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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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Distribution of Mizotropis pellita (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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MMaatteerriiaallss

aanndd

MMeetthhooddss

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