Genetic Effects in Petunia hybrida induced by Pollination with Pollen Treated with Lac Transducing Phages

Lehrstuhl flir Botanische Entwicklungsphysiologie Universitat Hohenheim, Federal Republic of Germany

Genetic Effects in Petunia hybrida induced by Pollination with Pollen Treated with Lac Transducing Phages DIETER HESS

With 7 figures Received May 18, 1978 . Accepted June 7, 1978

Summary A new system for introducing exogenous gene material into higher plants was tested. Pollen of Petunia hybrida were incubated in vitro with phages transducing the E. coli p-galactosidase gene, and in the controls with homologous Petunia DNA and with phages lacking the p-galactosidase gene, respectively. The pretreated pollen was used for pollination and fertilization of Petunia flowers. Growth and development of the seeds obtained were tested on lactose agar. There were statistically significant growth differences even in the first generation: the seedlings derived from pollen treated with phages transducing the p-galactosidase gene grew better than the controls. The growth difference could be maintained over the generations, in one series till to the fifth generation (the sixth generation was not yet tested). On the other hand, however, the growth characteristic could also be lost in subsequent generations. The experimental details, for instance the selection of the best growing plants in the controls as well, are not consistent with an eventual selection of preexisting mutants. The role of bacteria could be excluded by plating and by using rifamycin. Rifamycin, an inhibitor of bacterial RNA polymerases, was without any effect on the growth difference between experiments and controls. Reciprocal crosses revealed en intermediate behaviour of the growth characteristic. Therefore, the eventually transfered gene material should be loca:ed on the chromosomes. p-Galactosidase activity was measured under conditions optimal for the bacterial and for the plant enzyme, respectively. The activity measured under conditions optimal for the bacterial enzyme was in plants derived from pollen treated with transducing phage usually higher than in the controls. In reciprocal crosses, p-galactosidase activity under conditions optimal for the bacterial enzyme corresponded to the growth behavior, e. g. showed an intermediate inheritance as well. The results presented here give no full proof for a gene transfer. They justify, however, further investigations testing the pollen system which is so close to the natural process or gene recombination. Key words: p-Galactosidase, gene transfer, pollen, Petunia hybrida.

Introduction

Recently, several attempts were made to introduce exogenous gene material into higher plants (reviews: HESS, 1977, 1979; KLEINHOFS and BEHKI, 1977;

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KUNGMULLER, 1976). Successful gene transfer was reported especially from work with Petunia (HESS, 1972) and Arabidopsis (LEDOUX et aI., 1974) embryos and seedlings, with tomato (Doy et aI., 1973) and sycamore cells (JOHNSON et aI., 1973) in culture, and with barley grains (TURBIN et aI., 1975, SOYFER et aI., 1976). In none of these cases, however, the artifial gene transfer seems to be proven beyond any doubt (HESS, 1977, 1979). Isolated plant protoplasts, at first glance most promising systems, take up at least bacterial DNA into their cytoplasm (HESS, 1976, LIEBKE and HESS, 1977; SUZUKI and TAKEBE, 1976; UCHIMIYA and MURASHIGE, 1977) and probably also into their nuclei (BLASCHEK, 1979; LIEBKE et aI., 1977; LURQUIN and KADO, 1977). There is, however, up till now no substantiated report on the transcription, integration and replication of this DNA. One just knows that isolated Petunia nuclei are able to transcribe linear E. coli DNA (BLASCHEK and HESS, 1977) but wether this holds true also for nuclei inside the protoplasts is an open question. During the last years a new system was developed considering the difficulties to introduce the foreign gene material and to obtain high numbers of progenies especially from protoplast culture: pollen incubated in vitro with the exogenous gene material were used to pollinate plants of the same species. The aim was to introduce the exogenous gene material by using pollen as carriers into the zygote, or at least into the embryo sac from which it could be taken up by the developing embryo. Using this system, high numbers of seeds could be obtained easily and tested for genetic alterations (HESS, 1974, 1975). DNA (HESS et aI., 1974 a) and phages (HESS et aI., 1974 b) are taken up or at least very firmly fixed to swelling and germinating pollen. Following pollination with phage incubated pollen, phages could be recovered from the ovaries, although in minute amounts (HESS, unpublished results). Nicotiana glauca and N.langsdorffii are two species which form genetic tumors in the hybrids. It was possible to stimulate tumor formation in progenies of N. glauca derived from N. glauca pollen treated with N.langsdorffii DNA (HESS et aI., 1976). The tumor system, however, does not favor further analysis, because the biochemical background for tumor development is unknown. The work to be reported here takes advantage of transducing phage, successfully used in gene manipulations with human fibroblasts (HORST et aI., 1975, MERRIL et aI., 1971). In this paper we were dealing with phages transducing the E. coli lac-operon. Materials and Methods Plant material Petunia hybrida «Cyanidintyp» (HESS, 1963), a pure line selected from a commercial variety and maintained by selfing for more than 3S generations. Phage material

