Effects of kanamycin on pollen germination and tube growth of pollen from non-transformed plants and from transgenic tomato plants containing a chimaeric kanamycin resistance gene were determined. Germination of pollen was not affected by the addition of kanamycin to the medium in both genotypes. Kanamycin, however, severely affected tube growth of pollen from non-transformed plants, while pollen from plants containing the chimaeric gene were less sensitive and produced significantly longer tubes at kanamycin concentrations between 200-400 mg l-1. Apparently, this resistance for kanamycin correlates with the expression of the chimaeric gene during male gametophytic development.

AP Czernilofsky

D Zamir

FA Hoekstra

Gorla M Sari

J Tupy

JP Mascarenhas

JR Laughnan

KB Searcy

M Koornneef

NT Mascarenhas

P Elzen Van den

P Zambryski

R Hain

RB Horsch

RB Knox

RG Stanley

RP Willing

SD Tanksley

T Hodgkin

TJ Franklin

English

Effects of kanamycin on pollen germination and tube growth of pollen from non-transformed plants and from transgenic tomato plants containing a chimaeric kanamycin resistance gene were determined. Germination of pollen was not affected by the addition of kanamycin to the medium in both genotypes. Kanamycin, however, severely affected tube growth of pollen from non-transformed plants, while pollen from plants containing the chimaeric gene were less sensitive and produced significantly longer tubes at kanamycin concentrations between 200-400 mg l-1. Apparently, this resistance for kanamycin correlates with the expression of the chimaeric gene during male gametophytic development

Bino, R.J.

Hille, J.

Franken, J.

University of Groningen

   University of GroningenKanamycin resistance during in vitro development of pollen from transgenic tomato plantsBino, R.J.; Hille, J.; Franken, J.Published in:Plant Cell ReportsDOI:10.1007/BF00269553IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1987Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bino, R. J., Hille, J., & Franken, J. (1987). Kanamycin resistance during in vitro development of pollen fromtransgenic tomato plants. Plant Cell Reports, 6(5). https://doi.org/10.1007/BF00269553CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 27-04-2023Plant Cell Reports (1987) 6:333-336 Plant Cell Reports © Springer-Verlag 1987 Kanamycin resistance during in vitro development of pollen from transgenic tomato plants R.J.  Bino ~, J. HiUe 2. , ,  and J. Franken 1 Institute for Horticultural Plant Breeding (IV'I), P.O.B. 16, 6700 AA Wageningen, The Netherlands 2 Agricultural University, Department of Molecular Biology, De Dreijen 11, 6703 BC Wageningen, The Netherlands Received May 26, 1987 - Communicated by I. Potrykus ABSTRACT Effects of kanamycin on pollen germination and tube growth of pollen from non- transformed plants and from transgenic tomato plants containing a chimaeric kanamycin resistance gene were determined. Germination of pollen was not affected by the addition of kanamycin to the medium in both genotypes. Kanamycin,however, severely affected tube growth of pollen from non- transformed plants, while pollen from plants containing the chimaeric gene were less sensitive and produced significantly longer tubes at kanamycin concentrations between 200-400 mgl-l. Apparently, this resistance for kanamycin correlates with the expression of the chimaeric gene during male gametophytic development. ABBREVIATIONS ATW, Agrobacterium Tomato Wageningen; KAN, Kanamycin; KANr, Kanamycin resistant; KANs, Kanamycin sensitive; mRNA, messenger RNA; NPT, Neomycin phosphotransferase. INTRODUCTION Male gametophytic functioning is involved with microsporogenesis, pollen germination, pollen tube elongation, and the transmission of gametes to the embryo sac. During these processes, the male haploid genome plays an important role. Willing and Mascarenhas (1984) estimated that mRNAs present in mature pollen grains of Tradescantia paludosa are the products of approximately 20,000 different genes. Large numbers of these mRNAs are translated into proteins during pollen germination and early pollen tube elongation (Mascarenhas and Mermelstein, 1981). similar results were obtained using the pollen of Zea mays (Mascarenhas et al., 1984) and Nicotiana tabacum (Tupy, 1982). RNA synthesis and protein synthesis are independent during the early stages of in vitro pollen tube development, as was demonstrated by the use of specific inhibitors (see review Knox, 1984). These inhibition studies showed that RNA transcription in most plant species is not essential for germination and early pollen tube growth, whereas both processes are severely affected by translation inhibitors. Most genes transcribed and translated during pollen development are also expressed in vegetative parts of the plant. Using RNA saturation hybridizations, Willing and Mascarenhas (1984) reported the expression of at least 64% of the pollen sequences in shoot tissues, and a maximum of about 60% of shoot mRNAs was also present in pollen of Tradescantia paludosa. Based on isozyme profiles, Tanksley et al. (19SI) ahd Sari Gorla et al. (1986), found in Lycopersicon esculentum and Zea mays a comparable percentage of genetic overlap between the sporophytic and gametophytic phase of a plant. The parallel expression of genes in the sporophyte and the gametophyte suggests that a selection pressure applied to the pollen is likely to alter the expression of genes in the sporophyte. Indeed, sporophytic gene expression is modified by chilling stress during pollen development in tomato species (Zamir et al., 1982) and after heavy metal stress during in vivo pollen germination in Silene dioica and Mimulis guttatus (Searcy and Mulcahy, 1985). The development of Agrobacterium tumefaciens derived gene vectors have enabled the transfer of new genetic information into the cells of various plant species (Zambryski et al., 1983). The use of selectable marker genes which are constitutively expressed allows a convenient selection of transformed tissues (Horsch, 1985). Such an approach was also used by Koornneef et al. (1986), who introduced a chimaeric kanamycin resistance gene into tomato. The present study is undertaken to determine if this marker gene is also expressed in the gametophytic phase of the tomato plants and can be used for pollen selection. * Present address: Vrije Universiteit, Department of Genetics, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands Offprint requests to: R. J. Bino 3 3 4  MATERIALS AND METHODS Plant material. Tomato plants used in this study derived from material developed by Koornneef et al. (1986) and are indicated as MsK. This genotype is selected for outstanding regeneration capacity and originally derived from the cross Lycopersicon peruvianum x L. esculentum. Transformants were obtained following leaf disc transformation with either the Agrobacterium tumefaciens strain C58Cl(pGV3850::l103neodim) (czernilofsky et al., 1986) or the strain LBA 4404 (pAL4404, pAGSII2) (Van den Elzen et al., 1985) as described by Koornneef et al. (1986). In between the T-region border sequences, both strains contain the NPT II gene from Tn5 under transcriptional control of the regulatory elements of the nopaline gene from A. tumefaciens. Expression of the chimaeric gene confers resistance of transformed plant cells to kanamycin (Hain et al., 1985). Transformed plants were named ATW and the transgenic character was confirmed both by Southern blot analyses and by segregation ratios of the KANr trait of seeds obtained from selfed plants (i.e., resistant : sensitive = 3 : i) (Koornneef et al., 1986). As a control for kanamycin sensitivity , pollen of MsK plants and two commercial tomato cultivars (viz. Moneymaker and Delicates) were used. In vitro pollen germination. Plants were grown in glasshouses at 18-22 °C, under daylight conditions. Pollen was collected from flowers at anthesis and germinated in solidified media containing 3 mM H~B03, 1.7 mM Ca(NOi)z, 10% sucrose, 0.7% agar, with different concentrations of kanamycin sulphate (Sigma) at pH 5.8. Pollen was incubated for 2 h at 24 °C in the dark, and both stained and fixed with aceto-carmine (1%). Germination and pollen tube length were determined for separate samples of 5-7 complete replicates. Per flower, and for each concentration, germination of at least 200 pollen grains was scored. Since stress induces the extrusion of short pollen tubes (Stanley and Linskens, 1974), pollen was recorded as germinated only when the pollen tube length was twice the diameter of the pollen grain. Pollen tube length was measured using an ocular micrometer for 50 tubes per flower at each concentration. The results were expressed as the mean + standard deviation, significant differences among means (P < 0.05) were partitioned by an analysis of variance. C [o0~ o 1A 90 C • ~ 60 % ol  7o  6o  4,6 I f 30 ~ I i i i i i i I t i i I t t L o loo 200 400 8oo 16oo Concentration k~namycin (mgl -I) 1°6%~-'T~. I 1 B 4J g 06 0 g 66 66 ¢- 4 J  ~ 66 I Ill 4~0 3 4.1 C: tO0 ZOO 4:00 800 1600 O,I ,-4 Concentration kBnamycin (mEl -I) o o. Figs. 1 A and B. Effects of increasing KAN concentrations (mgl-l) on germination and tube growth of pollen from non-transformed MsK ( ¢ ) and transgenic ATW ( + ) plants. Pollen from ATW plants, transformed with C58CI and pollen from plants transformed with LBA 4404, behaved similar and are therefore combined. Germination percentages and tube lengths were determined for 5 (MsK) and 7 (ATW) complete replicates of pollen from different flowers. Results are expressed as mean + standard deviation. Fig. 1 A. Percentage of germination. Per flower, and for each concentration, germination of at least 200 pollen grains was scored. Fig. 1 B. Pollen tube lenghts expressed as percentages of the control value (0 mgl-i KAN). Per flower, and for each concentration, length of at least 50 pollen was scored. 335 C Ok . . . .  Fig. 2. ATW pollen, in vitro germinated at 0 mgl-i KAN (A), 400 mgl-i KAN (B) and 800 mgl-I KAN (C). Bar = 0.I mm. RESULTS The effects of increasing KAN concentrations on germination of pollen from MsK and ATW plants are presented in Fig. 1 A. Pollen germination of both genotypes was not significantly affected by the addition of KAN to the medium. Fig. 1 B shows the effects of the addition of KAN to the pollen germination medium on pollen tube length, a microscopic view of the results is presented in Fig. 2. After two hours of incubation, at 0 mgl-l, mean pollen tube length was 2.0 + 0.3 - 10-4 m. Inhibition of tube growth of pollen from non-transformed (KANs) MsK plants was evident at i00 mgl-i KAN, while at 400 mgl-i KAN, the tube length was about 50% of the value obtained at 0 mgl-l. In contrast, inhibition of pollen tube growth of transformed (KANr) ATW plants was manifest at 200 mgl-l, while the 50% value occured at about 800 mgl-l. Significant differences between pollen tube lengths of MsK and ATW plants were obtained at 200 mgl- 1 (P < 0.01) and 400 mgl-i (P < 0.05). No different effects of KAN were observed between pollen tube growth of ATW plants transformed with the pGV3850::l103neodim construct or with the pAGSlI2 plasmid. Pollen tube growth of the cultivars Moneymaker and Delicates was similarily affected by KAN as pollen of MsK plants. DISCUSSION Tomato pollen tube growth is inhibited by the addition of KAN to the germination medium. Initial effects of KAN on pollen of non-transformed MsK plants were already apparent at I00 mgl-l. Similar KAN concentrations have been reported to inhibit MsK callus formation and seedling growth (Koornneef et al., 1986). The effects on vegetative growth, however, became evident only after several weeks in culture, while effects on pollen tube development were manifest 2 hours after pollen germination. Toxicity of KAN is due to interference with the translation system of the 70S ribosomes as present in mitochondria and chloroplasts of eucaryotic cells (Franklin and Snow, 1975). Since pollen tubes lack chloroplasts, the effect of KAN might indicate that an active mitochondrial translation is important during tomato pollen tube growth. Similar effects on pollen tube growth of several other species were obtained by Hoekstra (1979), using chloramphenicol, another inhibitor of 70S ribosomal protein synthesis. KAN did not significantly affect tomato pollen germination. For this, at least two explanations are possible: A) the antibiotic reagent is only gradually absorbed during pollen germination, or, B) mitochondrial translation is not essential for tomato pollen germination. Pollen tube growth of plants containing the chimaeric resistance gene is less sensitive for KAN than pollen of non- transformed plants. Since ATW plants are heterozygous for the KANr trait, only half of the pollen population is expected to be resistant. Hence, the difference between pollen from ATW and MsK plants is even more evident considering the heterogenous character of the ATW pollen sample. 336 Resistance to aminoglycoside antibiotics such as KAN, is due to expression of the NPT II gene (Hain et al., 1985). During early tube growth in vitro, however, genes appear not to be transcribed (Knox, 1984). Probably, in pollen, mRNAs of the chimaeric gene are presynthesized concurrently with the other mRNAs during microsporogenesis, while KAN resistance is induced by gene translation during pollen tube elongation. The largest difference between pollen tube growth of ATW and MsK plants occured at 200- 400 mgl-i KAN. Here, tubes of ATW pollen are about 1.4 times longer than the pollen from MsK plants. Since the level of pollen resistance probably correlates with the amount of NPT present in the cells, the use of a stronger promoter may enhance selection efficiency. The gametophytic phase of a plant is characterized by large populations and haploid genomes. Expression of selection sensitive genes by the male haploid genome opens the possibility to make pollinations with pre-selected pollen. For example, the toxin from Drechslera maydis prevents germination of pollen from maize plants susceptible to the disease but not of pollen from resistant plants (Laughnan and Gabay, 1973), and, compounds present in Alternaria brassicicola culture filtrates inhibit brassica pollen germination (Hodgkin and MacDonald (1986). However, linkage of agronomic important traits with a newly introduced marker gene, would be of general interest for the application of pollen selection methods in plant breeding programmes. ACKNOWLEDGMENTS We thank J. Jansen for helping with the statistical analyses, and J.J.M. Dons and A.G. Stephenson for critically reading the manuscript. REFERENCES Czernilofsky AP, Hain R, Herrera-Estrella L, Lorz H, Goyvaerts E, Baker B, Schell J (1986) DNA 5:103-113 Franklin TJ, Snow GA (1975) Biochemistry of antimicrobial action, Chapman and Hall Ltd., London, pp 224 Hain R, Stabel P, Czernilofsky AP, Steinbiss HH, Herrera-Estrella L, Schell J (1985) Mol Gen Genet 199:166-168 Hodgkin T, MacDonald MV (1986) New Phytol 104:631-636 Hoekstra FA (1979) Planta 145:25-36 Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT (1985) Science 227: 1229-1232 Koornneef M, Hanhart C, Jongsma M, Toma I, Weide R, Zabel P, Hille J (1986) Plant Sci 45:201-208 Knox RB (1984) In: HF Linskens, Heslop- Harrison J (eds), Cellular interaction. Encycl Plant Physiol 17:508-608 Laughnan JR, Gabay SJ (1973) Crop Sc 13: 681-684 Mascarenhas JP, Mermelstein J (1981) Acta Soc Bot Pol 50:13-20 Mascarenhas NT, Bashe D, Eisenberg A, Willing RP, Xiao CM, Mascarenhas JP (1984) Theor Appl Genet 68:323-326 Sari Gorla M, Frova C, Binelli G, Ottaviano E (1986) Theor Appl Genet 72:42-47 Searcy KB, Mulcahy DL (1985) Amer J Bot 72: 1695-1699 Stanley RG, Linskens HF (1974) Pollen: biology, biochemistry, management. Springer-Verlag, Berlin, 329-334 Tanksley SD, Zamir D, Rick CM (1981) Science 213:453..-455 Tupy J (1982) Biol Plant 24:331-340 Van den Elzen P, Lee KY, Townsend J, Bedbrook J (1985) Plant Molec Biol 5: 149-154 Willing RP, Mascarenhas JP (1984) Plant Pbysiol 75: 865-868 Zambryski P, Joos H, Genetello C, Van Montagu M, Schell J (1983) Embo J 2: 2143-2150 Zamir D, Tanksley SD, Jones RA (1982) Genetics i01:129-137 

