Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with A Gene (CsTL1) Encoding a Chestnut Thaumatin-Like Protein.

We present a reproducible procedure for transforming somatic embryos of cork oak with the CsTL1 gene that codes for a thaumatin-like protein, in order to confer tolerance to Phytophthora cinnamomi. Different concentrations/combinations of the antibiotics carbenicillin and cefotaxime, as bacteriostatic agents, and kanamycin, as a selective agent, were tested. A lethal dose of 125 mg/L kanamycin was employed to select transgenic somatic embryos, and carbenicillin was used as a bacteriostatic agent at a concentration of 300 mg/L, which does not inhibit somatic embryo proliferation. The transformation efficiency was clearly genotype-dependent and was higher for the TGR3 genotype (17%) than for ALM80 (4.5%) and ALM6 (2%). Insertion of the transgenes in genomic DNA was confirmed by PCR analysis, whereas expression of the CsTL1 gene was evaluated by semi-quantitative real-time PCR (qPCR) analysis. A vitrification treatment successfully cryopreserved the transgenic lines generated. The antifungal activity of the thaumatin-like protein expressed by the gene CsTL1 was evaluated in an in vitro bioassay with the oomycete P. cinnamomi. Of the eight transgenic lines analyzed, seven survived for between one or two times longer than non-transgenic plantlets. Expression of the CsTL1 gene and plantlet survival days were correlated, and survival was generally greater in plantlets that strongly expressed the CsTL1 gene.

GAL4/GFP enhancer-trap lines for identification and manipulation of cells and tissues in developing Arabidopsis leaves.

BACKGROUND: Understanding developmental processes requires the unambiguous identification of cells and tissues, and the selective manipulation of the properties of those cells and tissues. Both requirements can most efficiently be satisfied through the use of GAL4/GFP enhancer-trap lines. No such lines, however, have been characterized for the study of early leaf development in the Columbia-0 reference genotype of Arabidopsis. RESULTS: Here we address this limitation by identifying and characterizing a set of GAL4/GFP enhancer-trap lines in the Columbia-0 background for the specific labeling of cells and tissues during early leaf development, and for the targeted expression of genes of interest in those cells and tissues. CONCLUSIONS: By using one line in our set to address outstanding questions in leaf vein patterning, we show that these lines can be used to address key questions in plant developmental biology.

Regulation of Somatic Embryo Development in Norway Spruce.

Somatic embryogenesis in Norway spruce combined with reverse genetics can be used as a model to study the regulation of embryo development in conifers. The somatic embryo system includes a sequence of developmental stages, which are similar in morphology to their zygotic counterparts. The system can be sufficiently synchronized to enable the collection and study of a large number of somatic embryos at each developmental stage.Here we describe a protocol for establishing transgenic cell lines in which genes of interest are upregulated or downregulated. Furthermore, we present methods for comparing embryo morphology and development in transgenic and control cell lines, including phenotyping the embryos, histological analysis, and tracking embryo development. The expression pattern of different genes is determined by GUS reporter assays.

The functional analysis of a wheat group 3 late embryogenesis abundant protein in Escherichia coli and Arabidopsis under abiotic stresses.

Late embryogenesis abundant (LEA) proteins are highly hydrophilic and thermostable proteins that could be induced by abiotic stresses in plants. Previously, we have isolated a group 3 LEA gene WZY3-1 (GenBank: KX090360.1) in wheat. In this study, the recombinant plasmid with WZY3-1 was transformed into Escherichia coli BL21 for protein expression. Furthermore, we transformed WZY3-1 into Arabidopsis. Overexpression of WZY3-1 in E.coli enhanced their tolerance to mannitol and NaCl. WZY3-1 protein could protect lactate dehydrogenase (LDH) under freeze and heat stress. Overexpression of WZY3-1 showed that WZY3-1 could help to improve the drought tolerance of transgenic Arabidopsis. In summary, our works show that WZY3-1 plays an important role in abiotic stress resistance in prokaryotic and eukaryotic organisms.

The ScFRK2 mitogen-activated protein kinase kinase kinase (MAP3K) is involved in early embryo sac development in Solanum chacoense.

