EXERCISE Exercise

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					Exercise 1: Tools and equipments of use in fruit crop breeding


Objective

To acquaint with different tools, equipments, chemical reagents and other accessories of
use in fruit breeding.


Materials

Various tools, equipments and chemicals which would be used to conduct different
experiments along with their specific use (s) are listed below:
               ARTICLE                         MAJOR USES

I.     Materials required in the field

1.     Notched scissors                      Flower structure, emasculation

2.     Forceps/tweezers                      Flower structure, emasculation, pollination,
                                             stigma receptivity
3.     Needles                               Flower structure, anther dehiscence

4.     Camel’s hair brush                    Flower structure, pollination

5.     Butter paper bags                     Emasculation, pollination

6.     Muslin cloth bags                     Self-pollination

7.     Metallic/labels                       Emasculation, pollination,         fruit   set,
                                             sampling
8.     Cages                                 Self-pollination

9.     Wooden boxes                          Seed stratification

10.    File/hammer                           Seed scarification

11.    Polythene bags                        Sampling of flower/ fruits/ seed

12.    Magnifying lens                       Flower structure, stigma           receptively,
                                             pollination.
13.    Jars/cans/boxes                       Hybrid fruit/seed collection

14.    Crochet thread                        Bagging and labeling
15.   Sterilized sand/ sphagnum moss        Seed stratification/sowing

II.   Equipments of use in the laboratory

16.   Dissecting microscope                 Flower structure

17.   Simple microscope

18.   Microscopic/ cavity slides            Pollen viability

19.   Cover slips/ watch glasses

20.   Pollen sieves

21.   Desiccator                            Pollen preparation, storage

22.   Glass vials

23.   Incubator

24.   Burner                                Pollen Viability

25.   Refrigerator/freezer/seed             Pollen/seed storage, seed stratification/
      germinator                            germination
26.   X-ray machine

27.   Gamma cell/UV light                   Irradiation
Exercise 2: Studies on bearing habit and flower structure

Objective

       To generator know how on floral biology of fruit crops.

Materials

      Notched scissors, forceps, needles, camel’s hair brush, polythene bags,
magnifying lens, dissecting microscope, sharp blade.


Procedure / Observations

1.     Bearing habit

       During the flowering time, record the following observations on a sample tree:

i)     Plant parts bearing flower buds

a)     on shoots of current year growth
b)     on shoots of previous year growth
c)     on shoots of two or more than two years old growth
d)     on spurs of old growth

ii)    Nature of buds

a)     Sample
b)     Mixed
c)     compound

iii)   Arrangement of Flower buds

a)     Opposite
b)     Alternate
c)     Collateral
d)     Superposed

iv)    Number of flower buds/location

a)     Solitary
b)     Two or three
c)     Clusters
2.     Flower structure

       Randomly collect 20-30 fully opened flowers from a sample tree and record the
following observations:

i)     Colour/number of sepals, petals and stamens

ii)    Type of flowers (hermaphrodite, hermaphrodite and staminate, hermaphoradite

       and pistillate, staminate, pistillate).

iii)   heterostyly (mid, thrum, pin) – relative position of anther/stigma

iv)    flower type (perigynous, epigynous, hypogynous) – on the basis of position of

       ovary (inferior/superior)

v)     presence or absence of hair on pistils

vi)    any other floral abnormality

a)     rudimentary pistil
b)     twin pistils
c)     neutral flowers
d)     twin flowers on single pedicel
Exercise 3: Pollen collection, its preparation and storage

Objective

       To carry out pollen studies and controlled hybridization.

Materials

       Scissors, forceps, pollen sieve, desiccator, glass vials, refrigerator.

Collection

       The easiest way of pollen collection is to gather them from fresh flowers with
dehiscing anthers. However, it is not desirable, considering possible contamination in
wind/insect-pollinated species. Therefore, collect anthers in the morning hours from
flowers about to open or from opened flowers protected from insect visits. Separate
anther lobes from filaments with the help of a tweezer/forceps and collect over a
nonsticking paper in a petridish.

Preparation

         Dry the anthers until dehiscence (indicated by appearance of dust/powder along
the slits) in a warm wind-free area for 12-24 h. Keep the upper lid of petridish slightly
titled to allow exchange of air to avoid condensation of vapours. Gently tap the dehisced
anthers on a non-sticking clean paper (sides folded to make a workable tray) to dust the
pollen or rub the dehiscing anthers over a fine mesh screen/pollen sieve and collect the
pollen underneath on a clean non-sticking paper. The later approach is desirable as pollen
so collected in free from debris or accessory floral tissues. If required, use a source of
heat (table lamp) or an incubator at 20-25º C to enhance drying of anthers.


