central cotton research report)

8/4/2019 Central Cotton Research Report)

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INTERNSHIP REPORTINTERNSHIP REPORT

CCRI MultanCCRI Multaninin

Plant Breeding and GeneticsPlant Breeding and Genetics

ByBy

Rabia FarooqRabia Farooq

Ag.2005-114Ag.2005-114

88thth semester 2009semester 2009

University College of AgricultureUniversity College of Agriculture

MULTANMULTAN


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Dedication

To my parents and respected teachers by virtue of whose

prayers

I am able to reach at this position and especially my

mothers whose hand are always raised for pray for me."


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It is certified that Rabia Farooq D/o. Muhammad

Farooq Anwer,

B.Sc (Hons) Agriculture, Major PBG, Registration No. ag-

2005-114, has successfully completed her internship work

during her stay (from January 19 to March 31, 2009) at

Central Cotton Research Institute, Multan

Senior SupervisorSupervisorMr. Muhammad Arshad Mr. PeerIdrees KhanSenior Scientific Officer / Head Scientific OfficerPlant Breeding & Genetics Section Plant

Breeding & Genetics SectionCCRI, MultanCCRI, Multan


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Acknowledgements

Pakistan being an agricultural country is facing a decline in the Agriculture

Sector. Many reasons are behind this decline but the most important one is the lack of

technical expertise in this sector. Many efforts are being conducted (suggested) to over-

come this problem. Many Universities are now facilitating their students with the as far

as possible practical work. Generally, at Graduate level, more emphasis is being given to

the theoretical work. This attitude is destroying the quality of education and also general

concepts are becoming deteriorated. Since the 2002, University of Agriculture,

Faisalabad has started to make a struggle to provide quality education by sending their

Graduates to different institutions in order to develop their minds to practical side before

they actually enter into practical sector.

Being a student ofUniversity College of Agriculture BZU Multan, I will also

appreciate this activity. It has not only improved my basic concepts but also given me a

broad spectrum about agriculture sector. I am extremely thankful to Dr. Mushtaq

Saleem, Principal, University College of Agriculture Multan andDr. Syed Bilal Hussain,

Associate Professor, College of Agriculture Multan for selecting me for Central Cotton

Research Institute Multan. I feel myself luckiest student as working with most intelligent

and hardworking Researchers and Scientists of the country. I am extremely thankful to

my supervisorMr. Peer Idrees Khan for his keen interest, kind cooperation and valuable

suggestion throughout the course of my research.

I m highly indebted to Mr. Ch Rehmat Ali, head of the Plant Breeding and

Genetics section, Mr. Mohammad Afzal (S.S.O), Mr. Noor Ilahi (S.S.O),

Mr Mohammad Akbar(S.O), Mr. Khadim Hussain (S.O)

I wish to express my unfathomable sense of gratitude to Mr. Mohammad

Arshad, director, CCRI, Multan for his keen interest, Guidance and pleasant

temperament. Special thanks for him will always be due.


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Rabia FarooqAg-2005-114

8th semester

University College OfAgriculture

BZU Multan

Contents:

What is Agriculture

About cotton

Role of cotton in Pakistan Agriculture

Introduction of PCCC

Introduction of CCRI

Objectives of CCRI

Achievements of CCRI Multan

Sections at CCRI

Varieties released by Plant Breeding Section of CCRI

Multan. Brief discussion about sections

Breeding and genetics section

Cotton breeding methods

Cotton breeding technology

Cultivar maintenance

Variety evaluation

Maintenance of World Cotton Gene Pool:

Desirable features of cotton cultivars / Breeding objectives

of cotton plant

Activities performed at CCRI Multan


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What is Agriculture:

Agriculture is the branch of science which deals with the raising of crops and

rearing of animal for benefit of mankind.

Farming: the practice of cultivating the land or raising stock

The growing of crops and rearing of animals

the science or process of farming or cultivating the soil for the production of plants

and animals that will be useful to humans in some way.

