<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

Agrobacterium-mediated Japonica rice transformation: a procedure assisted by an antinecrotic treatment

An Agrobacterium-mediated transformation protocol for Japonica rice (cv. R321), using conventional genetic vectors and explants pretreated with antinecrotic compounds is presented. We evaluated the effect of two compounds with known antioxidant activity (ascorbic acid and cysteine) and silver nitrate on the viability of stem sections taken from in vitro rice plantlets, and on their interaction with Agrobacterium tumefaciens (At 2260) containing a shuttle vector bearing the gusand bar genes. After co-culture, calli formed on the callus-induction medium were supplemented with phosphinotricin and cefotaxime; putative transgenic plants were recovered on the regeneration medium after three months. All recovered plants were challenged with the herbicide BASTA under greenhouse conditions, and some resistant individuals were analyzed using PCR and a histochemical GUS test. Southern blot analysis of several R₁ transgenic plants indicated the presence of at least two intact bar gene copies per genome. Inheritance of the bar gene at the R₂ generation was confirmed. Antinecrotic pretreatment of the explants provides an adequate environment for the interaction of A. tumefacienswith the plant cells, thus allowing satisfactory transformation performance without the need of super-binary vectors and hyperinfective A. tumefaciens strains. This revised version was published online in June 2006 with corrections to the Cover Date.     

The expression and perpetuation of inherent somatic variation in regenerants from embryogenic cultures of Pennisetum glaucum (L.) R. Br. (pearl millet)

Summary Genetic analysis was conducted on the qualitative and quantitative traits of sexual progeny derived from embryogenic cultures of two inbred lines of Pennisetum glaucum (L.) R. Br. (pearl millet). These lines included a genetically stable inbred of Tift 23 BE and a genetic marker line, derived from Tift 23BE, which bore qualitative genetic markers for a dominant purple plant trait (P) and two recessive traits, early flowering (e₁) and yellow stripe (ys). Tissue culture regenerant populations (R₀) and progeny populations (R₁) produced from these plants by selfing showed no qualitative genetic variation when derived from the genetically stable inbred Tift 23BE. In contrast, stably inherited qualitative variation for a number of genetic markers was observed in R₀, R₁, and R₂ progeny of the genetic marker line. In a population of 1,911 plants regenerated over a 12-month period, 0.02% of the population lost or showed reduced expression of the purple plant trait and 92% of plants were chlorophyll deficient. Plants showing reduction or loss of anthocyanin synthesis also flowered later. None of the purple plants showed any significant variation in flowering time. The incidence of chlorophyll deficiency increased with time in culture, 51 % of the progeny regenerated after 1 month were chlorophyll deficient, while 100% of the plants regnerated after 12 months were chlorophyll deficient. Qualitative variation was also observed in control populations of the genetic marker line where 1 plant in a total of 1,010 lacked purple pigmentation and a total of 6% showed chlorophyll variation in the first generation (S₀). The presence of qualitative variation in controls suggests that the inherent variation present in the original explant was expressed and perpetuated in vitro. Quantitative variation was observed for a number of traits in the first sexual cycle (R₁) of the marker line but did not occur in a subsequent generation, suggesting that this variation was epigenetic.     

Rapid and reproducible Agrobacterium-mediated transformation of sorghum

A rapid and reproducible Agrobacterium-mediated transformation protocol for sorghum has been developed. The protocol uses the nptII selectable marker gene with either of the aminoglycosides geneticin or paromomycin. A screen of various A. tumefaciens strains revealed that a novel C58 nopaline chromosomal background carrying the chrysanthopine disarmed Ti plasmid pTiKPSF₂, designated NTL₄/Chry5, was most efficient for gene transfer to sorghum immature embryos. A NTL₄/Chry5 transconjugant harboring the pPTN290 binary plasmid, which carries nptII and GUSPlus ^TM expression cassettes, was used in a series of stable transformation experiments with Tx430 and C2-97 sorghum genotypes and approximately 80% of these transformation experiments resulted in the recovery of at least one transgenic event. The transformation frequencies among the successful experiments ranged from 0.3 to 4.5%, with the average transformation frequency being approximately 1% for both genotypes. Over 97% of the transgenic events were successfully established in the greenhouse and were fully fertile. Co-expression of GUSPlus ^TM occurred in 89% of the transgenic T₀ events. Seed set for the primary transgenic plants ranged from 145 to 1400 seed/plant. Analysis of T₁ progeny demonstrated transmission of the transgenes in a simple Mendelian fashion in the majority of events.     

