Transformation and regeneration of loblolly pine: shoot apex inoculation with Agrobacterium

Loblolly pine (Pinus taeda L.) is the mostimportant tree species in US commerce and has much to gain through geneticengineering. This species can be transformed using particle bombardment andAgrobacterium; however, the regeneration of plants fromtransgenic tissues has been difficult and the recovery of transgenic plants hasbeen rare. A shoot-based and genotype-independent transformation methodemploying Agrobacterium tumefaciens was used to facilitaterecovery of plants and permit the transformation of elite germplasm. Shootsfrom4–6 week old seedlings and adventitious shoots from culture wereinoculated with A. tumefaciens EHA101 (pGUS3), or EHA105(pSSLa.3), subjected to selection and regenerated. Shoots that survivedexhibited expression of the uidA gene (GUS) in a patterncharacteristic of the either the CaMV35S promoter (pGUS3), or the larch RbcSpromoter (pSSLa.3) transferred. Recovered plants were screened using PCRamplification. Southern DNA analyses and amplification of the T-DNA borderjunction confirmed genomic integration of both transferreduidA and nptII genes. In this proofofconcept study, the overall recovery of P. taeda shoots wasfair (10–20%), while recovery of intact rooted plants was poor (>1%)due to difficulty in rooting. Recovery of intact rooted plants from inoculatedshoots of P. eldarica and P. radiatawas more efficient (10–30%). The addition of a shoot multiplication stepand effective rooting protocols will improve the efficiency of this genotypeindependent transformation method in P. taeda, and inotherPinus spp.     

Agrobacterium-mediated transformation of Vicia faba

Among the major grain legume crops, Vicia faba belongs to those where the production of transgenic plants has not yet convincingly been reported. We have produced stably transformed lines of faba bean with an Agrobacterium tumefaciens-mediated gene transfer system. Stem segments from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA101 or EHA105, carrying binary vectors conferring (1) uidA, (2) a mutant lysC gene, coding for a bacterial aspartate kinase insensitive to feedback control by threonine, and (3) the coding sequence for a methionine-rich sunflower 2S-albumin, each in combination with nptII as selectable marker. Kanamycin-resistant calluses were obtained on callus initiation medium at a frequency of 10–30%. Shoot regeneration was achieved on thidiazuron containing medium in a second culture step. A subsequent transfer of shoots to BA-containing medium was necessary for stem elongation and leaf development. Shoots were rooted or grafted onto young seedlings in vitro and mature plants were recovered. Molecular analysis confirmed the integration of the transgenes into the plant genome. Inheritance and expression of the foreign genes was demonstrated by Southern blot, PCR, western analysis and enzyme activity assays. Although at present the system is time-consuming and of relatively low efficiency, it represents a feasible approach for the production of genetically engineered faba beans.     

Efficient genetic transformation of red raspberry,Rubus ideaus L.

We have developed an efficient transformation system for red raspberry (Rubus ideaus L.) using Agrobacterium mediated gene transfer. Using this system we have successfully introduced a gene that encodes an enzyme, S-adenosylmethionine hydrolase (SAMase), in raspberry cultivars Meeker (MK), Chilliwack (CH) and Canby (CY). Leaf and petiole expiants were inoculated with disarmed Agrobacterium tumefaciens strain EHA 105 carrying either of two binary vectors, pAG1452 or pAG1552, encoding gene sequences for SAMase under the control of the wound and fruit specific tomato E4 promoter. Primary shoot regenerants on selection medium were chimeral containing both transformed and non-transformed cells. Non-chimeral transgenic clones were developed by iterative culture of petiole, node and leaf explants, on selection medium, from successive generations of shoots derived from the primary regenerants. Percent recovery of transformants was higher with the selection marker gene hygromycin phosphotransferase (hpt), than with neomycin phosphotransferase (nptII). Transformation frequencies of 49.6%, 0.9% and 8.1% were obtained in cultivars Meeker, Chilliwack and Canby respectively from petiole expiants using hygromycin selection. Genomic integration of transgenes was confirmed by Southern hybridization. Transgenic plants from a total of 218 independent transformation events (161 MK, 4 CH, 53 CY) have been successfully established in soil.Abbreviations ACCO amincocyclopropane-1-carboxylic acid oxidase - AS acetosyringone - BA 6-benzylaminopurine - CH cultivar Chilliwack - CY cultivar Canby - cv cultivar - hpt hygromycin phosphotransferase - IBA indolebutyric acid - MK cultivar Meeker - npt II neomycin phosphotransferase - SAMase S-adenosylmethionine hydrolase - TDZ Thidiazuron (N-phenyl-N'-l,2,3-thidiazol-5-ylurea)     

Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane

A reproducible method for transformation of sugarcane using various strains of Agrobacterium tumefaciens (A. tumefaciens) (AGL0, AGL1, EHA105 and LBA4404) has been developed. The selection system and co-cultivation medium were the most important factors determining the success of transformation and transgenic plant regeneration. Plant regeneration at a frequency of 0.8–4.8% occurred only when callus was transformed with A. tumefaciens carrying a newly constructed superbinary plasmid containing neomycin phosphotransferase (nptII) and β-glucuronidase (gusA) genes, both driven by the maize ubiquitin (ubi-1) promoter. Regeneration was successful in plants carrying the nptII gene but not the hygromycin phosphotransferase (hph) gene. NptII gene selection was imposed at a concentration of 150 mg/l paromomycin sulphate and applied either immediately or 4 days after the co-cultivation period. Co-cultivation on Murashige and Skoog (MS)-based medium for a period of 4 days produced the highest number of transgenic plants. Over 200 independent transgenic lines were created using this protocol. Regenerated plants appeared phenotypically normal and contained both gusA and nptII genes. Southern blot analysis revealed 1–3 transgene insertion events that were randomly integrated in the majority of the plants produced.     

An efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of “Egusi” melon (<Emphasis Type="Italic">Colocynthis citrullus</Emphasis> L.)

Cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were co-cultivated with Agrobacterium tumefaciens strain EHA101 carrying either plasmid pIG121-Hm harbouring genes coding for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII) or plasmid pBBRacdS harbouring these same genes along with a gene coding for 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Six weeks after co-cultivation, more than 35% of explants produced shoots in both cultivars. A DNA fragment corresponding to the gus gene or the selection marker nptII was amplified from genomic DNA extracted from leaves of regenerated plant clones rooted on hormone-free MS medium containing 100 mg/l kanamycin, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of one to three copies of the gus gene. Transformation efficiencies of cultivar NHC1-130 with EHA101(pIG121-Hm) and EHA101(pIG121-Hm, pBBRacdS) were 3.8% and 10%, respectively, which were higher than those obtained for cultivar ‘Ejagham’ of 2.4% and 5.7%, respectively. Co-cultivation medium containing 5 mg/l BA was effective for obtaining high transformation efficiency for both cultivars as compared with that without it.     

Agrobacterium tumefaciens-mediated transformation of Oncidium and Odontoglossum orchid species with the ethylene receptor mutant gene etr1-1

Oncidium and Odontoglosum orchid species have reduced display lives and are thus commercially less important than Phalaenopsis. One approach to prolonging display life permanently is to transform Oncidium and Odontoglossum with the ethylene receptor mutant gene etr1-1 from Arabidopsis under control of a flower specific promoter; this should reduce their sensitivity to exogenous ethylene. To achieve this it will be necessary to establish an efficient regeneration protocol using somatic embryogenesis and a routine Agrobacterium tumefaciens-mediated transformation procedure. Protocorm-like bodies (PLBs) of both orchid genera were regenerated from leaf tip explants. Leaf tips and PLBs, cultured in liquid and solid media, were compared as targets for genetic transformation. No transgenic shoots were obtained from leaf tips, while PLBs of Oncidium and Odontoglossum cultured on solid medium were successfully transformed with an expression vector containing nptII and gus genes driven by the cauliflower mosaic virus (CaMV) 35S promoter. Applying the A. tumefaciens strain EHA 105, transformation efficiencies of 1.3–2.7% were achieved for the investigated genotypes. Transformation with etr1-1 gene was achieved subsequently. Oncidium ‘Sweet Sugar’ has been successfully transformed and validated by PCR and Southern analysis.     

