Abstract
Low transformation efficiency and long generation time for production of transgenic Gerbera jemosonii plants leads to vulnerable gene function studies. Thus, transient expression of genes would be an efficient alternative. In this investigation, a transient expression system for gerbera petals based on the Agrobacterium infiltration protocol was developed using the reporter genes β-glucuronidase (gus) and green florescence protein (gfp). Results revealed the incapability of using the gfp gene as a reporter gene for transient expression study in gerbera flowers due to the detection of green fluorescent color in the non-infiltrated gerbera flower petals. However, the gus reporter gene was successfully utilized for optimizing and obtaining the suitable agroinfiltration system in gerbera flowers. The expression of GUS was detectable after three days of agroinfiltration in gerbera cultivars “Express” and “White Grizzly” with dark pink and white flower colors, respectively. The vacuum agroinfiltration protocol has been applied on the cultivar “Express” for evaluating the transient expression of the two genes involved in the anthocyanin pathway (iris-dfr and petunia-f3'5'h), which is responsible for the color in flowers. In comparison to the control, transient expression results showed change in the anthocyanin pigment in all infiltrated flowers with color genes. Additionally, blue color was detected in the stigma and pollen grains in the infiltrated flowers. Moreover, blue colors with variant intensities were observed in produced calli during the routine work of stable transformation with f3'5'h gene.
Introduction
The floricultural industry has focused its attention primarily on the development of novel colored and long living cut flowers. Gerbera is one of the most economically important cut flowers in the world. Egypt is considered a good location for cut flowers production. The European market has increased its demand of gerbera; therefore the gerbera growers are looking for a novel gerbera generation with excellent color characteristics, stem length, petal shape, vase life and others.1 Most of the plant pigments varied in pigmentation of floral parts ranging from white to red and purple colors, which belong to the anthocyanin group of flavonoids.2
Dihydroflavonol 4-reductase (DFR) is one of the enzymes specific to anthocyanin synthesis, which catalyzes the production of leucoanthocyanidins from dihydroflavonols. It can be hydroxylated on the 3′ or 5′ position of the B-ring by flavonoid 3′-hydroxylase (F3′H) to produce dihydroquercetin or by flavonoid 3′5'hydroxylase (F3′5'H) to form dihydromyricetin. The latter two compounds are involved in the production of flavonoid precursors and in the formation of particular “decorated” anthocyanin molecules.1,2 It is clear that the genes dfr and f3′5'h play important roles in the flavonoid biosynthetic pathway3 and in floral anthocyanin.4
Progress in gerbera transformation has been hampered by long-term regeneration protocols, which are requested for successful transformation5,6 as well as low regeneration frequencies. A long period of selection and regeneration of putative transgenic gerbera plants needs a long time to analyze the function of the introduced transgenes. Therefore, transient expression of genes was used as an efficient alternative to determining gene function while avoiding the low efficiency of transformation and the length of time of producing transgenic plants. It was stated that the genes could be transiently expressed up to 1,000-fold higher than in stable transformants.7 Moreover, it is possible to measure gene expression within a very short time, independent of the regeneration of a transformed cell in transient assays.8
Agrobacterium tumefaciens infiltration (agroinfiltration) is used as an efficient method for transient gene expression in intact plant leaves. This method is rapid and useful for gene expression analysis. Many different heterologous proteins can be produced without the need to generate transgenic plants, which is difficult for many plant species by agroinfiltration.9,10 Use of agroinfiltration for transient assays has become established for processes such as assigning gene function,11 element analysis12 and inducible gene studies.13
This study aims to develop and optimize gene expression via an agroinfiltration in gerbera flowers and investigate the capability of the two synthesized color genes, i.e., iris-dfr and petunia-f3'5'h, to change flower color in gerbera.
Results
Optimization of agroinfiltration system in gerbera flower
To adopt an efficient agroinfiltration system in gerbera flowers, the feasibility and efficiency of several factors—i.e., reporter genes (gfp and gus), acetosyringen and Agrobacterium density—have been studied. In addition, two ways of infiltration, i.e., syringe and vacuum, were evaluated with different infiltration times.
