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Concept based notes

Plant Secondary Metabolites

[B.Sc. (Biotechnology) Part-II]

Meesha Srivastava (M.Sc Biotech)

Revised by: Richa Tyagi

Lecturer

Deptt. of Science

Biyani Girls College, Jaipur

0.5lant Secondary Metabolites 2


Published by :

Think Tanks Biyani Group of Colleges Concept & Copyright :

Biyani Shikshan Samiti Sector-3, Vidhyadhar Nagar, Jaipur-302 023 (Rajasthan)

Ph : 0141-2338371, 2338591-95 Fax : 0141-2338007 E-mail : [email protected] Website :www.gurukpo.com; www.biyanicolleges.org ISBN: 978-93-81254-25-7 Edition : 2011 Price : Leaser Type Setted by : Biyani College Printing Department

While every effort is taken to avoid errors or omissions in this Publication, any mistake or

omission that may have crept in is not intentional. It may be taken note of that neither the publisher nor the author will be responsible for any damage or loss of any kind arising to anyone in any manner on account of such errors and omissions.



Preface

I am glad to present this book, especially designed to serve the needs of the

students. The book has been written keeping in mind the general weakness in understanding the fundamental concepts of the topics. The book is self-explanatory and adopts the “Teach Yourself” style. It is based on question-answer pattern. The language of book is quite easy and understandable based on scientific approach.

Any further improvement in the contents of the book by making corrections, omission and inclusion is keen to be achieved based on suggestions from the readers for which the author shall be obliged.

I acknowledge special thanks to Mr. Rajeev Biyani, Chairman & Dr. Sanjay Biyani, Director (Acad.) Biyani Group of Colleges, who are the backbones and main concept provider and also have been constant source of motivation throughout this Endeavour. They played an active role in coordinating the various stages of this Endeavour and spearheaded the publishing work.

I look forward to receiving valuable suggestions from professors of various educational institutions, other faculty members and students for improvement of the quality of the book. The reader may feel free to send in their comments and suggestions to the under mentioned address.

Note: A feedback form is enclosed along with think tank. Kindly fill the feedback form and submit it at the time of submitting to books of library, else NOC from Library will not be given.

Meesha Srivastava



Syllabus PLANT SECONDARY METABOLITES

BT - 601

Note : Question No. 1 shall consisit of wuestions requiring short answers and shall cover

entire paper . The paper is divided into four sections. Student are required to attempt

five questions in all, selecting not more than one question from each section. All

question carry equal marks.

Section -A

1. Introduction, secondary plant products in nature and their occurrence, type and

uses. Basic tools and techniques used in isolation and separation.

Section -B

2. Production in vitro- optimization selection effect of metabolism on secondary

metabolite- production, production under stress factors.

3. Production of alkaloids, steroids and saponins. Mechanism and control by

different factors, detoxification of secondary metabolites.

Section -C

4. Production of secondary metabolites by bioconversion genetic transformation for

production of secondary metabolite. Large scale production in bioreactor.

Commercial production of secondary metabolites using cell cultures. Use of

biorectors, immobilized cells. Biotransformation. Applications and limitations.

Cryopreservation and ex situ conservation of germplasm.

Microbial secondary metabolites, their occurrence types and uses.



Section -D

5. Sources and type of antitumor compounds, food additives and insecticides.

6. Products obtained in traditional medicinal systems and their significance in plant

biotechnology

7. Biotechnological approach on production of secondary metabolites e.g. ginkolides

from cell cultures of Ginkgo biloba L.

8. Use of immobilized cell svstems for the production of industrially important

chemicals

9. Genetic regulation of metabol. Gene expression in response to environmental

stimuli, Regulation of gene expression

10. Analysis of metabolic control and the structure metabolic.



Contents

S. No. Name of Topic

1. Introduction

1.1 Secondary Metabolites

1.2 Types & Uses of Secondary Metabolites

1.3 Techniques used for its Separation

2. Production of Secondary Metabolites

2.1 In In-Vitro Conditions

2.2 Optimization

2.3 Selection of Cells

2.4 Production under Stress Factor

3. Production of Alkaloids, Saponins and Steroids

3.1 Production of Alkaloids, Saponins and Steroids

3.2 Detoxification of Secondary Metabolites

4. Production of Secondary Metabolites by Bioconversion

4.1 Production of Secondary Metabolites by Bioconversion

4.2 Genetic Transformation for Production of Secondary Metabolite

4.3 Large Scale Production in Bioreactor and Uses.

4.4 Commercial Production of Secondary Metabolites using Cell Culture

4.5 Immobilized Cells

4.6 Biotransformation

4.7 Cryopreservation and Ex-Situ Conservation of Germplasm

4.8 Microbial Secondary Metabolites, Their Occurrence, Types and Uses

5. Sources and Types of Antitumor Compoud, Food Additives and Insecticides

6. Products Obtained in Traditional Medicinal and

the Significance in Plant Biotechnology

7.

Unsolved Paper



Introduction

Q.1 What are Plant Secondary Metabolites?

Ans.: Introduction : A plant cells produces two types of metabolites :

Metabolities

Primary Metabolites Secondary Metabolites

These are involved directly in growth & metabolism.

Example : Carbohydrates, Lipids, Oils etc.

These are the end products of primary metabolism. These are synthesized in specialized cells at particular development stages.

Example : Alkaloids, Steroids, Lignin, Resins.

Production of Secondary Metabolites Glucose

Pentose Phosphate Pathway Phosphoenol Pyruvate

Erythrose – 4 – PO42-

6 – Deoxyxylulose Pyruvate Shikimate Pathway Aliphatic Amino Acid

TCA Cycle Aromatic Acetyl CoA Amino Acids

Alkaloids Phenyl Propanoids

Flavonoids Terpenoids Mevalonic Malonyl – CoA Complex Acid Polykitides Alkaloids Comlex Complex Terpenoids Flavonoids

Q.2 Name the types and uses of Secondary Metabolites?

Ans.: Different Secondary Metabolites and their use can be tabulated as :-

S. Name of Use or Activity S. Name of Use or Activity

CHAPTER-1

C



No. Secondary Metabolite

No. Secondary Metabolite

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21))

(22)

Pyrethnins

Nicotine

Rotenoids

Azadirochtin

Phytocedysones

Baccharine

Bruceantine

Gsaline

3-Deoxycolchicine

Ellipticine

9-methoxyelliptcine

fagaronive

Tlarringtovinl

Jandicine N-oxide

Maytansive

Podophyllotoxin

Taxol

Thalicarpine

Tripdiolide

Vinblastine

Quinine

Digoxin

Insecticidal

Insecticidal

Insecticidal

Insecticidal

Insecticidal

Antineoplastic

Antineoplastic

Antineoplastic

Antineoplastic

Antineoplastic

Antineoplastic

Antineoplastic

Antineoplastic

Antineoplastic

Antineoplastic

Antineoplastic

Antineoplastic

Antineoplastic

Antineoplastic

Antineoplastic

Antimatarial

Cardiac tonic

(23)

(24)

(25)

(26)

(27)

(28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38)

(39)

(40)

(41)

(42)

Diosgunin

Morphine

Thebaine

Suopolanine

Alropine

Codeine

Shikonin

Anthroquinones

Rosamarinic

Acid

Jasmini

Stevioside

Croun

Capsacin

Vanillin

Gutla percha

Terpendids

Papaverive

Ajmalicive

Caffeine

Birberine

Antifertility

Analgesic

Source of codeine

Antihypertension

Muscle Relaxant

Analgesic

Dye,

Pharmaceutical

Dye, Laxative

Spice, Antioxident

Perfume

Sweetner

Saffron

Chilli

Vanilla

Rubber

Essential Oils

Spasmolytic

Hypertensive

Stimulant

Antispasmadic

Q.3 What are the basic techniques used for the separation of Secondary Metabolites?

