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Plant Molecular Biology Reporter 21: 31–41, March 2003 © 2003 International Society for Plant Molecular Biology. Printed in Canada.

Commentary

In Vitro Pollen Germination and Transient Transformation of Zea mays and Other Plant Species DANIELA N. SCHREIBER and THOMAS DRESSELHAUS* Biocenter Klein-Flottbek, AMP II, University of Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany Abstract. To study pollen-specific In vitro pollen gene germination expression,and fast and transformation convenient methods Schreiber involving and in vitro pollen germination and bombardment with promoter deletion constructs are Dresselhaus needed. Unfortunately, because of variation of pollen germability and tube growth, conducting these experiments is often unsatisfying for many plant species, including maize, especially when pollen is collected at different times of the day or season. We have overcome these problems by defining a novel medium (PGM) that guarantees germination efficiencies of more than 90% for maize pollen from at least 7 genotypes (A188, AC 3572 C, B73, H99, Hi-II, Q2, Tx232). This medium is also suitable to germinate pollen of other monocot species, such as Pennisetum americanum and Tradescantia species, and dicot species, such as Arabidopsis thaliana, Arachis hypogaea, Columnea oesterdiana, Nicotiana tabacum, Phaseolus vulgaris, Pisum sativum, Solanum lycopersicum, Solanum tuberosum, and Vicia faba. On average, reproducible germination rates ranging from 50-100% were observed with all plant species tested. In addition, we report a transient transformation assay using the luciferase (Luc) reporter gene. Biolistic parameters were defined to obtain reproducible Luc activity measurements after bombarding thick-walled pollen, such as maize pollen. For comparison, samples of germinated maize and tobacco pollen were bombarded with the reporter gene under control of the constitutive ubiquitinand pollen-specific ZmMADS2 maize promoters. The important parameters necessary to apply both in vitro pollen germination and transient transformation for a large range of plant species are discussed. Key words: Arabidopsis, biolistic transformation, germination, Gus, Luc, maize, pollen tube growth, tobacco Abbreviations: PGM, pollen germination medium; RT, room temperature; ZmMADS2, Zea mays MADS-box gene 2.

Introduction Male gametophyte development in higher plants is a complex process regulated by a series of coordinated gene expression events (Bedinger and Edgerton, 1990). After meiosis, pollen mother cells (PMC) produce a tetrad of microspores. These *

Author for correspondence. e-mail: [email protected]; fax: +49-40-42816-229; ph: +49-40-42816-312.

J:\PMBR\Pmbr 2101 mar\R03-011.vp Monday, April 07, 2003 10:11:46 AM


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Schreiber and Dresselhaus

microspores mature after an asymmetric division called microspore mitosis to form pollen grains consisting of a large vegetative cell enclosing a small generative cell inside the cytoplasm. In about 70% of plant species, dinucleate pollen is released from the anthers, and the second mitotic division of the generative cell takes place inside the pollen tube to generate 2 sperm cells (McCormick, 1993). Zea mays sheds trinucleate pollen (Bedinger, 1992). The vegetative cell’s main function is to form a pollen tube that grows toward the female gametophyte (embryo sac) to release the 2 sperm cells needed for double fertilization. Pollen- and pollen tube–specific promoters can be used as tools to analyse and modify these complex processes. To examine promoter elements responsible for tissue- or developmental stage–specific gene expression, transient transformation assays with promoter deletion constructs are fast and convenient. Stable germination rates are required to routinely perform pollen transformation experiments. In Z. mays, many problems have been reported in defining conditions under which stable germination rates occur (Hamilton et al., 1992; Heuer, 1999). Because optimal conditions could not be defined, maize pollen promoters have been studied in heterologous systems such as Tradescantia (Hamilton et al., 1992) or Arabidopsis (Hamilton et al., 1998). We report a novel in vitro pollen germination medium that can be used as a standard medium for germination and transformation of maize pollen. This medium also supports reproducible germination rates of other species, significantly higher than previously used media. Biolistic transformation of pollen compared to that of vegetative tissues has been shown to resemble pollen-specific expression in planta. Transgenic plants containing promoter deletions fused to reporter genes support this finding (Twell et al., 1989; Hamilton et al., 1992, 1998; Eyal et al., 1995; Bate and Twell, 1998). All pollen-specific promoters investigated are also active in pollen of other species, such as tobacco, regardless of whether they are monocots or dicots (Hamilton et al., 1998). A cis-element defined as a general pollen box could not be identified. This finding led researchers to believe it is sufficient to perform transient assays with promoter constructs of all plants in those species, like Tradescantia or Nicotiana, which germinate in vitro and are easily transformed because of a thin pollen wall. On the other hand, differences in activity of monocot and dicot promoters are reported, as well as altered expression patterns in species of different genetic relatedness (Xu et al., 1992; Gallusci et al., 1994; Miyoshi et al., 1995). Custers et al. (1997) reported a timeshift in expression of an early pollen gene of Brassica napus in tobacco. These findings make it desirable to study the function of a given promoter in the species of origin. In addition, large collections of male sterile (MS) mutants are available for maize and other species (Neuffer et al., 1997). For the study of MS genes and their corresponding promoters, fast and reproducible assays are necessary to avoid the time- and space-consuming examination of stably transformed plants, especially when high numbers of deletion constructs need to be studied to identify pollen-specific cis-elements. Here we report both a medium for reproducible germination of pollen (PGM) from maize and other plant species and a transient transformation assay. To our knowledge, this is the first report of a pollen transformation system for Z. mays that leads to reproducible results.