Cotton:Cotton is a natural fiber that finds use in many products. These range from

clothing to home furnishings to medical products. As a result, cotton is always in

demand though its use is subject to the strengths and weaknesses of the overall

economy. It accounts for 8.2 percent of the value added in agriculture and about 2

percent to GDP.


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Cotton has 51 species in world, out of which 4 are cultivated and 47 are wild.These species are found in four countries of the world. Out of 51 spp. 3 are Asian,

12 are African (A,B,E or F genome), 17 are Australian(C or D genome), 19 are

American(AADD or D genome).

Four cultivated species are

G.hirsutum,

G.barbadense,


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G.arboretum,

G.herbaceum.

Importance: Cotton is the most important fiber crop ( silver fiber ) crop in the world. It

consist of hairs growing from the epidermis of the seed. The fiber is woveninto fabrics either solely or in combination with other twins.

Cotton replaces the wool during the 19th century but declined during the

last 50 years. Due to impact of synthetic fiber.

Its seed is the important source of oil, which can be used in the cooking &

margarine manufacturing.

The Role of Cotton in PakistanPakistan is the fifth largest producer of cotton in the world, the third largest

exporter of raw cotton, the fourth largest consumer of cotton, and the largest

exporter of cotton yarn. 1.3 million farmers (out of a total of 5 million) cultivatecotton over 3 million hectares, covering 15 per cent of the cultivable area in thecountry.

Cotton and cotton products contribute about 10 per cent to GDP and 55 per cent to

the foreign exchange earnings of the country.

Cotton production supports Pakistans largest industrial sector, comprising some

400 textile mills, 7 million spindles, 650 dyeing and finishing units, nearly 1,000

ginneries, 300 oil expellers, small scale oil expellers (kohlus). It is by any measurePakistans most important economic sector..


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Introduction of PCCC:

PAKISTAN CENTRAL COTTON

COMMITTEENazar Muhammad

GondalPresident

Federal Minister for Food & Agriculture,

Govt. of Pakistan

Dr.Zahoor Ahmad Baloch Vice President

Dr.Zahoor Ahmad Baloch Director Research

Gul Muhammad Secretary

The Pakistan Central cotton Committee (PCCC) emerged as incorporatedinstitution on the national horizon in 1948,with broad objective to concentrate its

efforts on bringing an improvement in growing , marketing and manufacturing ofcotton and cotton by-products through an extensive program of Research &

Development (R&D) in all its conceivable aspects.

The Committee is semi-autonomous body with the Federal Minister of Food ,

Agriculture and Livestock as the president.

SUBCOMMITTEES:

The PCCC is guided by four specialized Sub-Committees comprising members,

nominated by the Federal Ministry of Agriculture representing the Ministries of

Agriculture, Textile, Commerce, Finance and Industries.

The Four Specialized Sub-Committees namely

(a) Executive Sub-Committee (ESC),

(b) Agriculture Research Sub-Committee (ARSC),

(c) Technological Research Sub-Committee (TRSC),

(d) Marketing & Economic Research Sub-Committee MERSC)

are responsible for management as well as Research & Development (R & D) work of

PCCC


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INTRODUCTION TO CENTRAL

COTTON RESEARCH INSTITUTE

MULTAN The Central Cotton Research Institute, Multan was established in 1970 underthe supervision of Pakistan Central Cotton Committee, Karachi. This Institute was

established with the objectives of fastening both scientific and extension activities.

As Multan region is the core and central cotton producing area of Pakistan so this

Institute is not only working on increasing the yield of cotton crop but the qualityas well. The Institute has three stories building with offices and well-equipped

centrally air-conditioned research laboratories. There is separate section for stores,

ginning laboratories and, stores for implements. One green house, three-glass

houses are also available for conductance of research work off-season.

In order to facilitate the researcher to outside research activities, a well-

established library having 7000 books; and 32 national and international journals,is also available.

Front view of CCRI Multan


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OBJECTIVES OF CCRI

The main objectives of the Institute are as follows:

Study the cotton plant from botanical, genetical, pathological,entomological, physiological & chemical, and other relevant stand

points in a coordinated manner.