Storage

       Transfer the pollen into small loosely stoppered glass vials. Keep them in a
desiccator over anhydrous calcium sulphate, until use. For long-term storage, keep the
desiccators containing pollen vials in a deep freezer below 20ºC or store the pollen in
small gelatin capsules or Teflon vials placed in cryoflasks containing liquid nitrogen at -
196ºC.
Exercise 4: Determination of pollen viability

Objective

       To assess the potential of a given pollen parent to serve as a pollinizer.

Materials

         Microscope, microscopic and cavity slides, watch glasses, cover slips, stain,
distilled water, measuring cylinder, scissors, forceps, pollen source(s).

1.     ACETO/PROPIONO CARMINE (1-2%) TEST

         Weigh 1 or 2 g of carmine powder, dissolve it in 95 ml of glacial acetic
acid/propionic acid. Add distilled water to make a total of 100 ml solution. Boil it, cool
and filter. Add few drops of 10% ferric chloride solution (10 g ferric chloride dissolved in
distilled water to make a final volume of 100 ml), mix it and store in a refrigerator.

Testing

        Shed the freshly collected pollen on a clean slide and put a drop or two of freshly
prepared stain over it. Agitate the pollen grains with a needle for about 30 seconds. Place
the cover slip, held in forceps, gently to avoid air bubbles. After few minutes, examine
the slide under a microscope. Coloured pollen grains are considered viable. Take 2-3
readings, avoid taking observations towards the edges of cover slip. Calculate average
pollen viability percentage.
Exercise 5: In vitro pollen germination tests

Objective

       To find out the actual geminability of pollen of a given parent.

Materials

        Scissors, forceps, glass vials, microscope, cavity slides, microscopic slides, cover
slips, watch glasses, regent bottles, beakers, agar agar, sucrose, burner, pollen sources.

General method

        Germinate a small sample of pollen on a suitable medium. In most cases sucrose
(10-30%) alone or combination with boron (10-100 ppm) is used. Agar (1-2%) is also
used sometimes as a solid geminating medium. Count the number of pollen grains
producing pollen tubes after 2, 4, 6, 8, or more hours under microscope. Calculate
percentage of such pollen grains which is usually considered as an index of its viability.
But consider only those pollen grains viable in which the pollen tube has grown double
the size of the pollen grain.

Hanging drop method

        Put 1-2 drops of germination medium in the cavity of a microscopic slide. Dust
the pollen over it and place a cover slip. Invert the slide instantly and suspend it over a
watch glass or petridish containing water. Cover the whole structure with a larger
petridish or glass jar to reduce evaporation. For simultaneous testing of several samples
use a microscopic slide with multiple cavities.


Agar test

        Pour the agar on a Petri dish or a microscopic slide. Add sucrose prior to
solidification. Dust the pollen on solidified medium. Agar slides serve wells as permanent
mounts.
Exercise 6: Methods of emasculation and pollination

Objective

       To learn how to make controlled crosses between any two parents.

Materials

       Notched scissors, forceps, camel’s hair brush, ladder, labels, magnifying lens,
crochet thread, glass rod, butter paper bags, eye lash brush, muslin cloth bags.

Emasculation

        Select appropriate branches on all the sides of a tree where emasculation is to be
done. Remove already opened flowers or immature flower buds and leave only suitable
flower buds. In self – pollinating or in cleistogamous crops, perform emasculation 3-4
days prior to day of anthesis. All the emasculation work should be done in morning
hours. Hold the shoot/spur gently avoiding breakage, and in one attempt remove petals,
distal ends of sepals in addition to stamens (radical emasculation). Do this using either
fingernails, forceps or modified scissors with notches cut in the blades and a screw for
adjusting the degree of closure. Check if any anther has fallen into calyx cup during
emasculation if so, remove it. Do not emasculate flowers towards the periphery of the
tree. Cover the emasculated flowers with insect – proof bags until pollination to avoid
contamination. Inspect the trees after 1-2 weeks to remove unemasculated flowers, if any.

Pollination

       Apply fresh or stored pollen from a desired male parent on to receptive stigmas of
emasculated flowers using camel’s hair brush/sterilized glass rod/cotton plug/velvet-
coated cork. Judge the stigma receptivity by confirming the exudation of watery fluids
from stigmatic surface as seen by naked eye or through a magnifying lens. Do not
overload the stigma with pollen. To avoid this, use an eye lash brush (single eye lash
picks up 5-6 pollen grains). After pollination, replace the bags to protect from winds/rain.
Repollinate the next day, if need be.