Fertilization-related kinase (FRK) is a group of the mitogen-activated protein kinase kinase kinase (MAP3K or MEKK) that has proliferated in Solanaceae species. Studies on the wild potato Solanum chacoense have shown that three ScFRKs are directly involved in female gametophyte development. Decreasing the expression of ScFRK1 and ScFRK3 by RNA interference lead to embryonic sac development arrest at the functional megaspore (FM) stage. As for ScFRK2, the first FRK studied, antisense and co-suppression lines showed no abnormality, while overexpression lines lead to a drastic decrease in seed numbers, presumably caused by a conversion of the ovule into a carpel-like structure. Here we show that in ScFRK2 overexpression lines, carpel-like structures from the ovule cannot explain the drastic decrease in seeds considering the low percentage of these carpel-like structures but occurs in early ovule development as observed in Scfrk1 and Scfrk3 knockdown mutants were most ovules are arrested at the FM stage. The highly similar phenotype from knockdown mutants (Scfrk1 and Scfrk3) and ScFRK2 overexpression lines suggests that these MAP kinases could operate antagonistically through a balance between ScFRK1 and 3 on one side and ScFRK2 on the other. This study strongly suggests the importance of the FRK family expression levels during early stages of ovule development in Solanum chacoense embryo sac.

Infection of Embryonic Callus with Agrobacterium Enables High-Speed Transformation of Maize.

Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of maize; however, about eight months of in vitro culture are still required to isolate transgenic plants. Furthermore, genetic transformation of maize depends on immature embryos, which greatly increases costs. Here, we report a method that ensures the competency of an embryogenic callus secondary culture under laboratory conditions for Agrobacterium-mediated transformation. Moreover, pretreatment of the cell wall with a mixed lytic enzyme solution prior to Agrobacterium infection, significantly improved transformation efficiency and stability. Average stable transformation efficiency was approximately 30.39%, with peaks of 94.46%. Expression and phenotypic analysis of the Rsc reporter gene were tested in the T0 generation of transgenic plants. Using this system, we successfully regenerated transgenic maize plantlets within three months of the emergence of the embryogenic callus. Additionally, we reduced somaclonal variation accompanying prolonged culture of maize cells in the dedifferentiated state, thus facilitating the molecular breeding of maize.

Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred.

Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell's ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10-14 days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agent bialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocols described here, average transformation frequencies (number of bialaphos resistant T0 callus events per 100 explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16-21 weeks is needed using either protocol from the start of transformation experiments to obtaining putative transgenic plantlets with established roots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immature ears as transformation starting material and caring for transgenic plants for seed production are also described.

Advances in Agrobacterium-mediated Maize Transformation.

One of the major limitations of maize transformation is the isolation of a large number of immature embryos using the time-consuming manual extraction method. In this article, we describe a novel bulk embryo extraction method for fast isolation of a large number of embryos suitable for both biolistic- and Agrobacterium-mediated transformation. Optimal gene delivery and tissue culture conditions are also described for achieving high efficiency in Agrobacterium-mediated maize transformation using phosphomannose isomerase (PMI) as a selectable marker.

Tissue Culture (Somatic Embryogenesis)-Induced Tnt1 Retrotransposon-Based Mutagenesis in Brachypodium distachyon.

Brachypodium distachyon is a model grass species for economically important cereal crops. Efforts are in progress to develop useful functional genomic resources in Brachypodium. A tobacco retrotransposon, Tnt1, has been used successfully in recent past to generate insertional mutagenesis in several dicot plant species. Tnt1 retrotransposon replicates, transposes, and inserts at multiple random genomic locations in the plant genome. Transposition occurs only during somatic embryogenesis but not during seed transmission. We developed Brachypodium transgenic plants that can express the Tnt1 element. Here, we describe an efficient tissue culture-based approach to generate Tnt1 insertional mutant population using transgenic Brachypodium line expressing the Tnt1 retrotransposon.

Methods for Functional Transgenics: Development of Highly Efficient Transformation Protocol in Brachypodium and Its Suitability for Advancing Brachypodium Transgenics.

