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REVIEW OF LITERATURE
2.1 Meizotropis pellita
Characteristic features:
Meizotropis pellita known as Patwa was recognized by YR Roskov, FA
Bisby, JL Zarucchi, BD Schrire & RJ White (eds), ILDIS world Database of
Legumes. Patwa was first reported by Osmonston in 1925 at Patwadanger (1530 m).
He had also seen this plant in Kali Kumaun and subsequently its presence was also
reported from Doti district of Nepal. This plant is a shrub with stout, woody
perennial rootstock from which several erect shoots up to 6 feet high and 0.75 inch
diameter are annually produced. Stems are ribbed with large pith. Leaves, stem,
inflorescence and pods are densely clothed with spreading white or pale brown
tomentum. Leaves are 18-30 inches long. Flowers are 0.5-1 inch long in fascicles of
usually 3-5, arranged in erect terminal and axillary simple raceme. Corolla has
bright red wings, keel changing to orange towards the base inside. The plant will
reappear in a year from the root stock in April / May (Sanjappa, 1987).
Classification:
Domain - Eukaryota
Kingdom - Plantae
Phylum - Magnophyta
Class - Magnoliopsida
Suborder - Fabanae
Order - Fabales
Family - Fabaceae
Genus - Meizotropis
Specific epithet - pellita
Chapter 2
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2.2 Geographical distribution and temperature:
Patwdanger is a village situated at 12 km. away from Nainital. Nainital is
the Indian state of Uttarakhand and headquarters of Nainital district in the
Kumaun foothills of the outer Himalayas. Patwadanger is located at latitude
29.33(290
19’’60’N) and longitude 79.43(79025’’60’E). It has an average
elevation of 1530 m. Patwadanger has maximum temperature 28 °C and
minimum temperature 17 °C in summers. In winters Patwadanger receives
snowfall between December and February with the temperatures varying
between a maximum of 15 °C and a minimum of −3 °C.
Earlier the distribution of this species was also reported in specific areas
of Nepal and Kali-Kumaun, presently it can’t be traced in those regions.
Distribution of the Genera Meizotropis, Butea and Spatholobus
(Ridder-Numan, 1998)
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2.3 Habitat
This species occurs more gregariously on flat hill tops as well as on the
valley slopes near dry rides and in open chir forest at around 5000 feet in May-
June.
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2.4 Family Fabaceae
Fabaceae or Leguminosae is a large and economically important family
of flowering plant, which is commonly known as the legume family, pea family,
bean family or pulse family. The name 'Fabaceae' comes from the defunct genus
Faba, now included into Vicia. Leguminosae is an older name still considered
valid, and refers to the typical fruit of these plants, which are called legumes.
Fabaceae is the third largest family of angiosperms after Orchidaceae (orchids)
and Asteraceae (daisies, sunflowers), and second only to Poaceae (grasses) in
terms of agricultural and economic importance. Legumes includes a large
number of domesticated species harvested as crops for human and animal
consumption as well as for oils, fiber, fuel, fertilizers, timber, medicinals,
chemicals, and horticultural varieties (Lewis et al., 2005). In addition, the family
includes several species studied as genetic and genomic model systems (e.g., pea
(Pisum sativum), barrel medic (Medicago truncatula), and trefoil (Lotus
corniculatus).
2.4.1. Characteristics
Morphologically, Fabaceae is characterized by leaves simple to compound
(pinnate, rarely palmate, or bipinnate), unifoliate, trifoliate (Medicago,
Trifolium), sometimes phyllodic (many species of Acacia), or reduced to a
tendril (as in Lathyrus), spirally arranged, with stipules present that are
sometimes large and leaf-like (Pisum) or developed into spines (Prosopis,
Robinia). Flowers are usually regular or irregular (i.e., actinomorphic to
zygomorphic in symmetry, respectively), bisexual, with a single superior carpel
(hypogynous to perigynous), pentamerous, arranged singly or in racemes, spikes,
or heads. The principal unifying feature of the family is the fruit, the legume
(Polhill, 1994). With a few exceptions, legumes are typically one-chambered
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pods (one locule), with parietal placentation along the adaxial suture, ovules two
to many, in two alternating rows on a single placenta, typically dry and dehiscent
along one or both sutures (legume), occasionally constricted into 1-seeded
sections (loments) or indehiscent (samara, drupe, achene).
2.4.2 Habitat and distribution
Legumes vary in habit from annual and perennial herbs to shrubs, trees,
vines/lianas, and even a few aquatics, the family is cosmopolitan in distribution.
Ranging in size from some of the smallest plants of deserts and arctic/alpine
regions to the tallest of rain forest trees, legumes are a conspicuous, and often
dominant, component of most of the vegetation types distributed throughout
temperate and tropical regions of the world (Rundel, 1989). Legumes are
particularly diverse in tropical forests with a seasonally dry aspect and temperate
shrublands tailored by xeric climates. The preference of legumes for semi-arid to
arid habitats is related to a nitrogen-demanding metabolism, which is thought to
be an adaptation to climatically variable or unpredictable habitats whereby leaves
can be produced economically and opportunistically (McKey, 1994). A hallmark
of legume biology, the fixation of atmospheric nitrogen via root-nodulating
rhizobial bacteria, is just one of several ways (in addition to arbuscular
mycorrhizas, ectomycorrhizas, and uptake of inorganic nitrogen compounds) in
which legumes obtain high levels of nitrogen to meet the demands of their
metabolism (Sprent, 2001).
2.4.3. Taxonomy
Taxonomically, Fabaceae has been traditionally divided into three
subfamilies, the Caesalpinioideae, Mimosoideae, and Papilionoideae (although
sometimes these have been ranked as separate families, as in Caesalpiniaceae,
Mimosaceae, and Papilionaceae), and considered most closely related to the
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Connaraceae and Sapindaceae on the basis of anatomy, morphology, and
biogeographic distributions (Polhill and Raven, 1981). The recognition of three
subfamilies is based on characteristics particularly of the flower, including size,
symmetry, aestivation of petals, sepals (united or free), stamen number and
heteromorphy, pollen (single or polyads), but also presence of a pleurogram,
embryo radicle shape, leaf complexity, and presence of root nodules (Lewis et
al., 2005). Differences in these characteristics led to the view that the
Mimosoideae and Papilionoideae are unique and distinct lineages in the family
which arose independently within a paraphyletic "basal" caesalpinioid
assemblage. The recent update of the tribal and generic classification of the
family, recognizes 36 tribes, 727 genera and 19,327 species (Lewis et al., 2005).
The family contains at least four genera of 500 or more species (Acacia,
Astragalus, Crotalaria, and Indigofera) and at least 40 genera with 100 spp. or
more. At the other extreme, nearly 500 genera are small, either being
monospecific or containing up to 10 species (Lewis et al., 2005). The legumes
being one monophyletic family (Doyle et al., 2000; Kajita et al., 2001;
Wojciechowski, 2003; Wojciechowski et al., 2004) that is more closely related
to Polygalaceae, Surianaceae, and Quillajaceae, which together form the order
Fabales (Angiosperm Phylogeny Group, 2003).
