Agrobacterium-mediated genetic transformation of oilseed Brassica campestris: Transformation frequency is strongly influenced by the mode of shoot regeneration

Summary Protocols were developed for efficient shoot regeneration from hypocotyl and cotyledon explants of oilseed Brassica campestris (brown sarson) cv. Pusa Kalyani. These were used for genetic transformation by an Agrobacterium based binary vector carrying neomycin phosphotransferase (npt) gene and -glucuronidase (gus)-intron gene for plant cell specific expression. Transformed plants were recovered from hypocotyl explants at a frequency of 7–13%. Addition of silver nitrate markedly enhanced shoot regeneration in hypocotyl explants under non-selection conditions and was found to be an absolute requirement under selection conditions. Cotyledon explants, inspite of being more regenerative, proved to be highly refractory to transformation. Only two chimeric transformed shoots were obtained from more than 10,000 cotyledons treated with Agrobacterium. In hypocotyl explants, shoot regeneration occurred from the vascular parenchyma both with and without the intervention of callus phase. Only the shoot buds differentiating from callus tissue were positive for GUS activity. In cotyledons, shoot buds originated only directly from the vascular parenchyma, generally at a distance of about 450–625 from the cut surface. Such shoots were negative for GUS activity.     

Transgenic poplars: expression of chimeric genes using four different constructs

Summary Leaf or stem explants of a hybrid poplar clone (Populus tremula X Populus alba), sensitive to Agrobacterium tumefaciens, were co-cultivated either by an octopine or a nopaline disarmed A. tumefaciens modified strain. Transformed poplar shoots were readily regenerated from explants. The protocol was improved using the nopaline disarmed strain C58/pMP90 with the binary vector pBI121. This protocol was then used to test three other vectors. The first one, possessing a nptII gene fused to the CaMV 19S promoter, permitted regeneration of transformed shoots in presence of 50 to 100 mg/l kanamycin. The two other vectors carried an additional nptII gene under the control of the CaMV 35S or CaMV 35S promoter with a double enhancer sequence (CaMV 70). CaMV 70 promoter provided consistently higher level of gene expression than the other promoters in both callus and leaf tissues.Abbreviations CaMV Cauliflower Mosaïc Virus - 2iP 2-isopentenyladenine - GUS and gus ß-glucuronidase - NAA 1-naphthaleneacetic acid - NPTII and nptII neomycin phosphotransferase II - NOS Nopaline synthase, X-Gluc: 5-bromo-4-chloro-3-indolyl ß-D glucuronide - Ap ampicillin - Gn gentamycin - Km kanamycin - Rf rifampicin - St streptomycin This work is dedicated to the late Marie France Michel who initiated the poplar biotechnology project at INRA.     

Transient gene expression in electroporated Solanum protoplasts

Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts –250 V/cm; Désirée mesophyll protoplasts –225 V/cm; Désirée suspension culture protoplasts –225 V/cm; and Désirée tuber protoplasts –150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36–48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the -glucuronidase (gus) gene, showed expression (at DNA concentrations between 0–10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20–30 pmol/ml) the patatin promoter directed 4–5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.     

A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica

Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. We found three key factors for enhancing successful regeneration: (1) tissue source of explants, (2) combination and concentration of growth regulators, and (3) culture conditions. The highest frequency of shoot regeneration was achieved using hypocotyl segments cultured on MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk under subdued light at 25±2°C; under these conditions, each hypocotyl segment produced over 30 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Regenerated shoots rooted easily on half-strength basal MS medium and were successfully established in the greenhouse. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole, cotyledon and hypocotyl with A. tumefaciens strains AGL1 and LBA4404. Transient expression was observed in all recipient tissues. Stable transgenic calli originating from petiole were obtained.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

Conditions of transformation and regeneration of `Induka' and `Elista' strawberry plants

Efficiency of plants' transformation depends on many factors. The genotype, applied techniques and conditions of plant's modification and modified plant regeneration are the most important among them. In our studies regeneration and transformation conditions for two strawberry cultivars were determined and compared. Plants were transformed by Agrobacterium tumefaciens LBA₄₄₀₄ strain containing plasmid pBIN19 with nptII and gus-reporter genes. Experiment was carried out on more than 1300 leaf explants from each cultivar. Generally, `Induka' plants characterized with higher regeneration potential than `Elista'. The highest number of regenerated shoots was obtained on MS medium with 0.4 mg l ^–1 IBA and 1.8 mg l^–1 BA (3.5 and 1.8 shoots/explant for `Induka' and `Elista', respectively). After plant transformation number of regenerated, transgenic shoots was higher for `Elista' (on the average: 8.3 shoots/100 explants). The number of transgenic `Induka' shoots, obtained at the same conditions, was twice lower (4.2). Simultaneously `Induka' plants needed higher kanamycin concentration for transgenic explants selection than `Elista' (25 mg l^–1). Preliminary incubation of A. tumefaciens in LB or MS medium with acetosyringone and IAA resulted in increasing transgenic shoots number (per 100 explants: `Induka' 4.5, `Elista' 8.0–9.5 shoots). After using untreated bacteria for plants' transformation, number of transgenic plants varied (dependently on cultivar) from 3.8 to 7.0/100 explants. Applying LB or MS as basic medium as well as adding tobacco plant extract to these media did not significantly influence transformation efficiency.     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

