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Department of Biotechnology (Government of India) New Delhi

Popular Lectures on Biotechnology

30.01.2013

Lecture Notes

PG & Research Department of Zoology

Rajah Serfoji Government College (Autonomous), Thanjavur 613 005 Tamilnadu

DBT Popular Lectures on Biotechnology

Zoology Dept, Rajah Serfoji Govt. College, Thanjavur

Pheromones in Rodent and Insect Pest Management

Dr. G. Archunan

(School of Biotechnology - Centre for Pheromone Technology)

Bharathidasan University, Tiruchirappalli - 620 024

Introduction

Pheromones are “molecules that are evolved signals, in defined ratios in the case of multiple

component chemicals, which are emitted by an individual and received by a second individual of

the same species, in which they cause a specific reaction, for example, a stereotyped behavior or a

developmental process” (Wyatt 2010, modified after Karlson and Luscher 1959).

Pheromones, derived from the Greek words meaning “too carry” and “to excite”, are a

special class of non-toxic, semiochemicals that attract other individuals of the same species, or

sometimes a closely related species, but have no effect on non target organisms. Karlson and

Luscher intended that pheromones should include chemical signals in animals of all kinds. These

compounds are extensively used in agriculture and forestry to detect the presence of pests, and to

monitor population increase or decrease over time.

Discovery of Pheromone

Adolph Butenandt, a German Biochemist aimed to discover the substance that female moths

use to attract males. He began by snipping off the abdominal tips of virgin female silkworm moths

and grinding them up. Then, using analytical chemistry techniques, he separated the moth slurry

into various extracts and tested each one on male silkworm moths. The domesticated silkworm

moth has lost its ability to fly. But the male will flutter his wings when excited by a nearby female

and when fooled by one of Butenandt’s extracts.

Working over the course of nearly three decades, Butenandt ground up about half a million

female silk-worm moths in his quest to identify their alluring perfume. At last in 1959, he

announced that the substance was a kind of alcohol (trans-10, cis-12-hexadecadien-1-ol) that

Butenandt christened bombykol, after the moth’s Latin name, Bombyx mori.

That same year, Peter Karlson, German biochemist and Swiss entomologist Martin Lüscher

introduced the term “pheromone” (Greek for “carrier of excitement”). Behavioral assays, such as

the wing-fluttering response used by Butenandt, remained key to identification of pheromones

throughout the 1960s.

Milestones in Pheromone Research

Insect pheromones (Table 1)

1870 French naturalist Jean-Henri Fabre notices a female peacock moth is able to

attract 150 male peacock moths from miles away.

New York entomologist Joseph A. Lintner suggests the chemical scents emitted

by insects could be used to control insect pests.

1957 German biologist Dietrich Schneider develops the electroantennogram (EAG), a

method for using the antenna of a moth to detect pheromones electrically.

1959 German chemist Adolf Butenandt isolates and characterizes the first insect

pheromone, that of the domestic silkworm moth.



German biochemist Peter Karlson and Swiss entomologist Martin Lüscher coin

the term “pheromone” to describe a compound an animal gives off that triggers a

specific behavioral or developmental reaction in a member of the same species.

1960 Pheromone researchers begin to use gas chromatography, mass spectometry,

and nuclear magnetic resonance along with EAG to identify insect pheromones.

U.S. Department of Agriculture chemist Morton Beroza reports his idea of using

sex pheromones to disrupt insect mating.

1961 Colin G. Butler identifies the pheromone of the honey bee, the first pheromone

that regulates the development of an insect.

1966 Chemist Robert Silverstein and entomologist David Wood demon strate that all

three components of the bark beetle’s pheromone blend are required to attract the

beetles—a phenomenon known as synergism.

1967 Entomologist Harry Shorey shows that pheromones can be used to disrupt the

mating of cabbage looper moths in the field.

1970 British biologist John Kennedy develops the wind tunnel assay.

Farmers begin to use pheromones for monitoring insect pests in order to reduce

insecticide use.

1971 Wendell Roelofs uses EAG as an analytical tool to identify the codling moth

pheromone.

1978 First pheromone is registered in the United States for commercial use in

mating disruption against the pink bollworm on cotton.

1980 Pheromones are used in more than a million traps to capture more than four

billion beetles, curbing an epidemic of bark beetles in the forests of Norway and

Sweden.

1990 Pheromones used for mating disruption effectively help curb insect damage in

stone-pitted fruit orchards, and tomato, rice, cotton, and grape fields.

2000 Pheromones used for mating disruption and mass trapping effectively help

Scirpophaga incetuals and Helicoverpa armigera insects damage in Sugarcane.

Spic Science Foundation (Chennai), NCL Pune and DRR, Hyderabad

Rodent pheromones (Table 2)

1956 Primer pheromone involvement was noted in estrous cycle disruption in group-

housed mice by the Netherland scientists Van den Hurk and Boot.

1959 Primer pheromone involvement was identified in estrus induction and pregnancy

block in mice by Wesley Whitten and Hilda Bruce respectively.

1967 Primer pheromone involvement was identified in acceleration of puberty in

female mice by American scientist John Vandenberg.

1978 Dimehtyl disulphide a male attractant of hamster vaginal secretion was

characterized

1984-

1986

Female attractants (2-sec-butyl-4,5-hydrothiazole & exo-brevicomin); oestrous

induction; intermale aggression compounds were identified in male mouse urine

1989&

1999

Pheromonal compounds for puberty acceleration were identified in male mouse

urine

1999 Dodecyl proprionate, a Pheromonal compound for anogenital licking was

identified from rat pup preputial gland

Identification of volatile compounds from cheek glands of lesser bandicoot rats



and assessment of their behavioural response

2001 Attractant compounds from rat clitoral and preputial gland were identified and

confirmed by behaviour assay

2002 Male and female urinary compounds in Indian wild type common house rat

(Rattus rattus) was identified by Archunan and his group.

2005 Pheromone compound were identified in Tatera indica, Indian gerbil

2006 Female sex pheromone (1-iodo-2 methyl undecane) was identified in swiss mouse

2010 Farnesol, attractant compound form the preputial gland of Indian wild type

common house rat (Rattus rattus) was characterized

Classification of Pheromones

Pheromones may elicit a behavioral response, longer-lasting developmental effects mediated

via hormones, or both. Pheromones are often described by function, by the effect they have. Based

on the types of response by recipients, the pheromones are classified as ‘‘primer’’, ‘‘releaser’’ and

‘‘imprinting’’. Primer pheromones induce a delayed response to prolonged stimulation mediated

through central nervous system (CNS) and endocrine system. A number of primer pheromonal

effects have been studied leading to the establishment of concrete ideas regarding their influences

on reproductive functions. Releaser pheromones induce a rapid behavioural response in the

recipients, generally mediated through the CNS. Sexual attraction, evocation of aggression,

recognition, alarming behaviour and mother-young interactions are examples of releaser

pheromones. Imprinting pheromones organizes the CNS of the pre-weaning off-springs at a critical

period and cause permanent alterations of adult behaviour.

Insect Pests

Insects, the most diverse groups of animals on the planet, including more than a million

described species and representing more than half of all known living organisms. Many insects are

considered pests by humans. Any animal which becomes a source of trouble or loss to human is

called a pest. Among insects such pests are numerous and are of different kinds. An insect is usually

called as a pest when it causes appreciable damage and loss to the crops or other belongings. Insects

commonly regarded as pests include those that are parasitic (mosquitoes, lice, bed bugs), transmit

diseases (mosquitoes, flies, cockroaches), damage structures (termites), or destroy agricultural

goods (locusts, weevils).

The pests may be classified as major or occassional. The insects damaging standing crops

cereals, fruits and other plant products of commercial importance are designated as crop pests.

Common pests of crops includes Pink boll worm, Spotted bollworm, Rice bug, Red cotton bug, The

red pumpkin beetle, Brinjal shoot and fruit borer, Sugar cane top shoot borer, Rhinoceros beetle.

Those insects destroying stored grains are called the store pest (e.g. Rice weevil, Khapra beetle,

Pulse beetle etc.,). Insects causing damage to household articles are called the household pests (e.g.

Silver fish, Termite etc).

Insect Pest Management

Identification

The first step in any integrated pest management program is to correctly identify the pest

and to determine the size/location of the infestation.

Understand Pest Biology



Growers should become familiar with the biology and life-cycle of the major insect pests

that attack their crops. Understanding some basic insect biology often reveals when the pest is most

vulnerable to control measures and helps lead to successful management.

Monitoring for Pests and Beneficials

Among the applications of pheromones, monitoring a population of insects to determine if

they are present or absent in an area or to determine if enough insects are present to warrant a costly

treatment is the most important one. This monitoring function is the keystone of integrated pest

management.

Conventional methods in Insect pest management

Cultural controls involve the adjustment of standard farm practices to avoid pests or to make

the environment less favorable for them. There are several types of cultural controls; Crop rotations,

sanitation, Companion planting, Trap cropping are a few examples of commonly used methods.

Soil tillage a method of mechanical control is used in which it can destroy insects and expose them

to their predators i.e., birds. In chemical control method a wide range of insecticides were used.

Insecticides do not work as well as often believed, and a long-term population decrease has never

been achieved, not in any species. Resistance against the available insecticides continues to be an

important issue. Insecticide sprays harm the terrestri and aquatic fauna.

Biological control is the use of natural enemies or beneficial insects, to reduce or delay pest

outbreaks. The goal of ecological pest management is to increase biological control and hold a

target pest below economically damaging levels – not to eliminate it completely – since decimating

the population also removes a critical food resource for the natural enemies that depend on it.