C/) 80 lac+, A-plac cI877(Z+) (A lac+), A-placS (A lac+S, a phage containing a terminal part of the lac repressoir, a part of the permease gene y, the intact lac controlling region p and 0, and the z gene for /I-galactosidase), A W8 (Alac-, a wild type phage, lacking any genetic information for /I-galactosidase), A-pgaI8cI857(K+T-E+) (Agal-). Phages and host bacteria Z. Pflanzenphysiol. Ed. 90. S. 119-132. 1978.

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were generously supplied by Drs. K. BEYREUTHER/K6In, P. M. GRESSH.opp/Canberra, and C. R. MERRILIBethesda. Preparation of phage

In the first experiments (Nrs. 1-3 in tab. 1) phages were grown by confluent lysis and prepared according to DoY et al. (1973). Phage pellets were finally suspended in MgTris (0.1 M Tris-HCI, containing 20 mM MgS04 X 7 H 20, pH 7.4). In all the other experiments phages were grown in liquid media and purified by CsCI gradient centrifugation according to HORST et al. (1975). The phage containing fractions were finally dialysed against MgTris. Otherwise standard bacteriological methods were used. Preparation of Petunia DNA Petunia DNA (Cya-DNA) was prepared by a modiiied MARMUR procedure including CsCI density gradient centrifugation (LIEBKE and HESS, 1977). As determined by sucrose gradient centrifugation (MARTIN and AMES, 1961) the sedimentation values of the DNA obtained were about 13 S, corresponding to ca. 1.9 X 106 daltons. Incubation of pollen

1 vol. of phage suspension was mixed with 9 vols of pollen germination medium (0.7 M sucrose, 0.001 M borate, pH 5.2 (FAHNRICH 1964); cloxacillin and ampicillin 50,ug/ml each. The resulting incubation medium usually contained 109_10 11 pfu/ml. In control experiments using homologous Petunia DNA 1 vol of DNA in 0.1 SSC was mixed with 9 vols. of the pollen germination medium to give a final DNA concentration of 50 ,ug/ml. 150 closed anthers of Petunia, collected one day before anthesis, were squashed in 1 ml of the incubation medium and kept for 5 hrs. in the dark at 27 DC, while being gently shaken. Pollination

24 hrs. before pollination anthers and petals were removed from receptor flower buds ca. 1-2 days before anthesis (HESS, 1975). One squashed anther and an additional drop of pollen suspension were put onto each stigma. 50-100 flowers were pollinated per assay. Biological tests