Kanamycin resistance during in vitro development of pollen from transgenic tomato plants

ARTS repository - University of Groningen

   University of GroningenKanamycin resistance during in vitro development of pollen from transgenic tomato plantsBino, R.J.; Hille, J.; Franken, J.Published in:Plant Cell ReportsDOI:10.1007/BF00269553IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1987Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bino, R. J., Hille, J., & Franken, J. (1987). Kanamycin resistance during in vitro development of pollen fromtransgenic tomato plants. Plant Cell Reports, 6(5). https://doi.org/10.1007/BF00269553CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 09-09-2023Plant Cell Reports (1987) 6:333-336 Plant Cell Reports © Springer-Verlag 1987 Kanamycin resistance during in vitro development of pollen from transgenic tomato plants R.J.  Bino ~, J. HiUe 2. , ,  and J. Franken 1 Institute for Horticultural Plant Breeding (IV'I), P.O.B. 16, 6700 AA Wageningen, The Netherlands 2 Agricultural University, Department of Molecular Biology, De Dreijen 11, 6703 BC Wageningen, The Netherlands Received May 26, 1987 - Communicated by I. Potrykus ABSTRACT Effects of kanamycin on pollen germination and tube growth of pollen from non- transformed plants and from transgenic tomato plants containing a chimaeric kanamycin resistance gene were determined. Germination of pollen was not affected by the addition of kanamycin to the medium in both genotypes. Kanamycin,however, severely affected tube growth of pollen from non- transformed plants, while pollen from plants containing the chimaeric gene were less sensitive and produced significantly longer tubes at kanamycin concentrations between 200-400 mgl-l. Apparently, this resistance for kanamycin correlates with the expression of the chimaeric gene during male gametophytic development. ABBREVIATIONS ATW, Agrobacterium Tomato Wageningen; KAN, Kanamycin; KANr, Kanamycin resistant; KANs, Kanamycin sensitive; mRNA, messenger RNA; NPT, Neomycin phosphotransferase. INTRODUCTION Male gametophytic functioning is involved with microsporogenesis, pollen germination, pollen tube elongation, and the transmission of gametes to the embryo sac. During these processes, the male haploid genome plays an important role. Willing and Mascarenhas (1984) estimated that mRNAs present in mature pollen grains of Tradescantia paludosa are the products of approximately 20,000 different genes. Large numbers of these mRNAs are translated into proteins during pollen germination and early pollen tube elongation (Mascarenhas and Mermelstein, 1981). similar results were obtained using the pollen of Zea mays (Mascarenhas et al., 1984) and Nicotiana tabacum (Tupy, 1982). RNA synthesis and protein synthesis are independent during the early stages of in vitro pollen tube development, as was demonstrated by the use of specific inhibitors (see review Knox, 1984). These inhibition studies showed that RNA transcription in most plant species is not essential for germination and early pollen tube growth, whereas both processes are severely affected by translation inhibitors. Most genes transcribed and translated during pollen development are also expressed in vegetative parts of the plant. Using RNA saturation hybridizations, Willing and Mascarenhas (1984) reported the expression of at least 64% of the pollen sequences in shoot tissues, and a maximum of about 60% of shoot mRNAs was also present in pollen of Tradescantia paludosa. Based on isozyme profiles, Tanksley et al. (19SI) ahd Sari Gorla et al. (1986), found in Lycopersicon esculentum and Zea mays a comparable percentage of genetic overlap between the sporophytic and gametophytic phase of a plant. The parallel expression of genes in the sporophyte and the gametophyte suggests that a selection pressure applied to the pollen is likely to alter the expression of genes in the sporophyte. Indeed, sporophytic gene expression is modified by chilling stress during pollen development in tomato species (Zamir et al., 1982) and after heavy metal stress during in vivo pollen germination in Silene dioica and Mimulis guttatus (Searcy and Mulcahy, 1985). The development of Agrobacterium tumefaciens derived gene vectors have enabled the transfer of new genetic information into the cells of various plant species (Zambryski et al., 1983). The use of selectable marker genes which are constitutively expressed allows a convenient selection of transformed tissues (Horsch, 1985). Such an approach was also used by Koornneef et al. (1986), who introduced a chimaeric kanamycin resistance gene into tomato. The present study is undertaken to determine if this marker gene is also expressed in the gametophytic phase of the tomato plants and can be used for pollen selection. * Present address: Vrije Universiteit, Department of Genetics, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands Offprint requests to: R. J. Bino 3 3 4  MATERIALS AND METHODS Plant material. Tomato plants used in this study derived from material developed by Koornneef et al. (1986) and are indicated as MsK. This genotype is selected for outstanding regeneration capacity and originally derived from the cross Lycopersicon peruvianum x L. esculentum. Transformants were obtained following leaf disc transformation with either the Agrobacterium tumefaciens strain C58Cl(pGV3850::l103neodim) (czernilofsky et al., 1986) or the strain LBA 4404 (pAL4404, pAGSII2) (Van den Elzen et al., 1985) as described by Koornneef et al. (1986). In between the T-region border sequences, both strains contain the NPT II gene from Tn5 under transcriptional control of the regulatory elements of the nopaline gene from A. tumefaciens. Expression of the chimaeric gene confers resistance of transformed plant cells to kanamycin (Hain et al., 1985). Transformed plants were named ATW and the transgenic character was confirmed both by Southern blot analyses and by segregation ratios of the KANr trait of seeds obtained from selfed plants (i.e., resistant : sensitive = 3 : i) (Koornneef et al., 1986). As a control for kanamycin sensitivity , pollen of MsK plants and two commercial tomato cultivars (viz. Moneymaker and Delicates) were used. In vitro pollen germination. Plants were grown in glasshouses at 18-22 °C, under daylight conditions. Pollen was collected from flowers at anthesis and germinated in solidified media containing 3 mM H~B03, 1.7 mM Ca(NOi)z, 10% sucrose, 0.7% agar, with different concentrations of kanamycin sulphate (Sigma) at pH 5.8. Pollen was incubated for 2 h at 24 °C in the dark, and both stained and fixed with aceto-carmine (1%). Germination and pollen tube length were determined for separate samples of 5-7 complete replicates. Per flower, and for each concentration, germination of at least 200 pollen grains was scored. Since stress induces the extrusion of short pollen tubes (Stanley and Linskens, 1974), pollen was recorded as germinated only when the pollen tube length was twice the diameter of the pollen grain. Pollen tube length was measured using an ocular micrometer for 50 tubes per flower at each concentration. The results were expressed as the mean + standard deviation, significant differences among means (P < 0.05) were partitioned by an analysis of variance. C [o0~ o 1A 90 C • ~ 60 % ol  7o  6o  4,6 I f 30 ~ I i i i i i i I t i i I t t L o loo 200 400 8oo 16oo Concentration k~namycin (mgl -I) 1°6%~-'T~. I 1 B 4J g 06 0 g 66 66 ¢- 4 J  ~ 66 I Ill 4~0 3 4.1 C: tO0 ZOO 4:00 800 1600 O,I ,-4 Concentration kBnamycin (mEl -I) o o. Figs. 1 A and B. Effects of increasing KAN concentrations (mgl-l) on germination and tube growth of pollen from non-transformed MsK ( ¢ ) and transgenic ATW ( + ) plants. Pollen from ATW plants, transformed with C58CI and pollen from plants transformed with LBA 4404, behaved similar and are therefore combined. Germination percentages and tube lengths were determined for 5 (MsK) and 7 (ATW) complete replicates of pollen from different flowers. Results are expressed as mean + standard deviation. Fig. 1 A. Percentage of germination. Per flower, and for each concentration, germination of at least 200 pollen grains was scored. Fig. 1 B. Pollen tube lenghts expressed as percentages of the control value (0 mgl-i KAN). Per flower, and for each concentration, length of at least 50 pollen was scored. 335 C Ok . . . .  Fig. 2. ATW pollen, in vitro germinated at 0 mgl-i KAN (A), 400 mgl-i KAN (B) and 800 mgl-I KAN (C). Bar = 0.I mm. RESULTS The effects of increasing KAN concentrations on germination of pollen from MsK and ATW plants are presented in Fig. 1 A. Pollen germination of both genotypes was not significantly affected by the addition of KAN to the medium. Fig. 1 B shows the effects of the addition of KAN to the pollen germination medium on pollen tube length, a microscopic view of the results is presented in Fig. 2. After two hours of incubation, at 0 mgl-l, mean pollen tube length was 2.0 + 0.3 - 10-4 m. Inhibition of tube growth of pollen from non-transformed (KANs) MsK plants was evident at i00 mgl-i KAN, while at 400 mgl-i KAN, the tube length was about 50% of the value obtained at 0 mgl-l. In contrast, inhibition of pollen tube growth of transformed (KANr) ATW plants was manifest at 200 mgl-l, while the 50% value occured at about 800 mgl-l. Significant differences between pollen tube lengths of MsK and ATW plants were obtained at 200 mgl- 1 (P < 0.01) and 400 mgl-i (P < 0.05). No different effects of KAN were observed between pollen tube growth of ATW plants transformed with the pGV3850::l103neodim construct or with the pAGSlI2 plasmid. Pollen tube growth of the cultivars Moneymaker and Delicates was similarily affected by KAN as pollen of MsK plants. DISCUSSION Tomato pollen tube growth is inhibited by the addition of KAN to the germination medium. Initial effects of KAN on pollen of non-transformed MsK plants were already apparent at I00 mgl-l. Similar KAN concentrations have been reported to inhibit MsK callus formation and seedling growth (Koornneef et al., 1986). The effects on vegetative growth, however, became evident only after several weeks in culture, while effects on pollen tube development were manifest 2 hours after pollen germination. Toxicity of KAN is due to interference with the translation system of the 70S ribosomes as present in mitochondria and chloroplasts of eucaryotic cells (Franklin and Snow, 1975). Since pollen tubes lack chloroplasts, the effect of KAN might indicate that an active mitochondrial translation is important during tomato pollen tube growth. Similar effects on pollen tube growth of several other species were obtained by Hoekstra (1979), using chloramphenicol, another inhibitor of 70S ribosomal protein synthesis. KAN did not significantly affect tomato pollen germination. For this, at least two explanations are possible: A) the antibiotic reagent is only gradually absorbed during pollen germination, or, B) mitochondrial translation is not essential for tomato pollen germination. Pollen tube growth of plants containing the chimaeric resistance gene is less sensitive for KAN than pollen of non- transformed plants. Since ATW plants are heterozygous for the KANr trait, only half of the pollen population is expected to be resistant. Hence, the difference between pollen from ATW and MsK plants is even more evident considering the heterogenous character of the ATW pollen sample. 336 Resistance to aminoglycoside antibiotics such as KAN, is due to expression of the NPT II gene (Hain et al., 1985). During early tube growth in vitro, however, genes appear not to be transcribed (Knox, 1984). Probably, in pollen, mRNAs of the chimaeric gene are presynthesized concurrently with the other mRNAs during microsporogenesis, while KAN resistance is induced by gene translation during pollen tube elongation. The largest difference between pollen tube growth of ATW and MsK plants occured at 200- 400 mgl-i KAN. Here, tubes of ATW pollen are about 1.4 times longer than the pollen from MsK plants. Since the level of pollen resistance probably correlates with the amount of NPT present in the cells, the use of a stronger promoter may enhance selection efficiency. The gametophytic phase of a plant is characterized by large populations and haploid genomes. Expression of selection sensitive genes by the male haploid genome opens the possibility to make pollinations with pre-selected pollen. For example, the toxin from Drechslera maydis prevents germination of pollen from maize plants susceptible to the disease but not of pollen from resistant plants (Laughnan and Gabay, 1973), and, compounds present in Alternaria brassicicola culture filtrates inhibit brassica pollen germination (Hodgkin and MacDonald (1986). However, linkage of agronomic important traits with a newly introduced marker gene, would be of general interest for the application of pollen selection methods in plant breeding programmes. ACKNOWLEDGMENTS We thank J. Jansen for helping with the statistical analyses, and J.J.M. Dons and A.G. Stephenson for critically reading the manuscript. REFERENCES Czernilofsky AP, Hain R, Herrera-Estrella L, Lorz H, Goyvaerts E, Baker B, Schell J (1986) DNA 5:103-113 Franklin TJ, Snow GA (1975) Biochemistry of antimicrobial action, Chapman and Hall Ltd., London, pp 224 Hain R, Stabel P, Czernilofsky AP, Steinbiss HH, Herrera-Estrella L, Schell J (1985) Mol Gen Genet 199:166-168 Hodgkin T, MacDonald MV (1986) New Phytol 104:631-636 Hoekstra FA (1979) Planta 145:25-36 Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT (1985) Science 227: 1229-1232 Koornneef M, Hanhart C, Jongsma M, Toma I, Weide R, Zabel P, Hille J (1986) Plant Sci 45:201-208 Knox RB (1984) In: HF Linskens, Heslop- Harrison J (eds), Cellular interaction. Encycl Plant Physiol 17:508-608 Laughnan JR, Gabay SJ (1973) Crop Sc 13: 681-684 Mascarenhas JP, Mermelstein J (1981) Acta Soc Bot Pol 50:13-20 Mascarenhas NT, Bashe D, Eisenberg A, Willing RP, Xiao CM, Mascarenhas JP (1984) Theor Appl Genet 68:323-326 Sari Gorla M, Frova C, Binelli G, Ottaviano E (1986) Theor Appl Genet 72:42-47 Searcy KB, Mulcahy DL (1985) Amer J Bot 72: 1695-1699 Stanley RG, Linskens HF (1974) Pollen: biology, biochemistry, management. Springer-Verlag, Berlin, 329-334 Tanksley SD, Zamir D, Rick CM (1981) Science 213:453..-455 Tupy J (1982) Biol Plant 24:331-340 Van den Elzen P, Lee KY, Townsend J, Bedbrook J (1985) Plant Molec Biol 5: 149-154 Willing RP, Mascarenhas JP (1984) Plant Pbysiol 75: 865-868 Zambryski P, Joos H, Genetello C, Van Montagu M, Schell J (1983) Embo J 2: 2143-2150 Zamir D, Tanksley SD, Jones RA (1982) Genetics i01:129-137 