Ans.: The techniques which art applied for the separation are :-

(a) Chromatography - Chromatography is a family of analytical chemistry

techniques for the separation of mixtures. It involves passing the sample, a

mixture which contains the analyzed, in the "mobile phase", often in a stream of

solvent, through the "stationary phase." The stationary phase retards the passage

of the components of the sample. When components pass through the system at

different rates they become separated in time, like runners in a marathon. Ideally,



each component has a characteristic time of passage through the system, called a

"retention time."

A chromatograph takes a chemical mixture carried by liquid or gas and separates

it into its component parts as a result of differential distributions of the solutes as

they flow around or over a stationary liquid or solid phase. Various techniques

for the separation of complex mixtures rely on the differential affinities of

substances for a gas or liquid mobile medium and for a stationary adsorbing

medium through which they pass;

(i) Thin Layer Chromatography-

In paper chromatography, chemical interactions with the paper make compounds

travel at different rates. A small spot of solution containing the sample is applied

to a strip of chromatography paper about one centimeter from the base. This sample

is adsorbed onto the paper. This means that the sample will contact the paper and

may form interactions with it. Any substance that will react with (and thus bond

to) the paper cannot be measured using this technique. The paper is then dipped

in to a suitable solvent (such as ethanol or water) and placed in a sealed container.

As the solvent rises through the paper it meets the sample mixture which starts to

travel up the paper with the solvent. Different compounds in the sample mixture

travel different distances according to how strongly they interact with the paper.

Paper chromatography takes some time and the experiment is usually left to

complete for some hours.

(ii) Gas Liquid Chromatography



Gas-liquid chromatography is based on a partition equilibrium of analyte

between a liquid stationary phase and a mobile gas. It is useful for a wide range

of non-polar analytes, but poor for thermally labile molecules.

(iii) High Performance Liquid Chromatography

Modern HPLC systems are highly automated

High performance liquid chromatography, frequently referred to simply as

HPLC, is a form of column chromatography used frequently in biochemistry. The

analyte is forced through a column by liquid at high pressure, which decreases

the time the separated components remain on the stationary phase and thus the

time they have to spread out within the column, leading to broader peaks. Less

time on the column then translates to narrower peaks in the resulting

chromatogram and thence to better selectivity (it's easier to differentiate one peak

from another) and sensitivity (tall, narrow peaks can be easier to discriminate

from noise than shorter, broader peaks). Solvents used include any miscible

combination of water or various organic liquids (the most common are methanol

or acetonitrile). Often, a gradient over time in the solvent composition passing

through the column is used to separate analyte mixtures, as a function of how

well the changing solvent composition differentially mobilizes the analyte. For

instance, using a water/methanol gradient, the more hydrophobic components

will elute under conditions of relatively high methanol, whereas the more

hydrophilic will elute under conditions of relatively low methanol. Whether one

starts with high methanol or low methanol depends on the nature of the

stationary phase.

The quantification can be done by :-

(i) UV Spectrophotometer

(ii) Fluorescence



(iii) Calculation of optical Density

Note : The separation and purification process for the recovery of product is

called as Extraction.

□□□



Production of Secondary Metabolites

Q.1 What are the various Biotechnological Method for the Production of Secondary

Metabolites?

Ans. Various methods are:-

Biotechnological methods

Immobilization Hairy Roots Elicitation Bioreactor

This technique is based on the confinements of bio-catalyst on or within a matrix by entrapment, adsorption or covalent linkage

This a technique in which hairy roots profusely produced with a suspension of A. rizogenes

In this technique enhanced production of stress factors may be achieved by treating plt. cell with extracts prepared from pathogenic fungi in the absence of living organization

It‘s a large culture vessel used to scale up the culture. Its an equipment fitted with microprocessor control unit to control the …….. gas how rate, agitation speed

The cells are

physically

entrapped in a gel

structure and

substrate product

In hairy root culture

transformation may

be induced

Molecules …..

stimulate

secondary

metabolites

leading to the

induction of stress

metabolites

Q.2 What are the various factors which effect In-Vitro Products of Secondary

Metabolites?

CHAPTER-2

C



Ans.: Various factors are :-

(1) Physical Factors :

(i) Light : It indirectly affects the secondary metabolism like light

mediated enzyme metabolism. Various colour light effect

accordingly such as :

White Light – Anthocyanin Product

Blue Light - Chlorogenic Acid Products

Red Light - Indole Product

(ii) Temperature : Its affect depend accordingly on the cultures for

example –

In C. roseus when incubated at 160C than on 270C their

productivity increases two folds.

N. tabbacum productivity increases when incubated at high

temp.

(iii) pH : Plant cells are cultured usually at a PH of 5 to 6 range. PH has

a great influence on the culture and thus has to be carefully

calibrated.

(2) Effect of Nutrient : Cultured plant cells are usually grown on medium

containing all the elements required for their sustained growth eg-

essential minerals, vitamins & carbohydrate source.

(i) Carbon Source : 2-3% concentration in the medium and are known

to influence the products of phytochemicals and optimal growth.

(ii) Nitrogen Source : A mixture of nitrate & ammonium is used in all

the media as a source of nitrogen.

(iii) Phosphate Source : Inorganic phosphate is involved in metabolic

regulation in photosynthesis and respiration. The altered phosphate

level in the media profoundly effect the biosynthesis of

phytochemicals by cultured plant cells.

(iv) Plant Growth Regulators : Concentration and ratio of growth

regulators directly govern the differentiation.



(v) Precursors : These are the molecules which are directly incorporated

into secondary metabolites with structural change. Different class of

compounds hair diff precursors.

There may be several precursors of a compounds produced at

different stages of biochemical synthesis.

Q.3 What are the various methods employed for Selection of Cells?

Ans.: The techniques involved are done to select the cells with maximal yield, cells can

be selected for high yields of secondary metabolites from a heterogeneous

population to improve the overall quality of the cultures.

Various Techniques are :-

(i) In pigmented cells demarcation between high and low pigment containing

cell care be made very easily by naked eye.

(ii) Secondary metabolites constitute a fluorescent compound, the cells are

highly fluorescent under UV light. The cells with high amount fluoresce

more intensity.

(iii) Chemical tests like Dragendorff‘s reagents for alkaloids or Hibermann-

Buchards test for steroids.

(iv) Thin layer chromatography of crude samples and use of specific reagent as

used for culture of that particular species.

(v) GLC, HPLC & RJA used as partial analysis.

Q.4 Explain how the Elicitation Mechanism is related to Secondary Metabolite

Product?

Ans.: Elicitation is a mechanism which is initiated by elicitors which turn initiate

second. Metabolite production in plants.

Elicitors : These are the compounds of biological orgin involved in plant microbe

interaction. These are of two types :-

(i) Biotic/Stress Agents

(ii) Abiotic/ Stress Agents - UV Light, Alkalinity Osmotic Pressure, Chemical.



Molecules which stimulate secondary metabolism leading to the induction of

stress metabolites are k/as elicitors.

The discovery of elicitation response was done by Run and co-workers in 1972.

Elicitor treatment has resulted in an increase in amount & activity of M-RNA

encoding enzymes of phytoaluxin biosynthesis in cell cultures. It has been

demonstrated that stress induced in normal, intact plant tissues leads to the

induction and accumulation of sec. metabolites.

□ □ □



Production of Alkanoids, Saponins and Steroids

Q.1 Explain the basic pathway of production of Alkaloids Steroids and Saponins in

Plants.

Ans. Alkaloids : These are defined as ‗basic nitrogenous plant products mostly

optically active and possessing nitrogen hitirocycles as their structural unit with a

pronounced physiological action.

