Laboratory Manual

BiologyClass XII

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FOREWORD

The National Council of Educational Research and Training (NCERT) is theapex body concerning all aspects of refinement of School Education. It hasrecently developed textual material in Biology for Higher Secondary stagewhich is based on the National Curriculum Framework (NCF)2005. The NCFrecommends that childrens experience in school education must be linkedto the life outside school so that learning experience is joyful and fills the gapbetween the experience at home and in community. It recommends to diffusethe sharp boundaries between different subjects and discourages rote learning.The recent development of syllabi and textual material is an attempt toimplement this basic idea. The present Laboratory Manual will becomplementary to the textbook of Biology for Class XII. It is in continuationto the NCERTs efforts to improve upon comprehension of concepts andpractical skills among students. The purpose of this manual is not only toconvey the approach and philosophy of the practical course to students andteachers but to provide them appropriate guidance for carrying outexperiments in the laboratory. The manual is supposed to encourage childrento reflect on their own learning and to pursue further activities and questions.Of course the success of this effort also depends on the initiatives to be takenby the principals and teachers to encourage children to carry out experimentsin the laboratory and develop their thinking and nurture creativity.

The methods adopted for performing the practicals and theirevaluation will determine how effective this practical book will proveto make the childrens life at school a happy experience, rather thana source of stress and boredom. The practical book attempts to providespace to opportunities for contemplation and wondering, discussionin small groups, and activities requiring hands-on experience. It ishoped that the material provided in this manual will help studentsin carrying out laboratory work effectively and will encourage teachersto introduce some open-ended experiments at the school level.

YASH PALProfessor and Chairperson

National Steering CommitteeNational Council of Educational

Research and TrainingNew Delhi21 May 2008

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PREFACE

The development of the present laboratory manual is in continuation tothe NCERTs efforts to improve upon comprehension of concepts andpractical skills among the students. The present laboratory manual willbe complementary to the textbook of Biology for Class XII.

The expansion of scientific knowledge and consequently the changein the system of education has led to the development of new methods ofinstructions. Today the stress is laid on the enquiry approach anddiscussion method instead of lecture method of teaching. Biology is nowsomething more than observation of living organisms. Study of Biologyincludes microscopic observations to reveal minute internal details of theorganism, biochemical testing to understand complex reactions takingplace inside the organisms, experiments with live organism to understandvarious physiological processes and even much more. In other wordsexperiments in Biology truly represents an interdisciplinary approach oflearning.

The new syllabus of Biology has been designed to cater to the needs ofpupil who are desirous of pursuing science further. The fundamentalobjective of this course is to develop scientific attitude and desiredlaboratory skills required for pursuing Biology as a discipline at this level.A similar approach has been taken while formulating the practical syllabusof Biology for higher secondary stage. The practical syllabus includescontent based experiments, which help in comprehension of the concepts.There are altogether twenty-five exercises in the present manual whichare based on Biology curriculum for Class XII. For each practical work,principle, requirements, procedure, precautions, observations, discussionand the questions are given in the book. The methodology of preparationof any reagent, if required, has been given alongwith the requirements,for the convenience of students and teachers. The questions are aimed todevelop learners understanding of the related problems. However, teachermay provide help in case the problem is found to be beyond the capabilityof the learner. Precautions must be well understood by the learners beforeproceeding with the experiments and projects. In addition to the coreexperiments enlisted in the syllabus for Class XII emphasis has also beengiven for pursuing Investigation Project Work. It is expected that theseinformations will motivate the students to take up independent projectwork on topics of their interest.

Appropriate appendices related to the observation and study oforganisms are given along with the experiment. International symbols forunits, hazards and hazard warnings are given at appropriate places inthe book. It is expected that this will make the learners more careful aboutthe environment and make them careful while dealing with the equipmentsand chemicals in the laboratory.

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It gives me a pleasure to express my thanks to all those who have beenassociated at various stages of development of this laboratory manual. It ishoped that this practical book will improve teaching-learning process inBiology to a great extent. The learners will be able to understand the subjectwell and will be able to apply the acquired knowledge in new situations.I acknowledge with thanks the dedicated efforts and valuable contributionof Dr Dinesh Kumar, coordinator of this programme and other team memberswho contributed and finalised the manuscript. I especially thank ProfessorG. Ravindra, Director (Incharge), NCERT for his administrative support andkeen interest in the development of this laboratory manual. I am also gratefulto the participating teachers and subject experts who participated in thereview workshop and provided their comments and suggestions which helpedin the refinement of this manual. We warmly welcome comments andsuggestions from our readers for further improvement of this manual.

HUKUM SINGH

Professor and Head

Department of Education in

Science and Mathematics

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MEMBERS

Animesh K. Mohapatra, Associate Professor, Regional Institute of Education,

NCERT, Ajmer

B.K. Tripathi, Professor, DESM, NCERT, New Delhi

C.V. Shimray, Assistant Professor, DESM, NCERT, New Delhi

N.V.S.R.K. Prasad, Associate Professor in Botany, Sri Venkateshwara College,

New Delhi

P.K. Durani, Professor (Retired), DESM, NCERT, New Delhi

Sunita L. Varte, Assistant Professor, DESM, NCERT, New Delhi

S.P. Sinha, Professor of Zoology (Retired), TM Bhagalpur University, Bhagalpur

V.V. Anand, Associate Professor , Regional Institute of Education, Mysore

MEMBER-COORDINATOR

Dinesh Kumar, Associate Professor, DESM, NCERT, New Delhi

LABORATORY MANUAL DEVELOPMENT TEAM

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The National Council of Educational Research and Training (NCERT) acknowledges the valuable

contribution of the individuals and organisations involved in the development of this laboratory

manual. The council also acknowledges the valuable contribution of the following academics for

reviewing and refining the manuscript of the laboratory manual: A.K. Sharma, Reader in Zoology,

University of Lucknow, Lucknow; Iswant Kaur, D.M. School, RIE, Bhopal; K. Muralidhar,

Professor, Department of Zoology, University of Delhi, Delhi; K.K. Sharma, Professor Department

of Zoology, M.D.S. University, Ajmer; M.M. Chaturvedi, Professor Department of Zoology,University of Delhi, Delhi; Nazir Ahmad Kakpori, Department of Education, Govt of Jammu &

Kashmir, Srinagar; Reena Mohapatra, St. Stephens Senior Secondary School, Ajmer; Savita

Sharma, Mount Carmel School, Dwarka, New Delhi; Savithri Singh, Professor and Principal,

Acharya Narendra Dev College, New Delhi; Shalu Dhawan, Amity International School, Saket,

New Delhi; Shivani Goswami, Mothers International School, New Delhi; V.K. Srivastava, Reader

in Zoology, J.N. College, Pasighat; Vijay Kumar, Delhi State Science Teacher Forum, New Delhi.

We also acknowledge the contributions of Anil Kumar and Binita Kumari, Junior Project

Fellows, DESM, NCERT, New Delhi.

Special thanks are also due to Hukum Singh, Professor and Head, DESM, NCERT for his

interest in the work and administrative support.The Council also acknowledges the efforts of Surender Kumar, Narender Kumar Verma, Monika

Rajput and Girish Goyal, DTP Operators, for helping in shaping this laboratory manual. The

contributions of Publication Department of NCERT in printing out this laboratory manual are

also duly acknowledged.

ACKNOWLEDGEMENT

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FOREWORDPREFACE

Introduction

Exercise 1 : To study the reproductive parts of commonlyavailable flowers

Exercise 2 : To calculate percentage of pollen germination

Exercise 3 : To study pollen tube growth on stigma

Exercise 4 : To study the discrete stages of gametogenesis inmammalian testis and ovary

Exercise 5 : To study and identify various stages of femalegametophyte development in the ovary of a flower

Exercise 6 : Preparation and study of mitosis in onion roottips

Exercise 7 : Study of stages of meiosis using permanent slides

Exercise 8 : To study the blastula stage of embryonicdevelopment in mammals, with the help ofpermanent slide, chart, model or photograph

Exercise 9 : To verify Mendel's Law of Segregation

Exercise 10 : To verify the Mendels Law of IndependentAssortment

Exercise 11 : Preparation and analysis of Pedigree Charts

Exercise 12 : To perform emasculation, bagging and taggingfor controlled pollination

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CONTENTS

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xExercise 13 : Staining of nucleic acid by acetocarmine

Exercise 14 : To identify common disease-causing organismsand the symptoms of the diseases

Exercise 15 : To study the texture of soil samples

Exercise 16 : To determine the water-holding capacity of soils

Exercise 17 : To study the ecological adaptations in plants livingin xeric and hydric conditions

Exercise 18 : To study the adaptations in animals living in xericand hydric conditions

Exercise 19 : To determine the pH of different water and soilsamples

Exercise 20 : To study turbidity of water samples

Exercise 21 : To analyse living organisms in water samples

Exercise 22 : To determine the amount of SuspendedParticulate Matter (SPM) in air at different sites ina city

Exercise 23 : To study plant population density by quadratmethod

Exercise 24 : To study plant population frequency by quadratmethod

Exercise 25 : Study of homologous and analogous organs inplants and animals

Investigatory Project Work

Project 1 : To study the effect of pH on seed germination

Project 2 : Quantitative analysis of phytoplankton in a waterbody

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Laboratory is a place where ideas and concepts can be tested throughexperiments. Biology, like any other discipline of science, is based onexperimental work and therefore practical forms an integral part of learning.Biology laboratory provides a unique learning environment where learnersinculcate scientific temper, develop relevant skills and get exposed to realmsof techniques and methodologies of scientific investigations. Laboratoryinvestigations in Biology increase the reasoning abilities, bring scientificattitude in a learner and also help in acquisition of skills of scientific processes.Also, observation of nature and the living organisms found in it is no lessimportant for the understanding of many aspects of the subject especiallythe diversity of the living organisms, their systematic study, their relationshipsamong themselves and with the environment. Knowledge in the field ofBiology can be acquired or constructed only on the basis of correctobservations and experimentally verifiable processes.