Morphologic,cytogenetic, and enzymatic variation in tissue culture regenerated plants of apomictic old-world bluestem grasses (Bothriochloa sp.)

Somaclonal variant plants may be of use in broadening the germplasm base of plant species and providing useful stocks for cytogenetic investigations. This study was conducted to compare morphologic, cytogenetic and enzymatic characteristics of 21 R₁ (initial regenerate) bluestem,Bothriochloa sp., plants, visibly identified in a field-grown population of 522 plants as probable variants, with their respective R₀ (explant donor) progenitor. An R₂ seedling population was grown to ascertain the transmission of the variant R₁ phenotypes. All R₁ plants differed from their respective R₀ progenitors in one or more morphological characters. Foliage colour was the most pronounced difference in most cases. Four of the plants, three of which were dwarfed, produced no inflorescences. The R₁ plants tended to be shorter than R₀ progenitors and had corresponding decreases in lengths on inflorescences and lowest racemes. All R₁ plants of accessions 8911C and 8793 had an increase in chromosome number from2n=4x=40 to2n=5x=50. Three dwarfed R₁ plants, derived from accession 8873B, were aneuploids, two having2n=48 chromosomes and the third being a probable mixoploid with 55–58 chromosomes. Other plants of accession 8873B had the R₀ chromosome number. Fertility, as estimated by pollen stainability and seed set, generally was reduced in R₁ plants relative to the R₀. This reduction was not drastic, however, with all flowering plants having 45% or higher seed set. Apomixis apparently maintained fertility in all R₁ plants, including those with a pentaploid chromosome number. All R₁ plants differed from their respective R₀ plants in peroxidase and esterase banding patterns. All R₁ plants of accessions 8911C, and 8793, respectively, had identical peroxidase and esterase bands. For both enzyme systems two banding patterns were present in R₁ plants of accession 8873B, with 12 of 13 plants exhibiting common patterns. Examination of R₂ progeny plants confirmed the genetic transmission of the variant phenotypes and, by virtue of uniformity, indicated apomictic reproduction in the R₁ plants. The results demonstrate the production of potentially useful genetic and cytogenetic variant plants via tissue culture in these apomictic species.     

Co-transformation of gene expression cassettes via particle bombardment to generate safe transgenic plant without any unwanted DNA

The presence of resistant selectable marker genes and other added DNAs such as the vector backbone sequence in transgenic plant might be an unpredictable hazard to the ecosystem as well as to human health, which have affected the safe assessment of transgenic plants seriously. Using minimal gene expression cassette (containing the promoter, coding region, and terminator) without vector backbone sequence for particle bombardment is the new trend of plant genetic transformation. In the present paper, we co-transformed the selectable marker bar gene cassette and non-selected cecropinB gene cassette into rice (Oryza sativa L.) by particle bombardment, then eliminated the selectable marker bar gene in R₁ generation applying the hereditary segregation strategy and attained two safe transgenic plants only harboring cecropinB gene cassettes without any superfluous DNA. This is the fist report indicating that the combination of minimal gene cassettes transformation with the co-transformation and segregation strategy can generate selectable marker-free transgenic plants, which will promote the advancement in plant genetic engineering greatly.     

Assessment of leaf micromorphology after full desiccation of resurrection plants

Resurrection plants are unique among higher plants because of their ability to withstand long periods of dehydration without damages. In this study, leaf epidermis and palisade mesophyll of three resurrection species, Haberlea rodopensis, Ramonda serbica and Ramonda myconi, grown under full desiccation and benign conditions, were analyzed by differential interference contrast microscopy. Detailed investigation of adaxial and abaxial leaf surfaces revealed species-specific differences in the size and number of epidermal cells and stomatal density. The applied full desiccation did not cause any significant deviations of these parameters from the controls. There were no changes in the size and number of mesophyll cells as well. Analysis of stomatal patterning displayed essentially hypostomatic leaves, having stomata mainly abaxially positioned. The most significant change detected in the leaves of dehydration-treated plants was the increased formation of adaxially positioned trichomes. This increase was very high in R. myconi, where the adaxial leaf surface was fully covered by trichomes. Despite the existence of small species-specific differences, the results showed uniform desiccation-related responses of the studied resurrection species. The quantified leaf epidermal and mesophyll features are discussed with respect to their possible contribution to the desiccation tolerance of resurrection species.     