Examples : Morphine, Quinine, Coninel.

Biosynthesis : The fundamental skelctions of alkaloids are derived from common

acids & other small biological molecules. A two simple types of reactions occur to

form complete structures. But they all need precursors to initiate the rxns and

different alkaloids have different precursors such as indole alkaloid tryptophan is

the precursor, papavarine is obtained from tyrosine.

Example : for the production of indole alkaloids.

Tryptophan

Secologanin Tryptophan

Strictasidine

*Fig. : Biosynthesis of Indole Alkaloids

Cathenanine

Ajmalicine

Serpentine

Steroids : They belong to large group of compounds k/as terpenoids or

isoprioids. These are deravitives of cyclopentanpoperhy.

Biosyntesis : Steroids production occurs vehen terpenes are formed by the

polymerization of isoprene units and triperpenoids.

CHAPTER-3

C



The term terpene refers to a group of natural products containing 30 carbon

atoms which are derived from six isoprene units.

C D

A B

Cyclopentanoperhydrophenanethrane

They are generally found in association with fats and are non saponifiable.

Saponins: These are widely distributed in monocot families.

Biosynthesis : Sapogenins are the precursors for saponins synthesis. In this

sapogenins conjagate with sugars to form saponins.

These sapogenins are used in the preparation of steroidal hormones at

commercial level.

General commercial method of production all the above is through cell culture

method.

Q.2 What is the mechanism of Detoxification of Secondary Metabolites in

Herbivores?

Ans.: Plants produce a variety of secondary metabolites to protect themselves from

pathogens and herbivores and/or to influence the growth of neighboring plants.

Some of these metabolites are toxic to the producing cells when their target sites

are present in the producing organisms. Therefore, a specific self-resistance

mechanism must exist in these plants. Self-resistance mechanisms, including

extracellular excretion, vacuolar sequestration, vesicle transport, extracellular

biosynthesis, and accumulation of the metabolite in a non-toxic form, have been

proposed thus far. Recently, a new mechanism involving mutation of the target



protein of the toxic metabolite has been elucidated. The mechanisms that plants

use to prevent self-toxicity from the following representative compounds:

cannabinoids, flavonoids, alkaloids, benzoxazinones, phenylpropanoids,

cyanogenic glycosides, and glucosinolates.

INTRODUCTION

Herbivores are faced with a food resource of poor quality not only because plants

are low in nutrients but also because they produce plant secondary metabolites

(PSMs) that have wide-ranging physiological effects from direct toxicity to

digestion impairment. Herbivore offenses including numerous behavioral and

physiological mechanisms herbivores use to deal with PSMs.

MECHANISMS

In this section, we present three general mechanisms (avoidance, regulation,

biotransformation) used by vertebrate herbivores to mitigate the effects of PSMs.

Of the three sections, ecologists may be least familiar with biotransformation.

1. Avoidance

DECOMPOSITION Herbivores that cache food before consumption may

behaviorally circumvent or reduce the effects of PSMs if the compounds degrade

during storage. Behavioral reduction of PSMs prior to ingestion has a number of

advantages. The detoxification system is energetically demanding. If animals can

reduce the dose of toxins consumed through food storage, they may save

significant quantities of energy that would be lost during detoxification. Second,

ingestion of lower doses may reduce the likelihood of the formation of toxic

intermediate metabolites or free radicals. Lastly, behavioral manipulation of

PSMs may enhance diet breadth by allowing herbivores to consume plants

containing toxins or concentrations of toxins that they would otherwise be unable

to process. Example of behavioral manipulation is Meadow snip branches from

conifer trees and delay consumption for a few days, during which time the PSMs

decrease.

TANNIN-BINDING SALIVARY PROTEINS (TBSP) Many herbivores produce

salivary proteins that may assuage the effects of tannins. Some tannins (condensed



or hydrolyzable) react with proline-rich proteins secreted in saliva, binding to

form a complex that is usually insoluble and unabsorbed. Proline has a secondary

amine group that gives it a rigid conformation and disrupts the alpha-helical

structure of proteins. This makes carbonyl groups in the protein available for

hydrogen bonding with the phenolic group of tannins. The interactions are

stronger with high-molecular weight condensed tannins that possess many

phenolic groups.

PERMEABILITY GLYCOPROTEIN AND CYTOCHROME P4503A Regulated

absorption of PSMs by gut cells is a unique and only recently acknowledged

herbivore offense. Absorption of PSMs across intestinal cells can be regulated by

two molecular mechanisms, a glycoprotein transporter (permeability glycoprotein,

or P-gp) and a biotransformation enzyme (cytochrome P450 3A, or CYP3A) that

function either in tandem or independently to reduce the amount of PSMs

absorbed. P-gp is one of a group of transporters that remove foreign substances

from cells. It was originally discovered in tumor cells, in which it is responsible for

resistance to several anticancer drugs (e.g., Vinca alkaloids, taxanes). P-gp is one of

a large superfamily of ATP-binding cassette transporters that are highly conserved

and found in all species from bacteria to mammals. P-gp is highly expressed on

the apical surface of cells in the lower intestine with increasing levels from the

duodenum to colon. P-gp pumps PSMs (e.g., digoxin, morphine) back into the

lumen of the gut. P-gp acts on hydrophobic substances, usually also containing a

hydrophilic region, with a tertiary nitrogen and aromatic ring. P-gp is also present

in liver and renal tubular cells, where it pumps substances into the bile and urine,

respectively, and is in the luminal side of the capillary endothelium that forms the

blood-brain barrier. Overall, the role of P-gp seems to be a defense against

xenobiotics such as PSMs, by opposing their absorption, hastening their excretion,

and protecting the brain.

CYP3A is a biotransformation enzyme that metabolizes about 50% of all drugs and

many PSMs. It is expressed in large amounts in liver cells but also in intestinal

cells where it usually acts in concert with P-gp to reduce the absorption and

bioavailability of PSMs. This joint defense is most effective against toxins that are

taken in low doses and which are slowly absorbed, such as the fungal metabolite,

cyclosporine. PSMs present in high concentrations that rapidly enter intestinal

cells will saturate both transporter and enzyme, resulting in dose-dependent

absorption. For very lipophilic PSMs, diffusion into intestinal cells is likely to be

faster than P-gp can return them to the lumen; however, the effect of the pump



will be to increase the time that the PSM will spend in the cell, where it is exposed

to metabolism by CYP3A. The result is more extensive metabolism of the PSM

rather than it being excreted unchanged in the feces.

MICROBIAL DETOXIFICATION Many propose that PSMs are detoxified by

microbes in foregut fermenting herbivores (e.g., ruminants, kangaroos, hoatzin). The

diverse microbial populations in the foregut can perform many reactions, which can

both reduce and increase the toxicity of ingested PSMs. However, there remain too

few examples to judge whether foregut microbial detoxification is an important

driver of diet diversification in wild herbivores. The best example comes from

agricultural systems a specific microorganism enabled goats to consume greater

quantities of Leucaena leucocephala containing mimosine, a toxic nonprotein amino

acid.

2. Regulation of PSM Intake :

Browsing mammals encounter a diverse range of PSMs in the majority of the foods

they consume. Therefore, complete avoidance of PSMs in the diet is not likely to be a

realistic strategy, as animals would need to exclude most available plants from their

diet. The vast majority of PSMs that animals ingest are not so acutely toxic that a

single bite would be lethal or severely detrimental, but large amounts of even low-

potency compounds can be harmful. Consequently, animals must have mechanisms

that allow them to detect and regulate their intake of PSMs to ensure that low-

potency toxins do not cause damage. Understanding the sorts of mechanisms that

animals use to regulate toxin intake is vital if we are to integrate the effects of PSMs

with broader theories of feeding. Conditioned food aversions (CFAs) have long been

known to be powerful influences on the diets of herbivores. CFAs arise when an

animal makes an association between the taste or smell of a plant and some negative

consequence—usually illness. Many studies have demonstrated that foods can be

repellent to herbivores without inducing CFAs.