Biology laboratory thus provides the learners an environment where theprocess of learning is facilitated by hands-on experiments. Biology is aunique discipline in the sense that it does not merely deal with the study ofmorphology, anatomy, physiology and reproduction of the living organisms,rather, understanding of the subject requires understanding of a number ofinterdisciplinary areas and approaches. On one hand, a biologist needs tobe sufficiently skilled in handling the enormous diversity of the livingorganisms, be it plants, animals, fungi or even microscopic bacteria, whileon the other hand, a biologist should be able to understand the biochemical,molecular, physiological, behavioural, genetic and many other phenomenapertaining to the living organisms. The study of intricate relationship ofdifferent types of organisms among themselves and also with its environmentis an important concern of a biologist. Thus, experiments and exercises inBiology train a learner about skills of observations, manipulation of theorganisms for the study of internal details, biochemical as well as molecularcomposition and processes, investigation of the abiotic environment and even

analysis of phenomena like inheritance and evolution.As far as the study of the living organism is concerned, correctness of the

method is very important. Such a study may be very simple, e.g., study ofhabit, habitat and external features of the plants or animals, or, it may involvecertain manipulations like dissection and section cutting of the parts of theorganisms to study the minute details. Very often observation and study ofthe magnified image of the minute parts under a microscope provides abetter insight about the features of the organisms. However, microscopicstudy involves certain specific skills depending on type of the organisms/tissues/cells to be studied. It involves specific preparations (peeling, section

Introduction

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2LABORATORY MANUAL: BIOLOGY

cutting, fixation, staining, dehydration, mounting, etc.) so that microscopicexamination reveals the expected details. As histological and cytologicalobservations give us only static pictures of the continuous processes, analysisof biochemical, physiological and ecological aspects need certain other kindsof skills such as preparation of chemicals and reagents, designing andperforming an experiment, observation and recording of data and ultimatelyinterpretation and drawing conclusions. While performing experiments,honesty in recording of data and its correct presentation is very importantas it is not only useful in the logical interpretation but also helps in theidentification of errors.

In order to perform experiments successfully, a learner needs to go tothe Biology laboratory well prepared. This includes the following:

1. Laboratory Record Book: For maintaining all the information includingrecording of data and its interpretation.

2. Dissection Box: A dissection box is required in the Biology laboratoryfor various purposes like handling and manipulation of living materials,performing experiments, preparation of slide, etc. A dissection boxshould contain scissors (two pairs, one small with fine tip and onelarger), scalpels (one small and one medium sized), forceps (two, onesmall with sharp fine tips and the other medium sized with blunt tips),dissecting needles (two), razor, hand lens, dropper, fine brush, etc.

3. Laboratory Manual

4. A Laboratory Coat or Apron

5. A Hand Towel

While in the laboratory a student should be very careful and methodical.One should listen carefully to the instructions given by the teacher/instructor before performing an experiment. In the biology laboratory astudent has to handle a number of sharp objects and hence necessaryprecaution and care should always be taken while handling objects likescissors, forceps, needles, scalpel, razor, etc. It is also very important tofollow the safety instructions mentioned on the instruments and/or on thelabel of the reagent/chemical. Students should also be aware about theuse of the First-aid Box so that in case of any accident or injury thepreliminary aid can be provided to the affected person.

While describing the experiment students are expected to follow a patternin which the aim of the experiment, its principle, list of the materials to beused, procedure, observation table (if required), inference and discussionshould be given. Necessary precautions to be taken should also bementioned appropriately in the procedure or at the end. There are a fewexperiments in which field visit is essentially required. For this all thenecessary preparations (materials, equipments, reagents and chemicals)

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3EXERCISE 1

should be made in advance. Drawing of illustrations is also an importantcomponent of the practical in Biology. Students are expected to follow certainfundamental rules while drawing the illustrations so that it reflects yourobservations correctly. Make your illustrations using pencil only and always use white drawing

sheet. Illustration should be in the centre of the page. Drawing of an object (plant, animal or experimental set-up) should be

proportionate in size. Draw your illustrations keeping the object before you. Drawing must be clear with simple outlines. Appropriately label your drawing. Parts of the drawing should be

indicated by straight horizontal line or arrow. Two lines or arrow shouldnever cross each other. As far as possible, labelling should be done onthe right side of the drawing. An appropriate legend or heading of thedrawing should also be given below it.

About the Manual

The main objective of the manual is to introduce the students of highersecondary stage to the fascinating world of plants, animals and microbesand their complex biological phenomena. The manual covers a completedescription of the experiments and exercises. The suggested experimentscover almost all the units/topics including those on diversity in living world,plant, animal and human physiology, genetics, bio-technology and humanwelfare and environment. A standard format has been used to describe eachexperiment which includes

Aim: It gives a brief title of the experiment under investigation. Principle: It is a very brief introduction of the experiment under

investigation and explains the biological phenomenon involved. It givesbrief but comprehensive ideas about the design of the experiment andexplains the significance of the phenomenon being studied.

Materials required: This includes the names of plants/animals to beused as 'samples', the type of apparatus, the type and quantity ofglasswares required, reagents, chemicals and solutions needed, theirconcentration and other specifications, method of preparations ofsolutions and reagents. If a particular material/chemical/glassware isnot available, sufficient alternatives have been suggested.

Procedure: This section includes full details of experimental procedureexplained stepwise, including special precautions necessary to be takenwhile the experiment is being conducted. Drawings of the samples,apparatus and the experimental setup, wherever found necessary, havebeen included to facilitate the students to perform the experiment asaccurately as possible.

INTRODUCTION

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Observation and Results: This section deals with the recording of allobservations made during the experiment. Students are advised toconsider the entire data. Data can be represented in the form of tables,graphs and histograms wherever possible. Use of units in which variousquantities are measured has been indicated in the manual.

Discussion: Included in this heading is a statement of the conclusionsdrawn from the experimental results and compared thesis (whereverpossible) with any comparable data from other sources. The relevanceof the conclusions drawn from the experimental results to the variousprocesses under investigation and to the life of plant, animal and microbeshas been prompted out.

Precaution: This section contains all the necessary precautions to betaken during experimentation to obtain results free of errors. However,attempts have been made to mention required precautions along withthe procedure also.

A great emphasis has been laid on a student getting valid results andinterpreting them. It is essential that the teacher should properly explaineach experiment so that inexperienced students will be able to obtain accurateresults within a reasonable time. Teachers are also expected to help studentsin identifying errors and mistakes committed during experiments and waysfor correcting them. It is possible that some of the students may undoubtedlybe capable of doing more sophisticated work than that represented in themanual. But introductory course of this sort has been designed to help allstudents for some useful and joyful experience by conducting theexperiments of their own. The manual also aims that students and teachersnot be discouraged by either incomplete experiments or experiments whichyield apparently meaningless results.

With the objectives of inculcating scientific temper among learners andproviding them an opportunity to undertake independent scientificinvestigation, Investigatory Project Work has been included as an integralpart of the practical curriculum of Class XII. Such investigatory projectsare expected to provide thrill in the learning process. It is also expected toserve the real purpose of practicals, i.e., developing an ability to hypothesiseand design experiments to address certain problems, to make observationsmethodically and to draw conclusions out of the experimental data. Acomprehensive idea about undertaking investigatory project has been givenin the book with a list of a few problems on which investigatory projectwork can be undertaken. However, the list is only suggestive and consideringthe wider scope students can undertake any kind of investigatory projectwork depending on their region, its climatic condition, availability ofresources, etc.

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Aim: To study the reproductive parts of commonly available f lowers

Principle : The male reproductive parts of a flower are the stamens collectively calledandroecium and the female reproductive parts are the carpels/pistils collectively calledgynoecium. The individual units of stamen consist of a filament, which supports the antherlobes. Gynoecium consists of stigma, style and ovary. Many variations are found in differentcharacteristics of both the stamens and carpels. We shall try to study these variations in thereproductive parts of flowers in the exercise.

Requirement: Commonly available flowers, needles, forceps, razor/scalpel blade, brush, slides,cover slip, watch glass, magnifying lens, dissecting microscope, compound microscope, etc.

Procedure(i) Familiarise with the terms to describe the reproductive parts of flowers

given in annexures of Exercise No. 11 of Laboratory Manual: Biology(Class XI) and at the end of this experiment.

(ii) Observe the flower with the naked eye, hand lens or under a dissectingmicroscope. Study their reproductive parts and count the number ofstamens and record their cohesive and adhesive features.

(iii) Cut L.S. of the flower and place it on a slide to observe the followingcharacters:

(a) Placement of anthers

(b) Position of the ovary: epigynous/perigynous/hypogynous.

(iv) Mount one stamen on a slide and study the following characters:

(a) Attachment of filament to anther

(b) Dehiscence pattern of the anther lobes for discharge of pollen.

(v) Cut T.S. of anther lobe to observe the number of pollen sacs.

(vi) Mount the pistil on a slide and study style, stigma and ovary. Recordthe number of stigma and nature of pistil.

(vii) Cut T.S. of ovary, mount it on a slide and observe

(a) Number of locules in the ovary

Exercise 1

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6LABORATORY MANUAL: BIOLOGY

(b) Type of placentation

(c) Number of ovules per locule

(viii) Draw labelled figures of your preparation and observations.

Questions

1. Name the most common type of placentation observed.

2. What is the most common type of dehisence pattern in anthers?

3. Name a few unisexual flower-bearing plants studied by you.

4. Flower is a modified shoot. Justify the statement based on your observation.

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Annexure 1

Description of reproductive parts of flowers

Androecium

Number of stamens The number of stamens may vary from a few to many in dif-ferent flowers

Stamens may be free or united. If united they can be of thefollowing type:

( i ) Syngenesious : Filaments free and anthers united,e.g., Sunflower.

( i i ) Synandrous : Stamens fused all through their length,e.g., Cucurbita

( i i i ) Adelphous: Anthers remain free and filaments are united.Adelphous condition can be

(a) MonoadelphousUnited to form 1 bundle, e.g.,China rose

(b) DiadelphousUnited to form 2 bundles, e.g., Pea

(c) PolyadelphousUnited into more than twobundles, e.g., Lemon

Fusion of stamens with other parts of the flower

( i ) Epipetalous : Stamens fused with petals,e.g., Sunflower, Datura

( i i ) Epiphyllous : Stamens fused with perianth,e.g., Lily

( i ) Basifixed: Filament attached to the base of anther,

e.g., Mustard

( i i ) Adnate : Filament attached along the whole length ofanther, e.g., Michelia, Magnolia

(i i i ) Dorsifixed : Filament attached to the back of anther,e.g., Passion flower

(iv) Versatile : Anther lobes attached with filament in themiddle portion with both ends free, e.g., Gramineaefamily

( i ) Porous : Pollens released through pores, e.g., Brinjal,Potato

( i i ) Longitudinal: Pollens released through the longitudinalslit of another lobes, e.g., China rose, Cotton

Cohesion(Fig. 1.1 ae)

Adhesion(Fig. 1.2)

Attachment of filament toanther(Fig.1.3 ad)

Dehiscence pattern

(Fig. 1.4 a,b)

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Gynoecium

Number of stamens The number of stamens may vary from a few to many indifferent flowers

(i) Epigynous: Position of ovary inferior to other floral parts,

e.g., Mustard, China rose

( i i ) Perigynous : Other floral parts are attached around theovary, e.g., Apple, Guava

(ii i ) Hypogynous: Position of ovary superior to other floralparts, e.g., Sunflower

If number of carpels is more than one, they may be

(i ) Apocarpous : Carpels are free. Each carpel has its ownstyle and stigma, e.g., Rose

( i i ) Syncarpous: Carpels are united, e.g., Ladys finger, Tomato

Vary from one to many

(i ) Unilocular : One locule, e.g., Rose, Pea

( i i ) Bilocular: Two locules, e.g., Datura

(ii i ) Multilocular : Many locules, e.g., Ladys finger, China rose

(i ) Marginal : The placenta forms a ridge along the ventralsuture of the ovary and the ovules are borne on this ridge,e.g., Pea

( i i ) Axile: The ovary is partitioned into several chambers orlocules and the placentae are borne along the septa ofthe ovary, e.g., Tomato, China rose

(ii i ) Parietal: The ovules develop on the inner wall of theovary or on peripheral part. Ovary unilocular but in somecases becomes two chambered due to formation of a falseseptum, e.g., Mustard

(iv) Free central : Ovules are borne on the central axis andsepta are absent, e.g., Carnation, Chilly

(v) Basal: Placenta develops at the base of the ovary,e.g. ,Sunflower.

Position of ovary(Fig. 1.5 ad)

Cohesion(Fig. 1.6 ac)

Number of locules in ovary

Placentation(Fig. 1.7 ae)

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Fig.1.1 Cohesion of stamens: (a) Syngenesious (b) Synandrous(c) Monoadelphous (d) diadelphous (e) Polyadelphous

(a) (b) (c)

(d)

(e)

Fig.1.2 Adhesion of Stamens: Epipetalous/Epiphyllous

Fig.1.3 Attachment of filament to anther: (a) Basifixed (b) Adnate(c) Dorsifixed (d) Versatile

(a) (b) (c) (d)

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LABORATORY MANUAL: BIOLOGY

Fig.1.5 Position of ovary: (a) Epigynous (bc) Perigynous (d) Hypogynous

(b) (c) (d)

Fig.1.4 Dehiscence pattern of anther: (a) Porous (b) Longitudinal

(a) (b)

Fig.1.6 Cohesion of carpels: (a) Apocarpous (bc) Syncarpous

(a) (b) (c)

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EXERCISE 1

Fig.1.7 Placentation: (a) Marginal (b) Axile (c) Parietal (d) Free central (e) Basal

(a) (b) (c)

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Aim: To calculate percentage of pollen germination

Principle: In nature, pollen grains germinate on the compatible stigmas of the carpel. Pollengrains can also be induced to germinate in a synthetic medium. During germination, intine(inner wall) of pollen grain emerges out as pollen tube through one of the germ pores in exine(outer wall).

Requirement: Calcium nitrate, boric acid, sucrose, distilled water, petridish, slides, coverslips,brush, needle, microscope, and mature pollen grains of Tradescantia/balsam/Jasmine/lily/pomegranate/grass/Vinca/China rose/Petunia.

Exercise 2

Procedure(i) Prepare the pollen germination medium by dissolving 10g sucrose,

30mg calcium nitrate and 10mg boric acid in 100ml of distilled water.Alternatively 10% sucrose solution can also be used.

(ii) Take a drop of medium or 10% sucrose solution on a cover slip andsprinkle mature pollen grains on the drop.

(iii) Invert the cover glass on to a slide

(iv) After 10 minutes, observe the slide under microscope.

(v) Count (a) total number of pollen grains seen in the microscope field,and (b) the number of pollen grains that have germinated.

ObservationSeveral pollen grains germinate and put forth pollen tubes. Count the totalnumber of pollen grains and the number of germinated pollen grains in 3-5different microscope fields. Tabulate your observations and calculate thepercentage of pollen germination.

Name of the plant used as source of pollenNumber of pollen grains in a field of microscope = N

Number of germinated pollen grains in a field of microscope = n

Percent pollen germination = 100nN

or 100Nn

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EXERCISE 2

DiscussionAlthough pollen grains of many species germinate in this medium, thepercentage of germinations and the time taken for germination varies indifferent species. Draw a germinating pollen grain and label.

Number of Total number of pollen (N) Total number of pollen germinated (n) % pollen germination

observation 100nN

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Average

Questions

1. How many pollen tubes emerge from a pollen grain?

2. What does the pollen tube carry?

3. Can you explain as to why some pollen grains fail to germinate?

4. Why do we use sucrose as the medium for pollen germination?

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Aim: To study pollen tube growth on stigma

Principle: Pollen grains germinate and form pollen tubes after they get deposited by the processof pollination on compatible stigma. Pollen tube, made up of cellulose, is an extension of theinner wall of pollen grain (intine). It emerges through one of the germ pore and passes throughtissues of stigma and style to reach the ovule. The growing pollen tube is observed by stainingwith cotton blue.

Requirement: 56 excised styles with stigma of Petunia/grass/maize/sunflower/Abelmoschus(Lady's finger), beaker, water, slides, cover slips, cotton blue stain, microscope, brush, needle.

Exercise 3

Fig.3.1 Growth of pollen tubein the style of a carpel

Pollen grains

Pollen tube

Style

Procedure(i) Place the stigmas in boiling water in a beaker

for softening the tissues for 510 minutes.

(ii) Stain with cotton blue for 35 minutes andwash with water to remove excess stain.

(iii) Mount one stigma in a drop of glycerine on aslide. Place a cover slip on the stigma and gentlypress the cover slip on the material. Observethe slide under a microscope.

(iv) If you fail to observe pollen tubes mountanother stigma.

ObservationLook for long blue-coloured tubular structurestraversing through the tissues of stigma andstyle (Fig. 3.1).

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EXERCISE 3

DiscussionPollen tubes are seen amidst the stylar tissue. Many pollen tubes may beseen. Trace the origin of pollen tubes to the pollen grains present aroundthe surface of the stigma.

Questions

1. Can pollen grains of one plant species germinate on stigma of other species?Give reasons.

2. Do all pollen tubes reach the ovules?

3. Are all the pollen tubes of equal length? If not, why?

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Aim: To study the discrete stages of gametogenesis in mammalian testis and ovary

Principle: In all male and female organisms gamete formation takes place in their gonads, i.e .,testis and ovary respectively. The process of gamete formation, called gametogenesis involvesmeiotic cell division. The gametogenic development in testis is called spermatogenesis and inovary it is oogenesis. They exhibit marked differences and can be examined in transverse section(T.S.) of these organs.

Requirement: Permanent slides of T.S. of testis and ovary, compound microscope,lens-cleaning paper and cleaning fluid

Procedure(i) Clean the slide and microscopes eye and objective lenses with the

help of lens cleaning paper using any cleaning fluid.(ii) Place the slide on the stage of the microscope and observe first under

lower magnification and then in higher magnification. Observe variousstages of gamete development.

(iii) Record your observations in the notebook and draw labelled diagrams.

ObservationT.S. of testis

Exercise 4

Fig. 4.1 T.S. of mammalian testis

Seminiferous tubule

Spermatozoa

GerminalEpithelium

Spermatogonia

(i) You will observe a large numberof seminiferous tubules underlower magnification. Observe acomplete tubule in highermagnification and view variousstages of gamete developmentfrom periphery towards lumen(Fig. 4.1) and identify thefollowing types of cells namely,Germinal epithelium,Spermatogonial cells, Primaryspermatocytes, Secondaryspermatocytes, Spermatids andSpermatozoa.

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EXERCISE 4

(ii) In T.S. of testis the space between tubules are filled with blood vesselsand a specific cell type called Leydig's cell or Interstitial cells.

T.S. of Ovary

(i) In the section of ovary, there is a massof tissue lined with germinalepithelium. Inside that you willobserve an ovum, which is a cellsurrounded by one to several layersof follicular cells. As the ovummatures, the number of surroundingfollicular cell layer increases (Fig. 4.2).

(ii) In the later stage of folliculardevelopment a cavity called antrumappears.

(iii) The cavity gets further enlarged andthe follicle grows bigger. This is thestage of Graafian follicle ready to release the ovum (ovulation).

(iv) In the next stage, you may notice a Corpus luteum, and/or Corpusalbicans, which differ from each other and also from Graafian follicle intheir features.

DiscussionSpermatogenesis is a continuous process after attainment of puberty, andthat is why gamete development and spermatozoa are observed in a singleseminiferous tubule. In case of ovary, the follicular development stages areobserved.

Questions

1. What would happen if meiosis fails to occur in gametocyte?

2. At which stage of follicular development, is ovum released?

3. Spermatogenesis is a continuous process. Justify the statement.

4. Draw a labelled diagram of T.S. of testis.

5. Draw a labelled diagram of T.S. of ovary.

6. What would happen if sperms are devoid of their tail?

7. What are the consequences of failure of ovulation?

Fig. 4.2 Section of mammalian ovary

Graafian Follicle

Corpus luteum

Corpus albicans

Antrum

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Aim: To study and identify various stages of female gametophyte development in the ovaryof a flower

Principle: In flowering plants, female gametophyte (embryo sac) is a microscopic structuresituated deep inside the ovule. An ovule generally has one female gametophyte. Developmentof female gametophyte begins with megaspore mother cell. Most common type of femalegametophyte is the monosporic, 8-nucleate, 7-celled type.

Requirements: Permanent slides of V.S. of ovary, photographs/chart or models showingstages of female gametophyte development and microscope

Procedure(i) In a V.S. of ovary we generally find several ovules. Carefully observe

each ovule and locate as many stages of female gametophytedevelopment as possible.

(ii) Draw the diagrams as observed under microscope.

Exercise 5

Embryo sac

Fig. 5.1 V.S. of an ovule

Chalaza

Outer integument

Inner integument

Micropyle

Funiculus

Observation(i) Record the features of ovule

like number of integuments,nucellus and micropylarand chalazal poles. Fig 5.1shows the femalegametophyte (embryo sac)as seen in a V.S. of an ovule.Different stages ofdevelopment of femalegametophyte are shown inFig. 5.2.

(ii) Observe the placement ofembryo sac close to themicropylar pole.

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EXERCISE 5

Fig. 5.2 Stages of gametophyte development: (a) megaspore with 2 nucleus(b) 4-nucleate stage (c) 8- nucleate stage (d) 8- nucleate stageshowing 3+2+3 distribution of nuclei (e) mature embryo sac.

(a) (b) (c)

Egg

Synergids

Central cell

Secondary nucleus

Antipodals(d) (e)

(iii) Note the contents of embryo sac, namely, an egg apparatus(2 synergids and egg) at micropylar end, secondary nucleus in thecenter and three antipodal cells at the chalazal end (Fig. 5.2).

Questions

1. Explain the difference between gamete and a gametophyte.

2. Name two differences between synergids and egg.

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Aim: Preparation and study of mitosis in onion root tips

Principle: Somatic growth in plants and animals takes place by the increase in the number ofcells. A cell divides mitotically to form two daughter cells wherein the number of chromosomesremains the same (i.e., unchanged) as in the mother cell. In plants, such divisions rapidly takeplace in meristematic tissues of root and shoot apices, where the stages of mitosis can beeasily observed. In animals, mitotically dividing cells can be easily viewed in the bone marrowtissue of a vertebrate, epithelial cells from gills in fishes and the tail of growing tadpole larvaeof frog.

Requirement: Onion bulbs, wide mouth glass tubes/jar/bottle, glacial acetic acid, ethanol2-4% acetocarmine/acetoorcein stain, N/10 HCl, spirit lamp/hot plate, slide, cover slips,blotting paper, molten wax/nail polish and compound microscope

ProcedureGrowing of root tips

Select a few medium-sized onion bulbs. Carefully remove the dry rootspresent. Grow root tips by placing the bulbs on glass tubes (of about 34cm. diameter) filled with water. Care should be taken so that the stem portionof the bulb (basal part) just touches the water. A few drops of water may beadded periodically to compensate evaporation losses. New roots may take36 days to grow. Cut 23 cm long freshly grown roots and transfer them tofreshly prepared fixative, i.e., aceto-alcohol (1:3:: glacial acetic acid : ethanol).Keep the root tips in the fixative for 24 hours and then transfer them to 70%ethanol (for preservation and use in future). Onion root-tip cells have a cellcycle of approximately 24-hour duration, i.e., they divide once in 24 hours,and this division usually takes place about two hours after sunrise. Therefore,roots grown on water should be cut only at that time to score maximumnumber of dividing cells.

Preparation of slide

Take one or two preserved roots, wash them in water on a clean and grease-free slide. Place one drop of N/10 HCl on the root tip followed by 23 dropsof aceto-carmine or aceto-orcein stain on it. Leave the slide for 510 minutes

Exercise 6

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EXERCISE 6

on a hot plate (or warm it slightly on spirit lamp). Care should be taken thatthe stain is not dried up. Carefully blot the excess stain using blotting paper.Now cut the comparatively more stained (23 mm) tip portion of the rootand retain it on the slide and discard the remaining portion. After(1020 seconds) put one or two drops of water and blot them carefully usingblotting paper. Again put a drop of water on the root tip and mount a coverslip on it avoiding air bubbles. Place the slide in between the folds of blottingpaper using the fingers in such a way that the cover slip mounted on theslide is properly held. Now slowly tap the cover slip using the blunt end of apencil so that the meristematic tissue of the root tip below the cover slip isproperly squashed and spread as a thin layer of cells. Carefully seal themargins of the cover slip using molten paraffin wax or nail polish. Thispreparation of onion root tips cells is now ready for the study of mitosis.

Study of slide

Place the slide on the stage of a good quality compound microscope. Firstobserve it under the lower magnification (10 X objective) to search for thearea having a few dividing cells. Examine the dividing cells under highermagnification of the microscope to observe the detailed features of mitosis.

ObservationThe stages of mitosis can be broadly categorised into two parts: karyokinesis(division of nucleus) followed by cytokinesis (division of cytoplasm, andultimately of the cell). Those cells, which are not in the phases of cell divisionare considered to be in interphase. You may observe that most of the cellsin a microscope field are in interphase

InterphaseThe cells are mostly rectangular, oval or even circular in shape, with almostcentrally situated densely stained nucleus. The chromatic (coloured) materialof the nucleus is homogeneous and looks granular. The boundary of thenucleus is distinct. One or few nucleoli (sing: nucleolus) can also be observedinside the nucleus (Fig. 6.1a).

Stages of Mitosis(a) Prophase

Intact nuclear outline is seen. The chromatin (seen as a homogeneousmaterial in the nucleus at interphase) appears as a network of fine threads(chromosomes). Nucleoli may or may not be visible (Fig. 6.1b).

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LABORATORY MANUAL: BIOLOGY

If the cell under observation is in the early stage of prophase then thechromatin fibres (chromosomes) are very thin. However, in the cellsat late prophase, comparatively thicker chromatin fibres would bevisible. Besides this, in the late prophase the nuclear membranemay not be noticed.

(b) Metaphase

The nuclear membrane disappears. Chromosomes are thick and are seenarranged at the equatorial plane of the cell (Fig. 6.1c). Each chromosome at

Fig.6.1 Interphase (a) and stages of mitosis (b - e) actual microscopicview on left side and its diagrammatic representation on theright hand side

a. Interphase

b. Prophase

c. Metaphase

d. Anaphase

e. Telophase

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EXERCISE 6

this stage has two chromatids joined together at the centromere, which canbe seen by changing the resolution of the microscope. Nucleolus is notobserved during metaphase.

(c) Anaphase

This stage shows the separation of the chromatids of each chromosome. Thechromatids separate due to the splitting of the centromere. Each chromatidnow represents a separate chromosome as it has its own centromere. Thechromosomes are found as if they have moved towards the two poles of thecell. The chromosomes at this stage may look like the shape of alphabets 'V','J' or 'I' depending upon the position of centromere in them. Different anaphasecells show different stages of movement of chromosomes to opposite poles,and they are designated to represent early, mid and late anaphase (Fig. 6.1d).

(d) Telophase

Chromosomes reach the opposite poles, lose their individuality, and looklike a mass of chromatin (Fig. 6.1e). Nuclear membrane appears to form thenuclei of the two future daughter cells.

CytokinesisIn plants, a cell plate is formed in the middle after telophase. The plate canbe seen to extend outwards to ultimately reach the margin of the cell anddivide the cell into two. Such cell plates are characteristic of plant cells(Fig. 6.2). However, in an animal cell, the two sides of the cell show inpushingsor constrictions formed from the peripheral region in the middle of the cell,which grow inward and meet to divide the cell into two daughter cells.

Draw labelled diagrams of all the phases of mitosis.

Fig. 6.2 Cytokinesis

DiscussionMitotic index (MI) is defined as a ratio of the total number of dividing cells (n)and the total number of cells (N) in a particular focus chosen randomly under

the microscope and is calculated as MI = 100n

N . By randomly selecting

5 to 10 such foci, one can estimate the mitotic index for a given type.

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LABORATORY MANUAL: BIOLOGY

Questions

1. Suggest names of a few tissues, which are suitable for the study of mitosis.

2. Why is mitosis also known as equational division?

3. What shape would a metacentric and a sub-metacentric chromosome exhibitduring the anaphase stage?

4. How does cytokynesis differ in plant and animal cells?

Features Interphase Karyokinesis Cytokinesis

Prophase Metaphase Anaphase Telophase

1. Cell morphology

2. Nuclear morphology

3. Chromosomes/chromatids

Tabulate your observations in the tabular form given below

The effect of different samples of water (polluted or contaminated) can beassayed on the mitotic-index (an indicative feature of somatic growth rate inthem).

Further, the impact of different types of pollutants on different phases ofmitosis can also be assayed.

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Aim: Study of stages of meiosis using permanent slides

Principle: Meiosis is a type of cell division in which the number of chromosomes is halved(from diploid to haploid) in the daughter cells, i.e., the gametes. The division is completed intwo phases, meiosis I and meiosis II. Meiosis I is a reductional division in which the chromosomesof homologous pairs separate from each other. Meiosis II is equational division resulting in theformation of four daughter cells. Stages of meiosis can be observed in a cytological preparationof the cells of testis tubules or in the pollen mother cells of the anthers of f lower buds.

Requirement: Permanent slides of meiosis and compound microscope

Exercise 7

ProcedurePlace the slide on the stage of the microscope and search for the dividingcells using lower magnification. When dividing cells are located observe themunder higher magnification.

ObservationObserve various stages of meiosis and identify them on the basis of the specificfeatures given in the table 7.1. A significant number of cells will be in theInterphase. These cells have a centrally positioned densely stained nucleus.In case of slide of animal tissue a few mitotically dividing spermatogonialcells may also be seen.

Unlike the prophase of mitosis, it is a comparatively complex phasecharacterised by a number of events. Five sub-phases can beidentified in it.

(a) Leptotene (leptos = slender tene = band or thread)

(i) The nuclear membrane and nucleolus are not distinctlyobservable (Fig. 7.1 a).

(ii) Fine network of thin threads are seen uniformly distributedin the nucleus.These are chromatin threads, which may beobserved as more prominent structures in the later stages.

Meiosis I

1. Prophase I

Table 7.1 Different stages of meiosis and their features

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Fig. 7.1 Sub-phase of Prophase I (a-d) actual microscopic view on leftside and its diagrammatic representation on the right hand side

(a) Leptotene

(b) Zygotene

(c) Pachytene

(d) Diplotene-Diakinesis

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EXERCISE 7

2. Metaphase I

3. Anaphase I

4. Telophase I

Meiosis II

1. Prophase II

(b) Zygotene (Zygon = paired)

This stage is characterised by the pairing of the homologouschromosomes, which can be seen as paired chromatin threads(bivalents) (Fig. 7.1b).

(c) Pachytene (pachy = thick)

The chromatin threads get condensed and appear shortened andthick. Pairs of homologous chromosomes can be seen. Eachchromosome has two chromatids and thus each bivalent consistsof four chromatids. This configuration is called tetrad (Fig. 7.1c).

(d) Diplotene (diplos = double)

The homologous chromosomes (each made up of two chromatids)show distinct separation from each other except at few regions whereattachments are seen (Fig. 7.1d). These are chiasmata (sing.chiasma) representing the site of exchange of the parts betweentwo homologous chromosomes (i.e. crossing over).

(e) Diakinesis (Dia = opposite; kinesis= separation or movement)

(i) The homologous pair of chromosomes appear more shortened,thick and prominent (Fig. 7.1d).

(ii) Chiasmata can be still observed.

(iii) All the homologous pairs appear scattered in the cell.

Homologous chromosomes are still in pairs, and are arranged alongthe equatorial plane of the cell (Fig. 7.2a). At this stage, the numberof bivalents can be counted. Chiasmata may still be seen in a fewbivalents.

The chromosome pairs appear to have moved towards the twoopposite poles of the cell. At the later stage, the anaphase - I mayshow the assembly of chromosomes at two poles (Fig. 7.2b). Thisresults into the reduction of number of chromosomes to half.

This stage can be identified by the presence of two chromatids ineach chromosome.

The chromosomes present at the two poles appear decondensedand form two distinct nuclei (Fig. 7.2c).

Note: After the telophase I stage there may or may not be cytokinesis.Thereafter the cell enters into the second meiotic division.

(i) Distinct thread- like chromatin fibres or rod- shaped chromosomeare seen.

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Fig.7.3 Phases of Meosis II (a,b) actual microscopic view on left sideand its diagrammatic representation on the right hand side.

(a) Metaphase II

(b) Anaphase II

(a) Metaphase I(b) Anaphase I

(c) Telophase I

Fig. 7.2 Phases of Meosis I (a-c) actual microscopic view on left sideand its diagrammatic representation on the right hand side.

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EXERCISE 7

2. Metaphase II

3. Anaphase II

4. Telophase II

Questions

1. What is the significance of meiosis?

2. What is synapsis and crossing over?

3. How can anaphase I and anaphase II be distinguished from each other?

4. Indicate distinguishing feature of metaphase I of meiosis and metaphase of mitosis.

5. How many daughter cells are produced at the end of meiosis?

6. The daughter cells produced at the end of meiosis are genetically different. Explain.

7. What is the significance of synapsis?

This phase is similar to that of mitotic division

(i) The chromosomes having two chromatids attached at thecentromere are observed arranged at the equatorial plane of thecell.

Note: Metaphase II of meiosis can be differentiated from metaphase-Ion the basis of the following features:

(ii) Each chromosome of metaphase II has two chromatid(Fig. 7.3a) whereas in metaphase I these are pairedhomologous chromosomes each having two chromatids thusforming tetrad.

(iii) In the metaphase I of meiosis, a few chiasmata are observed,where as no chiasmata are observed during metaphase II.

The two chromatids of each chromosome after separation appearto lie at the two poles of the cell (Fig. 7.3b).

Note: Anaphase II can also be distinguished from the anaphase I ofmeiotic division on the basis of chromatids: In anaphase I, eachchromosome has two distinct chromatids, but in anaphase II, eachchromosome is represented by one chromatid only.

The separated chromosomes appear decondensed and form nuclei(Fig. 7.3c).

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Aim: To study the blastula stage of embryonic development in mammals, with the help ofpermanent slide, chart, model or photograph

Principle: The zygote undergoes a few cycles of mitotic divisions to form a solid ball of cells calledmorula. The cells continue to divide and at a later stage a cavity is formed within it. This stage isblastula. The internal structural details of blastula can be observed in its transverse section.

Requirement: Permanent slide, chart/model of T.S. of blastula, compound microscope,lens cleaning fluid and paper

Exercise 8

ProcedureObserve the slide under lower magnification of the microscope. In case ofchart/models/photographs, note the feature of blastula in your practicalrecord and draw labelled diagram.

ObservationIn transverse section, the blastula appears as a sphere with a cavity, calledblastocoel within it (Fig. 8.1). Notice an outer layer of blastomeres calledtrophoblasts. A cellular mass, adhered to the trophoblast is present on oneend of the blastula. It is called inner cell mass.

Fig.8.1 Blastula stage of a mammal

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EXERCISE 8

Questions

1. What are the differences between blastula and morula?

2. What are the main structures you observe in T.S. blastula?

3. Match the stages in column I with features in column II

Column I Column II

(a) Trophoblast (i) Dividing cells of the morula(b) Morula (ii) Outer layer of blastula(c) Blastocoel (iii) Solid ball of cells

(iv) Cavity

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Aim: To verify Mendel's Law of Segregation

Principle: When two pure lines with contrasting forms of a particular character (phenotypes) arecrossed to produce the next generation (F1 generation), all the members of the progeny are of onlyone phenotype i.e. of one of the two parents. The phenotype that appears is called dominant, andthe one that does not appear is called recessive. When the F1 plants are selfed, the progeny i.e. theF2 generation is in the ratio of 3 dominant: 1 recessive (: or 75%: 25%). This reappearance ofthe recessive phenotype in F2 generation verifies law of segregation.

Requirement: 64 yellow and 64 green plastic beads, all of exactly same shape and size, (whenbeads are not available, pea seeds may be coloured using paint, these beads represent thegametes of a specific trait), plastic beakers/petri dishes and a napkin/hand towel

ProcedureStudents have to work in pairs to perform the experiment. The followingsteps are to be strictly followed in the sequence mentioned below.

(i) Put 64 yellow beads in one beaker/petridish and 64 green beads inthe other to represent respectively male and female gametes. Let theyellow bead be indicated by Y and green bead by y.

(ii) Take a bead from each container and place them together (it representsfertilisation) on the napkin spread before you on the table. (One studentto take out beads and to put in the hands of the other student who willput them on the table).

(iii) Just like the previous step, continue to pick beads and arrange themin pairs. Thus 64 pairs of beads are obtained representing the 64heterozygous F

1 progeny.

Note that all the F1 individuals are represented by one yellow and onegreen bead.

(iv) Put 32 F1 progeny in one petridish and the remaining 32 in another

petridish (representing the F1 males and females).

(v) Stir the beads of each petridish with a pencil/pen for about 10 timestaking care that no bead falls off.

Exercise 9

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EXERCISE 9

(vi) To obtain the F2 generation, one student would withdraw one beadfrom one beaker labelled male and one from the other beaker labelledfemale keeping his/her eyes closed (to ensure randomness), and put

them together in the stretched palm of the partner, who will put themtogether on the napkin spread over the table. Continue this process till

all the beads are paired. Thus 64 offsprings of F2 are obtained.

(vii) Note the genotype (YY or Yy or yy) of each pair, and their possible

phenotype.

(viii) Have six repeats of the experiment (steps i to vii) with partners changing

their roles. Pool all the data from the six repeats together.

(ix) Calculate the genotypic and phenotypic ratios of your pooled data.

Note that larger the sample size, more accurate is the result.

ObservationRecord the result in the following table:

Generation Repeat No. Total no. of Genotype (s) Phenotype (s)

individuals YY Yy yy

F1 1.

2.

3.

4.

5.

6.

Total

F2 1.

2.

3.

4.

5.

6.

Total

Phenotypic Ratio: in F1.

in F2.

Genotypic Ratio: in F1.

in F2.

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Questions

1. Do you expect the same results in terms of 3:1 ratio in F2 if you had started with

smaller number of beads (say 10 beads)?

DiscussionThe results are so because each diploid individual contains two copies ofevery gene - one copy on each of the two homologous chromosomes. Thesetwo copies of the gene may be of similar type (YY or yy) or are dissimilar Yy.The former (YY or yy) are called homozygous for that particular character,and the Yy are called heterozygous ones. The pure lines in the above crossare homozygous ones, which contributed only one copy of their gene (as aresult of meiosis) to their F1 progeny to restore its diploid nature with genotypeYy (heterozygous) where only one form (allele) is expressed (dominant) andthe other form (allele) is not expressed (recessive). This is the phenomenonof Dominance.

When the F1 individuals are crossed together to raise the F2 generation,each F

1 individual produces two types of gametes: 50% having dominant

allele, and the remaining 50% having recessive allele. These gametes undergorandom fusion during fertilisation to produce the F2 generation. Accordingto simple probability of mixing of opposite sex gametes (sperms and ova),offsprings of three genotypes are likely to appear as follows: [(half of gametesof Y type + half of remaining gamete y type) X (half gametes of Y type + halfof remaining gamete of y type)] = One-fourth of F2 individuals of YY phenotype+ half of F

2 individual Yy type + one-fourth of F

2 individul of yy type. Among

these proportion of dominant phenotype would be YY+ Yy = yellowand recessive phenotype yy i.e. green phenotypes in 3:1 or 75%:25%ratio.

This ratio of 3:1 in the F2 suggests that in the F

1 heterozygotes, the

recessive allele does not get destroyed and remains only in the recessive(dormant) state to get an opportunity to express itself when it has separatedfrom the influence of the dominant allele (Y). This is called Law of Segregationof the alleles.

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Aim: To verify the Mendels Law of Independent Assortment

Principle: In a dihybrid cross, the segregation of one gene pair is independent of the segregationof the other pair. It means that genes of two different traits assort independently to give aprobability ratio equal to segregration probability ratio of one allele pair X segregation probabilityratio of other allele pair, which comes to, (3:1) X (3:1) = 9:3:3:1

Requirement: Plastic beakers; 64 plastic beads each of yellow, green, red and white to represent,yellow and green colour of seed coat and red and white flowers respectively and napkin/handtowel

Exercise 10

ProcedureStudents are to work in pair.

The following steps are to be followed sequentially:

(i) Place 64 beads of each colour in four separate beakers.

(ii) Put the beakers containing the yellow and red beads on your left side,and those containing the green and white beads on your right side.The beakers on your left side represent plants bearing yellow seed andred flower (dominant character YY, RR). Beakers on the right siderepresent plants bearing green seeds and white flowers (recessivecharacter yy, rr). These are the two parental types having contrastingforms of two different characters.

(iii) Stir the beads in each beaker with a pencil/pen. Each bead nowrepresents alleles in the male and female gametes.

(iv) Pick up one yellow, one green, one red and one white bead, and putthem together on the napkin spread on the table.

(v) Continue picking up and putting together of the beads of all coloursas mentioned in the previous step, till all the beads are utilised.

(vi) Note that in all, 64 such 4-bead clusters are obtained representing theF

1 individuals. Ascertain their genotype and phenotype.

(vii) Next step is to cross these F1 individuals to raise the F

2 generation. Let

us suppose half of the 4-bead clusters (32 clusters) represent the maleparents and the remaining half (32 clusters) the female parents. Nowput the 32 red and 32 white beads together in one beaker (numbered-I), and similarly put 32 yellow and 32 green beads together in other

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beaker (numbered-II). These two beakers represent F1 female. Similarlyput remaining 32 red + 32 white beads in beaker numbered-III, and32 yellow and 32 green one in beaker numbered-IV to represent the F

1

male. The arrangement can be presented as below

(viii) Stir the beads in each beaker with a pencil. In order to raise the F2generation, pick up (with eyes closed) one bead from the beaker-I offemale and one bead from the beaker-III of the male, and put into thepalm of the partner student. Similarly, pick up one bead each from thebeaker-II of female and beaker IV of male to put in the palm of thepartner. This partner would now keep all the four beads together (torepresent the F2 individual). Continue this process till all beads areutilised. At the end, 64 F

2 individuals (each represented by a 4-bead

cluster) are obtained.

(ix) Determine the genotype and phenotype of each of the 64 F2 individualsand write down the number of individuals of different genotypes andphenotypes in the tabular form (given below), remembering that Y(yellow seed colour) is dominant over y (green seed) and R (red flower)is dominant over r (white flower).

(x) Repeat the whole procedure (steps i to ix) six times, and tabulate yourresults.

ObservationTabulate the results as follow:Symbol (-) indicates the presence of corresponding dominant or recessiveallele e.g. Y or y and R or r.

Summarise your results (adding together the data of all the six repeats)

F1 Generation

(a) Total number of individuals: _________________________

(b) Phenotype (s) _________________________

(c) Genotype (s) _________________________

Female F1 Male F1

32 red + 32 white (Beaker I) 32 red+32 white (Beaker III)

32 yellow + 32 green (Beaker II) 32 yellow + 32 green (Beaker IV)

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EXERCISE 10

Generation Total No. Genotype Phenotype

& repeat of offsprings Y-R- Y-rr yyR- yyrr Yellow Yellow Green Green

No. Red White Red white

F1

1.

2.

3.

4.

5.

6.

Total

F2

1.

2.

3.

4.

5.

6.

Total

F2 Generation

(a) Total number of individuals _________________________

(b) Phenotypes _________________________

(c) Number of individuals in each phenotypic class:

Number Phenotype

__________________ _____________________

__________________ _____________________

__________________ _____________________

__________________ _____________________

(d) Phenotypic ratio _____________________

(e) Genotypic ratio _____________________

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Questions

1. Linked traits fail to assort independently. Explain.

2. How is independent assortment of alleles important from the point of view ofvariation?

(f) Number of individuals of each genotypic class:

Number Genotype

__________________ _____________________

__________________ _____________________

__________________ _____________________

__________________ _____________________

__________________ _____________________

(g) Genotypic Ratio ______________________________________

DiscussionThe four phenotypic classes in the F2 generation are in ratio of 9:3:3:1 asexpected from the Law of Independent Assortment. The genotypic ratiowould be (1:2:4:2): (2:1):(2:1):1.

Note

1. In case six repeats of the experimental procedure are not feasible dueto time limitations, either the number of repeats be slashed down tothree or the data from single repeat of six different pair of students maybe pooled together to make the final calculations.

2. This Law of Independent Assortment was later found to be true only fortraits present on two different homologous pair of chromosomes, thatis, the two are not linked together. The linked traits do not assortindependently, rather they are inherited together (linked) except whencrossingover separates them.

3. It is quiet likely that you may not find your data exactly in theexpected ratio, instead almost approximate to it. The statisticalsignificance of this deviation from the exact expected ratio due to

probality can be checked using chi-square (2) test, about whichyou will study in higher classes.

Aim: Preparation and analysis of Pedigree Charts

Principle: The Mendelian concept of dominance and segregation can also be studied in humansby preparing and then analysing the pedigree charts. The internationally approved symbols forindicating males and females, marriages, various generations (I, II, III), etc., are given below.

Exercise 11

Requirement: Information about characters/traits in a family for more than one generation

ProcedureSelect a family in which any one of the monogenic traits such as tonguerolling, widow's peak, blood groups, red-green colour blindness, dimple in

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the cheek, hypertrichosis of ear, hitch-hiker's thumb, etc., is found. Ask theperson exhibiting the trait to tell in which of his/her parents, grand parents(both maternal and paternal), their children and grand children the trait inquestion is present. Among surviving individuals the trait may also beexamined. The information made available is the basis for the preparation ofpedigree chart using the appropriate symbols. A careful examination of thepedigree chart would suggest whether the gene for the character is autosome-linked dominant or recessive, X - chromosome linked dominant or recessive,

Y- chromosome linked or not.

Explanation

1. Autosome Linked Dominant traits: These are the traits whoseencoding gene is present on any one of the autosomes, and the wild-type allele is recessive to its mutant allele, i.e., the mutant allele isdominant.

The pedigree-chart can be of the undernoted pattern (Fig. 11.2), wherethe female being interviewed is exhibiting the trait, and is indicated byan arrow-mark in the chart.

The characteristic features of inheritance of such type of traits are:

(a) Transmission of traits occurs from parents of either sex.

(b) Males and females are equally affected.

(c) The pedigree is vertical, i.e., the trait is marked to be present in each ofthe generations.

(d) Multiple generations are characteristically affected.

Brachydactyly, polydactyly, dimple in the cheek are some of the commontraits of this type.

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EXERCISE 11

2. Autosomal Recessive trait: These are the traits whose mutant allele isrecessive to its wild type allele.

The pedigree chart can be more or less of the pattern given below (Fig.11.3), where the lady (marked by the arrow) is showing the trait. The bar

in the example represents the presence of corresponding dominant orrecessive allele for the specific trait.

Suppose the given trait is albinism. Denote its dominant allele as Athat produces pigments, and the recessive allele as a that fails to synthesisethe pigment, melanin. The female (our subject in generation III) is thereforeof genotype aa. She must have received each of her a allele from both theparents (generation-II), who are therefore themselves normal but are definitelyof genotype Aa, and are carriers of the trait. The allele a must also have beenpresent in her grand parents too, of course in heterozygous condition alsoto make them carriers (generation-I)

Albinism in the subjects children (generation-IV) suggests her husbandtoo to be of genotype Aa, a carrier. Marriage of her albino daughter to analbino man is bound to produce all her grand-children albino (gen-V).

The following are the salient features of the inheritance of such type of traits.

(a) Occur in equal proportions in multiple male and female siblings, whoseparents are normal but carriers;

(b) The siblings are homozygous for the defective allele, but their parents,though some may appear normal, are obviously heterozygous, i.e.,are merely carriers of the trait.

(c) Consanguinity (marriage between man and woman genetically relatedto each other, such as cousins) occasionally results in the appearanceof such traits.

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3. X-Linked Dominant traits: These are the traits whose encoding geneis present on the X- chromosome, and the mutant allele of which isdominant over its wild-type allele.

Such traits are very rare, and are almost difficult to find in thepopulation. One example is oral-facial-digital syndrome (DucheneMuscular Dystrophy), which results in absence of teeth, cleft (bifid) tongueassociated with mental retardation. The pedigree chart may appear asfollows (Fig. 11.4):

The possible genotypes of the above pedigree can be written as follows(Fig 11.5):

Fig. 11.5 Genotypes of individuals shown in Fig. 11.4

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EXERCISE 11

Here, the dominant mutant allele is denoted by D, and its recessive wildtype allele is denoted by d. Remember that human females have twoX-chromosomes (XX), and the males have only one X and one Y chromosome.Males receive their lone X-chromosome from their mother, and theY-chromosomes from their father, whereas females receives one of herX-chromosome from her mother, and the other X from her father.

The characteristics of such inheritance are:

(a) The trait appears in almost all the generations, and the inheritance isvertical.

(b) If the female is affected, then about half of her sons are affected.

(c) If the male is affected then all of his daughters would be affected, butnone of his sons are affected.

(d) In short, the pedigree resembles the pattern of inheritance of autosomaldominants, except that there is no male-to-male transmission.

4. X-linked Recessive traits: These are the traits whose encoding gene ispresent on the X-chromosome and its mutant allele is recessive to itswild-type allele.

Red-green colour blindness and hemophilia, are some of its well knownexamples. The characteristic features of such inheritance are:

(a) Females express the trait only when they are homozygous for themutant allele, whereas the males do so even when they are hemizygousfor it.

The pedigree chart would appear as the following one (Fig. 11.6):

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(b) About half of the sons of the carrier (heterozygous for the trait) femalesare affected. In case of homozygous females showing the trait, fiftypercent of her daughters and all of her sons are likely to be affected.Therefore, the males are most affected in the population.

(c) Affected persons are related to one another through the maternal sideof their family.

(d) Any evidence of male-to-male transmission of the trait rules out theX- linked inheritance.

5. Y-chromosome linked traits: These are the traits whose gene is presenton the Y-chromosome. The females do not have any Y-chromosome,whereas all the males must have a Y-chromosome to be a male, and thisY-chromosome they get from their father. Therefore, any trait linked tothe Y- chromosome must be present only in males, and certainly not inany of the females. This is why these traits are also called male-sex limitedtraits. All the sons of the affected male would express the trait whereasnone of his daughters would do so.

The pattern of the pedigree chart would be as follows (Fig 11.7):

Hypertrichosis of the ear (presence of hairs on pinna) is one most commonexample of such traits.

Questions

1. How will you differentiate between autosome linked dominant and sex chromosomelinked dominant pedigree chart? Explain.

2. Discuss the differences in the patterns of autosome linked recessive and sex-chromosome linked pedigree.

Note: Students may be asked to prepare the pedigree-chart from given data and analyse thepattern of inheritance. The work may be done as a project.

Aim: To perform emasculation, bagging and tagging for controlled pollination

Principle: Conventional plant breeding programmes involve bringing under human controlreproductive processes that lead to seed and fruit formation. For this controlled pollination isdesirable using male and female parent having desired traits. One of the process that can be easilybrought under human control is emasculation. For this the knowledge of flower structure,mechanism of pollination, fertilisation and physiology of f lowering is essential for this. Inemasculation technique the stamens are removed before anthesis to obtain female parent and pollenfrom the desired male parent is transferred on to its stigma.

Requirement: Ornamental plants/ wild plants bearing large bisexual flower, magnifying lens,tweezers, small sharp scissors, brush, alcohol, rubber bands, paper bags, paper clips and tags

Procedure(i) Select a flower in bud condition where antheses has not occurred. Open

the bud carefully and remove the stamens (Fig. 12.1). Mark this asfemale parent plant.