The seeds collected were allowed to ripe for at least three weeks at room temperature and then sown onto 9 em plastic petri dishes (40 seeds/petri dish) on agar nutrient medium (WAGNER and HESS, 1963) containing 1, 2, and 4 0 10 lactose respectively. Sugars were added by sterile filtration to avoid decomposition. Previous experiments with untreated seedlings had shown that there were nearly no growth differences on the lactose concentrations indicated. Higher concentrations, however, could not be used because even sucrose shows at concentrations of more than 4 % inhibiting effects on seedling growth due to the increased osmolarity of the medium. To get comparable results it is absolutely necessary to store the seeds of a given experimental series under exactly the same conditions for the same time because seedling development is influenced by age and after ripening conditions. The petri dishes were kept in light chambers for 7 days at 25°C under 10 hrs. daily illumination (ca. 5000 lux). Thereafter, the seedlings with three visible leaflets were counted. Tricotyledonous seedlings, in experiments as well as in controls ca. 1 %, were counted as three leaflet seedlings. Previous experiments had demonstrated that the number of visible leaflets could be used as a measure of growth and development. Progenies

The best growing seedlings (usually 10 per assay) were selected from experiments and controls and transplanted into 100 ml Erlenmeyer flasks containing the same lactose agar

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medium. After 4-6 weeks under the conditions indicated above the best developed surviving plants were transfered to the soil, allowed to grow under green house conditions and then selfed. The following generations were tested and selected in the same way. Preparation and test on fJ-galactosidase

fJ-Galactosidase was extracted from freeze-dried plant material as described earlier and HESS, 1977). Crude extracts (step 1 of the preparation procedure) were tested under conditions optimal for plant and bacterial p-galactosidase, respectively (KOMP and (KOMP

HESS,

1977).

Results and Discussion

Heritable growth effects in progenies derived from phage treated pollen Growth effects induced in the first generation derived from phage treated pollen are shown in tab. 1. In the first experiment (N r. 1 in tab. 1) several - seven out of 652 - extremely well growing seedlings apeared which could be easily differentiated from smaller ones and from the controls. Usually, however, the differences are not so distinct (fig. 1). Therefore, and especially under statistical aspects, it seemed preferable to count the seedlings with three leaflets. As may seen from tab. 1, in four out of six major experiments the number of three leaflet seedlings was significantly higher following a treatment with phage carrying the E. coli p-galactosidase gene than in the respective controls. In the controls we used Petunia DNA (eya-DNA), phage A gal-, and phage A lac As for the transducing phage A gal- it should be remembered that only the transferase gene and not the other genes of the gal - operon were inactivated by mutation. The progenies derived from Agal- treated pollen, however, showed in these experiments no growth differences on 0.1 % galactose agar when compared with untreated controls. Therefore, it seems unlikey that they could contribute to a better growth on

a

b Fig. 1: Growth differences in the first generation derived from phage treated pollen. Pollen were treated with phage Alac+ (a), and with A gal- (b), respectively. Photographs were taken 14 days after sowing on 4 % lactos2 agar m~dium. In the experiment (a) especially the root system is much better developed than in the control (b). Z. Pflanzenphysiol. Bd. 90. S. 119-132. 1978.

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Tab. 1: Effects of pollen treatments with transducing phage on the first progenies. Pollen of Petunia hybrida var. «Cyanidintyp» were treated with phage C/J 80 lac+ and }. lac+, respectively, and in the controls with homologous Petunia DNA (= Cya-DNA), phage .Ie gal- and .Ie lac-, respectively. Phage titre 109 _10 10 pfu/ml. Each experiment (E) was paralleled by a control (C). The seeds obtained by pollination of Petunia flowers with the pretreated pollen were tested on 1 % (Nrs. 1-3) or 4 % (Nrs. 4-7) lactose agar. 7 days after sowing the plantles with three visible leaflets were counted. Nr. (year)

Pollen treatment

1 (1974) 2 (1974) 3 (1974) 4 (1977) 5 (1976 ) 6 (1977) 7 (1977)

E: C: E: C: E: C: E: C: E: C: E: C: E: C:

C/J 801ac+ Cya-DNA C/J 801ac+ I. galC/J 801ac+ AgalAlac+ AgalAlac+ s J.lacAlac+ s AlacAlac+ s Alac-