Agrobacterium suspensions
Overnight cultures of Agrobacterium with all constructs under study were grown at 28°C in liquid LB medium, harvested and diluted to reach final concentration of 1.0 at OD 600 by resuspending either in liquid LB or in MES buffer. It was observed that all the infiltrated flowers with Agrobacterium suspended in LB medium wilted and died due to the severe contamination. Conversely, the infiltrated flowers with Agrobacterium suspended in MES buffer survived up to three weeks post infiltration. Thus the Agrobacterium culture in MES buffer was used in agroinfiltration process.
GFP expression
Agrobacteriun GV3101::pEarleyGate103::gfp-intron was used for agroinfiltration of white and dark pink flowers of gerbera. Results showed that the difficulty for detecting GFP expression in infiltrated gerbera flowers was due to the fluorescent background observed in the non-agroinfiltrated flowers. Figure 1 illustrates the non-agroinfiltrated petals and pollen grains under the fluorescent stereo microscope. This result indicates the inefficacy of using the gfp reporter gene in the optimization of the agroinfiltration system in gerbera flowers.
Published online:
01 January 2013Figure 1. The green fluorescent color detected in (A) non-infiltrated petal and (B) the pollen grain in non-infiltrated under the fluorescent UV microscope.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kgmc20/2013/kgmc20.v004.i01/gmcr.23925/20141028/images/medium/kgmc_a_10923925_f0001.gif"})
Figure 1. The green fluorescent color detected in (A) non-infiltrated petal and (B) the pollen grain in non-infiltrated under the fluorescent UV microscope.
GUS expression
Agrobacterium GV3101::pISV2678 with the gus-intron gene was used for agroinfiltrating both colors of gerbera flower. Results revealed different levels of expression among the treatments and the floret levels. The GUS expression could be detected three days post infiltration in both methods and in the flower colors. Blue color appeared at the base and middle of the petals in all infiltrated flowers (Fig. 2). Generally, highest GUS expression among treatments was detected in infiltrated petals for 30 min after 10 d for both flower colors and in infiltration methods (vacuum and syringe, Table 1). Furthermore, inner florets in the two flower colors, pink and white, revealed higher expression than the outer ones.
Published online:
01 January 2013Figure 2. GUS expression in agroinfiltrated gerbera flower in (A) dark pink and (B) white flower colors.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kgmc20/2013/kgmc20.v004.i01/gmcr.23925/20141028/images/medium/kgmc_a_10923925_f0002.gif"})
Figure 2. GUS expression in agroinfiltrated gerbera flower in (A) dark pink and (B) white flower colors.
Table 1. Level of blue color intensities for GUS transient expression in agroinfiltrated gerbera flower petals
In the case of the syringe method, there was fast flower death due to contamination after infiltration, and the infiltrated flowers could not survive more than two weeks even though there were not sufficient petals in each infiltrated flower for investigation. Therefore, the expression was detected in both outer and inner florets together. The gus gene activity showed high expression between 3–10 d post infiltration. Expression in both methods started to decrease after the second week.
Effect of the acetosyringone on the transient gene expression
Different concentrations (0 to 150 µM) of acetosyringone showed the same level of GUS expression in the vacuum infiltrated flower petals. This indicates that acetosyringone has no effect on agroinfiltration for gerbera flowers.
Agrobacterium density on the transient gene expression
Different densities of Agrobacterium suspensions used for infiltration showed no detectable effect on expression level, as the blue color of GUS expression was similar for all the densities of bacterial suspensions.
Transient expression of iris-dfr and petunia f3′5'h gens on gerbera flower
To investigate the ability of the iris-dfr and petunia-f3′5'h genes in changing the flower color, vacuum agroinfiltration protocol was applied on gerbera “Express” with pink flower color. Agrobacterium GV3101::dfr or f3′5'h individually or in combination were performed in this investigation. The expression was detected during the 7th to the 10th day post infiltration. Results showed ranges of purple color present in the petal of all infiltrated flowers with different treatments. In contrary, the control had the pink color in uniformed pattern (Fig. 3). Furthermore, blue color was observed in the stigma and pollen grain of the infiltrated flowers (Fig. 4).
