Protocols. Protocol for PEG-induced fusion and plant regeneration (CREC)

Protocol for PEG-induced fusion and plant regeneration (CREC)

Citrus somatic hybrids are most often pro- duced from the fusion of protoplasts iso- lated from embryogenic callus or suspension cultures of one parent with leaf-

derived protoplasts of the second parent. At least one parent in any fusion combination must be embryogenic to provide the capac- ity for plant regeneration in the somatic hybrid progeny. This protocol has been used successfully to produce somatic hybrid plants from more than 120 parental combinations at the University of Florida’s Citrus Research and Education Center (CREC), Lake Alfred, Florida, USA.

Protoplast isolation: embryogenic parent

Embryogenic callus or suspension cultures are generally maintained on either EME (MT basal medium containing 0.5 g/l malt extract (Murashige and Tucker, 1969)) or H+H (modifi ed MT basal medium contain- ing 1.55 g/l glutamine, 0.5 g/l malt extract and 50% less KNO3 and NH4NO3). Cultured cells used for protoplast isolation should be in the log phase of growth. For protoplast isolation, transfer 1–2 g of friable callus tissue into a 60 ´ 15 mm Petri dish (for sus- pension, transfer ~2 ml of suspension with a wide-mouth pipette and drain off the liquid using a Pasteur pipette). Resuspend the cells in 2.5 ml of 0.7 M BH3 medium (Grosser and Gmitter, 1990) (BH3 is a nitrate-free MT basal medium containing

3.1 g/l glutamine, 20 ml/l coconut water and the Kao and Michayluk (1974) organic addenda), and then add 1.5 ml of fi lter-ster- ilized enzyme solution containing 0.7 M mannitol, 12.0 mM CaCl2, 6.0 mM MES buffer, 1.4 mM NaH2PO4, 1% Onozuka RS cellulase, 1% macerase, 0.2% pectolyase Y- 23, pH 5.6. Seal dishes with Nescofi lm (or Parafilm) and incubate overnight on a rotary shaker at 20 r.p.m., in either low light or darkness.

Leaf parent

The best results are generally obtained using fully expanded leaves of new fl ush that have not fully hardened, taken from seedlings or recently budded plants maintained in a growth chamber or heavily shaded green- house. Use of in vitro grown leaf material precludes the need for decontamination prior to isolation. Leaf material is decontam- inated by immersion in 1 N HCl for a few sec- onds followed by a 12–15 min immersion in 10–15% commercial bleach containing three drops of Liquinox soap or other surfactant, followed by a 5 min rinse and two 10 min rinses in double-distilled H2O. Damaged vas- cular tissue and the midvein region are removed with a sharp scalpel. Remaining leaf material is feathered or cut into thin strips with a sharp scalpel and incubated in 3 ml of enzyme solution combined with 8 ml of 0.7 M BH3 medium in a 125 ml side-armed Erlenmeyer fl ask (with the side arm covered with Miracloth to prevent contamination). Leaf material in the enzyme cocktail is then evacuated for 15 min at 50 kPa to facilitate enzyme infi ltration. Preparations are then sealed and incubated as above.

Protoplast purifi cation (using a sucrose/mannitol gradient)

Following incubation, preparations are passed through a 45 mm stainless steel (or nylon) mesh screen to remove undigested cell clumps and debris. Protoplast-containing fi ltrates are then centrifuged for 4–10 min at

100 g in 15 ml calibrated screw-top cen- trifuge tubes. The supernatant is removed

with a Pasteur pipette, and the pellet con- taining the protoplasts is gently resuspended in 5 ml of a 25% sucrose solution containing CPW nutrients (27.2 mg/l KH2PO4, 100 mg/l KNO3, 150 mg/l CaCl2, 250 mg/l MgSO4, 2.5

mg/l Fe2(SO4)3·6H2O, 0.16 mg/l KI, 0.00025

CuSO4, pH 5.8) (Frearson et al., 1973). This is followed by slowly pipetting 2 ml of a 13%

mannitol solution (containing CPW salts) directly on top of the sucrose layer (avoid mixing). Centrifuge for 6 min at 100 g. Viable protoplasts form a band at the interface between the sucrose and the mannitol. Carefully remove the protoplasts from this interface and resuspend them in the appro- priate volume of BH3 medium as necessary for further manipulation.