Production of soft rot resistant calla lily by expressing a ferredoxin-like protein gene (<Emphasis Type="Italic">pflp</Emphasis>) in transgenic plants

An efficient protocol for the Agrobacterium tumefaciens-mediated transformation of calla lily (Zantedeschia elliottiana (W. Wats.) Engl. cultivar ‘Florex Gold’) is described. Shoot basal discs were co-cultivated with A. tumefaciens C58C1 carrying a plasmid containing neomycin phosphotransferase (nptII) and plant ferredoxin-like protein (pflp) genes. After Agrobacterium co-cultivation, the shoot basal discs were exposed to 100 mg l⁻¹ kanamycin for selection. Twenty-eight out of 260 discs (10.8%) were found to have survived and produced shoot clusters. Twenty-six of these were confirmed to contain the pflp transgene by PCR, ending up in 10% transformation efficiency. The disease resistance investigation revealed that 18 transgenic plants exhibited resistance to soft rot disease caused by Erwinia carotovora subsp. carotovora. The presence of pflp gene was demonstrated by PCR, and its accumulation and activity was confirmed by Western blot and disease resistance assay. This was the first report to show the successful transformation and resistance to a bacterial pathogen in Zantedeschia. The protocol is useful for the quality improvement of calla lily through genetic transformation.     

Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture

Summary Previous experiments have revealed that the maize transposable element Activator (Ac) may become active during tissue culture. The objective of the present study was to determine whether a second transposable element, Suppressor-mutator (Spm), could also be activated in tissue culture and detected in regenerated maize plants. Approximately 500 R₁ progeny of 143 regenerated plants (derived from 49 embryo cell lines) were crossed as males onto an Spm-responsive tester stock. Spm activity was observed in two R₁ progeny of a single regenerated plant. This plant had been regenerated from Type II (friable embryogenic) callus of an A188 × B73 genetic background after 8 months in culture; the absence of Spm activity in four other plants regenerated from this same callus demonstrates that Spm activity was not present before culturing. Approximately 20 Spm-homologous DNA sequences were detected in each of the inbreds used to initiate the tissue cultures; it is presumed that one of these became active to give rise to Spm activity.     

An efficient grafting system for transgenic plant recovery in cotton (Gossypium hirsutum L.)

A successful transformation program relies on the number of survival plants in soil that can be obtained. Low recovery of transgenic plants is still a key restrictive factor for transgenic cotton production. In order to utilize genetic transformation in cotton breeding program effectively, an efficient grafting system for recovering plants derived from somatic embryogenesis following Agrobacterium infection and kanamycin selection was developed. Various aspects of in vitro grafting were examined in efforts to improve the efficiency of transformant recovery. Using strong seedling rootstocks was the first important step to obtain high rate of successful grafts. Scion size >0.6 cm and seedling rootstock at age of 6–12 days were appropriate for grafting. The successful grafting ratio was higher when using hypocotyls without radicle. Shoot-tip and shoot stem with axillary bud were also suitable for in vitro grafting, which meant we could significantly improve the survival ratio of transgenic plantlets, because one plantlet has a shoot-tip but several axillary buds. Based on our data, the period from in vitro seedling rootstock germination to transplant of grafts to field usually takes one month. Over 90% successful grafting ratio could be obtained under optimal conditions, which represented a significant improvement over currently available methods for recovery of cotton plantlet from somatic embryogenesis after transformation. Ex vitro grafting could also be used for plant recovery, which gave an average of successful grafting ratio of 71.9%. However, this method was strongly affected by environmental factors.     

Segregation analysis and RFLP mapping of the R1 and R3 alleles conferring race-specific resistance to Phytophthora infestans in progeny of dihaploid potato parents