Identify problems of cotton growers and find out the remedial

measures.

Provide education and training in cotton production to extension,

research and teaching staff of government and non-government

organizations.

Educate, motivate and transfer cotton production technology from

research Institute to the cotton growers.

Provide training in cotton production technology to the developingcountries.

Provide technical support to the Pakistan Central Cotton Committee

in coordinating and developing a national programme for cotton

research and development


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Achievements of CCRI Multan

Followings are the major achievements of CCRI, MULTAN:

* Development of cotton varieties

* Cotton gene pool

* Wild species collection

* Cotton leaf curl virus resistance

* Chemical control of weeds

* Ridge planting

* IPM for cotton

* Consumptive use of water

* Fertilizer requirements

* Heat stress and yield analysis

* Transfer of technology

Sections at CCRI:

The Central Cotton Research Institute, Multan is working with following sections:


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Name of Section Year of Establishment

Cytogenetics 1970

Entomology 1970

Pathology 1970

Physiology 1970

Statistics 1970

Breeding 1973

Agronomy 1975

Fibre Technology 1976

Transfer of Technology 1983

1: CYTOGENETICS:The Section was established in 1970 with an objective of conducting fundamental

studies on the cytogenetic aspects of the genus Gossypium,to which belongscotton.

Transfer desirable genes of virus species in the cultivated species of cotton.

Study inter and intra genomic relationships in the genus Gossypium.

Create new variation in the Gossypium germplasm

Evolution of cotton varieties resistant to pest and diseases.

Maintenance of genetic stock of world collection of wild species

2: ENTOMOLOGY:

The Section was established in 1970. Its programme of work included both

fundamental and applied research as mentioned below:

Monitoring of bollworms population with sex pheromones and light trap.

Performance of different strains of cotton from CCRI against insect pests.

Training on cotton plant protection imparted to farmers and staff of

Agriculture Extension and Pesticide Companies etc. Evaluating performance of new insecticides received from pesticide

companies.

3: PLANT PATHOLOGY:

Cotton leaf curl virus was first observed in Pakistan on few plants during 1967


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Investigating were made on leaf curl virus of cotton revealed that evolving

resistant varieties could only the control of the disease.

Investigations were made to check the effect of CLCuV on yield, fibre

characteristic, sowing dates, and seed treatment.

Molecular characterization of CLCuV was done by PCR.

Investigation was also made on different inoculation methods on different

cultivars against bacterial blight of cotton and survival of bacterium

4: PLANT PHYSIOLOGY AND

CHEMISTRY:

Studies were carried out on plant nutrition, soil-plant-water relationships, heat

tolerance and plant-pesticides relationships.

Screening of heat resistant varieties

Development of drought tolerance

Nutrients availability in soil of different regions.

5: STATISTICS:

This Section assists all the sections of the Institute in experimental design and

statistical analysis of data. Report on prevailing drawbacks and quality of data of

NCVT is prepared annually. Computer programmes are written for the analysis of

data.

Designing and statistical analysis of the experiments.

To conduct research in sampling method.

To conduct survey on cotton production practices.

6: PLANT BREEDING AND GENETICS

Breeders ListName Qualification Designation


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Ch. Rehmat Ali M. Sc.(Hons) Agri. S.S.O / Head

Mr. Muhammad Afzal M. Sc.(Hons) Agri. S.S.O

Mr. Noor Illahi M. Sc.(Hons) Agri. PBG S.S.O

Mr. Muhammad Idrees Khan M. Sc. (Hons) Agri. PBG S.O

Mr. Muhammad Akbar M. Sc. (Hons) Agri. PBG S.OMr. Khadim Hussain M. Sc. (Hons) Agri. PBG S.O

The Section was established in 1973 and now working on following objectives:

Development of high yielding, early maturing, short statured, heat tolerant,

climate adaptive varieties with high lint percentage, desirable fibrecharacteristics and genetic inbuilt resistance against insect pests and

diseases especially cotton leaf curl virus.