   University of GroningenKanamycin resistance during in vitro development of pollen from transgenic tomato plantsBino, R.J.; Hille, J.; Franken, J.Published in:Plant Cell ReportsDOI:10.1007/BF00269553IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1987Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bino, R. J., Hille, J., & Franken, J. (1987). Kanamycin resistance during in vitro development of pollen fromtransgenic tomato plants. Plant Cell Reports, 6(5). https://doi.org/10.1007/BF00269553CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 20-06-2022Plant Cell Reports (1987) 6:333-336 Plant Cell Reports © Springer-Verlag 1987 Kanamycin resistance during in vitro development of pollen from transgenic tomato plants R.J.  Bino ~, J. HiUe 2. , ,  and J. Franken 1 Institute for Horticultural Plant Breeding (IV'I), P.O.B. 16, 6700 AA Wageningen, The Netherlands 2 Agricultural University, Department of Molecular Biology, De Dreijen 11, 6703 BC Wageningen, The Netherlands Received May 26, 1987 - Communicated by I. Potrykus ABSTRACT Effects of kanamycin on pollen germination and tube growth of pollen from non- transformed plants and from transgenic tomato plants containing a chimaeric kanamycin resistance gene were determined. Germination of pollen was not affected by the addition of kanamycin to the medium in both genotypes. Kanamycin,however, severely affected tube growth of pollen from non- transformed plants, while pollen from plants containing the chimaeric gene were less sensitive and produced significantly longer tubes at kanamycin concentrations between 200-400 mgl-l. Apparently, this resistance for kanamycin correlates with the expression of the chimaeric gene during male gametophytic development. ABBREVIATIONS ATW, Agrobacterium Tomato Wageningen; KAN, Kanamycin; KANr, Kanamycin resistant; KANs, Kanamycin sensitive; mRNA, messenger RNA; NPT, Neomycin phosphotransferase. INTRODUCTION Male gametophytic functioning is involved with microsporogenesis, pollen germination, pollen tube elongation, and the transmission of gametes to the embryo sac. During these processes, the male haploid genome plays an important role. Willing and Mascarenhas (1984) estimated that mRNAs present in mature pollen grains of Tradescantia paludosa are the products of approximately 20,000 different genes. Large numbers of these mRNAs are translated into proteins during pollen germination and early pollen tube elongation (Mascarenhas and Mermelstein, 1981). similar results were obtained using the pollen of Zea mays (Mascarenhas et al., 1984) and Nicotiana tabacum (Tupy, 1982). RNA synthesis and protein synthesis are independent during the early stages of in vitro pollen tube development, as was demonstrated by the use of specific inhibitors (see review Knox, 1984). These inhibition studies showed that RNA transcription in most plant species is not essential for germination and early pollen tube growth, whereas both processes are severely affected by translation inhibitors. Most genes transcribed and translated during pollen development are also expressed in vegetative parts of the plant. Using RNA saturation hybridizations, Willing and Mascarenhas (1984) reported the expression of at least 64% of the pollen sequences in shoot tissues, and a maximum of about 60% of shoot mRNAs was also present in pollen of Tradescantia paludosa. Based on isozyme profiles, Tanksley et al. (19SI) ahd Sari Gorla et al. (1986), found in Lycopersicon esculentum and Zea mays a comparable percentage of genetic overlap between the sporophytic and gametophytic phase of a plant. The parallel expression of genes in the sporophyte and the gametophyte suggests that a selection pressure applied to the pollen is likely to alter the expression of genes in the sporophyte. Indeed, sporophytic gene expression is modified by chilling stress during pollen development in tomato species (Zamir et al., 1982) and after heavy metal stress during in vivo pollen germination in Silene dioica and Mimulis guttatus (Searcy and Mulcahy, 1985). The development of Agrobacterium tumefaciens derived gene vectors have enabled the transfer of new genetic information into the cells of various plant species (Zambryski et al., 1983). The use of selectable marker genes which are constitutively expressed allows a convenient selection of transformed tissues (Horsch, 1985). Such an approach was also used by Koornneef et al. (1986), who introduced a chimaeric kanamycin resistance gene into tomato. The present study is undertaken to determine if this marker gene is also expressed in the gametophytic phase of the tomato plants and can be used for pollen selection. * Present address: Vrije Universiteit, Department of Genetics, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands Offprint requests to: R. J. Bino 3 3 4  MATERIALS AND METHODS Plant material. Tomato plants used in this study derived from material developed by Koornneef et al. (1986) and are indicated as MsK. This genotype is selected for outstanding regeneration capacity and originally derived from the cross Lycopersicon peruvianum x L. esculentum. Transformants were obtained following leaf disc transformation with either the Agrobacterium tumefaciens strain C58Cl(pGV3850::l103neodim) (czernilofsky et al., 1986) or the strain LBA 4404 (pAL4404, pAGSII2) (Van den Elzen et al., 1985) as described by Koornneef et al. (1986). In between the T-region border sequences, both strains contain the NPT II gene from Tn5 under transcriptional control of the regulatory elements of the nopaline gene from A. tumefaciens. Expression of the chimaeric gene confers resistance of transformed plant cells to kanamycin (Hain et al., 1985). Transformed plants were named ATW and the transgenic character was confirmed both by Southern blot analyses and by segregation ratios of the KANr trait of seeds obtained from selfed plants (i.e., resistant : sensitive = 3 : i) (Koornneef et al., 1986). As a control for kanamycin sensitivity , pollen of MsK plants and two commercial tomato cultivars (viz. Moneymaker and Delicates) were used. In vitro pollen germination. Plants were grown in glasshouses at 18-22 °C, under daylight conditions. Pollen was collected from flowers at anthesis and germinated in solidified media containing 3 mM H~B03, 1.7 mM Ca(NOi)z, 10% sucrose, 0.7% agar, with different concentrations of kanamycin sulphate (Sigma) at pH 5.8. Pollen was incubated for 2 h at 24 °C in the dark, and both stained and fixed with aceto-carmine (1%). Germination and pollen tube length were determined for separate samples of 5-7 complete replicates. Per flower, and for each concentration, germination of at least 200 pollen grains was scored. Since stress induces the extrusion of short pollen tubes (Stanley and Linskens, 1974), pollen was recorded as germinated only when the pollen tube length was twice the diameter of the pollen grain. Pollen tube length was measured using an ocular micrometer for 50 tubes per flower at each concentration. The results were expressed as the mean + standard deviation, significant differences among means (P < 0.05) were partitioned by an analysis of variance. C [o0~ o 1A 90 C • ~ 60 % ol  7o  6o  4,6 I f 30 ~ I i i i i i i I t i i I t t L o loo 200 400 8oo 16oo Concentration k~namycin (mgl -I) 1°6%~-'T~. I 1 B 4J g 06 0 g 66 66 ¢- 4 J  ~ 66 I Ill 4~0 3 4.1 C: tO0 ZOO 4:00 800 1600 O,I ,-4 Concentration kBnamycin (mEl -I) o o. Figs. 1 A and B. Effects of increasing KAN concentrations (mgl-l) on germination and tube growth of pollen from non-transformed MsK ( ¢ ) and transgenic ATW ( + ) plants. Pollen from ATW plants, transformed with C58CI and pollen from plants transformed with LBA 4404, behaved similar and are therefore combined. Germination percentages and tube lengths were determined for 5 (MsK) and 7 (ATW) complete replicates of pollen from different flowers. Results are expressed as mean + standard deviation. Fig. 1 A. Percentage of germination. Per flower, and for each concentration, germination of at least 200 pollen grains was scored. Fig. 1 B. Pollen tube lenghts expressed as percentages of the control value (0 mgl-i KAN). Per flower, and for each concentration, length of at least 50 pollen was scored. 335 C Ok . . . .  Fig. 2. ATW pollen, in vitro germinated at 0 mgl-i KAN (A), 400 mgl-i KAN (B) and 800 mgl-I KAN (C). Bar = 0.I mm. RESULTS The effects of increasing KAN concentrations on germination of pollen from MsK and ATW plants are presented in Fig. 1 A. Pollen germination of both genotypes was not significantly affected by the addition of KAN to the medium. Fig. 1 B shows the effects of the addition of KAN to the pollen germination medium on pollen tube length, a microscopic view of the results is presented in Fig. 2. After two hours of incubation, at 0 mgl-l, mean pollen tube length was 2.0 + 0.3 - 10-4 m. Inhibition of tube growth of pollen from non-transformed (KANs) MsK plants was evident at i00 mgl-i KAN, while at 400 mgl-i KAN, the tube length was about 50% of the value obtained at 0 mgl-l. In contrast, inhibition of pollen tube growth of transformed (KANr) ATW plants was manifest at 200 mgl-l, while the 50% value occured at about 800 mgl-l. Significant differences between pollen tube lengths of MsK and ATW plants were obtained at 200 mgl- 1 (P < 0.01) and 400 mgl-i (P < 0.05). No different effects of KAN were observed between pollen tube growth of ATW plants transformed with the pGV3850::l103neodim construct or with the pAGSlI2 plasmid. Pollen tube growth of the cultivars Moneymaker and Delicates was similarily affected by KAN as pollen of MsK plants. DISCUSSION Tomato pollen tube growth is inhibited by the addition of KAN to the germination medium. Initial effects of KAN on pollen of non-transformed MsK plants were already apparent at I00 mgl-l. Similar KAN concentrations have been reported to inhibit MsK callus formation and seedling growth (Koornneef et al., 1986). The effects on vegetative growth, however, became evident only after several weeks in culture, while effects on pollen tube development were manifest 2 hours after pollen germination. Toxicity of KAN is due to interference with the translation system of the 70S ribosomes as present in mitochondria and chloroplasts of eucaryotic cells (Franklin and Snow, 1975). Since pollen tubes lack chloroplasts, the effect of KAN might indicate that an active mitochondrial translation is important during tomato pollen tube growth. Similar effects on pollen tube growth of several other species were obtained by Hoekstra (1979), using chloramphenicol, another inhibitor of 70S ribosomal protein synthesis. KAN did not significantly affect tomato pollen germination. For this, at least two explanations are possible: A) the antibiotic reagent is only gradually absorbed during pollen germination, or, B) mitochondrial translation is not essential for tomato pollen germination. Pollen tube growth of plants containing the chimaeric resistance gene is less sensitive for KAN than pollen of non- transformed plants. Since ATW plants are heterozygous for the KANr trait, only half of the pollen population is expected to be resistant. Hence, the difference between pollen from ATW and MsK plants is even more evident considering the heterogenous character of the ATW pollen sample. 336 Resistance to aminoglycoside antibiotics such as KAN, is due to expression of the NPT II gene (Hain et al., 1985). During early tube growth in vitro, however, genes appear not to be transcribed (Knox, 1984). Probably, in pollen, mRNAs of the chimaeric gene are presynthesized concurrently with the other mRNAs during microsporogenesis, while KAN resistance is induced by gene translation during pollen tube elongation. The largest difference between pollen tube growth of ATW and MsK plants occured at 200- 400 mgl-i KAN. Here, tubes of ATW pollen are about 1.4 times longer than the pollen from MsK plants. Since the level of pollen resistance probably correlates with the amount of NPT present in the cells, the use of a stronger promoter may enhance selection efficiency. The gametophytic phase of a plant is characterized by large populations and haploid genomes. Expression of selection sensitive genes by the male haploid genome opens the possibility to make pollinations with pre-selected pollen. For example, the toxin from Drechslera maydis prevents germination of pollen from maize plants susceptible to the disease but not of pollen from resistant plants (Laughnan and Gabay, 1973), and, compounds present in Alternaria brassicicola culture filtrates inhibit brassica pollen germination (Hodgkin and MacDonald (1986). However, linkage of agronomic important traits with a newly introduced marker gene, would be of general interest for the application of pollen selection methods in plant breeding programmes. ACKNOWLEDGMENTS We thank J. Jansen for helping with the statistical analyses, and J.J.M. Dons and A.G. Stephenson for critically reading the manuscript. REFERENCES Czernilofsky AP, Hain R, Herrera-Estrella L, Lorz H, Goyvaerts E, Baker B, Schell J (1986) DNA 5:103-113 Franklin TJ, Snow GA (1975) Biochemistry of antimicrobial action, Chapman and Hall Ltd., London, pp 224 Hain R, Stabel P, Czernilofsky AP, Steinbiss HH, Herrera-Estrella L, Schell J (1985) Mol Gen Genet 199:166-168 Hodgkin T, MacDonald MV (1986) New Phytol 104:631-636 Hoekstra FA (1979) Planta 145:25-36 Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT (1985) Science 227: 1229-1232 Koornneef M, Hanhart C, Jongsma M, Toma I, Weide R, Zabel P, Hille J (1986) Plant Sci 45:201-208 Knox RB (1984) In: HF Linskens, Heslop- Harrison J (eds), Cellular interaction. Encycl Plant Physiol 17:508-608 Laughnan JR, Gabay SJ (1973) Crop Sc 13: 681-684 Mascarenhas JP, Mermelstein J (1981) Acta Soc Bot Pol 50:13-20 Mascarenhas NT, Bashe D, Eisenberg A, Willing RP, Xiao CM, Mascarenhas JP (1984) Theor Appl Genet 68:323-326 Sari Gorla M, Frova C, Binelli G, Ottaviano E (1986) Theor Appl Genet 72:42-47 Searcy KB, Mulcahy DL (1985) Amer J Bot 72: 1695-1699 Stanley RG, Linskens HF (1974) Pollen: biology, biochemistry, management. Springer-Verlag, Berlin, 329-334 Tanksley SD, Zamir D, Rick CM (1981) Science 213:453..-455 Tupy J (1982) Biol Plant 24:331-340 Van den Elzen P, Lee KY, Townsend J, Bedbrook J (1985) Plant Molec Biol 5: 149-154 Willing RP, Mascarenhas JP (1984) Plant Pbysiol 75: 865-868 Zambryski P, Joos H, Genetello C, Van Montagu M, Schell J (1983) Embo J 2: 2143-2150 Zamir D, Tanksley SD, Jones RA (1982) Genetics i01:129-137 

