Decisiones judiciales en torno a la libertad anticipada.

Previous workers have found 0.35 M CaClg to be the optimal osmoticum for protoplasting mycelia of P. infestans and P. parasitica (Pesti & Ferenczy, 1979; Jahnke et aL, 1987). Protoplast formation from mycelia of P. infestans isolate ATCC48720 in 0.35 M CaCl2 and 0.71 M KCl; 0.2 M CaCl2 was compared. Approximately equivalent wet weights of 4 day mycelia were digested in 25 ml of 0.35 M CaCl2 or 0.71 M KCl; 0.2 M CaCl2 plus 10 mg/ml NovoZym 234. After digestion for 3 h in 0.35 M CaCl2 virtually no mycelial debris remained and 4.1 x 10^ protoplasts had been produced. Large mycelial fragments remained in the KCl + CaCl2

osmoticum after the same time and the protoplast yield was only 5.9 x 10®. Therefore 0.35 M CaCl2 was selected as the protoplasting osmoticum in further experiments. The regeneration of mycelial protoplasts in various liquid and solid regeneration media was also compared (Fig. 4.11). Protoplasts were washed three times in 0.9 M mannitol (isotonic to PB + 0.8 M mannitol) to remove CaCl2 and NovoZym 234 prior to inoculation into regeneration media. Of the liquid media PB + sorbitol or mannitol in the concentration range 0.8 M-1.0 M gave the highest regeneration efficiencies. Due to clumping of some cells it was not possible to count the actual number of regenerated protoplasts on solid media. No significant differences in the proportion of clumped cells or the sizes of the clumps was apparent between the media so the results in Figure 4.11, although underestimates of actual regeneration frequencies, were presumed to reflect actual differences in the ability of the different media to support regeneration. The highest regeneration frequencies in liquid media were observed in PB emended with sorbitol or mannitol in the concentration range 0.8 - 1.0 M. In contrast to the findings of Pesti & Ferenczy (1979) and Jahnke et al. (1987), both liquid and solidified PB + 0.4 M mannitol + 0.1 M CaCl2 supported only low levels of regeneration.

4.3 DISCUSSION

Encysted zoospores, sporangia and mycelia of P. infestans isolate ATCC48720 can be protoplasted and regenerated at high efficiencies. The optimal conditions for each system are summarised in Table 4.2.

Table 4.2 Summary of ontimal nrotonlastinff and regenemtion conditions.

Cell type Encysted

zoospores

Sporangia Mycelia

Protoplasting 0.71 M KCl; 0.71 M KCl; 0.35 M CaCl2

osmoticum 0.2 M CaClg 0.2 M CaCl2

NovoZym 234 50 20 10

concentration (mg/ml)

Digestion time (h) 2 24 1.5

Yield 50% 70% -3.6 X 10®

per culture Regeneration 0.8 M sorbitol 1.0 M sorbitol 0.8 M mannitol osmoticum

Regeneration -90 -80 9& -27 96

efficiency

Many isolates of P. infestans sporulate poorly but as very high protoplast viability is possible the operation of a transformation system in such isolates should not be compromised by low regeneration frequencies, unless the transformation protocol itself is seriously detrimental to cell viability. Although precise figures for protoplast yield and regeneration vary between experiments the overall conclusions reached in these investigations have been confirmed in transformation experiments employing these protoplasting and regeneration systems.

The batch number of the NovoZyme 234 was found to be very important for the formation of protoplasts from sporangia. Batch no. 1961 (no longer available) was used in all the experiments on sporangia described in this chapter. A more recent batch (no, 2416) did not protoplast sporangia of isolate ATCC48720 even after 48 h incubation but was equally efficient as batch no. 1961 in protoplasting encysted zoospores and mycelia.

The suggestion that inclusion of CaClg in regeneration media is detrimental to colony establishment was not unambiguously confirmed in the experiments with sporangial protoplasts as osmolality was not a controlled parameter. However, growth inhibition appears to be a general effect of the presence of mineral salts in regeneration media on protoplasts of isolate ATCC48720, as maximum regeneration frequencies are not achieved if there is significant carry over of (NovoZym 234 - free) protoplasting osmoticum into the regeneration medium. The best way to ensure this is to wash the protoplasts by centrifugation with a suitable sorbitol or mannitol solution prior to inoculation into regeneration medium. Washing the protoplasts with PB + sorbitol or mannitol results in the formation of a precipitate, caused by calcium, in which the protoplasts become embedded making accurate haemocytometer counts impossible.

Jahnke et al, (1987) reported decreases in the ability of regenerated mycelial protoplasts of P. parasitica to establish macroscopic colonies on solid media the longer they were incubated in liquid medium before hand. Such a test was not applied to P.

infestans protoplasts in this work but protoplasts continued to grow vigorously in liquid regeneration media for at least several days. Growth appeared just as abundant as in media inoculated with similar numbers of intact sporangia. Although this assessment was subjective substantial reduction in cell viability on prolonged incubation in liquid media was not obvious.

Regeneration of cyst and sporangial protoplasts sufficient for the cells to survive inoculation onto plant material is achieved within 18 h, allowing rapid manipulation and screening of isolates. A protoplasting and regeneration system for sporangia and

encysted zoospores has the advantage of permitting transform ation and other protoplast manipulations to be performed on cells harvested from sporulating lesions on plant stems and leaves. This approach may be useful for Oomycetes, such as the downy mildews, which are obligate pathogens.

The improvements in regeneration efficiencies for P. infestans m ycelium makes the use of these cells in transformation experiments viable. The cultures used to produce mycelium for protoplasts in these and other experiments were each inoculated with sporangia harvested from one Rough Rye Agar culture of isolate ATCC48720. It was apparent that inoculum density was a major limiting factor on protoplast yield. This is the principal reason for the modest mycelial protoplast yields obtained in this work. Other forms of inoculum such as mycelial blocks or mycelium fragmented in a blender may be used. Mycelial block inocula are unsuitable for this work because relatively little new mycelium is produced in the short incubation periods appropriate for protoplasting. This is probably due to a slow initial growth rate from the mycelial blocks and the relatively low numbers of growth points. Exponentially growing mycelial fragments obtained by successive homogenisation of liquid cultures (e.g. Jahnke et al., 1987) are probably the most reliable and powerful form of inoculum since they can be easily produced in large quantities from even the poorest sporulating isolates. One drawback of this technique is the ease with which cultures can be contaminated, particularly in laboratories where other highly sporulating and more vigorous fungi are being cultured.

5 0 - Q _i y 4 0 - H ^ 3 0 - CL g 20- O DC CL 1 0 - 0.8 1.0 1.2 1 . 4 0.2 0 . 4 0.6 KCl CONCENTRATION (M)

Figure 4.1. Effect of KCl concentration on cyst protoplast yield. Data are means of two counts. 1 0 0 - I e z

_o

g

s

Figure 4.2. Regeneration of cyst protoplasts in PB emended with various concentrations of mannitol. Data are means of two counts.

1001