In vitro pollen germination and transformation

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Materials and Methods Pollen collection Pollen of Arabidopsis thaliana, Arachis hypogaea, Columnea oesterdiana, Nicotiana tabacum, Pennisetum americanum, Solanum lycopersicum, Solanum tuberosum, Tradescantia species, and Z. mays was collected from nonstressed greenhouse plants. Pollen of Phaseolus vulgaris, Pisum sativum, and Vicia faba was collected from the botanical garden in Hamburg. To study germination efficiencies, pollen was collected in paper bags and spread on PGM plates with 0.3% noble agar. Pollen grains were picked with a brush and shaken off to spread equally over the plate. For transformation, N. tabacum pollen was collected directly from mature flowers into microfuge tubes and mixed with 2 mL of PGM. We pipetted 200 µL onto PGM plates and left them at room temperature (RT) for 45-60 min. A relatively even spreading of maize pollen was achieved by placing Petri dishes (3 cm in diameter) containing PGM and 0.3% noble agar side by side, 30 cm under tassels that were shaken. This method gave reproducible results during transient transformation studies. For Z. mays, it was important to use anthers that had not been in contact with pest control. Pollen germination medium (PGM) 2 X PGM contained the following:

• • • • •

10% sucrose (Roth) 0.005% H3BO3 (Sigma-Aldrich) 10 mM CaCl2 (Sima-Aldrich) 0.05 mM KH2PO4 (Merck) 6% PEG 4000 (Merck-Schuchardt)

After adding all components, PGM was heated to 70°C for 10 min on a stirring heater and sterile filtrated. For preparation of germination plates, an equal volume of autoclaved 0.6% noble agar (Agar Agar Molecular biology grade, AppliChem) to a final concentration of 0.3% was added and poured into 3-cm Petri dishes. It is important to use agar instead of other gelling agents such as agarose or phytagel. Biolistic transformation To determine optimal conditions for biolistic transformation of pollen, bombardment with pUbi::Luc was performed using a particle gun (PDS 1000/He gun, BioRad) at 1350, 1550, 1800, and 2000 psi. Distances were 2 cm between rupture disk and macrocarrier, 1.5 cm between macrocarrier and stopping screen, and 5.5 cm from stopping screen to surface of the plate. Best results were obtained with 1800 and 2000 psi. Reduction of the distance between rupture disk and stopping screen to 1.8 cm and bombardment with 1500 psi gave reproducible results resembling transformation at higher pressures. Transient transformation studies with pUbi::Luc, pZmMADS2::Luc, promless::Luc, pUbi::Gus, and pZmMADS2:: Gus were performed at 1500 psi under optimized conditions. Gold particles (0.2-2 µm, Heraeus, Karlsruhe) were covered with 500 ng plasmid DNA per