Plant transformation is an invaluable technique in plant genomics by which an extra foreign DNA sequence is introduced into a plant genome. Changing the plant genome is leading to owning new genetic characteristics. Model plant is a keystone in a study of the comprehensive plant phylum. Here, I describe an efficient method to transform the plant species Brachypodium distachyon which, due to its characters, is developing to be an important plant model.

Application of Tissue Culture and Transformation Techniques in Model Species Brachypodium distachyon.

Brachypodium distachyon has recently emerged as a model plant species for the grass family (Poaceae) that includes major cereal crops and forage grasses. One of the important traits of a model species is its capacity to be transformed and ease of growing both in tissue culture and in greenhouse conditions. Hence, plant transformation technology is crucial for improvements in agricultural studies, both for the study of new genes and in the production of new transgenic plant species. In this chapter, we review an efficient tissue culture and two different transformation systems for Brachypodium using most commonly preferred gene transfer techniques in plant species, microprojectile bombardment method (biolistics) and Agrobacterium-mediated transformation.In plant transformation studies, frequently used explant materials are immature embryos due to their higher transformation efficiencies and regeneration capacity. However, mature embryos are available throughout the year in contrast to immature embryos. We explain a tissue culture protocol for Brachypodium using mature embryos with the selected inbred lines from our collection. Embryogenic calluses obtained from mature embryos are used to transform Brachypodium with both plant transformation techniques that are revised according to previously studied protocols applied in the grasses, such as applying vacuum infiltration, different wounding effects, modification in inoculation and cocultivation steps or optimization of bombardment parameters.

Retarded germination of Nicotiana tabacum seeds following insertion of exogenous DNA mimics the seed persistent behavior.

Tobacco seeds show a coat-imposed dormancy in which the seed envelope tissues (testa and endosperm) impose a physical constraint on the radicle protrusion. The germination-limiting process is represented by the endosperm rupture which is induced by cell-wall weakening. Transgenic tobacco seeds, obtained by insertion of exogenous genes codifying for seed-based oral vaccines (F18 and VT2eB), showed retarded germination with respect to the wild type and modified the expression of endogenous proteins. Morphological and proteomic analyses of wild type and transgenic seeds revealed new insights into factors influencing seed germination. Our data showed that the interference of exogenous DNA influences the germination rather than the dormancy release, by modifying the maturation process. Dry seeds of F18 and VT2eB transgenic lines accumulated a higher amount of reserve and stress-related proteins with respect to the wild type. Moreover, the storage proteins accumulated in tobacco F18 and VT2eB dry seeds have structural properties that do not enable the early limited proteolysis observed in the wild type. Morphological observations by electron and light microscopy revealed a retarded mobilization of the storage material from protein and lipid bodies in transgenic seeds, thus impairing water imbibition and embryo elongation. In addition, both F18 and VT2eB dry seeds are more rounded than the wild type. Both the morphological and biochemical characteristics of transgenic seeds mimic the seed persistent profile, in which their roundness enables them to be buried in the soil, while the higher content of storage material enables the hypocotyl to elongate more and the cotyledons to emerge.

Microprojectile Bombardment Transformation of Date Palm Using the Insecticidal Cholesterol Oxidase (ChoA) Gene.

The overall objective of this work is to optimize the transformation system for date palm as a first step toward production of date palm clones resistant to noxious pests. A construct harboring the cholesterol oxidase (ChoA) gene, which renders plant resistance against insect attack, is introduced into embryogenic date palm callus using the PDS-1000/He particle bombardment system. The process involves the establishment of embryogenic callus cultures as well as immature embryo-derived microcalli that are used as target tissues for shooting and optimization of transformation conditions. This chapter in addition explains molecular and histochemical assays conducted to confirm gene integration and expression.

Genetic analyses of the inheritance and expressivity of autonomous endosperm formation in Hieracium with different modes of embryo sac and seed formation.