In vitro Crown Galls Induced by Agrobacterium tumefaciens Strain A281 (pTiBo542) in Trigonella foenum-graecum

Transformation of fenugreek (Trigonella foenumgraecum) was carried out with A281 oncogenic strain of Agrobacterium tumefaciens using root, cotyledon and hypocotyl explants excised from 1-week-old seedlings, which showed that the plant was highly susceptible to transformation. Tumors (calli) were selected on 50 mg dm^–3 kanamycin. They were analyzed for -glucuronidase (GUS) expression. Presence of uidA (gus) gene, was confirmed by polymerase chain reaction (PCR) amplification.     

A biolistic approach for the transfer and expression of a gusA. reporter gene in embryogenic cultures of Pinus radiata

Summary The biolistic® particle delivery system was used for the delivery of DNA into embryogenic tissue culture cells of Pinus radiata D. Don. Several experiments with varying parameters were performed to increase the delivery efficiency. Six different controlling elements were cloned upstream of the ß-glucuronidase coding sequence (gusA reporter gene) and transient expression of the gusA reporter gene was compared three days after bombardment. The results clearly indicate a decrease in transient expression as follows: pEmu-derivatives with the ocs-enhancer-element > 2x CaMV 35S (with Kozak consensus-sequence) > 2x CaMV 35S (without Kozak consensus sequence) > CaMV 35S (with Kozak consensus-sequence) > CaMV 35S (without Kozak consensus sequence). Time course experiments monitoring gusA expression showed a significant decrease in the number of blue spots 10–14 days after bombardment. A few blue clumps however, were still detected 35 days after shooting. Embryo initials expressing the gusA gene in all cells were also detected. The results suggest that it will be possible to develop a reliable biolistic protocol for stable integration of genes into Pinus radiata embryogenic cultures which are capable of plant regeneration.Abbreviations ccc covalently closed circular DNA - lin linearised DNA - E restriction enzyme Eco RI - Sph restriction enzyme SpH I - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Chromosomal integration is required for spatial regulation of expression from the β-phaseolin promoter

The stringency of spatial expression of phaseolin, the major storage protein of bean (Phaseolus vulgaris) seeds, has been rigorously evaluated using stable and transient transformation techniques. Transgenic tobacco plants known to be homozygous for the β-glucuronidase (gus) reporter sequence under the regulation of various lengths of the β-phaseolin gene (phas) promoter were shown to express gus only in developing seed tissues. No expression was detected in calli initiated from stems, leaves and immature seeds, showing that expression was not leaky in undifferentiated tissues. Control plants and cultures containing gus fused to the CaMV 35S promoter actively expressed gus under identical conditions. It was not possible to induce expression in phas/gus calli with ABA, GA or jasmonic acid. Treatment of the cultures with 5-azacytidine did not result in expression, excluding methyletlon as the major factor regulating the phas promoter. However, strong gus expression was detected in seed of plants regenerated from these callus cultures, confirming that neither gene rearrangements nor deletion were responsible for the lack of activity seen in tissues other than the developing seed. In contrast to the above observations, strong transient expression of gus was detected in tobacco, bean and soybean leaves following introduction of the phas/gus fusion constructs via biolistic approaches and in electroporated bean leaf and hypocotyl protoplasts. These experiments show unequi-vocally that the phas promoter is under rigorous spatial control when integrated into the genome, but lacks spatial control when present as extrachromosomal naked DNA. A putative model explaining these differences is presented.     

Transforming embryogenic cell lines of Gladiolus with either a bar-uidA fusion gene or cobombardment

Summary Embryogenic cell lines of Gladiolus were bombarded with the bar-uidA fusion gene under the cauliflower mosaic virus (CaMV) 35S promoter (pDM327) or cobombarded with uidA under the CaMV 35S promoter (pBCG) and bar under the CaMV 35S promoter (pDM307). Over 500 cell lines were isolated for either the fusion gene or cobombarded cells following selection on Murashige and Skoog's medium supplemented with 2 mg 1⁻¹ (9 μM) 2,4-dichlorophenoxyacetic acid and 6 mg 1⁻¹ phosphinothricin. The optimum DNA concentration for, isolating stable transformants was one-tenth that for optimal isolation of lines with gus expression, and three times as many cell lines were isolated following cobombardment as compared to bombardment with the bar-uidA fusion gene. Three times as many cell lines (72% of the cell lines) containing the bar-uidA fusion gene expressed gus as compared to cobombarded cell lines (23%) following histological staining. Gus expression ceased after 1 yr in culture for 5% of the cell lines containing the fusion gene and 3% of the cobombarded cell lines. The bifunctionality and utility of the bar-uidA fusion gene were demonstrated, accompanied by enhanced gus expression.     

