Leucine and spermidine enhance shoot differentiation in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Enhanced numbers of multiple shoots were induced from shoot tip explants of cucumber. The effects of amino acids (leucine, isoleucine, methionine, threonine, and tryptophan) and polyamines (spermidine, spermine, and putrescine) along with benzyladenine (BA) on multiple shoot induction were investigated. A Murashige and Skoog (MS) medium containing a combination of BA (4.44 μM), leucine (88 μM), and spermidine (68 μM) induced the maximum number of shoots (36.6 shoots per explant) compared to BA (4.44 μM) alone or BA (4.44 μM) with leucine (88 μM). The regenerated shoots were elongated on the same medium. Elongated shoots were transferred to the MS medium fortified with BA (4.44 μM), leucine (88 μM), and putrescine (62 μM) for root induction. Rooted plants were hardened and successfully established in soil with a 90% survival rate.     

Long-term suspension cultures of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) with high embryogenic potential

Cucumber (Cucumis sativus L.) cytokinin-independent embryogenic cell suspension cultures were derived and maintained for more than 3.5 years without losing the embryogenic potential. The preparation and the characteristics of the cucumber embryogenic cell suspension possess many similarities to that of carrot. The cultures were induced from hypocotyl explants of in vitro grown cucumber plants in liquid MS media containing 2,4-dichlorophenoxyacetic acid as the sole growth regulator during 6 weeks and they contained a heterogeneous array of several different types of single cells and cell clusters (PEMs). The established cell suspensions were subcultured in 1-week interval, while the inoculation density was optimized to 2.0 × 10⁵ cells ml⁻¹ using cell viability as a marker. Somatic embryos were obtained after the transfer of the proembryogenic masses to a hormone-free semisolid MS medium with a frequency of 388 ± 57 somatic embryos per 1 ml of packed cell volume of the established cucumber embryogenic culture within 7 days. The frequency of normal somatic embryos with two cotyledons was found to be 78%. Such embryos possessed the potential of spontaneous maturation and the embryo conversion rates were 87%. The yield of normally growing plants was much higher compared with that previously described for cucumber systems. Somatic embryo-derived plants were successfully transferred to the greenhouse where they flowered and fruited.     

Proanthocyanidins accelerate the germination of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) seeds

Proanthocyanidins (PAs) are the end products of the flavonoid biosynthetic pathway in many seeds, but their biological function is rarely unknown during seed germination. In the present study, we observed that PAs pretreatment accelerated cucumber seeds germination with maximum efficiency at 0.15% by measuring germination percentage and radical length. Using inhibitors of abscisic acid (ABA), gibberellins (GA) and alternative oxidase (AOX) and H₂O₂ scavenger pretreatment and gene expression analysis, we found that the accelerated effect of 0.15% PAs on seed germination was due to the decreased ABA biogenesis and enhanced GA production. ROS are induced by PAs pretreatment. Then, the enhanced ROS contributed to GA and ethylene accumulation and ABA decrease in seeds. Moreover, the improvement of GA was involved in the further induction of antioxidant enzymes activities. Therefore, our findings uncover a novel role of PAs in seed germination and clarify the relationships between ROS, ABA, GA and ethylene during seed germination.     

Effects of ultraviolet-B irradiation on the cuticular wax of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) cotyledons

Cucumber seedlings were grown under three doses of supplemental ultraviolet-B (UV-B) irradiation to examine the effects on the surface structure of the cotyledons. Medium and high doses of irradiation induced glazing (formation of translucent, glossy layers) on the adaxial surfaces of cotyledons, especially those exposed to a high dose of UV-B. Observation with a scanning electron microscope revealed that the adaxial surfaces of cotyledons exposed to a medium dose of UV-B and controls became rough in appearance, but unevenness of the surface was not apparent in cotyledons irradiated with a high dose of UV-B. UV-B irradiation affected the types and amounts of alkanes and primary alcohols, the main components of cucumber cuticular wax. Based on cotyledon area, the amounts of these components were significantly higher in cotyledons irradiated with a medium dose of UV-B than in controls. This effect could be a consequence of small cotyledon area and constant wax production in the cotyledons irradiated with a medium dose of UV-B. The distribution patterns of homologs within the alkane and primary alcohol fractions shifted during growth to longer alkyl chain length in the control cotyledons. UV-B irradiation repressed these changes, suggesting that UV-B acts on cuticular wax biosynthetic pathways.     