Background and Aims: Apomixis, or asexual seed formation, in polyploid Hieracium subgenus Pilosella species results in clonal progeny with a maternal genotype. An aposporous embryo sac forms mitotically from a somatic cell, without prior meiosis, while embryo and endosperm formation is fertilization independent (autonomous). The latter two developmental components are tightly linked in Hieracium . Recently, two plants, AutE196 and AutE24, were identified from two different crosses. Both form embryo sacs via the sexual route by undergoing meiosis, and embryo development requires fertilization; however, 18 % of embryo sacs can undergo autonomous endosperm (AutE) formation. This study investigated the qualitative and quantitative inheritance of the AutE trait and factors influencing phenotype expressivity. An additional focus was to identify the linkage group bearing the AutE locus in AutE196. Methods: Crosses and cytology were used to examine the inheritance of AutE from AutE24 and AutE196, and to reintroduce apomictic components into AutE plants, thereby changing the ploidy of developing embryo sacs and increasing the dosage of AutE loci. Markers from a Hieracium apomict linkage map were examined within a backcrossed AutE196 mapping population to identify the linkage group containing the AutE196 locus. Key Results: Qualitative autonomous endosperm in the AutE24 line was conferred by a single dominant locus, and the trait was transmitted through male and female gametes in AutE196 and AutE24. Expressivity of the trait did not significantly increase when AutE loci from AutE196 and AutE24 were both present in the progeny, within embryo sacs formed via apospory, or sexually derived embryo sacs with increased ploidy. It remains unclear if these are identical loci. Conclusions: The qualitative trait of autonomous endosperm formation is conferred by single dominant loci in AutE196 and AutE24. High expressivity of autonomous endosperm formation observed in apomicts requires additional genetic factors. Potential candidates may be signals arising from fertilization-independent embryo formation.

Protocols for In Vitro Propagation, Conservation, Synthetic Seed Production, Embryo Rescue, Microrhizome Production, Molecular Profiling, and Genetic Transformation in Ginger (Zingiber officinale Roscoe.).

Ginger is a rhizomatous plant that belongs to the family Zingiberaceae. It is a herbaceous perennial but cultivated as annual, with crop duration of 7-10 months. Ginger is native to India and Tropical South Asia. The tuberous rhizomes or underground stems of ginger are used as condiment, an aromatic stimulant, and food preservative as well as in traditional medicine. Ginger is propagated vegetatively with rhizome bits as seed material. Cultivation of ginger is plagued by rhizome rot diseases, most of which are mainly spread through infected seed rhizomes. Micropropagation will help in production of disease-free planting material. Sexual reproduction is absent in ginger, making recombinant breeding very impossible. In vitro technology can thus become the preferred choice as it can be utilized for multiplication, conservation of genetic resources, generating variability, gene transfer, molecular tagging, and their utility in crop improvement of these crops.

Transfer of Dicamba Tolerance from Sinapis arvensis to Brassica napus via Embryo Rescue and Recurrent Backcross Breeding.

Auxinic herbicides (e.g. dicamba) are extensively used in agriculture to selectively control broadleaf weeds. Although cultivated species of Brassicaceae (e.g. Canola) are susceptible to auxinic herbicides, some biotypes of Sinapis arvensis (wild mustard) were found dicamba resistant in Canada. In this research, dicamba tolerance from wild mustard was introgressed into canola through embryo rescue followed by conventional breeding. Intergeneric hybrids between S. arvensis (2n = 18) and B. napus (2n = 38) were produced through embryo rescue. Embryo formation and hybrid plant regeneration was achieved. Transfer of dicamba tolerance from S. arvensis into the hybrid plants was determined by molecular analysis and at the whole plant level. Dicamba tolerance was introgressed into B. napus by backcrossing for seven generations. Homozygous dicamba-tolerant B. napus lines were identified. The ploidy of the hybrid progeny was assessed by flow cytometry. Finally, introgression of the piece of DNA possibly containing the dicamba tolerance gene into B. napus was confirmed using florescence in situ hybridization (FISH). This research demonstrates for the first time stable introgression of dicamba tolerance from S. arvensis into B. napus via in vitro embryo rescue followed by repeated backcross breeding. Creation of dicamba-tolerant B. napus varieties by this approach may have potential to provide options to growers to choose a desirable herbicide-tolerant technology. Furthermore, adoption of such technology facilitates effective weed control, less tillage, and possibly minimize evolution of herbicide resistant weeds.