Development of F1 commercial cotton hybrids. Maintenance of Cotton Gene Pool containing 1800 cotton genotypes (Local

and Exotic).

Multiplication of Basic seeds for seed companies (Government/Private).

Training of student (Internship) from various Universities/Colleges to get

experience for their fresh job.

To train the officers from various organizations (Government/Private)

about the updated application technologies.

To evaluate the performance of promising strains of different breeders of

Pakistan in National Coordinated varietal trial (NCV)

To evaluate the performance of commercial hybrids in National CommercialHybrids Trail (NCHT).

7: AGRONOMY:

Various agronomic practices were studied to develop most appropriate technologyof cotton production with minimum expenses. These included method of

cultivations, fertilizer and irrigation requirements, time of application of nitrogen

and method of application of nitrogen.

Soil management

Agronomic practices for cotton
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Land preparation

Optimum sowing time

Plant population

Irrigation

Fertilizer application Weed control

Organic farming

Low cost production technology

Relay cropping to get maximum profit

8: FIBER TECHNOLOGY

The Section was established in 1976. The main objectives of the section were toprovide the fibre testing facilities.

Testing of lint sample

Study the effect of environment on fiber quality in relation to boll setting.

Testing of fiber length. Fiber Strength, fineness, trash percentage in the lint

and color of the fiber

9: TRANSFER TECHNOLOGY:

The function of the Transfer of Technology Section is to disseminate research

findings of the Institute to various target groups including Agricultural ExtensionWorkers and Cotton Growers through multi-media publicity campaigns

comprising of training courses, on-farm training programs, printed material, radio

and TV broad-casts and press releases for the benefit of the farming community.

Dissemination of information on cotton production technology based on the

research findings of the Institute was continued to be delivered to varioustarget groups through multimedia integrated approach.

To carry out audience research for being feed back

produce source material for use in the training programme

Varieties released by Plant Breeding

Section of CCRI, MultanSince 1973 to update 10 varieties were released by Breeding Section their morphological

features are as under:


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Varieties Year of release1-CIM70 1986

2-CIM109 1990

3-CIM240 1992

4-CIM1100 1996

5-CIM448 19966-CIM443 1998

7-CIM446 1998

8-CIM482 2000

9-CIM473 2002

10-CIM499 2003

11-CIM506 2004

12-CIM707 2004

13-CIM496 2005

14-CIM534 2006

Cotton breeding methods:

There are basically three methods used throughout the world for any crop

improvement.

1. Introduction


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2. Selection

3. Hybridization

(i) Direct crossing

(ii) Three way crossing

(iii) Double crossing

(iv) Multiple crossing(v) Back crossing

Cotton Breeding Technology:

Pedigree method of breeding as detailed below was mainly used for development

for new varieties.

Single, three way and double crosses were attempted with specific

objectives. The bolls of each cross combination were bulked to make F1 seed.

F1 seed is planted to raise F2 crop.

Maximum single plant selection of desirable segregates is made in F2

because maximum variation occurs in F2.

The plants selected in F2 generation are planted in plant to progeny rows in

F3.

The plants selected from F3 are planted to progeny row in F4 to F6

generation.

In F6 desirable homozygous lines are selected as new strains.

The strains are extensively tested in micro-varietal & varietal trials at

CCRI, Multan and at Testing Centers

If the performance of a new strain is better compared with commercial

varieties, then it is registered with Federal Seed Certification and

Registration Department and its performance is further tested in National

Coordinated Varietal Trial, Director Cotton Research Trial.

On the basis of performance in VT, DCR, NCVT the expert

sub-committee consider the case and forward it for final

approval to Punjab Seed Council Chaired by Minister of

Agriculture, Punjab. After the approval by Punjab Seed

Council, the seed is given for general cultivation

The schematic diagram of variety development and

improvement is given as under:

Year Ist. Crossing generation P1 x P2

Year 2nd F1 generation Hybrid production


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Year 3rd. F2 generation Single plant selection

Year 4th. F3 generation Single plant selection

Year 5th. Year

7th.

