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Tissue Preparation Techniques






A general protocol for cytogenetic investi- gations is summarized in Table 1. To study the tissue by light microscopy the specimen is generally sliced into thin sections or


 
 

Table 1. A slide preparation protocol for observation of Citrus mitotic fi gures.

1. Collect root tips with 3-5 mm in length with vigorous growth and soak in water in refrigerator at 6 oC for 24 hours.

2. Fix with ice-cold fl uid of methanol: acetic acid (3: 1) for 1 day.

3. Transfer roots to ice-cold citrate buffer (0.01 M citric acid/sodium citrate, pH4.6).

4. Excise 10 root tips with @ 1mm long and transfer into enzyme solution (7-8% Pectinase Sigma + 2% Cellulase Onozuka in the citrate buffer) in an Eppendorf tube.

5. Spin down specimens by centrifugation and incubate at 37oC for 1.5 to 2.5 hours.

6. Centrifuge at 2500 rpm to discard supernatant, add new citrate buffer and suspend with gentle pipet- ting.

7. After washing with citrate buffer 3 times, add 100-200 µl cold methanol: acetic acid (3: 1) and resus- pend.

8. Drop cell maceration mixture on slide glass and air-dry.

9.

 
 

Confi rm the preparation by phase contrast microscopy.

Modifi ed after Ito et al., (1992)


 

 

 
 

 


 

Fig. 6.1. Chromosomal variability in Citrus species. (a and b) Metaphases of Citrus depressa showing

(a) no secondary constrictions (SECs) and (b) one proximal and two terminal SECs. Prophase (c), prometaphase (d) and metaphase (e) chromosome complement of C. deliciosa. Metaphase of

C. pennivesiculata (f), C. volkameriana (g) and Poncirus trifoliata (h). Larger arrows, proximal SECs; small arrows, terminal SECs; arrowheads,

heteropycnotic blocks. The bar in (h) represents 5 mm for all fi gure parts (Guerra et al., 1997).

 

 

squashed and contrast within tissues is induced using dyes or fluorochromes. Citrus chromosome preparations for routine examination can be made from pollen mother cells, root or shoot meristem and embryos (Guerra et al., 1997). The metaphase stage is arrested by pre-treat- ment solutions before the fi xation (Fig. 6.1). Mitotic analysis of the samples can be improved by pretreatments with 0.002 M 8- hydroxyquinoline (8HQ) for 1 h at room temperature and then 20-23 h at 10°C (Oiyama and Okudai, 1986). Also, 1, 4- dichlorobenzene has been employed suc- cessfully as a pretreatment reagent in citrus karyotype studies (Jaskani, 1998). It straightens of the chromosome arms and prevents excessive clumping. Louzada et al. (2002) improved chromosome scattering and micronuclei visualization by treating


Fig. 6.2. (a) Diploid cell (2n = 18) of Swinglea glutinosa with chromosomes scattered;

(b) multinucleated protoplast of ‘Ruby Red’ grapefruit; (c) cell from ‘Ruby Red’ grapefruit + ‘Succari’ sweet orange with 22 chromosomes; (d) a cell from Swinglea glutinosa + sour orange with 22 chromosomes (Louzada et al., 2002).

 

 

suspension cells for 2 h with 1, 4- dicholorobenzene (Fig. 6.2). Storage of root tips at 6°C enhanced mitotic index in dif- ferent Citrus species (Ito et al., 1993). The combination of 8HQ and 4oC treatment resulted in increased resolution of mitotic fi gures in the root tips (Roose et al., 1998).

 

 

Fixation

The aim of fi xation is to preserve the tissue in a state that most closely refl ects a living cell. The choice of fi xative is generally dependent on the tissue of interest as dif- ferent fixatives can preserve particular tissue elements. However, the most com- monly used fi xatives for general plant his- tology are buffered aldehyde and formalin/acid/alcohol mixtures (FAA). FAA fi xes nucleic acids very well but gives poor morphological preservation and makes the tissue hard (Spence, 2001). Chen et al. (2004) fi xed fl ower buds for 48 h in Carnoy’s solution of ethanol-acetic acid (3: 1) at room temperature and kept in 70%


 


 

ethanol at 4°C until use, while, Louzada et al. (2002) fi xed suspension cells in the same fi xative but for 24 h.

 

 

Maceration

Softening of meristematic tissues is tradi- tionally obtained by soaking in 1N HCl at 60°C for 10 min. Enzymatic digestion has been reported to improve karyotype analy- sis (Jamieson et al., 1986). Fixed anthers macerated in a 100 µl enzyme mixture (2% cellulase and 0.5% pectolyase) in 75 mM KCl, pH 4.8, for 20 min at 37°C digested cell wall and improved mitotic index. Compared to the traditionally used macera- tion by soaking samples in 1N HCl at 60oC and squashing them on a glass slide, the enzymatic digestion of cell wall produces stable specimen for the karyotype analysis including in situ hybridization (Jamieson et al., 1986). Citrus root tips fi xed with in Carnoy’s solution, are rinsed with water or

0.1 M citrate buffers and then macerated with the combination of cellulase and pectinase (Ito et al., 1992). The optimum combinations of enzymes were different among experiments. For example, the root tip cells of C. junos seedling macerated in 2% cellulase Onozuka RS and 6% pecti- nase (Sigma) in citrate buffer, generated better preparation index with digestion at 37 oC for 2-2.5 hr (Ito et al., 1992). A com- bination of 3% cellulase RS and 1% pecti- nase Y-23 were used for the observation of young leaf mitosis (Befu et al., 2000).

 

 

Staining

Specifi c staining is obtained by using dye which has an affi nity for a particular cell type or tissue element. The root meristems are squashed in a drop of 45% acetic acid and then stained in a 1% aceto-carmine or aceto-orcine (Rao et al., 1992) or 2% Giemsa solution (Guerra et al., 1997). Staining root tips in Schiff”s solution (leuco-basic fucsin) for 45 min in the dark enhances chromosome visualization.


 

Another dye reported to stain anther squashes is Carbol Fuchsin solution (Chen et al., 2004). To visualize karyotypes under fl uorescence microscope, Louzada et al. (2002) stained digested cell suspensions with 4, 6-diamidino2-phenylindole dihy- drochloride (DAPI) @ 0.4 µg/ml.

 

 


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