Increasing plant and animal diversity supports the abundance and effectiveness of natural enemies.

Insect pheromones

Over the last few decades of research, scientists have identified pheromones from over 1,600

different species of insects, that are produced by special glands in the abdomen of insects and it

attracts the opposite gender of the same species. In recent years, pheromone research directed

toward the elucidation of the chemical identity and behavioral role of pheromones usually have

been concerned with the economically injurious species in the expectation that the synthetic

pheromone could be applied in Insect Pest Management.

The regulation of the biology of the social insects was constituted by these compounds,

the pheromones, and the elucidation of their chemistry has also provided behaviorists to study

the variety of social behaviors. Insects produce pheromones for attracting the mate (e.g., most

moths), for marking foraging routes (e.g., ants) or to signal alarm to neighbors (e.g., aphids).

Pheromones have also been isolated from many higher animals. With insects, though, pheromones

have found wide application in the fields of agriculture, forestry, and urban pest management, and

there are companies that specialize in the discovery, manufacturing, and sales of pheromone-related

products.

Identified and synthesized insect sex attractant

Chemical attractants have been synthesized for some of the most important insect pests,

including cabbage looper, boll weevil, gypsy moth, pink bollworm, tobacco budworm, European

corn borer, codling moth, several species of bark beetles attacking forest trees, Douglas-fir tussock

moth, and house fly, ((2)-9-tricosene, Carlson et al., 1971), Mediterranean fruit fly (Q6-nonenoate

and (E)-6-nonen-l-o1). Encouraging results have been reported by the use of Pheromone technique



for a number of species including the gypsy moth, the boll weevil, leafroller, western pine beetle,

elm bark beetle.

Pheromone trap for insect pest management

Detection and survey of insect pests has been considered as the significant and the most

obvious use of pheromones. In relation to this principle, the utilization of a synthetic pheromone-

baited trap for monitoring the presence and abundance of an economically important species does

not differ from the early naturalists' use of female-baited traps for collecting. Pheromone traps are

very effective monitoring devices and are relatively cheap to purchase. In case of pheromone traps,

the lure slowly releases synthetic attractants that helps in detection of a single species of insect.

Product assembly is very easy. Remember that the accuracy of traps depends on their number and

correct deployment in field. There are different types of pheromones viz. Sex attractant

pheromones, Aggression pheromones, Trail pheromones, Alarm pheromones have been identified

in the individual insect species and utilized the pheromone compounds for their respective trap

development method (see in detail Table 3)

Table 3: Insect pheromone compounds for their respective trap development methods

Name of the

insect

Host plant Pheromonal

compound

Type of the trap

Stink bug,

Euschistus

conspersus

Cotton,soyabean

etc.

Methyl(2E,4Z)deca

dienonate

Aggregation pheromone

Aggregation

pheromone

Currents,

goosetarries

{Z}-9-octadecen-4-

olide(9)

Pheromone-baited sticky traps.

(sex pheromone) attracticide or

matting disruption

Plum curculio,

Contrachelus

nenuphar

Stone, pome

fruit

Grandisoic acid Aggregation pheromone

Cone gall midge,

Contarinia

aregonensis

--------------------

brinjal shoot and

shoot borer,

Leucinodes

orbonalis

Douglas-fir

--------------------

Brinjal

(Z,Z)-4,7 –

tridecadier-(s)-2-yl

acetate (female)

-----------------------

(E) –11-

hexadecenyl

acetate (male)

Sex attractants

-----------------

sex attractants (man trapping)

Disruption of mating

Mating disruption has been effectively used with agriculturally important moth pests, flies

and beetles that had become immune to broad-spectrum insecticides. In this scenario, relatively

large quantity of synthetic pheromones (combination of several pheromone components) are more

or less evenly distributed throughout the field to adapt sensory receptors or habituate behavioral

response (confusion) or to exhaust the individuals in orientation attempts (wild goose chases) and

the false odor plumes attract males away from females that are waiting to mate. Various methods of

disseminating the pheromones or mating inhibitors into the air may be involved.



(Jay F. Brunner and Alan Knight, 1993)

The pheromone may be released by natural volatilization or released as an aerosol by

spraying devices. The pheromone may be encapsulated and the amount that permeates the air will

be governed by the rate of release from the capsules. This causes a reduction of mating, and thus

reduces the population density of the pests. The reduction rate of mating is observed in pinworm

population in Mexico when the pinworm pheromone is dispersed. Only about 3 - 4 percent

pinworm are able to mate under pheromone treated condition with the reduced level of loss to the

crops than the use of conventional methods (i.e., Insecticides) from 80% to 30%.

Pheromones as Insect detectors

Pheromones play a prominent role in insect detection systems. Pheromones are already in

use by Federal and State regulatory agencies for surveys and detection of the gypsy moth, pink

bollworm, boll weevil, and other pests. Investigations are underway on pheromones of various other

species in efforts to improve pest management systems by determining where and when control

measures need to be applied. The availability of these new and powerful attractants for early

detection of insects in new areas of spread will in time add a new dimension to insect eradication

and containment programs (Knipling, 1976).

Rodent Pest

Rodents (animals that have continually growing incisor teeth and no canine teeth) are a

dominant group of mammals. There are more than 2700 species of rodents worldwide; in fact, 42% of all the mammal species on Earth are rodents. Two-thirds of living rodent species belong to just

one family, the Muridae, and most of the rodents found in Asia, both pests and non-pests, also

belong to this family.

Among vertebrate pests, rodents are the most destructive to the agriculture in India. The

rodent fauna of the Indian sub-continent is represented by 46 genera and 128 species. Of these 18

species are commensal and agricultural pests. Their habitat, distribution, abundance and economic



significance varies in different crops, seasons and geographical regions of the country. (Parshad

V.R, 1999) Some species are widely distributed while others are locally important. Rats and mice

have adapted well to the diversity of agricultural habitats created by humans.

Rodents have three major impacts. The first is the substantial damage they can cause at any

stage of the growing crop. The second is the losses they cause post-harvest to stored grain and

vegetables. The third, and often overlooked, impact is on the health of smallholder farmers - rodents

are carriers of at least 20 severely debilitating human diseases (Meerburg et al. 2009).

Losses due to rodent pests

In agriculture, rodents cause direct damage to various commodities by gnawing and feeding

and indirect damage by spoilage, contamination, deterioration and enhancing susceptibility to

fungal and bacterial infestations during pre- and post-harvest stages. Almost all field crops are

affected by rodents. Rodents are the major production constraint of rice. Irrespective of the type of

rice production system, rodents cause considerable damage to rice crops in India and other South

Asian countries. The commonest species infesting rice fields are Bandicota bengalensis, Rattus

meltada and Mus booduga.

Several species are involved in damage to wheat which includes, B. bengalensis, R. meltada,

T. indica and Mus spp. Damage occurs throughout the crop growth period but is greater at the

ripening stages. The pattern of damage to the cereal crops is different to that in wheat and rice. The

rodents attack the seeds after sowing and the seedlings more than the subsequent growth stages. R.

gleadowi, T. indica, R. meltada, R. nitidus and Mus spp. are the most common species infesting in

cereal crop such as Maize, pearl millet and sorghum. Rodents inflict direct and secondary damage to

sugarcane. Direct damage is caused mainly by B. bengalensis, and N. indica. Rodents prefer to

colonize cane fields because they provide an undisturbed habitat for their burrowing, feeding and

breeding activities, a protective cover from avian predators and an abundant amount of high energy

food for most of the year.

Among the oil seed crops, groundnut is severely attacked by rodents. M. musculus and M.

booduga are abundant in groundnut fields and major damage is caused by T. indica, R. meltada and

B. bengalensis. Cotton bolls provide rodents with seeds for feeding and fibre for making nests. R.

meltada, T. indica and B. bengalensis are the major rodent pests of cotton. Rodents attack almost all

vegetable such as Tomato, Chilli, Bottlegourd, Cucumber, Cabbage, Pea crops mostly at the

seedling and mature stages.

Apart from agricultural losses, rodents cause severe economic losses to Animal houses

particularly in poultry production they cause both by direct damage and also indirectly by spreading

several diseases among the birds and to poultry keepers themselves. Poultry farms, provide a most

favourable and stable habitat throughout the year for large rodent populations which by their

burrowing, nibbling, feeding, defecation, urination and extensive movements damage the poultry

farm environment thereby leads to dramatic economic loss.

While considering the post harvest damages food stores, godowns, grocery shops grain

markets, food processing units such as bakeries and floor mills are infested by the house rat R. rattus

and the house mouse M. musculus throughout the country in both rural and urban situations and by

the lesser bandicoot rat B. bengalensis in major metropolitan cities such as Bombay, Calcutta, Delhi

and Madras

Rodent pest management

Due to variations in geographical and climatic factors; systems of crop production and post-

harvest storage; carrying capacity of the environment; biology of the pest rodent species; the nature



and extent of rodent problems and the perceptions and socioeconomic conditions of the people, no

single strategy or method of control is feasible or applicable in all different pest situations.

Knowledge of the characteristics, extent of damage and the situations vulnerable to attack by

rodents in different crops and regions is important in planning management strategies. Developing

an effective integrated management plan requires a good understanding of the basic ecology of

individual rodent pest species. This in turn is dependent on access to field methodologies that

enable us to understand the population dynamics and field ecology of rodents. In order to establish

an effective ecologically-based pest management (EPM) with integrated pest management (IPM), it

is mandatory to know the ecology, diversity and population status of rodent pests.