3. Biotransformation :

PSMs can be eliminated from the body by excretion or chemical change

(biotransformation), or a combination of these processes. Because of the way the

terrestrial kidney works to conserve water, lipophilic substances are poorly excreted.

In the mammalian kidney, the plasma filtered at the glomerulus is nearly completely



reabsorbed from the renal tubules, and lipophilic substances are extensively

reabsorbed across the renal tubular epithelium leaving polar molecules that cannot

permeate the epithelial cells to be excreted in the urine. The most lipophilic PSMs

(e.g., monoterpenes) are not excreted without transformation, whereas more polar

PSMs (e.g., quercetin, gallic acid) are excreted unchanged to varying extents.

Terrestrial animals have evolved a powerful suite of biotransformation enzymes to

convert lipophilic PSMs into more polar metabolites readily excreted in urine or bile.

These biotransformation enzymes are broadly categorized into two groups:

functionalization (also called Phase 1), in which functional groups are introduced

into metabolites, and conjugation (Phase 2), in which adducts are formed with

endogenous compounds to further increase polarity. Functionalization and

conjugation enzymes can work alone or in tandem, depending on the substrate. The

most important are the mixed-function oxidases of the cytochrome P450 family

(P450s) . These enzymes are considered to have evolved in part as a response to

dietary plant toxins. Although most drugs lose their activity after biotransformation,

there are examples of PSMs whose toxicity is mediated by metabolites.

□ □ □



Production of Secondary Metabolites by Bioconversion

Q.1 What is Bioconversion and how is it applicable for the production of Secondary

Metabolites?

Ans.: Bioconversion is also called as biotransformation and it can be defined as the

conversion of one chemical into another i.e. of a precursor (or substrate) into a

penal product using a cell suspension acting as biocatalyst.

Biocatalyst can be micro-organism plant or animal cells, either growing in

quiescent state or an extract from such cells or a purified enzymes.

Bioconversion is the process in which genes on coding relevant enzymes can be

introduced in the hast cells by the means of Recombinant DNA technology.

The two basic mechanism employed are :

(i) Freely Suspended Plant Cells

(ii) Immobilized Plant Cells

(i) Freely Suspended Plant Cells : In this freely suspended cells form the

biocatalytic system. Once they are added many reactions takes place such as

hydroxylation, glycosylation.

The precursors (Plant cell enzyme) are added after the inoculation of cells,

the bioconversions occurs readily in the plant cells, a precursor has to pass

the cell wall and cell member barrier.

(ii) Immobilized Plant Cells : In this the cells are immobilized and entrapped.

By entrapment the cells. Become protected against shear a damage.

Entrapment of cells may result in a kind of micro-environment, resembling that of

the organized tissue in the intact plant.

Entrapment of plant cells is one of the means to create non-growth conditions

under which the production of secondary metabolites may be improved.

CHAPTER-4

C



For entrapment different kind of matrix are being used these are alginate, the

most popular matrix.

The basic steps involved in production of sec. metabolites through bioconversion

are :-

(i) Plant Cell Culture

(ii) Addition of Precursors

Freely suspended Immobilized Cells

Precursors or Enzyme Extraction :

Guide Enzyme

Homogenisation

Purification by (NH4)SO4 ppt.

Coloumn chromatography

Characterization

Application of Enzyme in the Culture

(iii) Production of Secondary Metabolites

(iv) Bioconversion of Synthetic Precursors

Example : Strictosidine Synthase – Condensation – Tryptamine – Strictosidine

Enzyme RXN Type Precursor Product

Q.2 Explain the various techniques through which Plants can be genetically

transformed to Product Secondary Metabolites.

Ans.: Various techniques are :-

(a) Agrobacterium tumefaciens

(b) Liposomes

(c) Microinjection

(d) Sonication

(e) Electroporation



(f) Chemically Stimulated DNA Uptake

(g) Laser Microbeam

(h) Silicon Carbide Fibres

(a) Agrobacterium tumefaciens causes crown gall disease in dicotyledonous

sps. Crown Gall leads to the isolation of plasmid for genetic manipulation.

Oncogenic strains of a tumefaciens possess a large plasmid known as the

tumor inducing (Tc) plasmid transformation is associated with and

accomplished by transfer of a stable, replicating portion of plasmid DNA

to plant cell.

(b) These are used for introduction of nucleic acid into plants. Liposomes are

small lipid sacs containing plasmids are prepared artificially.

Figure depicting basic structure of liposomes

(c) Microinjection : Delivery of nucleic acid to cells is done through special

capillary needles, pumps, micromanipulators inverted microscope and

other equipment.



(d) Mild sonication (20Ktp3) has been used to facilitate the uptake and

transient expression of a chloramphenical acetyltranoterase (CAT) gene

inprotoplast, or intact cells.

(e) This method is based on the use of the short electrical pulses of high field

strength. Electroporation causes the uptake of DNA into protoplast by

temporary permeabilization of the plasma member to macromolecules.

(f) Direct uptake it stimulated by PEG. It involves mixing of freshly isolated

cells

Addition of 15-20% PEG

Mixture incubated for 30 mins.

washed & plated for growth

(g) An UV laser microbeam is used. A 343 nm beam is directed through an

adjustable vents into the optical path of on inverted microscope. This laser

beam can make holes in any part of cell which is in focus. Laser

micropuncture of the cell wall and plasma membion allows uptake

plasmid DNA.

(h) In this vortexing plasmid DNA and plant cells with silicon carbide fibre

(0.6 um and 10-80 um in length). During vortex, silicon fibres penetrate

cells and create five holes permitting entry of DNA.

Q.3 What is Cell Immobilization and give its application.



Ans.: Cell Immobilization : This technique is based on the confinement of biocatalyst

on or within a matrix by entrapment, adsorption or covalent linkage.

Methods on immobilization are : -

(1) Chemical Binding

(a) Binding to Carrier

(i) Covalent

(ii) Adsorption

(b) Cross-linking

(i) Pure Cross Linking

(ii) Co-Cross Linking

(2) Physical Retention

(a) Matrix Entrapment

(i) Fibre

(ii) Beads

(b) Membrane Enclosing

(i) Membrane Reactors

(ii) Encapsulation



Three types of Entrapment System are used with Chemical Binding :-

(a) Gel formation by iomic linking on a charged polymer (Covalent linkage to

a polymer).

(b) Gel formation by cooling off a heated polymer (entrapment within a

polymer).

(c) Gel formation by chemical rxns. (Gross linking, radical polymer).

Some of the Matrix which is being used for the Entrapment are :-

(a) Agarose (c) Carrageenan

(b) Alginate (d) Llrethane Polymers

(e) Phenylene Oxide

This Immobilization Process is being carried out in Bio-Reactors. These are:-

(i) Packed bed Reactors

(ii) Well mixed Reactors

(ii) Fluidized Bed Reactors

(iv) Membrane Type Reactors

The advantages of using Immobilization Technique are :-

(a) This increase the cell viability and stability in the cultures.

(b) Immobilization provides protection from mechanical and osmotic stress.

(c) Plating efficiency of immobilized cells is increased to money folds.



(d) Isolated and immobilized single cells can be cultured as single cells for

prolonged period.

(e) This is an carrier way of separation of biocatalyst from the product.

The major disadvantage of this technique are :-

(i) This technique is applicable for selected cell lines.