   University of GroningenKanamycin resistance during in vitro development of pollen from transgenic tomato plantsBino, R.J.; Hille, J.; Franken, J.Published in:Plant Cell ReportsDOI:10.1007/BF00269553IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1987Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bino, R. J., Hille, J., & Franken, J. (1987). Kanamycin resistance during in vitro development of pollen fromtransgenic tomato plants. Plant Cell Reports, 6(5). https://doi.org/10.1007/BF00269553CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019Plant Cell Reports (1987) 6:333-336 Plant Cell Reports © Springer-Verlag 1987 Kanamycin resistance during in vitro development of pollen from transgenic tomato plants R.J. Bino ~, J. HiUe 2. ,, and J. Franken 1 Institute for Horticultural Plant Breeding (IV'I), P.O.B. 16, 6700 AA Wageningen, The Netherlands 2 Agricultural University, Department of Molecular Biology, De Dreijen 11, 6703 BC Wageningen, The Netherlands Received May 26, 1987 - Communicated by I. Potrykus ABSTRACT Effects of kanamycin on pollen germination and tube growth of pollen from non- transformed plants and from transgenic tomato plants containing a chimaeric kanamycin resistance gene were determined. Germination of pollen was not affected by the addition of kanamycin to the medium in both genotypes. Kanamycin,however, severely affected tube growth of pollen from non- transformed plants, while pollen from plants containing the chimaeric gene were less sensitive and produced significantly longer tubes at kanamycin concentrations between 200-400 mgl-l. Apparently, this resistance for kanamycin correlates with the expression of the chimaeric gene during male gametophytic development. ABBREVIATIONS ATW, Agrobacterium Tomato Wageningen; KAN, Kanamycin; KANr, Kanamycin resistant; KANs, Kanamycin sensitive; mRNA, messenger RNA; NPT, Neomycin phosphotransferase. INTRODUCTION Male gametophytic functioning is involved with microsporogenesis, pollen germination, pollen tube elongation, and the transmission of gametes to the embryo sac. During these processes, the male haploid genome plays an important role. Will ing and Mascarenhas (1984) estimated that mRNAs present in mature pollen grains of Tradescantia paludosa are the products of approximately 20,000 different genes. Large numbers of these mRNAs are translated into proteins during pollen germination and early pollen tube elongation (Mascarenhas and Mermelstein, 1981). similar results were obtained using the pollen of Zea mays (Mascarenhas et al., 1984) and Nicotiana tabacum (Tupy, 1982). RNA synthesis and protein synthesis are independent during the early stages of in vitro pollen tube development, as was demonstrated by the use of specific inhibitors (see review Knox, 1984). These inhibition studies showed that RNA transcription in most plant species is not essential for germination and early pollen tube growth, whereas both processes are severely affected by translation inhibitors. Most genes transcribed and translated during pollen development are also expressed in vegetative parts of the plant. Using RNA saturation hybridizations, Wil l ing and Mascarenhas (1984) reported the expression of at least 64% of the pollen sequences in shoot tissues, and a maximum of about 60% of shoot mRNAs was also present in pollen of Tradescantia paludosa. Based on isozyme profiles, Tanksley et al. (19SI) ahd Sari Gorla et al. (1986), found in Lycopersicon esculentum and Zea mays a comparable percentage of genetic overlap between the sporophytic and gametophytic phase of a plant. The parallel expression of genes in the sporophyte and the gametophyte suggests that a selection pressure applied to the pollen is likely to alter the expression of genes in the sporophyte. Indeed, sporophytic gene expression is modified by chil l ing stress during pollen development in tomato species (Zamir et al., 1982) and after heavy metal stress during in vivo pollen germination in Silene dioica and Mimulis guttatus (Searcy and Mulcahy, 1985). The development of Agrobacterium tumefaciens derived gene vectors have enabled the transfer of new genetic information into the cells of various plant species (Zambryski et al., 1983). The use of selectable marker genes which are constitutively expressed allows a convenient selection of transformed tissues (Horsch, 1985). Such an approach was also used by Koornneef et al. (1986), who introduced a chimaeric kanamycin resistance gene into tomato. The present study is undertaken to determine if this marker gene is also expressed in the gametophytic phase of the tomato plants and can be used for pollen selection. * Present address: Vrije Universiteit, Department of Genetics, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands Offprint requests to: R. J. Bino 334 MATERIALS AND METHODS Plant material. Tomato plants used in this study derived from material  developed by Koornneef et al. (1986) and are indicated as MsK. This genotype is selected for outstanding regenerat ion capacity and or ig inal ly derived from the cross Lycopers icon peruvianum x L. esculentum. Transformants were obtained fol lowing leaf disc transformat ion with either the Agrobacter ium tumefaciens strain C58Cl(pGV3850:: l103neodim) (czerni lofsky et al., 1986) or the strain LBA 4404 (pAL4404, pAGSII2) (Van den Elzen et al., 1985) as descr ibed by Koornneef et al. (1986). In between the T-region border sequences, both strains contain the NPT II gene from Tn5 under transcr ipt ional  control of the regulatory elements of the nopal ine gene from A. tumefaciens. Expression of the chimaeric gene confers resistance of t ransformed plant cells to kanamycin (Hain et al., 1985). Transformed plants were named ATW and the transgenic character was conf irmed both by Southern blot analyses and by segregation ratios of the KANr trait  of seeds obtained from selfed plants (i.e., resistant : sensi t ive = 3 : i) (Koornneef et al., 1986). As a control for kanamycin sensit iv i ty , pol len of MsK plants and two commercial  tomato cult ivars (viz. Moneymaker  and Delicates) were used. In vitro pol len germination. Plants were grown in glasshouses at 18-22 °C, under dayl ight conditions. Pol len was col lected from flowers at anthesis and germinated in sol id i f ied media containing 3 mM H~B03, 1.7 mM Ca(NOi)z, 10% sucrose, 0.7% agar, with di f ferent concentrat ions of kanamycin sulphate (Sigma) at pH 5.8. Pol len was incubated for 2 h at 24 °C in the dark, and both stained and fixed with aceto-carmine (1%). Germinat ion and pol len tube length were determined for separate samples of 5-7 complete replicates. Per flower, and for each concentration, germinat ion of at least 200 pol len grains was scored. Since stress induces the extrusion of short pol len tubes (Stanley and Linskens, 1974), pol len was recorded as germinated only when the pol len tube length was twice the diameter of the pol len grain. Pol len tube length was measured using an ocular micrometer  for 50 tubes per flower at each concentration. The results were expressed as the mean + standard deviation, s ignif icant d i f ferences among means (P < 0.05) were part i t ioned by an analysis of variance. C [o0~ o 1A 90 C • ~ 60 % ol 7o 6o 4,6 I f 30 ~ I i i i i i i I t i i I t t L o loo 200 400 8oo 16oo Concentration k~namycin (mgl -I) 1°6%~-'T~. I 1 B 4J g 06 0 g 66 66 ¢- 4 J  ~ 66 I Ill 4~0 3 4.1 C: tO0 ZOO 4:00 800 1600 O,I ,-4 Concentration kBnamycin (mEl -I) o o. Figs. 1 A and B. Effects of increasing KAN concentrat ions (mgl-l) on germinat ion and tube growth of pol len from non-transformed MsK ( ¢ ) and transgenic ATW ( + ) plants. Pol len from ATW plants, t ransformed with C58CI and pol len from plants t ransformed with LBA 4404, behaved simi lar and are therefore combined. Germinat ion percentages and tube lengths were determined for 5 (MsK) and 7 (ATW) complete repl icates of pol len from dif ferent flowers. Results are expressed as mean + standard deviation. Fig. 1 A. Percentage of germination. Per flower, and for each concentration, germinat ion of at least 200 pol len grains was scored. Fig. 1 B. Pol len tube lenghts expressed as percentages of the control value (0 mgl- i  KAN). Per flower, and for each concentration, length of at least 50 pol len was scored. 335 C Ok . . . .  Fig. 2. ATW pollen, in vitro germinated at 0 mgl- i  KAN (A), 400 mgl- i  KAN (B) and 800 mgl- I  KAN (C). Bar = 0.I mm. RESULTS The effects of increasing KAN concentrat ions on germinat ion of pol len from MsK and ATW plants are presented in Fig. 1 A. Pol len germinat ion of both genotypes was not s igni f icant ly affected by the addit ion of KAN to the medium. Fig. 1 B shows the effects of the addit ion of KAN to the pol len germinat ion medium on pol len tube length, a microscopic  v iew of the results is presented in Fig. 2. After  two hours of incubation, at 0 mgl-l, mean pol len tube length was 2.0 + 0.3 - 10-4 m. Inhibit ion of tube growth of pol len from non-transformed (KANs) MsK plants was evident at i00 mgl- i  KAN, whi le at 400 mgl- i  KAN, the tube length was about 50% of the value obtained at 0 mgl-l. In contrast, inhibit ion of pol len tube growth of t ransformed (KANr) ATW plants was manifest  at 200 mgl-l, whi le the 50% value occured at about 800 mgl-l. S igni f icant  di f ferences between pol len tube lengths of MsK and ATW plants were obtained at 200 mgl-  1 (P < 0.01) and 400 mgl- i  (P < 0.05). No di f ferent effects of KAN were observed between pol len tube growth of ATW plants t ransformed with the pGV3850: : l103neodim construct or with the pAGSlI2 plasmid. Pol len tube growth of the cult ivars Moneymaker  and Del icates was s imi lar i ly  affected by KAN as pol len of MsK plants. DISCUSSION Tomato pol len tube growth is inhibited by the addit ion of KAN to the germinat ion medium. Initial ef fects of KAN on pol len of non-transformed MsK plants were already apparent at I00 mgl-l. S imi lar  KAN concentrat ions have been reported to inhibit  MsK cal lus formation and seedl ing growth (Koornneef et al., 1986). The effects on vegetat ive growth, however, became evident only after several weeks in culture, whi le effects on pol len tube development were mani fest  2 hours after pol len germination. Toxic i ty  of KAN is due to interference with the translat ion system of the 70S r ibosomes as present in mitochondr ia  and chloroplasts  of eucaryot ic  cells (Franklin and Snow, 1975). Since pol len tubes lack chloroplasts,  the effect of KAN might indicate that an active mitochondr ia l  t rans lat ion is important during tomato pol len tube growth. Simi lar effects on pol len tube growth of several other species were obtained by Hoekstra (1979), using chloramphenicol ,  another inhibitor of 70S r ibosomal protein synthesis. KAN did not s igni f icant ly  affect tomato pol len germination. For this, at least two explanat ions are possible: A) the ant ibiot ic  reagent is only gradual ly  absorbed during pol len germination, or, B) mitochondr ia l  t ranslat ion is not essential  for tomato pol len germination. Pol len tube growth of plants containing the chimaeric resistance gene is less sensit ive for KAN than pol len of non- t ransformed plants. Since ATW plants are heterozygous for the KANr trait, only half  of the pol len populat ion is expected to be resistant. Hence, the di f ference between pol len from ATW and MsK plants is even more evident consider ing the heterogenous character  of the ATW pol len sample. 336 Resistance to aminoglycoside antibiotics such as KAN, is due to expression of the NPT II gene (Hain et al., 1985). During early tube growth in vitro, however, genes appear not to be transcribed (Knox, 1984). Probably, in pollen, mRNAs of the chimaeric gene are presynthesized concurrently with the other mRNAs during microsporogenesis, while KAN resistance is induced by  gene translation during pollen tube elongation. The largest difference between pollen tube growth of ATW and MsK plants occured at 200- 400 mgl-i KAN. Here, tubes of ATW pollen are about 1.4 times longer than the pollen from MsK plants. Since the level of pollen resistance probably correlates with the amount of NPT present in the cells, the use of a stronger promoter may enhance selection efficiency. The gametophytic phase of a plant is characterized by large populations and haploid genomes. Expression of selection sensitive genes by the male haploid genome opens the possibil ity to make pollinations with pre-selected pollen. For example, the toxin from Drechslera maydis prevents germination of pollen from maize plants susceptible to the disease but not of pollen from resistant plants (Laughnan and Gabay, 1973), and, compounds present in Alternaria brassicicola culture filtrates inhibit brassica pollen germination (Hodgkin and MacDonald (1986). However, linkage of agronomic important traits with a newly introduced marker gene, would be of general interest for the application of pollen selection methods in plant breeding programmes. ACKNOWLEDGMENTS We thank J. Jansen for helping with the statistical analyses, and J.J.M. Dons and A.G. Stephenson for critically reading the manuscript. REFERENCES Czernilofsky AP, Hain R, Herrera-Estrella L, Lorz H, Goyvaerts E, Baker B, Schell J (1986) DNA 5 :103-113 Franklin TJ, Snow GA (1975) Biochemistry of antimicrobial ac t ion ,  Chapman and Hall Ltd., London, pp 224 Hain R, Stabel P, Czernilofsky AP, Steinbiss HH, Herrera-Estrella L, Schell J (1985) Mol Gen Genet 199:166-168 Hodgkin T, MacDonald MV (1986) New Phytol 104:631-636 Hoekstra FA (1979) Planta 145:25-36  Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT (1985) Science 227: 1229-1232 Koornneef M, Hanhart C, Jongsma M, Toma I, Weide R, Zabel P, Hille J (1986) Plant Sci 45:201-208 Knox RB (1984) In: HF Linskens, Heslop- Harrison J (eds), Cellular interaction. Encycl Plant Physiol 17:508-608 Laughnan JR, Gabay SJ (1973) Crop Sc 13: 681-684 Mascarenhas JP, Mermelstein J (1981) Acta Soc Bot Pol 50 :13-20  Mascarenhas NT, Bashe D, Eisenberg A, Willing RP, Xiao CM, Mascarenhas JP (1984) Theor Appl Genet 68:323-326 Sari Gorla M, Frova C, Binelli G, Ottaviano E (1986) Theor Appl Genet 72 :42-47  Searcy KB, Mulcahy DL (1985) Amer J Bot 72: 1695-1699 Stanley RG, Linskens HF (1974) Pollen: biology, biochemistry, management. Springer-Verlag, Berlin, 329-334 Tanksley SD, Zamir D, Rick CM (1981) Science 213:453..-455 Tupy J (1982) Biol Plant 24 :331-340 Van den Elzen P, Lee KY, Townsend J, Bedbrook J (1985) Plant Molec Biol 5: 149-154 Will ing RP, Mascarenhas JP (1984) Plant Pbysiol 75: 865-868 Zambryski P, Joos H, Genetello C, Van Montagu M, Schell J (1983) Embo J 2: 2143-2150 Zamir D, Tanksley SD, Jones RA (1982) Genetics i01 :129-137 

