Study Notes on Chromosome Technique

The below mentioned article provides a study note on Chromosome Technique used in Plants:- 1. Study of Mitotic Chromo­somes 2. Study of Meiotic Chromo­somes 3. Identifying Features of Cytological Plates.

Study of Mitotic Chromo­somes:

Mitosis occurs in all meristematic cells which are located in root tip, shoot tip, young leaf tip and other growing organs of plant. Of all these organs, root tip is the most actively grow­ing part.

A. Root Tip Squash Technique:

(a) Acetic-orcein staining:

1. Pretreatment:

Fresh young root tips of 1 cm long are taken and washed with water and transferred to corked glass tube containing a suitable pre-treatment agent. The tube is kept at 10-12°C for a specified period.

2. Fixation:

Pretreated root tips are fixed in a suitable fixative (acetic-ethanol 1:2 or 1:3) for 1-24 hr.

3. Staining-cum-maceration:

Fixed root tips are treated in 45% acetic acid for 5-10 min and are transferred to stain mixture 2% acetic-orcein and NHCI (9:1). The tube is gently heated over the flame for 5-10 seconds and kept for 45 min – 1 hr.

4. Squashing:

A root tip is taken in a drop of 45% acetic acid on a slide. Only the tip region is taken discarding the other tissue. A cover-slip is placed over the root tip and squashed by applying uni­form pressure with the thumb through a piece of blotting paper.

5. Sealing:

The preparation is perfectly sealed with paraffin wax.

6. Permanent mounting:

The temporary preparation can be made permanent following butanol schedule (vide mounting process).

(b) Feulgen staining:

1. Pretreatment:

Fresh young root tips of 1 cm long are taken and washed with water and transferred to a suitable pre- treating agent in a tube. The corked tube is kept at low temperature (10-12°C) for a specific period.

2. Fixation:

After washing with water, the pretreated root tips are fixed in a suit­able fixative (acetic ethanol 1:2 or 1:3) for 1 hr. – 24 hr. and gradually brought down to water passing through 90%, 80%, 70%, 50%, 30% ethanol.

3. Hydrolysis:

Root tips are hydrolyzed in NHCI at 56-58°C for 10-12 min. (prefe­rably 10 min).

4. Washing:

Root tips are washed with distilled water.

5. Staining:

Root tips are transferred to leucobasic fuchsin solution and kept in it for 30-45 min at 10-12°C under dark condition; the tips appear magenta coloured. 6&7. Squashing and sealing are to be carried out in the same way as described under acetic-orcein staining.

8. Permanent mounting:

The temporary preparation can be made permanent following acetic-alcohol schedule.

B. Leaf Tip Squash Technique:

1. Pretreatment:

Very young leaf tips are dissected out, washed in water and transferred to a corked glass tube con­taining a suitable pretreating agent such as para-dichlorobenzene at a suitable temperature for a specific period.

2. Fixation:

Pretreated leaf tips are fixed in a suitable fixative such as acetic-ethanol (1:2, 1:3) for a specific period.

3. Staining-cum-maceration:

Perform with acetic-orcein: NFHCI mixture (9:1).

4. Squashing:

Single leaf tip is taken in a drop of 45% acetic acid on a slide. Only the tip region is taken discarding the other tissue. A cover-slip is placed^ over the leaf tip and squashed by applying uniform pressure with the thumb

6. Permanent mounting:

Butanol schedule is followed for this purpose.

C. Root Tip Section Technique:

1. Fixation:

Fresh young root tips of 1 cm long are taken and washed with water. These are transferred to a corked glass tube containing a mixture of 1 % chromic acid and 10% formalin mixture (1:1) and kept for 12-24 hr.

2-8.

Washing, dehydration, clearing, infiltra­tion, embedding, section cutting, and removal of embedding material are to be carried out in the same way as described under Processing.

9. Pre-mordanting:

The slides are kept in 1% aqueous chromic acid solution overnight and washed in running water for 3 hr.

10. Staining:

The slide is placed in 1% aque­ous crystal violet stain for 30 min and the excess dye is washed off by rinsing in dis­tilled water.

11. Mordantin:

The slide is processed through 1% I2 and 1% Kl mixture in 80% ethanol for 45 sec.

12. Dehydration:

The slide is passed through absolute ethyl alcohol I, II, III, keeping 2-3 sec in each.