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bombardment and plate. Before transformation, pollen was left for 10 (maize) or 60 min (tobacco) on PGM plates until the first pollen tubes were visible under a microscope. Transformation of young leaves was performed at 1350 psi. Detection of luciferase and Gus activity Detection of luciferase activity was performed with the Luciferase Assay System (Promega). Pollen was carefully scraped off PGM plates with a spatula and transferred to ice-cold microfuge tubes 16 h after germination at RT. Samples were homogenized with a micropistil (Eppendorf) for about 1 min, combined with 100 µL of cell culture lysis reagent (Promega), and vortexed. After 1 min of centrifugation at 12,000 g, supernatants were used for luciferase measurements using a Lumometer (Berthold). Histochemical localization of Gus activity was performed using 1 mg/mL X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) as described by Jefferson et al. (1987) in staining buffer containing 0.5 mM potassium ferri/ferrocyanide after 16 h of germination. Generation of constructs for transformation PUbi::Luc contains a modified ubiquitin promoter of maize, the firefly luciferase coding region, and the nopaline synthase terminator (Christensen and Quial, 1996). This construct was used as a positive control for maize and as a negative control for tobacco in transient transformation assays. PZmMADS2::Luc was constructed by inserting a fragment containing 1804 bp upstream of the ZmMADS2 AUG START codon (D. Schreiber and T. Dresselhaus, unpublished results, GenBank accession no. AY227363) into a promoterless luciferase vector (de Wet et al., 1987). This vector was slightly modified by inserting an Nhe I–adapter in front of the luciferase coding sequence (R. Brettschneider, unpublished results). A 1758-bp fragment of genomic DNA was generated by genome walking with primer tnorf2 (5’-TAA GGA GCG AGA GGT TGT GGT TGT GG-3’) at the 3’ end in a Pvu II “Genome Walker” library (Clontech) of Z. mays. This fragment contains 1502 bp upstream of the transcription start site of the ZmMADS2 gene and 256 bp of the ZmMADS2 5’UTR. This fragment was ligated into the pCR–Blunt II TOPO vector (Invitrogen), restricted with Hind III and Nhe I to cut off the adapter primer and the 5’UTR of ZmMADS2, and inserted into the promoterless luciferase vector. We isolated 255 bp of the ZmMADS2 5’-UTR with Nhe I from a cDNA clone of a Z. mays mature pollen library (Heuer et al., 2000) and integrated in correct orientation. The resulting plasmid, pDNS-5b, was used as a template for amplifying the complete ZmMADS2 promoter and the complete 5’UTR of ZmMADS2 ranging from position -1502 to +291 relative to the transcription start site. For construction of pZmMADS2::Luc, a Hind III restriction site was introduced using primer Prom1 (5’-AAG CTT GTA AAG ACC TCG ACC GGA A-3’) at the 5’ end. The opposite primer LUC2 (5’-GCC TTA TGC AGT TGC TCT CC-3’) was directed inside the luciferase coding sequence behind an Xba I site. PCR products were restricted with Xba I and Hind III and ligated into the corresponding linearized vector. PZmMADS2::Gus was generated after amplifying the ZmMADS2 promoter with 5’UTR from pZmMADS2::Luc with primers Prom1 and Nco-rev (5’-CTT TCC




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CCT CCC CAT GGA TC-3’), introducing an Nco I restriction site around the AUG START codon. The Ubiquitin promoter was exised from pUbi::Gus (Christensen et al., 1996) with Hind III and Nco I and the ZmMADS2 promoterfragment with 5’UTR inserted instead. All constructs were fully sequenced before usage.