F4 F6 generation Progeny row selection

Year 8th. F7 Better PRS grown in MVT

Year 9th. F8 Better MVT grown in VT

Year 10th. Year11th.

F9 F10 Better VT grown in ZVT,DCR, NCVT

Year 12th. Year13th.

F11 F-12 Seed multiplication and production of pre-basic

seed

Year 14th. F13 Basic seed

Fig--Schematic diagram of variety development using pedigree

selection

Cultivars maintenance:

The development of a cotton variety by Breeding Section and its maintenance

thereafter is an integral part of the whole breeding procedure, which can not be

set apart from breeder.

Maintenance is a cyclic repeated activity carried out during the

lifetime of a variety for preservation of its original characteristics specially

distinguishes features and economic traits. The cotton varieties deteriorate

in the absence of a proper system of maintenance and renewal of pre-basicseed is therefore essential.The deterioration occurs mainly due to mechanically as well as Genetically

controlled factors. The mechanical factors include mechanical mixing of seeds of

different varieties due to careless handling during planting, picking, marketing,ginning processing and storage. The genetic factors mainly include the

accumulation of negative mutations and spontaneous out crossing. There is

evidence that varieties which are more uniform in plant type deteriorate lessrapidly.

Variety evaluation:

Procedure:There we use Introduction, Selection and Hybridization methods.

Introduction:In introduction the genotype is introduced from the field.

Selection:In selection two types of selection is done:


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Mass selection

Pure line selection

Mass selection:In mass selection, we select superior plants on phenotypic basis.

Pure line selection:Pure lines are selected having desirable characteristics.

Hybridization:Cross between two different parents is hybridization

Direct crossing

Three way crossing

Double crossing

Multiple crossing

Back crossing

Maintenance of World Cotton Gene Pool:The genetic stocks of world Cotton collections comprising of about 1800

(Local and Exotic) genotypes were also maintained at breeding andGenetics Section. These genotypes are supplied free of cost to all cotton

breeders (to be utilized in breeding for variety evolution but not use as a

variety) /university students/private seed companies in the country and

abroad.

Purpose:

For obtaining mike, G.O.T, staple length, strength.

To incorporate desirable characters in one variety.

In CCRI 2000 cotton genotypes are present. These include varieties,

cultivars, promising strains collectively all called as genotype.

Desirable features of cotton cultivars /

Breeding objectives of cotton plant:

Cotton plant should have following desirable features:

High seed cotton yield

(a) more no. of bolls

(b) large boll size.

Earliness


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Tolerance to various insect pests. .

Resistant to various diseases.

Tolerance to high temperature and drought.

Adoptive to salinity and water logging.

Good seed qualities

(a) high oil contents

(b) high germination and vigor

(c) high maturity

(d) high seed index(e) gossypol free.

Adaptation to mechanical harvesting

(a) erect plant

(b) even distribution

(c) less lateral spread

(d) long fiber

(e) early and uniform maturity(f) large well opened fluffy bolls.

(g) Smooth leave.

Excellent fiber characteristics

(a) high GOT

(b) long staple length

(c) high fiber maturity

(d) high fiber fineness(e) high fiber strength

Activities performed at CCRI Multan

A. Field visits:

During my stay at CCRI, Multan following activities were also performed.

Practical demonstration about lay out.

Primary and secondary thinning activities.

Weed control by hoeing and weedicides.

Determination of CLCV in the field.

Checking nectariless, hairy, gossypol glands in cotton varieties.


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Demonstration of different instruments for ginning.

Checking the morphological characters of cotton plant.

Emasculation and pollination

Cotton picking

Ginning

Delinting of cotton seed

Seed index

Seed germination

Seed kernel weight

Bt test

Growing of plant in glass house:There in glass house pots were placed at different distance. Pot length was

1ft. The upper portion was of two inch and the lower one was 4inch.