Published online:
01 January 2013Figure 3. Cross sections in gerbera petals under 40× light microscope for Nonagroinfiltrated flower (A) with uniform color, agroinfiltrated petal with dfr gene (B), f3′5'h/dfr(C) and f3′5'h(D) showing ranging of color variation petal compared with the non-infiltrated.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kgmc20/2013/kgmc20.v004.i01/gmcr.23925/20141028/images/medium/kgmc_a_10923925_f0003.gif"})
Figure 3. Cross sections in gerbera petals under 40× light microscope for Nonagroinfiltrated flower (A) with uniform color, agroinfiltrated petal with dfr gene (B), f3′5'h/dfr(C) and f3′5'h(D) showing ranging of color variation petal compared with the non-infiltrated.
Published online:
01 January 2013Figure 4. Arrows pointed to the blue stigma (A) blue pollen grain (B) after agroinfiltration with color involved genes
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kgmc20/2013/kgmc20.v004.i01/gmcr.23925/20141028/images/medium/kgmc_a_10923925_f0004.gif"})
Figure 4. Arrows pointed to the blue stigma (A) blue pollen grain (B) after agroinfiltration with color involved genes
Three weeks post infiltration, the base of the flower petals turned blue with different intensity, the darkest blue color was detected in flowers infiltrated with the dfr/f3′5'h followed by dfr and finally with the f3′5'h (Fig. 5).
Published online:
01 January 2013Figure 5. The color petal's bases of the (A) non-agroinfiltrated gerbera flower controlled compared with gerbera with (B)dfr gene, (C) a combination of dfr and f3′5'h gens showing dark bases color and (D)f3′5'h gene
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kgmc20/2013/kgmc20.v004.i01/gmcr.23925/20141028/images/medium/kgmc_a_10923925_f0005.gif"})
Figure 5. The color petal's bases of the (A) non-agroinfiltrated gerbera flower controlled compared with gerbera with (B)dfr gene, (C) a combination of dfr and f3′5'h gens showing dark bases color and (D)f3′5'h gene
Expression of petunia-f3′5'h gene in stable transformation of gerbera
During the gerbera transformation routine work for cultivar “Express” with f3′5'h gene, different levels of blue color were observed in numbers of produced calli after one week of subculture into fresh regeneration medium (Fig. 6).
Published online:
01 January 2013Figure 6. Light and dark blue gerbera callus generated after the transformation with the f3'5'h gene.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kgmc20/2013/kgmc20.v004.i01/gmcr.23925/20141028/images/medium/kgmc_a_10923925_f0006.gif"})
Figure 6. Light and dark blue gerbera callus generated after the transformation with the f3'5'h gene.
Discussion
For particular target traits, transient assays are less time-consuming, less laborious and cost-effective.11 Agroinfiltration is the infiltration process of Agrobacterium suspensions into plant organs, which is a fast and highly efficient method for transient expression of desired gene(s). This method is an indispensable analytical tool for studying gene(s) function in plants that does not require expensive equipment.14,15
The procedure of producing transgenic Gerbera jemosonii plants needs long time. Gerbera is known by low transformation efficiency.6 Therefore, the agroinfiltrated method for gerbera flowers has been established. Transient gene expression via agroinfiltration facilitated the study of the effect of color involved genes on flowers before undergoing the production of transgenic gerbera. Transient gene expression has been evaluated16 using the agroinfiltarted method with gus reporter gene in rose petals. Yasmin and Debener16 reported that the efficiency of expression was found to be dependent on the rose genotype, flower age, position of petals within a flower, Agrobacterium strain and temperature of co-cultivation.
