Title: Generative reproduction of Vaccinium myrtillus in laboratory conditions and the influence of zinc-smelter emissions on it

The germination of Vaccinium myrtillus seeds and the development of the seedlings in laboratory conditions are described. The dynamics, the power of germination and Pieper coefficients for the seeds were strongly influen­ ced by the intensity of zinc-smelter pollution of the forest stand from which they originated. The rates of survival were also estimated for seedlings transferred from filter-ipaper into polluted and unpolluted samples of soil and litter. The differences noticed in the latter parameter point to the possibility of formation in the polluted stand of ecotypes more resistant to the influence of pollution than the original ones.


INTRODUCTION
There are only few incomplete data in the literature concerning ge nerative reproduction of Vaccinium species and particularly of the eco logy of Vaccinium myrtillus seedlings (K o z i r a c k i and Targoński 1978). The authors undertook, therefore, laboratory investigations of the germination of this species and described the seedlings grown during three years. The experience gained in this way was afterwards utilized in research of the influence of zinc-smelter emissions on the generative reproduction of Vaccinium myrtillus. In particular the dynamics and the power of germination of the seeds and the survival rates of the seedlings depending on the pollution level of the stands were investigated. MATERIAL The seeds of Vaccinium myrtillus, the germination of which on fil ter-paper and on soil and litter samples was studied in the laboratory, originated from three stands of a forest ecosystem (Żyglinek, Brynica II and KokOtek), representing different pollution degrees by the emis sions of the neighbouring zinc-works at Miasteczko Śląskie, mainly by some heavy metals (Pb, Zn, Cd, Cu) contained in the dustfall (C z uchajowska et al. 1980(C z uchajowska et al. , Czuchajowska 1981. The investigations were carried out in 1978-1980, in the same manner as described for Vaccinium vitis-idaea (Czuchajowska and Strączek 1981). The mixture of soil and litter samples, collected from the three investigated stands in which the seedlings primarily developed on filter-paper were transferred, contained both components in a 1:1 ratio by volume.
All laboratory germinations were carried out in four replications. The following determinations were made: 1) the percentage of seeds ger minating on every subsequent day, called the germination dynamics, 2) germination power, 3) Pieper coefficients giving the average number of days required for single seed germination and 4) survival rates of seed lings, described by the percentage of the total number of seedlings which survived after a given number of days. The reproducibility of results was excellent, the data in Figs. 2-4 and Table 1 represent mean values.