Rodent control methods

There are several traditional techniques used by farmers for controlling rodents, which can

be grouped into two basic approaches: i. lethal or reductional ii. Non-lethal or preventive. The

lethal approach, particularly the use of rodenticides and trapping, which provides an immediate

solution to the problem, is often considered the most effective method of controlling rodents; while

non-lethal or preventive measures involving environmental, cultural and biological methods, which

may produce a more lasting effect, are seldom adopted. Both the methods are having their own

advantages and disadvantages. Though the lethal approach considered as the effective and

economical method the hazardous nature of its component to environment is to be realized.

Environmental methods

The maintenance of clean, hygienic environment leads to the reduction of rodent

harbourage. Garbage, junk and other hiding and nesting materials provide harbourage to rodents in

animal and human dwellings and in stores and godowns. The periodic removal of rubbish and good

hygiene discourage rodents in that premises. Since weeds provide an important component of

rodent diet, the practice of weed control with chemicals and other techniques, which also reduced

rodent pest problems in crop fields.

Banding of rodent infesting trees (e.g. coconut trees) with metallic sheet, use of lethal and

sub-lethal electric barriers or fences around the fields, placing of screw-pine leaves along the edges

of paddy fields, flagging of palm leaves or polythene pieces in rice fields, or plant material which

makes rattling sound, electronic or chemical repellents are few methods used to prevent the entry of

rodents, denying them access to food in agricultural fields and during post-harvest storage.

Cultural methods

Rodent pests and their damage to crops can also be reduced conventionally by some cultural

practices. Deep tillage destroys rodent burrows and drives away rodents. Rodents cause more

damage in fields where shelter and food has been available to them earlier and they can then shift to

fields where harvesting is late or delayed. Severe damage due to such practices can be prevented by

adopting synchronous sowing or transplantation and harvesting schedules for the same variety over

a large field area.

Biological methods

The most obvious method for rodent control is the biological methods in which predators,

parasites, pathogens and reproductive inhibitors are used against rodent pest. Cats, mongeese,

jackals, foxes, owls, hawks, kites, monitor lizard and snakes are the major predators of rodents.

Encouragement of these predators may provide significant role in rodent control. Micro and

macroparasites are considered as bio-control agents of rodents; due to its adverse potential health

risk to livestock and humans, has been ignored. Since the higher rate of reproduction among rodents



is the cause for abundance a number of chemicals such as clomiphene, tetradifon, furadantin and

colchicine, glyzophrol, ethyl methanesulphonate, alpha-chlorohydrin has been applied for its

reproduction inhibition property. Though the natural control of rodents is an important method due

to over exploitation of land and forest resources and developments in transport, urbanization has

been disrupted.

Mechanical methods

Mechanical methods such as hunting, killing and trapping are in general methods. These

practices are significant only over small areas due to high labor cost. Two basic types of traps are

being used, the snap or kill trap and the live trap. Among the snap traps the Tanjore Bow trap, arrow

traps, break-back spring loaded snap traps have been used traditionally. Among these the break-

back snap trap is most popular. Live traps include the primitive type pit fall or pot trap, foldable

iron sheet boxes with a spring loaded shutter commonly called as Sherman traps, small sized

wooden boxes, also called single-rat traps and multi-catch wonder traps of different sizes and

shapes.

Chemical methods

Zinc phosphide, aluminium phosphide, barium carbonate, arsenic trioxide, strychnine

alkaloid, thalliumsulphate, alphanapthyl thiourea, norbormide, scillirocide, sodium fluroacetate,

vacor and a gophacide are used as acute rodenticide against Indian rodent pests. bromethalin,

flupropadine, calciferol (ergocalciferol, vitamin D2) and cholecalciferol (vitamin D3) are the

subacute rodenticide where death is delayed for several days after ingestion of a lethal amount.

The anticoagulant rodenticides are either hydroxycoumarins or their related indane-dione

compounds (e.g. warfarin, fumarin, coumatetralyl, diphacinone and chlorophacinone) also used

against most of the species of Indian rodents. Since 1988 for the control of agricultural and

commensal rodents, the second generation anticoagulant namely difenacoum, brodifacoum,

bromadiolone, flocoumafen and difethiolone, has been used for rodent control.

Bait shyness and role of pheromones

Rats are instinctively cautious. It will take them a night or two before they’ll sample a new

bait placed in their environment or sniff around a trap. They may eat very small amounts at first,

like a taste-tester, and any subsequent feedings depend on the food and its physiological effect. If a

poison sickens but does not kill them, they will associate food with the illness and avoid it. This is

why sub-lethal dosages of single-dose acute toxicants are a problem: They result in “bait shyness.”

Bait shyness can persist for weeks or months and may be transferred to nontoxic foods of similar

types. Today’s anticoagulant rodenticides are slow acting so there are no symptoms of poisoning

even if sub-lethal does is consumed.

The studies of Selvaraj and Archunan (2002) revealed that the preputial and cheek glands

extracts are effective in improving the acceptance of poisoned bait by the laboratory rat (Rattus

norvegicus). In same way the male preputial gland extract appears to be more effective than cheek

gland extract in masking of the bait shyness behaviour of female rats (Selvaraj and Archunan ,

2006).

Allelochemical odour for rodent pests Apart from species-species odour i.e. Pheromone to develop pheromone trap for rodents,

inter-species communication, i.e. Kairomone, substances need to be identified fro rodent

management purpose. For instance, rat/mouse is afraid of cat odour, at the same time cat/snake is



attracted by rat odour. Identification of the specific odorants of predator and make use of them as

aversive agent is another alternative method for rodent pest management program.

Rodent Pheromones: future prospect to develop biotrap

Archunan and Achiraman (2006) discussed in detail about rodent pheromones and reviews

its possibility for application in rodent pest management programme. In mammals, urine and faeces

are the most primitive and common source of pheromones. It was identified that there are about 40

different specialized scent glands in mammals. Many different chemicals have been reported as

pheromones that include alcohols, aldehydes, acids, saturated or unsaturated aliphatic or aromatic

compounds from non-polar molecules such as alkenes to very polar compounds which may be

acidic or basic. Most studies on pheromones in rodents have been done with house mice. Novotny and co-

workers are the pioneers in the field of rodent pheromone identification and functioning. They have

demonstrated that 2-sec-butyldihydrothiazole and 2,3-dehydro-exo-brevicomin from male mouse

urine potentiate inter-male aggression, attracts females and induce oestrus. Interestingly, the same

urinary compounds are also present in Indian commensal male house mouse and further work is

under process in our research group. Additionally, they have shown that the sequiterpenes E,E,-

alpha-faranesene and E-beta-farnesene from male mouse preputial gland exert the same effect.

Futhermore, n-pantyl actate, and cis-2-penten-1-ylacetate,2-heptanone,trans-4-hepten 2-one,trans-5-

hepten-2-one,2,5-dimethyl pyrazine, present in the urine of adult females, have been found to delay

puberty in juvenile females, while 6-hydroxy-6-methyl-3-hepanone from male mouse urine

accelerates it (Archunan, 2009 for review). The volatiles identified in albino mice need to be

extended in wild type mice or rat and use of them for rodent pest programme.

The GC-MS analysis of urine carried out by Selvaraj and Archunan (2002) revealed that

male house rat urine contained ethanol, 2-(octylthio), 1,3,5 triazone-2,4-diamine, and 1-

chlorodecane. Similarly, female urine (during estrus) had the 3 compounds hydroperoxide, 1-

nitropentane, and 4-azidoheptane. Furthermore, the bioactivity of these identified compounds was

assayed and odor preference test revealed that the identified compounds show opposite-sex as well

as same-sex attraction. The ligand carrier for pheromone communication, a 18 KDa protein was

identied in rat (Rattus norvegicus) preputial gland (Ponmanickam and Archunan, 2006), rat pup,

which involves in mother young interaction (Ponmanickam and Archunan, 2009), Indian wild type

common house rat, (Rattus rattus) (Rajkumar et al., 2009). The farnesol, a volatile compound,

characterized in the house rat (Rattus rattus) preputial gland (Rajkumar et al., 2011) is showing a

promising candidate to develop 'Biotrap' for rat pest.

The volatile compounds have been identified and confirmed their functional aspect by

bioassay in several wild type rodents including Bandicoot bengalensis (Kannan and Archunan,

2007), Tatera indica (Palanivel, 2005), Rattus rattus (Achiraman, 2008 - M.phil thesis) and so on.

Advantages of pheromones in rodent pest mananagement

1. Minute quantities of pheromones are enough to attract and kill large number of rodents and

hence they are economical.

2. They are non-pollutant and ecologically acceptable

3. It is labour saving

4. It is species specific and non-targets

5. Easy handling

Disadvantages of pheromones in rodent pest management



1. Rodent pheromones have to be identified in many wild species

2. Sex pheromone can attract only one sex

3. Farmers should have the knowledge of these pheromones

4. Quick results cannot be obtained

Pheromone-binding protein and its impact on pheromone trap

The pheromone-binding protein, a lipocalin superfamily is identified from several sources of

mammalian species including rat (α2U-globulin) and mouse (MUP- Major Urinary Protein). The

α2U globulin is very well characterized in house rat in both urine and preputial gland sources and it

is important to note that the volatile pheromone are identified as bound form with α2U-globulin.

The characteristics features of α2U-globulin is to release the volatile (pheromone compounds)

slowly in the environment. It provides circumstantial evidence that the volatile compound

responsible for attractant in house rat has been confirmed as promising candidate to develop a bio-

trap for rodent pest management programme.