Seeds tested 652 455 263 196 388 497 1587 659 405 929 1588 1731 688 549

Statistics Seedlings with three leaflets number 0/0 P (Ofo)

Signif.

not counted; selection of the best growing seedlings; d. the text 1-0.10f0 77 203 + 65 127 10-5 Ofo 49 191 213 43 861 54 < 0.1 Ofo + 282 43 30-10 % 191 47 52 322 460 29 < 0.1 Ofo + 342 20 235 34 < 0.1 Ofo + 100 18

lactose media indirectly via a better utilization of the galactose set free by the seedlings own f3-galactosidase. Nevertheless, the best control seemed to be a phage differing just in the interesting operon. Tab. 1 demonstrates that in two out of three experiments using the combination .Ie lac+5 / .Ie lac- the number of three leaflet seedlings was significantly higher in the .Ie lac+5 experiments than in the .Ie laccontrols. The results obtained with the first progenies derived from phage treated pollen, however, could not be interpreted in the way that all these better growing seedlings where genetically altered. Experiments with Nicotiana had demonstrated that a DNA treatment of pollen may trigger an unspecific tumor stimulation maintained over mitotic divisions but lost by sexual propagation (HESS et all., 1976). Therefore, in all experiments where one is dealing with growth characteristics, it seems absolutely necessary to test the sexual progenies. The best growing seedlings were selected from experiments and controls and used for selfing. The second seed generation was tested on lactose agar as before. The results are presented in figs. 2-4. In the first generation, five out of seven major experiments were successful (Nrs. 1, 2, 4, 6, and 7 in tab. 1). Fig. 2 summarizes the results obtained by testing the second progenies derived from the best growing first generation plants. The growth differences found in the first generation were maintained.

z. Pjlanzenphysiol. Bd. 90. S. 119-132. 1978.

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Nr. IGen.

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3367 1 202

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Fig. 2: Growth differences in the second generation derived from phage treated pollen. Nr.lgeneration refers to the numbering in tab. 1. For instance, 212 means that we were dealing with experiment Nr. 2 from tab. 1, second generation. Overall values from repeated biological tests are given. The seedlings were grown on 4 % lactose agar. Statistics: X2 test for the evaluation of percentage frequencies. In all these experiments, plants and capsules from one given plant were tested separately. As an example, figs. 3 and 4 demonstrate the situation for the sin g 1 e plant and its capsules tested. In several cases the better growth characteristic was lost. In no case, however, the growth of the control capsules was significantly higher than in the experiment despite the fact that we selected the best growing plants from controls as well as from experiments. In most cases, the third generation is now growing up. In the series, however, where we compared the efficacy of phage ¢ 80 lac+ and Petunia DNA (Nr. 1 in tab. 1, Nr. 1 in fig. 2, and fig. 3) even the fifth generation was already tested. 7 plants had been selected from the first generation. 4 of them maintained the better growth characteristic also in the second generation, although not in all their capsules tested (fig. 3). In the third and forth generation the situation was unchanged compared with the second generation. In all the four cases, the better growth characteristic was maintained even in the fifth generation. There were, however, quantitative differences to the preceding generations. Fig. 5 gives an example: Several plants where selected from the best growing forth generation progeny (numbered «8») and selfed to get the fifth generation. In the experiment the overall percentage of three leaflet seedlings was lower than in the second to forth generation (cf. Nr. 1 in fig. 2). The same holds true if one looks at the single capsules tested. The high values of more than 80 Ufo three leaflet plantlets found from several capsules of the second to forth generation were no more reached (cf. fig. 3). The seeds from some experiment capsules grew even worse than the controls. Whether this is due to a gradual loss of the growth characteristic ore to other, unknown reasons will remain an open question till we have tested the sixth generation. Z. Pflanzenphysiol. Ed. 90. S. 119-132. 1978.