2.4.4. Fossil Record
The Fabaceae contains over 19,000 extant species widely distributed
throughout the world in many ecological settings, from deserts of high latitudes
to seasonally dry and wet tropical forests of equatorial regions (Lewis et al.,
2005). Legumes appear to have diversified during the Early Tertiary (Herendeen
et al., 1992) to become a ubiquitous feature of modern terrestrial biotas, similar
to the timing of diversification of several other modern families of angiosperms.
The fossil record of the Fabaceae is abundant and diverse, particularly in the
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Tertiary, with fossil flowers, fruits, leaflets, wood, and pollen known from
numerous localities (Crepet and Taylor, 1985; Crepet and Herendeen, 1992;
Herendeen, 1992; Herendeen et al., 1992). The first definitive legumes appear
during the Late Paleocene (Herendeen, 2001; Herendeen and Wing, 2001;
Wing et al., 2004). Attempts to estimate the age of legumes and diversification
in the family, based on molecular sequence data, have been published in recent
years (Wikstrom et al., 2001).
2.4.5. Agricultural & Economic Importance of Legumes
Legumes have demonstrated agricultural importance for thousands of
years, beginning with the domestication of lentils (Lens esculenta) in Iran dating
to 9,500 to 8,000 years ago, their use as a food source during the prehistory of
North and South America (beans, more than 3,000 years ago), and their use by
the Roman Empire as a food source and for soil improvement (Graham and
Vance, 2003). Today legumes are an increasingly invaluable food source not just
for humans, accounting for 27% of the world's primary crop production, but also
for farm animals (Graham and Vance, 2003). Grain legumes alone contribute
33% of the dietary protein nitrogen needs of humans, while soybeans (Glycine
max) and peanut (Arachis hypogeae) provide more than 35% of the world's
processed vegetable oil and a rich source of dietary protein for the poultry and
pork industries (Graham and Vance, 2003). While they produce nitrogen-
containing protein in abundance, legumes are deficient in sulfur containing
amino acids and other nutrients needed by people and animals. For this reason,
legumes and cereal crops are often raised together to account for the amino acids
and other elements which they are lacking (Gepts et al., 2005).
Many legumes form root nodules to fix atmospheric nitrogen in a
symbiotic relationship with the soil bacteria 'rhizobia'. Legumes are extremely
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diverse in their abilities to nodulate, not all species can and there is a wide
variety of nodules that form, depending on the species in symbiosis. Industrially,
legumes have many uses in making biodegradable plastics, oils, dyes, and
biodiesel fuel. Legumes are used traditionally in folk medicines, but also
demonstrate importance in modern medicine. Isoflavones commonly found in
legumes are thought to reduce the risk of cancer and lower cholesterol and
soybean phytoestrogens are being studied for use in postmenopausal hormone
replacement therapy (Graham and Vance, 2003). Legumes also produce a
hypoglycemic effect when eaten, making them a recommended food for diabetics
(Gepts et al., 2005).
2.5. Seed germination
2.5.1. Viability and germination
Regeneration from seeds is the most commonly used method of
propagation in many plant species. Germination incorporates all those events that
commence with the uptake of water by the quiescent dry seed and terminate with
the elongation of the embryonic axis. The visible sign that germination is
complete is usually the penetration of the structure surrounding the embryo by
the radicle (Bewley and Black, 1994).
In general the seeds of many alpine and subalpine plant species are viable
at maturity and with time the viability decreases. Proper storage conditions do
enhance the period of viability to a certain extent. Studies on germination,
viability and vigour of fresh and aged seeds of some endangered medicinal plant
species of western Himalayas like Achilla mellefolium, Gentiana kurroo and
Podophylum hexadrum showed that germination of fresh seeds was higher
compared to aged seeds. Significantly positive correlation was observed between
germination percentage, vigour index and viability. A negative correlation was
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observed between electrical conductivity and germination percentage and rate
(Thakur et al., 2004). In a similar study germination potential , speed of
germination and vigour index of medicinal plants, Eruca sativa Lam and
Anthenis altissima L., under cold room(40C) and dry room(room temperature
storage) conditions were recorded (Alizadeh and Isvand, 2004).
The use of plant growth regulators (PGRs) and chemical compounds as
pre soaking treatment to influence seed germination is well known. Gibberellins
(GA3), cytokinins (BA, Kn, thiourea, potassium nitrate have been reported to
promote germination in some medicinal plants of Indian Himalayan region.
Prasad (1999) reported enhancement of seed germination of Podophylum
hexandrum and Aconitum heterophyllum by different treatments. It was observed
that in Podophyllum hexandrum, seed germination improved to 70% following
scarification and 50% following treatment with PGR, namely gibberellic acid
(GA3). In Aconitum heterophyllum, germination improved to 65% following GA3
treatment whereas 20% germination was found in control. Nadeem et al. (2000)
reported a nearly two fold improvement in germination of Podophyllum
hexandrum seeds following treatment with GA3 alone or a combination of GA3
and BAP. GA3 significantly enhanced seed germination (42.5% compared to
27.5% in control) in Aconitum balfourii while BAP promoted (42.5% compared
to 25% in control) in Aconitum hetrophyllum. Although the nitrogenous
compound thiourea increased the rate as well as the germination percentage in
both species but KNO3 enhanced germination in Aconitum balfourii only
(Pandey et al., 2000).
Germination test at temperature range from 5-350C, either continuous or
alternative high and low temperatures have been assessed for germination of
many alpine plants generally non dormant seeds show the requirement of
relatively high temperatures for Germination (Baskin and Baskin, 1998). Cold
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Stratification was found to lower the temperature requirement of seeds of
Saxifraga Tricuspidata (Densmore, 1997) and Caltha introloba (Wardlaw et
al., 1989).
2.5.2. Seed dormancy
Seed dormancy is a state in which seeds are prevented from germinating
even when environmental conditions are normally favourable for germination
(Copeland and McDonald, 1985). There are two classes of seed dormancy: coat
imposed and embryo dormacy. In coat imposed dormacy, radicle emergence is
blocked by the physical barrier of the testa, aleurone or endosperm layer. Seed
coat imposed dormancy can be overcome by cutting the seedcoat (Debeaujon
and Koornneef, 2000). Conversely, seed with embryo dormancy will not
germinate when the seedcoat is cut. Dormancy may also be due to the presence
of germination inhibitors such as abscisic acid (Bewley, 1997; Baskin and
Baskin, 1998).