(ii) Knowledge of matrix is highly recommended.

The two most important applications are :-

(a) Enhanced production of secondary metabolites

(b) Biotransformation can be carried out.

Q.4 Explain Bioreactor, its applications and advantages.

Ans.: Bioreactor is a large culture vessel made up of glass for use at laboratory scale but large scale bioreactors are made up of stainless still.

This instrument is with:-

(a) pH controller

(b) Control unit for

(i) Dissolved Oxygen

(ii) Gas Flow Rate

(iii) Agitation

(c) Temperature

(d) Foaming



Fig. : Depicting Various Parts of Bioreactor

The various factors which affect the growth in bioreactor are :-

(a) Gas - Liquid Mass Transfer : Maintaining a constant Oxygen mass

transfer coefficient is basis of Many scale-up bio-processes.

(b) Shear : It refers to forces exerted on the surface of a body in direction

parallel to surface.

(c) Mixing : Mixing of the dissolved nutrients of the culture medium, is an

important factor.

Bioreactors can be basically classified into following two types :-

(A) Mechanically Agitated Bioreactors :

These are most commonly used for large scale culture of plant,

animal and microbial cells.

In this type, the medium is agitated with the help of a mechanically

driven impeller.

Two types of impeller used are :-



(i) Flat Blade Turbine Impeller : Used in bactrial culture

(ii) Marine Propeller : Used when low shear mixing is required.

(B) Preumatically Agitated Bioreactors : These are of two types :-

(i) Bubble Coloumn : In this, air is bubbled at the base of the coloumn

and medium is agitated with this.

(ii) Air Lift : Gas is sparged in the riser section and after the gas

disengages at the top of the column at the medium then flows

downward in the down corner section.

Circulation in the air-lift bioreactor promotes better mixing.

Bioreactors are used for the production of secondary metabolites, which can be

done as :-

(a) Shikonin Production :

Used as a remedy for various skin ailments and as a dye for skin &

cosmetics.

It‘s synthesized in cells of lithospermum crythrorhizon.

In this from shikonic acid and isoprenoid pathway.

Pathway for Shikonin Production

(b) Anthocyanin Production :

They are natural pigments and are safe for human health.

Have a high potential utility value as a food additive and marker.

They accumulate in small amounts.

Generally, production requires irradiation of light.

Some of the sps from which extraction is being done.

(i) Dacus Carota

(ii) Vitis Uinifera

(iii) Ajuga Reptans

(iv) Vitis Hybrid

Various method applied for its production are :-



(i) Shake Flask Cultures

(ii) Continuous Cell Aggregate Cloning

(iii) Biomass Production in Fermentors

(c) Organ Culture :

It‘s the micro-propagation of number of plant sps and various parts

of plants.

The culture of differentiated plant tissues is strongly affected by

four factors :-

(i) Moisture (ii) Temperature

(iii) Soluble Nutrients (iv) Agar

(d) Hairy Root Culture :

(i) It‘s an alternative approach.

(ii) Hairy root culture has a high biosynthetic capacity as compared to

unorganized cultures.

(e) Indole Alkaloids Production :

(iii) Produced by in-vitro cultures of cantharanthus rouses.

(iv) Two stage culture systems were used.

(f) Rosamarinic Acid Production :

(i) It has been found in families lamacal and 60 raginaceal.

(ii) This only occurs in colues blumee.

The limitations of bioreactors‘ are :-

(a) This is a costly technique

(b) Plant cells are large in size, sensitive to shear, this limits them to be used in

bioreactors.

Q.5 Explain the Commercial Production of Secondary Metabolites using Cell

Cultures.

Ans.:

Plant cell suspension culture is potential technique for synthesis of secondary

metabolites.



It a plant cell are cultured under conditions that allow the expression of a

certain degree of differentiation, they have the potential to produce either de

novo or by biotransformation of specific precursors, a wide range of

secondary products.

Types of suspension cultures :-

(1) Batch Culture :

It‘s a cells suspension culture grown in a fixed volume of nutrient culture

medium.

Cell suspension increases in biomass by cell division and cell growth until

a factor in the culture environment + (nutrient or Oxygen availability)

becomes limiting and the growth ceases.

The cells in culture exhibit the 5 phases :-

(i) Lag Phase

(ii) Exponential Phase

(iii) Linear Phase

(iv) Deceleration Phase

(v) Stationary Phase

(2) Continuous Culture :

A culture is continuously supplied with nutrients by the in flow of fresh

medium but the culture volume is normally constant.

Its of two types :-

(i) Open Continuous Culture : This is the culture in which inflow of fresh

medium is balanced by outflow of corresponding volumes of culture

including harvest of cells.

These may be of 2 types :-

(a) Chemostat : Growth rate and cell density are held constant.

Growth limiting nutrients are present.

(b) Turbidostat : Medium flows in order to increase turbidity so as to

maintain culture at fixed.



Optical density of suspension :

(ii) Closed Continuous Culture : In this cells are retained and inflow of fresh

medium is balanced by outflow of corresponding volumes of spent medium

only.

(3) Semi – Continuous Culture : In this type of culture, the inflow of fresh medium

is manually controlled at infrequent intervals by ―drain and refill‖ process, such

that the volume of culture removed is always replaced by an equivalent volume

of fresh medium.

Q.6 Define –

(a) Cryopreservation

(b) Ex-Situ Conservation of Germplasm

Ans.: Cryopreservation : It means preservation in the frozen states. This is generally

meant solid CO2 (-79˚C), in low temperature deep freezers (-80˚C).



Fig. : Standard Protocol for Cryopreservation of Shoot Tips

Ex-Situ Conservation of Germplasm: Chief mode of preservation of germplasm,

providing the suitable condition.

Ex – Situ

Botanical

Gardens

Nurseries Seed Bank In-Vitro Freezing

Method

At Room

Temperature

At Cold Temperature

Slow Growth Technique Cryopreservation

Cells or tissues can be stored at non-freezing conditions in slow growth state rather than at optimum level of growth.

This can be applied to tissue culture by :-

(a) Temperature

(b) How Oxygen Pressure

(c) Hormones

(d) Osmotic Inhibitors

Preservation in the frozen state.

Application of this techniques are:-

(a) Conservation of genetic material.

(b) Freeze storage of cell cultures.

(c) Maintenance of disease free stocks.

(d) Cold acclimation and frost resistance.

Q.7 What are Microbial Secondary Metabolites?

Ans.:

Secondary metabolites are the microbial products which are produced during

the idiophase of microbial growth.



These are secreted when depletion of one or more nutrients is caused in the

culture medium.

Secondary metabolites are not required by the micro – organism for their

growth.

These are called Growth Independent Metabolites.

The two major Secondary Metabolites :

(a) Toxins

(b) Antibiotics

(a) Toxins : Types of toxins are :-

(i) Bacterial Toxins :

There are proteinaceous in nature.

They interrupt the metabolism of host cells.

Example : Streptomyces Insecticidal Activity

(ii) Fungal Toxins (Mycotoxins) :

It plays an important role in production of may products sucha s

enzymes, organic acids etc.

Mycotoxins refers to secondary metabolites.

Example : Claviceps Fusarium etc.

(b) Antibiotics :

The growth inhibition of the former micro-organization was mediated

by secretion of toxic metabolites.

The metabolite was termed as ‗antibiotic‘ and the phenomenon of act of

growth inhibition by antibiotics as ‗antibiosis‘.

The antibiotics are defined as ―the complex chemical substance‖, the

secondary metabolites which are produced by micro-organism and act

against other micro-organism.

The antibiotics are produced in culture medium during idiophase due

to depletion caused by one or more nutrients in the medium.

Example : Penicillin, Cephalosporin, Tetracycline‘s etc.



