The document presents two methods for preparing mitotic chromosomes from plant tissues for analysis. The first method uses young leaves from citrus plants, applying enzymatic maceration with a low concentration of enzymes over a long incubation period. This yielded well-spread metaphase chromosomes suitable for karyotyping. The second method uses young leaf buds from birch, applying a cold treatment, enzymatic digestion of protoplasts, and dropping the protoplasts to obtain complete, distinguishable metaphase spreads for analysis and fluorescence in situ hybridization. Both methods provide alternatives to using root tips that produce better quality chromosome spreads.

1. Preparation of Mitotic Chromosomes in Plants:
Presented by :NOORANI GUL NABI KHAN
Status :M1
Advisor: Dr. Chitose HONSHO
Date : 18 December 2014
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2. Today’s presentation Agenda:
• Introduction
• Summary of research paper 1
• Summary of research paper 2
• Gained knowledge
• Questions and discussion section
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3. Introduction:
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• Chromosomes were observed by Nageli in 1842.
• Chromosome researches had already found a large
variation in the number of chromosomes among
plant species.
• Thus, determination of chromosome number is an
important subject of the time.

4. What is the importance of studying the chromosome
number of plants nowadays?
• Genetic and breeding investigations often require karyotype
analysis, chromosome counting and banding.
• Chromosome number data remain valuable today in
systematics.
• It allows us for better selection of individuals to be included
in phylogenetic.
• DNA sequence – studies
• Cytogeographic studies etc.
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5. What kind of methods used for the determination
of mitotic chromosome no. in plants?
Early Methods Conventional methods
• Smear or sectioning method
In this method the plant tissue cut into thin slices
by hand or microtome. Counting the
chromosomal regions in the sliced sections.
Limitations:
1. Time consuming
2. Most fresh tissue is very delicate, easily
distorted and damaged
3. Laborious
4. Difficulties in hard materials
Squash method with several associate techniques:
It widely used for chromosome preparation but
require skill and experience too.
• Pretreatment and softening methods
• 1944, Emsweller and Stuart suggest Enzymatic
maceration for plant tissues.
• Enzymatic maceration with air-drying
• Giemsa staining method
Now these together are the current standard
method.
Advantages: chromosomes are free from
cytoplasmic debris and spread evenly on the glass
slide.
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Keep in mind that Cytological procedures for chromosome preparation depend upon
on plant species, the objective of the experiments and personal preference of the
cytologists but basic technique is same.
Purposes of pretreatment.
stop the formation of spindle, increase the number
of metaphase cells by arresting the chromosomes at
the metaphase plate, contracts the chromosome
length with distinct constrictions, and increase the
viscosity of the cytoplasm.

6. What kind of plant materials can be used?
• Root tips are the most convenient source of mitotic cells.
Limitations:
1. Root-tips metaphase chromosomes are highly condensed.
2. Difficult of counting small and numerous chromosomes.
3. Impossible to get on field young roots from large woody plants.
4. Root-tips from germinated seeds do not represent the same
genomic constitution as their mother tress (seed or pollen) due to
introgressive hybridization.
5. Metaphase index is low as compared to young leaves chromosome.
6. Not well spread on glass slides.
• Young flower buds, shoot tips, young leaves ,cell
suspension and callus can be substituted for
chromosome samples preparation.
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7. Young leaves mitotic chromosome
• Available in the most growing seasons.
• Well spread metaphase chromosomes
• Suitable for small chromosomes sizes and
numerous species.
• High mitotic index
• Perfect for karyotyping, gene mapping and
genome analysis
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Let us study together about young
leaves mitotic chromosome

8. A Chromosome Preparation Method Using
Young Leaves of Citrus (Kitajima. A et al,. 2001)
Objective of research paper:
• Oiyama (1981) squash method Chromosome no.
but not suitable for chromosome identification
of their morphology.
• Root tips of seedlings have been used but aerial
tissues like young leaves are more desirable for
accurate cytological and karyotyping studies.
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Metaphase chromosome

9. Materials and methods:
Basic procedures of chromosome preparation:
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Pretreatment 2mM 8-hydroxyquinoline, 3h 20C Fix in Fixative
solution, Store at 5C
Washing
Universal enzyme cocktail
with hypotonic solution
100µl enzyme
solution, 37 C
incubated
After incubation, 900µl
hypotonic solution added
and keep for one hour
Cell suspension is centrifuged at
1500 rpm/5min. Removal of
900µl supernatant
Washing and centrifuge twice as last step
60µl drop form 30cm
Air-drying overnight,
15min 2% Giemsa
staining