Present status of Pheromone research in India

Rajagopal et al., (2012) aimed to map pheromone biology research in India over a period of

31 years (1978–2008) as reflected through web of science. As per their report the first paper in the

area of pheromones was published in 1978 and it rose to 27 research articles, the highest during a

year 2006. 330 papers have been published by Indian pheromone researchers in journals having

impact factors between 0.20 and 4.14. In recent years, there are over 200 Institutions are being

actively engaged in pheromone research. Their study also concludes that the output of pheromone

biology research in India has gradually increased over the years.

Conclusion

Health hazards, environmental issues, resistance, effect on non-target organisms, marketing

opportunity for organically-produced food are well-known arguments against the use of chemicals

and other conventional methods over the control of insects and rodents. Very few environmentally

safe control methods against insect species and that biological control methods are often quite

expensive or less reliable than pheromones. The potentials of pheromone-based methods, in the

control of insect and rodent, are to be recognized and utilized where the knowledge on

identification, isolation and characterization of pheromones, chemistry of pheromone, biology and

population dynamics of the pest species, synthesis of pheromone, appropriate techniques to use in

the fields to be revealed. Furthermore, the extension of the use of pheromones to other species for

other purposes i.e., stimulation of spawning and early puberty, in stimulation and synchronization of

oestrus in non-breeding season, in the identification of oestrus/heat period, in improving the

maternal bonding, in enhancing the wild animals conservation, in regulating the human fertility also

possible by the use of the already available database on identification and bioassay of insect and

rodent pheromones.

References

Achiramam S & Archunan G (2005) 3–Ethyl-2,7- Dimethyl octane, a testosterone dependent unique

urinary sex pheromone in male mouse (Mus musculus): Anim. Reprod. Sci., 87: 151-161



Achiraman S and Archunan G (2002) Characterization of urinary volatiles in Swiss male mice

(Mus musculus): bioassay of identified compounds; J. Biosci. 27 679–686

Achiraman S. Archunan G. Ponmanickam P. Rameshkumar K. Kannan S and George J (2010) 1-

Iodo-2 methylundecane [1I2MU]: An estrogen-dependent urinary sex pheromone of female

mice. Theriogenology, 74: 345-354.

Archunan G and Achiraman S (2006) Pheromones in rodent pest management, Vertebrate pests in

agriculture – Indian scenario (ed) S. Sridhara. Scientific publishers, Jodhpur 365-386 pp

Archunan G (2009) Vertebrate pheromones and their biological importance. J. Exp. Zool. India,

12: 227-239.

Carlson DA. Mayer MS. Silhacek DL. James JD. Morton Beroza and Bierl BA (1971) Sex

Attractant Pheromone of the House Fly: Isolation, Identification and Synthesis Science,

New Series. 174: 76-78.

Karlson P and Luscher M (1959) ‘Pheromones’: a new term for a class of biologically active

substances. Nature 183:55–56

Knipling EF (1976) Environmental Health Perspectives 14: 145-152

Meerburg GM. Singleton GR and Kijlstra A (2009) Rodent-borne diseases and their risks for

public health. Crit. Rev. Microbiol. 35: 221–270.

Parshad VR (1999) Rodent control in India. Integrated Pest Management Reviews 4: 97–126, 1999.

Peter Witzgall (2001) Pheromones - future techniques for insect control? IOBC wprs Bulletin . 24:

114-122

Ponmanickam P and Archunan G (2006) Identification of α-2u globulin in the rat preputial gland

by MALDI-TOF analysis. Indian J. Biochem Biophys., 43: 319- 322.

Ponmanickam P. Jebamercy T. Archunan G. and kannan S (2009) Detection of alpha-2u globulin

in the rat pup preputial gland. Curr. Zool., 55: 296 – 300.

Rajkumar R. Ilayaraja R. Mucignat C. Cavaggioni A and Archunan G (2009) Identification of α2u-

globulin and bound volatiles in the Indian common house rat (Rattus rattus): Indian J

Biochem Biophys., 46: 319-324.

Rajagopal T. Archunan G. Surulinathi M. Ponmanickam P (2012) Research output in pheromone

biology: a case study of India. Scientometrics 012: 0788-4.

Selvaraj R and Archunan G (2002) Role of male scent glands in improving poison bait acceptance

in female rats, Rattus norvegicus . Int. J Exp. Biol. 40: 53-57.

Selvaraj R and Archunan G (2006) Efficacy of male scent glands and urine in masking poison bait

odour in female house rats, Rattus rattus. J Pest Sci. 79:255-258.

Wyatt TD (2010) Pheromones and signature mixtures: defining species-wide signals and variable

cues for individuality in both invertebrates and vertebrates. J. Comp. Physiol. A 196:685–

700



Plant Cell, Tissue and Organ Culture: Advances in Biotechnological Application

Dr. A. Muthusamy

Division of Biotechnology, Manipal Life Sciences Centre

Manipal University, Planetarium Complex, Manipal – 576 104. Karnataka

Introduction:

Plants are the most attractive and imperative groups of autotrophic organisms which produce the

oxygen and sustains all life forms on the earth. The plant systems act as the channel of energy into

the biosphere, convert the sun light energy into carbohydrate and provide food for all organisms

(prokaryotes and eukaryotes) either directly or indirectly, and shape our environment. Human

populations has grown nearly ten-fold over the past 3 centuries, increasing further and therefore,

demand for food, feed and fodder is ever increasing. Current World population is 6.8 billion and is

expected to reach 9 billion in 2045 (Lidder & Sonnino 2012). In recent years, land area of

cultivation and rate of growth in agricultural productivity has declined while the demand for food

continues to escalate. Broad range of agricultural genetic diversity needs to be available and utilized

in order to feed the growing population. Besides the prime energy, the carbohydrate, plant

communities are also act as the sources of the number of drugs (plant secondary metabolites), and

great parts of the pharmaceuticals available in modern medicine are directly or indirectly derived

from plant sources. Furthermore, a growing world-wide interest in the use of phytopharmaceuticals

as complementary or alternative medicine, either to prevent or to ameliorate many diseases, has

been noted in recent years. Therapeutic properties of medicinal plants have been significantly

redounded to prevent and cure various human ailments. Among the 7000 species of plants used by

humans since from the beginning of agriculture, only 150 crop species of plant system being

cultivated for the majority of its calories. Most important of the stable foods are the cereals,

particularly wheat and rice, with more than one-third of all cultivated land used to produce these

two crops.

There will be a three major challenges before ever increasing human population in the 21st

Century are food, energy and the environment, including drastic climate change and environmental

degradation due to pollution and severe losses in different habitats collectively known as

sustainability challenges. Plant life, the growth and development plays an indispensable role in all

three of these challenges. Plant communities are key performers in defining our climate, and

agricultural development is a major factor in habitat intrusion and pollution of waterways by

indiscriminate application of different fertilizer and runoff. In addition, these challenges are

complex and dependent with each other; as the climate changes, additional challenges are

positioned on plant growth and development which leads to drastic reduction in food supply and

severe losses in habitat. Hence, the research program on plants in multiple directions is needed to

arrange and deliver keys to these major challenges. Plant tissue culture and biotechnology has

developed as a stimulating area of plant sciences as it can generate unique and multiple

opportunities for the manipulation of plants to boost the productivity of all groups of plant systems.

Concept of Plant Tissue Culture:

Plant tissue culture is a state of the art techniques and practice used for in vitro propagation and

large scale production of plantlets under sterile conditions. Growth of the science of tissue culture

was historically linked to the discovery of cell and cell theory. More than 240 years ago, Henri-



Louis Duhamel du Monceau’s (1756) have observed the spontaneous callus formation on the

decorticated region of elm plants during the experiments on wound healing in plants. His studies,

according to noted biologist Gautheret (1985), could be considered a ‘foreword’ for the discovery of

plant tissue culture. The development of the multicellular eukaryotic higher organisms from a

single-celled zygote chains the totipotent behavior of a cell. The concept of cell culture was

proposed by Gottlieb Haberlandt (1902), a German Botanist who first tried to initiate the plant cell

culture and recognized as the founder of plant cell culture concept evolve into a powerful tool

utilized throughout the plant sciences. Plant tissue culture broadly refers to growing plant cells,

tissues, organs, seeds, or other plant parts in a sterile environment on a nutrient medium. Tissue

culture is being used for an increasing variety of purposes. Originally used largely for fundamental

research to study cell division, plant growth, and biochemistry, the technology has grown and is

being widely implemented on a more applied scale. In many cases, protocols have been developed

and refined so that they have become a standard and commercially viable practice for propagating

many important horticultural crops. The key to the successful application of tissue culture is the

manipulation of media compositions to achieve desired outcomes. By altering media components,

tissue can be induced to produce shoots, roots, callus, or somatic embryos or inhibit growth for

long-term storage. The most common application of tissue culture is micropropagation, which

usually involves growing plants in an agar solidified nutrient media. Micropropagation can facilitate

the rapid production and propagation of plant species.

Totipotency is one of the unique and specific properties of plant cells and tissues, which

confirms the opportunity of existence of the plants under different conditions of attached mode of

life and plants are maintaining some special cell files are known as meristematic cells at four

different places (shoot and root tip, nodal and intermodal regions). In nature, the unique potential of

totipotency is the capacity of plants for various pathways of vegetative reproduction and in the

possibility of fast reinstatement of stress damaged parts of shoots and roots, and this unique

capacity of plant cells and tissues confirmed a wide range of use of plants cells, tissues and organs

in multi directional application in biotechnology. Under in vitro conditions, the matured living cells

first undergo dedifferentiation (cancellation of existing metabolism and decondensation of

chromosome) to become normal cell and they will divide continuously to form callus which leads to

organogenesis or somatic embryogenesis (redifferentiation) under the influence of components of

nutrient medium, vitamins, concentration and combination of plant growth regulators (Figure 1).