H S CH3

R – CO – NH CH3

O N

β- Lactam Ring

Penicillin

□ □ □



Sources and Types of Antitumor Compound, Food Additives and

Insecticides

Q.1 What are the sources and types of –

(i) Antitumor Compounds

(ii) Food Additives

(iii) Insecticides

Ans.: Antitumor Compounds : Plants have been used in the treatment of cancer,

concentrated crude plant extract is used for their inhibitory activity against tumor

systems.

Plant tissue culture provides and excellent system to produce such

compound in controlled conditions and to study their production,

regulation, bio-synthesis and bio-transformation.

Among the anticancer agents of natural origin vincristine vinblastine,

podo-phyllotoxins and taxol are currently used clinically.

Examples :

o Baccharis Megapotamica Baccharin

o Brucea Antisenterica Bauceantine

o Catharanthus Roseus Vincristine & Vinblastine

o Taxus Brevifolia Taxol

Food Additives : These are the compounds which are natural origin and are used

as additional flavours, sueuctner and colours.

The synthetic food additive are harmful to body so natural are produced

through conventional methods.

CHAPTER-5

C



But conventional methods were not successful due to several reasons such

as :-

(i) Agroclimatic Conditions

(ii) Seasonal Specificity

(iii) Diseases

Biotechnological methods of production of foods additives are :-

(i) Plant All Culture

(ii) Tissue Culture

(iii) Organ Culture

Examples :

Plant Sps. Product

(A) Colours

Dacus Cartota Anthocyanin

Ceuphorbia Mili Anthocyanin

(B) Flavours

Allium Cepa Onion Flavour

Vanilla Planifolia Vanilla

(C) Sweetner

Stevia Rebandiana Stevioside

Thauimatococcus Danielli Thaumatin

Insecticides : Natural insecticides of plant origin are effective against a wide of range of

insects, many of which cannot be successfully controlled by synthetic insecticides.

Commercial insecticides of plant origin include pyrethrins, notmnoids

nictonine, neemix, azatin and quassin.

Unlike synthetic insecticides natural compounds are relatively non-

harzadesus to human beings and other mannals.



Plant insecticides generally exert their effect by interfering with physiology

of insects, affecting their nervous system, hampering their normal

development and acting as antifeedents.

Insecticides produced through in vitro method are :-

Plant Species Insecticide

o Azadirachta Indica Azadirachtin

o Tagetes Erecta Pyrethins

□ □ □



Products of Traditional Medical System

and Significance in Plant Biotechnology

Q.1 Name the products of Traditional Medicinal System and their significance in Biotechnology.

Ans.:

The medicinal plants in secondary plant products are termed as medicinal or officinal plants.

These secondary metabolites or products exert a profound physiological effect on the mammalian system and thus are known as active principles of plant.

The physiological effect of these active principles is used for curing ailments and therefore these are drugs of plant origin or natural drugs.

The use of crude drugs of plant origin is used in the Indian system of medicine or ‗Ayurveda‘.

Crude Drugs : Unpurified preparations of active principles, plant extract or sometimes powdered plant material.

Efforts are made to know the exact chemical nature of these drugs.

To determine the chemical nature of such compound, isolation of the substance in pure form using various separation techniques, chemical properties and spectral characteristics.

Purified secondary products are used in exact proportions in allopathic medicines.

Examples :

Plant Species Insecticide

o Acetyldigioxin Digitoxin

o Aescin Digioxin

o Allontoin Ephedrine

□ □ □

CHAPTER-6

C



Multiple Choice Questions

1. Secondary metabolites are produced by-

(a) Carbohydrates

(b) Primary metabolite

(c) Lipid

(d) Proteins

2. Morphine is an alkaloid obtained from-

(a) Opium poppy

(b) Papairs

(c) Catharanthus roseus

(d) Voacanga Africana

3. The precursors for biosynthesis of pyrrolidine alkaloids are

(a) Lysine

(b) Tyrosine

(c) Quindic acid

(d) N-methyl-s-pyrollinium

4. Tryptophan is the precursor of biosynthesis of-

(a) Cinchona alkaloide

(b) Tyrosine

(c) Indole alkaloide

(d) Catharanthine

5. Papavarine is obtained from-

(a) Tyrosine nicotine

(b) Nicotine

(c) Ornithine

(d) Pyrrolidine

6. Nicotine is extracted from which part of the plant.

(a) Roots

(b) Leafs

(c) Steam



(d) Hairs

7. Resins are produced in-

(a) Roots

(b) Ducts or glands

(c) Steams

(d) Bark

8. ―Osmophoses‖

(a) Special glands or cells

(b) Bark of medicinal plant

(c) Medicine obtained from poppy

(d) Essential oil from same plant

9. Anthocyanins are indicators of-

(a) Stress

(b) Salt resistance

(c) Temperature

(d) Water resistance

10. Anthocyanins are stable at-

(a) Acidic pH

(b) Basic pH

(c) Netural at 7

(d) None of the above

11. Vanilla flavor is obtained from ………………… (vanilla planifolia)

12. Lemonades are-

(a) Insecticidal compounds

(b) Antitumor

(c) Sweeteners

(d) None of the above

13. ……………. are used as insecticides and as fish poison in tropical countries

(Retinoid).

14. In animal ………… is converted into cholesterol (steroids).



15. …………. dissolve in water to give a soapy solution (saponine)

16. Saponins are soluble in water and can be extracted with …………. (Water).

17. The genus agave is a member of the family ………. (Agavaceae).

18. Micrometry is a process ……….. (by which the measurement of cells is carried out

through a microscope).

19. The unit of measurement of micrometry is ………. (0.001 mm or 1 micrometer).

20. Autoradiography is a technique based on the use of …………. (Radioactive

isotopes).

21. …………. is a technique to separate molecules on the basis of difference in size,

shape, mass, charge and adsorption properties (chromatography).

22. Ant metabolic compounds are inhibitors of ………….. (Nucleic acid synthesis).

23. Betalaines are characteristic pigments of …….. (angiosperms)

24. Thaumatins are sweet proteins (proteins) present in fruits.

25. Carbohydrates are incorporated at ………. % concentration in the medium.



Key Terms

1. Plant Secondary metabolites- PSM are a large group of different chemical

compounds which after perform vital function in the plant.

2. Elicitor- A molecule produced by the host which induces a response by the pathogen, conversely, an elicitor can be produced by a pathogen eliciting a response in the host.

3. Enzyme- A protein produced by living cells that can speed up specific biochemical reaction but which itself remain unaltered.

4. Primary metabolites- A primary metabolite is a kind of metabolite that is directly involved in normal growth, development.

5. Antioxidant- A substance that inhibits oxidation.

6. Chromatography- Separation of a mixture by passing it is a solution or

suspension or as a vapor through a medium in which the components moves at different rates.

7. Immobilization- Fixation of a body part in order to promote proper healing. 8. Enzyme immobilization- The covalent attachment of an enzyme to solid matrix

so that it cannot move. 9. Hairy root- A phase of crown gall during which there is abnormal development

of fine fibrous roots. 10. Bioreactor- An apparatus in which a biological reaction or process is carried out. 11. Alkaloids- Any of various organic compounds normally with basic chemical

properties and usually containing at least one nitrogen atom is a heterocyclic ring. 12. Flavonoids- Any of a large class of pigments having a structure based on or

similar to that of flavonoids. 13. Steroids- Any of a large class of organic compounds with a characteristic

molecular structure containing four rings of carbon atom. 14. Detoxifications- The process of, removing toxic substances or qualities. 15. Cell culture- Growing cell artificially in lab or inverted. 16. Cryopreservation- is a process where whole tissue or cell is preserved by cooling

to low sub zero temperature.