Agrobacterium tumefaciens-mediated transformation of Rhipsalidopsis gaertneri

A protocol for Agrobacterium tumefaciens-mediated genetic transformation of Rhipsalidopsis cv. CB5 was developed. Calluses derived from phylloclade explants and sub-cultured onto fresh callus induction medium over a period of 9–12 months were co-cultivated with A. tumefaciens LBA4404. Plasmid constructs carrying the nptII gene, as a selectable marker, and the reporter uidA gene were used. Transformed Rhipsalidopsis calluses with a vigorous growth phenotype were obtained by extended culture on media containing 600 mg l⁻¹ kanamycin. After 9 months of a stringent selection pressure, the removal of kanamycin from the final medium together with the culture of the transformed calluses under nutritional stress led to the formation of several transgenic adventitious shoots. Transformation was confirmed by GUS staining (for uidA gene), ELISA analysis and Southern blot hybridization (for the nptII gene). With this approach, a transformation efficiency of 22.7% was achieved. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for this cactus species.     

Stable transformation of peanut callus viaAgrobacterium-mediated DNA transfer

Transformed callus was produced from peanut (Arachis hypogaea L. cv. Okrun) hypocotyl explants after four days of co-cultivation withAgrobacterium tumefaciens strains EHA101, LBA4404 or ASE1 carrying the binary vector pKYLX71GUS on a defined medium followed by selection with kanamycin (200 mg l^–1). Transformed calluses were cultured as independent cell lines potentially derived from a single transformation event. Stable integration and expression of foreign gene(s) in the callus was confirmed by Southern and western blot analyses and enzyme assays. A few cell lines showed a single insert of the foreign gene. Using the above protocol, transformed peanut callus expressing the peanut stripe virus coat protein gene was obtained.     

Transformation of a recalcitrant grain legume, Vigna mungo L. Hepper, using Agrobacterium tumefaciens-mediated gene transfer to shoot apical meristem cultures

The efficiency of Vigna mungo L. Hepper transformation was significantly increased from an average of 1% to 6.5% by using shoot apices excised from embryonic axes precultured on 10 M benzyl-6-aminopurine (BAP) for 3 days and wounded prior to inoculation in Agrobacterium tumefaciens strain EHA105 carrying the binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a -glucuronidase (GUS) gene (gusA) interrupted by an intron. The transformed green shoots that were selected and rooted on medium containing kanamycin, and which tested positive for nptII gene by polymerase chain reaction, were established in soil to collect seeds. GUS activity was detected in whole T₀ shoots and T₁ seedlings. All T₀ plants were morphologically normal, fertile and the majority of them transmitted transgenes in a 3:1 ratio to their progenies. Southern analysis of T₁ plants showed integration of nptII into the plant genome.     

Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration

A reproducible and efficient transformation method was developed for the banana cv. Rasthali (AAB) via Agrobacterium-mediated genetic transformation of suckers. Three-month-old banana suckers were used as explant and three Agrobacterium tumefaciens strains (EHA105, EHA101, and LBA4404) harboring the binary vector pCAMBIA1301 were used in the co-cultivation. The banana suckers were sonicated and vacuum infiltered with each of the three A. tumefaciens strains and co-cultivated in the medium containing different concentrations of acetosyringone for 3 days. The transformed shoots were selected in 30 mg/l hygromycin-containing selection medium and rooted in rooting medium containing 1 mg/l IBA and 30 mg/l hygromycin. The presence and integration of the hpt II and gus genes into the banana genome were confirmed by GUS histochemical assay, polymerase chain reaction, and southern hybridization. Among the different combinations tested, high transformation efficiency (39.4 ± 0.5% GUS positive shoots) was obtained when suckers were sonicated and vacuum infiltered for 6 min with A. tumefaciens EHA105 in presence of 50 μM acetosyringone followed by co-cultivation in 50 μM acetosyringone-containing medium for 3 days. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into banana has been developed and that this transformation system could be useful for future studies on transferring economically important genes into banana.     