Figure 1. Pant protoplast system used to study cellular dedifferentiation (Zhao et al., 2001).



The in vitro culture can be initiated using any one of the explants from leaf, stem, root,

embryo, ovary and anther part of herb, shrub and tree plants except treachery elements and sieve

elements since these two cells permanently lose their developmental potentialities and the

differentiation is terminal. The dedifferentiation, division and re-differentiations of mature plant

cells pave the possibilities for multidirectional application in biotechnology with prime aim of large

scale production of rare, endangered and threatened plant species via organogenesis and somatic

embryogenesis, development of disease resistant and stress tolerant crop plants and production of

commercially valuable plant secondary metabolites for human health (Figure 2).

Figure 2. Major areas of plant cell and tissue cultures, and some fields of application (Neumann et

al., 2009).

Biotechnological application of plant cell, tissue and organ culture:

I. Germplasm collection and conservation:

Biotechnology is a pool of technological innovation in the world of agriculture; however it

has variant levels of scope and contents in different group of plant systems. During last century, the

plant cell, tissue and organ culture (PCTOC) has developed as a potential technique to produce

genetically pure line of important crop plant species using in vitro technique rather than existing

indifferent plant populations. Hence, the in vitro cell and tissue culture techniques is envisioned to

target germplasm collection and conservation of rare, endangered and threatened plant species for

rapid mass micropropagation in large scale in order to 1) introduce in their natural ecosystem which

was degraded by man-made activities or natural disasters, and 2) large scale in vitro production and

conservation of important medicinal plantlets which are over exploited since they have higher

pharmaceutical industrial value. Germplasm conservation refers to protection of genetic diversity of

crop plants from genetic erosion. There are two important methods of germplasm conservation or

preservation. i) In-situ conservation and ex situ conservation (Table 1) and the following flow-chart

depicts the role of plant cell, tissue and organ culture for large scale production of plantlets from

stored germplasm..



Table 1. Types of conservation.

In situ conservation Ex situ conservation

Natural park,

Botanical garden

Biosphere reserve,

Gene sanctuary

Seed banks,

Plant Bank

Shoot tip bank, Cryopreservation

Cell and organ bank, DNA bank

II. Agriculture and Horticulture:

Biotic, abiotic factors and climate change which threats to biodiversity and significantly

impact genetic resources of food and agriculture (GRFA) and food production. No simple, all-

encompassing solution to the challenges of increasing productivity while conserving genetic

diversity. Sustainable management of GRFA requires a multipronged approach, conventional,

PCTOC and powerful biotechnological tools can provide for the management of GRFA. Further,

advances in biotechnologies are occurring at a rapid pace and provide novel opportunities for more

effective and efficient management of GRFA (Merillon et al., 2012). Plant diseases worldwide are

responsible for billions of dollars’ worth of crop losses every year due to biotic stresses and total

potential loss from world-wide is 25 to 40%. Globally, enormous losses of the crops are caused by

the plant diseases since from time of seed sowing, pre and post-harvest and storage. Based on the

primary importance of plant tissue culture and biotechnology to solve the number of sustainability

challenges, I would like to narrate the lecture on importance of plant cell, tissue and organ culture in

various biotechnological application i.e. conservation, large scale in vitro production of plantlets,

development of disease resistance and stress tolerance plants, development of drugs, novel

chemicals including antibodies from plant tissue culture through three broad range of approaches

for manipulation of plants for various application (Figure 3). Hence, plant cell, tissue and organ

culture act as a platform to develop variant/modified crop plants to meet the present need of human

populations.

1. Micropropagation by organogenesis and somatic embryogenesis:

Micropropagation, also known as in vitro multiplication of plants, is the foremost

application of plant tissue culture. For many species, micropropagation provides a means for rapid

Germplasm Collection & conservation

Explants (seeds, stem, leaf, root)

Organogenesis & somatic embryogenesis

Large scale production of plantlets

Introduction to original habitat and new habitat



propagation and commercial production of crop, medicinal and aromatic plants compared to

traditional propagation techniques. Like, clonal propagation, axillary shoot multiplication, direct

(adventitious) organogenesis, callus to organogenesis, somatic embryogenesis, virus elimination, in

vitro grafting, in vitro gene banks, stock plant banks, somatic variation, managing ‘natural’

variation, induced mutation, in vitro screening and selection, anther or microspore culture-

production of haploids, leading to double haploids, protoplast culture and somatic fusion, DNA

transformation systems, recovery of regenerated from transformed cells, cell culture and

biosynthesis in bioreactors (production of secondary metabolites). The development and large scale

production of plantlets by direct organogenesis (using shoot tip, auxiliary buds and nodal explants)

as well as somatic embryogenesis (any explant) whereas the indirect organogenesis and somatic

embryogenesis via callus intermediate (using any explant) will be achieved with supplementation of

appropriate concentration and combination of plant growth regulators.

Figure 3. Biotechnological application and crop improvement through plant tissue culture

technology (www.biocyclopedia.com).

2. Development and Improvement of Hybrids:

Isolation of protoplast from desired two varieties, development of cell fusion and

hybridization techniques has solved the problem of incompatibility of plants and widened the scope

of production of new varieties within a short time. The somatic hybrid plantlets inherited many

characters viz., intermediate leaf morphology, stomata, forms and color of tubers, prolonged

flowering, large and fertile pollen grains, high yield and resistance/tolerance against biotic and

abiotic stresses. One focus of our research is to develop improved, non-invasive, seedless nursery

crops. One of the most effective means for developing seedless plants is to create triploids (plants

with three sets of chromosomes) While triploids grow normally, they are unable to divide equally

during meiosis and typically fail to produce viable gametes. Triploids have been developed for

many food crops including watermelon, bananas and grapes. Triploids can be bred by hybridizing

tetraploids with diploids. The following flow-chart depicts the step-by-step methods for isolation of

protoplast from different cultuvars, fusion and development of plantlets for hybrid vigor.

http://www.biocyclopedia.com/



Isolation of Protoplast

Fusion of the protoplasts of desired species or varities

Identification and selection of somatic hybrid cells

Culture of hybrid cells

Regeneration of hybrid plants

3. Production of artificial seeds:

The concept of artificial seeds for the first time was proposed by T. Murashige at a

Symposium in Belgium in 1977. Artificial seeds are the encapsulated or immobilized somatic

embryos with a protecting gel. The encapsulating gel act as seed coat and nutritive tissue

(endosperm) which provides nutrient to developing plantlets similar to natural seeds. The sodium

alginate or calcium alginate used to encapsulate the somatic embryos, stored at suitable temperature

and germination of somatic embryos, the artificial seeds (Figure 4).

Figure 4. Stages of somatic embryo, encapsulated somatic embryo (artificial seed) and germination

of artificial seed (Saiprasad, 2001).

4. Induction of somaclonal and gametoclonal variations, and polyploid:

Somaclonal and gametoclonal variations are natural phenomenon in plant cell cultures when

the callus and gametic cells are being cultured for repeated number of cycles. The variation includes

among plant cells and tissues are known tissue or culture-induced variations. Alternatively, the

combined use of induced mutation techniques with in vitro culture methods to induce somaclonal

and gametoclonal variations has great potential in breeding programs. In the past few years ionizing

radiation and chemical mutagens used extensively along with plant tissue culture, and their response

on in vitro mutation efficiency has been reported in many major crops. Induced mutations using

irradiation or chemical mutagens is another advance in biotechnology that may have potential

benefits for the production of sterile plants and novel forms (dwarfs) and foliage types (variegation).

Mutation breeding is also beneficial to increase variability in species with low genetic diversity

such as Hypericum frondosum. Gamma irradiation has been used for several decades for whole

plants and seed; however, more recently the procedures have been used to induce mutations in

tissue cultures. It is particularly desirable to treat callus cultures and to regenerate plants from single

cell lines to eliminate chimera tissue. The importance of in vitro mutagenesis, plant tissue culture

and its vital role in inducing somatic embryogenesis (Figure 5) agronomic characters (Figure 6),

disease resistance, tolerance to abiotic stress like, salinity, NaCl-tolerant mutant in Chrysanthemum,

low-temperature-tolerant mutants have been reported in major food crops. The development of new

polyploids, through chromosome doubling, may increase ornamental characteristics, expand



breeding opportunities, and restore fertility in sterile hybrids ultimately leading to the development

of improved cultivars. Induction of polyploidy has been employed to improve ornamental

characteristics and facilitate breeding programs for a wide range of plant taxa.

The use of physical energy in the field of agriculture has enhanced the genetic diversity and

agronomical characters of different crop plants. The recent reports on the use of physical energy,

especially laser irradiation on plants shows significant changes in the thermodynamic characters of

laser irradiated seeds which eventually triggers internal energy and lead to enhanced seed

germination, biochemical, physiological and growth characters of the seedlings/plants. The

preliminary study was undertaken in our Manipal Life Sciences Centre, Manipal University to

assess the effect of He-Ne laser irradiation on in vitro seed germination, physiological and

biochemical characters in the seedlings of brinjal (Solanum melongena L.) var. Mattu Gulla, a

unique variety being cultivated in Mattu Village, Udupi, Karnataka (Figure 7). The results indicates

improved in vitro germination of seeds, growth, physiological and biochemical characters in

seedlings of eggplant at 25 & 30 J/cm2 of laser irradiation (Muthusamy et al., 2012). The

established method could be used for pre-sowing treatment of eggplant seeds with laser rays for

enhanced germination and growth; also it may be applied essentially to improved agronomical

characters and fungal disease resistance in Mattu Gulla.