17. Food additives- are substances that are added to enhance food‘s taste and appreonce.

18. Photochemical- The Chemistry of the effects of light on chemical systems. 19. Saponins- A toxic compound that is present is a soapwort and makes foam when

shaken with water. or

Any of various plant glycosides that form soapy lathers when mixed and agitated with wates, used in detergent

20. Microbial secondary metabolism- Secondary metabolites are produced by microbes, plants and fungi and animals but not by all of them.



Case Study

Case Study Of Plant Secondary Metabolites

Although plant secondary metabolites are generally associated with plant defense responses

against herbivores and pathogens, these unique compounds can be involved in a broad array of

ecological functions (Bertin et al. 2003). Plants release secondary compounds into their

environment that change soil chemistry, thus increasing nutrient uptake or protecting against

metal toxicity (Dakora and Phillips 2002; Hawes et al. 2003). Chemical signals are essential for

mediating interactions between plant roots and nonpathogenic soil bacteria, including dinitrogen-

fixing bacterial symbionts (Dakora and Phillips 2002; Marx 2004). However, microbes and

herbivores are not the only organisms that are influenced by plant secondary metabolites. There is

considerable evidence to suggest that plant-derived chemical compounds are also involved in

direct communication between plants (Singh et al. 1999; Chamberlain et al. 2001; Bertin et al.

2003; Palmer et al. 2004; Weir et al. 2004). While this phenomenon was first documented in

approximately 300 BC by Greek and Roman writers, it was not until 1937 that Hans Molisch

coined the term "allelopathy" to describe such plant-plant interactions (Rice 1984; Willis 2004).

In the current literature, the term allelopathy can describe any direct or indirect effect of plant

chemical compounds on another plant or microbe, although it is most often used to refer to

chemical-mediated negative interference between plants (Willis 2004).

Plants use a variety of mechanisms to release secondary compounds into their surrounding

environment (Singh et al. 2003). Numerous compounds are released from decomposing plant

litter as leachates into the soil. Nearly all living plant tissues actively release secondary

compounds as volatiles, such as compounds that generate the scents associated with flowers or

pungent leaves, although roots can also actively release volatiles (Steeghs et al. 2004). Finally,

plants use up to 30% of their photosynthate in the production of root exudates, which affect the

local soil environment, termed the rhizosphere (Bertin et al. 2003). Each of these processes may

release chemicals that mediate allelopathic interactions between plants.

Renewed interest in allelopathy has stemmed from the discovery that spotted knapweed

(Centaurea maculosa), an invasive exotic weed introduced to North America, releases phytotoxic

secondary metabolites into the environment that inhibit the growth and germination of North

American plant species (Bais et al. 2002, 2003; Perry et al. 2005b). Spotted knapweed is an

outcrossing perennial of the aster family. The weed is native to Europe where it is not a dominant

or problematic species. However, in the northwestern United States it has become one of the

worst invasive weeds, infesting over 4.5 million acres in Montana alone (Mauer et al. 2001).

Spotted knapweed often colonizes disturbed areas in North America, but it also invades

rangelands, pastures and prairies, where it displaces native species and establishes dense

monocultures (Figure 1). As early as 1962, allelopathic interactions were proposed to be

responsible for the aggressive behavior of this weed (Suchy and Herout 1962; Kelsey and Locken



1986). Mounting evidence suggests that spotted knapweed, in addition to containing potentially

toxic compounds in its leaves (Kelsey and Locken 1986), also exudes phytotoxins from its roots

(Bais et al. 2001, 2002; Ridenour and Callaway 2001).

Allelopathy in Centaurea maculosa

Figure 1 Spotted knapweed (Centaurea maculosa) populations. In its native

European habitat, spotted knapweed (center of photograph) coexists with

numerous other plant species (left panel). In its introduced range of North

America, spotted knapweed is capable of displacing natives and forming dense

monocultures (right panel). (Photographs taken by Giles Thelen and Dean

Pearson; www.plantecology.org.) (Click image to enlarge.)

Phytotoxins produced by invasive exotic plants such as spotted knapweed may explain the

remarkable success of some of these plants when transported to a new continent. The "novel

weapons hypothesis" for plant invasions proposes that some invasive plants produce novel

chemicals that are more effective against neighboring species in their new environment than in

their native environment (Callaway and Aschehoug 2000; Callaway and Ridenour 2004). While

species in the native environment may have evolved resistance to the phytotoxins, species in the

new environment have only recently come into contact with the phytotoxins and may not have

had time to evolve resistance. Lack of allelochemical resistance in the new range may give some

exotic plants a competitive advantage, allowing them to displace native species. To examine the

effects of spotted knapweed root exudates on native North American plants, Ridenour and

Callaway (2001) used activated carbon to adsorb organic compounds in the soil. Activated carbon

reduced the negative effect of spotted knapweed on Idaho fescue (Festuca idahoensis), suggesting

that chemicals in spotted knapweed root exudates may give knapweed a competitive advantage

over native species. However, to demonstrate the role of allelopathy in spotted knapweed

invasion, allelochemicals produced by knapweed had to be identified.



Identifying Allelopathic Compounds

Identifying compounds involved in allelopathic interactions requires complex experimentation,

especially when the chemicals of interest are exuded belowground. To isolate and characterize

compounds released in spotted knapweed root exudates, plants were grown in sterile liquid media

under laboratory conditions (Bais et al. 2002). A bioassay was developed in which media from

spotted knapweed cultures was applied to other plants, and effects of the media were recorded

over time. Media containing knapweed exudates inhibited the growth and germination of several

plant species, suggesting the presence of phytotoxins. To determine which compound(s) in the

exudates were responsible for the phytotoxic activity, the media was extracted with organic

solvents and fractionated by high performance liquid chromatography (HPLC). These fractions

were used in a similar bioassay and the phytotoxic activity was found to be confined to one

chromatographic fraction containing a single peak, suggesting that a single compound was

responsible for the phytotoxicity. The active fraction was analyzed by nuclear magnetic resonance

(NMR) and mass spectrometry (MS) to identify the specific compound of interest.

Using this approach, the phytotoxin present in spotted knapweed root exudates was identified as a

racemic mixture of (±)-catechin (hereafter catechin) (Figure 2). Bioassays with the two

enantiomers revealed that they have different effects. (−)-Catechin is a potent phytotoxin,

whereas (+)-catechin is a weaker phytotoxin with some antimicrobial activity (Bais et al. 2002,

2003; Veluri et al. 2004). Purified catechin from spotted knapweed root exudates and

commercially available catechin acted similarly against a wide variety of plant species in

bioassays, suggesting the chemical identification was correct. While catechin was identified as

the principle phytotoxin in spotted knapweed root exudates, other chemicals in spotted knapweed

root exudates or plant tissue may have similar phytotoxic properties. Further, spotted knapweed

may produce other phytotoxic compounds when grown under more realistic field conditions, as

opposed to the highly artificial laboratory conditions used in these experiments. In the model

plant Arabidopsis thaliana, distinct root exudation profiles are associated with different stages of

development (Walker et al. 2003); thus it is possible that spotted knapweed may secrete different

phytotoxic compounds at different stages of development.

Figure 2 Chemical structure of (+)-catechin and (−)-catechin. (Click image to

enlarge.)

Ecological Relevance of Allelopathic Interactions



Identification of the phytotoxin catechin in spotted knapweed root exudates suggests that

knapweed may participate in allelopathic interactions. However, it is also necessary to assess the

ecological relevance of catechin production. To this end, a number of studies have examined soil

catechin concentrations in areas invaded by spotted knapweed. Several studies reported high soil

catechin concentrations (mean > 0.5 mg g-1

) in spotted knapweed infestations (Bais et al. 2002,

2003; Perry et al. 2005a; Thelen et al. 2005; Weir et al. 2006). However, recent studies have

found very low concentrations or no catechin in knapweed-infested soils (Blair et al. 2005; Perry,

unpublished data). These inconsistencies could be due to seasonal or annual variation in secretion

patterns, differences in soil physical or chemical properties, variation in soil microbial

communities, or differences in soil collection, storage and extraction methodologies.