Bino, R.J.,

Hille, J.,

Franken, J.,

University of Groningen Digital Archive

Plant Cell Reports (1987) 6:333-336  Plant Cell 
Reports 
© Springer-Verlag 1987 
Kanamycin resistance during in vitro development of pollen 
from transgenic tomato plants 
R.J. Bino ~, J. HiUe 2. ,, and J. Franken 
1  Institute for Horticultural Plant Breeding (IV'I), P.O.B.  16, 6700  AA  Wageningen, The Netherlands 
2  Agricultural University, Department of Molecular Biology, De Dreijen 11, 6703  BC Wageningen, The Netherlands 
Received May 26,  1987 -  Communicated by I. Potrykus 
ABSTRACT 
Effects  of kanamycin  on pollen  germination 
and  tube  growth  of  pollen  from  non- 
transformed  plants  and  from  transgenic 
tomato  plants  containing  a  chimaeric 
kanamycin  resistance  gene were  determined. 
Germination  of  pollen was not  affected  by 
the  addition  of kanamycin to the medium  in 
both genotypes.  Kanamycin,however,  severely 
affected  tube  growth of pollen  from  non- 
transformed  plants,  while  pollen  from 
plants  containing  the chimaeric  gene  were 
less  sensitive  and  produced  significantly 
longer  tubes  at  kanamycin  concentrations 
between  200-400  mgl-l.  Apparently,  this 
resistance  for kanamycin  correlates  with the 
expression  of the chimaeric  gene during male 
gametophytic  development. 
ABBREVIATIONS 
ATW,  Agrobacterium  Tomato Wageningen;  KAN, 
Kanamycin;  KANr,  Kanamycin  resistant;  KANs, 
Kanamycin  sensitive;  mRNA,  messenger RNA; 
NPT,  Neomycin phosphotransferase. 
INTRODUCTION 
Male  gametophytic  functioning  is  involved 
with microsporogenesis,  pollen germination, 
pollen tube elongation,  and the transmission 
of  gametes  to the embryo  sac.  During these 
processes,  the male haploid genome plays  an 
important  role.  Willing  and  Mascarenhas 
(1984)  estimated  that  mRNAs  present  in 
mature  pollen  grains  of  Tradescantia 
paludosa  are the products  of  approximately 
20,000  different  genes.  Large numbers  of 
these  mRNAs  are translated  into  proteins 
during  pollen germination  and early  pollen 
tube  elongation  (Mascarenhas  and 
Mermelstein,  1981).  similar  results  were 
obtained  using  the  pollen  of  Zea  mays 
(Mascarenhas  et  al.,  1984)  and  Nicotiana 
tabacum  (Tupy,  1982).  RNA  synthesis  and 
protein  synthesis  are  independent  during the 
early  stages  of  in  vitro  pollen  tube 
development,  as was demonstrated  by the use 
of  specific  inhibitors  (see  review  Knox, 
1984).  These  inhibition  studies  showed that 
RNA  transcription  in most plant  species  is 
not  essential  for  germination  and  early 
pollen  tube growth,  whereas  both processes 
are  severely  affected  by  translation 
inhibitors. 
Most  genes  transcribed  and  translated 
during pollen development  are also expressed 
in vegetative  parts  of the plant.  Using RNA 
saturation  hybridizations,  Willing  and 
Mascarenhas  (1984)  reported the  expression 
of  at least  64% of the pollen  sequences  in 
shoot tissues,  and a maximum  of about  60%  of 
shoot  mRNAs was  also present  in  pollen  of 
Tradescantia  paludosa.  Based  on  isozyme 
profiles,  Tanksley  et al.  (19SI)  ahd Sari 
Gorla  et al.  (1986),  found  in Lycopersicon 
esculentum  and  Zea  mays  a  comparable 
percentage  of genetic  overlap  between  the 
sporophytic  and  gametophytic  phase  of  a 
plant. 
The  parallel  expression  of genes  in  the 
sporophyte  and the gametophyte  suggests  that 
a  selection pressure  applied  to the  pollen 
is  likely to alter the expression  of  genes 
in the sporophyte.  Indeed,  sporophytic  gene 
expression  is  modified by chilling  stress 
during pollen development  in tomato  species 
(Zamir  et al.,  1982)  and after heavy metal 
stress  during  in vivo pollen germination  in 
Silene  dioica  and Mimulis  guttatus  (Searcy 
and Mulcahy,  1985). 
The  development  of  Agrobacterium 
tumefaciens  derived  gene  vectors  have 
enabled  the  transfer  of  new  genetic 
information  into the cells  of various  plant 
species  (Zambryski  et al.,  1983).  The use  of 
selectable  marker  genes  which  are 
constitutively  expressed  allows  a  convenient 
selection  of  transformed  tissues  (Horsch, 
1985).  Such an approach was  also used  by 
Koornneef  et al.  (1986),  who  introduced  a 
chimaeric  kanamycin  resistance  gene  into 
tomato.  The present  study  is undertaken  to 
determine  if  this  marker  gene  is  also 
expressed  in the gametophytic  phase  of  the 
tomato  plants  and can be used  for  pollen 
selection. 
* Present address: Vrije Universiteit, Department of Genetics, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands 
Offprint requests to: R. J. Bino 334 
MATERIALS  AND METHODS 
Plant  material.  Tomato plants  used  in  this 
study  derived  from material  developed  by 
Koornneef et al.  (1986)  and are  indicated as 
MsK.  This  genotype  is  selected  for 
outstanding  regeneration  capacity  and 
originally  derived  from  the  cross 
Lycopersicon  peruvianum  x  L.  esculentum. 
Transformants  were obtained  following  leaf 
disc  transformation  with  either  the 
Agrobacterium  tumefaciens  strain 
C58Cl(pGV3850::l103neodim)  (czernilofsky  et 
al.,  1986)  or the strain LBA 4404  (pAL4404, 
pAGSII2)  (Van den Elzen et al.,  1985)  as 
described  by Koornneef  et  al.  (1986).  In 
between the T-region border sequences,  both 
strains  contain  the NPT II gene  from  Tn5 
under  transcriptional  control  of  the 
regulatory  elements  of the  nopaline  gene 
from  A.  tumefaciens.  Expression  of  the 
chimaeric  gene  confers  resistance  of 
transformed  plant cells to kanamycin  (Hain 
et al.,  1985).  Transformed plants were  named 
ATW  and  the  transgenic  character  was 
confirmed both by Southern blot analyses  and 
by segregation ratios of the KANr trait  of 
seeds  obtained  from selfed  plants  (i.e., 
resistant  : sensitive =  3  : i)  (Koornneef et 
al.,  1986).  As  a  control  for  kanamycin 
sensitivity  ,  pollen  of MsK plants  and  two 
commercial tomato  cultivars  (viz.  Moneymaker 
and Delicates)  were used. 
In  vitro  pollen germination.  Plants  were 
grown  in  glasshouses at  18-22  °C,  under 
daylight  conditions.  Pollen was  collected 
from  flowers at anthesis  and germinated  in 
solidified media  containing  3 mM  H~B03, 
1.7  mM Ca(NOi)z,  10%  sucrose,  0.7%  agar, 
with  different concentrations  of  kanamycin 
sulphate  (Sigma)  at pH  5.8.  Pollen  was 
incubated  for 2  h  at 24  °C  in the dark,  and 
both  stained  and  fixed with  aceto-carmine 
(1%). 
Germination  and  pollen  tube  length  were 
determined  for  separate  samples  of  5-7 
complete  replicates.  Per  flower,  and  for 
each concentration,  germination of at  least 
200  pollen grains was scored.  Since  stress 
induces the extrusion of short pollen  tubes 
(Stanley  and Linskens,  1974),  pollen  was 
recorded  as germinated only when the pollen 
tube  length was twice the diameter  of  the 
pollen  grain.  Pollen  tube  length  was 
measured  using an ocular micrometer  for  50 
tubes per  flower at each concentration.  The 
results  were  expressed  as  the  mean  + 
standard deviation,  significant differences 
among  means  (P <  0.05)  were partitioned  by 
an analysis of variance. 
C  [o0~ 
o  1A 
90 
C 
•  ~  60 
% 
ol  7o 
6o 
4,6 
I 
f 
30 
~  I  i  i  i  i  i  i  I  t  i  i  I  t  t  L 
o  loo  200  400  8oo  16oo 
Concentration  k~namycin  (mgl  -I) 
1°6%~-'T~.  I  1 B 
4J 
g  06 
0 
g  66 
66  ¢- 
4J 
~  66 
I 
Ill  4~0 
3 
4.1 
C:  tO0  ZOO  4:00  800  1600 
O,I 
,-4 
Concentration  kBnamycin  (mEl  -I) 
o 
o. 
Figs.  1 A  and B.  Effects  of  increasing KAN 
concentrations  (mgl-l)  on germination  and 
tube  growth of pollen  from  non-transformed 
MsK  ( ¢  ) and transgenic ATW  (  +  )  plants. 
Pollen  from ATW plants,  transformed  with 
C58CI  and  pollen  from  plants  transformed 
with  LBA  4404,  behaved  similar  and  are 
therefore combined.  Germination percentages 
and tube  lengths were determined  for  5  (MsK) 
and  7  (ATW)  complete replicates  of  pollen 
from  different  flowers.  Results  are 
expressed as mean +  standard deviation. 
Fig.  1  A.  Percentage  of germination.  Per 
flower,  and  for  each  concentration, 
germination  of at least 200  pollen  grains 
was  scored. 
Fig.  1  B.  Pollen tube  lenghts expressed as 
percentages  of  the control value  (0  mgl-i 
KAN).  Per  flower,  and  for  each 
concentration,  length of at  least 50 pollen 
was  scored. 335 
C Ok  .... 
Fig.  2.  ATW pollen,  in vitro germinated  at  0 
mgl-i  KAN  (A),  400 mgl-i  KAN  (B)  and  800 
mgl-I  KAN  (C).  Bar =  0.I mm. 
RESULTS 
The effects  of  increasing KAN concentrations 
on  germination  of pollen  from MsK and  ATW 
plants  are presented  in Fig.  1  A.  Pollen 
germination  of  both  genotypes  was  not 
significantly  affected by the  addition  of 
KAN to the medium. 
Fig.  1  B  shows  the  effects  of  the 
addition  of  KAN to the pollen  germination 
medium  on  pollen  tube  length,  a 
microscopic view of the results  is presented 
in Fig.  2.  After two hours  of  incubation,  at 
0 mgl-l,  mean pollen tube  length  was  2.0  + 
0.3  - 10-4 m.  Inhibition of tube growth  of 
pollen  from  non-transformed  (KANs)  MsK 
plants was  evident  at  i00 mgl-i  KAN,  while 
at 400 mgl-i KAN,  the tube  length was about 
50%  of  the value obtained  at  0  mgl-l.  In 
contrast,  inhibition of pollen tube  growth 
of  transformed  (KANr)  ATW  plants  was 
manifest  at 200 mgl-l,  while the  50%  value 
occured  at  about  800  mgl-l.  Significant 
differences  between pollen tube lengths  of 
MsK and ATW plants were obtained at 200 mgl- 
1  (P <  0.01)  and 400 mgl-i  (P <  0.05).  No 
different  effects  of  KAN  were  observed 
between  pollen  tube growth of  ATW  plants 
transformed  with  the  pGV3850::l103neodim 
construct  or  with  the  pAGSlI2  plasmid. 
Pollen  tube  growth  of  the  cultivars 
Moneymaker  and  Delicates  was  similarily 
affected by KAN as pollen of MsK plants. 
DISCUSSION 
Tomato  pollen tube growth  is  inhibited  by 
the  addition  of  KAN  to  the  germination 
medium.  Initial effects  of KAN on pollen  of 
non-transformed  MsK  plants  were  already 
apparent  at  I00  mgl-l.  Similar  KAN 
concentrations have been reported to  inhibit 
MsK  callus  formation and  seedling  growth 
(Koornneef et al.,  1986).  The effects  on 
vegetative growth,  however,  became  evident 
only  after several weeks  in culture,  while 
effects  on  pollen  tube  development  were 
manifest  2 hours  after pollen  germination. 
Toxicity  of KAN  is due to  interference with 
the translation  system of the  70S  ribosomes 
as  present  in mitochondria  and chloroplasts 
of  eucaryotic  cells  (Franklin  and  Snow, 
1975).  Since pollen tubes  lack chloroplasts, 
the  effect  of KAN might  indicate  that  an 
active  mitochondrial  translation  is 
important during tomato pollen tube  growth. 
Similar  effects  on pollen tube  growth  of 
several  other  species  were  obtained  by 
Hoekstra  (1979),  using  chloramphenicol, 
another  inhibitor of  70S  ribosomal  protein 
synthesis.  KAN did not  significantly affect 
tomato  pollen  germination.  For  this,  at 
least two explanations  are possible:  A)  the 
antibiotic  reagent  is  only  gradually 
absorbed during pollen germination,  or,  B) 
mitochondrial  translation  is  not  essential 
for tomato pollen germination. 
Pollen tube  growth  of plants  containing 
the  chimaeric  resistance  gene  is  less 
sensitive  for  KAN  than  pollen  of  non- 
transformed  plants.  Since ATW  plants  are 
heterozygous  for the KANr trait,  only half 
of  the pollen population  is  expected to  be 
resistant.  Hence,  the  difference  between 
pollen  from ATW and MsK plants  is even  more 
evident  considering  the  heterogenous 
character  of  the  ATW  pollen  sample. 336 
Resistance  to  aminoglycoside  antibiotics 
such  as KAN,  is due  to  expression  of the NPT 
II  gene  (Hain  et al.,  1985).  During  early 
tube  growth  in vitro,  however,  genes  appear 
not  to  be  transcribed  (Knox,  1984). 
Probably,  in pollen,  mRNAs  of the  chimaeric 
gene  are presynthesized  concurrently  with 
the  other  mRNAs  during  microsporogenesis, 
while  KAN  resistance  is  induced  by  gene 
translation  during  pollen  tube  elongation. 
The  largest  difference  between  pollen  tube 
growth  of ATW  and MsK plants  occured  at  200- 
400 mgl-i  KAN.  Here,  tubes  of ATW pollen  are 
about  1.4  times  longer  than  the pollen  from 
MsK  plants.  Since  the  level  of  pollen 
resistance  probably  correlates  with  the 
amount  of NPT present  in the  cells,  the use 
of a  stronger  promoter  may  enhance  selection 
efficiency. 
The  gametophytic  phase  of  a  plant  is 
characterized  by  large  populations  and 
haploid  genomes.  Expression  of  selection 
sensitive  genes  by the male  haploid  genome 
opens  the possibility  to make  pollinations 
with  pre-selected  pollen.  For example,  the 
toxin  from  Drechslera  maydis  prevents 
germination  of  pollen  from  maize  plants 
susceptible  to the  disease  but not  of pollen 
from  resistant  plants  (Laughnan  and  Gabay, 
1973),  and,  compounds  present  in Alternaria 
brassicicola  culture  filtrates  inhibit 
brassica  pollen  germination  (Hodgkin  and 
MacDonald  (1986).  However,  linkage  of 
agronomic  important  traits  with  a  newly 
introduced  marker  gene,  would  be of general 
interest  for  the  application  of  pollen 
selection  methods  in  plant  breeding 
programmes. 
ACKNOWLEDGMENTS 
We  thank  J.  Jansen  for helping  with  the 
statistical  analyses,  and J.J.M.  Dons  and 
A.G.  Stephenson  for critically  reading  the 
manuscript. 
REFERENCES 
Czernilofsky  AP,  Hain  R,  Herrera-Estrella  L, 
Lorz  H,  Goyvaerts  E,  Baker  B,  Schell  J 
(1986)  DNA  5:103-113 
Franklin  TJ,  Snow  GA  (1975)  Biochemistry 
of  antimicrobial  action,  Chapman  and 
Hall  Ltd.,  London,  pp  224 
Hain  R,  Stabel  P,  Czernilofsky  AP,  Steinbiss 
HH,  Herrera-Estrella  L,  Schell  J  (1985) 
Mol  Gen Genet  199:166-168 
Hodgkin  T,  MacDonald  MV  (1986)  New  Phytol 
104:631-636 
Hoekstra  FA  (1979)  Planta  145:25-36 
Horsch  RB,  Fry JE,  Hoffman  NL,  Eichholtz  D, 
Rogers  SG,  Fraley  RT  (1985)  Science  227: 
1229-1232 
Koornneef  M,  Hanhart  C,  Jongsma  M,  Toma  I, 
Weide  R,  Zabel  P,  Hille  J  (1986)  Plant 
Sci  45:201-208 
Knox  RB  (1984)  In:  HF  Linskens,  Heslop- 
Harrison  J  (eds),  Cellular  interaction. 
Encycl  Plant  Physiol  17:508-608 
Laughnan  JR,  Gabay  SJ  (1973)  Crop  Sc  13: 
681-684 
Mascarenhas  JP,  Mermelstein  J  (1981)  Acta 
Soc Bot  Pol  50:13-20 
Mascarenhas  NT,  Bashe  D,  Eisenberg  A, 
Willing  RP,  Xiao  CM,  Mascarenhas  JP 
(1984)  Theor  Appl  Genet  68:323-326 
Sari  Gorla  M,  Frova  C,  Binelli  G,  Ottaviano 
E  (1986)  Theor  Appl  Genet  72:42-47 
Searcy  KB,  Mulcahy  DL  (1985)  Amer J  Bot  72: 
1695-1699 
Stanley  RG,  Linskens  HF  (1974)  Pollen: 
biology,  biochemistry,  management. 
Springer-Verlag,  Berlin,  329-334 
Tanksley  SD,  Zamir  D,  Rick  CM  (1981)  Science 
213:453..-455 
Tupy  J  (1982)  Biol  Plant  24:331-340 
Van  den  Elzen  P,  Lee  KY,  Townsend  J, 
Bedbrook  J  (1985)  Plant Molec  Biol  5: 
149-154 
Willing  RP,  Mascarenhas  JP  (1984)  Plant 
Pbysiol  75:  865-868 
Zambryski  P,  Joos  H,  Genetello  C,  Van 
Montagu  M,  Schell  J  (1983)  Embo  J  2: 
2143-2150 
Zamir  D,  Tanksley  SD,  Jones  RA  (1982) 
Genetics  i01:129-137 