Green-fluorescent protein (gfp) is one of widely visualize tools used for establishing gene transfer and studying gene function. GFP emits a green colored light when it absorbs UV light.17,18 Although the gfp is efficiently used in plant cells as a reporter gene, our study clarifies that the gfp gene is not a suitable reporter gene used for optimizing the agroinfiltration condition in gerbera flowers, as the florescent greenish color was detected in the non-agroinfiltrated gfp-transgenic flowers under the UV light. Mercuri et al.19 previously reported that detecting macroscopic green fluorescence in transgenic Limonium flowers was difficult due to the presence of various floral pigments.
Transient expression is varied in different floret positions, as the inner florets of pink and white flower revealed highest expression. It was previously recorded that the middle petals of stage 2 in rose flowers were found to be optimal for the transient expression studies.16
Although the ability of acetosyringone to induce the virulence genes of Agrobacterium necessary to transfer T-DNA is known,20 the presence of acytosyringone in this study did not show any improvement in transient expression of gerbera petals. This is completely confirmed in previous studies, which investigated these factors in Arabidopsis and lettuce11 and in rose petals.16 The Agrobacterium density ranging between 0.1 and 1.7 at OD600 did not show differences in the GUS expression level; this is in agreement with the results of Yasmin and Debener16 who found that the density of Agrobacterium suspensions had no significant effect over a broad range of OD values (0.5 to 4.0). Only densities less than 0.5 did not lead to visible GUS expression. In contrary, it was observed that at least weak expression down to densities of OD600 = 0.1.14,21
Anthocyanins are found in different plant species, representing some of the most important natural pigments, which are responsible for the wide range of red to purple colors in flowers, fruits, seeds, leaves and stems.22,23 Aizza and Dornelas2 reported that Dihydroflavonol 4-reductase (DFR) and flavonoid 3′5’ hydroxylase (F3′5'H) are responsible for the anthocyanin synthesis.
In this study, the positive expression of both genes iris-dfr and petunia-f3'5'h represented by the color variations generated in petals, stigma and pollen grains indicated that both anthocyanin involved genes have a potential ability to change the color of gerbera flower and can be used in gerbera stable transformation to produce novel colors.
Furthermore, different levels of blue color that obtained in the produced gerbera calli after transforming with petunia-f3'5'h gene via Agrobacterium, indicated the successful expression of the gene f3′5'h. This positive expression indicates the capability of the introduced gene in changing the gerbera flower to blue or darker color. This result declares that the color responsible gene(s) under a constitutive promoter could be used as a proof of gene expression in early stage of stable transformation in gerbera.
Different levels of blue color could be reflected to the sucrose concentrations. Hiratsuka et al.24 reported that the soluble sugar content affect the anthocyanin biosynthesis in grape berry. In addition, previous reports25,26 stated that the different sucrose concentrations (20%, 40% and 60%) in tissue culture medium released more anthocyanin.
Previous reports27 proved that f3'5'h triggered the production of precursor delphindin which is derived anthocyanin that usually needs to produce blue flower. Moreover, blue rose production has been successfully developed via biotechnology by using the iris-dfr gene and viola f3'5'h gene after silencing the own rose-dfr gene by the RNAi strategy.28 The blue rose was produced after 13 y of collaborative research by an Australian company, Florigene, and a Japanese company, Suntory, creating a rose in 2004 by employing genetic engineering with blue pigments.
Conclusion
In the current study, several factors were investigated for adopting an agroinfiltration protocol for gerbera flowers, acetosyringone, Agrobacterium suspensions and its density, vacuum and syringe methods. It can be concluded from results that the gus reporter gene is a better tool for optimizing the gerbera flower agroinfiltration protocol rather than gfp. In addition, acetosyrengion and the density of the Agrobacterium culture were not considered as important factors when adopting an agroinfiltrated gerbera flowers protocol.
Furthermore, the best agro-infiltration protocol was obtained when using the vacuum method under the following condition: placing flowers upside down on a wetted filter paper for two days before infiltration and then soaking flowers in the Agrobacterium suspension using a desiccator for 30 min. The infiltrated flowers were then replaced onto a wetted filter paper and incubated at 22°C ± 2 at dark for three days, then transferred to the light. The high expression was detected after the 10 days. Finally, iris-dfr and petunia-f3'5'h genes can be successfully used for changing the flower color in the gerbera.