10. Condition of enzymatic maceration:
A preliminary experiment on enzymatic
maceration was conducted while using
pummelo plant materials.
• Different strength of enzyme cocktail
• Incubation time
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11. Results and Discussion:
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Pummelo leaves by 1(A), 1/10(B) and 1/100(C)
strength of 3% cellulase +1% pectolyase

12. Continues …
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13. Continues …
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• Compared to pervious literature, in this
experiment lower enzyme concentrations
with longer incubation yielded a better
chromosome preparation.
• Pre-fixation help the pre-metaphase and
metaphase visible.
• Leaf size and fineness influence the
degree of enzymatic maceration.
• Tapping of slides and dropping has same
result.
• Using root-tips of citrus seedling, it is
necessary to distinguish the seedling is
zygotic or not. In monoembryonic
pummelo , the chromosome compositions
were different from that of the seed or
pollen parent.
Conclusion: The chromosome composition of zygotic seedlings in fruit tress
may not be the same as that of the mother tree.

14. Preparation of Chromosomes from plant leaf meristems
for karyotype analysis and in situ hybridization.
Objective: Root-tip chromosome is often impossible to make karyotypic
analysis, gent mapping or genome analysis because of small and
numerous chromosome are highly condensed.
Therefore, young leaf tissue are a reliable source of metaphases in
any plant species (small and numerous, cutting does not produce
roots, introgressive hybridization)
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By: Anamathawat. K,. 2003.

15. Materials:
• Plant materials: shoot-tips or leaf buds (2-3mm) of birch
• Chemicals and reagents:
1. Ice-water: For cold treatment
2. Fixative: glacial acetic acid : ethanol (1:3 v/v)
3. Digestion enzyme mixture: store -20C.
4. 75mM KCl solution, ph. 7.
5. Chromo sulfuric acid: For slides cleaning
• Small equipment and consumables:
• Microscope items: slides, coverslips, forceps, coplin jar
• Molecular items: 0.5ml, 1.5ml microtubules, micropipettes and
disposable pipette tips, nylon mesh
• Equipment: Phase-contrast microscope and micro-centrifuge.
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16. Procedure:
1. Tissue collection: leaf buds from long shoots 2-4mm, place ice-water (0-
4c) for 23-27h to arrest metaphases
2. Fixation: place 2-3 leaf buds in each micro tube containing 1ml cold
fixative ,2h/R, Store -20c.
3. Enzyme digestion: place 2-3 buds in 100µl of cellulase/pectinase,
incubate 3-5h at RT
4. Protoplast isolation: make suspension in enzyme mixture by breaking the
leaf tissue, filter with nylon mesh into new 1.5ml tube, keep the filtered
cell/protoplast suspension at 4C for some hours.
5. Hypotonic treatment of protoplasts: add 1.5ml of cold 75mM KCl into
protoplast suspension, gently mix and stand for 5-7min.
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17. Continuous…
6. Cleaning and fixation of the protoplasts: spin down at 7000 rpm for
5min, discard supernatant and add 1ml of ice-cold fixative to protoplast
pellet, re-suspend and spin down again at 7000 rpm , remove
supernatant and repeat this fixative treatment twice more.
7. Protoplast dropping: add 50µl fresh and cold fixative onto the protoplast
suspension and gently mix, drop on wet slide from 10-20cm height.
8. Treatment of chromosome for in situ hybridization: this step involves
further fixation and drying
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18. Results and discussion:
• This protocol is highly effective for studying Cytogenetic
and population genetics of plants in the field or in nature
especially those with small and numerous chromosomes.
• Root-tips metaphase chromosomes are highly condensed
and difficult to count.
• Leaf chromosome are well spread easier to get an
accurate count.
• Conventional squashing technique make poor quality
spreads , chromosome stick together or some are lost or
float away between cells. Aneuploidy
• Metaphase chromosome by protoplast dropping method
is usually complete.
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19. Results and discussion:
• Leaf chromosomes can be differentiable by size; in this
case (0.4-5µm long)
• Leaf buds or shoot-tips are convenient and available
for most of the growing season.
• Metaphase index is high
• Leaf buds chromosomes can avoid those difficulties
which arise in root-tips chromosomes.
• It is tool to asses cytogenetic status of field plants.
• Also, this protocol is suitable for fluorescence in situ
hybridization (FISH).
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20. Metaphase chromosomes:
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21. References:
• Kitajima, A., et al (2001) A Chromosome preparation method
using young leaves of citrus. (J. Japan. Soc. Hort. Sci.) 70 (2):
191-194. 2001
• Anamathawat, K. (2003) Preparation of chromosomes from
plant leaf meristems for karyotype analysis and in situ
hybridization. Methods in Cell Science 25: 91-95 (2003).
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Thank you for your kind attention
Cheers to new the year 2015 because it bring another chance
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