Dormancy, germination and early events in seedling development are
thought to be regulated by the interaction of various plant growth regulators like
gibberellins, cytokinins, brassinosteroides, auxins and abscisic acid (Bewley,
1997; Kucera et al., 2005). Seed dormancy is subject to hormonal control. The
plant hormone abscisic acid (ABA) is needed to induce seed dormancy during
embryo maturation, whereas the hormone gibberellin (GA3) is needed to
stimulate seed germination (Ariizumi and Steber, 2007). There are different
methods to overcome dormancy, which vary from species to species, such as
heating (Herranz et al., 1998), stratification, scarification (Narbona et al., 2003)
and gibberellin application (Demirsoy et al., 2010). Gibberellic acid is known to
break dormancy of several types of seeds (a) light promoted seeds (b) light
inhibited seeds (c) seeds requiring stratification (storage at low temperature in a
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moist condition) (d) seeds requiring after ripening (storage at room temperature
in dry condition) (Shepley et al., 1972). Breaking of seed dormancy by GA3 or
cytokinins like BA has been reported in many plant species including those from
alpine regions (Arnold et al., 1996; Rascio et al., 1998; Nadeem et al., 2000;
Pandey et al., 2000). GA3 is known to eliminate the chilling requirement often
needed by certain seeds (Bewley and Black, 1994; Arnold et al., 1996). The
promotory role of GA3 with BA attributed to a permissive role of cytokinins
(Khan, 1975; Walker et al., 1989). It has been reported that GA3 and ABA act
at different times and sites; GA3 play key role in dormancy and in promotion or
germination while ABA induces dormancy during seed germination (Kucera et
al., 2005).
2.6. Tissue culture technology
All plant cells have the potential to be totitpotent, i.e., to be able to
dedifferentiate, divide and regenerate into whole plants (Loidl, 2004). This was
the idea that Gottlieb Haberlandt had in mind when he first attempted plant tissue
culture in the early 20th
century (Caponetti et al., 2004). Although he failed in
his venture to regenerate plants from isolated tissues, his work attracted the
attention of the scientific world and, consequently, abundant research was
developed on the topic.
2.6.1. Tissue culture concepts as applied to the Fabaceae Family
Tissue culture is usually defined as a heterogeneous group of techniques
in which explants (protoplasts, cells, tissues or organs) are aseptically placed
onto a culture medium of defined chemical composition, and incubated under
controlled conditions (Mroginski et al., 2004b). There are three types of plant
regeneration systems that are used most frequently: micropropagation,
organogenesis and somatic embryogenesis. Micropropagation consists of the in
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vitro propagation of selected genotypes through improved axillary shoot
production from explants with pre-existing meristems (Kane, 2004). In contrast,
the other two regeneration schemes are based on the use of nonmeristematic
tissues as explants: organogenesis is the de novo formation of organs (shoots,
roots or flowers), and somatic embryogenesis is the production of embryos
without a previous fusion of gametes (Radice, 2004). Members of the Fabaceae
family have traditionally been regarded as recalcitrant to in vitro regeneration,
particularly in the case of cultivated grain legumes (Griga, 1999; Veltcheva et
al., 2005; Mundhara and Rashid, 2006). Veltcheva et al. (2005) suggest that
recalcitrance in grain legumes could be caused by the narrow genetic base of the
cultivated varieties that have undergone inbreeding and selection for long periods
of time. In addition, they suggest that in forage species the outbreeding and lower
genotype selection may account for easier identification of responsive genotypes.
Some of the factors that affect in vitro response of a given species are genotype,
explant, composition of the culture medium and conditions under which explants
are incubated (Radice, 2004). The genotype of the donor plant is one of the most
critical factors since it influences in vitro responses, from the establishment of
the explant to the regeneration of whole plants, as well as ex vitro, during the
acclimatization of regenerated plants. The importance of the genotype on in vitro
plant regeneration of cultivated peanut (Arachis hypogaea L.) has been
demonstrated by Chengalrayan et al. (1998), who assessed 16 genotypes for
responsiveness in vitro using a protocol to induce somatic embryogenesis. These
authors found differences in the frequency of response at each stage of the
process and suggested that genotype could be the primary factor influencing
conversion of somatic embryos to plantlets. A similar experiment was carried out
in soybean (Glycine max (L.) Merrill), in which 17 breeding lines were evaluated
for their response and ability to regenerate plants through somatic embryogenesis
(Tomlin et al., 2002). Among these lines, a significant difference in the
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percentage of responsive explants, number and quality of somatic embryos were
observed. Other legume species in which differences among genotypes were
reported, particularly regarding somatic embryogenesis are Medicago sativa L.
and Trifolium pratense L. (Lakshmanan and Taji, 2000). For Trifolium
pratense, Quesenberry and Smith (1993) increased genotype regeneration
frequency from less than 5% to almost 70%, after five cycles of recurrent
selection. The explant type used for culture establishment depends on the
objectives that are pursued since it determines responsiveness of the plant
material in vitro (Lakshmanan and Taji, 2000). The aspects that should be
considered in explant selection are: explant tissue (leaves, petals, anthers, roots,
meristems, cotyledons, epicotyls, hypocotyls), explant size, explantation time,
topophysis and polyphenol oxidation (Kane, 2004).
It is widely accepted that immature zygotic embryos and young seedlings
are the most responsive explants to induce somatic embryogenesis in legume
species. This is because areas where cells show active division are more
responsive to the embryogenic stimulus (Griga, 1999; Mundhara and Rashid,
2006). However, a range of explants have been used with success to induce
somatic embryogenesis in Fabaceae family. These have included mature seeds,
shoot apices, seedlings, hypocotyls, cotyledons, leaves, petioles, internodes,
roots, endosperms, cell suspensions and protoplasts (Lakshmanan and Taji,
2000). For the induction of organogenesis in legume species, a similar variety of
explants has been used. As an example, in Arachis, several explants have been
capable of regenerating plants: fully expanded leaves (Dunbar and Pittman,
1992), leaflets from young seedlings (Akasaka et al., 2000), epicotyls, petioles
(Cheng et al., 1992), cotyledons, embryo-axis, mature whole seeds
(Radhakrishnan et al., 2000), protoplasts (Li et al., 1993), mature zygotic
embryo-derived leaflets (Chengalrayan et al., 2001) and shoot apices
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(Radhakrishnan et al., 1999). Culture medium composition is determined by
the type and concentration of inorganic salts (macro- and micro-nutrients),
organic compounds (sugar, vitamins, activated charcoal, etc.), plant growth
regulators (mainly auxins and cytokinins), gelling agents or other support system
and the gaseous atmosphere inside the culture vessel (Radice, 2004). The most
widely used basal medium for legume regeneration is MS medium developed by
Murashige and Skoog (1962) for callus cultures of tobacco (Murashige and
Skoog, 1962). This basal medium, which has a high salt concentration, has been
used to achieve plant regeneration in several legume genera, such as Arachis
(Rey and Mroginski, 2006), Astragalus (Luo et al., 1999), Cajanus (Singh et
al., 2003), Cassia (Agrawal and Sardar, 2006), Cicer (Chakraborti et al.,
2006), Dalbergia (Singh and Chand, 2003), Glycine (Tomlin et al., 2002),
Lathyrus (Barik et al., 2005), Lotus (Akashi et al., 2003), Phaseolus (Delgado-
Sanchez et al., 2006), Pisum (Loiseau et al., 1998), Trifolium (Ding et al., 2003)
and Vigna (Saini and Jaiwal, 2002). However, other basal media have been
specifically developed for certain legume species, such as Glycine max
(Gamborg et al., 1968) and Trifolium pratense (Collins and Phillips, 1982).
Several plant growth regulators have been used with success in plant
regeneration protocols for legume species, but the type of response and
effectiveness of the compounds are highly dependent on the species and even on
genotypes within a species. In general, auxins are used to induce somatic
embryogenesis, whereas cytokinins are used to induce organogenesis.