Materials and Methods
Plant material
Establishment of the agroinfiltration protocol was performed on the flowers of Gerbera jamesonii cultivars “Express” and “White Grizzly” with dark pink and white flowers, respectively. Routine transformation treatments were performed on in vitro cultures of Gerbera jamesonii cultivar “Express” with dark pink flowers.
Agrobacterium strains, genes and constructs
To establish the agroinfiltration protocol in gerbera petals, Agrobacterium strains GV3101 harboring ISV2678::35S::gus-intron or pEarleyGate103::35S::gfp-intron were used as source of the visual marker genes gus and gfp. To study the effect of iris-dfr and petunia-f3'5'h genes on changing the gerbera flower color, both genes according to the sequence in the GenBank were synthesized and cloned into the plasmid pBluescript II by the eurofins: mwg/operon.
Construction of dfr and f3'5'h into the binary vectors
The iris-dfr gene was subcloned into pCambia1390 under control of 35S promoter and nos terminator creating pCambia/dfr. This was performed by releasing the BamHI/EcoRI-dfr gene fragment from the pBluescript/dfr and ligated into the lineariezed vector by the same enzymes creating the binary vector pCambia/dfr. However, the petunia f3'5'h gene has been cloned into the binary vector pFGC5941 under the control of 35S promoter and ocs3 terminator. This was done by releasing the BamHI/XbaI-f3'5'h gene fragment from the pBluescript/f3′5’h and ligated into the lineariezed pFGC5941 vector by the same enzyme creating pFGC5941/ f3'5'h. The constructed plasmids were then transformed individually to Agrobacterium strain GV3101 via elctroproration. This was performed by adding 10–50 ng to chilled competent cells and transferred to pre-chilled electroporation cuvettes 2 mm gab sizes (Bio-rad), under the following condition: Gene Pulser unit “set volts” at setting to 2.5 KV, the “CAP” setting to 25 uFD and the resistance to 400 Ohm on the pulse controller unit.
Preparation of Agrobacterium cultures for agroinfiltration
Overnight cultures of Agrobacterium harboring gus, gfp, dfr or f3′5'h were grown at 28°C in liquid LB medium supplemented with 25 mg/l gentamycin. When cultures reached about 0.8 at OD600, cells were harvested and diluted to reach final concentration to 1.0 at OD600 by resuspending either in liquid LB or in a MES buffer composed of 10 mM MES/KOH pH5.6 and 10 mM MgCl2.29 Cultures were incubated for 2 h at room temperature before infiltration. For infiltration with both 35S::dfr and 35S::f3′5'h, Agrobacterium suspensions in MES buffer were mixed in a 1:1 ratio.
For increasing the Agrobacterium efficiency, bacterial suspensions were supplemented with 0, 100 and 150 µM of asetosyringone just before the infiltration. In addition, four different density of bacterial culture suspension (0.1, 0.5, 1.0 and 1.7 OD600) were used.
Flowers agroinfiltration protocol
Agroinfiltration was performed on both gerbera cultivars “Express” with dark pink color and “White Grizzly” with white color. Two different methods for infiltration, i.e., vacuum and syringe, were applied. For the syringe method, bacterial suspension was infiltrated from a hole punctured at the base of the petal using a one ml needless syringe11,30 . In the case of vacuum method, flowers were pre-treated through placing upside down on a wetted Whatman filter paper for two days. Flowers were submerged in the Agrobacterium suspension in Sigma Plastic boxes and placed inside a desiccator. Infiltration was then performed at 200 mbar for 5, 10, 20 and 30 min. The infiltrated petals were returned to a wet filter paper in a temperature-controlled incubator in the dark. The infiltrated petals were assayed for GUS and GFP expression after 3, 7, 10 and 14 d. The vacuum agroinfiltration protocol has been applied on the gerbera with dark pink flower color for studying the transient expression of the iris-dfr and petunia-f3′5'h genes.
