**6. Isolation and culture of garlic protoplasts**

Garlic genotypes derived from 'Perla' (C-37-1/8, C-3-1/25 and C-CN-95/2), 'Chino', 'Coreano' and 'Criollo Aguascalientes' were used for protoplast preparation. Cloves of these genotypes were peeled and disinfected (briefly: cloves were soaked in 70% ethanol 1 min and 20% commercial bleach 20 min, after three rinses with sterile distilled water the cloves were soaked again in 70% ethanol 1 min and rinsed again with sterile distilled water). Surface-sterilized cloves were cut into pieces 0.5 to 1.0 cm long, and were inoculated aseptically on MS (Murashige & Skoog, 1962) medium pH 5.8 containing 0.15 mg L-1 2,4-D (Dichlorophenoxy acetic acid), 5.0 mg L-1 BA (Benzyl adenine), 50 g L-1 glucose and 3.5 g L-1 Phytagel. Incubation conditions were 28 ±2 °C, with a light intensity of 59 Em2s.

Protoplasts were isolated from leaves and callus using a modification of the method from Spangenberg (1997). Briefly: 1 g of tissue was placed in petri dish together with 5 ml of enzyme mixture (2% Onozuka R-10, 1% macerozyme, 0.5% pectinase, 0.5% mannitol and 0.9% CaCl2); dishes were incubated on orbital shaker at 5 rpm, about 4-6 h (Novák et al., 1983). Callus tissues were best for protoplast isolation within the range 106-108 counts/ml (Fig. 3). Protoplasts were isolated from the debris and inoculated on semi-solid MS medium. Unfortunately, whole plant regeneration remained elusive.

photosynthesis assimilation µM/m2/s (A) and CO2 internal concentration ppm (CI). From these measurements, it was found that for some genotypes like 'C-CN-9/2' evapotranspiration was the highest at 30 d, as opposed to 'Criollo Aguascalientes' and 'Chino Jaspeado' with the lowest value at 90 d. Stomatal conductance was high 'C-CN-9/25', mainly after 90 d. Most genotypes showed negative photosynthesis rate, and internal CO2 showed no clear tendency within genotypes. Weight remained stable during the first 60 d, but after that period, it decreased about 1/3 of the initial values. The bulbs behavior at the

Fig. 2. View of stored garlic at 90 d. Sprouted heads are from short-shelf life cultivars. Heads

Garlic genotypes derived from 'Perla' (C-37-1/8, C-3-1/25 and C-CN-95/2), 'Chino', 'Coreano' and 'Criollo Aguascalientes' were used for protoplast preparation. Cloves of these genotypes were peeled and disinfected (briefly: cloves were soaked in 70% ethanol 1 min and 20% commercial bleach 20 min, after three rinses with sterile distilled water the cloves were soaked again in 70% ethanol 1 min and rinsed again with sterile distilled water). Surface-sterilized cloves were cut into pieces 0.5 to 1.0 cm long, and were inoculated aseptically on MS (Murashige & Skoog, 1962) medium pH 5.8 containing 0.15 mg L-1 2,4-D (Dichlorophenoxy acetic acid), 5.0 mg L-1 BA (Benzyl adenine), 50 g L-1 glucose and 3.5 g L-1

Protoplasts were isolated from leaves and callus using a modification of the method from Spangenberg (1997). Briefly: 1 g of tissue was placed in petri dish together with 5 ml of enzyme mixture (2% Onozuka R-10, 1% macerozyme, 0.5% pectinase, 0.5% mannitol and 0.9% CaCl2); dishes were incubated on orbital shaker at 5 rpm, about 4-6 h (Novák et al., 1983). Callus tissues were best for protoplast isolation within the range 106-108 counts/ml (Fig. 3). Protoplasts were isolated from the debris and inoculated on semi-solid MS medium.

Phytagel. Incubation conditions were 28 ±2 °C, with a light intensity of 59 Em2s.

with low number of sprouting bulbs are from 'Perla' (bottom left corner).

**6. Isolation and culture of garlic protoplasts** 

Unfortunately, whole plant regeneration remained elusive.

final of postharvest period is show in the Fig. 2.


Table 2. Photosynthetic and respiration rate of cloves from six garlic genotypes after 90 d storage.

Fig. 3. Protoplast isolation from P-C-3 1/25 genotype: A,B) Bulb ('Perla' type), C) *In vitro*  callus culture and D) Protoplasts at 40x magnification.