Furthermore, phenolic compounds such as catechin tend to oxidize very rapidly (Appel 1993)

making quantification of soil catechin difficult and unreliable. The development of new

methodologies to sample the actual secretion of plant secondary metabolites over time would

improve our ability to measure these compounds in the field.

Laboratory experiments examining the effects of catechin on plant germination and growth

suggest that native North American grassland species vary considerably in sensitivity to catechin

(Perry et al. 2005b). Further work on two catechin-resistant native species, Gaillardia grandiflora

and Lupinus sericeus, suggests that these species may produce root exudates that detoxify

catechin (Weir et al. 2006). When catechin-sensitive species are grown in Gaillardia root

exudates under laboratory conditions, they become less sensitive to catechin. Further, catechin-

sensitive species growing in spotted knapweed-infested fields are more successful when near

Lupinus plants. Both Gaillardia and Lupinus exude relatively high concentrations of the organic

acid oxalate, which may act as an antioxidant to reduce catechin toxicity. Oxalate concentrations

in Gaillardia root exudates increased in the presence of catechin, and application of oxalate to the

roots of catechin-susceptible species reduced the effects of catechin (Weir et al. 2006).

Identifying catechin-resistant species and understanding the mechanisms of catechin resistance

may facilitate the development of more effective spotted knapweed management strategies.

Molecular Mechanisms of Allelochemicals

The mechanism by which catechin acts as a phytotoxin at the molecular level remains unknown.

Some clues have been obtained by analyzing the effects of catechin on plant roots in the

laboratory (Bais et al. 2003). When catechin is applied to susceptible plant roots, cytoplasmic

condensation—a signature of cell death—initiates at the root tip and spreads upwards in a wave.

After 45 minutes, the entire root tip is dead, as visualized by fluorescence vitality staining. It was

hypothesized that this wave of cell death might be accompanied by the accumulation of reactive

oxygen species (ROS), as is often found in the plant hypersensitive response to pathogen

infections. Using a fluorescent dye to visualize ROS production, it was found that catechin

treatment caused an immediate increase in ROS that cascaded up the root, preceding cell death by

ten minutes. When strong antioxidants were applied to roots in addition to catechin, no increases

in ROS or cell death were observed, suggesting that catechin causes root cell death indirectly by

facilitating ROS production. In addition, a spike in intracellular calcium ions (Ca2+

) was found 30

seconds after catechin treatment. Ca2+

is an important secondary messenger in many signaling

pathways including plant stress response (Orrenius et al. 2003). To understand more about the



effect of catechin on these signaling pathways, gene expression analysis was employed (Bais et

al. 2003). The model plant, A. thaliana, is susceptible to catechin, making it possible to use

microarray analysis to identify gene expression changes in response to this phytotoxin. Ten genes

were strongly upregulated in A. thaliana roots ten minutes after catechin treatment, but returned

to normal levels after one hour, suggesting that these genes are involved in the plant response to

catechin that leads to ROS production and eventually cell death. Of the ten genes identified, four

encode transcripts of unknown function. A. thaliana t-DNA mutant lines corresponding to eight

of the identified genes are available and may prove useful in understanding how catechin leads to

cell death.

Understanding plant secondary metabolite biosynthesis, release, and mechanistic action can

provide insights into larger ecological questions. By using spotted knapweed as a model system,

our laboratories are attempting to understand allelopathic interactions and their influence on plant

invasion at molecular, physiological, and ecological levels. Ultimately, the ability to silence the

production and secretion of catechin or any other allelochemicals produced by spotted knapweed

using transgenic or RNA interference technology will provide a conclusive test of whether

allelopathy plays a strong role in knapweed invasion.



M.Sc./B.Sc. (Part II) Examination, 2011

(Faculty of Science)

(Common to Three and Five Year Integrated Course)

BIOTECHNOLOGY

Paper BT-601

Plant Secondary Metabolites

Time.: 3 Hours Max. Marks : 50 Attempt FIVE questions in all, including question No. 1 which is compulsory, selecting ONE question from each Section. Each question carries equal 10 marks. 1. Answer the following in maximum two lines:

(i) Define photochemical

(ii) Write two functions of steroids.

(iii) Name different types of chromatography

(iv) What are immobilized cells?

(v) Who discovered Penicillin? 1 x5

Fill in the blanks:

(vi) ………….and ………….are natural insecticides.

(vii) Taxol is………….compound.

(viii) Secondary metabolites are obtained from………..

(ix) Immobilization of invertase was first of all developed by

…………and………….

(x) ……………..is a technique to enhance the accumulation of secondary

metabolites in plant tissue cultures. 1 x 5

Section-A

Year-2011



2. Write down the basic tools and techniques used for the isolation and separation of

secondary metabolites. 10

3. Write short notes on the following:

(i) Thin layer chromatography

(ii) Spectrophotometry 5+5

Section-B

4. What are the different types of alkaloids extracted from plants? Write down their

functions in plants and uses for human beings. 10

5. Describe briefly :

(a) Saponins

(b) Production of secondary metabolites under stress conditions. 5+5

Section-C


(a) Microbial secondary metabolites

(b) Cryopreservation 5+5

7. What is genetic transformation for the production of secondary metabolites?

Describe its application for the commercial production of secondary metabolites.

10

Section-D

8. Describe the biotechnological approaches on the production of secondary

metabolites using cell cultures taking Ginkgo biloba as an example. 10




(a) Products from traditional medicinal system

(b) Gene expression in response to environmental stimuli. 5+5

****************



Bibliography

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730–741.

Bais, H. P., Vepachedu, R., Gilroy, S., Callaway, R. M., and Vivanco, J. M. (2003) Allelopathy

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1380.

Bais, H. P., Walker, T. S., Stermitz F. R., Hufbauer R. A., and Vivanco J. M. (2002)

Enantiomeric-dependant phytotoxic and antimicrobial activity of (±)-catechin: A rhizosecreted

racemic mixture from spotted knapweed. Plant Physiol. 128: 1173–1179.

Bertin, C., Yang, X., and Weston, L. A. (2003) The role of root exudates and allelochemicals in

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Blair, A., Hanson, B., Brunk, G., Marrs, R., Westra, P., Nissen, S., and Hufbauer, R. A. (2005)

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success. Ecol. Lett. 8: 1039–1047.

Callaway, R. M., and Aschehoug, E. T. (2000) Invasive plants versus their new and old

neighbors: A mechanism for exotic invasion. Science 290: 521–523.

Callaway, R. M., and Ridenour, W. M. (2004) Novel weapons: Invasive success and the evolution

of increased competitive ability. Front. Ecol. Environ. 2: 436–443.

Chamberlain, K., Guerrieri, E., Pennacchio, F., Pettersson, J., Pickett, J. A., Poppy, G. M.,

Powell, W., Wadhams, L. J., and Woodcock, C. M. (2001) Can aphid-induced plant signals be

transmitted aerially and through the rhizosphere? Biochem. Syst. Ecol. 29: 1063–1074.

Dakora, F. D., and Phillips, D. A. (2002) Root exudates as mediators of mineral acquisition in

low-nutrient environments. Plant and Soil 245: 35–47.

Hawes, M. C., Bengough, G., Cassab, G., and Ponce, G. (2003) Root caps and rhizosphere. J.

Plant Growth Regul. 21: 352–367.

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