Phytophthora infestans (Mont.) de Bary is the most important fungal pathogen of the potato (Solanum tuberosum). The introduction of major genes for resistance from the wild species S. demissum into potato cultivars is the earliest example of breeding for resistance using wild germplasm in this crop. Eleven resistance alleles (R genes) are known, differing in the recognition of corresponding avirulence alleles of the fungus. The number of R loci, their positions on the genetic map and the allelic relationships between different R variants are not known, except that the R1 locus has been mapped to potato chromosome V The objective of this work was the further genetic analysis of different R alleles in potato. Tetraploid potato cultivars carrying R alleles were reduced to the diploid level by inducing haploid parthenogenetic development of 2n female gametes. Of the 157 isolated primary dihaploids, 7 set seeds and carried the resistance alleles R1, R3 and R10 either individually or in combinations. Independent segregation of the dominant R1 and R3 alleles was demonstrated in two F₁ populations of crosses among a dihaploid clone carrying R1 plus R3 and susceptible pollinators. Distorted segregation in favour of susceptibility was found for the R3 allele in 15 of 18 F₁ populations analysed, whereas the RI allele segregated with a 1:1 ratio as expected in five F₁ populations. The mode of inheritance of the R10 allele could not be deduced as only very few F₁ hybrids bearing R10 were obtained. Linkage analysis in two F₁ populations between R1, R3 and RFLP markers of known position on the potato RFLP maps confirmed the position of the R1 locus on chromosome V and localized the second locus, R3, to a distal position on chromdsome XI.     

Agrobacterium-mediated transformation of the winter jujube (Zizyphus jujuba Mill.)

Winter jujube, a species that originated in China, is the most prominent elite variety of jujube (Zizyphus jujuba Mill.). Due to its economic value and its recalcitrance to improvements through traditional plant breeding approaches, genetic transformation techniques may have a great potential in providing the means to transfer one or more selected desirable traits into the plant genome. We reported here an improved protocol for the Agrobacterium-mediated transformation of shoot tips of winter jujube. We have identified a set of optimum transformation conditions that take into account Agrobacterium inoculum density, Agrobacterium incubation period, co-cultivation conditions, and vacuum (use of a vacuum pump to create a negative-pressure environment). The highest transformation frequency (5.2%) was obtained when the shoot-tip explants were infected for 10 min and co-cultured for 4 days with Agrobacterium at OD₆₀₀ 0.8 under a negative pressure of 0.5 × 10⁵ Pa. PCR and southern blot analyses confirmed the presence of transgenic plants and the stable integration of the target gene into the genome of regenerated plants. A histochemical staining analysis for GUS activity in the transgenic shoot tips also validated the efficiency of the transformation system.     

Analysis of a <Emphasis Type="Italic">LEA</Emphasis> gene promoter via <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the desiccation tolerant plant <Emphasis Type="Italic">Lindernia brevidens</Emphasis>

An Agrobacterium tumefaciens-based transformation procedure was developed for the desiccation tolerant species Lindernia brevidens. Leaf explants were infected with A. tumefaciens strain GV3101 harbouring a binary vector that carried the hygromycin resistance gene and an eGFP reporter gene under the control of a native dehydration responsive LEA promoter (Lb2745pro). PCR analysis of the selected hygromycin-resistant plants revealed that the transformation rates were high (14/14) and seeds were obtained from 13/14 of the transgenic lines. A combination of RNA gel blot and microscopic analyses demonstrated that eGFP expression was induced upon dehydration and ABA treatment. Comparison with existing procedures used to transform the well studied resurrection plant and close relative, Craterostigma plantagineum, revealed that the transformation process is both rapid and leads to the production of viable seed thus making L. brevidens a candidate species for functional genomics approaches to determine the genetic basis of desiccation tolerance.     

Expression of a C3 plant Rubisco SSU gene in regenerated C4 Flaveria plants

We report the successful transformation, via Agrobacterium tumefaciens infection, and regeneration of two species of the genus Flaveria: F. brownii and F. palmeri. We document the expression of a C₃ plant gene, an abundantly expressed ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit gene isolated from petunia, in these C₄ plants. The organ-specific expression of this petunia gene in Flaveria brownii is qualitatively identical to its endogenous pattern of expression.     

Transgenic mimicry of pathogen attack stimulates growth and secondary metabolite accumulation

Plant secondary metabolites, including pharmaceuticals, flavorings and aromas, are often produced in response to stress. We used chemical inducers of the pathogen defense response (jasmonic acid, salicylate, killed fungi, oligosaccharides and the fungal elicitor protein, cryptogein) to increase metabolite and biomass production in transformed root cultures of the medicinal plant, Withania somnifera, and the weed, Convolvulus sepium. In an effort to genetically mimic the observed effects of cryptogein, we employed Agrobacterium rhizogenes to insert a synthetic gene encoding cryptogein into the roots of C. sepium, W. somnifera and Tylophora tanakae. This genetic transformation was associated with stimulation in both secondary metabolite production and growth in the first two species, and in growth in the third. In whole plants of Convolvulus arvensis and Arabidopsis thaliana, transformation with the cryptogein gene led, respectively, to increases in the calystegines and certain flavonoids. A similar transgenic mimicry of pathogen attack was previously employed to stimulate resistance to the pathogen and abiotic stress. In the present study of biochemical phenotype, we show that transgenic mimicry is correlated with increased secondary metabolite production in transformed root cultures and whole plants. We propose that natural transformation with genes encoding the production of microbial elicitors could influence interactions between plants and other organisms.     