Nevertheless, there are some exceptions such as in Trifolium repens L.,
Medicago sativa and Phaseolus spp., in which it was possible to achieve somatic
embryogenesis by using cytokinins instead of auxins (Lakshmanan and Taji,
2000). In addition to media composition factors, incubation conditions under
which explants are incubated must be controlled. These include temperature,
light quality and intensity, photoperiod, humidity and hygiene (Mroginski et al.,
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2004b). In general, the temperature for incubation of cultures is between 23-29
ºC, depending on optimal growth requirement of the species (Radice, 2004). In
some cases, when the process to be induced is somatic embryogenesis, cultures
are incubated in the dark, since light is not required for this developmental
pathway. In contrast, when organogenesis is to be induced, cultures are usually
kept under light conditions with a specific photoperiod. In addition, light quality
(spectral quality) and quantity (photon flux) are reported to have an important
role in morphogenetic processes in vitro and on the subsequent growth of the
regenerated structures (Lian et al., 2002).
Since its origin in the early 20th century, tissue culture procedures have
been used for a variety of purposes, such as basic studies of particular
physiological processes because the use of tissues instead of whole plants usually
simplifies the study of the phenomenon (Mroginski et al., 2004b). Another use
of tissue culture is the production of plants free from certain specific pathogens,
generally viruses, through meristem or shoot tip culture alone or combined with
thermo/chemotherapy. However, the most important application of these
techniques from an economic point of view is related to micropropagation. This
method is particularly important in horticulture, since it generally maintains
genetic stability (Kane, 2004), and allows propagation of periclinal chimeras.
This kind of chimera may be important in ornamental species and cannot be
propagated through organogenesis or somatic embryogenesis. Tissue culture may
also be used for the production of interspecific hybrids where zygotic embryos
abort early in their development and have to be rescued, or in the case of plants
with a rudimentary embryo (Mroginski et al., 2004b). The production of
dihaploid, homozygous plants is also possible through anther or ovule culture,
which reduces the time required to achieve homozygosis in breeding programs.
Other applications of tissue culture include the induction of somaclonal variation,
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production of secondary metabolites using cell culture, generation of somatic
hybrids through protoplast fusion, and plant regeneration after transformation
protocols. From the species preservation point of view, tissue culture constitutes
a valuable technique for medium and long-term germplasm conservation (in vitro
and cryoconservation), as well as plant material exchange since pathogen-free
plants are used for this purpose (Mroginski et al., 2004b).
Since plants were first domesticated, diseases and pests have threatened
crop productivity. Some diseases caused by fungi and bacteria may be controlled
if certain practices are used during the cultivation of the crop. However, in the
case of viruses, the control is usually more difficult and in many cases the only
indication of the presence of a virus is a reduction in crop yields. Viral diseases
are transmitted rapidly particularly when the crop is vegetatively propagated
(Kartha, 1984). Meristem culture is one of the tools to eliminate viruses from
plant material, provided that the rate of virus multiplication and movement in the
plant is lower than the rate at which the meristematic region elongates. This is
often the case since vascular tissues do not reach the meristem. In the Fabaceae
family, meristem culture has been applied successfully to the rescue of
interspecific hybrids between Arachis hypogaea and Arachis stenosperma
Krapov & W.C. Gregory, and Arachis hypogaea and Arachis. otavioi, which
showed symptoms of peanut stripe virus (Radhakrishnan et al., 1999).
Meristem culture with or without thermo/chemotherapy has also been used to
eliminate peanut mottle virus, peanut stripe virus and tomato spotted wilt virus
from interspecific hybrids of Arachis that were maintained vegetatively in the
germplasm collection at the Southern Regional Plant Introduction Station
(Griffin, GA) (Dunbar et al., 1993a). In contrast, shoot tip culture was not an
effective procedure to regenerate plants free of the peanut mottle viruses.
Prasada et al. (1995) excised seed axis from peanut stripe virus infected seed
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and cultured them on a medium containing ribavirin to obtain peanut plants free
of the virus. Meristem culture has also been successfully applied to other legume
genera, such as Trifolium and Phaseolus, for the production of plants free of
common viruses (Phillips and Collins, 1979; Veltcheva et al., 2005).
Micropropagation, the true-to-type propagation of a genotype through
tissue culture techniques, is a useful tool in breeding programs. Among other
advantages, it enables the production of uniform plants from a selected genotype
at a high multiplication rate (Olmos et al., 2004). The stages for
micropropagation from shoot explants are: a) donor plant selection and
preparation, b) axillary shoot proliferation, c) pre transplant or rooting, and d)
transfer to the natural environment (Kane, 2004). Cultivated peanut has been
reported to have limited reproductive efficiency, which is a drawback when large
populations are required for breeding purposes (Radhakrishnan et al., 2000).
Micropropagation may be used to overcome this situation, provided that an
efficient in vitro protocol is available. For this species, Radhakrishnan et al.
(2000) developed a high frequency micropropagation protocol from embryo axes
and plant regeneration from other juvenile explants. Successful micropropagation
protocols have also been developed for other species such as Vigna mungo (L.)
Hepper, a grain legume important in South Asia and Australia, where plants were
regenerated from shoot tips, embryo axes and cotyledonary nodes (Saini and
Jaiwal, 2002).
As civilization advances, the centers of diversity of many important plants
for food and forage are threatened. This situation implies the loss of valuable
genes contained in the wild relatives of the cultivated species that could be used
in breeding programs. For this reason, germplasm is kept in storage facilities,
mainly as seeds, which require considerable land and labor to be renewed. For
many species belonging to the genus Arachis, seed viability decreases abruptly
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after 2-3 years of storage. However, some protocols have been developed to
recover plants from seeds that would not germinate by themselves through the in
vitro culture of embryonic axes (Dunbar et al., 1993b; Morris et al., 1995).
Cryopreservation constitutes an alternative to the laborious and time consuming
storage of seeds. Not only does it allow for long-term storage, but it also ensures
genetic stability, requires little space and low maintenance (Gagliardi et al.,
2003). The ultralow temperatures of liquid nitrogen cause interruption of all
biochemical reactions protecting the plant material from physiological and
genetic changes (Yamada et al., 1991). In addition, plants are kept free from
pathogens when propagated from plants that have been indexed for the presence
of specific microorganisms. Protocols for cryopreservation have been developed
for several species: Arachis burchellii Krapov. & W.C. Greg., Arachis hypogaea,
Arachis retusa Krapov et al., (Gagliardi et al., 2003), Arachis macedoi Krapov.
& W.C. Greg., Arachis pietrarellii Krapov. & W.C. Greg., Arachis prostrata
Benth. Arachis villosulicarpa (Gagliardi et al., 2002) and Trifolium repens
(Yamada et al., 1991) among others. Medium-term conservation of germplasm
can also be done by maintaining plants under in vitro conditions, which has
similar advantages as cryopreservation: little space, low maintenance, and
protection from pathogens. Moreover, plants kept in vitro are a ready. source of
material in case the production of a large number of plants is required
(Bhojwani, 1981).