Results and Discussion Optimization of in vitro pollen germination Many efforts have been made to analyse pollen-specific promoters from different plant species and to determine cis-elements responsible for pollen specificity. Transient transformation of in vitro–germinated pollen with marker genes under the control of promoter deletion constructs is a fast method to study many constructs in one experimental series. For Z. mays, in vitro pollen germination has been reported to be the most critical point because of high variations of pollen germability (Hamilton et al., 1992, 1998; Heuer et al., 2000). We examined maize pollen tube growth on different germination media described in the literature (Hamilton et al., 1992; Walden, 1993; Torres et al., 1995). None of the protocols gave stable and reproducible germination rates, even under controlled greenhouse conditions. Walden (1993) described a principle strategy for maize pollen germination with the need to alter components of the medium to optimize it for specific genotypes. Walden (1993) reported germination rates above 90% when pollen was germinated for 30 min on plates containing 50 mM sucrose (nearly 12%), 0.01% boric acid, 0.03% CaCl2, and 0.7% noble agar for a number of inbred lines like Seneca 60, Oh43, and others. Torres et al. (1995) used a slightly modified medium containing 0.2% DMSO and described that most germination took place within 30 min for the line B73. Using these media, Heuer (1999) reported highly variable pollen germination rates with the inbred line A188. Often, pollen did not germinate at all. Neither collection of pollen at different times of the day or season nor alteration of humidity or temperature led to stable germination rates. For a detailed analysis of the pollen-specific Zm13 promoter from maize, Hamilton et al. (1992) established a transient transformation protocol with Tradescantia paludosa as a model system because analyses of maize pollen was unsatisfying. The germination medium used for Tradescantia and Z. mays, initially described by Mascarenhas (1966), contained 10% sucrose, 0.01% boric acid, 0.1% yeast extract, 10 mM CaCl2, and 0.05 mM KH2PO4. Using this pollen germination medium as a basis, we altered concentrations of all components until more than 90% of maize pollen showed long and stable tubes 16 h after germination. Supposing that pollen tube bursting was caused by low osmotic pressure in the medium, we examined sucrose contents ranging from 6-20% in 2% steps. On average, germination rates of 50% were obtained with 16% sucrose. A closer examination in 0.5% steps confirmed this result. In a second step, concentrations of H3BO3 were investigated analogously in a range from 0.02-0.0025%. We found a concentration of 0.005% boron to result in pollen germination rates of more than 90%. Concentrations of CaCl2 below 10 mM reduced germination rates to zero. In some experiments, a high calcium concentration of



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about 100 mM resulted in pollen tube branching. Alteration of KH2PO4 concentration and yeast extract showed no remarkable effects. About 1 h after germination on the improved medium, pollen tubes burst on plates containing 1% noble agar. A content of 0.5% noble agar led to accumulation of liquid on the surface, and pollen burst immediately. To stabilize the medium, we substituted a portion of sucrose with PEG 4000 and found an optimal content of 10% sucrose and 6% PEG 4000. Substitution of sucrose with PEG 4000 was examined in 1% steps from 1-10% PEG 4000 (corresponding to 6%15% sucrose). Application of PEG with higher or lower molecular weights did not result in satisfying germination rates. With this new medium, PGM, using plates with 0.3% noble agar was possible. Fresh maize pollen or pollen dried for up to 2 d at RT germinated well on PGM plates or in liquid PGM medium; long and stable tubes were visible even 16 h after germination was initiated (Figure 1a). Germination rates of 90-100% were observed for maize pollen of all tested genotypes in liquid and on solid medium (Table 1). Although germination rates were stable when pollen was collected at different times of the day or season, transient transformation showed best results when pollen was collected in the afternoon from 2-5 PM and applied directly on PGM plates. On average, water content of pollen collected in the afternoon is lower, cytoplasm appeared thicker, and, thus, fewer pollen tubes were damaged by particle bombardment. Pollen of different monocot and dicot plant species was tested to investigate the general applicability of PGM. Germination rates of 70-100% were obtained for A. thaliana, different species of Tradescantia (T. paludosa, T. Spathacea, T. zebrina), C. oesterdiana, N. tabacum, and members of the Fabaceae, such as A. hypogaea, P. sativum, and V. faba (Table 1 and Figure 1). Pollen of P. americanum, P. vulgaris, S. lycopersicum, and S. tuberosum showed 50-80% germination rates (Table 1). A difference in germination rates was not observed, even when pollen was collected at different times of the day. Although PGM is not a general germination medium for all plant species, it enables the study of pollen from many monocot and dicot species of different families under equal conditions. For successful application of PGM to plant species that germinated at unsatisfying rates, such as lily, rice, and wheat, we recommend modifying the PEG and boric acid content as described above. Pollen that burst on PGM (e.g., wheat) may be stabilized by a higher PEG content. Germination rates of stable pollen (e.g., lily, rice) should be stimulated by higher concentrations of H3BO3. Transient transformation of maize and tobacco pollen using the luciferase reporter gene Biolistic transformation of maize pollen was found to be highly dependent on helium pressure. Best results were obtained after transformation at 1800-2000 psi. The average activity of pUbi::Luc was 132 times higher than the control (promoterless construct, Figure 2). Young leaves were bombarded for comparison. In leaves, activity of pUbi::Luc at 1350 psi was 147 times higher than the control. To establish a standard protocol for transient transformation of maize pollen, reducing the helium pressure was necessary because 1800-2000 psi can only be reached with a full helium gas bottle. After conducting a few experiments, helium pressure decreased in the bottle to generate 1550 psi at the rupture disk.