Stable transformation with petunia-f3'5'h
A regeneration system for transformed gerbera was performed according to Hussein et al.6 The juvenile explants of gerbera cultivar Express were soaked with the Agrobacterium culture that contains f3′5'h, for 5–10 min. Subsequently, the explants were dried on sterile filter paper, then transferred to the regeneration medium Ge36 composed of MS medium31 2.0 BA mg/l and 0.25 mg/l ABA for 3 d under dark conditions. Thereafter, the explants were transferred to the regeneration medium with 500 mg/l cefotaxim. One week post-culturing the explants enlarged and calli were formed on the petiole bases, then callus were enlarged during a month. Subsequently, these calli were transferred to shoot formation media (MS containing 2 mg/l BA and 2 mg/l IAA), calli were transferred to a fresh medium for another 3–4 weeks.
GFP expression detection
Visual detection of GFP was performed using the fluorescent microscope (Leica MZ FLIII) with filters 450–490 nm. The transient expression in infiltrated gerbera flower petals was photographed with a Digital Camera Sony, Cybershot.
GUS expression detection
GUS expression was visually determined using the histochemical assay.32 An average of 20 petals in seven replicated experiments was evaluated. Samples were incubated in staining solution overnight at 37°C and chlorophyll was then bleached by fixation in 70% ethanol. GUS expression levels were visually rated on a scale from (0 to ++++), starting from no expression (score 0) to very high expression (++++).
Detection of iris-dfr and petunia-f3′5'h genes expression
Visual detection of the iris-dfr and petunia-f3′5'h genes expression was performed on the cross section of infiltrated and non-infiltrated petals using light microscope and was photographed with magnification of 40×.
| Flower color | Expression detection day | 3 d | 7 d | 10 d | 14 d | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| White Flowers | Floret levels | Outer floret | Disks florets | Outer floret | Disk florets | Outer floret | Disk florets | Outer floret | Disk florets | ||
| 10 min | + | ++ | ++ | +++ | ++++ | ++++ | + | + | |||
| 20 min | ++ | +++ | ++ | +++ | +++ | ++++ | + | ++ | |||
| 30 min | ++ | ++++ | ++ | ++++ | ++++ | ++++ | ++ | + | |||
| Syringe | ++++ | +++ | ++++ | +++ | |||||||
| Control | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |||
| Pink Flowers | 10 min | ++ | ++ | +++ | +++ | +++ | ++++ | ++ | + | ||
| 20 min | ++ | ++ | ++ | +++ | ++++ | ++++ | + | + | |||
| 30 min | ++ | ++++ | +++ | ++++ | ++++ | ++++ | +++ | ++ | |||
| Syringe | ++++ | +++ | ++++ | +++ | |||||||
| Control | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |||
Acknowledgments
We thank the Science, Technology and Development Fund (STDF) for funding this investigation under a project entitled “Engineering of gerbera plants for producing novel flower colors,” Dr P. Ratet, Institut des Sciences Vegetales (ISV), Centre National de la RechercheScientifique (CNRS), Gif-Sur-Yvette, France for kindly obtaining pISV2678 and the Modern Agriculture Company (PICO) for providing the in vitro cultures of Gerbera jamesonii cultivar “Express.”
No potential conflicts of interest were disclosed.