13. Differentiation:

The slide is transferred to clove oil I, kept for 2-5 min (observation under microscope is required for satisfac­tory staining) and then passed through clove oil II for 10-15 min.

14. Clearing:

The slide is kept in xylol I, II and III for 1 hr each.

15. Mounting:

Finally mounted in Canada balsam under a cover-slip and the slide is allowed to dry overnight on a hot plate.

Determination of Mitotic Index and Metaphase Frequency:

Mitotic index is the frequency of the dividing cells in a plant tissue at a time. It reflects the rate of growth of the plant. Mitotic index is calculated as the percentage of dividing cells among the total number of cells in a tissue (Fig. 21.1). For the study of chromosome number and chromo­some morphology, metaphase stage is worked out because at this stage, chromosome remains in maximum condensed state.

Frequency of metaphase can be calculated as the percentage of cells in metaphase among the total number of dividing cells.

Procedure:

Root tip is the ideal material for the study, it contains the population of dividing cells at different stages. Acetic-orcein squash technique is followed (pretreatment not neces­sary).

Karyotype and Idiogram Prepa­ration:

Karyotype is the phenotypic appearance of the entire chromosome complement of the species, it represents all the chromosome types based on their morphology in the total comple­ment. The number of chromosomes and the mor­phology of each chromosome pair is normally constant and characteristic for a species (Fig. 21.2).

The diagrammatic representation of the karyotype is the idiogram. On the other hand, karyogram is the actual representation of the karyotype performed from the microphotograph drawing.

The criteria needed to prepare karyotype are:

(i) The number of chromosomes in the cell;

(ii) The length of each chromosome including the individual length of short arm and long arm;

(iii) The position of the primary constriction that is centromere;

(iv) The existence and the localiza­tion of secondary constriction.

Procedure:

1. Pretreatment, fixation and staining are done.

2. The slide is prepared through squashing technique.

3. Well scattered metaphase plate with distinct constriction of chromosomes is found out under oil immersion objective and drawn with camera lucida or drawing prism.

4. Magnification under oil immersion objective is worked out with the help of a stage micrometer and camera lucida.

10 stage division = 10 x 10 µm= 100 µm

= 0.1 mm.

1 stage division = 0.01 mm.

0.01 mm is magnified into x mm.

1 mm is magnified into x/0.01mm = X x 100 times.

So, magnification of microscope under oil immersion objective is X x 100.

5. The length of short arm of each chromosome is measured in mm from the drawing and its actual length in pm is determined as follows:

6. Then the length of long arm of each chro­mosome is determined as above.

7. The whole length of the chromosomes is then calculated by adding short arm length and long arm length.

8. Centromeric Index i.e. ‘i’ value is calculated using the formula:

9. Chromosomes are classified into the follow­ing groups on the basis of the location of the constrictions:

10. Karyotype table is made (Table 21.5) and Idiogram or karyogram is prepared conside­ring the length as well as ‘i’ value of chromo­somes. They are grouped as A, B, C, type and arranged from longest to smallest in each group (Fig. 21.3).

Some Common Materials:

1. Allium cepa (2n=16)

2. Allium sativum (2n=16)

3. Aloe vera (2n=14)

4. Lens culinaris (2n=14)

5. Nigella sativa (2n=12)

6. Pisum sativum (2n=14)

7. Sagittaria sagittifolia (2n=22)

8. Trigonella foenum-graecum (2n=16)

9. Viciafaba (2n=12)

10. Hordeum vulgare (2n = 14)

Study of Meiotic Chromo­somes:

In higher plants, meiosis occurs in pollen mother cells (PMC) which are present in the anthers and in the megaspore mother cell which lies within the ovule. An ovule containing only one megaspore mother cell and surrounded by several layers of vegetative cells, is rather difficult to work out. Anthers, on the other hand, contain large numbers of pollen mother cells which can be very easily exposed by bursting the anthers.

So, usually meiosis is studied from pollen mother cells. Meiosis occurs only once in the life cycle of an annual plant. To study the meiotic behaviour of chromosomes of a species, prophase I, metaphase-l and metaphase-II are the proper stages (Fig. 21.4).

Hence, flower buds of appro­priate size are to be selected to work out the suitable stages of meiosis; otherwise the stages may be too prometaphase or too mature. Different schedules followed for studying meiosis are – temporary smear technique, permanent smear technique and section technique.