Acta Soc Bot Pol 50:13-20 Mascarenhas

DNA 5:103-113 Franklin TJ, Snow GA

Science 213:453..-455 Tupy J

University of Groningen Research Database

  
 University of Groningen
Kanamycin resistance during in vitro development of pollen from transgenic tomato plants
Bino, R.J.; Hille, Jacob; Franken, J.
Published in:
Plant Cell Reports
DOI:
10.1007/BF00269553
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
1987
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Bino, R. J., Hille, J., & Franken, J. (1987). Kanamycin resistance during in vitro development of pollen from
transgenic tomato plants. Plant Cell Reports, 6(5). DOI: 10.1007/BF00269553
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
Plant Cell Reports (1987) 6:333-336 Plant Cell 
Reports 
© Springer-Verlag 1987 
Kanamycin resistance during in vitro development of pollen 
from transgenic tomato plants 
R.J. Bino ~, J. HiUe 2. ,, and J. Franken 
1 Institute for Horticultural Plant Breeding (IV'I), P.O.B. 16, 6700 AA Wageningen, The Netherlands 
2 Agricultural University, Department of Molecular Biology, De Dreijen 11, 6703 BC Wageningen, The Netherlands 
Received May 26, 1987 - Communicated by I. Potrykus 
ABSTRACT 
Effects of kanamycin on pollen germination 
and tube growth of pollen from non- 
transformed plants and from transgenic 
tomato plants containing a chimaeric 
kanamycin resistance gene were determined. 
Germination of pollen was not affected by 
the addition of kanamycin to the medium in 
both genotypes. Kanamycin,however, severely 
affected tube growth of pollen from non- 
transformed plants, while pollen from 
plants containing the chimaeric gene were 
less sensitive and produced significantly 
longer tubes at kanamycin concentrations 
between 200-400 mgl-l. Apparently, this 
resistance for kanamycin correlates with the 
expression of the chimaeric gene during male 
gametophytic development. 
ABBREVIATIONS 
ATW, Agrobacterium Tomato Wageningen; KAN, 
Kanamycin; KANr, Kanamycin resistant; KANs, 
Kanamycin sensitive; mRNA, messenger RNA; 
NPT, Neomycin phosphotransferase. 
INTRODUCTION 
Male gametophytic functioning is involved 
with microsporogenesis, pollen germination, 
pollen tube elongation, and the transmission 
of gametes to the embryo sac. During these 
processes, the male haploid genome plays an 
important role. Will ing and Mascarenhas 
(1984) estimated that mRNAs present in 
mature pollen grains of Tradescantia 
paludosa are the products of approximately 
20,000 different genes. Large numbers of 
these mRNAs are translated into proteins 
during pollen germination and early pollen 
tube elongation (Mascarenhas and 
Mermelstein, 1981). similar results were 
obtained using the pollen of Zea mays 
(Mascarenhas et al., 1984) and Nicotiana 
tabacum (Tupy, 1982). RNA synthesis and 
protein synthesis are independent during the 
early stages of in vitro pollen tube 
development, as was demonstrated by the use 
of specific inhibitors (see review Knox, 
1984). These inhibition studies showed that 
RNA transcription in most plant species is 
not essential for germination and early 
pollen tube growth, whereas both processes 
are severely affected by translation 
inhibitors. 
Most genes transcribed and translated 
during pollen development are also expressed 
in vegetative parts of the plant. Using RNA 
saturation hybridizations, Wil l ing and 
Mascarenhas (1984) reported the expression 
of at least 64% of the pollen sequences in 
shoot tissues, and a maximum of about 60% of 
shoot mRNAs was also present in pollen of 
Tradescantia paludosa. Based on isozyme 
profiles, Tanksley et al. (19SI) ahd Sari 
Gorla et al. (1986), found in Lycopersicon 
esculentum and Zea mays a comparable 
percentage of genetic overlap between the 
sporophytic and gametophytic phase of a 
plant. 
The parallel expression of genes in the 
sporophyte and the gametophyte suggests that 
a selection pressure applied to the pollen 
is likely to alter the expression of genes 
in the sporophyte. Indeed, sporophytic gene 
expression is modified by chil l ing stress 
during pollen development in tomato species 
(Zamir et al., 1982) and after heavy metal 
stress during in vivo pollen germination in 
Silene dioica and Mimulis guttatus (Searcy 
and Mulcahy, 1985). 
The development of Agrobacterium 
tumefaciens derived gene vectors have 
enabled the transfer of new genetic 
information into the cells of various plant 
species (Zambryski et al., 1983). The use of 
selectable marker genes which are 
constitutively expressed allows a convenient 
selection of transformed tissues (Horsch, 
1985). Such an approach was also used by 
Koornneef et al. (1986), who introduced a 
chimaeric kanamycin resistance gene into 
tomato. The present study is undertaken to 
determine if this marker gene is also 
expressed in the gametophytic phase of the 
tomato plants and can be used for pollen 
selection. 
* Present address: Vrije Universiteit, Department of Genetics, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands 
Offprint requests to: R. J. Bino 
334 
MATERIALS AND METHODS 
Plant material. Tomato plants used in this 
study derived from material  developed by 
Koornneef et al. (1986) and are indicated as 
MsK. This genotype is selected for 
outstanding regenerat ion capacity and 
or ig inal ly derived from the cross 
Lycopers icon peruvianum x L. esculentum. 
Transformants were obtained fol lowing leaf 
disc transformat ion with either the 
Agrobacter ium tumefaciens strain 
C58Cl(pGV3850:: l103neodim) (czerni lofsky et 
al., 1986) or the strain LBA 4404 (pAL4404, 
pAGSII2) (Van den Elzen et al., 1985) as 
descr ibed by Koornneef et al. (1986). In 
between the T-region border sequences, both 
strains contain the NPT II gene from Tn5 
under transcr ipt ional  control of the 
regulatory elements of the nopal ine gene 
from A. tumefaciens. Expression of the 
chimaeric gene confers resistance of 
t ransformed plant cells to kanamycin (Hain 
et al., 1985). Transformed plants were named 
ATW and the transgenic character was 
conf irmed both by Southern blot analyses and 
by segregation ratios of the KANr trait  of 
seeds obtained from selfed plants (i.e., 
resistant : sensi t ive = 3 : i) (Koornneef et 
al., 1986). As a control for kanamycin 
sensit iv i ty , pol len of MsK plants and two 
commercial  tomato cult ivars (viz. Moneymaker  
and Delicates) were used. 
In vitro pol len germination. Plants were 
grown in glasshouses at 18-22 °C, under 
dayl ight conditions. Pol len was col lected 
from flowers at anthesis and germinated in 
sol id i f ied media containing 3 mM H~B03, 
1.7 mM Ca(NOi)z, 10% sucrose, 0.7% agar, 
with di f ferent concentrat ions of kanamycin 
sulphate (Sigma) at pH 5.8. Pol len was 
incubated for 2 h at 24 °C in the dark, and 
both stained and fixed with aceto-carmine 
(1%). 
Germinat ion and pol len tube length were 
determined for separate samples of 5-7 
complete replicates. Per flower, and for 
each concentration, germinat ion of at least 
200 pol len grains was scored. Since stress 
induces the extrusion of short pol len tubes 
(Stanley and Linskens, 1974), pol len was 
recorded as germinated only when the pol len 
tube length was twice the diameter of the 
pol len grain. Pol len tube length was 
measured using an ocular micrometer  for 50 
tubes per flower at each concentration. The 
results were expressed as the mean + 
standard deviation, s ignif icant d i f ferences 
among means (P < 0.05) were part i t ioned by 
an analysis of variance. 
C [o0~ 
o 1A 
90 
C 
• ~ 60 
% 
ol 7o 
6o 
4,6 
I 
f 
30 
~ I i i i i i i I t i i I t t L 
o loo 200 400 8oo 16oo 
Concentration k~namycin (mgl -I) 
1°6%~-'T~. I 1 B 
4J 
g 06 
0 
g 66 
66 ¢- 
4 J  
~ 66 
I 
Ill 4~0 
3 
4.1 
C: tO0 ZOO 4:00 800 1600 
O,I 
,-4 
Concentration kBnamycin (mEl -I) 
o 
o. 
Figs. 1 A and B. Effects of increasing KAN 
concentrat ions (mgl-l) on germinat ion and 
tube growth of pol len from non-transformed 
MsK ( ¢ ) and transgenic ATW ( + ) plants. 
Pol len from ATW plants, t ransformed with 
C58CI and pol len from plants t ransformed 
with LBA 4404, behaved simi lar and are 
therefore combined. Germinat ion percentages 
and tube lengths were determined for 5 (MsK) 
and 7 (ATW) complete repl icates of pol len 
from dif ferent flowers. Results are 
expressed as mean + standard deviation. 
Fig. 1 A. Percentage of germination. Per 
flower, and for each concentration, 
germinat ion of at least 200 pol len grains 
was scored. 
Fig. 1 B. Pol len tube lenghts expressed as 
percentages of the control value (0 mgl- i  
KAN). Per flower, and for each 
concentration, length of at least 50 pol len 
was scored. 
335 
C Ok . . . .  
Fig. 2. ATW pollen, in vitro germinated at 0 
mgl- i  KAN (A), 400 mgl- i  KAN (B) and 800 
mgl- I  KAN (C). Bar = 0.I mm. 
RESULTS 
The effects of increasing KAN concentrat ions 
on germinat ion of pol len from MsK and ATW 
plants are presented in Fig. 1 A. Pol len 
germinat ion of both genotypes was not 
s igni f icant ly affected by the addit ion of 
KAN to the medium. 
Fig. 1 B shows the effects of the 
addit ion of KAN to the pol len germinat ion 
medium on pol len tube length, a 
microscopic  v iew of the results is presented 
in Fig. 2. After  two hours of incubation, at 
0 mgl-l, mean pol len tube length was 2.0 + 
0.3 - 10-4 m. Inhibit ion of tube growth of 
pol len from non-transformed (KANs) MsK 
plants was evident at i00 mgl- i  KAN, whi le 
at 400 mgl- i  KAN, the tube length was about 
50% of the value obtained at 0 mgl-l. In 
contrast, inhibit ion of pol len tube growth 
of t ransformed (KANr) ATW plants was 
manifest  at 200 mgl-l, whi le the 50% value 
occured at about 800 mgl-l. S igni f icant  
di f ferences between pol len tube lengths of 
MsK and ATW plants were obtained at 200 mgl-  
1 (P < 0.01) and 400 mgl- i  (P < 0.05). No 
di f ferent effects of KAN were observed 
between pol len tube growth of ATW plants 
t ransformed with the pGV3850: : l103neodim 
construct or with the pAGSlI2 plasmid. 
Pol len tube growth of the cult ivars 
Moneymaker  and Del icates was s imi lar i ly  
affected by KAN as pol len of MsK plants. 
DISCUSSION 
Tomato pol len tube growth is inhibited by 
the addit ion of KAN to the germinat ion 
medium. Initial ef fects of KAN on pol len of 
non-transformed MsK plants were already 
apparent at I00 mgl-l. S imi lar  KAN 
concentrat ions have been reported to inhibit  
MsK cal lus formation and seedl ing growth 
(Koornneef et al., 1986). The effects on 
vegetat ive growth, however, became evident 
only after several weeks in culture, whi le 
effects on pol len tube development were 
mani fest  2 hours after pol len germination. 
Toxic i ty  of KAN is due to interference with 
the translat ion system of the 70S r ibosomes 
as present in mitochondr ia  and chloroplasts  
of eucaryot ic  cells (Franklin and Snow, 
1975). Since pol len tubes lack chloroplasts,  
the effect of KAN might indicate that an 
active mitochondr ia l  t rans lat ion is 
important during tomato pol len tube growth. 
Simi lar effects on pol len tube growth of 
several other species were obtained by 
Hoekstra (1979), using chloramphenicol ,  
another inhibitor of 70S r ibosomal protein 
synthesis. KAN did not s igni f icant ly  affect 
tomato pol len germination. For this, at 
least two explanat ions are possible: A) the 
ant ibiot ic  reagent is only gradual ly  
absorbed during pol len germination, or, B) 
mitochondr ia l  t ranslat ion is not essential  
for tomato pol len germination. 
Pol len tube growth of plants containing 
the chimaeric resistance gene is less 
sensit ive for KAN than pol len of non- 
t ransformed plants. Since ATW plants are 
heterozygous for the KANr trait, only half  
of the pol len populat ion is expected to be 
resistant. Hence, the di f ference between 
pol len from ATW and MsK plants is even more 
evident consider ing the heterogenous 
character  of the ATW pol len sample. 
336 
Resistance to aminoglycoside antibiotics 
such as KAN, is due to expression of the NPT 
II gene (Hain et al., 1985). During early 
tube growth in vitro, however, genes appear 
not to be transcribed (Knox, 1984). 
Probably, in pollen, mRNAs of the chimaeric 
gene are presynthesized concurrently with 
the other mRNAs during microsporogenesis, 
while KAN resistance is induced by  gene 
translation during pollen tube elongation. 
The largest difference between pollen tube 
growth of ATW and MsK plants occured at 200- 
400 mgl-i KAN. Here, tubes of ATW pollen are 
about 1.4 times longer than the pollen from 
MsK plants. Since the level of pollen 
resistance probably correlates with the 
amount of NPT present in the cells, the use 
of a stronger promoter may enhance selection 
efficiency. 
The gametophytic phase of a plant is 
characterized by large populations and 
haploid genomes. Expression of selection 
sensitive genes by the male haploid genome 
opens the possibil ity to make pollinations 
with pre-selected pollen. For example, the 
toxin from Drechslera maydis prevents 
germination of pollen from maize plants 
susceptible to the disease but not of pollen 
from resistant plants (Laughnan and Gabay, 
1973), and, compounds present in Alternaria 
brassicicola culture filtrates inhibit 
brassica pollen germination (Hodgkin and 
MacDonald (1986). However, linkage of 
agronomic important traits with a newly 
introduced marker gene, would be of general 
interest for the application of pollen 
selection methods in plant breeding 
programmes. 
ACKNOWLEDGMENTS 
We thank J. Jansen for helping with the 
statistical analyses, and J.J.M. Dons and 
A.G. Stephenson for critically reading the 
manuscript. 
REFERENCES 
Czernilofsky AP, Hain R, Herrera-Estrella L, 
Lorz H, Goyvaerts E, Baker B, Schell J 
(1986) DNA 5 :103-113 
Franklin TJ, Snow GA (1975) Biochemistry 
of antimicrobial ac t ion ,  Chapman and 
Hall Ltd., London, pp 224 
Hain R, Stabel P, Czernilofsky AP, Steinbiss 
HH, Herrera-Estrella L, Schell J (1985) 
Mol Gen Genet 199:166-168 
Hodgkin T, MacDonald MV (1986) New Phytol 
104:631-636 
Hoekstra FA (1979) Planta 145:25-36  
Horsch RB, Fry JE, Hoffman NL, Eichholtz D, 
Rogers SG, Fraley RT (1985) Science 227: 
1229-1232 
Koornneef M, Hanhart C, Jongsma M, Toma I, 
Weide R, Zabel P, Hille J (1986) Plant 
Sci 45:201-208 
Knox RB (1984) In: HF Linskens, Heslop- 
Harrison J (eds), Cellular interaction. 
Encycl Plant Physiol 17:508-608 
Laughnan JR, Gabay SJ (1973) Crop Sc 13: 
681-684 
Mascarenhas JP, Mermelstein J (1981) Acta 
Soc Bot Pol 50 :13-20  
Mascarenhas NT, Bashe D, Eisenberg A, 
Willing RP, Xiao CM, Mascarenhas JP 
(1984) Theor Appl Genet 68:323-326 
Sari Gorla M, Frova C, Binelli G, Ottaviano 
E (1986) Theor Appl Genet 72 :42-47  
Searcy KB, Mulcahy DL (1985) Amer J Bot 72: 
1695-1699 
Stanley RG, Linskens HF (1974) Pollen: 
biology, biochemistry, management. 
Springer-Verlag, Berlin, 329-334 
Tanksley SD, Zamir D, Rick CM (1981) Science 
213:453..-455 
Tupy J (1982) Biol Plant 24 :331-340 
Van den Elzen P, Lee KY, Townsend J, 
Bedbrook J (1985) Plant Molec Biol 5: 
149-154 
Will ing RP, Mascarenhas JP (1984) Plant 
Pbysiol 75: 865-868 
Zambryski P, Joos H, Genetello C, Van 
Montagu M, Schell J (1983) Embo J 2: 
2143-2150 
Zamir D, Tanksley SD, Jones RA (1982) 
Genetics i01 :129-137 