| Abbreviations: | |
| DFR | Dihydroflavonol 4-reductase |
| F3′5'H | Dihydroquercetin or by flavonoid 3′5'-hydroxylase |
| GFP | green-fluorescent protein |
| GUS | β-glucuronidase |
References
- Holton TA, Brugliera F, Tanaka Y. Cloning and expression of flavonol synthase from Petunia hybrida. Plant J 1993; 4:1003 - 10; http://dx.doi.org/10.1046/j.1365-313X.1993.04061003.x; PMID: 7904213 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Aizza LCB, Dornelas MC. A genomic approach to study anthocyanin synthesis and flower pigmentation in Passion flowers. J Nucleic Acids 2011; 2011:1 - 17; http://dx.doi.org/10.4061/2011/371517 [Crossref], [Google Scholar]
- Tanaka Y.. Flower color and cytochromes P450. Photochemistry review 2006; 5:283 - 91; http://dx.doi.org/10.1007/s11101-006-9003-7 [Crossref], [Google Scholar]
- Zabala G., Vodkin L.O.. A rearrangement resulting in small tandem repeats in the f3′5’h gene of white flower genotypes is associated with the Soybean W1 locus. Plant genome 2007; 2:113 - 24 [Google Scholar]
- Orlikowska T, Nowak E, Marasek A, Kuchrska D. Effect of growth regulators and incubation period on in vitro regeneration of adeventitious shoots from gerbera petioles. Plant Cell Tissue Organ Cult 1999; 59:95 - 102; http://dx.doi.org/10.1023/A:1006453905086 [Crossref], [Web of Science ®], [Google Scholar]
- Hussein G.M., Ismail I.A., Hashem M.E., ElMiniawy S.M., Abdallah N.A.. In vitro regeneration of gerbera. Landbauforschung-agriculture and forestry research 2008; 58:97 - 102 [Web of Science ®], [Google Scholar]
- Janssen BJ, Gardner RC. Localized transient expression of GUS in leaf discs following co-cultivation with Agrobacterium.. Plant Mol Biol 1989; 14:61 - 72; http://dx.doi.org/10.1007/BF00015655 [Crossref], [Web of Science ®], [Google Scholar]
- Kapila J, Rycke RD, Montagu MV, Angenon G. An Agrobacteriummediated transient gene expression system for intact leaves. Plant Sci 1997; 122:101 - 8; http://dx.doi.org/10.1016/S0168-9452(96)04541-4 [Crossref], [Web of Science ®], [Google Scholar]
- Fischer R, Vaquero-Martin C, Sack M, Drossard J, Emans N, Commandeur U. Towards molecular farming in the future: transient protein expression in plants. Biotechnol Appl Biochem 1999; 30:113 - 6; PMID: 10512789 [PubMed], [Web of Science ®], [Google Scholar]
- Horn ME, Woodard SL, Howard JA. Plant molecular farming: systems and products. Plant Cell Rep 2004; 22:711 - 20; http://dx.doi.org/10.1007/s00299-004-0767-1; PMID: 14997337 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Wroblewski T, Tomczak A, Michelmore R. Optimization of Agrobacterium mediated transient expression assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol J 2005; 3:259 - 73; http://dx.doi.org/10.1111/j.1467-7652.2005.00123.x; PMID: 17173625 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Hellens R.P., Allan A.C., Friel E.N., Bolitho K., Grafton K., Templeton M.D., Karunairetnam S., Gleave A.P., Laing W.A.. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Bio Med central plant methods 2005; 1:1 13 [PubMed], [Google Scholar]
- Lee MW, Yang Y. Transient expression assay by agroinfiltration of leaves. Methods Mol Biol 2006; 323:225 - 9; PMID: 16739580 [PubMed], [Google Scholar]
- Santos-Rosa M, Poutaraud A, Merdinoglu D, Mestre P. Development of a transient expression system in grapevine via agro-infiltration. Plant Cell Rep 2008; 27:1053 - 63; http://dx.doi.org/10.1007/s00299-008-0531-z; PMID: 18317773 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Zottini M, Barizza E, Costa A, Formentin E, Ruberti C, Carimi F, et al. Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells. Plant Cell Rep 2008; 27:845 - 53; http://dx.doi.org/10.1007/s00299-008-0510-4; PMID: 18256839 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Yasmin A, Debener T. Transient gene expression in rose petals via Agrobacterium infiltration. Plant Cell Tissue Organ Cult 2010; 102:245 - 50; http://dx.doi.org/10.1007/s11240-010-9728-2 [Crossref], [Web of Science ®], [Google Scholar]