Molecular cloning and structural analysis of the cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) phosphoenolpyruvate carboxykinase (<Emphasis Type="Italic">CsPCK</Emphasis>) gene

Genomic sequence of the ATP-dependent phosphoeno/pyruvate carboxykinase (CsPCK) gene has been determined first from cucumber. Several putative clones were isolated in three rounds of genomic library screening with designated cDNA probes. These clones were analyzed via restriction digests, Southern hybridization, and nucleotide sequencing to ascertain the structure of theCsPCK gene. Analysis of a selected positive clone (λcscpk-4A) demonstrated that this gene consists of 13 exons and 12 introns, spanning 9 kb in the cucumber genome. Exon 1 contains only 23 nucleotides of the 5′-noncoding region of cucumberPCK cDNA, whereas Exon 2 comprises 12 nucleotides of the S′-noncoding region with an N-terminal PEPCK coding sequence. All the exon-intron junction sequences agree with the GT/AG consensus, except for the 5 donor site of Intron 7, where GC replaces the GT consensus. As with rice (Oryza sativa), cucumber contains only one copy of theCsPCK gene in its haploid genome. The overall number of exons and the structure of this gene are similar to those for bothArabidopsis Chromosome 4 (Atg4)PCK and the rice PCX genes, which contain 13 and 12 exons, respectively. Two additionalArabidopsis PCK genes can be found in the fifth chromosome (Atg5), which contains 9 exons and 8 introns (with 628 and 670 amino acids, respectively) of the PEPCK peptide. TheCsPCK gene promoter has conserved plant-specific as-acting elements within 2 kb of the 5’ flanking region. Several common cis-acting elements of the isocitrate lyase (icl) and malate synthase(ms) gene promoters, identified in theCsPCK gene, are responsible for the sugar response during plant development, especially at germination. These conserved elements are discussed here.     

Characterization of PSI recovery after chilling-induced photoinhibition in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) leaves

By simultaneously analyzing the chlorophyll a fluorescence transient and light absorbance at 820 nm as well as chlorophyll fluorescence quenching, we investigated the effects of different photon flux densities (0, 15, 200 μmol m⁻² s⁻¹) with or without 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the repair process of cucumber (Cucumis sativus L.) leaves after treatment with low temperature (6°C) combined with moderate photon flux density (200 μmol m⁻² s⁻¹) for 6 h. Both the maximal photochemical efficiency of Photosystem II (PSII) (F _v/F _m) and the content of active P700 (ΔI/I _o) significantly decreased after chilling treatment under 200 μmol m⁻² s⁻¹ light. After the leaves were transferred to 25°C, F _v/F _m recovered quickly under both 200 and 15 μmol m⁻² s⁻¹ light. ΔI/I _o recovered quickly under 15 μmol m⁻² s⁻¹ light, but the recovery rate of ΔI/I _o was slower than that of F _v/F _m. The cyclic electron transport was inhibited by chilling-light treatment obviously. The recovery of ΔI/I _o was severely suppressed by 200 μmol m⁻² s⁻¹ light, whereas a pretreatment with DCMU effectively relieved this suppression. The cyclic electron transport around PSI recovered in a similar way as the active P700 content did, and the recovery of them was both accelerated by pretreatment with DCMU. The results indicate that limiting electron transport from PSII to PSI protected PSI from further photoinhibition, accelerating the recovery of PSI. Under a given photon flux density, faster recovery of PSII compared to PSI was detrimental to the recovery of PSI or even to the whole photosystem.     

Fine mapping of a male sterility gene <Emphasis Type="Italic">ms</Emphasis>-<Emphasis Type="Italic">3</Emphasis> in a novel cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) mutant

Key message

The cucumber male sterility gene ms - 3 was fine mapped in a 76 kb region harboring an MMD1 -like gene Csa3M006660 that may be responsible for the male sterile in cucumber.

Abstract

A cucumber (Cucumis sativus L.) male sterile mutant (ms-3) in an advanced-generation inbred line was identified, and genetic analysis revealed that the male sterility trait was controlled by a recessive nuclear gene, ms-3, which was stably inherited. Histological studies suggested that the main cause of the male sterility was defective microsporogenesis, resulting in no tetrad or microspores being formed. Bulked segregant analysis (BSA) and genotyping of an F₂ population of 2553 individuals were employed used to fine map ms-3, which was delimited to a 76 Kb region. In this region, a single non-synonymous SNP was found in the Csa3M006660 gene locus, which was predicted to result in an amino acid change. Quantitative RT-PCR analysis of Csa3M006660 was consistent with the fact that it plays a role in the early development of cucumber pollen. The protein encoded by Csa3M006660 is predicted to be homeodomain (PHD) finger protein, and the high degree of sequence conservation with homologs from a range of plant species further suggested the importance of the ms-3 non-synonymous mutation. The data presented here provide support for Csa3M006660 as the most likely candidate gene for Ms-3.