PEG-induced protoplast fusion

This protocol is simple, effi cient, inexpen- sive and non-toxic to citrus protoplasts. Mix approximately equal volumes of puri- fi ed protoplasts from each parental source in 0.6 M BH3 medium and centrifuge for 4 min at 100 g. Resuspend the pellet of mixed protoplasts in a volume of BH3 medium equal to 4–20 times the volume of the orig- inal pellet (10´ is recommended for initial experiments, followed by fine tuning). Pipette two drops of the resuspended mix- ture into 60 ´ 15 plastic Petri dishes (the number of dishes determined by the volume of mixed protoplasts). Immediately add two drops of PEG solution (40% poly- ethylene glycol 8000, 0.3 M glucose and 66 mM CaCl2 at pH 6.0) to each fusion dish and incubate for 8 min. Note that the PEG solu- tion rapidly acidifi es over time – the pH should be checked prior to use. Add two drops of A + B solution (9: 1 (v/v), A = 0.4 M glucose, 66 mM CaCl2 and 10% dimethyl- sulphoxide at pH 6.0; and B = 0.3 M glycine at pH 0.5 using KOH pellets) to each fusion dish. To avoid precipitation, the A + B solu- tion should be mixed just prior to use. Following another incubation of 12 min, add 12–15 drops of BH3 medium around the periphery of the fusing protoplasts. After incubating for 5 min, carefully remove the PEG plus [A + B] solution with

a Pasteur pipette and replace it with 15 drops of BH3 medium. After incubating for another 10 min, remove the BH3 medium with a Pasteur pipette and replace it with 12–15 drops of fresh BH3 medium. Repeat this washing step twice more, carefully avoiding the loss of protoplasts. After the fi nal wash, protoplasts are cultured directly in the fusion Petri dish in either a shallow pool (8–12 drops of medium) or thin-layer culture (1.5 ml of medium) in either BH3 medium, EMEP medium or a 1: 1 (v/v) mix- ture of BH3 and EMEP (Grosser and Gmitter, 1990). Seal plates with Nescofi lm, and culture in either darkness or low light. This protocol is not only useful for generating somatic hybrids, but also fre- quently produces cybrids as a by-product (as does the electofusion protocol below). The cybrids generally contain the nucleus of the leaf parent, the mitochondrial genome of the callus parent, and the

chloroplast genome is randomly inherited.

Protoplast culture and plant regeneration

Following incubation for 4–6 weeks, cul- tures can be supplemented with medium containing reduced osmoticum, which is accomplished by adding 10–12 drops of a 1: 2 (v/v) mixture of 0.6 M BH3 medium and

0.38 M EME medium (MT basal containing

125 g/l sucrose and 0.5 g/l malt extract). After another two weeks, cultures can be transferred to solid medium in 100 ´ 20 Petri dishes with further osmoticum reduc- tion as follows: add 2 ml of a 1: 2 (v/v) mix- ture of BH3 medium and 0.15 M EME medium (MT basal containing 50 g/l sucrose and 0.5 g/l malt extract) to each fusion dish and pour the entire contents on to solid medium plates containing standard agar-solidifi ed EME medium. The liquid medium containing the protoplast-derived colonies should be spread evenly over the entire plate. Vigorously growing cultures may require dilution in order to achieve somatic embryo induction. Cultures recalci- trant to embryo induction can also be trans- ferred to EME medium containing 50 g/l maltose instead of sucrose as the carbohy-

drate source. Recovered somatic embryos are enlarged and germinated using any standard citrus somatic embryogenesis media sequence (Grosser and Gmitter, 1990). Niedz et al. (2002) have recently shown that the use of certain semi-perme- able membranes (i.e. cellulose acetate) can positively affect the normalizing of citrus embryogenesis, thereby improving the effi - ciency of plant recovery. Abnormal embryos that fail to germinate can be dis- sected into large sections and cultured on DBA3 medium (X.X. Deng et al., 1992) for shoot induction. Resulting shoots can be rooted on RMAN medium (Grosser and Gmitter, 1990). Rooted plants can be trans- ferred to any suitable commercial potting mixture and maintained under cover at high humidity for 2–3 weeks for acclimati- zation.