- Soboleski MR, Oaks J, Halford WP. Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells. FASEB J 2005; 19:440 - 2; PMID: 15640280 [PubMed], [Web of Science ®], [Google Scholar]
- Prakesh CS. Green fluorescent protein. (1997). <http://199.88.21.38/AgHe%20Website/Biotech%20Website/Prakash_Publications% 7f%7f%7f%7f%7f/green_protein.html> Accessed 1998 Feb 18. [Google Scholar]
- Mercuri A, Sacchetti A, De Benedetti L, Schiva T, Alberti S. Green fluorescent flowers. Plant Sci 2001; 161:961 - 8; http://dx.doi.org/10.1016/S0168-9452(01)00497-6 [Crossref], [Web of Science ®], [Google Scholar]
- McCullen CA, Binns AN. Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annu Rev Cell Dev Biol 2006; 22:101 - 27; http://dx.doi.org/10.1146/annurev.cellbio.22.011105.102022; PMID: 16709150 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Kim MJ, Baek K, Park CM. Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis. Plant Cell Rep 2009; 28:1159 - 67; http://dx.doi.org/10.1007/s00299-009-0717-z; PMID: 19484242 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Grotewold E. The genetics and biochemistry of floral pigments. Annu Rev Plant Biol 2006; 57:761 - 80; http://dx.doi.org/10.1146/annurev.arplant.57.032905.105248; PMID: 16669781 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Rausher MD. Evolutionary transitions in floral color. Int J Plant Sci 2008; 169:7 - 21; http://dx.doi.org/10.1086/523358 [Crossref], [Web of Science ®], [Google Scholar]
- Hiratsukaa S, Onoderaa H, Kawaia Y, Kuboa T, Itohb H, Wada R. ABA and sugar effects on anthocyanin formation in grape berry cultured in vitro.. Sci Hortic (Amsterdam) 2001; 90:121 - 30; http://dx.doi.org/10.1016/S0304-4238(00)00264-8 [Crossref], [Web of Science ®], [Google Scholar]
- Zhong JJ, Xu GR, Yoshida T. Effects of initial sucrose concentration on excretion of anthocyanin pigments in suspended cultures of Perilla frutescens cells. World J Microbiol Biotechnol 1994; 10:590 - 2; http://dx.doi.org/10.1007/BF00367674 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Tsai PJ, Delvaa L, Yua TY, Huanga YT, Dufosséb L. Effect of sucrose on the anthocyanin and antioxidant capacity of mulberry extract during high temperature heating. Food Res Int 2005; 38:1059 - 65; http://dx.doi.org/10.1016/j.foodres.2005.03.017 [Crossref], [Web of Science ®], [Google Scholar]
- Tanaka Y, Katsumoto Y, Brugliera F, Mason J. Genetic engineering in floriculture. Plant Cell Tissue Organ Cult 2005; 80:1 - 24; http://dx.doi.org/10.1007/s11240-004-0739-8 [Crossref], [Web of Science ®], [Google Scholar]
- Katsumoto Y, Fukuchi-Mizutani M, Fukui Y, Brugliera F, Holton TA, Karan M, et al. Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin. Plant Cell Physiol 2007; 48:1589 - 600; http://dx.doi.org/10.1093/pcp/pcm131; PMID: 17925311 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Bazzini AA, Mongelli VC, Hopp HE, del Vas M, Asurmendi S. A practical approach to the understanding and teaching of RNA silencing in plants. Electron J Biotechnol 2007; 10:178 - 90; http://dx.doi.org/10.2225/vol10-issue2-fulltext-11 [Crossref], [Web of Science ®], [Google Scholar]
- Schöb H, Kunz C, Meins F Jr.. Silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigating sequence requirements for gene silencing. Mol Gen Genet 1997; 256:581 - 5; http://dx.doi.org/10.1007/s004380050604; PMID: 9413443 [Crossref], [PubMed], [Google Scholar]
- Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 1962; 15:473 - 97; http://dx.doi.org/10.1111/j.1399-3054.1962.tb08052.x [Crossref], [Web of Science ®], [Google Scholar]
- Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 1987; 6:3901 - 7; PMID: 3327686 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]