Promoterless gus gene shows leaky β-glucuronidase activity during transformation of tomato with bspA gene for drought tolerance

Transformation of tomato (Lycopersicon esculentum Mill.) was carried out using disarmed Agrobacterium tumefaciens strain EHA 105 harboring a binary vector pBIG-HYG-bspA. The plasmid contains the bspA (boiling stable protein of aspen) gene under the control of a CaMV35S promoter and nopaline synthase (NOS) terminator, hygromycin phosphotransferase gene (hpt) driven by nopaline synthase promoter and polyadenylation signal of Agrobacterium gene7 as terminator and a promoterless gus gene. Very strong β-glucuronidase (GUS) expression was observed in transformed tomato plants but never in non-transformed (control). Since GUS expression was observed only in transformed plants, the possibility of the presence of endogenous GUS enzymes was ruled out. Possibility of false GUS positives was also ruled out because the GUS positive explants reacted positively to polymerase chain reaction (PCR) and PCR-Southern tests carried out for the presence of bspA gene, which indicated the integration of T-DNA in tomato genome. The promoterless GUS expression was hypothesized either due to leaky NOS termination signal of bspA gene or due to different cryptic promoters of plant origin. It was concluded that GUS expression was observed in the putative transgenics either due to the read through mechanism by the strong CaMV35S promoter or due to several cryptic promoters driving the gus gene in different transgenic lines.     

Transgenic grasspea (Lathyrus sativus L.): Factors influencing Agrobacterium-mediated transformation and regeneration

A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD₆₀₀0.6 and diluted to a cell density of 10⁹ cells ml^–1 for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 g ml^–1). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l^–1 indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T₁ seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.     

High frequency organogenesis in hypocotyl,cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.     

Patterns of transformation intensity on flax hypocotyls inoculated with Agrobacterium tumefaciens

Summary Cellular transformation intensities on flax (Linum usitatissimum) hypocotyl explants using disarmed Agrobacterium tumefaciens were investigated through various preculture durations, cocultivation durations and removal of epidermis. The expression of an intron-containing -glucuronidase (GUS) gene driven by CaMV 35S promoter served as a reporter for determination of transformed tissues on hypocotyls. The binary plasmid p35SGUSINT in octopine-type Agrobacterium strain GV2260 was used as the vector system. A prolonged cocultivation duration (5–7 days) resulted in a much higher transformation staining intensity (frequency * tissue area) than 2- or 3-day-cocultivation duration on hypocotyls variously precultured prior to inoculation. A high staining intensity on the two cut ends was obtained from nonprecultured hypocotyls. A reduction in intensity on the upper cut end of hypocotyls was observed with preculture times greater than 6 days. Peeled hypocotyls with a post-peeling preculture of 2 or 3 days had a high proportion of superficial area covered by transformed tissues after a 7 day-cocultivation duration. These results will help to improve the efficiency of recovery of transgenic plants by increasing the proportion of transformation in the regenerable tissues.     

Studies on genetic transformation of olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) somatic embryos: I. Evaluation of different aminoglycoside antibiotics for <Emphasis Type="Italic">nptII</Emphasis> selection; II. Transient transformation via particle bombardment

As a first step to the establishment of a genetic transformation protocol for olive somatic embryos obtained from the seeds of cv. ‘Picual’, the efficiencies of different aminoglycoside antibiotics as selective agents to be used with the nptII marker gene, and the particle bombardment technique for transient transformation have been evaluated. Among the three antibiotics tested, paromomycin and kanamycin showed a similar inhibitory effect and, at 200 mg l⁻¹, both of them impaired callus growth after 8 weeks of culture. However, when isolated embryos were cultured in the presence of these antibiotics, a 20% of the embryos still remained viable at 400 mg l⁻¹. Neomycin was discarded as a selective agent since it showed only a moderate toxic effect. Contrary to solid medium, when olive callus was cultured in liquid medium supplemented with different paromomycin concentrations for 3 weeks, the callus growth was impaired at the lowest antibiotic concentration, 3 mg l⁻¹. Best conditions for transient transformation of olive callus using PDS-1000/He system were a 6 cm target distance and a 900 psi bombardment pressure. pCGU∆1 plasmid, containing the gus gene under the control of sunflower ubiquitin promoter yielded a significantly higher number of gus expression areas per bombarded explant than pGUSINT or pJGUS5 plasmids, where the gus gene is driven by CaMV35S promoter or CaMV35S with enhancer, respectively. Almost 45% of bombarded explants showed gus expression 12 weeks after bombardment.     