A. Temporary Smear Technique (Acetic-carmine Staining):

1. Selection of bud:

Flower buds are taken serially from an inflorescence, starting from the smallest to the largest, until the correct bud hav­ing divisional stages is found. Excision of buds during the day time approximately 8 a.m. to 2 p.m. of a bright sunny day gives good result.

If the flower buds are collected in the field, they may be kept in acetic acid-ethyl alcohol (1:2) mixture. In such cases, just before smearing, these buds are transferred to 45% acetic acid and kept for 5-10 min. The fixed buds can be stored in 70% ethanol.

2. Fixation-cum-staining:

A drop of 1% acetic-carmine solution is taken on a clean grease-free slide, 2-3 anthers are dissected out from a suitable sized flower bud with a needle and put them in the fixation-cum-staining mixture.

3. Smearing:

With the help of a clean iron scalpel, the anthers are smeared by gently pressing anthers to squeeze out the content. The debris are removed and a cover-slip is put on it. The slide is slightly heated over a flame. Excess fluid is blot­ted off giving gentle pressure on the cover-slip.

4. Observation:

The cover-glass is sealed with paraffin and observed under the micro­scope. The slide can be kept in temporary condi­tion for 1-4 weeks if it is sealed perfectly and stored in a cool place.

5. Permanent mounting:

The preparation can be made permanent following the butanol schedule.

B. Permanent Smear Technique (Crystal Violet Staining):

1. Selection of bud:

Flower buds of diffe­rent sizes are taken. Single anther is dissected out from each bud and smeared following acetic- carmine method until meiotic divisional figures are observed.

2. Smearing:

Remaining anthers of the selected bud are dissected out and transferred on a clean, dry, grease-free slide. With the help of a clean scalpel the anthers are pressed to squeeze out the content and empty anther lobes are dis­carded. The content is quickly drawn into a thin smear on the slide with the scalpel.

3. Fixation:

The slide is immediately immersed in a tray containing suitable fixative such as Navashin’s fluids A and B, freshly mixed in equal proportion and kept for 3hr to overnight.

4. Washing:

The slide is placed in a wash­ing trough under running water for 1-3 hr.

5. Staining:

The slide is kept in 1% aque­ous crystal violet stain for 30 min and the excess dye is washed off by rinsing in distilled water.

6. Mordanting:

The slide is kept in 1% I₂ and 1% Kl mixture in 80% ethanol for 45 sec.

7. Dehydration:

The slide is passed through ethanol I, II, and III keeping 2-3 sec in each.

8. Differentiation:

The slide is transferred to clove oil I, kept for 2-5 min (observation under microscope is required for satisfactory staining) and then passed through clove oil II keeping 10- 15 min.

9. Clearing:

The slide is kept in xylol -1, II and III for 1 hr. each.

10. Mounting:

Finally, the slide is mounted in Canada balsam under a cover-slip and is allowed to dry overnight on a hot plate.

Some Common Materials:

1. Allium cepa (n=8)

2. Phlox drummondii (n=7)

3. Rhoeo discolor (n=6)

4. Sagittaria sagittifolia (n=11)

5. Setcreasea brevifolia (n=12)

6. Solanum torvum/nigrum (n=12)

7. Datura stramonium (n = 12)

8. Hordeum vulgare (n = 7)

9. Tradescantia virginiana (n = 6)

10. Allium tuberosum (n = 2x = 16)

Identifying Features of Cytological Plates:

A. Meiosis (Normal Stages):

1. Diplotene stage of Prophase-) of Meiosis (Fig. 21.4. j, k, I)

i. Cells are spherical in shape and loosely arranged.

ii. Absence of cell plate or partition wall.

iii. Bivalents are comparatively less con­densed, homologues are separating showing several intercalary chiasmata along the length.

iv. Nucleolus (faintly stained) present.

1. Diakinesis stage of Prophase-I of Meiosis (Fig. 21.4h)

a. Cells are spherical in shape and loosely arranged.

b. Chromosomes are short and thickened.

c. Chromosome constrictions are not clear.

d. Absence of cell plate or partition wall.

e. Bivalents are comparatively less con­densed.

f. Bivalents are scattered throughout the cytoplasm.

g. Bivalents showing different configurations, e.g., X, Y or lens shaped due to different degrees of chiasma terminalization.

h. Bivalents are countable.

i. Nucleolus absent.