Bagging

        Cover the recently pollinated flowers with proper insect-proof butter paper bags
or muslin cloth bags to avoid damage due to frost, strong winds, hail-storm or heavy
rains. Remove the bags after some time when young fruitlets start developing.
Labelling

        Tie a suitable label (metallic, wooden or wax – coated luggage label) on each
shoot/spur below the last pollinated flower. Put the following information on the
respective labels.

   -   Parentage (female x male)
   -   Number of flowers emasculated and pollinated
   -   Date of emasculation and pollination
   -   Name of the breeder and location
Exercise 7: Determination of stigma receptivity

Objective

   i)       To find out actual time and duration for which pollination are effective.
   ii)      To work out cross compatibility/incompatibility reaction between two parents.


Materials

        Notched scissors, forceps camel’s hair brush, eye lash, ladder, labels, magnifying
lens, crochet thread, glass rod, butter paper bags, muslin cloth bags.

Simple Test

         Visually observe through naked eye or with the help of a magnifying lens, several
stigmas at random. Presence of exudates (water fluid) on stigmatic surface would indicate
that stigma is receptive.

Fruit set test

        However, more reliable test for determination of stigma receptivity is by making
controlled pollinations at different staged of flower development i.e.

   -     Two days before anthesis
   -     One day before anthesis
   -     Day of anthesis
   -     One day after anthesis
   -     Two days after anthesis

        At each of the described stages, emasculate 40-50 randomly selected flowers and
pollinate immediately. Count the number of fruits set after 4 – 6 weeks in each case. On
the basis of percentage of fruit set, work out the initiation and duration for which stigma
remains receptive.

        Following the same procedure as described previous exercise apply pollen from
different male parents to a separate set of 50 flowers emasculated on a female parent.
Like the above, count number of fruits setting after 4-6 weeks. Development of fruits in a
given combination is an indication of cross-compatibility between the two varieties,
otherwise not.
Exercise 8: Quantitative determination of fruit set through self-pollination

Objective

        To investigate whether a particular variety of a given crop requires cross-
pollination or not, to set fruits.

Materials

            Forceps, camel’s hair brush, selfing bags, ladder, metallic labels, cages, crochet
thread.

Self-pollination

       Self-pollination is the process of obtaining seeds through pollination of stigma of
a flower by pollen of the same flower or from a flower on the same tree.

Self-pollination tests are carried out in two ways:

      i)       Natural self-pollination (bagging/caging)
      ii)      Artificial self-pollination (by hand)

i)          Natural self – pollination

a)          Bagging

       Select four healthy flowering branches on all the four sides of a tree. Remove
damaged flowers and those which have already opened or are about to open. Count the
remaining unopened flower buds on each branch. Cover each branch with a specially
ringed muslin bag (insect-proof). Label all the branches indicating the date of bagging
and number of flower buds bagged. Remove the bags after 4-6 weeks and count the
number of fruits set on each tree. Then calculate the average fruit set percentage.

b)          Caging

       Enclose the trees in cages prior to initiation of flower opening. Cages should have
top and side walls of plastic with windows for ventilation, ensuring them insect-proof.
After a stipulated period (generally 4-6 weeks) depending upon the crop, remove the
cages. Observe the development of fruits or no fruits.

ii)         Artificial (hand self-pollination

       These tests are more reliable than bagging/caging as the fruits could develop due
to apomixis or parthenocarpy. In this test, flowers are hand pollinated with pollen
obtained from the same tree. Collect the pollen, emasculate and pollinate selected number
of flowers on four branches of the tree. After 4-6 weeks, count the number of developing
fruits on each branch and work out the per cent fruit set average.
Exercise 9: Quantitative determination of fruit-set through open and cross-
pollination

Objective

       To estimate and compare productivity achieved through natural and artificial
cross-pollination.

Materials

       Forceps, camel’s hair brush, butter paper bags, metallic labels, crochet thread,
notched scissors.

Open-pollination

       Mark four healthy and flowering branches in all the four directions of a tree.
Remove the damaged or diseased flowers. Count the total number of flowers (including
already opened and unopened) on each branch. Put a label at the proximal end of each
branch to indicate number of flowers and date of record. Observe the trees after 40 days
and count the number of resulting fruits developing on each branch. Calculate the average
percentage of fruit set.

Cross-pollination

        Select four healthy and flowering branches on a tree in all the four-directions.
Retain and count the flower buds at ‘balloon’ stage and remove all opened and immature
flower buds. Perform emasculation and subsequently pollinate with the desired male
parent. Tag all the branches with appropriate labels depicting information regarding
parents (male and female), date of emasculation/pollination and number of flowers
pollinated. Count the number of fruits set after 40 days on each branch and thus calculate
the percentage.
Exercise 10: Pre-sowing treatments for seeds germination

Objective

       To overcome seed germination barriers such as hard seed coat, dormancy

Materials

        File, wooden boxes, tin boxes, plastic containers, refrigerator, sterilized sand,
sphagnum moss, mercuric chloride, gibberellins, thiourea, sulphuric acid, labels, hammer,
nut cracker.

In many fruit crops, seeds required pre-sowing treatments to overcome dormancy or to
enhance germination rate. These are as follows:

1.     Scarification

i)     Crack the hard seed coats with a hammer or completely remove the shells.
ii)    Use of file to soften the hard seed coat.
iii)   Dip the seed in hot boiling water for 5-10 minutes, transfer to tap water for 3-4
       days.
iv)    Chemical

       Dip the seed in concentrated sulphuric acid or hydrochloric acid for few to tea
minutes. Alternatively, treat the seed with appropriate doses of growth regulators like
gibberellins and thiourea.

2.     Stratification

       Keep the fungicide treated soaked seeds in wooden boxes or in soil pits
containing moistened sand in alternate layers (seed followed by sand and so on)
maintained at a low temperature (below 7.2ºC). Generally, do this in the months of
November-December. For small quantities of seed, use tin cases or rigid plastic
containers (used ice cream or freezer containers). Make holes in their base and place the
seed in alternate layer of stratification medium (sphagnum moss/sand/perlite). Keep the
containers in a refrigerator/deep freezer maintaining temperature below 7.2ºC for varied
period depending upon the fruit crop. Ensure that the site of stratification is rodent – free,
shady and adequate moisture is maintained. Prepare a map indicating which seed lot is in
each box/pit container in addition to labels tagged on them.
Exercise 11: Induction of mutations through use of physical / chemical mutagens


Objective

       To create genetic variation (mutants) for improvement

Materials

a)     Physical mutagens (sources):

       X-ray machine
       Gamma cell
       Thermal neutrons

b)     Chemical mutagens:

       N-nitroso – N- Methyl urea
       Ethyl methane sulphonate

c)     Plant Parts:

       Dormant bud scions
       Stem or root cutting/active summer buds
       Seeds/pollen grains/ Pollen mother cells

Exposure / Application Procedures

1.     Physical mutagens (Irradiation)

        Arrange the dormant bud scions or cuttings horizontally in front of the source or
radiations. In case of a gamma source, place the plant material to be treated in the space
provided in the gamma cell. Lower it into the source where inside, the material gets
exposed to the gamma rays.

2.     Chemical mutagens

        Select suitable well developed axillary buds on shoots of current season or
previous year growth. Cut the apical bud and exclude immature buds. Treat 3-4 buds per
shoot. If buds are large then remove the scales a day prior to treatment. Immerse the
whole shoots/cuttings or seeds as the case may be in the desired chemical solutions. Add
dimethyl sulphoxide (1-2%) to improve the penetration of chemicals into plant tissue.
Alternatively, place cotton wicks saturated in chemical solution on the buds. Preferably
wrap the cotton wick around the axillary bud and swab it with chemical mutagen. After a
day or so, remove the cotton wicks and wash the buds. In case of seeds, remove the seed
Coats and use stratified seeds instead of dry ones. Thoroughly wash the seeds
immediately after treatment with chemicals.

Post treatment handling

        Immediately propagate the treated plant material and allow the grafted plants to
grow under optimum conditions. Ensure active growth through adequate supply of water,
nutrients and light. In order to identify/select the mutants, either propagate (budding) the
primary shoot or do repeated heading back of the primary shoot. Leave the buds basal to
those propagated with the original graft to produce shoots for selection.
Exercise 12: Induction of polyploidy by colchicine


Objective

       To obtain somatic polyploids via disrupting mitosis in somatic cells or tissues.

Materials

       Meristematic tissues, seeds, colchicines, dimethyl sulphoxide, sucrose or glucose.

Procedure

        Select axillary meristems or sub axillary meristems on current season shoots.
Remove the apical meristem and all leafy tissue around the axillary bud. Apply
colchicines (0.1-0.9%) to buds using cotton, agar or lanolin along with 2% dimethy
sulphoxide or 5% glucose. Remove the source of colchicines after one or few days to
permit the growth of lateral shoots. Screen out the shoots showing signs of polyploidy
and propagate the potential polyploidy buds for further evaluation. Somatic polyploidy or
giant sports are easily recognizable by their various growth, thicker leaves and larger
fruits.

       Seeds, if are to be used, should be treated with a dilute solution of colchicines
(0.2%). Rinse the seeds after few hours or even days. Then sow the seeds for raising
seedlings so as to screen somatic polyploids.

				
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