Clitoria ternatea L. also known as “butterfly pea” is a multipurpose
forage legume belonging to family Fabaceae. It is distributed in tropical Asia, the
Philippine Islands and Madagascar (Anonymous, 1988). Seeds were germinated
on MS basal medium supplemented with gibberellic acid (1.0 µM) within one
week of inoculation. Cotyledonary node of Clitoria ternatea L. gave rise to
multiple shoots (number of shoots varied) when cultured on MS medium
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fortified with different concentrations of BA and Kn. The highest rate of shoot
multiplication with 90% response was obtained on MS medium containing BA
(5.0 µM). Regenerated and elongated shoots were rooted on half strength MS
medium fortified with IBA (2.0 µΜ) after two weeks of sub-culturing. Complete
plantlets were then hardened, acclimatized and transplanted to natural conditions,
where they exhibited 80% survivability (Ismail et al., 2011).
Crotalaria retusa Linn. (family Fabaceae), distributed throughout India
especially open forest and other tropical and sub-tropical regions of the world,
which can be used as antagonistic to nematodes in sustainable crop production
systems. High frequency of multiple shoots was induced on MS medium
supplemented with BAP (13.31 µM). 100% cultures responded with an average
number of 11.6 shoots per explants. However, the average shoot length was
limited to 3.2 cm. The addition of 2.15 µM NAA along with 13.31 µM BAP gave
an average number of (12.4) shoots with an average shoot length of 6.2 cm.
Callus was obtained on MS medium supplemented with BAP (2.21 µM) and
2,4-D (13.57 µM). Optimum callus regeneration obtained on MS medium
supplemented with 13.31 µM BAP and 2.15 µM NAA. On this medium, 96.4 %
cultures responded with an average number of 12.6 shoots per culture. The
shoots obtained via multiple shoot induction and organogenesis was rooted on
half-strength MS medium supplemented with IBA (7.38 µM). The rooted shoots
were successfully transplanted to soil with 90% success (Devendra and
Srinivas, 2011).
Albizia lebbeck (L.) Benth., a potential medicinal plant, commonly known
as Shirish or Woman’s Tongue tree, is an erect, deciduous, mimosoid legume. It
is distributed throughout India, Bangladesh, tropical and subtropical regions of
Asia and Africa. Root explants from 15 day-old-aseptic seedlings were cultured
on Murashige and Skoogs (MS) medium supplemented with different
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concentrations BA, Kn, 2-iP singly as well as in combination with NAA. The
highest rate of shoot multiplication (16.0 ± 1.87 for the average shoot number
and 5.16 ± 0.38 cm for shoot length) was achieved on MS medium supplemented
with 7.5 µM BA and 0.5 µM NAA. Rooting was achieved on microshoots using
half strength MS medium with 2.0 µM IBA after four weeks of culture. The in
vitro raised healthy plantlets were established in earthen pots containing garden
soil and grown in greenhouse with >80% survival rate (Shahnaz et al., 2011).
Abrus laevigatus E. May. commonly known as 'Kunch' in Bengali is a
deciduous woody climber of the family Fabaceae. Different plant parts of this
species contain various kinds of alkaloids such as glycerrhizin, precol, abrol,
abrasine, abrin A and abrin B, which indicate its medicinal value. (Biswas and
Ghosh, 1973; Joshi, 2000). Yellowish green, fragile, nodular callus was induced
at the cut surface of the nodal segments cultured on MS fortified with 5.0 mg/1
BA, 0.2 mg/l Kn and 0.1 mg/1 IBA. The callus differentiated into adventitious
shoots when it was sub-cultured on to MS supplemented with 3.0 mg/l BAP +
0.5 mg/1 Kin + 0.5 mg/1 NAA. On an average 6.87 ± 0.26 shoots developed.
These micro-shoots were rooted in half-strength MS containing 1.0 mg/1 IBA
and the rooted plantlets were transferred to soil after acclimatization (Pandhure
et al., 2010).
Taverniera abyssinica A. Rich., a medicinal plant species belonging to the
Fabaceae family commonly known as dingetegna, literally meaning, remedy
against sudden illness. The species is known to occur in Northeast Africa and
Southeast Asia. It is a threatened medicinal plant that usually grows in a bush
land limestone areas with an altitude range of 1700 to 2300 above sea level
(Thulin, 1989). The in vitro germination of seeds was obtained on Murashige
and Skoog medium supplemented with 12 g 1-1
phytoagar without sucrose. Light
green compact calli from node, petiole and shoot meristem explants were
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efficiently induced on Gamborg medium containing (0.90 or 1.80 µM) 2,4-D
combined with 2.22 µM BAP, and supplemented with 30 g 1-1
sucrose and 5 g 1-1
phytagel. Callus initiation from shoot meristems and nodes was faster and
occurred with a higher frequency than callus initiation from petiole and leaf
segments (P<0.05). A high frequency of shoot regeneration (100%) was obtained
upon transfer of calli onto regeneration medium containing 8.88 µM BAP
combined with 1.14 µM IAA. Regenerated shoots were transferred to root
induction medium, which turned out to be optimal when half strength B5
medium was supplemented with 9.84 µM IBA. Upon transfer to glasshouse, 86%
survived and grew vigorously (Balcha et al., 2010).
Seedlings of the leguminous shrub; Colutea istria Mill. were used as
explants for the micropropagation of this vulnerable species. Cotyledonary nodes
stem node sections and shoot tips from the in vitro germinated seedlings were
examined for micropropagation. For multiplication, the explants were cultured
on MS medium containing BA at concentrations of 0.25, 0.5 and 1 mg/L either
individually or in combination with 2iP at a concentration of 0.5 mg/L. The
combination of BA and 2iP was recommended for multiplying the established
shoots produced from colyledonary nodes and stem node sections due to the
significantly higher average number of shoots/explant comparing to the media
containing only BA. Explants rooted on MS medium containing 0.5 mg/L of both
Indole-3-butyric acid (IBA) and NAA and plantlets with well developed shoots
and roots were transferred to soil and grew normally without loss of green colour
and wilting (Hegazi and Gabr, 2010).
Grasspea (Lathyrus sativus L.) is a self-pollinated, annual, herbaceous
legume rich in protein in the tribe Vicieae (Adan.) de Candolle of the family
Fabaceae. Grasspea is cultivated in many countries of the world as animal feed.
An efficient and reproducible protocol for in vitro rapid and large-scale
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propagation of the plant has been developed from immature zygotic embryos
using various concentrations of TDZ and BAP+NAA with and without ascorbic
acid. The results showed that TDZ and BAP+NAA without ascorbic acid were
ineffective to induce shoot regeneration from explants due to excretion of
phenolic compounds. TDZ (with ascorbic acid) was more potent for axillary
shoot regeneration compared to BAP+NAA (with ascorbic acid) with the highest
shoot regeneration on MS medium containing 0.45 mg/l TDZ. The shoots
regenerated on MS medium containing 0.45 mg/l TDZ (with ascorbic acid) were
rooted on MS medium supplemented with 0.90 mg/l NAA. It was not difficult to
acclimatize all of the rooted plants in soil in greenhouse (Kendir et al., 2009).