3. Metaphase-I of Meiosis (in polar view) (Fig. 21.4 d, e, f, g)

(i) Cells are spherical in shape and loosely arranged.

(ii) Chromosomes are short and thickened.

(iii) Chromosome constrictions are not clear.

(iv) Absence of cell plate or partition wall.

(v) Bivalents are highly condensed, distinct and countable indicating the ‘n’ number.

(vi) Bivalents are scattered in the cytoplasm (polar view).

(vii) Nuclear membrane and nucleolus absent.

4. Metaphase-I of Meiosis (in lateral view) (Fig. 21.4a)

(i) Cells are spherical in shape and loosely arranged.

(ii) Chromosomes are short and thickened.

(iii) Chromosome constrictions are not clear.

(iv) Absence of cell plate or partition wall.

(v) Bivalents are highly condensed, distinct and countable indicating the ‘n’ number.

(vi) Bivalents are arranged linearly along the equatorial plane (lateral view).

(vii) Nuclear membrane and nucleolus absent.

(iv) Marginal outline of separated chromo­somal mass is smooth.

6. Telophase-I of Meiosis:

(i) Cells are spherical in shape and loosely arranged.

(ii) Cell plate or partition wall is absent.

(iii)Two compact lumps of chromosomal masses are visible in the two poles.

(iv) Marginal outline of separated chromo­somal mass is smooth.

(v) Distinct chromosomes are absent and not countable.

7. Prophase-II of Meiosis:

(i) Cells are spherical in shape and loosely arranged.

(ii) Presence of cell plate or partition wall.

(iii) P.M.C. in diad (2-celled) condition.

(iv) Each cell of the diad contains less distinct chromosomes.

(v) Chromosomes are comparatively less condensed and not countable.

8. Metaphase-II of Meiosis (Fig. 21.4c):

(i) Cells are spherical in shape and loosely arranged.

(ii) Chromosome constrictions are not clear.

(iii) Chromosomes are short and thickened.

(iv) Presence of cell plate or partition wall.

(v) PM.C. in diad (2-celled) condition.

(vi) Two haploid sets of condensed chromo­somes are visible in the two separate equatorial planes of the diad in a line (lateral view)/scattered (polar view).

(viii) Chromosomes are countable.

9. Anaphase-II of Meiosis:

i. Cells are spherical in shape and loosely arranged.

ii. Chromosomes are short and thickened.

iii. Presence of cell plate or partition wall.

iv. PM.C. in diad (2-celled) condition.

v. Nucleolus and nuclear membrane absent.

vi. Each cell of the diad-contains 2 sets of comparatively less condensed separating daughter chromosomes.

vii. Each set of chromosomes lies in between the equatorial plane and the pole, with the arms projecting towards the equa­torial plane.

10. Telophase-II of Meiosis:

i. Cells are spherical in shape and loosely arranged.

ii. Presence of cell plate or partition wall.

iii. PM.C. in diad (2-celled) condition.

iv. Two compact chromosomal masses in the opposite poles are visible in the indi­vidual cells of the diad.

v. Marginal outline of each chromosomal mass is smooth.

B. Meiosis (Abnormalities):

11. Meiosis showing Laggard Bivalent/Univalent: in Anaphase-I or Telophase-I (Fig. 21.5a, b)

i. Cells are spherical in shape and loosely arranged.

ii. Cell plate or partition wall is absent.

iii. Two sets of short and thickened chromo­somes are present away from equatorial plane (Anaphase-I).

iv. Two compact and highly condensed chromosomal masses are present in the two poles (Telophase-I).

v. One bivalent/univalent is visible solitarily near the equatorial plane and away from the poles.

vi. Two sets of daughter chromosomes are visible towards the opposite poles and are away from the equatorials plans ( Anaphase)

vii. Centromeres lie at the equator with arms projecting towards poles (metaphase)

12. Meiosis showing Chromosome Bridge in Anaphase l/Telophase-l (Fig. 21.5c)

i. Cells are spherical in shape and loosely arranged.

ii. Cell plate or partition wall absent.

iii. Two sets of short and thickened chromo­somes are present away from equatorial plane (Anaphase-I).

or

(iii) Two compact and highly condensed chromosomal masses are present in the two poles (Telophase-I).