Figure 5 Figure 6

Figure 5. Somatic embryogenesis in peanut and germination of plantlets from irradiated embryonic

callus (Muthusamy et al., 2007)

Figure 6. Induction of somaclonal variations in cotton. a) Mutagen induced early flowering mutant

line, b-d) tall, number of branch and high yielding mutants (Muthusamy & Jayabalan, 2011)



Figure 7. Laser irradiation and in vitro seed germination of brinjal (Muthusamy et al. 2012).

5. Development of disease resistant and stress tolerance of crop plants:

Most of the crop plants are suffered from pathogenic agents i.e. fungus, bacteria, virus,

nematodes etc. and abiotic stresses i.e. soil, water, salt, metal, pollutants, etc. The biotic and abiotic

stresses affecting the primary growth and development of important crop plants which leads to

severe reduction yield characters. It is very essential to develop disease free/tolerant stock plants for

farmers in order to ensure maximum yield and quality of agricultural products. Now plant cell and

tissue culture techniques act as wide range of platform to develop disease resistance and stress

tolerant plants in laboratory within short span of time compare with conventional breeding

programs. The development of transgenic plants for fungal disease resistance in cotton and abiotic

stress in black gram through plant tissue culture technique. The following flow-chart depicts the

development of disease resistant and stress tolerant plant.

1. Selection of crop plant

2. Identification of biotic/abiotic stress

3. Extraction of biotic/abiotic compounds

6. Addition of biotic/abiotic

compound

5. Callus development

4. Inoculation of explants

7. Selection of resistant/tolerant

callus

8. Regeneration of plantlets

9. Bioassay for resistance/tolerance



5. Production of plant secondary metabolites:

Plants are the planet’s most sophisticated producer of chemicals by converting sunlight into

chemical energy as primary products in the biosphere and encompassing to the extremely wide

diverse and complex array of unique secondary metabolites they synthesize as bi-products of

primary metabolites. The techniques and tools of biochemistry has been important classic discipline

in plant research. While we animals were busy nailing down things like locomotion and

consciousness, the plants, without ever lifting a finger or giving it a thought, acquired an array of

extraordinary and occasionally diabolical powers by discovering how to synthesize remarkably

complicated molecules (secondary metabolites) which are the ones designed expressly to act on the

brains of animal, sometimes to attract their attention, but more often to repel and sometimes even

destroy them (Pollan, 2002). Plant cell and tissue cultures can be established routinely under sterile

conditions from explants, such as plant leaves or stems for the production secondary metabolites.

Strain improvement, methods for the selection of high-producing cell lines, and medium

optimizations can lead to an enhancement in secondary metabolite production. Organ cultures often

have sites of synthesis and storage of secondary metabolites in separate compartments. Elicitors,

compounds triggering the formation of secondary metabolites, can be abiotic or biotic. Natural

elicitors include polysaccharides such as pectin and chitosan, which are also used in the

immobilization and permeabilization of plant cells. Immobilization provides several advantages,

such as continuous process operation, but for the development of an immobilized plant cell culture

process natural or artificially induced secretion of the accumulated product into the surrounding

medium is necessary. Hence, the plant cell, tissue and organ culture has great potential to produce

number useful secondary metabolites. Table 2 predicts the various aspects of biotechnological

application for plant-derived secondary metabolites.

Methods Techniques

Cell, tissue and organ

culture

Micropropagation of

medicinal and aromatic

plants

Transgenic

plants/organisms

Newer sources

Safety consideration

Cell culture, shoot and root culture

Rare, endangered and threatened plants, high

yielding varieties and metabolically engineered

plants

Expression/over expression, metabolic

engineering, heterologous expression and

molecular farming

Algae and other photosynthetic marine forms

Ethical and biosafety asses

(c.f. Ramachandra Rao and Ravishankar, 2002)

Currently we have number of methods and techniques for plant cell, tissue and organ culture

for the specific in vitro production of food additives, pharmaceuticals (medicines for different

diseases), insecticides, perfumes, oils and dyes etc (Figure 8). Besides these usual methods of

production, we can use specific elicitors of both biotic and abiotic. The biotic elicitors include,

directly released by microorganisms and recognized by the plant cell (enzymes, cell wall

fragments), formed by action of microorganisms on plant cell wall (fragments of pectins etc.),

formed by the action of plant enzymes on microbial cell walls (chitosan, glucans) and compounds,

endogenous and constitutive in nature, formed or released by the plant cell in response to various

stimuli. The abiotic elicitors include physical or chemical nature working via endogenously formed

biotic elicitors, i.e. UV light, windfall, denatured proteins (RNase), freezing and thawing cycles,

non-essential components of media (agarose, tin, etc.), heavy metals, chemicals with high affinity to



DNA and with membrane-destroying activities like detergents i.e. xenobiochemicals, fungicides

(maneb, butylamine, benomyl) and herbicides (acifluorofen).

Figure 8 Application of plant tissue culture and biotechnology in Agriculture and Industry.

Conclusion and future prospects:

The plant cell culture and biotechnology has progressed as new era in the field of

biotechnology with special focus on the production of enormous plant secondary metabolites during

past decades. The development of genetic engineering and molecular biology techniques endorsed

the remarkable appearance of enhanced biosynthesis of several agricultural products including

pharmaceuticals for human health during second half of 20th

century. The several products from

plant cell culture have played important role in human health and the demand is increasing every

year in several countries. However, these successes would have been incredible without the

techniques of plant tissue culture which act as platform for the introduction of genetic information

into plants from closed relative or non-relative species. Currently, the production of novel chemicals

includes recombinant proteins, such as vaccines and antibodies with the use of transgenic plants

which represents economical and sustainable alternative to fermentation-based production systems.

The production of vaccines and antibodies (plantibodies) are emerging area of research, since plants

are free from human diseases thus further help to reduce the screening cost for viruses and bacterial

toxins. The plant cell, tissue and organ culture-derived food additives, pharmaceuticals (medicines

for different diseases), insecticides, perfumes, oils and dyes are available in markets and the demand

is increasing every year. Conceivably some of the greatest influence of plant tissue culture is yet to

come in future. The use of the technology for genetic modification of plant cells provides a

powerful tool for both fundamental and applied research. The application of gene transfer

technology has already been successfully applied to many crops species to increase resistance to



herbicides and pests and to increase yields. Research on genetic modification on ornamentals may

also lead to improved characteristics and to induce sterility. Further, we need to select the most

appropriate techniques from the pool of several conventional as well as sophisticated

biotechnological and molecular biological techniques for each genus of important crop, medicinal

and aromatic plants for improved plant products for food, fiber, timber, pigments, dyes, oils and

medicinally important compounds to meet the demands of ever growing human population.

Suggested Reading:

Bhomkar P, Upadhyay CP, Saxena M, Muthusamy A, Shiva Prakash N & Sarin NB. 2008.. Mol.

Breed. 22(2): 169-181.

Chawla HS. 2002. Introduction to plant biotechnology, Chawla HS (Ed). Oxford & IBH Publ Co.

Pvt. Ltd. New Delhi.

Ehrhardt DW and Frommer WB. 2012. Plant Cell. 24(2): 374-394.

Gagik SS. 1990. In: Melhods in Molecular Biology Vol. 6, Plant Cell and Tissue Culture, Pollard

JW and Walker JM (Eds), The Humana Press. pp. 1-12.

Ganesan M and Jayabalan N. 2006. Plant Cell Tiss Organ Cult. 87: 273–284.

Gideon Grafi. 2004. Developmental Biology 268: 1– 6.

Lidder P and Sonnino A. 2012. Adv Genet. 78: 1-167.

Merillon, Jean Michel; Ramawat, Kishan Gopal (Eds.). 2012. Plant Defence: Biological Control.

Springer

Muthusamy A and Jayabalan N. 2011. Acta Physiologiae Plantarum. 33(5): 1793–1801.

Muthusamy A., Pratibha PK., Vijendra Prabhu., Mahato KK., Vidhu SB., Radhakrishna Rao M.,

Gopinath PM and Satyamoorthy K. 2012. Photochemistry and Photobiology, 88(5), 1227-

1235

Muthusamy A, Vasanth K, Sivasankari D, Chandrasekar BR and Jayabalan N. 2007. Biol.

Plantarum 51(3): 430-435.

Neumann KH, Ashwani Kumar and Imani J. 2009. Plant Cell and Tissue Culture - A Tool in

Biotechnology. Principles and Practice. Neumann KH, Ashwani Kumar and Imani J. (Eds.),

Springer-Verlag, Heidelberg.

Petolino JF, Roberts JL and Jayakumar P. 2003. In: Handbook of industrial cell culture:

mammalian, microbial, and plant cells, Vinci VA and Parekh SR (Eds). Humana Press Inc.

New Jersey. pp. 243-258.

Pollan M. 2002. The Botany of Desire: A plant’s Eye view of the World, Random House Publishing

Group, US.

Ramachandra Rao S and Ravishankar GA. 2002. Biotechnology Advances 20: 101–153.

Razdan MK. 2003. Introduction to plant tissue culture, 2nd

edition. Razdan MK (Ed). Oxford & IBH

Publishing Co. Pvt. Ltd. New Delhi.

Saiprasad GVS. 2001. Resonance 39-47.

Steeves TA and Sussex IM. 1989. Patterns in Plant Development. Cambridge Univ. Press,

Cambridge, UK.

Wilson SA and Roberts SC. 2011. Plant Biotechnology J. 1–20.

Yadav K, Narender S and Sharuti V. 2012. Journal of Agricultural Technology 8(1): 305-318.