Bambara groundnut (Vigna subterranea (L.) Verdc.) is essentially grown
for human consumption. The seed makes a rich food, as it contains sufficient
quantities of protein, carbohydrate and fat (Rowland, 1993). In vitro
regeneration via direct organogenesis in Bambara groundnut using hypocotyl and
epicotyl cuttings was done using Basal MS medium supplemented with BAP,
kinetin or TDZ with or without NAA. Multiple shoots were induced from both
explants but regeneration efficiency was higher when epicotyl cuttings were
used. BAP (2mg/l) gave the highest response (73.33 - 97.77%) with the
regeneration of 3.7 shoots per explant with hypocotyl and 5.8 shoots per explant
with epicotyl. The regenerated shoots were readily elongated on the same
medium as used for induction and rooted on half-strength MS basal medium
without any growth regulators. 62% of the plantlets were successfully
acclimatized and potted plants were established in soil with 73% survival rate
(Kone et al., 2009).
Thermopsis turcica Kit Tan, Vural & Kucukoduk is the sole endemic
representative of the genus Thermopsis R. Br. in Turkey. The clonal propagation
of endangered T. turcica using rhizome cutting and epicotyl explants. Rhizome
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cuttings were treated with NAA or IBA before planting for vegetative
multiplication. Rhizome cuttings pretreated with NAA (10 mg/L) were both
rooted and sprouted (66.6%) after 100 days. Application of NAA induced callus
and adventitious root formation in epicotyl explants and BA induced production
of microshoots. Low levels of NAA (0.5-1 µM) together with BA promoted
shoot initiation and development. The highest regeneration rate (86.6%), with a
mean number of shoots (3.05) and a mean length of shoots (2.3 cm) per epicotyl,
was achieved at 10 µM BA and 0.5 µM NAA. About 83% of in vitro regenerated
shoots rooted on ½ MS medium supplemented with 0.3 µM NAA. In vitro
plantlets were morphologically normal and a uniform chromosome complement
of 2n = 18 were detected in root tips (Cenkci et al., 2009).
Mucuna pruriens Bak (Fabaceae) commonly known as Kivach, Alkusi,
Cowhage, Kaunch, Velvet bean is an economically important medicinal plant
found in bushes and hedges and dry deciduous, low forests throughout the plains
of India. It is a wild plant and its every part is full of medicinal value. Its most
important parts are seeds and roots which are good source of giving vital
energies. Seeds are excellent source of L-DOPA (lavodopa 3,4- dihydroxyphenyl
alanine) which is precursor of dopamine a neurotransmitter used in the treatment
of Parkinson’s disease. Callus proliferation was studied on cotyledon, leaf and
stem explant of Mucuna pruriens Bak. cultured on MS medium supplemented
with 2,4-D, IBA, NAA and BAP alone or in combination. Light brown callus
formation was followed by formation of milky white callus on the surface of
young excised shoots and leaf tissues of Mucuna pruriens. Sometimes green
callus was also observed. Development of root and stem with leaves were
investigated from excised stem, leaf and cotyledon tissues (Patel et al., 2007).
Psoralea corylifolia Linn., commonly known as Babchi, is an endangered
and medicinally important plant belonging to the family Fabaceae. The plant is
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well recognized in Chinese and Indian folkloric medicine as a laxative,
aphrodisiac, anthelmintic, diuretic and diaphoretic in febrile conditions. The
seeds have been recommended in the treatment of leucoderma, leprosy, psoriasis
and inflammatory diseases of the skin. Embryogenic callus was induced from
hypocotyl segments on Murashige and Skoog medium supplemented with 2.7–
10.8 mM NAA and 2.2 mM BAP. High-frequency somatic embryogenesis was
achieved after transfer of embryogenic callus clumps to MS medium
supplemented with 1.4 mM NAA and 2.2 mM BAP, alone or in combination
with 0.9–2.8 mM abscisic acid (ABA). The addition of 0.6 or 1.2mM L-
glutamine to the MS medium containing 2.7 mM NAA, 2.2 mM BAP, and 0.9
mM ABA significantly enhanced maturation of somatic embryos to
cotyledonary-stage. Well developed embryos germinated on ½ MS, MS medium
without any growth regulator and also on MS medium supplemented with BAP
(2.2–8.8 mM). Somatic embryo-derived plants were transferred to pots, where
they grew well and attained maturity (Sahrawat and Chand, 2001).
Sophora tomoiro (Phil.) Skottsb. (leguminosae) is an endemic endangered
woody species of Chile is in imminent danger of extinction. Since natural
regeneration by seeds is poor and plant growth is very slow, asexual propagation
is necessary. In vitro regeneration from 3-4 month old aseptic seedlings was
achieved. A range of NAA and BA concentrations induced root formation in
nodal segment explants, developing plantlets and also promoted axillary bud
development. In subculture, nodal sections derived from axillary growth initiated
multiple shoot formation and roots in a liquid medium leading to plantlet
formation (Jordan et al., 2001).
A comparison of resistance to root rot caused by Phytophthora cinnamoni
Rands cultivars propagated from tissue culture and rooted cutting was done. The
method of propagation did not significantly affect root rot ratings. This result is
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encouraging because micropropagation is essential for the rapid multiplication of
new woody plant materials to the nursery industry (Krebs and Wilson, 2002).
Kings Park and botanic garden in south west Australia is responsible for
developing specialized collections of rare and endangered indigenous flora.
Macro and micropropagation procedures are used including conventional cutting
and seed propagation, grafting and in the in vitro programme whole seeds
(asymbiotic and symbiotic germination), excised seed embryos, shoot apices and
inflorescence sections. Wherever possible, explants are collected from major
provenances of the species and a wide cross section of a species population.
Although many of the rare flora of Western Australia are now in the ex situ
collection maintained by Kings park and Botanic garden attempts are being made
to develop slow growth storage for in vitro cultures and cryostorage. Trial
recovery programmes have commenced with a number of species including the
rare and endangered Purdie’s donkey orchid (Diuris purdiei) (Dixon, 1994).
In vitro propagation of Rhododendron ponticum L. subsp. Baeticum, an
endangered species of Portugal was attained. Several cytokinins: IAA ratios and
a range of zeatin concentrations were evaluated for their effect on shoot
multiplication from apical shoots and nodal segments. The type of cytokinin and
the origin of the explant affected shoot multiplication. Increasing zeatin
concentration promoted shoot multiplication independently of explant type. The
best in vitro rooting was observed in Anderson’s modified medium with
macrosalts reduced to one-half. Best results (100% rooting and survival) were
observed for ex vitro rooting. The micropropagated plants from this study were
successfully reintroduced into their natural habitat (87% of survival after 8
months) (Almeida et al., 2005).