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Figure 1. Germinated pollen on PGM plates showing germination rates of about 100%. (A) Zea mays, (B) Tradescantia paludosa, (C) Nicotiana tabacum, (D) Columnea oesterdiana. Bars = 50 µm.

Table 1. In vitro germination rates with pollen of different plant species. Germination frequencies were compared between PGM described in this paper and a germination medium for Tradescantia species originally described by Mascarenhas (1966). More than 10 experiments were conducted with pollen of species listed. Species

PGM1

Mascarenhas2

Arachis hypogaea Columnea oesterdiana Nicotiana tabacum Pennisetum americanum Phaseolus vulgaris Pisum sativum Solanum lycopersicum Solanum tuberosum Tradescantia paludosa Vicia faba Zea mays

80 95 80 40

– – – –

100% 100% 100% 50%

10 95 80 20

50 75 50 50 80 90 80

– – – – – – –

60% 100% 80% 80% 100% 100% 100%

30 – 40% 70 – 80% 30 – 60% 30 – 60% 80 – 100% 50 – 70% 0 – 80%

1PGM

= pollen germination medium described in this paper. after Mascarenhas (1966).

2Medium


– – – –

50% 100% 100% 30%


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Figure 2. Luciferase activity after particle bombardment. PUbi::Luc, pZmMADS2::Luc, and a promoterless construct were bombarded in maize and tobacco pollen as well as young leaves of maize. Relative percentage activities compared to highest expressed constructs are shown. The ZmMADS2 promoter of Zea mays is exclusively active in pollen. The maize ubiquitin promoter is not active in tobacco. Measured Luc activity in maize pollen was about 30 times lower than in tobacco pollen.

Bombarding maize pollen at 1550 psi was not sufficient to obtain significant Luc activity. The protocol was modified by reducing the distance between rupture disk and stopping screen. With this modification, luciferase activity under control of the ubiquitin promoter was at 1550 psi, 66 times higher than that of the control. Transformation at 1350 psi led to unreproducible results. To standardize Luc activity in different transformation events, an internal control with pUbi::Gus was performed. Determination of Gus activity in chemoluminescent assays was possible, but results did not always correlate with Luc activity (data not shown). In comparison to Gus, the luciferase assay is extremely sensitive (Ow et al., 1986; de Wet et al., 1987), and light production by luciferase has the highest quantum efficiency of any known chemoluminescent reaction (Seliger and McElroy, 1960). Only a few bombarded pollen tubes expressed Gus (Figures 3a, c), which is not sufficient because a relatively high activity is needed for reproducible measurements. Therefore, Gus expression in maize and tobacco pollen was compared by staining with X-Gluc. After transforming the Gus marker gene under control of the pollen-specific ZmMADS2 promoter, an intense blue staining of individual pollen tubes was observed in tobacco, but only slight signals were visible in maize pollen tubes (Figures 3a, c). Gus activity directed by the maize ubiquitin promoter was mainly observed in pollen grains of Z. mays, even when most of the cytoplasm of the vegetative cell was outside of the grain in the pollen tube (Figure 3b). To prove pollen specificity of the ZmMADS2 promoter, transformation of maize and tobacco pollen and young leaves was performed with a pZmMADS2:: Luc, pUbi::Luc, and a promoterless construct at the above defined parameters. To ensure that the same amount of pollen was transformed in each bombardment, maize pollen was spread in PGM on plates to reveal an equal distribution of pollen grains. In later experiments, pollen was collected directly on PGM plates, as described. In each assay, 4-6 plates were bombarded with each of the constructs. Luc activity directed by the ubiquitin promoter was set to 100% in each