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Fig. 3: Growth differences in the second generation derived from phage treated pollen. For the experiment (treatment of pollen with phage C/J 80 lac+) the values for each plant and its capsules tested are given. 211, for instance, means "Plant Nr. 2, capsule Nr. h. The overall values could be seen from fig. 2, Nr. 1/2. Statistics: X2 test for the evaluation of percentage frequencies; comparison with the mean value of the controls (treatment of pollen with homologous Petunia DNA), see on the top). + = statistically significant at the 0.10/0 level (P 0.1), (+) = statistically significant at the 1 % level (1 % P 0.1 0/0), - = no statistical significance, ? = only 80 seeds could be tested, value questionable.

<

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For practical reasons, it was not possible to follow a II the better growing plants through the generations. Therefore, the percentage of genetically altered plants could not be determined.

Exclusion of bacterial contamination All the growth tests were performed under strictly sterile conditions. By plating seedling extracts On lactose agar containing several indicator dyes for the detection of lactose utilizing bacteria (bromothymol-blue, lacmoid, china blue, and fuchsin, respectively) no bacteria could be detected or only the bacteria brought into the assay be the nOn sterile preparation of the plant extracts. Nevertheless, to be sure that nO bacteria were involved, Rifamycin, a well known inhibitor of bacterial RNA polymerases was used. Even the lowest concentration of Z. Pf/anzenphysiol. Bd. 90. S. 119-132. 1978.

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Fig. 4: Growth differences in the second generation derived from phage treated pollen. For the experiment (treatment of pollen with phage Alac+5) the values for each plant and its capsules tested are given and compared with the mean value of the controls (treatment of pollen with phage Alac-). The overall value of the experiment could be seen from fig. 2, Nr. 6/2. Statistics as in fig. 3. ? = only 58 seeds could be tested, value questionable.

Rifamycin tested (50 .ug/ml) impaired also the seedling growth on lactose agar. If the number of three leaflet seedlings on 2 % lactose agar without Rifamycin was set to 100 Ofo, 50 ,ug/ml of the antibiotic reduced the percentage to 69 Ofo. On 50 ,ug/ml, however, the appearance of the seedlings was normal whereas on higher concentrations the plantlets were yellowish and died early. Using 50 .ug/ml Rifamycin it could be demonstrated that the better growth in the experimental series (pollen treated with phage Cb 80 lac+) compared with the controls (pollen treated with A gal-) was maintained (fig. 6). Therefore, the role of bacteria could be excluded.

Intracellular localisation of the eventually transfered gene material An uptake of exogenous genetic material including phages into plant cells and protoplasts seems to be proven (references in the introduction). Up till now, however, even in the system most propagated for uptake studies, isolated protoplasts, an uptake into the nuclei can neither be proved nor disproved. To decide this question for our experiments reciprocal crosses between one of the best growing plants derived from pollen treated with phage Cb 80 lac+ and the best growing control derived from Z. Pjlanzenphysiol. Bd. 90. S. 119-132. 1978.

Genetic effects in Petunia hybrida Capsule Cya-DNA

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1 2 3 4 5 6 7 8

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Fig. 5: Growth differences in the fifth generation derived from phage treated pollen (first generation: tab. 1, Nr. 1; second generation: fig. 2, Nr. 112, and fig. 3; forth generation: cf. fig. 7). In the experiment one of the best growing plants of the forth generation derived from pollen treated with phage Cb 80 lac+ was selfed and the progenies tested on 4 % lactose agar. The same was done in the control. For the experiment the values for each capsule tested are given and compared with the mean value of the control capsules. Statistics as in fig. 3. X = values statistically significant lower than the mean value of the controls (treatment of pollen with homologous Petunia DNA). In two of the four cases indicated, however, there was no statistical significant difference to the relevant special control which showed by unknown reasons reduced growth as well.