     

Ultrastructure analysis of cadmium-tolerant and -sensitive cell lines of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Previously, a stable cell suspension culture of cucumber tolerant to cadmium (Cd) was established (Gzyl and Gwóźdź, Plant Cell Tissue Organ Cult 80:59–67, 2005). In this study, ultrastructures of Cd-tolerant and -sensitive cells were analyzed by transmission electron microscopy (TEM). Ultrastructural differences between cell lines exposed to 100 μM CdCl₂ were observed both at cellular and organelle levels. Tolerant cells exposed to Cd exhibited well-preserved cellular structures in comparison with sensitive cells. Increased numbers of osmiophilic globules in the cytoplasm and nucleolus-associated bodies as well as electron dense material in vacuoles were observed in cadmium tolerant cells. In contrast, ultrastructure of sensitive cells following exposure to Cd exhibited distinct disturbances including vacuolation, disintegration of cytoplasm, and structural changes in both mitochondria and endoplasmic reticulum. TEM observations confirmed the adaptation of tolerant cells to Cd.     

Effect of root applied 24-epibrassinolide on carbohydrate status and fermentative enzyme activities in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) seedlings under hypoxia

The effects of 24-epibrassinolide (EBR) added to nutrient solution on growth of cucumber (Cucumis sativus L.) under root-zone hypoxia were investigated. Cucumber seedlings were hydroponically grown for 8 days in normoxic and hypoxic nutrient solutions with and without addition of EBR at 1 μg l⁻¹. EBR exerted little influence on plant performance in the normoxic nutrient solution, while the chemical alleviated root-zone hypoxia-induced inhibition of root and shoot growth and net photosynthetic rate (Pn). EBR added to hypoxic nutrient solution caused an increase in the concentration of fructose, sucrose, and total soluble sugars in the roots but not in the leaves. Root-zone hypoxia enhanced the activities of lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), and pyruvate decarboxylase in the roots. Interestingly, EBR further enhanced ADH activity but lowered LDH activity in hypoxic roots. These results suggest that EBR added to hypoxic nutrient solution may stimulate the photosynthate allocation down to roots and the shift from lactate fermentation to alcohol fermentation in hypoxic roots, resulting in the increase in ATP production through glycolysis and the avoidance of cytosolic acidosis and eventually enhanced tolerance of cucumber plants to root-zone hypoxia.     

Identification and functional characterization of <Emphasis Type="Italic">APRR2</Emphasis> controlling green immature fruit color in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Nitrogen (N) is a macronutrient essential for plant growth and development. Meanwhile, grafting is a method used to alleviate stress tolerance of various biotic and abiotic factors. This study aims to investigate how pumpkin grafting (PG) improves N use efficiency of watermelon. A commercial watermelon cultivar “Zaojia 8424” [Citrullus lanatus (Thunb.) Matsum. and Nakai.] was self-grafted and then grafted onto pumpkin (Cucurbita maxima?×C. moschata) rootstock cv. Qingyan Zhenmu No. 1. The grafted plants were exposed to two levels of N (9 and 0.2 mM) under hydroponic conditions. The grafted plants were harvested at days 11 and 22 after low N (0.2 mM) treatment. PG improved the N use efficiency of watermelon scion through the vigorous root system of pumpkin rootstock that enhanced the uptake and accumulation of N, P, K, Ca, Mg, B, and Mn in watermelon. Gene expressions of nitrate reductase (Cla002787, Cla002791, and Cla023145) and nitrite reductase (Cla013062) genes were increased, promoting N assimilation. Mesophyll thickness and SPAD index (relative chlorophyll measurement) were also improved. Furthermore, pumpkin rootstock also enhanced the supply of zeatine riboside (ZR) and isopentenyl adenosine (iPA) in the leaves, promoting shoot growth. All these lead to improved plant growth and nitrogen use efficiency of pumpkin rootstock-grafted watermelon plants.     

Ontogeny of somatic embryos in cucumber (<Emphasis Type="Italic">cucumis sativus</Emphasis> L.)

Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion. Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium containing 175.2 mM sucrose and 0.5 gl⁻¹ activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6 mM sucrose and 1.4 μM gibberellic acid (GA₃) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants.     

Salicylic acid and phenolic compounds under cadmium stress in cucumber plants (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

In this pot experiment, cucumbers (Cucumis sativus L.) were grown in a model soil contaminated by three different concentrations of cadmium (40, 160, and 320 mg.kg^?1) with different accompanied anions (Cl^?, SO₄ ^2?). In all variants, the most Cd (90 %) was accumulated in the roots, but higher content in the case of Cl^?. The distribution of Cd in various cucumber organs was as follows: root > stem > leaf > fruits. However, in variants with higher doses of Cd with SO₄ ^2?, the ratio was changed as follows: root > leaf > stem > fruits. In all variants, least of Cd (max. 1 %) was found in fruits. Variants with the highest Cd doses were significantly different by comparison with all other variants, but higher content was in the case of Cl^? anion. Stimulation effect on the biomass production and growth of aerial parts and roots of plants in all variants with Cd was observed. Toxicity symptoms, mainly in the presence of leaf chlorosis and yellowing, were more visible in the variants with Cl^?, in comparison with SO₄ ^2?. The amounts of phenol compounds in leaves rose almost in all variants. Only the variants with higher Cd content with SO₄ ^2? showed slight reduction. One possible explanation of reduced content may be their bounding on Cd. The content of salicylic acid was reduced in all variants with Cd treatment. However, it is difficult to conclude their role in plant defence responses to heavy metal, because their actual defence mechanism is still unclear. However, from these results, we can suggest that the accompanying anion and the form in which Cd exists may have an impact on the involvement of various antioxidant systems.     

A mutant in the <Emphasis Type="Italic">CsDET2</Emphasis> gene leads to a systemic brassinosteriod deficiency and <Emphasis Type="Italic">super compact </Emphasis>phenotype in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Key message

A novel dwarf cucumber mutant, scp-2, displays a typical BR biosynthesis-deficient phenotype, which is due to a mutation in CsDET2 for a steroid 5-alpha-reductase.

Abstract

Brassinosteroids (BRs) are a group of plant hormones that play important roles in the development of plant architecture, and extreme dwarfism is a typical outcome of BR-deficiency. Most cucumber (Cucumis sativus L.) varieties have an indeterminate growth habit, and dwarfism may have its value in manipulation of plant architecture and improve production in certain production systems. In this study, we identified a spontaneous dwarf mutant, super compact-2 (scp-2), that also has dark green, wrinkle leaves. Genetic analyses indicated that scp-2 was different from two previously reported dwarf mutants: compact (cp) and super compact-1 (scp-1). Map-based cloning revealed that the mutant phenotype was due to two single nucleotide polymorphism and a single-base insertion in the CsDET2 gene that resulted in a missense mutation in a conserved amino acid and thus a truncated protein lacking the conserved catalytic domains in the predicted steroid 5α-reductase protein. Measurement of endogenous hormone levels indicated a reduced level of brassinolide (BL, a bioactive BR) in scp-2, and the mutant phenotype could be partially rescued by the application of epibrassinolide (EBR). In addition, scp-2 mutant seedlings exhibited dark-grown de-etiolation, and defects in cell elongation and vascular development. These data support that scp-2 is a BR biosynthesis-deficient mutant, and that the CsDET2 gene plays a key role in BR biosynthesis in cucumber. We also described the systemic BR responses and discussed the specific BR-related phenotypes in cucumber plants.

     

The levels of melatonin and its metabolites in conditioned corn (<Emphasis Type="Italic">Zea mays</Emphasis> L.) and cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) seeds during storage

The efficiency of pre-sowing conditioning/priming methods used to apply melatonin into seeds was verified: osmopriming in the case of dicot Cucumis sativus and hydropriming of monocot Zea mays seeds. Both priming techniques were selected experimentally as optimal for the studied plant species. Four different seed variants were compared: control non-treated ones, and seeds conditioned with water or with 50 or 500 μM melatonin water solutions. The HPLC–MS quantitative and qualitative analyses were used to determine the content of melatonin and of its potential metabolites in the seeds during 1 year following the conditioning. The control seeds and those conditioned with water contained small amount of endogenous melatonin in both species. However, the level of this indoleamine increased markedly in cucumber and corn seeds primed with exogenous melatonin and it was always correlated with the concentration of melatonin applied. It was noted that melatonin was metabolized during seed storage by its gradual oxidation, thus it protects dry seeds against oxidative stress, prevents potential injuries and significantly increases seeds quality. Interestingly, in the control and water-primed seeds, seasonal fluctuations of endogenous melatonin concentration were noted and significant increase in this indoleamine in the winter month was observed. This suggests that in seeds endogenous melatonin could play a crucial role in seasonal rhythms independently of environmental conditions.     