Protocol for protoplast electrofusion and plant regeneration

Suspension and leaf protoplasts were iso- lated and purifi ed as described by Grosser and Gmitter (1990). The purifi ed protoplast bands are transferred to new centrifugation tubes, and then washed twice by centrifu- gation at 100 g for 10 min in electrofusion solution containing 0.6 M mannitol and

0.25 mM CaCl2, pH 5.8. Fusions are con- ducted using an SSH-2 instrument

(Shimadzu Somatic Hybridizer-2, Japan). The electrofusion chamber is the FTC-03 with 0.8 ml volume. A protoplast mixture (0.8 ml) containing 3–5 ´ 105 callus proto- plasts and 10–15 ´ 105 mesophyll proto- plasts was transferred to the FTC-03 chamber, sealed by parafi lm and kept still for 5 min before fusion treatment. The elec- trical fusion parameters were carefully determined prior to fusion (Guo et al., 1998). For most fusion combinations, the following parameters can work well: AC (alternate current) fi eld, 60 s, 125 V/cm; DC (direct current) fi eld, 1250 V/cm, 30 ms in duration, fi ve times at 0.5 s intervals; fi nal time, 5 s. For some fusion combination such as red tangerine + trifoliate orange,

where the protoplast size of the latter was only 1/3–1/5 the size of the former, altered parameters (i.e. AC fi eld, 5 s, 200 V/cm; then AC fi eld, 30 s, 100 V/cm; DC fi eld, 1250 V/cm, 30 ms in duration, fi ve times at

0.5 s intervals; fi nal time, 5 s) are required, which not only resulted in good formation of protoplast pearl chains, and a 5–6% bin- uclear heterokaryon rate, but also circum- vented the negative effect of the long duration time of high AC fi eld strength on the viability of protoplasts (Guo et al., 2002). After fusion treatment, the proto- plast mixtures are incubated for at least 10 min before being transferred to 10 ml cen- trifuge tubes, and then centrifuged at 100 g for 4 min. This results in an optimum rate of binuclear heterokaryons. The super- natant is then discarded, and the fusion products are resuspended at a density of 1–2 ´ 105 cells/ml in BH3 medium (Grosser and Gmitter, 1990) by liquid thin-layer cul- ture. The subsequent protoplast culture and plant regeneration procedures are the same as mentioned above.

Protocol for in situ hybridization in citrus (CIRAD, from D’Hont et al., 1996, 1998)

Small fragments (1 mm2) of leaf or root mitotic areas are digested in pectinase + cellulase solution before performing chro- mosome squashes. For GISH, total genomic DNA of each species is used as a probe. DNA probes of approximately 300 bp are marked by nick translation of dNTPs linked with two kinds of molecule (biotin or digoxigenin) that should be revealed by two florochromes with different emission colours. For rDNA study, the probe pTa71 (18S–25S rDNA) marked with digoxigenin is used. The chromosome squashes are hybridized with probes after a step of denaturation. Then the biotin probes are revealed by avidin marked with Texas red (red coloration), while digoxigenin probes are revealed with anti-digoxigenin antibod- ies marked with fl uorescein isothiocyanate (FITC; green coloration). After an amplifi ca- tion step, a counterstaining of chromo-

somes with 4’, 6-diamino-2-phenylindole (DAPI) is performed. See D’Hont et al. (1996, 1998) for detailed protocols.