Genetic transformation of Coffea canephora by vacuum infiltration

Agrobacterium-mediated plant transformation protocol was evaluated as a fast method to obtain genetically modified Coffea canephora plantlets. Leaf explants were used as source material for Agrobacterium tumefaciens-mediated transformation involving a vacuum infiltration protocol, followed by a step of somatic embryogenesis induction and a final selection of the transformed plants. A. tumefaciens strain C58CI containing the binary vector pER10W-35SRed was used. PCR amplification of DsRFP gene and visual detection of the red fluorescent protein demonstrated 33% transformed embryos. The protocol presented here produces reliable transgenic coffee embryos in two months.     

Desiccation of plant tissues post-<Emphasis Type="Italic">Agrobacterium</Emphasis> infection enhances T-DNA delivery and increases stable transformation efficiency in wheat

Summary Factors influencing the Agrobacterium-mediated transformation of both monocotyledonous and dicotyledonous plant species have been widely investigated. These factors include manipulating Agrobacterium strains and plasmids, growth conditions for vir gene induction, plant genotype, inoculation and co-culture conditions, and the selection agents and their application regime. We report here a novel physical parameter during co-culture, desiccation of plant cells or tissues post-Agrobacterium infection, which greatly enhances transfer DNA (T-DNA) delivery and increases stable transformation efficiency in wheat. Desiccation during co-culture dramatically suppressed Agrobacterium growth, which is one of the factors known to favor plant cell recovery. Osmotic and abscisic acid treatments and desiccation prior to inoculation did not have the same enhancement effect as desiccation during co-culture on T-DNA delivery in wheat. An efficient transformation protocol has been developed based on desiccation and is suitable for both paromomycin and glyphosate selection. Southern analysis showed approximately 67% of transgenic wheat plants received a single copy of the transgene.     

The effect of co-cultivation and selection parameters on <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Chinese soybean varieties

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and l-cysteine (600 mg l⁻¹) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone. The optimized temperature for infected explant co-cultivation was 22°C. Regenerated transgenic shoots were selected and produced more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with 3 mg l⁻¹ hygromycin for 10 days, 5 mg l⁻¹ hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l⁻¹ hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. T₁ plants were analyzed and transmission of transgenes to the T₁ generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of a recalcitrant grain legume,lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medik)

A simple and reproducible Agrobacterium-mediated transformation protocol for a recalcitrant legume plant, lentil (Lens culinaris M.) is reported. Application of wounding treatments and efficiencies of three Agrobacterium tumefaciens strains, EHA105, C58C1, and KYRT1 were compared for T-DNA delivery into lentil cotyledonary node tissues. KYRT1 was found to be on average 2.8-fold more efficient than both EHA105 and C58C1 for producing transient β-glucuronidase (GUS) gene (gus) expression on cotyledonary petioles. Wounding of the explants, use of an optimized transformation protocol with the application of acetosyringone and vacuum infiltration treatments in addition to the application of a gradually intensifying selection regime played significant roles in enhancing transformation frequency. Lentil explants were transformed by inoculation with Agrobacterium tumefaciens strain, KYRT1 harboring a binary vector pTJK136 that carried neomycin phosphotransferase gene (npt-II) and an intron containing gusA gene on its T-DNA region. GUS-positive shoots were micrografted on lentil rootstocks. Transgenic lentil plants were produced with an overall transformation frequency of 2.3%. The presence of the transgene in the lentil genome was confirmed by GUS assay, PCR, RT-PCR and Southern hybridization. The transgenic shoots grafted on rootstocks were successfully transferred to soil and grown to maturity in the greenhouse. GUS activity was detected in vegetative and reproductive organs of T₀, T₁, T₂ and T₃ plants. PCR assays of T₁, T₂ and T₃ progenies confirmed the stable transmission of the transgene to the next generations.