(iv) Thread-like chromosome bridge passing through the equatorial plane is clearly visible connecting the two sets of separating chromosomes (Anaphase I)/ chromosomal masses (Telophase-I).

13. Meiosis showing Multivalent Ring in Metaphase-I (Fig. 21.5d):

i. Cells are spherical in shape and loosely arranged.

ii. Chromosome constrictions are not clear.

iii. Chromosomes are short and thickened.

iv. Absence of cell plate or partition wall.

v. Nuclear membrane and nucleolus are absent.

vi. A large multivalent ring of 12 chromo­somes is visible within the P.M.C.

vii. Chromosomes are found attached end to end.

viii. This is due to reciprocal translocation heterozygote.

C. Mitosis (Abnormalities):

14. Mitotic Metaphase/Anaphase showing Early Separation (Fig. 21.6g):

i. Cells are of different sizes and compactly arranged.

ii. Nucleolus and nuclear membrane are absent.

iii. Chromosomes are long.

iv. All chromosomes lie at the equatorial plane (Metaphase).

v. Centromeres are towards the pole with arms projecting towards the equatorials plane(Anaphase)

vi. One or a few chromosomes are noticed slightly preceding other daughter chromosomes and are present very close to pole.

15. Mitotic Anaphase showing Late Separation (Fig. 21.6e):

(i) Cells are of different sizes and compactly arranged.

(ii) Nucleolus and nuclear membrane are absent.

(iii) Chromosomes are long.

(iv) Two sets of daughter chromosomes are visible near the two opposite poles and are away from the equatorial plane.

(v) Centromeres are towards the poles with arms projecting towards the equatorial plane.

(vi) One or a few chromosomes are visible soli­tarily out of separating chromosomes in between the equatorial plane and the pole.

16. Mitotic Anaphase showing Sticky Bridge (Fig. 21.6a):

i. Cells are of different sizes and compactly arranged.

ii. Nucleolus and nuclear membrane are absent.

iii. Chromosomes are long. Two sets of daughter chromosomes are visible near the two opposite poles and are away from the equatorial plane.

iv. Centromeres are towards the poles with arms projecting towards the equatorial plane.

v. Chromosomes are sticky in nature.

vi. Thread-like chromosome bridge is visible connecting the two sets of separating chromosomes passing through the equa­torial plane.

vii. This abnormality may be due to effect of some physical or chemical agent.

17. Mitotic Metaphase showing Fragmentation:

(i) Cells are of different sizes and compactly arranged.

(ii) Nucleolus and nuclear membrane are absent.

(iii) Chromosomes are long, condensed and scattered in the cytoplasm/arranged in the equatorial plane.

(iv) One (or a few) small chromosomal frag­ment is visible.

(v) The chromosomal fragment is much smaller in size than the rest of the chro­mosomes and are possibly acentric.

(vi) This abnormality may be due to effect of some physical or chemical agent.

18. Mitotic Anaphase showing Multi-polarity (Fig. 21.6 b,f):

(i) Cells are of different sizes and compactly arranged.

(ii) Nucleolus and nuclear membrane are absent.

(iii) Chromosomes are long.

(iv) Daughter chromosomes are moving towards poles.

*(v) More than two poles are visible within a dividing cell.

*(vi) Each pole is star shaped, consists of dau­ghter chromosomes with radiating arms.

*(vii) Uneven distribution of chromosomes is noticed in the different poles.

*(viii) This abnormality may be due to effect of some chemical agent.

D. Pollen Mitosis:

19. Pollen Mitosis in Metaphase (Fig. 21.6h):

(i) Cells are boat shaped, loosely arranged and-‘with prominent exine and intine.

(ii) Nucleolus and nuclear membrane are absent.

(iii) Chromosomes are condensed, long, distinct and countable; constrictions are visible.

(iv) Haploid set of chromosomes, indicating the ‘n’ number, are scattered in the cyto­plasm.

20. Pollen Mitosis in Anaphase:

(i) Cells are boat shaped, loosely arranged and with prominent exine and intine.

(ii) Nucleolus and nuclear membrane are absent.

(iii) Two haploid sets of daughter chromosomes are visible towards the two opposite poles and are away from the equatorial plane.

(iv) Chromosomes are comparatively less condensed, longer, not countable.

(v) Centromeres are towards the poles with arms projecting towards the equatorial plane.