In India several institutes are involved in ex situ, in situ conservation
research and development efforts on Himalayan plant species. Their studies vary
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from studies on phenology, seed germination, polysaccharide estimations,
microbiological studies, Genetic variation studies, protected area network
program and the institutes involved are High Altitude Plant Physiology Centre,
HNBGU, Srinagar, Uttarakhand, GBPIHED, Kosi Katarmal, Almora,
Uttarakhand, Tropical Botanical Garden and Research Institute, Palode,
Thiruvananthpuram, GBPIHED, Tadong, Gangtok, Sikkim, IHBT, Palampur,
Himanchal Pradesh and Indian Institute of Remote Sensing, Dehradun to name a
few. However till date there is no report of in vitro micropropagation of
Meizotropis pellita of an endemic species of Uttarakhand hills.
2.7. Antioxidants and Antimicrobials
An antioxidant is a molecule capable of inhibiting the oxidation of other
molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen
from a substance to an oxidizing agent. Oxidation reactions can produce free
radicals. In turn, these radicals can start chain reactions. When the chain reaction
occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate
these chain reactions by removing free radical intermediates, and inhibit other
oxidation reactions. They do this by being oxidized themselves, so antioxidants
are often reducing agents (Sies, 1997). A paradox in metabolism is that while the
vast majority of complex life on Earth requires oxygen for its existence, oxygen
is a highly reactive molecule that damages living organisms by producing
reactive oxygen species (ROS) (Davies, 1995). Consequently, organisms contain
a complex network of antioxidant metabolites and enzymes that work together to
prevent oxidative damage to cellular components such as DNA, proteins and
lipids. In general, antioxidant systems either prevent these reactive species from
being formed, or remove them before they can damage vital components of the
cell (Sies, 1997; Davies, 1995; Vertuani et al., 2004). However, since reactive
oxygen species do have useful functions in cells, such as redox signaling, the
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function of antioxidant systems is not to remove oxidants entirely, but instead to
keep them at an optimum level (Rhee, 2006).
Obtaining adequate nutrients from various foods plays a vital role in
maintaining normal function of the human body. With recent advances in
medical and nutrition sciences, natural products and health-promoting foods have
received extensive attention from both health professionals and the common
population. New concepts have appeared with this trend, such as nutraceuticals,
nutritional therapy, phytonutrients and phytotherapy (Bland, 1996; Berger and
Shenkin, 2006; Bagchi, 2006). These functional or medicinal foods and
phytonutrients or phytomedicines play positive roles in maintaining well being,
enhancing health and modulating immune function to prevent specific diseases.
They also hold great promise in clinical therapy due to their potential to reduce
side effects associated with chemotherapy or radiotherapy and significant
advantages in reducing the health care cost (Ramaa et al., 2006). The history of
plants being used for medicinal purpose is probably as old as the history of
mankind. Extraction and characterization of several active phyto-compounds
from these plants have given birth to many drugs. The potential natural
anticancer drugs like vincristine, vinblastine and taxol can be the best examples
(Huie, 2002). Free radicals are found to be a product of normal metabolism.
Although oxygen is essential for aerobic forms of life, oxygen metabolites are
highly toxic. As a consequence, reactive oxygen species (ROS) are known to be
implicated in many cell disorders and in the development of many diseases
including cardiovascular diseases, atherosclerosis, chronic inflammation etc
(Gutteridge, 1993; Knigh, 1995). Although organisms have endogenous
antioxidant defences produced during normal cell aerobic respiration against
ROS, other antioxidants are taken both from natural and synthetic origin
(Rechner, 2002). Antioxidants that can inhibit or delay the oxidation of an
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oxidizable substrate in a chain reaction, therefore, appear to be very important
(Halliwell, 1992). Synthetic antioxidants are widely used but their use is being
restricted nowadays because of their toxic and carcinogenic effects. Thus,
interest in finding natural antioxidants, without any undesirable effect, has
increased greatly (Rechner, 2002).
In plants, the term antioxidant often refers to a wide range of phenolic
compounds that vary from simple phenolic acids to highly polymerized
compounds such as tannins. Phenolic compounds or polyphenols are categorized
into 15 main classes with over 8,000 identified compounds. The largest category
is the flavonoid group, comprising 13 classes with over 5,000 compounds (Fine
and Candidate, 2000). In plants, polyphenols are important for structural
support, as antiherbivorous substances, for attracting pollinators, for protection
from ultraviolet radiation and for wound repair (Harborne, 1998). The human
body also synthesizes endogenous antioxidants such as superoxide dismutases,
glutathione peroxidases, alpha-tocopherol and melatonin to counteract cellular
damage by active oxygen and free radicals (Manchester et al., 2000; Mojzisova
and Kuchta, 2001; Oktay et al., 2003). Many studies suggest that endogenous
antioxidants, or exogenous antioxidants supplied by diet, can function as free
radical scavengers and improve human health. Consumption of a variety of plant
foods may provide additional health benefits (Connor et al., 2002; Oktay et al.,
2003; Parr and Bolwell, 2000). Antioxidants that retard the oxidation process
may additionally exhibit antimicrobial activity (Cutter, 2000; Hao et al., 1998).
Natural antioxidants can be an alternative to the use of synthetic compounds in
food and pharmaceutical technology or serve as lead compounds for the
development of new drugs. Due to complex composition of different plant
products more than one method is recommended for the antioxidant activity
(Chu et al., 2000). Methods currently used include DPPH (1,1-diphenyl-2-
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picryl-hydrazyl assay (Brand-william et al., 1995) and bleaching of β- carotene
(Marco, 1968).
Psoralea corylifolia is a medicinally important plant, belongs to family
Fabaceae. The seeds are used in indigenous medicine as laxative, aphrodisiac,
anthelmintic, diuretic and diaphoretic in febrile conditions. The antioxidant
properties of aqueous and solvent extract of seeds of P. corylifolia L. were
evaluated in vitro employing different standard assays. All the extracts tested
were effective in quenching superoxide anion. Maximum 1, 1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging activity of 89.0 % was observed at
25µg/ml in alcohol water extract when compared with standard tocopherol and
BHA. The results suggest strong antioxidant potential of alcohol and water (1:1)
extract of seeds of P. corylifolia that could play an important role in the
modulation of oxidative stress (Kiran and Raveesha, 2010).
Abrus pulchellus Wall is a twinning shrub belonging to the family
Fabaceae. DPPH radical scavenging assay and Fe+3
reducing assay were carried
to determine antioxidant activity of methanolic extract of A. pulchellus leaves
extract. The extract exhibited marked antioxidant activity by scavenging DPPH
free radical in a concentration dependent manner. In Fe+3
reducing assay,
increase in the absorbance revealed the reducing power of extracts (Vinayaka et
al., 2009).
The antioxidative potential of essential oil and methanol extracts from
aerial parts of Pimpinella aurea (Apiaceae) were evaluated using inhibition of
free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and β- carotene/linoleic acid
assay. The polar subfraction of the methanol extract was able to reduce the stable
free radical DPPH with IC50 =108 ±0.5 µg/ml, which was higher than that of
synthetic antioxidant butylated hydroxyl toluene (BHT) with IC50=19.8 ± 0.5
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µg/ml. In contrast the nonpolar subfraction showed major effectiveness s in β-
carotene/linoleic acid assay with 65.87% inhibition. The amounts of total
phenolic compounds were also determined (Javad et al., 2009).
