MMCT in Citrus – Detailed Procedure

The following is the complete protocol for microprotoplast isolation. It is important to emphasize that the suspension cells to be used as donor must be fast growing ones for the isolation of a large quantity of micro- protoplasts, since the micronucleation occurs during cell division.

1. Grow suspension cells in a 3-4 day sub-

culture cycle in H+H medium (Grosser and Gmitter, 1990) containing 50% of macronu- trients, under appropriated lighting condi- tions.

2. One day after subculturing, add 10 mM of Hu for 24 h.

3. Wash out the HU four times with H+H medium.

4. Add 32 mM APM for 24 h. Cells with chromosomes scattered (Fig. 11.1) or with several micronuclei (Fig. 11.2) can be visu- alized by staining fi xed cells with 4¢, 6- diamidino-2-phenyindole (DAPI) at 0.4

mg/ml.

5. Drain and divide the cells into approxi- mately 300 mg amounts in 60 ´ 15 mm Petri dishes and add 32 mM APM + 10 mM CB + one part of enzyme solution to three parts of

0.6 M BH3 medium (enzyme and 0.6 M BH3, as described by Grosser and Gmitter, 1990). Incubate in the dark with gentle agi- tation for 15–18 h.

6. Filter the micronucleated protoplasts through a 45 mm stainless steel screen (Small Parts, Inc.,) and pellet them at 100. g to remove enzyme. Purify the protoplasts by centrifugation through a 25% sucrose – 13% mannitol gradient and wash once with

Fig.11.6. A root tip cell containing 24 chromo- somes, 2 n + 6.

0.4 M mannitol. Remove most of the man- nitol and maintain the protoplasts as a dense solution. All solutions must contain 32 mM APM + 10 mM CB during the whole process of protoplast isolation and purifi ca- tion.

7. Prepare an iso-osmotic gradient of per- coll by adding 7.2% mannitol to a percoll solution (Amersham Pharmacia Biotech), and spin at 100, 000. g at 20°C for 30 min in a swinging bucket rotor.

8. To the top of the pre-formed percoll gra- dient add the dense solution of micronucle- ated protoplasts and spin at 100, 000. g for 2 h at 20°C in a swinging bucket rotor.

9. After centrifugation, usually nine bands are obtained (Fig. 11.3). The number of bands will depend on the amount of proto- plasts loaded and on the efficiency of micronucleation. Band ‘a’, forms at the sur- face of the gradient and is usually com- posed of a sticky material. This band should not be collected since it will con- taminate the microprotoplast suspension. A glass rod or the tip of a Pasteur pipette can be used to remove the central part of this band to facilitate the collection of the others. Band ‘b’ is normally very thick and contains a large amount of microproto- plasts, and is usually collected together with band ‘c’. In all microprotoplast isola- tions performed so far, for citrus, the amount and quality of microprotoplasts originated from these two bands has been more than can be handled by one person per day in fusion experiments. The other bands below ‘c’ contain very clean and good quality small size microprotoplasts; however, the amount of mannitol collected together with the bands makes it very diffi - cult to precipitate the microprotoplasts, even when diluted with large amounts of

0.6 M BH3.

10. Collect bands ‘b’ and ‘c’ (Fig. 11.3) and dilute it to at least 15 ml with 0.6 M BH3.

11. Filter the microprotoplasts suspension sequentially through a 20 mm nylon mesh screen, 14 mm nucleopore membrane, and 10 and 5 mm nylon mesh screens. The nylon screens are manufactured by Small parts

Inc., and the Nucleopore membranes by Corning. If necessary, a light pressure can be applied to facilitate the fl ow; if high pressure is applied, the microprotoplasts may be damaged and larger microproto- plasts will fl ow through.

12. The microprotoplasts can be visualized under fl uorescence after staining with 3 ml of an acridine orange solution at 10 mg/ml. A size comparison of a protoplast (P) and microprotoplasts (M) is shown in Fig. 11.4.

13. Protoplasts from a recipient species should by this time already be isolated and purified as described by Grosser and Gmitter (1990).

14. Protoplasts and microprotoplasts are mixed in a proportion of 1: 2 or 1: 3 and fused by polyethylene glycol (PEG) method as described by Grosser and Gmitter (1990).

15. The fusion products are cultured as described by Louzada et al. (2002) and plants are regenerated using B germination medium (Grosser and Gmitter, 1990) with half strength of macronutrients.

16. Regenerated plants are rooted or micro- grafted on suitable rootstock.

17. Chromosome counting is performed according to Louzada et al. (2002), or by any other cytological method. Molecular characterization can be carried out by polymerase chain reaction (PCR)-based methods or amplifi ed fragment length poly- morphism (AFLP). If the species are dis- tantly related, probably GISH (genome in situ hybridization) will be feasible. Fluorescence in situ hybridization (FISH) or (GISH) for MMCT between closely related citrus species is very diffi cult to accomplish.