University of GroningenKanamycin resistance during in vitro development of pollen from transgenic tomato plantsBino, R.J.; Hille, J.; Franken, J.Published in:Plant Cell ReportsDOI:10.1007/BF00269553IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1987Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bino, R. J., Hille, J., & Franken, J. (1987). Kanamycin resistance during in vitro development of pollen fromtransgenic tomato plants. Plant Cell Reports, 6(5). https://doi.org/10.1007/BF00269553CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 30-12-2025Plant Cell Reports (1987) 6:333-336 Plant Cell Reports © Springer-Verlag 1987 Kanamycin resistance during in vitro development of pollen from transgenic tomato plants R.J.  Bino ~, J. HiUe 2. , ,  and J. Franken 1 Institute for Horticultural Plant Breeding (IV'I), P.O.B. 16, 6700 AA Wageningen, The Netherlands 2 Agricultural University, Department of Molecular Biology, De Dreijen 11, 6703 BC Wageningen, The Netherlands Received May 26, 1987 - Communicated by I. Potrykus ABSTRACT Effects of kanamycin on pollen germination and tube growth of pollen from non- transformed plants and from transgenic tomato plants containing a chimaeric kanamycin resistance gene were determined. Germination of pollen was not affected by the addition of kanamycin to the medium in both genotypes. Kanamycin,however, severely affected tube growth of pollen from non- transformed plants, while pollen from plants containing the chimaeric gene were less sensitive and produced significantly longer tubes at kanamycin concentrations between 200-400 mgl-l. Apparently, this resistance for kanamycin correlates with the expression of the chimaeric gene during male gametophytic development. ABBREVIATIONS ATW, Agrobacterium Tomato Wageningen; KAN, Kanamycin; KANr, Kanamycin resistant; KANs, Kanamycin sensitive; mRNA, messenger RNA; NPT, Neomycin phosphotransferase. INTRODUCTION Male gametophytic functioning is involved with microsporogenesis, pollen germination, pollen tube elongation, and the transmission of gametes to the embryo sac. During these processes, the male haploid genome plays an important role. Willing and Mascarenhas (1984) estimated that mRNAs present in mature pollen grains of Tradescantia paludosa are the products of approximately 20,000 different genes. Large numbers of these mRNAs are translated into proteins during pollen germination and early pollen tube elongation (Mascarenhas and Mermelstein, 1981). similar results were obtained using the pollen of Zea mays (Mascarenhas et al., 1984) and Nicotiana tabacum (Tupy, 1982). RNA synthesis and protein synthesis are independent during the early stages of in vitro pollen tube development, as was demonstrated by the use of specific inhibitors (see review Knox, 1984). These inhibition studies showed that RNA transcription in most plant species is not essential for germination and early pollen tube growth, whereas both processes are severely affected by translation inhibitors. Most genes transcribed and translated during pollen development are also expressed in vegetative parts of the plant. Using RNA saturation hybridizations, Willing and Mascarenhas (1984) reported the expression of at least 64% of the pollen sequences in shoot tissues, and a maximum of about 60% of shoot mRNAs was also present in pollen of Tradescantia paludosa. Based on isozyme profiles, Tanksley et al. (19SI) ahd Sari Gorla et al. (1986), found in Lycopersicon esculentum and Zea mays a comparable percentage of genetic overlap between the sporophytic and gametophytic phase of a plant. The parallel expression of genes in the sporophyte and the gametophyte suggests that a selection pressure applied to the pollen is likely to alter the expression of genes in the sporophyte. Indeed, sporophytic gene expression is modified by chilling stress during pollen development in tomato species (Zamir et al., 1982) and after heavy metal stress during in vivo pollen germination in Silene dioica and Mimulis guttatus (Searcy and Mulcahy, 1985). The development of Agrobacterium tumefaciens derived gene vectors have enabled the transfer of new genetic information into the cells of various plant species (Zambryski et al., 1983). The use of selectable marker genes which are constitutively expressed allows a convenient selection of transformed tissues (Horsch, 1985). Such an approach was also used by Koornneef et al. (1986), who introduced a chimaeric kanamycin resistance gene into tomato. The present study is undertaken to determine if this marker gene is also expressed in the gametophytic phase of the tomato plants and can be used for pollen selection. * Present address: Vrije Universiteit, Department of Genetics, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands Offprint requests to: R. J. Bino 3 3 4  MATERIALS AND METHODS Plant material. Tomato plants used in this study derived from material developed by Koornneef et al. (1986) and are indicated as MsK. This genotype is selected for outstanding regeneration capacity and originally derived from the cross Lycopersicon peruvianum x L. esculentum. Transformants were obtained following leaf disc transformation with either the Agrobacterium tumefaciens strain C58Cl(pGV3850::l103neodim) (czernilofsky et al., 1986) or the strain LBA 4404 (pAL4404, pAGSII2) (Van den Elzen et al., 1985) as described by Koornneef et al. (1986). In between the T-region border sequences, both strains contain the NPT II gene from Tn5 under transcriptional control of the regulatory elements of the nopaline gene from A. tumefaciens. Expression of the chimaeric gene confers resistance of transformed plant cells to kanamycin (Hain et al., 1985). Transformed plants were named ATW and the transgenic character was confirmed both by Southern blot analyses and by segregation ratios of the KANr trait of seeds obtained from selfed plants (i.e., resistant : sensitive = 3 : i) (Koornneef et al., 1986). As a control for kanamycin sensitivity , pollen of MsK plants and two commercial tomato cultivars (viz. Moneymaker and Delicates) were used. In vitro pollen germination. Plants were grown in glasshouses at 18-22 °C, under daylight conditions. Pollen was collected from flowers at anthesis and germinated in solidified media containing 3 mM H~B03, 1.7 mM Ca(NOi)z, 10% sucrose, 0.7% agar, with different concentrations of kanamycin sulphate (Sigma) at pH 5.8. Pollen was incubated for 2 h at 24 °C in the dark, and both stained and fixed with aceto-carmine (1%). Germination and pollen tube length were determined for separate samples of 5-7 complete replicates. Per flower, and for each concentration, germination of at least 200 pollen grains was scored. Since stress induces the extrusion of short pollen tubes (Stanley and Linskens, 1974), pollen was recorded as germinated only when the pollen tube length was twice the diameter of the pollen grain. Pollen tube length was measured using an ocular micrometer for 50 tubes per flower at each concentration. The results were expressed as the mean + standard deviation, significant differences among means (P < 0.05) were partitioned by an analysis of variance. C [o0~ o 1A 90 C • ~ 60 % ol  7o  6o  4,6 I f 30 ~ I i i i i i i I t i i I t t L o loo 200 400 8oo 16oo Concentration k~namycin (mgl -I) 1°6%~-'T~. I 1 B 4J g 06 0 g 66 66 ¢- 4 J  ~ 66 I Ill 4~0 3 4.1 C: tO0 ZOO 4:00 800 1600 O,I ,-4 Concentration kBnamycin (mEl -I) o o. Figs. 1 A and B. Effects of increasing KAN concentrations (mgl-l) on germination and tube growth of pollen from non-transformed MsK ( ¢ ) and transgenic ATW ( + ) plants. Pollen from ATW plants, transformed with C58CI and pollen from plants transformed with LBA 4404, behaved similar and are therefore combined. Germination percentages and tube lengths were determined for 5 (MsK) and 7 (ATW) complete replicates of pollen from different flowers. Results are expressed as mean + standard deviation. Fig. 1 A. Percentage of germination. Per flower, and for each concentration, germination of at least 200 pollen grains was scored. Fig. 1 B. Pollen tube lenghts expressed as percentages of the control value (0 mgl-i KAN). Per flower, and for each concentration, length of at least 50 pollen was scored. 335 C Ok . . . .  Fig. 2. ATW pollen, in vitro germinated at 0 mgl-i KAN (A), 400 mgl-i KAN (B) and 800 mgl-I KAN (C). Bar = 0.I mm. RESULTS The effects of increasing KAN concentrations on germination of pollen from MsK and ATW plants are presented in Fig. 1 A. Pollen germination of both genotypes was not significantly affected by the addition of KAN to the medium. Fig. 1 B shows the effects of the addition of KAN to the pollen germination medium on pollen tube length, a microscopic view of the results is presented in Fig. 2. After two hours of incubation, at 0 mgl-l, mean pollen tube length was 2.0 + 0.3 - 10-4 m. Inhibition of tube growth of pollen from non-transformed (KANs) MsK plants was evident at i00 mgl-i KAN, while at 400 mgl-i KAN, the tube length was about 50% of the value obtained at 0 mgl-l. In contrast, inhibition of pollen tube growth of transformed (KANr) ATW plants was manifest at 200 mgl-l, while the 50% value occured at about 800 mgl-l. Significant differences between pollen tube lengths of MsK and ATW plants were obtained at 200 mgl- 1 (P < 0.01) and 400 mgl-i (P < 0.05). No different effects of KAN were observed between pollen tube growth of ATW plants transformed with the pGV3850::l103neodim construct or with the pAGSlI2 plasmid. Pollen tube growth of the cultivars Moneymaker and Delicates was similarily affected by KAN as pollen of MsK plants. DISCUSSION Tomato pollen tube growth is inhibited by the addition of KAN to the germination medium. Initial effects of KAN on pollen of non-transformed MsK plants were already apparent at I00 mgl-l. Similar KAN concentrations have been reported to inhibit MsK callus formation and seedling growth (Koornneef et al., 1986). The effects on vegetative growth, however, became evident only after several weeks in culture, while effects on pollen tube development were manifest 2 hours after pollen germination. Toxicity of KAN is due to interference with the translation system of the 70S ribosomes as present in mitochondria and chloroplasts of eucaryotic cells (Franklin and Snow, 1975). Since pollen tubes lack chloroplasts, the effect of KAN might indicate that an active mitochondrial translation is important during tomato pollen tube growth. Similar effects on pollen tube growth of several other species were obtained by Hoekstra (1979), using chloramphenicol, another inhibitor of 70S ribosomal protein synthesis. KAN did not significantly affect tomato pollen germination. For this, at least two explanations are possible: A) the antibiotic reagent is only gradually absorbed during pollen germination, or, B) mitochondrial translation is not essential for tomato pollen germination. Pollen tube growth of plants containing the chimaeric resistance gene is less sensitive for KAN than pollen of non- transformed plants. Since ATW plants are heterozygous for the KANr trait, only half of the pollen population is expected to be resistant. Hence, the difference between pollen from ATW and MsK plants is even more evident considering the heterogenous character of the ATW pollen sample. 336 Resistance to aminoglycoside antibiotics such as KAN, is due to expression of the NPT II gene (Hain et al., 1985). During early tube growth in vitro, however, genes appear not to be transcribed (Knox, 1984). Probably, in pollen, mRNAs of the chimaeric gene are presynthesized concurrently with the other mRNAs during microsporogenesis, while KAN resistance is induced by gene translation during pollen tube elongation. The largest difference between pollen tube growth of ATW and MsK plants occured at 200- 400 mgl-i KAN. Here, tubes of ATW pollen are about 1.4 times longer than the pollen from MsK plants. Since the level of pollen resistance probably correlates with the amount of NPT present in the cells, the use of a stronger promoter may enhance selection efficiency. The gametophytic phase of a plant is characterized by large populations and haploid genomes. Expression of selection sensitive genes by the male haploid genome opens the possibility to make pollinations with pre-selected pollen. For example, the toxin from Drechslera maydis prevents germination of pollen from maize plants susceptible to the disease but not of pollen from resistant plants (Laughnan and Gabay, 1973), and, compounds present in Alternaria brassicicola culture filtrates inhibit brassica pollen germination (Hodgkin and MacDonald (1986). However, linkage of agronomic important traits with a newly introduced marker gene, would be of general interest for the application of pollen selection methods in plant breeding programmes. ACKNOWLEDGMENTS We thank J. Jansen for helping with the statistical analyses, and J.J.M. Dons and A.G. Stephenson for critically reading the manuscript. REFERENCES Czernilofsky AP, Hain R, Herrera-Estrella L, Lorz H, Goyvaerts E, Baker B, Schell J (1986) DNA 5:103-113 Franklin TJ, Snow GA (1975) Biochemistry of antimicrobial action, Chapman and Hall Ltd., London, pp 224 Hain R, Stabel P, Czernilofsky AP, Steinbiss HH, Herrera-Estrella L, Schell J (1985) Mol Gen Genet 199:166-168 Hodgkin T, MacDonald MV (1986) New Phytol 104:631-636 Hoekstra FA (1979) Planta 145:25-36 Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT (1985) Science 227: 1229-1232 Koornneef M, Hanhart C, Jongsma M, Toma I, Weide R, Zabel P, Hille J (1986) Plant Sci 45:201-208 Knox RB (1984) In: HF Linskens, Heslop- Harrison J (eds), Cellular interaction. Encycl Plant Physiol 17:508-608 Laughnan JR, Gabay SJ (1973) Crop Sc 13: 681-684 Mascarenhas JP, Mermelstein J (1981) Acta Soc Bot Pol 50:13-20 Mascarenhas NT, Bashe D, Eisenberg A, Willing RP, Xiao CM, Mascarenhas JP (1984) Theor Appl Genet 68:323-326 Sari Gorla M, Frova C, Binelli G, Ottaviano E (1986) Theor Appl Genet 72:42-47 Searcy KB, Mulcahy DL (1985) Amer J Bot 72: 1695-1699 Stanley RG, Linskens HF (1974) Pollen: biology, biochemistry, management. Springer-Verlag, Berlin, 329-334 Tanksley SD, Zamir D, Rick CM (1981) Science 213:453..-455 Tupy J (1982) Biol Plant 24:331-340 Van den Elzen P, Lee KY, Townsend J, Bedbrook J (1985) Plant Molec Biol 5: 149-154 Willing RP, Mascarenhas JP (1984) Plant Pbysiol 75: 865-868 Zambryski P, Joos H, Genetello C, Van Montagu M, Schell J (1983) Embo J 2: 2143-2150 Zamir D, Tanksley SD, Jones RA (1982) Genetics i01:129-137 