The plant Abrus precatorius belongs to family Fabaceae popularly known
as Rati in Hindi, Crab’s eye in English. The in-vitro antioxidant activity of
ethanolic seeds extract of Abrus Precatorius (ASEt) was screened using tests
such as hydroxyl radical-scavenging activity, reducing power activity, and
hydrogen peroxide-scavenging activity. The in-vitro antioxidant assay showed
ASEt posses potent antioxidant activity when compared with reference
compound butylated hydroxytoluene (BHT) (Pal et al., 2009).
The free radical scavenging was used to test antimicrobial activity of
activity of the methanolic extract of Glycyrrhiza glabra (Fabaceae) using DPPH
bioassay. The free radical scavenging activity was found moderate having IC 50
value of 87.152 µg/ml (Sultana et al., 2010). Antioxidant activity of the bark
extracts of Bauhinia purpurea were evaluated in terms of inhibition of free
radicals by 2, 2’-diphenly-1-picrylhydrazyl (DPPH). Aqueous extract followed
by methanolic extract exhibited strong to moderate antioxidant activity (Avinash
et al., 2011).
The antimicrobial compounds found in plants are of interest because
antibiotic resistance is becoming a worldwide public health concern especially in
terms of food-borne illness and nosocomial infections (Anderson et al., 2001;
Hsueh et al., 2005; Lin et al., 2005; Mora et al., 2005; Navon-Venezia et al.,
2005; Vattem et al., 2004). Naturally occurring antimicrobials are being sought
as replacements for synthetic preservatives such as parabens (ethyl, methyl, butyl
and propyl parabens), butylated hydroxytoluene (BHT) and butylated
hydroxyanisole (BHA) that are under scrutiny as suspected cancercausing agents
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(Bergfeld et al., 2005; Byford et al., 2002; Sun et al., 2003; Wangensteen et
al., 2004). Plants produce a multitude of organic compounds that have
antimicrobial activity. The compounds are found in various plant parts such as
stems, roots, leaves, bark, flowers or fruits and seeds and include alliin/allicins,
isothiocyanates, plant pigments, hydrolytic enzymes, proteins, essential oils and
phytoalexins or phenolic compounds (Cutter, 2000; Smid and Gorris, 1999).
Licorice (Glycyrrhiza glabra L., Fabaceae) is a well-known medicinal
herb that grows in various parts of the world. The disc diffusion method was used
to test antimicrobial activity of activity of the methanolic extract of Glycyrrhiza
glabra (Fabaceae) against thirteen bacteria (Bacillus megaterium, Bacillus
subtilis, Staphylococcus aureus, Sarcina lutea, Escherichia coli, Pseudomonas
aeruginosa, Salmonella paratyphi, S. typhi, Shigella boydii, S. dysenteriae,
Vibrio mimicus & V. parahemolyticus). In antimicrobial screening, G. glabra
showed potent antimicrobial activity against almost all the test organisms except
Pseudomonas aeruginosa. It exhibited highest sensitivity against Staphylococcus
aureus with the zone of inhibition 22 mm (Sultana et al., 2010).
Antibacterial activity of ethanolic and aqueous extract of heart wood of
Aacacia catechu wild was screened against selected enteric pathogens
Salmonella typhi, Shigella flexneri, E.coli, Klebsiella pneumonia, Vibrio cholera,
Pseudomonas aeruginosa (Gram negative bacilli) and Staphylococcus aureus,
(Gram positive cocci) using agar well diffusion technique. The results of this
study showed that both the extracts at different concentrations exhibited anti
bacterial activity against the bacterial species tested. The ethanolic extract
showed higher degree of activity than aqueous extract when compared with the
standards. The ethanolic extract was more effective against Shigella flexneri,
Vibrio cholerae and Staphylococcus aureus with a zone of inhibition of 25mm,
25mm and 24 mm diameter (at conc. 200 µg.) respectively and was least
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effective against Pseudomonas aeruginosa with zone of inhibition of 10mm (at
conc. 200 µg.) Among the other bacterial species studied E.coli and Salmonella
typhi showed a zone of inhibition of 19mm diameter (at conc. 200 µg.) and
Klebsiella pneumoniae showed inhibition zone of 16mm diameter (at conc. 200 µg)
(Geetha et al., 2011).
Bark extracts of Bauhinia purpurea were evaluated for antimicrobial and
antioxidant activities. Among different solvent extracts, aqueous extract
exhibited a broad spectrum of antimicrobial activity. It showed strong
antibacterial activity against Gram positive bacterial strains like Bacillus subtilis,
Staphylococcus aureus and Gram negative strains like Escherichia coli and
Klebsiella pneumonia and antifungal activity against Candida albicans. While
methanolic extract showed moderate to strong antibacterial activity against B.
subtilis, E. coli and K. pneumonia, the extracts of hexane, chloroform and ethyl
acetate did not show any anti bacterial or antifungal activity against the tested
fungal and bacterial strains (Avinash et al., 2011).
The antibacterial effect of aqueous and methanolic extracts of 12 selected
Indian medicinal plants viz. Abrus precatorius L. (Fabaceae), Caesalpinia
pulcherrima Swartz (Caesalpiniaceae), Cardiospermum halicacabum L.
(Sapindaceae), Casuarina equisetifolia L. (Casuarinacea), Cynodon dactylon (L.)
Pers. (Poaceae), Delonix regia L.(Fabaceae), Euphorbia hirta L.
(Euphorbiaceae), Euphorbia tirucalli L. (Euphorbiaceae ), Ficus benghalensis L.
(Moraceae), Gmelina asiatica L. (Verbenaceae), Santalum album L.
(Santalaceae), Tecomella undulata (Sm.) Seem. (Bignoniaceae) was evaluated on
several Gram-positive and Gram-negative bacterial strains like Bacillus cereus,
Staphylococcus aureus, Enterobacter aerogenes), Escherichia coli and
Klebsiella pneumoniae using agar disc diffusion and agar well diffusion method.
The most susceptible Gram-positive bacterium was Bacillus cereus, while the
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most susceptible Gram-negative bacterium was Klebsiella pneumoniae. The
extracts of Abrus precatorius, Cardiospermum halicacabum and Gmelina
asiatica could not inhibit any of the bacterial strains. The most active
antibacterial plant was Caesalpinia pulcherrima. The significant antibacterial
activity of active extracts was in comparison with the standard antimicrobics,
piperacillin (100 µg/disc) and gentamicin (10 µg/disc). The results obtained in
the study suggest that Caesalpinia pulcherrima can be used in treating diseases
caused by the test organisms (Parekh and Chanda, 2007).
Methanol extracts of seeds of native and naturalized plants found in the
Mississippi river basin (United States) was tested for antimicrobial activity using
a disk diffusion assay against Staphylococcus aureus, Pseudomonas aeruginosa,
Escherichia coli and Candida albicans. Antimicrobial activities were observed in
the 158 species tested. Extracts of seeds from 35 species had antimicrobial
activity. L. salicaria L., Rumex crispus L., Rumex verticillatus L. and Spirea
tomentosa L. had high levels of antimicrobial activity against all four
microorganisms (Borchardt et al., 2009).
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