Martonvasar, Hungary

Hungarian Academy of Sciences

Dynamics of pH changes in corn (Zea mays L.) haploid cell suspension cultures --Kovács, G The loss of embryogenic competence of long term cell suspension cultures is one of the major problems faced in the development of in vitro regeneration systems for propagation as well as genetic manipulation. Currently, the causes for the loss of embryogenic competence, which is associated with the time of culture and the number of subcultures, are believed to be genetic or physiological. On the contrary, according to our hypothesis these changes are generated by the repeated stress caused by the suboptimal physical culture conditions, such as pH, dissolved oxygen, etc. The pH of a cell culture medium is one of the most important factors, as growth promoting properties and the selectivity of the culture media are pH dependent. In a majority of cases the pH of a culture medium lies between 5.5 and 6.0. Murashige and Skoog (1962) reported that a pH value of 5.7-5.8 is suitable for maintaining all the salts in soluble form, even with relatively high phosphate levels, and is enough to permit rapid growth and differentiation of the tissue.

Several workers observed that the presence of plant tissue affects the pH of the medium and in most cases there is a fast pH drop from 5.8 to around 4.5 during the first two days of cultivation. The pH optimum of cell growth is not a constant pH value. It changes with the medium composition, and probably there is a different optimum for maximum growth (cell proliferation) and for differentiation.

Supposing that plant cells have an active role in changing the medium pH, we should choose the initial pH of the medium so that the cells spend less energy on changing their circumstances. The aim of the present work is to determine the effect of the initial pH in consecutive subcultures of corn haploid cell suspensions in order to minimize the stress conditions caused by the periodic pH changes.

An anther culture originated haploid corn cell line was used throughout. This cell line was proved to be able to regenerate green plants in a separate experiment even in large scale cultures.

BM medium supplemented with 1 mg/l 2,4-D and 3% sucrose was used in all experiments. The pH of the medium was adjusted after heat sterilization by sterile 1N HCl or 0.5N KOH. The initial pH of the medium was adjusted to 5.8 in experiment A) and to 4.6 in experiment B). Cell suspension culture was cultivated in 250 ml Erlenmeyer flasks containing 100 ml medium prior to culture inoculation, and was rotated on a shaker at 100 rpm for 7 days at 27 C. The initial dry cell concentration at the time of inoculation was around 0.2 g/l. After 7 days the cells were settled and media was removed. Cell mass was divided and fresh media with adjusted pH was added for the next 7 days subculture.

A New Brunswick 1500 ml CelliGen Cell Culture System fermenter was used for determination of the pH profiles during 7 days of cultivations. Mixing was accomplished using Cell-lift impeller at an agitation rate of 100 rpm. Temperature was controlled at 27 C. Cultures were continuously sparged with air at a rate of 500 ml/min. The fermenter was exposed to warm white fluorescent light (45 mmol/m2s) for a 16 h photoperiod. pH was measured by sterilizable Ingold electrodes. The medium was the same as in shaking flask experiments, pH of the medium was adjusted after heat sterilization. The cells grown in 4 shaking flasks for 7 days were collected and used as inoculum for the fermentation process. pH electrode signal was recorded on-line by a computerized process monitoring system.

Cell suspension was poured into vacuum filter with a pre-weighed filter paper. Medium was removed by vacuum and fresh cell weight was measured.

The embryogenic competence of the cell cultures from pH 5.8 and 4.6 was tested in a hormone free liquid BM medium (pH: 5.8) at the end of the experiment.

Our first corn haploid cell suspension culture experiments were carried out in shaking flasks. The initial pH of the media was adjusted to the "traditional" pH 5.8. The pH of the medium at the end of one week of cultivation varied between 4.3 and 4.6. The cells formed 1-3 mm size aggregates and had a brownish color indicating pigment production.

A series of parallel shaking flask experiments were carried out with different initial pH values. The initial pH of the subculture media was 5.8 in experiment A) and 4.6 in experiment B). These initial pH values were used in all consecutive subcultures. Four subcultures were examined for each initial pH and the pH values at the end of the 7 days culture were recorded. (Fig. 1.)

Despite the fact that there was almost 1.5 pH unit difference between the initial adjusted pH of the subcultures there were no significant differences between the two experiments in final media pH values. However there were differences in the "behavior" of the cultures. Cultures on pH 4.6 have lost their bright-yellow color and formed lighter-color or white aggregates. Cell growth was also different. Fresh cell weight after 7 days was 4 times higher than the inoculum weight at pH 5.8 and 9 times higher at pH 4.6 .

The changes in medium pH were determined by taking sterile samples from both of the experiment series. Samples were taken and pH was measured once a day during the 7 days cultivation. Measured pH values are presented in Fig. 2.

The result of the pH measurements during 7 days cultivation reflects that pH dropped rapidly from 5.8 to around 4.3 during the first two days of cultivation. After that sudden change pH remains quite stable. On the contrary there was no such dramatic pH drop in the cultures initiated from pH 4.6 where pH slowly drifts to a pH value around 4.3. According to the results of embryoid induction, cell cultures originated from 5.8 media produced dramatically less embryoids than when the initial pH was 4.6. Under the given conditions used for embryo induction only globular embryos were observed in both cases.

More careful examination of the changes in medium pH were carried out in two experiments in a bioreactor. The initial pH of the media in the bioreactor was adjusted to 5.8 and to 4.6. Fig. 3. presents the pH changes during the cultivations.

The results of the bioreactor experiments show the same overall behavior as the shaking flask experiments. The pH changes during the first two days are even more striking. The pH drop at the beginning of the process seems to be a more complex process. After the inoculation at pH 5.8 the medium pH first drops to around pH 4.4 already in the first hours of the cultivation. This pH drop is followed by a short increase then pH monotonously decreases during the first two days reaching a steady state for the remaining time of the cultivation. This pH fluctuation does not occur in the experiments started from pH 4.6. In this case there is only a small but rapid drop to a value around 4.3 where pH stabilizes already after 12 hours.

It has to be noted that the final pH values of the cultures in bioreactors are a little bit lower than in the shaking flask experiments probably due to the different aeration.

The results of monitoring the changes in the cell culture medium pH suggest that the traditional method of starting subcultures from pH 5.8 without any attention paid to the final pH of the medium leads to periodically changing environment. Each subculture inoculation to high pH causes dramatic stress condition for the cells (Fig. 4.). The rapid pH changes during the first 2 days of the culture indicate that cells must spend energy on adjusting the medium pH to a more favorable value after each subculture inoculation. This energy consumption for maintenance purposes results in a lower cell production yield, and decreased embryogenic capacity.

The adjustment of the initial pH of the subculture media to 4.6 eliminates the periodic pH changes during the first two days of the culture. This also means eliminating stress conditions which result in higher cell production efficiency and embryogenic competence.

Figure 1. Final pH of the media after 7 days cultivation. Initial pH of the media was 5.8 for experiment A) and 4.6 for experiment B)

Figure 2. Changes in pH of the media during 7 days cultivation in shaking flasks. pH* in the legend indicates the initial pH of the culture media.

Figure 3. Changes in pH of the media during 7 days cultivation in bioreactor. pH* indicates the initial pH of the culture media.

Figure 4. Periodic changes in pH of the media during consecutive subcultures. pH* indicates the initial pH of the subculture media.

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