NARCIS 

Crossref

University of GroningenKanamycin resistance during in vitro development of pollen from transgenic tomato plantsBino, R.J.; Hille, J.; Franken, J.Published in:Plant Cell ReportsDOI:10.1007/BF00269553IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1987Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bino, R. J., Hille, J., & Franken, J. (1987). Kanamycin resistance during in vitro development of pollen fromtransgenic tomato plants. Plant Cell Reports, 6(5). https://doi.org/10.1007/BF00269553CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 24-12-2025Plant Cell Reports (1987) 6:333-336 Plant Cell Reports © Springer-Verlag 1987 Kanamycin resistance during in vitro development of pollen from transgenic tomato plants R.J.  Bino ~, J. HiUe 2. , ,  and J. Franken 1 Institute for Horticultural Plant Breeding (IV'I), P.O.B. 16, 6700 AA Wageningen, The Netherlands 2 Agricultural University, Department of Molecular Biology, De Dreijen 11, 6703 BC Wageningen, The Netherlands Received May 26, 1987 - Communicated by I. Potrykus ABSTRACT Effects of kanamycin on pollen germination and tube growth of pollen from non- transformed plants and from transgenic tomato plants containing a chimaeric kanamycin resistance gene were determined. Germination of pollen was not affected by the addition of kanamycin to the medium in both genotypes. Kanamycin,however, severely affected tube growth of pollen from non- transformed plants, while pollen from plants containing the chimaeric gene were less sensitive and produced significantly longer tubes at kanamycin concentrations between 200-400 mgl-l. Apparently, this resistance for kanamycin correlates with the expression of the chimaeric gene during male gametophytic development. ABBREVIATIONS ATW, Agrobacterium Tomato Wageningen; KAN, Kanamycin; KANr, Kanamycin resistant; KANs, Kanamycin sensitive; mRNA, messenger RNA; NPT, Neomycin phosphotransferase. INTRODUCTION Male gametophytic functioning is involved with microsporogenesis, pollen germination, pollen tube elongation, and the transmission of gametes to the embryo sac. During these processes, the male haploid genome plays an important role. Willing and Mascarenhas (1984) estimated that mRNAs present in mature pollen grains of Tradescantia paludosa are the products of approximately 20,000 different genes. Large numbers of these mRNAs are translated into proteins during pollen germination and early pollen tube elongation (Mascarenhas and Mermelstein, 1981). similar results were obtained using the pollen of Zea mays (Mascarenhas et al., 1984) and Nicotiana tabacum (Tupy, 1982). RNA synthesis and protein synthesis are independent during the early stages of in vitro pollen tube development, as was demonstrated by the use of specific inhibitors (see review Knox, 1984). These inhibition studies showed that RNA transcription in most plant species is not essential for germination and early pollen tube growth, whereas both processes are severely affected by translation inhibitors. Most genes transcribed and translated during pollen development are also expressed in vegetative parts of the plant. Using RNA saturation hybridizations, Willing and Mascarenhas (1984) reported the expression of at least 64% of the pollen sequences in shoot tissues, and a maximum of about 60% of shoot mRNAs was also present in pollen of Tradescantia paludosa. Based on isozyme profiles, Tanksley et al. (19SI) ahd Sari Gorla et al. (1986), found in Lycopersicon esculentum and Zea mays a comparable percentage of genetic overlap between the sporophytic and gametophytic phase of a plant. The parallel expression of genes in the sporophyte and the gametophyte suggests that a selection pressure applied to the pollen is likely to alter the expression of genes in the sporophyte. Indeed, sporophytic gene expression is modified by chilling stress during pollen development in tomato species (Zamir et al., 1982) and after heavy metal stress during in vivo pollen germination in Silene dioica and Mimulis guttatus (Searcy and Mulcahy, 1985). The development of Agrobacterium tumefaciens derived gene vectors have enabled the transfer of new genetic information into the cells of various plant species (Zambryski et al., 1983). The use of selectable marker genes which are constitutively expressed allows a convenient selection of transformed tissues (Horsch, 1985). Such an approach was also used by Koornneef et al. (1986), who introduced a chimaeric kanamycin resistance gene into tomato. The present study is undertaken to determine if this marker gene is also expressed in the gametophytic phase of the tomato plants and can be used for pollen selection. * Present address: Vrije Universiteit, Department of Genetics, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands Offprint requests to: R. J. Bino 3 3 4  MATERIALS AND METHODS Plant material. Tomato plants used in this study derived from material developed by Koornneef et al. (1986) and are indicated as MsK. This genotype is selected for outstanding regeneration capacity and originally derived from the cross Lycopersicon peruvianum x L. esculentum. Transformants were obtained following leaf disc transformation with either the Agrobacterium tumefaciens strain C58Cl(pGV3850::l103neodim) (czernilofsky et al., 1986) or the strain LBA 4404 (pAL4404, pAGSII2) (Van den Elzen et al., 1985) as described by Koornneef et al. (1986). In between the T-region border sequences, both strains contain the NPT II gene from Tn5 under transcriptional control of the regulatory elements of the nopaline gene from A. tumefaciens. Expression of the chimaeric gene confers resistance of transformed plant cells to kanamycin (Hain et al., 1985). Transformed plants were named ATW and the transgenic character was confirmed both by Southern blot analyses and by segregation ratios of the KANr trait of seeds obtained from selfed plants (i.e., resistant : sensitive = 3 : i) (Koornneef et al., 1986). As a control for kanamycin sensitivity , pollen of MsK plants and two commercial tomato cultivars (viz. Moneymaker and Delicates) were used. In vitro pollen germination. Plants were grown in glasshouses at 18-22 °C, under daylight conditions. Pollen was collected from flowers at anthesis and germinated in solidified media containing 3 mM H~B03, 1.7 mM Ca(NOi)z, 10% sucrose, 0.7% agar, with different concentrations of kanamycin sulphate (Sigma) at pH 5.8. Pollen was incubated for 2 h at 24 °C in the dark, and both stained and fixed with aceto-carmine (1%). Germination and pollen tube length were determined for separate samples of 5-7 complete replicates. Per flower, and for each concentration, germination of at least 200 pollen grains was scored. Since stress induces the extrusion of short pollen tubes (Stanley and Linskens, 1974), pollen was recorded as germinated only when the pollen tube length was twice the diameter of the pollen grain. Pollen tube length was measured using an ocular micrometer for 50 tubes per flower at each concentration. The results were expressed as the mean + standard deviation, significant differences among means (P < 0.05) were partitioned by an analysis of variance. C [o0~ o 1A 90 C • ~ 60 % ol  7o  6o  4,6 I f 30 ~ I i i i i i i I t i i I t t L o loo 200 400 8oo 16oo Concentration k~namycin (mgl -I) 1°6%~-'T~. I 1 B 4J g 06 0 g 66 66 ¢- 4 J  ~ 66 I Ill 4~0 3 4.1 C: tO0 ZOO 4:00 800 1600 O,I ,-4 Concentration kBnamycin (mEl -I) o o. Figs. 1 A and B. Effects of increasing KAN concentrations (mgl-l) on germination and tube growth of pollen from non-transformed MsK ( ¢ ) and transgenic ATW ( + ) plants. Pollen from ATW plants, transformed with C58CI and pollen from plants transformed with LBA 4404, behaved similar and are therefore combined. Germination percentages and tube lengths were determined for 5 (MsK) and 7 (ATW) complete replicates of pollen from different flowers. Results are expressed as mean + standard deviation. Fig. 1 A. Percentage of germination. Per flower, and for each concentration, germination of at least 200 pollen grains was scored. Fig. 1 B. Pollen tube lenghts expressed as percentages of the control value (0 mgl-i KAN). Per flower, and for each concentration, length of at least 50 pollen was scored. 335 C Ok . . . .  Fig. 2. ATW pollen, in vitro germinated at 0 mgl-i KAN (A), 400 mgl-i KAN (B) and 800 mgl-I KAN (C). Bar = 0.I mm. RESULTS The effects of increasing KAN concentrations on germination of pollen from MsK and ATW plants are presented in Fig. 1 A. Pollen germination of both genotypes was not significantly affected by the addition of KAN to the medium. Fig. 1 B shows the effects of the addition of KAN to the pollen germination medium on pollen tube length, a microscopic view of the results is presented in Fig. 2. After two hours of incubation, at 0 mgl-l, mean pollen tube length was 2.0 + 0.3 - 10-4 m. Inhibition of tube growth of pollen from non-transformed (KANs) MsK plants was evident at i00 mgl-i KAN, while at 400 mgl-i KAN, the tube length was about 50% of the value obtained at 0 mgl-l. In contrast, inhibition of pollen tube growth of transformed (KANr) ATW plants was manifest at 200 mgl-l, while the 50% value occured at about 800 mgl-l. Significant differences between pollen tube lengths of MsK and ATW plants were obtained at 200 mgl- 1 (P < 0.01) and 400 mgl-i (P < 0.05). No different effects of KAN were observed between pollen tube growth of ATW plants transformed with the pGV3850::l103neodim construct or with the pAGSlI2 plasmid. Pollen tube growth of the cultivars Moneymaker and Delicates was similarily affected by KAN as pollen of MsK plants. DISCUSSION Tomato pollen tube growth is inhibited by the addition of KAN to the germination medium. Initial effects of KAN on pollen of non-transformed MsK plants were already apparent at I00 mgl-l. Similar KAN concentrations have been reported to inhibit MsK callus formation and seedling growth (Koornneef et al., 1986). The effects on vegetative growth, however, became evident only after several weeks in culture, while effects on pollen tube development were manifest 2 hours after pollen germination. Toxicity of KAN is due to interference with the translation system of the 70S ribosomes as present in mitochondria and chloroplasts of eucaryotic cells (Franklin and Snow, 1975). Since pollen tubes lack chloroplasts, the effect of KAN might indicate that an active mitochondrial translation is important during tomato pollen tube growth. Similar effects on pollen tube growth of several other species were obtained by Hoekstra (1979), using chloramphenicol, another inhibitor of 70S ribosomal protein synthesis. KAN did not significantly affect tomato pollen germination. For this, at least two explanations are possible: A) the antibiotic reagent is only gradually absorbed during pollen germination, or, B) mitochondrial translation is not essential for tomato pollen germination. Pollen tube growth of plants containing the chimaeric resistance gene is less sensitive for KAN than pollen of non- transformed plants. Since ATW plants are heterozygous for the KANr trait, only half of the pollen population is expected to be resistant. Hence, the difference between pollen from ATW and MsK plants is even more evident considering the heterogenous character of the ATW pollen sample. 336 Resistance to aminoglycoside antibiotics such as KAN, is due to expression of the NPT II gene (Hain et al., 1985). During early tube growth in vitro, however, genes appear not to be transcribed (Knox, 1984). Probably, in pollen, mRNAs of the chimaeric gene are presynthesized concurrently with the other mRNAs during microsporogenesis, while KAN resistance is induced by gene translation during pollen tube elongation. The largest difference between pollen tube growth of ATW and MsK plants occured at 200- 400 mgl-i KAN. Here, tubes of ATW pollen are about 1.4 times longer than the pollen from MsK plants. Since the level of pollen resistance probably correlates with the amount of NPT present in the cells, the use of a stronger promoter may enhance selection efficiency. The gametophytic phase of a plant is characterized by large populations and haploid genomes. Expression of selection sensitive genes by the male haploid genome opens the possibility to make pollinations with pre-selected pollen. For example, the toxin from Drechslera maydis prevents germination of pollen from maize plants susceptible to the disease but not of pollen from resistant plants (Laughnan and Gabay, 1973), and, compounds present in Alternaria brassicicola culture filtrates inhibit brassica pollen germination (Hodgkin and MacDonald (1986). However, linkage of agronomic important traits with a newly introduced marker gene, would be of general interest for the application of pollen selection methods in plant breeding programmes. ACKNOWLEDGMENTS We thank J. Jansen for helping with the statistical analyses, and J.J.M. Dons and A.G. Stephenson for critically reading the manuscript. REFERENCES Czernilofsky AP, Hain R, Herrera-Estrella L, Lorz H, Goyvaerts E, Baker B, Schell J (1986) DNA 5:103-113 Franklin TJ, Snow GA (1975) Biochemistry of antimicrobial action, Chapman and Hall Ltd., London, pp 224 Hain R, Stabel P, Czernilofsky AP, Steinbiss HH, Herrera-Estrella L, Schell J (1985) Mol Gen Genet 199:166-168 Hodgkin T, MacDonald MV (1986) New Phytol 104:631-636 Hoekstra FA (1979) Planta 145:25-36 Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT (1985) Science 227: 1229-1232 Koornneef M, Hanhart C, Jongsma M, Toma I, Weide R, Zabel P, Hille J (1986) Plant Sci 45:201-208 Knox RB (1984) In: HF Linskens, Heslop- Harrison J (eds), Cellular interaction. Encycl Plant Physiol 17:508-608 Laughnan JR, Gabay SJ (1973) Crop Sc 13: 681-684 Mascarenhas JP, Mermelstein J (1981) Acta Soc Bot Pol 50:13-20 Mascarenhas NT, Bashe D, Eisenberg A, Willing RP, Xiao CM, Mascarenhas JP (1984) Theor Appl Genet 68:323-326 Sari Gorla M, Frova C, Binelli G, Ottaviano E (1986) Theor Appl Genet 72:42-47 Searcy KB, Mulcahy DL (1985) Amer J Bot 72: 1695-1699 Stanley RG, Linskens HF (1974) Pollen: biology, biochemistry, management. Springer-Verlag, Berlin, 329-334 Tanksley SD, Zamir D, Rick CM (1981) Science 213:453..-455 Tupy J (1982) Biol Plant 24:331-340 Van den Elzen P, Lee KY, Townsend J, Bedbrook J (1985) Plant Molec Biol 5: 149-154 Willing RP, Mascarenhas JP (1984) Plant Pbysiol 75: 865-868 Zambryski P, Joos H, Genetello C, Van Montagu M, Schell J (1983) Embo J 2: 2143-2150 Zamir D, Tanksley SD, Jones RA (1982) Genetics i01:129-137 

https://pure.rug.nl/ws/files/14695317/1987PlantCellRepBino.pdf

Kanamycin resistance during in vitro development of pollen from transgenic tomato plants

Abstract

Similar works

Full text

Available Versions

University of Groningen

ARTS repository - University of Groningen

ARTS repository - University of Groningen

University of Groningen

University of Groningen Digital Archive

University of Groningen Research Database

University of Groningen

NARCIS

Crossref

ARTS repository - University of Groningen