Protocol for CAPS analysis of citrus mitochondrial and chloroplastic genomes (CIRAD, from Lotfy et al., 2002)

DNA amplifi cation

The 13 pairs of universal cytoplasmic primers are described in Demesure et al. (1995). Some of them have been modifi ed to obtain better amplification with citrus (Lotfy et al., 2002). The primers and PCR conditions selected are described in Table

10.3. The PCR mixture (25 ml) contains 67

mM Tris–HCl (pH 8.8), 16 mM (NH4)2SO4,

0.01% Tween-20, 0.2 mM of each primer and

300 mM of dNTP (Eurobio), 1.5–2.5 mM MgCl2, 0 or 4% glycerol (depending on the primer pair used, Table 10.3), 0.5 U of Taq DNA polymerase (Eurobio) and 50 ng of citrus DNA sample. The mixture is covered with a drop of mineral oil, and the reaction is performed in a DNA thermal cycler (model PTC-100 MJ Research), programmed for an initial denaturing cycle of 4 min at 94°C then 30 cycles of 45 s denaturation at 92°C, 45 s annealing at 55 or 58°C (depend- ing on the primer pair used, Table 10.3), 3 min elongation at 72°C and a fi nal step of 10 min at 72°C to complete the synthesis of DNA strands.

DNA restriction

Amplified DNA fragments are digested using 4–6 base recognition restriction endonucleases (Dra I, Alu I, Bsp 143-I, Hae III, Rsa I, EcoR I, Mva I, Hin fI, Hin dIII, Ava II and Ama 87I) (Eurogentec or Amersham), in a fi nal volume of 25 ml containing 1´ specifi c buffer (Eurogentec or Amersham) for each restriction enzyme, 5 U of endonuclease and 15 ml of amplifi cation product. The reaction medium is incubated for 3 h at 37°C.

PCR conditions for Citrus

aUniversal primers previously described by Demesure et al. (1995) are used but some modifi cations are introduced.

bNew combinations.

cThe trnC2 sequence contains two different bases compared with Demesure et al. (1995).

dThe trnT3 sequence is inverted compared with Demesure et al. (1995).

DNA analysis

Native and digested amplifi cation products are separated by electrophoresis in a 1.8% agarose gel with 1´ TBE during 5 h, and then visualized by UV fl uorescence after staining with ethidium bromide (3 mg/ml). The size of the separated fragments is esti- mated by comparison with the 1 kb DNA ladder (0.5–10 kb) (Sigma).

Propidium iodide staining for fl ow cytometry (Yu et al., 1993)

Ploidy analysis and cell cycle distribution analysis

PROTOCOL

1. Pellet 2–3 ´ 106 cells in a 15 ml cen- trifuge tube and wash once with cold phos- phate-buffered saline (PBS)/azide solution.

2. Resuspend cells in 1 ml of low salt stain by gently vortexing. Cover the tube with foil and incubate in a 37˚ C water bath for 20 min with gentle mixing every 5 min.

3. Add 1 ml of high salt stain by gently vor- texing and store at 4˚ C for at least 1 h, and preferably overnight.

The Bauer DNA staining technique uses a hypotonic solution of Triton X-100 detergent to strip cell membranes and pro- duce a nuclear suspension. RNase digestion destroys RNA which also stains with pro- pidium iodide. The high salt buffer is added to re-establish an isotonic solution.

SOLUTIONS

1. Sterile PBS + 0.1% sodium azide. Store a 4˚ C.

2. Low salt stain (100 ml).

● 3.0 g of PEG 8000.

● 5.0 ml of propidium iodide solution (1 mg/ml). Dissolve 0.01 g of propidium iodide in 10 ml of autoclaved distilled water; use 5 ml for low salt stain and 5 ml for high salt stain. Cover the cylinder with foil.

● 18, 000 units of RNase A (Worthington Biochemicals supplied as DNase-free liquid) add 180 units/ml to staining solu- tion just before use.

● 1 ml of 10% (v/v) Triton X-100 with 9 ml of PBS/azide, vortex. Use 1 ml for low salt stain and 1 ml for high salt stain.

● Add 4 mM sodium citrate to bring the volume to 100 ml (~94 ml).

● Add the above reagents, bring the volume to 100 ml with citrate and cover the cylin- der with foil.

3. High salt stain (100 ml).

● 3.0 g of PEG 8000.

● 5 ml of propidium iodide solution (1 mg/ml).

● 1 ml of 10% (v/v) Triton X-100 (see above).

● Add 400 mM sodium chloride to bring the volume to 100 ml (~94 ml).

● Pipette 4 ml of each stain into sterile pop- top tubes and store at –20˚ C covered with foil.

The stain is stable for 1 year.