IVF: Medical Implications of Developmental Biology

Dr.P. Mariappan

PG and Research Department of Zoology

Rajah Serfoji Government College (Autonomous), Thanjavur 613 005, Tamil Nadu

e-mail: [email protected]

In Vitro Fertilization is a procedure in which sperms are allowed to fertilize eggs in a petri-

dish under laboratory conditions. The fertilized eggs are incubated in an environment favorable to

reproduction. Then the healthy developing embryos are carefully implanted into the woman's uterus

where, it completes the term. This technique is popularly known as ‘Test Tube Baby”. In 1978 July

25th

, the first test tube baby Louise Joy Borwn was born in England and following this so far 5

million children were born through this method. Millions of families are having a child and the

burden due to infertility is reduced a great extent. Louise Brown, the first test tube baby is said to

me a miracle baby in those days. But now a day there is no miracle in this aspect, since in each and

every town has a fertility clinic.

An estimation of WHO says that 13-19 million couples are infertile in India. There is no

clear cut definition for fertility. Infertility refers to the biological inability of a person to contribute

to conception. Infertility may also refer to the state of a woman who is unable to carry a pregnancy

to full term. Normally 80% of the couple are having pregnancy in one month of their married life,

another 10% have a child in a year. The remaining 10% are considered subfertile group since they

are not able to have a child. According to World Health Organization Infertility is the inability to

conceive a child. A couple may be considered infertile if, after two years of regular sexual

intercourse, without contraception, the woman has not become pregnant. Primary infertility is

referred as infertility in a couple who have never had a child. Secondary infertility is failure to

conceive following a previous pregnancy. Infertility may be caused by infection in the man or

woman, but often there is no obvious underlying cause. The infertility rate among the eligible

couple ranges from 1:10 to 1:6 which differs from culture to culture and country to country. Various

factors such as physical, psychological conditions contribute infertility.

The factors responsible for infertility are a) genetic b) general defects like diabetes mellitus,

thyroid disorders, adrenal diseases c) Hypothalamic-pituitary factors like hyperprolactinemia,

hypopituitarism, presence anti thyroid antibodies and d) environmental factors such as toxins glues,

volatile organic solvents and silicones, physical agents and chemical dusts and pesticides etc.

The above mentioned causes are common to male and female. Sex specific factors are also

influence the infertility. In the case of female the factors influence the fertility are ovulation

problems (e.g. polycystic ovarian syndrome), fallopian tube blockage, pelvic inflammatory disease,

age-related problems (fertility decreases after the age of 35), problems in uterine cavity, previous

tubal ligation, etc. Apart from this time of intercourse is also important. It must be done during the



release of egg from ovary. Low amount of sperm production or absence of sperm in semen is the

major problem in male infertility.

Some time the male and female partners may be infertile or sub-fertile and the infertility

arises from the combination of these factors. For others, it is suspected that the problem is due to

immunological or genetic. Such a couple can have child through assisted reproductive technology.

About 10% of the cases both partners may be normal or fertile as per the available methods of

assessment, still they are not able to have a child. Such infertility cases are referred as unexplained

infertility.

Infertility Assessment

It is generally considered in male dominant societies, the fertility as the “woman problem”

and even in well civilized and educated societies the males are not ready or volunteer themselves

for a medical check up for infertility treatment. About 40% of the infertility is assessed due to male

infertility and about 35% of the problem is because of low sperm count. Hence male partner must

be tested for infertility tests. At the same time assessment of male infertility is easy and doing so,

reduces the unnecessary female assessment and discomfort to them.

Male

In male, preliminary assessment includes collection of information about complete

reproductive and medical history along with medications, life style assessment which includes

exercise, smoking and alchocolic habits, physical exams and some personal questions about sexual

life. The preliminary assessment is usually done by an urologist. Following this semen analysis will

be carried out. The sample for semen analysis will be collected by masturbation, following 3 days

abstinence from ejaculation. The entire amount ejaculated semen will be collected in a sterile

specimen container and should be analyzed in andrology labs. The semen sample will be analysed

within an hour of collection in order to avoid compromising the sample. The sample may be even

collected in a private room at IVF clinic.

Semen will be assessed for appearance, colour, pH and leukocyte presence. The laboratory

persons count the sperm numbers and the sperm motility. In a normal person the sperm count is at

least 20 million sperm per mL, with 50% the sperms showing forward progressive movement. The

morphology (shape) of sperm is also assessed. If indicated, the sperm vitality can also be counted

(the percentage of immotile sperm that are alive or dead) based on the effects of a dye on the sperm.

Some clinics repeat the assessment if any problem is detected to confirm the findings. There are

several reasons noticed for low sperm in semen though production is there. These include 1)

retrograde ejaculation (backward ejaculation of sperm into the bladder), absence vas deferens

(genetic defect), Obstruction (anywhere between the testicles and the penis) and anti-sperm

antibodies (sperm is a potential antigen against which immune system produces antibodies

abnormally).



Figure 1:

Normal Sperm

Sperm Count

Male Infertility Problems

Female

Different types of infertility problems are experienced by women and many of these fertility

problems can be easily treated. The causes responsible for female infertility are a) acquired or

behavioural factors (age, tobacco smoking, alcohol, body weight and eating disorders, drugs,

diseases and surgery) b) genetic disorders (mutation of several genes, chromosomal abnormalities-

Turner syndrome, intersex conditions and androngen insensitivity conditions) and c) anatomic

factors. The female reproductive system is a very delicate structure and easily affected by several

factors such as hypothalamic-pituitary factors-Hypothalamic dysfunction, hyperprolactinemia,

ovarian factors-polycystic ovary syndrome, anovulation, diminished ovarian reserve, premature

menopause, menopause, luteal dysfunction, gonadal dysgenesis, ovarian cancer, tubal

(ectopic)/peritoneal factors-Endometriosis, pelvic adhesions, pelvic inflammatory disease (PID,

usually due to chlamydia), tubal occlusion, tubal dysfunction, uterine factors-Uterine

malformations, Uterine fibroids, Asherman's Syndrome; Cervical factors-Cervical stenosis,

Antisperm antibodies, Non-receptive cervical mucus; Vaginal factors-Vaginismus, Vaginal

obstruction.

Figure 2: Female reproductive tract



The diagnosis of infertility begins with a medical history and physical exam. The medical

laboratory tests include a) hormone testing, to measure levels of female hormones at certain times

during a menstrual cycle. On day 2 or 3 FSH and estrogen levels are quantified to assess ovarian

reserve. Level of TSH is measured to find out normal functioning of thyroid gland. In the second

half of the cycle the amount of progesterone is measured to confirm ovulation. Examination and

imaging of female reproductive system is done. This includes endometrial biopsy (to verify

ovulation and inspection of the lining of the uterus), laparoscopy (for inspection of pelvic organs),

fertiloscopy (a modern surgical technique used for early diagnosis), Pap smear (to check infection),

pelvic exam (for abnormalities or infection), postcoital test (peroform after intercourse to check

sperm survivability in cervical mucus) and special X-ray tests

In fertility Treatment

Treatment of infertility depends on the cause and includes counseling, fertility treatments.

Initial and laboratory assessment of the couples identifies the problem and following this fertility

treatment begins. In some cases the fertility problems are more easily treated. In general, as the

woman ages (especially after age 35), her chances of getting pregnant goes down and the risk of

miscarriage goes up. Hence the treatment for this couple differs from younger couples. In general

infertility can be treated with medicine, surgery, artificial insemination (AI), or assisted

reproductive technology (ART). Many times these treatments are combined. In most cases

infertility is treated with drugs or surgery.

Female

Treatments for women depend on the type of problem. When the problem is with ovulation

the treatment may include taking medicine, such as Clomiphene (stimulation of ovaries to release

eggs), metformin (treatment of polycystic ovary syndrome). Hormones such as Human Menopausal

Gonadotropin - hMG (Repronex, Pergonal), Follicle Stimulating Hormone - FSH (Gonal-F,

Follistim), Gonadotropin Releasing Hormone (GnRH) analog are used for ovulation. If the fallopian

tubes are blocked or damaged tubal surgery and grafting is done. In the case of mild to moderate

endometriosis the treatment may include laparoscopic surgery to remove endometrial tissue growth.

This treatment may not be an option is the case of severe endometriosis.

Male

In male the infertility treatment includes behavioural therapy for impotency and premature

ejaculation. When the sperm count is low surgical removal sperm is used. Low sperm count due to

infection is treated with antibiotics. Absence of sperms due to blockage in male reproductive tract is

corrected by surgery.

IVF Procedure

In vitro Fertilization treatment starts with taking the history of the infertile couples by the

medical experts which is followed by physical and laboratory examinations. This includes a test for

the sperm count in the case of male partner and a pelvic examination, cervical culturing, and

staining of cervical secretions for the female partner. Based on this findings treatment procedure is



recommended. In the case of male infertility due to azospermia the treatment is through donor

insemination. ICSI is followed for the persons with low sperm count. In the case of female partner,

depending up on the problem the treatment follows. When egg production is normal and problem is

with conception or development in the uterus treatment starts with administration of fertility drugs

to the woman to stimulate her ovarian follicles to produce as many healthy eggs as possible. This is

necessary because a single fertilized egg or pre-embryo has only a small chance of survival results

in low success rate. Eggs are retrieved 27 to 36 hours by a specific stimulation technique such as

ultrasonographically guided aspiration or laparoscopy, and as many eggs as possible are obtained

per single retrieval attempt. The harvested eggs are inseminated by a sample of semen that contains

sperm of good quality and prepared by washing to induce capacitation. When cleavage occurs, the

embryos are transferred to the woman’s uterus or surrogate mother’s uterus for further

development.