CLE19 expressed in the embryo regulates both cotyledon establishment and endosperm development in Arabidopsis.

Embryo and endosperm development are two well co-ordinated developmental processes in seed formation; however, signals involved in embryo and endosperm interactions remain poorly understood. It has been shown before that CLAVATA3/ESR-RELATED 19 (CLE19) peptide is able to trigger root meristem consumption in a CLV2-dependent manner. In this study, the role of CLE19 in Arabidopsis seed development was explored using antagonistic peptide technology. CLE19 is expressed in the epidermal layers of the cotyledon primordia, hypocotyl, and root cap in the embryo. Transgenic plants carrying an antagonistic CLE19 G6T construct expressed under the control of CLE19 regulatory elements exhibited a dominant seed abortion phenotype, with defective cotyledon establishment in embryos and delayed nuclear proliferation and cellularization in endosperms. Ectopic expression of CLE19 G6T in Arabidopsis under the control of an endosperm-specific ALE1 promoter led to a similar defect in cotyledon establishment in embryos but without an evident effect on endosperm development. We therefore propose that CLE19 may act as a mobile peptide co-ordinating embryo and endosperm development.

Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos.

Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.

Assessment of RNAi-induced silencing in banana (Musa spp.).

BACKGROUND: In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana. RESULTS: Using embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6-8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target sequences (26-nt and 19-nt). CONCLUSIONS: RNAi-induced silencing was achieved in banana, both at transient and stable level, resulting in significant reduction of gene expression and enzyme activity. The success of silencing was dependent on the targeted region of the target gene. The successful generation of transgenic ECS for second transformation with (an)other construct(s) can be of value for functional genomics research in banana.

Somatic embryogenesis, tetraploidy, and variant leaf morphology in transgenic diploid strawberry (Fragaria vesca subspecies vesca 'Hawaii 4').

BACKGROUND: The diploid (2n = 2x = 14) strawberry model plant Fragaria vesca ssp. vesca 'Hawaii 4' was employed for functional analysis of expressed DNA sequences initially identified as being unique to Fragaria and of unknown or poorly understood function. 'Hawaii 4' is prominent in strawberry research due to its ease of Agrobacterium-mediated transformation and regenerability, and its status as the source of the first complete strawberry genomic sequence. Our studies of a set of transformants have documented intriguing, construct-associated effects on leaf morphology, and provide important and unexpected insights into the performance of the 'Hawaii 4' transformation and regeneration system. RESULTS: Following Agrobacterium-mediated transformation of leaf explants with gene constructs carried by Gateway® vectors, plants were regenerated using a modified version of an established 'Hawaii 4' protocol. Expanding upon the findings of prior studies, we documented that plantlet regeneration was occurring via a somatic embryogenic rather than an organogenic developmental pathway. Among transformants, several variations in leaf morphology were observed. Unexpectedly, a particular leaf variant type, occurring in ~17% of all regenerants independent of construct type, was found to be attributable to tetraploidy. The tetraploidy-associated alteration in leaf morphology could be differentiated from the leaf morphology of diploid regenerants on the basis of a quantitative ratio of leaf dimensions: B/A, where B is the width of the central leaflet and A is the overall width of the trifoliate leaf. Variant effects on leaf morphology of four different transgenic constructs were also documented, and were in all cases distinguishable from the effects of tetraploidy. CONCLUSIONS: These results define opportunities to optimize the existing 'Hawaii 4' protocol by focusing on treatments that specifically promote somatic embryogenesis. The reported morphological metric and descriptions will guide future transgenic studies using the 'Hawaii 4' model system by alerting researchers to the potential occurrence of polyploid regenerants, and to differentiating the effects on leaf morphology due to polyploidy versus transgenic manipulations. Finally, an intriguing spectrum of leaf morphology alterations resulting from manipulation of expressed sequences of uncertain function is documented, providing a foundation for detailed studies of the respective genes and their functional roles.