T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices

This paper establishes that the isolated shoot meristem of monocotyledons can be infected and transformed using Agrobacterium. Since this explant from nearly any cereal cultivar can rapidly regenerate into a plant, using this explant effectively eliminates the genotype regeneration restrictions to cereal crop transformation allowing direct transformation of elite germplasm. Shoot apices of Oryza sativa L. Tropical Japonica, cv. Maybelle were explants used for cocultivation, and gene transfer was accomplished using Agrobacterium containing plasmids for the bar gene expression driven by the CaMV 35S promoter or by the rice actin 1 promoter. Experiments to determine the survival rates of isolated shoot apices on media containing the herbicide, glufosinate-ammonium (PPT), established that no shoot apices survived on 0.5 or 1.0 mg/l PPT. After shoot apices were cocultivated with Agrobacterium, 2.8% (overall 20 out of 721 shoot apices) survived on 0.5 mg/l PPT. Results demonstrated that the use of the actin 1 promoter-based expression vector and an extra-wounding treatment of the meristematic cells appeared to be most effective in promoting transformation. Integration, expression and transmission of the transferred foreign genes in primary, R1 and R2 generation plants were confirmed by molecular analyses and herbicide application tests. A germination test of R2 progeny from one of the transgenic plants (R1) established a phenotype segregation ratio showing a non-Mendelian inheritance pattern. Inactivation of the transferred foreign gene in R2 progeny appeared to result from transgene methylation.     

Regulation of the maizerab17 gene promoter in transgenic heterologous systems

The maizerab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardellet al., Plant Mol Biol 14 (1990) 423–432). Here we demonstrate that 5 upstream sequences of therab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5 upstream fragment ofrab17 (–1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused torab17 promoter deletions indicate that a 300 bp DNA fragment (–351/–102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (–219/–102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.     

Agrobacterium tumefaciens-mediated transformation of Solanum gilo Raddi as influenced by explant type

An efficient system for Agrobacterium tumefaciens-mediated transformation of Solanum gilo was established. The marker genes for kanamycin resistance and ß-glucuronidase expression were introduced. A comparison between cotyledon and hypocotyl explants showed that while regeneration was better from hypocotyl explants, cotyledon explants gave better transformation efficiency (46% vs. 32%). Four levels of kanamycin selection (100, 150, 200 and 250 mg/l) were tested for effect on transformation efficiency with each type of explant. Lower levels of kanamycin worked better using cotyledon explants, while higher levels of kanamycin worked better for hypocotyl explants. All nine t₀ plants tested for expression of the kan ^r gene were positive. The progeny of three of these plants showed a pattern of classical Mendelian inheritance (3 to 1) for both the kan ^r and the ß-glucuronidase genes.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - NPTII neomycin phosphotransferase - GUS ß-glucuronidase     

TheCaMV 35S promoter is highly active on floral organs and pollen of transgenic strawberry plants

We have evaluated the expression of the reporter -glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter in flowers and pollen from 14 independent transgenic strawberry lines. Of the 14 lines evaluated, 13 (92.8%) showed GUS activity—as estimated by the histochemical GUS assay—in some floral organs, with expression being most common in the flower stem, sepals, petals, ovary and stigma. Ten of these thirteen transgenic lines (77%) showed GUS activity in pollen, although the percentages of positive pollen per flower varied greatly among the different lines. A study of the GUS expression during pollen maturation showed that the (CaMV 35S) promoter showed low expression in pollen from flower buds before anthesis but was activated in mature pollen following anther dehiscence. The percentages of pollen grains that showed GUS activity ranged from 2.1% to 46.3%. These percentages were similar or even higher when mature pollen was stored dry at room temperature for 2 weeks. After 5 weeks of storage, the percentages of GUS-positive pollen decreased in two of the six lines analysed but remained at similar values in the other four lines. GUS activity was also measured in protein extracts of mature pollen by means of the fluorometric GUS assay, with the values obtained ranging from 3.8 mol MU mg protein^–1 h^–1 to 0.26 mol MU mg protein^–1 h^–1. Contrary to the generally held view that the CaMV 35S promoter is virtually silent in pollen, we conclude that it is highly expressed in transgenic strawberry pollen.Abbreviations CaMV 35S Cauliflower mosaic virus promoter - GUS -Glucuronidase (EC 3.2.1.31) - MU 4-Methyl umbelliferone - nos Nopaline synthase promoter - nptII Neomycin phosphotransferase - X-Gluc 5-Bromo-4-chloro-3-indolyl--d-glucuronic acid