pollen treated with homologous Petunia DNA were performed. Both parents were in the third generation after pollen treatments. Testing the progenies on 4 % lactose agar revealed an intermediaey growth behaviour of the reciprocal hybrids (fig. 7). Similar reciprocal crosses with a phage A gal- derived control plant gave comparable results. The absence of any maternal inheritance demonstrates that at least in the case of the plants tested the eventually transfered exogenous gene material must have been integrated into the chromosomes. fJ-Galactosidase activity

Biochemical evidence for a successful gene transfer would be the proof of bacterial fJ-galactosidase in the better growing plants. E. coli fJ-galactosidase is protected

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DIETER HESS

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+

<

Fig. 7: Growth and p-galactosidase activity In reciprocal crosses between experiment and control plants derived from phage treated pollen. Third generation plants were selfed (a X s, b X s) and crossed reciprocally (a X b, b X a). a = plants derived from pollen treated with phage (/)80 lac+ (experiment), b = plants derived from pollen treated with homologous Petunia DNA (control). Growth (black bars) was measured by the percentage of three test for the evaluation of percentage freleaflet seedlings on 4 % lactose agar. Statistics: quencies. The differences between a X sand b X s, between a X s and the reciprocal crosses, and between b X s and the reciprocal crosses are statistically significant at the 0.1 % level (P < 0.1 Ofo). The difference between a X band b X a is statistically not significant. At the top representative petridishes from the series tested. p-Galactosidase activity (white bars) under test conditions optimal for the E. coli enzyme. To overcome difficulties in the standardization of the extraction procedure of minute activities, aliquots of the same extract were measured under conditions optimal for the plant and the bacterial enzyme, respectively. The activity measured under conditions optimal for the bacterial enzyme was expressed in percentages of the activity measured under conditions optimal for the plant enzyme. Single plants were kept for 6 to 8 weeks on 4 Ofo lactose agar and then tested. Statistics: mean values and standard deviations are given. Standard deviation was much higher in the experiments (a X s) than in the controls (b X s). This reflects the fact that several experimental plants failed to show an higher «bacterial>, p-galactosidase activity, perhaps due to a loss of the eventually transfered gene material during vegetative development. On the other hand controls showed uniformly the expected low activity at conditions optimal for the bacterial enzyme. The reciprocal crosses reveal an intermediate inheritance of the growth characteristic as well as of the p-galactosidase activity measured under conditions optimal for the E. coli enzyme. Furthermore, an example for the behavior of the forth generation (a X s, b X s) is given.

,,2

Z. Pflanzenphysiol. Bd. 90. S. 119-132. 1978.

Genetic effects in Petunia hybrida

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against heat denaturation by the respective antibodies. Using this immunological test the presence of E. coli p-galactosidase in tomato cells treated with transducing phage ¢ 80 lac+ seemed to be proven (Doy et aI., 1973). Unfortunately, in comparable experiments with sycamore cells in suspension culture the heat protection test failed completely (JOHNSON et aI., 1973). Investigations on the Petunia p-galactosidase revealed further possibilities for the differentiation of plant and bacterial enzymes (KOMP and HESS, 1977). The simplest test system takes advantage of the differences in the temperature and pH optima: at 55°C and pH 4.3 the Petunia enzyme works optimally and the E. coli enzyme shows only slight activity whereas at 35°C and pH 7.2 the reverse is true. The activity of the plant enzyme (specific activity and total activity extractable per plantlet) in the better growing seedlings was partly higher than in the controls, and partly not. The activity of the plant enzyme, however, must be of secondary importance here. To overcome difficulties in standardization of the

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Z. P/lanzenphysiol. Bd. 90. S. 119-132. 1978.