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Figure 3. Histochemical localization of Gus expression in transiently transformed pollen. (A) Maize pollen showing weak signals in the tip of the pollen tube after bombardment with pZmMADS2::Gus. Inset: untransformed maize pollen. (B) Maize pollen after bombardment with pUbi::Gus showing weak signals both inside the pollen tube and pollen grain. (C) Tobacco pollen after bombardment with pZmMADS2::Gus. Intense staining of a couple of pollen grains and pollen tubes is visible. Bars = 50 µm.

experimental series to compare the activity of all constructs used. Activity was about the same in all experiments (Figure 2). Overall expression was higher when pollen was applied directly from tassels to the plates. Luciferase activity using pZmMADS2::Luc was 39% in maize pollen–compared with pUbi::Luc and 0.3% in Z. mays leaves. Background Luc activity directed by the promoterless construct was also about 0.3%, proving the pollen specificity of the ZmMADS2 promoter. In tobacco pollen, the ubiquitin promoter was not active. Luc activity directed by the maize ubiquitin promoter and that without promoters were both below 0.1% in tobacco pollen compared to a relative activity of 100% directed by the maize ZmMADS2 promoter. Activity of the ZmMADS2 could not be detected in young N. tabacum leaves (data not shown). This result clearly shows pollen-specific expression directed by the ZmMADS2 promoter both in tobacco and maize. Though



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luciferase activity directed by pZmMADS2::Luc was about 30 times lower in maize pollen compared to tobacco pollen, the obtained results were significant and reproducible, and the relative activity using pUbi::Luc as a standard was the same in all experiments. The endogenous ZmMADS2 transcript has been shown to be translocated into pollen tubes immediately upon germination (Heuer et al., 2000). After bombarding pollen with pZmMADS2::Gus, expression of the marker gene was displayed as expected in the pollen tubes of maize and tobacco pollen (Figures 3a, c). Although several monocot genes display regulated expression in tobacco reflecting the situation in the species of origin, some of them have been shown to respond to different regulatory mechanisms in the heterologous system (Takaiwa et al., 1991; Luan and Borgorad, 1992). In addition, the promoter of the endosperm-specific transcription factor gene Opaque 2 (O2), which controls the synthesis of a major storage protein in Z. mays, directs Gus expression in endosperm, embryo, and cotyledons of transgenic tobacco seeds (Gallusci et al., 1994). The authors postulate that expression of the O2 promoter in embryos may be due to the fact that in tobacco, unlike maize, storage proteins accumulate in the endosperm and embryo. These reports underline the necessity of conducting analyses of cis- elements in the species of origin. The results reported here demonstrate that pollen-specific promoter analyses are now applicable to maize, when luc is used as a marker system. The described method is fast and convenient and offers the opportunity to investigate many pollen- specific promoters now in a homologous system, such as Z. mays, in order to overcome species-dependent differences of regulatory elements in promoters. Acknowledgments We would like to thank Dr Reinhold Brettschneider for providing the promoterless luciferase construct and Dr Sigrid Heuer for fruitful discussions. The Südwestdeutsche Saatzucht (Rastatt) is acknowledged for financial support to D.N.S. References Bate N and Twell D (1998) Functional architecture of a late pollen promoter: pollenspecific transcription is developmentally regulated by multiple stage-specific and codependent activator elements. Plant Mol Biol 37: 859-869. Bedinger P (1992) The remarkable biology of pollen. Plant Cell 4: 879-887. Bedinger P and Edgerton MD (1990) Developmental staging of maize microspores reveals a transition in developing microspore proteins. Plant Physiol 92: 474-479. Christensen AH and Quial PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5: 213-218. Custers JBM, Oldenhof MT, Schrauwen JAM, Cordewener JHG, Wullems GJ, and van Lookeren Campagne MM (1997) Analysis of microspore-specific promoters in transgenic tobacco. Plant Mol Biol 35: 689-699. de Wet JR, Wood KV, DeLuca M, Helinski DR, and Subramani S (1987) Firefly lucirerase gene: structure and expression in mammalian cells. Mol Cell Biol 7: 725-737.