Agrobacterium-mediated transformation in Citrullus lanatus

Agrobacterium tumefaciens-mediated transformation was used to produce transgenic watermelon. Cotyledonary explants of Citrullus lanatus Thumb (cv. Daesan) were co-cultivated with Agrobacterium strains (LBA4404, GV3101, EHA101) containing pPTN289 carrying with bar gene and pPTN290 carrying with nptII gene, respectively. There was a significant difference in the transformation frequency between bacteria strains and selective markers. The EHA101/pPTN289 showed higher transformation frequency (1.16 %) than GV3101/pPTN289 (0.33 %) and LBA4404/pPTN289 or /pPTN290 (0 %). The shoots obtained (633 and 57 lines) showed some resistance to glufosinate and paromomycin, respectively. Of them, the β-glucuronidase positive response and PCR products amplified by bar and nptII specific primers showed at least 21 plants resistant to glufosinate and at least 6 plants to paromomycin. Southern blot analysis revealed that the bar gene integrated into genome of transgenic watermelon. Acclimated transgenic watermelons were successfully transplanted in the greenhouse and showed no phenotypic variation.     

Genetic transformation of selected mature cork oak (Quercus suber L.) trees

A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and -glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil.Abbreviations BA N⁶-Benzyladenine - GUS -Glucuronidase - MSSH Expression-proliferation medium - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase gene - uidA -Glucuronidase gene     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of blackgram: An assessment of factors influencing the efficiency of <Emphasis Type="Italic">uidA</Emphasis> gene transfer

Agrobacterium tumefaciens strain EHA105 carrying a binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron, was used for transformation of Vigna mungo cotyledonary node explants. Various factors such as preculture and wounding of explants, manipulations in inoculation and co-cultivation conditions were found to play a significant role in influencing tissue competence, Agrobacterium virulence and compatibility of both, for achieving the maximum transformation frequencies. The stable transformation with 4.31 % efficiency was achieved using the optimized conditions. The transformed green shoots that were selected and rooted on medium containing kanamycin and tested positive for nptII gene by polymerase chain reaction were established in soil to collect seeds. GUS activity was detected in leaves, roots, pollen grains and T₁ seedlings. Southern analysis of T₀ plants showed the integration of nptII into the plant genome.     

Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium

Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L⁻¹ gellan gum-solidified NDM containing 10 g L⁻¹ sucrose, 20 mg L⁻¹ hygromycin and 40 mg L⁻¹ meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 μM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.     

Genetic transformation and regeneration of rubber tree (Hevea brasiliensis Muell. Arg) transgenic plants with a constitutive version of an anti-oxidative stress superoxide dismutase gene

Agrobacterium tumefaciens-mediated genetic transformation and the regeneration of transgenic plants was achieved in Hevea brasiliensis. Immature anther-derived calli were used to develop transgenic plants. These calli were co-cultured with A. tumefaciens harboring a plasmid vector containing the H. brasiliensis superoxide dismutase gene (HbSOD) under the control of the CaMV 35S promoter. The -glucuronidase gene (uidA) was used for screening and the neomycin phosphotransferase gene (nptII) was used for selection of the transformed calli. Factors such as co-cultivation time, co-cultivation media and kanamycin concentration were assessed to establish optimal conditions for the selection of transformed callus lines. Transformed calli surviving on medium containing 300 mg l^-1 kanamycin showed a strong GUS-positive reaction. Somatic embryos were then regenerated from these transgenic calli on MS2 medium containing 2.0 mg l^-1 spermine and 0.1 mg l^-1 abscisic acid. Mature embryos were germinated and developed into plantlets on MS4 medium supplemented with 0.2 mg l^-1 gibberellic acid, 0.2 mg l^-1 kinetin (KIN) and 0.1 mg l^-1 indole-3-acetic acid. A transformation frequency of 4% was achieved. The morphology of the transgenic plants was similar to that of untransformed plants. Histochemical GUS assay revealed the expression of the uidA gene in embryos as well as leaves of transgenic plants. The presence of the uidA, nptII and HbSOD genes in the Hevea genome was confirmed by polymerase chain reaction amplification and genomic Southern blot hybridization analyses.Communicated by L. Peña     