RESULTS
Several years observation of seeds germinating on filter-paper in optimum conditions, show that an epigenic germination occurred. No differences between the seeds originating from the control area and the polluted ones were found. The radicle emerged from the seed around the 10th day, preceeding by about 14 days the appearance of the epicotyl outside the seed coat. The cotyledons were fully developed 20-25 days after sowing. The hypocotyl was then 1 cm in length, the cotyledons were green with a smooth margin. They were on the average about 3 mm in length and 1 mm in width. The main root was 1 cm long, it possessed 5-10 ramifications on. the average. Around the 40th day the first leaf appeared, which was ovate and serrate. The formation of every subsequent leaf took 7 to 10 days. After about 2 months a bud in the axil of one of the cotyledons appeared and started to elongate faster than the main shoot ' (Fig. la). The seedlings grown in the laboratory did not shed leaves. This was at first attributed by the authors to the artificial conditions of cultiva tion. However, it was found la'ter that the one-two-or even three--year-old seedlings of Vaccinium myrtillus of generative reproduction origin, also kept their leaves in natural conditions. Thus, it appeared that the seedlings behave differently than the vegetatively reproducing plants of this species. The one-and two-year-old seedlings growing in natural, pollution-free stands, had the same appearance as those grown in the laboratory on soil from the control area. They were only distinctly smaller (Fig. lb). Also smaller, though not to the same extent, were seedlings grown in the laboratory on polluted soils. Among the seedlings from the natural stand, formation of rhizomes during the third year was observed, indicating a possibility of vegetative propagation.
The dynamics of germination on filter-paper show that the newly collected seeds representing a highly differentiated level of pollution with heavy metals (Czucha jowska 1981) are characterized by an identical time-pattern of germination and differ only slightly in the percentage of seeds which germinated on the given days (Fig. 2a). In every case the percentage of germinating seeds decreased below 2°/« in the 19th day. Storage of the seeds for 3 months at 2° C, changed the dynamics of germination. The seeds with a definite level of pollution showed now only one dominating maximum of germination, which was shifted by 2-3 days as compared to that shown previously. The period after which the germination ability disappears is prolonged by a few days (Fig. 2b). A 6-mon' th storage of the seeds at 2° C results in a decrease of the intensity of maxima, e.g. for the control seeds by 50% on the 17th days and for the extremely polluted ones by 40%. An increase in the storage temperature from 2° C to 20° C does not influence the germination dynamics (Fig. 2c). When the germination of seeds on soil taken from the same stand was delayed by 3 months, it differed markedly from the process carried out on filter paper (Fig. 2d, cf. Fig. 2b).
Substitution of soil by litter from the same stand from which the seeds were taken, does not change essentially the time-pattern of ger mination. It only decreases the intensity of the early appearing maxima often by one half.  Dynamics of germination of Vaccinium myrtillus seeds On filter-paper: anewly collected seeds, bseeds kept for 3 months at 2°C, cseeds kept for 6 months at 20° C; germination on soil originating from the same stand as the seeds: d -seeds kept for 3 months at 2°C. Curve 1control stand at Kokotëk, 2 -not extremely polluted stand at Brynica II, 3extremely polluted stand at Zyglinek The picture of survival of the seedlings germinating on filter-paper (from seeds stored for 2.5 months) and then transplanted on soil or litter taken from the same stand as the seeds, points to a clear sequence of the rates of survival, decreasing in time {Fig. 3). It is in agreement with the increasing pollution degree, lower values beeing always found for the litter than for the soil. The above mentioned sequence is distur bed only after the 90th day from planting the seedlings on the soil/litter, this being particularly visible after 102 days. The differentiation of survival seems to be very substantial, e.g. on the 80th day the rate of survival of the extremely polluted seedlings equals 35% and of the less polluted and the control ones -72% and 79%, respectively. The prolongation of the time of storage of the seeds from 3 to 6 months does not influence essentially the survival pattern of the corresponding seeds on the soil in the period up to the 80th day. However, the scat tering of the survival rates of the extremely polluted seeds was remar kable. time, days Fig. 3. Rate of survival of seedlings on soil and litter from the same stand as the seeds Seedlings from 'control seeds on control soil (1) and litter (2), seedlings from less polluted seeds on less polluted soil (3) and litter (4), seedlings from extremely polluted seeds on extremely polluted soil (5) and litter (6) Germination of seeds extremely differing in their level of pollution, carried out on soil and litter well blended mixtures, also differing widely in their degree of pollution, shows the rates of survival decreasing in the following order: C^Cm, Exs/Cn" Cs/Exin and Exs/Exm, where C and Ex denote, respectively, the conditions of the control and extreme pollu tion for the seeds (s) and the soil-litter mixture (m) (Fig. 4a). These regularities are disturbed only after the 100th day. In the case of the less polluted seeds the picture becomes complicated (Fig. 4b), their rates of survival on the soil-litter mixtures of two different levels of pollu tion being higher than on the unpolluted soil-mixture composition. For the latter case the rate of survival on the 96th day is 2-3 times lower than for the system polluted seeds-polluted mixture of soil and litter. Fig. 4. Rate of survival of seedlings growing on mixtures of soil and litter of different degree of pollution a) Control seedlings on control soil-litter mixture (1), and on extremely polluted mixture (2), seedlings from extremely polluted seeds on control mixture (3) and on extremely polluted soil-litter mixture i(4). b) Seedlings from less polluted seeds on less polluted soil-litter mixture fl), on extremely polluted mixture (2) and on control mixture (3) The data concerning the germination power and Pieper coefficients listed in Table 1, show that an increase of the pollution degree of seeds changes to a rather small extent these parameters of germination on filter-paper. For the processes occurring on soil or litter, the diffe rences depending an the level of pollution were much higher. 670 Z. Czuchajowska From the point of view of mass exchange, seeds germinating on fil ter-paper represent a much simpler system than those germinating on soil or litter. The time-pattern of their germination dynamics appeared also much less complicaited. It was only slightly different for seeds origi nating from fruits collected on the stands representing a different degree of pollution. This fact could result, among other things, from limited differences in the level of heavy metals; from among the seven investigated metals, the differences concern Pb, Cd and Mn only (Czu chajowska 1981). A few-month storage of seeds inside the fruits be fore the germination process begun created, however, better conditions for the appearance of changes caused by contamination. Therefore, the pictures of germination dynamics are more changed, although still in a limited extent. When the seeds are planted on soil or litter with different content of heavy metals, which is always much higher than that in seeds, the penetration into the seeds of metals is favoured. This could be the reason of the drastic change in germination dynamics. The authors do not consider possible to go at the present stage of research beyond the purely phenomenological description of these chan ges. However, it has to be mentioned that a similar content of organic matter in the investigated soils representing different pollution degrees does not oppose at all the accepted opinion that organic matter favours germination (W o 11 a n et al. 1978). The different concentrations of me tals, influencing the course of several processes going on in the soil, among other things also by changing the pH and buffer capacity values (Abouguendia and Redmann 1979), can change the quality and concentration of organic substances in the soil, diffusing into the ger minating seeds. Also the microbial activity is quite different, bringing about several serious consequences.
The curves of survival of the seedlings prove that a decisive factor for this phenomenon is differentiation of the degree of pollution of the soil or litter but not that of the seeds, because the seedlings from the extremely polluted seeds show higher rates of survival on the control soil/litter than those from the control area grown on the extremely polluted soil/'litter. The differences in metal content in the seeds origi nating from different areas are very much lower than those in the soil and litter samples. Nevertheless, the observed decrease of the survival rate of the extremely polluted seedlings grown on the control soil-litter composition, is much more pronounced than that in the control seedlings-control soil/litter system, indicating thereby that a significant role of contamination of seeds has to be also considered.
In the case of non-extreme pollution of soil, litter and seeds, the regularities given above do not appear; after a sufficiently long period cf time, the rate of survival of the corresponding seedlings on the parent soil-litter mixture is distinctly higher than on the control soil/litter composition. This favours the opinion of the possible formation of ecoty pes of Vaccinium myrtïllus already resistant to pollution from the zinc--plant, which has affected the investigated ecosystem for more than 15 years. The authors have noticed the same behaviour of Vaccinium vitis-idaea (Czucha j o-wska and Strączek 1981), for which the phenomenon was more pronounced, probably because of the evergreen nature of that species.
Reverting to the fact described in Results, of non-shedding leaves by the laboratory-grown seedlings of Vaccinium myrtillus, correspon ding to that found in natural stands, one could give the following explanation. Small plants, well hidden in the litter and moss, could survive quite well the winter period. Their further growth in early spring was facilitated by the absence of rhizomes, which begun to de velop only when the seedlings were three or four years old. The fact that seedlings grown in the laboratory on highly polluted soils were larger than those originating from the unpolluted stands, resulted from the differentiation of germination and growth conditions in favour of the laboratory conditions, fully compensating the limitations connected with contamination of soil.