PROTOCOL 1

1. Pretreatment of root tips with 100ppm of the pyrethroid (pH 5.8 adjusted with 1N HCl) at 4 °C for 24 hrs.

2. Fix the samples with farmer solution (3:1 absolute ethanol and glacial acetic acid) for 24 hrs.3. Squash the sample and observe under microscope.

-Source: Orrillo, Matilde. 1993. An alternative pretreatment method for mitotic chromosome observation in potatoes. American Journal of Potato Research 70 (7): 543-548.

PROTOCOL 2

1. Pretreatment of toot tips with 0.0028M 8-hydroxyquinoline for 24h at 10 °C2. Fix the sample with Carnoy’s fixative (3:1 absolute ethanol and glacial acetic acid)3. Hydrolyze it in 5N HCl for 20 min.4. Squash in 45% (v/v) acetic acid and stain with 2% (v/v) Giemsa and observe under microscope.

-Source: BRASILEIRO, Ana Christina Rabello; WILLADINO, Lilia; CARVALHEIRA, Gianna Griz and GUERRA, Marcelo. Callus induction and plant regeneration of tomato (Lycopersicon esculentum cv. IPA

5) via anther culture. Cienc. Rural [online]. 1999, vol.29, n.4, pp. 619-623. ISSN 0103-8478. http://dx.doi.org/10.1590/S0103-84781999000400008.

PROTOCOL 3

1. Pretreatment of root tips with 2mM 8-hydroxyquinoline at room temperature for 2 h and then at 4 °C for 2 h (or 16 h?).

2. Fix the sample in acetic alcohol (1:3 v/v) at 4 °C for 2 h.3. (Soften the sample by 1N HCl for 3 h at room temperature and subsequent 10 min at 60 °C. And

either store in DD water in 4 °C or immediately follow next step).4. Wash the sample with distilled water and macerate in a solution of enzyme mixture for 30 to 60

min at 38 °C. The solution consisted of 4% cellulose RS (Yakult), 1% Pectolyase Y23 (Seishin Pharmaceutical), (2.1% macerozyme too?) 0.07 M KCl (not necessary?) and 0.075 M Na2EDTA (or 1 mM ETDA ?), and was adjusted to pH 4.0 with 0.1 N HCl (Zhuang et al., 1990).

5. Wash the macerated root tips with distilled water (not necessary?).6. Spread the macerated root tips on the slide by tapping with fine tweezers by adding a few drops

of acetic alcohol and air-dried under room conditions.7. Stain the air-dried specimens with 4% Giemsa diluted with 1/15 M phosphate buffer (pH 6.8) for

3 to 5 min, air-dried again, mounted in Permount and observed under a microscope.

Source: Park, S. M., M. Hiramatsu and A. Wakana. 1999. Aneuploid plants derived from crosses with triploid grapes through immature seed culture and subsequent embryo culture. Plant Cell, Tissue and

Organ Culture 59: 125-133

PROTOCOL 4

1. Pretreatment with ice-cold water for 8 to 24 h (TSUNEWAK1 • JENKINS, 1960), or with 0.002 M hydroxyquinoline for 3 to 4 h at room temperature (TJIO & LEVAN, 1950). Both for root and shoot tips.

2. Fix the materials in a 3:1 fixative solution (three parts 90% ethanol and one part glacial acetic acid) for 24 h or more. Root tip materials can be kept in the fixative solution for several months in a refrigerator.

(For meiotic study, suitable flower buds are collected and fixed in the 3:1 fixative solution. Although the meiotic materials may also be kept refrigerated in the 3:1 fixative for several months, it is recommended that they be transferred after a few days to 70% ethanol and refrigerated until used. A single anther is used to determine the desired meiotic stage. When the desired stage is found, the other four anthers are macerated then transferred into the staining solution or kept in 70% ethanol.)

3. Hydrolytic maceration is done for both mitotic and meiotic materials in 1N HCl at 60°C for 6 minutes. The root tips and anthers are macerated in a small watch glass by adding several drops of 1N HC1 and keeping in an incubator at 60°C for 6 minutes; then an equal amount of 1N NaOH is added, or simply remove the 1N HC1 by a small piece of blotting paper.

4. Stain the fixed materials in 0.8 to 1.0% acetocarmine by keeping them 3 or 4 days at room temperature, or for any longer period in a refrigerator.

5. Squash the stained materials as follows: a. Remove a root cap.b. Cut a small piece of the growing point or, if possible, squeeze out a small amount of

meristematic tissue from the stained root tip.c. Put a piece of cut meristematic tissue on a slide glass. For meiotic study, one anther is cut

transversely into two pieces on a slide glass if anthers are relatively large.d. When materials are over-stained, de-staining can be accomplished in the following way.

Apply a drop of a mixture of 1 N acetocarmine and 1N HC1 (1:1) and heat the slide over an alcohol flame for several seconds (depending upon the stain density), and then remove the solution quickly with a piece of blotting paper. This procedure also macerates materials. The de-staining time is a function of the density of the stain.

e. Apply a drop of 45% acetic acid, then put on a cover slip.f. Heat the slide gently for several seconds over a small alcohol flame. The use of 80%

ethanol is recommended for an alcohol lamp.g. Tap the cover slip very gently with the tip of a scalpel while holding the corner of the

cover slip with a finger or cork to prevent slipping. Tapping pressure should always be applied at the same place over the specimen.

h. Heat the slide again for a few seconds over an alcohol flame. i. Place blotting paper over the cover slip and press gently but quickly with a thumb or

index finger while holding the corner with another finger of the other hand. This procedure spreads and flattens the cells and removes the excess fluid.

6. Observe the preparation.

7. Apply a drop of mixture of 10 parts 45% acetic acid and one part glycerine (Rattenbury's fluid) to the corner of the cover slip to make the preparation semi- permanent.

8. If cells are crowded and/or the cytoplasm is still over-stained, apply a drop of the Rattenbury's fluid or 45% acetic acid and gently heat again (step f), and repeat the procedure from g) through i) to improve the quality of the preparation.

-Source: Tsuchiya, T. and C. Nakamura. 1978. Acetocarmine squash method for observing sugar beet chromosomes. Euphytica 28 (1979) 249-256

PROTOCOL 5

1. Collect the root samples and fix them with farmer’s solution.2. Wash in 2 or 3 changes of 70% ethanol for 1 hr or more in each change.3. Store in last change.4. Drain (with absorbent paper to remove most 70% ethanol).5. Cover material with 2-3 X as much staining solution as height of material.6. Leave 1-7 days Experience + bulk of material). [Putting in 60 °C paraffin oven hastens + is

innocuous]7. Retain excess staining solution (Snow’s Acidic Carmine) for future use.8. Rinse material with dH2O or 70% ethanol.9. Prepare slides by macerating the tissues in 60 °C with 45% acetic acid (include heating) by

squash method.10. Store material in ethanol.

(Note: Aceto-carmine stain is not required, and materials should not be hydrolyzed in 1N HCl, because HCl bleaches the stain.)

-Source: Singh, Ram J. Plant Cytogenetics 2nd ed.