Genetic transformation of golden pothos (<Emphasis Type="Italic">Epipremnum aureum</Emphasis>) mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

To establish a procedure for Agrobacterium tumefaciens-mediated transformation of golden pothos (Epipremnum aureum) plants, the effects of selection antibiotics and the preculture period of stem explants before A. tumefaciens infection were examined. Explants were co-cultivated with A. tumefaciens EHA105, harboring the plasmid pGWB2/cGUS, on a somatic embryo-inducing medium supplemented with acetosyringone. Resulting transgenic somatic embryos were screened on an antibiotic selection medium, and the transgenic pothos plants were regenerated on a germination medium. Hygromycin was the optimum selection antibiotic tested. The preculture period significantly affected the transformation efficiency, with explants precultured for one-day showing the best efficiency (5–30%). Both transformed hygromycin-resistant embryos and regenerated plants showed β-glucuronidase activity. Southern blot analysis confirmed transgene integration into the pothos genome. This reproducible transformation system for golden pothos may enable the molecular breeding of this very common indoor plant.     

Agrobacterium tumefaciens-mediated transformation of the aromatic shrub Lavandula latifolia

Transgenic plants of the aromatic shrub Lavandula latifolia (Lamiaceae) were produced using Agrobacterium tumefaciens-mediated gene transfer. Leaf and hypocotyl explants from 35–40-day old lavender seedlings were inoculated with the EHA105 strain carrying the nptII gene, as selectable marker, and the reporter gusA gene with an intron. Some of the factors influencing T-DNA transfer to L. latifolia explants were assessed. Optimal transformation rates (6.0 ± 1.6% in three different experiments) were obtained when leaf explants precultured for 1 day on regeneration medium were subcultured on selection medium after a 24 h co-cultivation with Agrobacterium. Evidence for stable integration was obtained by GUS assay, PCR and Southern hybridisation. More than 250 transgenic plants were obtained from 37 independent transformation events. Twenty-four transgenic plants from 7 of those events were successfully established in soil. -glucuronidase activity and kanamycin resistance assays in greenhouse-grown plants from two independent transgenic lines confirmed the stable expression of both gusA and nptII genes two years after the initial transformation. Evidence from PCR data, GUS assays and regeneration in the presence of kanamycin demonstrated a 1:15 Mendelian segregation of both transgenes among seedlings of the T1 progeny of two plants from one transgenic L. latifolia line.     

Evaluation of a cotyledonary node regeneration system forAgrobacterium-mediated transformation of pea (Pisum sativum L.)

Summary A rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed 6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression of the NOS gene in the selfed R₁ progeny. NPTII activity was observed in the R₂ generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this procedure were unsuccessful.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

Agrobacterium-mediated transformation and regeneration of cotton plants

Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and β-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as explants. Explants were cultured in a Murashige and Skoog (MS) medium containing various hormone combinations to induce shoot regeneration. The highest frequency of shoot formation was obtained from the shoot meristem. After selection in the MS medium containing kanamycin (50 mg/l), these tissues were tested by histochemical GUS assay. Shoots regenerated from excised shoot meristems or their halves were cultured for 4–6 weeks to obtain rooted plants, which then produced fully-developed plants and seeds in pots. Genomic integration of the kanamycin-resistance gene was detected by the PCR analysis. Seed germination percentage was 95% after the F1 seeds of transgenic cotton plants were cultured on half-strength MS medium supplemented with 50 mg/l kanamycin. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cotton was optimized. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 462–467. The text was submitted by the authors in English.