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DIETER HESS

extraction procedure especially of minute activities, aliquots of the same extract were measured under optimal conditions for plant and bacterial enzyme, respectively. The relation between both values was calculated. In several cases all the seedlings grown for 14 days in one petridish (40 seedlings) were tested. In the third and forth generation the specific activity at 35°C and pH 7.2, optimal for the bacterial p-galactosidase, was in 7 out of 8 tested samples higher than in the controls. It seems preferable, however, to test individual plants and not whole progenies representing eventually a mixture of genetically altered and unaltered plants. Therefore, single plants with special good growth were transfered 14 days after sowing to Erlenmeyer flasks and grown on the same lactose medium till they gave enough material to enable individual enzyme tests (further 4-6 weeks). Fig. 7 gives an example. p-Galactosidase activity under test conditions optimal for the bacterial enzyme parallelled with the growth behavior, e. g. showed higher values in the experiment than in the control and an intermediary inheritance in reciprocal crosses.

Conclusion The above results should not be interpreted as full proof for a successful gene transfer using the pollen system. On the one hand, the more "biological" evidence, based on statistically significant growth differences, seems to be sufficient. At least it seems to be difficult to find an other, better explanation. The biochemical evidence, however, has to be improved. The higher p-galactosidase activity in plants derived from pollen treated with phage transducing the p-galactosidase gene might be due to an indirect stimulation by quite other factors. A thorough investigation including immunological methods was initiated. Acknowledgements Mrs. U. FINKENMEYER and G. SCHNEIDER, Miss M. KLAIBER and M. DANNER are thanked for skilful technical assistance, W. PLISCHKE for the supervision of the English. Drs. K. BEYREUTHER/Koln, P. M. GRESSHoFF/Canberra, and C. R. MERRIL/Bethesda are thanked for generous gifts of phages and host bacteria. Dr. K. BEYREUTHER introduced one of us into phage liquid culture techniques. Part of the work was supported by a grant of the Deutsche Forschungsgemeinschaft.

References BLASCHEK, W.: Studies on association between chromatin of isolated cell nuclei of Petunia hybrida with exogenous DNA. Planta. In preparation (1979). BLASCHEK, W. and D. HESS: Transcription of bacterial DNA by isolated plant nuclei. Experientia 33,1594-1595 (1977). Doy, C. H., P. M. GRESS HOFF, and B. G. ROLFE: Biological and molecular evidence for the transgenosis of genes from bacteria to plant cells. Proc. Nat. Acad. Sci. U.S.A. 70, 723-726 (1973). FAHNRICH, P.: Untersuchungen liber den EinfluB des Bors bei der Pollenkeimung und beim Pollenschlauchwachstum. In: H. F. LINSKENS (Ed.): Pollen Physiology and Fertilization 120-127, North Holland, Amsterdam 1964.