Genetic mapping of <Emphasis Type="Italic">psl</Emphasis> locus and quantitative trait loci for angular leaf spot resistance in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

One of the most important cucumber diseases is bacterial angular leaf spot (ALS), whose increased occurrence in open-field production has been observed over the last years. To map ALS resistance genes, a recombinant inbred line (RIL) mapping population was developed from a narrow cross of cucumber line Gy14 carrying psl resistance gene and susceptible B10 line. Parental lines and RILs were tested under growth chamber conditions as well as in the field for angular leaf spot symptoms. Based on simple sequence repeat and DArTseq, genotyping a genetic map was constructed, which contained 717 loci in seven linkage groups, spanning 599.7 cM with 0.84 cM on average between markers. Monogenic inheritance of the lack of chlorotic halo around the lesions, which is typical for ALS resistance and related with the presence of recessive psl resistance gene, was confirmed. The psl locus was mapped on cucumber chromosome 5. Two major quantitative trait loci (QTL) psl5.1 and psl5.2 related to disease severity were found and located next to each other on chromosome 5; moreover, psl5.1 was co-located with psl locus. Identified QTL were validated in the field experiment. Constructed genetic map and markers linked to ALS resistance loci are novel resources that can contribute to cucumber breeding programs.     

Mutations in <Emphasis Type="Italic">CsPID</Emphasis> encoding a Ser/Thr protein kinase are responsible for round leaf shape in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Key message

Two round-leaf mutants, rl-1 and rl-2, were identified from EMS-induced mutagenesis. High throughput sequencing and map-based cloning suggested CsPID encoding a Ser/Thr protein kinase as the most possible candidate for rl-1. Rl-2 was allelic to Rl-1.

Abstract

Leaf shape is an important plant architecture trait that is affected by plant hormones, especially auxin. In Arabidopsis, PINOID (PID), a regulator for the auxin polar transporter PIN (PIN-FORMED) affects leaf shape formation, but this function of PID in crop plants has not been well studied. From an EMS mutagenesis population, we identified two round-leaf (rl) mutants, C356 and C949. Segregation analysis suggested that both mutations were controlled by single recessive genes, rl-1 and rl-2, respectively. With map-based cloning, we show that CsPID as the candidate gene of rl-1; a non-synonymous SNP in the second exon of CsPID resulted in an amino acid substitution and the round leaf phenotype. As compared in the wild type plant, CsPID had significantly lower expression in the root, leaf and female flowers in C356, which may result in the less developed roots, round leaves and abnormal female flowers, respectively in the rl-1 mutant. Among the three copies of PID genes, CsPID, CsPID2 and CSPID2L (CsPID2-like) in the cucumber genome, CsPID was the only one with significantly differential expression in adult leaves between WT and C356 suggesting CsPID plays a main role in leaf shape formation. The rl-2 mutation in C949 was also cloned, which was due to another SNP in a nearby location of rl-1 in the same CsPID gene. The two round leaf mutants and the work presented herein provide a good foundation for understanding the molecular mechanisms of CsPID in cucumber leaf development.

     

The heterochromatin as a marker for protoplast differentiation of <Emphasis Type="Italic">Cucumis sativus</Emphasis>

The protoplast cultures of Cucumis sativus in two culture systems were used to study heterochromatin reassembly during dedifferentiation of isolated protoplasts and their subsequent differentiation into calli and proembryos. Here we show that dedifferentiation of the cucumber mesophyll cells is accompanied by a dramatic reduction in size and numbers of nuclear chromocenters. Although chromocenters were newly established during protoplast culture, the measured relative heterochromatin content differed according to the culture system used. Protoplast culture leading to proembryo formation displayed a lower level of relative heterochromatin content than cultures resulting in calli and the relative heterochromatin content reached values close to those estimated for somatic embryos.     

Transfer and expression of <Emphasis Type="Italic">npt</Emphasis>II and <Emphasis Type="Italic">bar</Emphasis> genes in cucumber (<Emphasis Type="Italic">Cucumis satavus</Emphasis> L.)

Summary The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l⁻¹ phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT (2–6 mg l⁻¹). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l⁻¹) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization.