In vivo analysis of chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence induction

Quantitative characteristics of photosynthetic electron transport were evaluated in vivo on the basis of the multi-exponential analysis of OJIP fluorescence transients induced by saturating actinic light. The OJIP fluorescence curve F(t), measured in Chlamydomonas reinhardtii cells, was transformed into the (1 − F _O/F(t)) × (F _V /F _M)⁻¹ transient, which is shown to relate to PS 2 closure. We assumed that kinetics of PS 2 closure during OJIP rise reflects time-separated processes related to the establishment of redox equilibrium at the PS 2 acceptor side (OJ), PQ pool (JI), and beyond Cyt b/f (IP). Three-exponential fitting was applied to (1 − F _O/F(t)) × (F _V /F _M)⁻¹ transient to obtain lifetimes and amplitudes of the OJ, JI, and IP components of PS 2 closure, which were used to calculate overall rates of reduction and re-oxidation of the PS 2 acceptor side, PQ pool, and intermediates beyond Cyt b/f complex. The results, obtained in the presence of inhibitors, oxidative reagents, and under different stress conditions prove the suggested model and characterize the introduced parameters as useful indicators of photosynthetic function.     

<Emphasis Type="Italic">GmFtsH9</Emphasis> expression correlates with in vivo photosystem II function: chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence transient analysis and eQTL mapping in soybean

Filamentation temperature-sensitive H (FtsH) is an ATP-dependent zinc metalloprotease involved in diverse biological functions. There are 12 FtsH proteins in Arabidopsis, among which AtFtsH2 plays an important role in regulating the turnover of photosystem II (PSII) reaction center D1 protein and the development of the photosynthetic apparatus. Here, we have identified 11 FtsH genes in the soybean genome by a bioinformatics approach. These soybean FtsH genes corresponded to seven Arabidopsis FtsH genes, suggesting that the main characteristics of soybean FtsH genes were formed before the evolutionary split of soybean and Arabidopsis. Phylogenetic analyses allowed us to clone a soybean AtFtsH2-like gene designated as GmFtsH9. The predicted protein of GmFtsH9 consists of 690 amino acids and contains three typical FtsH proteins conserved domains. The expression level of GmFtsH9 was determined in a soybean recombinant inbred line population under a pot experiment conducted for measuring chlorophyll a fluorescence transient parameters, photosynthetic CO₂ fixation rate (P _N), and seed yield. Expression quantitative trait loci (eQTL) mapping revealed two trans-acting eQTLs for GmFtsH9. The significant correlation of gene expression level with chlorophyll a fluorescence transient parameters and the presence of overlapping eQTL (QTL) between gene expression level and chlorophyll a fluorescence transient parameters indicated that GmFtsH9 could be involved in regulating PSII function. These results further lead to the understanding of the mechanism underlying FtsH gene expression, and contribute to the development of marker-assisted selection breeding programs for modulating soybean FtsH gene expression.     

<Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll <Emphasis Type="Italic">a</Emphasis> for activity

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA ₆₈₀₃) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA ₆₈₀₃ gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA₆₈₀₃ was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA₆₈₀₃ was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA₆₈₀₃ is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.     

Polyamines stimulate non-photochemical quenching of chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence in <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis>

Polyamines (PAs) are small metabolites that are produced and oxidized in chloroplasts with an obscure mode of action. Recently, we showed that qE is stimulated by PAs in higher plants (Nicotiana tabacum) and in genetically modified plants with elevated thylakoid-associated PAs (Ioannidis and Kotzabasis Biochim Biophys Acta 1767:1371–1382, 2007; Ioannidis et al. Biochim Biophys Acta 1787:1215–1222, 2009). Here, we investigated further their quenching properties both in vivo in green algae and in vitro is isolated LHCII. In vivo spermine up-regulates NPQ in Scenedesums obliquus about 30%. In vitro putrescine—the obligatory metabolic precursor of PAs—has a marginal quenching effect, while spermidine and spermine exhibit strong quenching abilities in isolated LHCII up to 40%. Based on available 3D models of LHCII we report a special cavity of about 600 Å³ and a near-by larger pocket in the trimeric LHCII that could be of importance for the stimulation of qE by amines.     

Underestimated chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence measurements on <Emphasis Type="Italic">Buxus microphylla</Emphasis> red winter leaves

Leaves under stressful conditions usually show downregulated maximum quantum efficiency of photosystem II [inferred from variable to maximum chlorophyll (Chl) a fluorescence (F_v/F_m), usually lower than 0.8], indicating photoinhibition. The usual method to evaluate the degree of photoinhibition in winter red leaves is generally by measuring the F_v/F_m on the red adaxial surface. Two phenotypes of overwintering Buxus microphylla ‘Wintergreen’ red leaves, with different measuring site and leaf thickness, were investigated in order to elucidate how red pigments in the outer leaf layer affected the Chl a fluorescence (F_v/F_m) and photochemical reflectance index. Our results showed that the F_v/F_m measured on leaves with the same red surface, but different leaf thickness, exhibited a slightly lower value in half leaf (separated upper and lower layers of leaves by removing the leaf edge similarly as affected by winter freezing and thawing) than that in the intact leaf (without removing the leaf edge), and the F_v/F_m measured on the red surface was significantly lower than that on the inner or backlighted green surface of the same thickness. Our results suggest that the usual measurement of F_v/F_m on red adaxial surface overestimates the actual degree of photoinhibition compared with that of the whole leaf in the winter.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Spectrally decomposed dark-to-light transitions in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Photosynthetic activity and respiration share the thylakoid membrane in cyanobacteria. We present a series of spectrally resolved fluorescence experiments where whole cells of the cyanobacterium Synechocystis sp. PCC 6803 and mutants thereof underwent a dark-to-light transition after different dark-adaptation (DA) periods. Two mutants were used: (i) a PSI-lacking mutant (ΔPSI) and (ii) M55, a mutant without NAD(P)H dehydrogenase type-1 (NDH-1). For comparison, measurements of the wild-type were also carried out. We recorded spectrally resolved fluorescence traces over several minutes with 100 ms time resolution. The excitation light was at 590 nm so as to specifically excite the phycobilisomes. In ΔPSI, DA time has no influence, and in dichlorophenyl-dimethylurea (DCMU)-treated samples we identify three main fluorescent components: PB–PSII complexes with closed (saturated) RCs, a quenched or open PB–PSII complex, and a PB–PSII ‘not fully closed.’ For the PSI-containing organisms without DCMU, we conclude that mainly three species contribute to the signal: a PB–PSII–PSI megacomplex with closed PSII RCs and (i) slow PB → PSI energy transfer, or (ii) fast PB → PSI energy transfer and (iii) complexes with open (photochemically quenched) PSII RCs. Furthermore, their time profiles reveal an adaptive response that we identify as a state transition. Our results suggest that deceleration of the PB → PSI energy transfer rate is the molecular mechanism underlying a state 2 to state 1 transition.     

<Emphasis Type="Italic">Kretzschmaria varians</Emphasis> sp. nov., <Emphasis Type="Italic">Xylaria coremiifera</Emphasis> sp. nov. and <Emphasis Type="Italic">Xylaria umbonata</Emphasis> sp. nov. from Costa Rica

Kretzschmaria varians, a species apparently related to K. micropus, is described as new. It is distinguished primarily by having asci with 2 to 8 ascospores with inconstant germination slit length and remains of synnemata on stromata and surrounding substrate. Xylaria coremiifera, described here as new, bears small fragile coremia on pulvinate stromata and the surrounding substrate. Asci often have fewer than 8 ascospores, most frequently 4. Xylaria umbonata, described here as new, produces perithecia around a central umbo that appears to be the remains of a synnema. Ascospores have long spiralling germination slits.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Diurnal cycle of chlorophyll fluorescence in <Emphasis Type="Italic">Phalaenopsis</Emphasis>

Chlorophyll (Chl) fluorescence of warm day/cool night temperature exposed Phalaenopsis plants was measured hourly during 48 h to study the simultaneous temperature and irradiance response of the photosynthetic physiology. The daily pattern of fluorescence kinetics showed abrupt changes of photochemical quenching (q_P), non-photochemical quenching (NPQ) and quantum yield of photosystem II electron transport (Φ_PSII) upon transition from day to night and vice versa. During the day, the course of Φ_PSII and NPQ was related to the air temperature pattern, while maximum quantum efficiency of PSII photochemistry (F_v/F_m) revealed a rather light dependent response. Information on these daily dynamics in fluorescence kinetics is important with respect to meaningful data collection and interpretation.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Leaf gas exchange and chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence of <Emphasis Type="Italic">Eucalyptus urophylla</Emphasis> in response to <Emphasis Type="Italic">Puccinia psidii</Emphasis> infection

One of the most important diseases of eucalyptus plantations is caused by the rust fungus Puccinia psidii. While the genetic basis of rust resistance has been addressed recently, little is known about the physiological aspects of Eucalyptus–P. psidii interaction. In order to fill this gap, we undertook a study investigating the effects of P. psidii infection on photosynthetic processes of two E. urophylla clones with contrasting resistance to the pathogen. Our results show that gas exchange and chlorophyll a fluorescence parameters were virtually unaffected in the resistant clone. In the susceptible clone, photosynthetic rates were chiefly constrained by biochemical limitations to carbon fixation. Photosynthesis was impaired only in symptomatic tissues since the reductions in photosynthetic rates were proportional to the diseased leaf area. Rust infection provoked chronic photoinhibition to photosynthesis in the susceptible clone. Overall, differences in the ability for light capture, use and dissipation may play a significant role in explaining the clonal differences in Eucalyptus in response to P. psidii infection. To our knowledge, this is the first report of the effect of rust infection on gas exchange and chlorophyll a fluorescence parameters in Eucalyptus.     

The Heat Shock Gene, <Emphasis Type="Italic">htpG</Emphasis>, and Thermotolerance in the Cyanobacterium, <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The htpG null mutant was obtained by inserting a chloramphenicol resistance cassette (Cm ^r) in the htpG coding sequence. The htpG null mutant (htpG), hsp16.6, and the double mutant, htpG::hsp16.6 cells showed little growth disadvantage at 30°C and 37°C, but not at 40°C. This suggests that HtpG and HSP16.6 proteins do not have an essential role during growth at normal and mildly elevated temperatures. Cell growth, cell survival rate, and oxygen electrode measurements demonstrated that htpG, hsp16.6, and htpG::hsp16.6 cells were sensitive to heat stress. Decreased basal and acquired thermotolerance was observed when mutants were heat shocked, with htpG::hsp16.6 being the most sensitive. A comparison of mutants showed that hsp16.6 was more sensitive to heat shock than htpG. Received: 19 November 2002 / Accepted: 19 December 2002     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Overexpression of <Emphasis Type="Italic">flv3</Emphasis> improves photosynthesis in the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6803 by enhancement of alternative electron flow

Background

To ensure reliable sources of energy and raw materials, the utilization of sustainable biomass has considerable advantages over petroleum-based energy sources. Photosynthetic algae have attracted attention as a third-generation feedstock for biofuel production, because algae cultivation does not directly compete with agricultural resources, including the requirement for productive land and fresh water. In particular, cyanobacteria are a promising biomass feedstock because of their high photosynthetic capability.

Results

In the present study, the expression of the flv3 gene, which encodes a flavodiiron protein involved in alternative electron flow (AEF) associated with NADPH-coupled O₂ photoreduction in photosystem I, was enhanced in Synechocystis sp. PCC6803. Overexpression of flv3 improved cell growth with corresponding increases in O₂ evolution, intracellular ATP level, and turnover of the Calvin cycle. The combination of in vivo¹³C-labeling of metabolites and metabolomic analysis confirmed that the photosynthetic carbon flow was enhanced in the flv3-overexpressing strain.

Conclusions

Overexpression of flv3 improved cell growth and glycogen production in the recombinant Synechocystis sp. PCC6803. Direct measurement of metabolic turnover provided conclusive evidence that CO₂ incorporation is enhanced by the flv3 overexpression. Increase in O₂ evolution and ATP accumulation indicates enhancement of the AEF. Overexpression of flv3 improves photosynthesis in the Synechocystis sp. PCC6803 by enhancement of the AEF.

     

Electron self-exchange and self-amplified posttranslational modification in the hemoglobins from <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 and <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002

Many heme proteins undergo covalent attachment of the heme group to a protein side chain. Such posttranslational modifications alter the thermodynamic and chemical properties of the holoprotein. Their importance in biological processes makes them attractive targets for mechanistic studies. We have proposed a reductively driven mechanism for the covalent heme attachment in the monomeric hemoglobins produced by the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 (GlbN) (Nothnagel et al. in J Biol Inorg Chem 16:539–552, 2011). These GlbNs coordinate the heme iron with two axial histidines, a feature that distinguishes them from most hemoglobins and conditions their redox properties. Here, we uncovered evidence for an electron exchange chain reaction leading to complete heme modification upon substoichiometric reduction of GlbN prepared in the ferric state. The GlbN electron self-exchange rate constants measured by NMR spectroscopy were on the order of 10²–10³ M⁻¹ s⁻¹ and were consistent with the proposed autocatalytic process. NMR data on ferrous and ferric Synechococcus GlbN in solution indicated little dependence of the structure on the redox state of the iron or cross-link status of the heme group. This allowed the determination of lower bounds to the cross-exchange rate constants according to Marcus theory. The observations illustrate the ability of bishistidine hemoglobins to undergo facile interprotein electron transfer and the chemical relevance of such transfer for covalent heme attachment.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Inhibition of hydrogen uptake in <Emphasis Type="Italic">Escherichia coli</Emphasis> by expressing the hydrogenase from the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis>sp. PCC 6803

Background  

Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H⁺ + 2e^- ↔ H₂ (g).     

Anatomy and photosystem II activity of <Emphasis Type="Italic">in vitro</Emphasis> grown <Emphasis Type="Italic">Aechmea blanchetiana</Emphasis> as affected by 1-naphthaleneacetic acid

Auxins are one of the main regulators of in vitro plant growth and development. However, the mechanisms, by which auxins, such as 1-naphthaleneacetic acid (NAA), affect in vitro root and leaf anatomy and photosystem function, remain unclear. Accordingly, the aim of the present study was to analyze the effect of different NAA concentrations on the anatomy and photosynthetic performance of in vitro-propagated Aechmea blanchetiana and to determine whether such a treatment affects micropropagated plants after acclimatization. In vitro-established A. blanchetiana plants were transferred to culture media that contained 0, 2, 4, or 6 μM NAA, and after 50 d, they were transplanted into plastic seedling trays with a commercial substrate and cultivated for 60 d in a greenhouse. The plants were evaluated after a 50-d in vitro NAA exposure (growth traits, chlorophyll α fluorescence, and root and leaf anatomy) and after 60 d of acclimatization in the greenhouse (root and leaf growth). Changes induced by NAA in root anatomy might improve uptake of minerals and sugars from the medium, thereby increasing the in vitro growth. In the leaves, the lowest chlorenchyma thickness and sclerenchyma area were observed in plants grown without NAA, and NAA exposure also improved photosystem II activity. The highest ex vitro growth rate was observed for plants that were propagated with 4 μM NAA. Therefore, the use of NAA during in vitro propagation can improve the anatomical and physiological quality of A. blanchetiana plants, as well as to improve ex vitro transfer.