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HESS, D.: Die Bliite als Ort der Anthocyansynthese. Z. Bot. 51, 142-155 (1963). - Transformationen an hoheren Organismen. Naturwissenschaften 59, 348-355 (1972). - Neue Methoden der genetischen Kombination bei hoheren Pflanzen. BioI. Rdsch. 12, 297-311 (1974). - Uptake of DNA and bacteriophages inot pollen and genetic manipulations. In: L. LEDOUX (Ed.): Genetic manipulations with plant materials 519-537, Plenum Press, New York,1975. - Uptake and expression of foreign genetic material in plant protoplasts. In: J. F. PEBERDY, H. J. ROGERS, A. H. ROSE, and E. C. COCKING (Eds.): Microbial and plant protoplasts 125-144, Academic Press, London and New York, 1976. - Cell modification by DNA uptake. In: J. REINERT and Y. P. S. BAJAJ (Eds.): Applied and fundamental aspects of plant cell, tissue and organ culture. 506-577, Springer, Berlin-Heidelberg-New York, 1977. - Modification of the information content of plant cells. In: P. K. EVANS (Ed.): Plant improvement through in vitro - techniques, Academic Press, London, in press (1979). HESS, D., P. M. GRESSHOFF, U. FIELITZ, and D. GLEISS: Uptake of protein and bacteriophage into swelling and germinating pollen of Petunia hybrida. Z. Pflanzenphysiol. 74, 371-376 (1974 b). HESS, D., H. U:iRZ, and E. M. WEISSERT: Die Aufnahme bakterieller DNA in quellende und keimende Pollen von Petunia hybrida. Z. Pflanzenphysiol. 74, 52-63 (1974 a). HESS, D., G. SCHNEIDER, H. LORZ, and G. BLAICH: Investigations on the tumor induction in Nicotiana glauca by pollen transfer of DNA isolated from Nicotiana langsdorffii. Z. Pflanzenphysiol. 77, 247-254 (1976). HORST, j., F. KLUGE, K. BEYREUTHER, and W. GEROK: Gene transfer to human cells: transducing phage plac gene expression in GM r - gangliosidosis fibroblasts. Proc. Nat. Acad. Sci. U.S.A. 79, 3531-3535 (1975). JOHNSON, C. B., D. GRIERSON, and H. SMITH: Expression of plac5 DNA in cultured cells of a higher plant. Nature New BioI. 244,105-106 (1973). KLEINHOFS, A. and R. BEHKI: Prospects for plant genome modification by nonconventional methods. Ann. Rev. Genetics 11, 79-101 (1977). KLINGMULLER, W.: Genmanipulation und Gentherapie. Springer, Berlin-Heidelberg-New York,1976. KOMP, M. and D. HESS: p-Galactosidase from Petunia hybrida: characterization and differentiation from E. coli p-galactosidase activity. Z. Pflanzenphysiol. 81, 248-259 (1977). LEDOUX, L., R. HUART, and M. JACOBS: DNA-mediated correction of thiamineless Arabidopsis thaliana. Nature 249, 17-21 (1974). LIEBKE, B., W. BLASCHEK, and D. HESS: Studies on the uptake of exogenous DNA into isolated nuclei of Petunia hybrida. Z. Pflanzenphysiol. 84, 265-274 (1977). LIEBKE, B. and D. HEss: Uptake of bacterial DNA into isolated mesophyll protoplasts of Petunia hybrida. Biochem. Physiol. Pflanzen 171,493-501 (1977). LURQUIN, P. F. and C. 1. KADO: Escherichia coli plasmid pBR313 insertion into plant protoplasts and into their nuclei. Mole. gen. Genet. 154, 113-121 (1977). MARTIN, R. G. and B. N. AMES: A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J. BioI. Chern. 236, 1372-1381 (1961). MERRIL, C. R., M. R. GEIER, and J. C. PETRICCIANI: Bacterial gene expression in human cells. Nature 233, 398-400 (1971). SOYFER, V. N., N. A. KARTEL, N. M. CHEKALIN, Y. B. TITov, K. K. CIEMINIS, and N. V. TURBININ: Genetic modification of the waxy character in barley after an injection of wild type exogenous DNA. Analysis of the second seed generation. Mutation Res. 36, 303-310 (1976).

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TURBIN, N. V., V. N. SOYFER, N. A. KARTEL, N. M. CHEKALIN, Y. L. DOROHov, Y. B. TITOV, and K. K. CrEMINIS: Genetic modification of the waxy character in barley under the action of exogenous DNA of the wild variety. Mutation Res. 27, 59-68 (1975). SUZUKI, M. and 1. TAKEBE: Uptake of single-stranded bacteriophage DNA by isolated tobacco protoplasts. Z. Pflanzenphysiol. 78, 421-433 (1976). UCHIMIYA, H. and T. MURASHIGE: Quantitative analysis of the fate of exogenous DNA in Nicotiana protoplasts. Plant Physiol. 59, 301-308 (1977). WAGNER, G. and D. HESS: In vitro - Bcfruchtungen bei Petunia hybrida. Z. Pflanzenphysiol. 69, 262-269 (1973).

Prof. Dr. DIETER HESS, Lehrstuhl fi.ir Botanische Entwicklungsphysiologie der Universitat Hohenheim, Emil-Wolff-Str. 25, D-7000 Stuttgart 70.

z. PJlanzenphysiol. Bd. 90. S. 119-132. 1978.