Procedure:

In a pot there was a hole in lower surface and covered by a paper, the

basic purpose of this hole is aeration and the coming out of extra water. I

Took normal field soil or silt put it in the pot watered it. Stir it with

spatula. Sow seeds in it at different places. The depth of the seed should

be maintained for better germination. The seeds which were used forsowing firstly check out for virus presence. Add silt in it. Covered the pot

with plastic bag. When the germination started, than transfer the seedlings


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into field. When they start growing in the field thinning is done. There are

three types of thinning is used

Primary

Secondary

Tertiary

Primary:When the plant height goes to 6inches the primary thinning is done

Secondary:When the plant height goes to 9inches secondary thinning is done.

Tertiary:When the plant height goes to 12inches the tertiary thinning is done.

Primary and the final thinning is much more important than the second

one.

Fertilizer and spaying practices should be applied.

Temperature:35OC is maximum temperature and 22oC is minimum temperature which

should be maintained in the glass house.

Ideal temperature for boll setting is 25-28oC. Cotton is not a plant

basically it is a tree. It got germination after 3-4 days. After 30-40 days

squares come out. 20days are required for flowering and after flowering

30-35days are required for boll setting. Total days from germination to

maturity are actually 100.

Crossing techniques in glass house:

Cotton is an self pollinated crop. In cotton conventional emasculation and

pollination method is used.

Emasculation:

Firstly I selected a mature square, then I removed its petals and anthers

with the help of forceps. I covered the emasculated cotton square with

soda straw tube to prevent the entry of anthers.

Pollination:


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In the next morning I select a mature flower from another plant. I consider

that flower as a male and emasculated one was female. Male flower had

matured pollens like powdery appearance. Rubbed that flower on the

emasculated one and again covered with soda straw tube. I tagged that

pollinated flower with cross number and date.

After about 7 days mature boll was set.

Ginning:

Ginning is done to separate the seed from lint. Through lint and seed

weight we check the lint percentage of any sample. Often lint and seed

percentage are in the ratio of 40:60 respectively. For ideal variety lintweight should higher than seed weight.

Firstly I took sample of 100gm kapas. That sample had specific ginning

number. Ginning of that sample was done through ginning machine,

where seeds were separated from lint. Then seeds and lint were collected

separately. Weight the seed and lint individually.

From 100gm kapas 58.8gm seed and 40.2gm of lint was obtained. I repeat

that experiment on different varietal samples.

Precautions:Precautions should be taken during sowing, processing, bagging and seed

storage avoid to mechanical mixture.

Ginning scheme and evaluation of GOT %age:Theory:

GOT is the amount in %age of lint out of seed cotton. Generally GOT of

cotton equals to 40% or 49-50%


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Flow sheet diagram of ginning process:Sample cleaningweighing of sample saw machine/roller(ginning)

Lint(GOT)weighinglint+seed

Formula:GOT can be calculated by using the formula

GOT% = (weight of lint/weight of seed cotton) 100

Delinting of cotton seed:Commercial sulfuric acid is used for Delinting at the rate of 1 liter for Delinting of

10kg of fuzzy seed.

Took seed of cotton and delint it to remove the fuzz from it. Delinting was doneby dipping the seed in H2SO4 and stir it until the fuzz was removed. Delinting of

the seed with acid is done in order to remove fuzz as well as to remove seed born

disease.

Larva of pink boll worms was killed. After removing the fuzz washed the seed in

water again and again so that acid was removed from seed because acid may kill

the embryo. After that seed was dipped in water in a beaker for 6 hours to makethe testa of the seed softer. The seed which may settle down in the bottom are

good for germination called sinker however the seed which float on the top are not

good for germination are called floater seeds.

Seed index:

Weight of the 100 seeds in grams is called seed index. Seed are collected

randomly.

Variety having greater seed index it must have greater seed ratio and seed

rate.

Seed Germination:Theory:

Germination is a process in which a dormant seed renews its growth and

develops into seedling.

