still successful
germination of encapsulated tissues on various planting substrates has been reported
only for a few plant species either in a controlled culture room environment or
greenhouse conditions. The major limiting factor for reduced germination is the low-
nutrient availability. Therefore, it is necessary to build up a nutrient reservoir for the
encapsulated plant tissue, either endogenously or exogenously. Kavyashree et al.
(2006) exogenously supplied half-strength LS nutrients in horticultural grade soilrite
mix (peat: perlite: vermiculite 1:1:1) for ex vitro germination of mulberry synseeds
with healthy shoot and root systems. Similarly, Lata et al. (2009) reported 100%
conversion of synseeds on 1:1 potting mix-fertilome with coco natural growth
medium, moistened with full-strength MS medium with 3% sucrose and 0.5% PPM in
Cannabis sativa.
Synseed technology also acts as a tool of germplasm exchange and short term
conservation for rare and endangered plant species. For this purpose synseed storage
is a critical factor which determines their successful germination after transportation
abroad. Therefore, appropriate storage conditions and definite storage period are
prerequisites to maintain viability during exchange of germplasm for successful
commercialization of synseed technology. For short to medium-term storage, the aim
is to increase the interval between subculture by reducing growth. In this respect,
various strategies have been applied for slow growth maintenance of cultures.
Low temperature and light intensity induce modifications in the physiology of
stored explants, such as reduced respiration, water loss, wilting and ethylene
production, thus allowing the storage of cultures from several months to years without
51
the necessity of transferring to fresh medium (Ozudogru et al. 2010). The temperature
requirement for optimum viability varies from plant to plant. Generally, 4 ºC
temperature is found to be most suitable for synseeds storage (Saiprasad and Polisetty
2003, Kavyashree et al. 2006, Faisal et al. 2007, Singh et al. 2007, Pintos et al. 2008,
Sharma et al. 2009a & b, Ikhlaq et al. 2010, Tabassum et al. 2010). Gangopadhyay et
al. (2005) stored the synseeds of Ananas comosus in different racks of a refrigerator
with a range of temperature (4, 8, 12 and 16 ºC) for 60 days. Among the four
temperature regimes, the beads stored at 8 ºC showed maximum germination
frequency when allowed to re-grow again on nutrient media.
Ray and Bhattacharya (2010) optimized best storage environment for Eclipta
alba synseeds by changing in vitro physicochemical conditions. They extended
storage duration up to 12 weeks by decreasing the sucrose concentration in the
alginate matrix from 3 to 1 or 2%. Adriani et al. (2000) has also reported the
pronounced effect of sucrose on re-growth ability of synseed and suggested that the
sucrose availability can be a limiting factor in conversion ability of Actinidia
synseeds.
2.6 Acclimatization of plantlets
The ultimate success of micropropagation depends on the ability to transfer
and re-establish vigorously growing plants from in vitro to green house conditions.
This involves acclimatization or hardening-off plantlets to conditions of significantly
lower relative humidity and higher light intensity. Although, micropropagation has
been extensively used for the rapid multiplication of various plant species and
considerable efforts have been directed to optimize the conditions for in vitro stages
of micropropagation, but the process of acclimatization of tissue culture raised plants
to the natural environment has not been fully studied (Hazarika 2003).
Micropropagation is often restricted due to high percentage of plantlets’ death during
ex vitro transplantation (Pospisilova et al. 1999). The acclimatization of
micropropagated plants remains a critical stage; in the first week after transfer to ex
vitro conditions, plantlets cope with the different stresses and have to adapt to the
external environmental conditions (Aragon et al. 2005). In fact, microparopagated
plants are difficult to transplant due to following of two primary reasons:
52
i) A heterotrophic mode of nutrition
ii) Poor control of water loss (Kane 2000).
A number of researches have been conducted to solve various problems
related to acclimatization such as relative humidity and indicate that high humidity
increase the survival percentage of micropropagated plants (Kozai 1991). A
composite of anatomical and physiological features, characteristic of in vitro plant
produced in 100% relative humidity, contributes to the limited capacity of
microprapagted plants to regulate water loss immediately following transplanting.
These features include no epicuticular wax formation, poor cuticle development,
poorly differentiated mesophyll, poor connection between shoots and roots and
improper function of stomata resulted in excessive water loss and poor photosynthetic
capacity in ex vitro acclimatized plants (Ziv 1991, Kane 2000, Chen et al. 2006).
Leaf surface covering agents, such as glycerol, paraffin and grease also
promoted ex vitro survival of herbaceous species, but have not been evaluated over a
long-term or examined on woody species (Selvapandiyan et al. 1988). Several growth
retardants which reduce damage due to wilting without deleterious side effect have
been suggested to improve ex vitro survival of regenerants. Absiccic acid (ABA) is
considered as a growth retardant which may alleviate ‘transplantation shock’ and
speed up acclimatization of tobacco plantlets under ex vitro conditions (Pospisilova et
al. 1998). Ray and Bhattacharya (2008) established an efficient and simple protocol
for in vitro propagataion of Eclipta alba including successful transplantation by
priming the plantlets with a growth retardant, chlorocholine chloride (CCC) for the
first time. Among various concentrations of CCC, 6.33 µM was found most effective
for inducing certain beneficial changes. In 30 day-old treated shoots, they observed an
increased number of roots, elevation in chlorophyll content and plant biomass. They
reported that the arrested undesirable shoot elongation made the plants sturdy and
more suitable for acclimatization. The primed plants exhibited 100% survival
frequency as compared to control plants (84%). Priming of micropropagated
propagules has already been recommended for obtaining better acclimatized plants
(Nowak and Shulaev 2003, Hazarika 2003). The concept of priming the tissue culture
raised plants to improve acclimatization is based on the fact that certain chemicals
effectively pre-sensitize cellular metabolism of plants (Nowak and Shulaev 2003) and
increase the adaptive ability of in vitro cultures (Conrath et al. 2002, Nowak and
Pruski 2004).