The in vitro fertilization procedure has the five main steps: 1) Ovulation induction and Super

ovulation 2) Oocyte monitoring 3) Oocyte retrieval 4) Insemination and 5) Embryo transfer (Figure

3).

Figure 3: IVF procedure

1 Ovulation Induction and Super Ovulation

Ovulation induction and Super ovulation are the terms used to describe the drug-induced

ovulation and production of multiple eggs respectively for assisted reproductive technologies, such

as IVF. Fertility drugs (gonadotropins) are injected to stimulate the ovaries so as to produce mature

eggs. The aim of ovulation induction (OI) is to grow and ovulate an egg in a woman who normally

does not ovulate, while the goal of superovulation (SO) is to produce more than one egg to improve



fertility in a woman who already ovulates. In a natural menstrual cycle luteinizing hormone (LH)

and follicle stimulating hormone (FSH) are released from the pituitary gland and these hormones

stimulate the growth of a follicle – the fluid space in the ovary where the egg grows. Although

several follicles grow, in a natural cycle only one becomes mature enough to ovulate its egg. In

ovulation induction, women who do not ovulate at all take gonadotropins (forms of FSH and/or LH)

by injection to stimulate the growth of one or more eggs. In superovulation, women who usually

ovulate take these same gonadotropin injections to stimulate the growth of more than one egg

(Figure 4).

Figure 4: Normal and stimulated cycle of ovulation

Ovulation induction medications are referred as fertility drugs and the most common drugs

used in fertility treatment are clomiphene citrate, gonadotropins, Metformin and Parlodel.

Clomiphene Citrate (Clomid, Serophone) is available in a tablet form and recommended for women

who have infrequent periods or long menstrual cycles. Headaches, blurred vision and hot flashes are

some common side effects reported those who have taken these drugs. The injectable

Gonadotropins (Repronex, Follistim, Bravelle, Pergonal and GonalF) is used to release,

development and maturation of the egg. Abdominal distention/discomfort, bloating sensation, mood

swings, fatigue or restlessness are reported as side effects and these side effects are relieved by

follicular aspiration.

Super ovulation is the term used to describe the more aggressive level of ovulation

induction. Gonadotropins or combination gonodotopin with clomiphene is used for production of

multiple eggs. Patients undergoing superovulation must be closely monitored by blood tests and

ultrasounds. Monitoring ensures that the patient does not hyperstimulate and also helps the

physician administer the correct dosage of medication so that only a few follicles develop. This is a

critical step to keeping the multiple pregnancy rates low. A low dose of hCG prescribed at the end

of the superovulation treatment process to stimulate ovulation. Ovulation will occur between 24-36

hours after HCG.

2 Oocyte Monitoring

Monitoring ovarian response to the stimulation depends mainly on the biophysical

parameters of follicular growth, and hormonal parameters. Oocyte monitoring is done by a

physician to determine when the eggs are ready for retrieval. Hormonal level is assessed by frequent

blood sampling. Urine samples also used to check hormone levels. Another way of monitoring

involves the use of intravaginal ultrasound to track follicular growth. The eggs develop inside fluid-

filled sacs in the ovaries called follicles, which enlarge as the eggs mature. Ultrasound studies are

performed on a frequent basis until oocyte (egg) retrieval. Sonography can depict developing



follicles, beginning at the time they measure between 3 and 5 mm. when it reaches maturity the

inner dimensions range from 17 to 25 mm.

3 Oocyte Retrieval

Transvaginal oocyte retrieval (TVOR) oocyte retrieval (OCR) or Egg collection, is referred

as removal of oocytes from the ovary of the female, enabling fertilization outside the body. In this

method, under ultrasound guidance, a needle is inserted through the vaginal wall and into an ovarian

follicle (care should be taken not to injure organs located between the vaginal wall and the ovary).

The other end of the needle is attached to a suction apparatus. When follicle is entered, suction is

gently applied to aspirate follicular fluid with oocyte. The follicular fluid is observed under

microscope for separation/collection of ova by an embryologist. Once the ovarian follicles have

been aspirated on one ovary, the needle is withdrawn, and the procedure is repeated on the other

ovary. The entire procedure is usually completed within an hour and performed under a light

general anesthesia or local anesthesia. The eggs are then placed in an incubator at 37ºC.

Figure 5: Oocyte Retrieval

4. Insemination of eggs

The eggs are retrieved through the above method are placed in a special culture medium

(fluid) and allowed to remain for approximately 2 to 3 hours. While the eggs are in the process of

retrieval, the male partner is asked to provide sperm sample. Surgical sperm removal is used when a

man has had a vasectomy or his ejaculation consists no sperm. Within an hour of collection the

sperm are prepared and a small number of active sperm are placed in the medium (containing salt,

protein and antibiotics) with each egg. The sperm and eggs are placed in incubators, which enables

fertilization to occur. This process is known as insemination. In some cases where fertilization is

suspected to be low, intra cytoplasmic sperm injection (ICSI) may be used. In this method a single



sperm is injected directly into an egg in an attempt to achieve fertilization. If ICSI is to be done, it

will be performed approximately 4 hours after the egg retrieval. An embryologist carries out regular

checking of the developing embryo. Once cell division begins the fertilized eggs are considered as

embryos and it is ready for implantation after three to five days of healthy growth.

5. Embryo Transfer

Embryo transfer is the final and simple step of IVF in which embryos are placed into the

uterus of a female. Normally 2-4 cell stage embryos are transferred. This can be done at anytime

between day 1 through day 6 after the retrieval of the egg. But most clinics do it in between days 2 -

4. Embryos used for transfer may be either “fresh” from fertilized egg of the same menstrual cycle

or “frozen”. If freezed embryo is used, it must be thawed just prior to the transfer. For embryo

transfer the uterine lining (endometrium) needs to be appropriately prepared so as to the embryo(s)

can implant. In a natural or stimulated cycle, the embryo transfer takes place in the luteal phase at a

time where the lining is appropriately undeveloped in relation to the status of luteinizing hormone.

In a cycle where a "frozen" embryo is transferred, the recipient woman could be given first estrogen

preparations (about 2 weeks), then a combination of oestrogen and progesterone so that the lining

becomes receptive for the embryo. The time of receptivity is known as “implantation window”.

The embryo transfer procedure starts by placing a speculum in the vagina to visualize the

cervix, which is cleansed with saline solution or culture media. A soft transfer catheter loaded with

the embryos is inserted through the cervical canal and advanced into the uterine cavity. Ultrasound

guidance is used for correct placement of catheter, which is 1–2 cm from the uterine fundus.

Anesthesia is generally not required. After insertion of the catheter, the contents are expelled and

the embryos are deposited. Limited evidence supports making trial (mock trail) transfers before

performing the procedure with embryos. In zygote intra fallopian transfer (ZIFT) eggs are removed

from the woman, fertilised, and placed in the woman's fallopian tubes rather than the uterus.

Minimal risks are associated with the embryo transfer procedure which includes the loss of embryos

during transfer or implanting the embryos in the wrong place such as the fallopian tubes. Though

some women experience mild cramping in general the procedure is usually painless.

Ethics

This artificial conception raises the possibilities of myriad problems – social, ethical,

emotional, spiritual issues and also its own scientific issues. In India there are several social issues

associated with in vitro fertilization. One such a problem is surrogacy. India is considered as the

capital of surrogacy since the rent for womb is cheaper than other countries. An estimate says that

in India the cost of IVF is 1/3 of the UK. In Anand (Gujarat) where rent-a-womb is a thriving

industry, with no dearth of ignorant and poor women, and no laws to regulate the mushrooming

fertility clinics. Though the ICMR guidelines restricts the surrogacy the implementation of

guidelines are questioned. There is no published data in this regard. Most of the information

available in this aspect is the statements given by physicians and patients who haven’t succeeded in

having a child through this technique. Apart from these some of the women underwent IVF have



developed medical complications due to administration of hormones for super ovulation.

Administration hormones to induce the development of more number of oocytes than one in a cycle,

alters the normal hormonal harmony. This leads to development of cancer in some women. Having

a child in a later part of life is also posing severe health problems. Some IVF centres and genetic

labs have Pre-implantation Genetic Diagnosis (PGD) facilities. By using this facility these clinics

earn little more money than normal IVF to implant male babies. Like these several other issues are

also associated with IVF which will be strictly regulated through implementation ICMR guidelines.

Selected References

Geoffrey Sher, Virginia Marriage Davis, Jean Stoess. 1998. In Vitro Fertilization: The A.R.T. of

Making Babies. Facts on File, Incorporated.

Godwin Meniru. 2001. Cambridge Guide to Infertility Management and Assisted Reproduction.

Cambridge University Press.

Kay Elder, Brian Dale, Yves Ménézo and Joyce Harper . 2011. In-Vitro Fertilization. Cambridge

University Press, New York.

Lita Linzer Schwartz. Alternatives to Infertility: Is Surrogacy the Answer? (Frontiers in Couples

and Family Therapy, No 4). Brunner/Mazel Publishers.

Nagy, Zsolt Peter; Varghese, Alex C.; Agarwal, Ashok. 2011. Practical Manual of In Vitro

Fertilization: Advanced Methods and Novel Devices. Springer.

Peter R. Brinsden, Bourn Hall Clinic. 1999. A Textbook of in Vitro Fertilization and Assisted

Reproduction: The Bourn Hall Guide to Clinical and Laboratory Practice. Parthenon

Publishing Group.

Sandra Ann Carson, Peter R. Casson, Deborah J. Shuman. 1999. Complete Guide to Fertility. NTC

Publishing Group.

Nobel Prize for Medicine 2010-Robert G Edwards Louise Brown-the first test tube baby

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