Seed when soaked, testa turns soft, radical develop into root and plumules

turns into shoot, by taking food from cotyledon and by absorbing

moisture. In wild species seed testa is hard which hinders the proper


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germination so we have to follow some alternate ways to improve their

germination.

Conditions necessary for germination are:

moisture

oxygen

optimum temperature (32oc) time of incubation ( 72hours )

Materials: beaker

cotton seed

wet newspaper

Incubator

Method:

I took 100 seeds of cotton. Soaked that seeds in petri dish having water.

Took a newspaper and immersed it in water.

Then I placed that seeds on wet absorbing papers in a row with

radicals ends in the lower direction.

I fold that paper into a roll and placed each roll in a beaker and

kept that beaker into incubator for 72 hours at the temperature of

32oC.

After 72 hours I count the germinated seed

Through this germination percentage is calculated easily.

Seed kernal weight:

I took 100 seeds from different varieties.

Cut the seed into two half by using surgical blade.

Removed the testa/seed coat and separated the kernel of the seed.

I separate the 100 seeds kernel and weight them by using the same

method.

That was seed kernel weight.


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Bt test:Intended use:The envirologix quickstix combo kit for Cry1 Ac & Cry2 A is designed to

extract & detect the presence of endotoxins.

The combo strips will recognize both Cry1 Ac & Cry2 A endotoxins in

separate region of the same strips.

How the test works:

Cotton crop that have been genetically modified with stacked Bt genes

express Bt toxins in their leaves and seeds. To detect these Cry1 Ac &

Cry 2A proteins with the envirologix quickstix combo strips, the sample

must be extracted and the proteins solubilized in the extraction buffer

provided.

Each combo strip has an absorbent pad at each end. The protective tape

with the arrow indicates which end of the strip to insert into the extraction

tube. The sample travels up the membrane strip & is absorb into the larger

pad at the top of the strip. The portion of the strip between the protective

tape and the absorbent pad at the top of the strip is used to view the

reaction.

Sample preparation:

To extract cotton leaf tissue:

Sandwich a section of leaf tissue between the cap & body of the

disposable tissue extractor tube. Snap two circular tissue punches

by closing the cap. Push the leaf punches down into the tapered

bottom of the tube with pestle. Write the sample identification on

the tube with a water proof marker.

Insert the pestle into the tube and grind the tissue by rotating thepestle against the sides of the tube with the twisting motion.

Continue this process for 20-30 seconds until the leaf tissue is well

ground. Add 0.5ml extraction buffer.

Repeat the grinding step to mix tissue with extraction buffer.

Dispose of the pestle.

Dont reuse pestle on more than one sample.

To extract cotton seed:


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Crush a single cotton seed.

Transfer to an extraction tube marked with sample

identification.

Add 0.5 ml extraction buffer.

Close the tube cap securely. Shake the tube for 20-30 sec using up & down motion ensuring

the crushed seed and buffer are well mixed.

Allow the solid material to settle at the bottom of the tube

The extract take on yellow to brown color when the sample are

prepared properly.

How to run the Quickstix Strip test:

Allow refrigeration canisters to come to room temperature

before opening Avoid bending of the strip.

Place the strip into the extraction tube as the sample will travel

up the strip.

Allow the strip to develop for 10 mints before making final

interpretation.

To retain the strip cut of and discard the bottom section of the

strip covered by the arrow tape.

Interpreting the results:

Development of the control lines within 10 minutes indicates that the strip has

functioned properly. Any strip that doesnt develop a control line should be

discarded and the sample retested using another strip.

There develop three types of lines should be noted properly

One line

Two lines

Three lines

One line:If the extract is from negative sample the strip will only show the one control line.

Two lines:If the extract contain either Cry 2A or Cry 1Ac proteins, the strip will develop two

lines

Three lines:


29/29

If the extract is from a sample containing both Cry 2A and Cry 1Ac proteins, a

total of three lines will appear.

Precautions:

Be sure to use a new tube for each sample tested.

Protect all components from hot and cold extremes of temperature when not

in use.

Dont leave in direct sunlight.

central cotton research report)

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