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Eyal Y, Curie C, and McCormick S (1995) Pollen specificity elements reside in 30 bp of the proximal promoters of two pollen-expressed genes. Plant Cell 7: 373-384. Gallusci P, Salamini F, and Thompson RD (1994) Differences in cell type-specific expression of the gene Opaque 2 in maize and transgenic tobacco. Mol Gen Genet 244: 391-400. Hamilton DA, Roy M, Rueda J, Sindhu RK, Sanford J, and Mascarenhas JP (1992) Dissection of a pollen-specific promoter from maize by transient transformation assays. Plant Mol Biol 18: 211-218. Hamilton DA, Schwarz YH, and Mascarenhas JP (1998) A monocot pollen-specific promoter contains separable pollen-specific and quantitative elements. Plant Mol Biol 38: 663-669. Heuer S (1999) Isolierung und Charakterisierung von cDNAs aus Eizellen und Pollen von Mais (Zea mays L.) mit Homologie zu MADS-Box-Transkriptionsfaktoren. Ph.D. Dissertation. University of Hamburg, Shaker, Aachen. ISBN 3-8265-6603-3. Heuer S, Lörz H, and Dresselhaus T (2000) The MADS box gene ZmMADS2 is specifically expressed in maize pollen and during maize pollen tube growth. Sex Plant Reprod 13: 21-27. Jefferson RA, Kavanagh TA, and Bevan MW (1987) GUS fusions: β-glucoronidase as a sensitive and versatile gene marker in higher plants. EMBO J 6: 3901-3907. Luan S and Borgorad L (1992) A rice cab gene promoter contains separate cis-acting elements that regulate expression in dicot and monocot plants. Plant Cell 4: 971-981. Mascarenhas JP (1966) Pollen tube growth and ribonucleic acid synthesis by vegetative and generative nuclei of Tradescantia. Amer J Bot 53: 563-569. McCormick S (1993) Male gametophyte development. Plant Cell 5: 1265-1275. Miyoshi H, Usami T, and Tanaka I (1995) High levels of Gus gene expression driven by pollen-specific promoters in electroporated lily pollen protoplasts. Sex Plant Reprod 8: 205-209. Neuffer MG, Coe EH, and Wessler SR (1997) Mutants of maize, p 311. Cold Spring Harbor Laboratory Press. Ow DW, Wood KV, DeLuca M, DeWet JR, Helinski DR, and Howell SH (1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234: 856-859. Seliger HH and McElroy WD (1960) Spectral emission and quantum yield of firefly bioluminescence. Arch Biochem Biophys 88: 136. Takaiwa F, Oono K, and Kato A (1991) Analysis of the 5’ flanking region responsible for the endosperm-specific expression of a rice glutelin chimeric gene in transgenic tobacco. Plant Mol Biol 16: 49-58. Torres MA, Puigdomènech J, and Stiefel V (1995) Specific distribution of mRNAs in maize growing pollen tubes observed by whole mount in situ hybridization with non-radioactive probes. Plant J 8: 317-321. Twell D, Klein TM, Fromm ME, and McCormick S (1989) Transient expression of chimeric genes delivered into pollen by microprojectile bombardment. Plant Physiol 91: 1270-1274. Walden DB (1994) In vitro pollen germination. In: Freeling M and Walbot V (eds), The Maize Handbook, pp 723-724, Springer, New York, Inc. Xu H, Davies SP, Kwan BYH, O’Brien AP, Singh M, and Knox RB (1992) Haploid and diploid expression of a Brassica campestris anther-specific gene promoter in Arabidopsis and tobacco. Mol Gen Genet 239: 58-65.