Exercise 12

Fig. 12.1 Showing process of Emasculation

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Questions

1. Why is emasculation performed before anthesis?

2. What are the advantages of using a bag containing minute pores?

Fig. 12.2 Bagging of an emasculated flower

(ii) Cover the emasculated flower with a plasticbag to protect it from undesired pollen(Bagging) (Fig. 12.2). The bag should beheld securely in place with a paper clip/string/thread. Select the size of bag inaccordance with the flower size. Bagsmust be transparent with minute pores.

(iii) Bring into physical contact anthers of adesired male plant containing maturepollen grains with the stigmatic surfaceof emasculated female flower (Fig. 12.3).Use tweezers/brush if necessary to dustthe stigmatic surface with pollen.

(iv) Cover the pollinated flower again with thebag immediately. For identification, labelthe female parent (Tagging). Eachpollinated flower should bear a labelcontaining the name of the seed parent,the letter X (to signify a cross), the nameof the pollen parent, and the date on whichthe cross was effected.

Fig. 12.3 Showing cross pollination on anemasculated flower

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Aim: Staining of nucleic acid by acetocarmine

Principle: Acetocarmine combines with nucleic acid present in the nuclei of cells to form a deepred conjugate.

Requirements: Onion bulb, onion root tips, 2 to 4% acetocarmine/acetoorcein stains, slideand coverslips, brush/needle, pair of fine scissors, filter paper and microscope

Procedure(i) Peel off epidermis from the fleshy leaf of onion and put it on a slide.

Add a few drops of water over it to avoid desication.

(ii) Cut out a small piece (about 0.5 cm size) of the epidermal peel anddiscard the remaining portion.

(iii) Wipe out the water with a filter paper.

(iv) Put 2 drops of acetocarmine on the epidermal peel and heat gently ona spirit lamp.

(v) Apply a coverslip over the peel avoiding air bubbles and wrinkles ofthe material.

(vi) Wipe out the excess stain with help of blotting paper.

(vii) Examine the material under low magnification of a microscope.

ObservationRecord your observations with regard to shape of cell, the number of nucleiand their position in the cell. Draw a diagram based on your preparationand label its parts.

DiscussionNuclei in cells are extremely rich in nucleic acid which exist in a conjugatedform with protein to form nucleoproteinous structures, called chromatinfibres/chromosomes.

Exercise 13

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Questions

1. What are the building blocks of the nucleic acid?

2. What is DNA and how is it different from RNA?

3. Name different nitrogenous bases present in the nucleic acid.

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Aim: To identify common disease-causing organisms and the symptoms of the diseases

Principle: There are quite a large number of organisms that are parasitic/pathogenic to humans.These organisms substantialy damage the human body and cause diseases, which may even be fatalsometimes. These organisms exhibit characteristic features in their external morphology. Symptomsof the diseases caused by them are also specific.

Requirement : Preserved specimens/permanent slides/photographs of Ascaris, Entamoeba,Plasmodium, Ring-worm fungus and compound microscope

ProcedureObserve the preserved specimens/slides/photographs and note down thefeatures in the practical record book. Take care to observe all the minutedetails and draw labelled diagrams of the pathogens.

Exercise 14

Observation

A. EntamoebaObserve the following features of the parasite in the slide or photograph:

(i) It is unicellular.

(ii) Shape of the cell is irregular due topseudopodia.

(iii) A single nucleus is present eccentrically inthe cell.

(iv) *In the nucleus a peripheral ring of granuleof nucleoprotein and central karyosome areobserved. Rest of the space in the nucleuslooks empty (Fig. 14.1).

(v) A few food vacuoles may be seen in thecytoplasm. Contractile vacuoles are absent.

(vi) *Mature quadrinucleated cysts may bepresent.

Fig.14.1 An Entamoeba

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Note: Entamoeba is an intestinal parasite in humans and causes amoebicdysentery. The symptoms of the disease are frequent loose, mucus filledwatery stools, abdominal pain and spasms.

Systematic positionPhylum Protozoa

Class Rhizopoda

Type Entamoeba histolytica

* Distinctive feature of the pathogen

B. Plasmodium vivax(i) It is an intracellular endoparasite seen easily within the RBC of the

infected person.

(ii) It is unicellular.

(iii) The most diagnostic stage of the parasite is "signet ring" stage in theerythrocytes, within which it appears as a roundedbody (Fig. 14.2).

(iv) It has a big vacuole inside, and the cytoplasm is accumulated at oneplace containing the nucleus. Because of the above mentioned features,the parasite appears as a ring.

Search the stage in the blood film slide, find the signet-ring stage, anddraw its labeled diagram.

Note: It is a protozoan parasite causing malaria in humans. When an infectedfemale anopheles mosquito bites a healthy person, it injects the infectivestage, sporozoite, into the peripheral blood vessels. The infective stageundergoes several rounds of multiplication in liver and erythrocytes.

Symptoms: Intermittent high fever with chills followed by profuse sweatingat an interval of alternate days.

Systematic positionPhylum Protozoa

Class Sporozoa

Type Plasmodium vivax

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EXERCISE 14

C. AscarisThe external features of round worm are as follows:

(i) Body long (20 to 40 cm), cylindrical (5 to 6 mmdiameter) with no segmentation (Fig. 14.3).

(ii) Sexes are separate; the females are longer thanthe males.

(iii) Both the ends are pointed; posterior end of maleis ventrally curved.

(iv) Mouth is situated at the anterior end, and issurrounded by three lips, one present mid-dorsally and rest two lips are situatedventrolaterally (for viewing these lips a magnifyinglens is needed).

(v) Single longitudinal lines are present on the dorsal,ventral and on the two lateral sides, all along thelength of the body. Out of these the lateral linesare comparatively more distinct than the otherslines.

(vi) Excretory pore is present on the ventral surfaceslightly behind the anterior end.

(vii) In addition to the ventrally curved posterior tip,the male worm has a pair of penial spicules veryclose to the cloacal opening.

(viii) In case of female specimen a female genitalaperture is present mid-ventrally about one thirddistance from the anterior end.

Systematic positionPhylum Aschelminthes

Class Nematoda

Type Ascaris lumbricoides

Note: Round worm or Ascaris is one of the common parasite found in theintestine of human beings.

Symptoms: (a) Irregular bowel, (b) Occasional vomiting, (c) Anaemia

Fig.14.3 Ascaris (a) Female (b) Male

Female genitalaperture

Mouth

Penial spicule

(a)

(b)

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Trichophyton (Ringworm fungus)It is a fungus that feeds on keratin of the skin of human beings. The featuresas observed under the microscope are:

1. Texture of hyphae is waxy, glabrous to cotton like.

2. Unstained hyphae are white, yellowish brown to reddish brown in colour.

Systematic positionKingdom Fungi

Class Deuteromycetes

Type Trichophyton rubrum

SymptomsRingworm is a contagious fungal infection of the skin. Infected area of skinis itchy, red, raised, scaly patches (with sharply defined edges). It is morered on the periphery than in the center creating a ring like appearance.

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Aim: To study the texture of soil samples

Principle: Texture is one of the most important physical properties of soil. The soil texture is basedupon division of the size of soil particles into three size fractions viz., Sand (20.05mm averageparticle diameter), Silt (0.050.002mm) and Clay ( less then 0.002mm). If one of these fractionsdominates the properties of a soil, the name of that fraction is included in the name of the texture.A soil which has all of these fractions in nearly equal proportion is called a loam soil.

The four termssand, silt, clay andloam are combined in various ways to name12 different textural classes. The 12 texturalclasses and the percentages of sand, silt andclay fractions that are included in each areshown in textural triangle (Fig. 15.1).

Texture affects several physicochemicalproperties of soil like density, capillary andnon-capillary pore spaces, water holdingcapacity, aeration, temperature and also theroot penetration.

Requirement : Oven/stove dried soilsamples, balance, weights, mechanical sieveset and blotting sheets/old newspapers

ProcedureThree methods are suggested here. Any one of these may be followed.

Method I

(i) Collect about 300500g of soil from two different locations. Label them as sample A and B.

(ii) Dry the samples in an oven, or stove or in sun to remove the soil moisture (capillary andbound water).

(iii) Select the 3 sieves of different mesh sizes (2mm, 0.05mm and 0.002mm). Arrange them ina collecting chamber as shown in Fig. 15.2.

(iv) Place 200g of the soil in the Ist sieve (sieve of 2mm mesh) and close the lid. To sieve the soil,shake the set manually for 510 minutes and collect the three soil fractions.

Exercise 15

Fig.15.1 Soil Textural Triangle

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Soil sample Percentage (%) Texture class

Sand Silt Clay

A

B

Note for Teachers: The sieve sets contain a number and an abbreviation BSS/ASTM/ISS on each sieve. In the given table (Table No. 15.1) the corresponding aperture size ofthe sieves is listed. For example, BSS 30 sieve aperture size will be 500 microns.

Fig.15.2 Sieve set

2mm mesh

0.05mm mesh

0.002mm mesh

Collecting chamber

Lid

the precentage lines of silt run parallel to the clay side of the triangle and, (iii)

perentage lines of sand run parallel to the silt silde of the triangle. In reading

the textural triangle, any two particle fractions will locate the textural class at

the point where these two intersect.