Regeneration Of Transgenic Plants Research Articles

Optimization process for de-novo organogenesis and regeneration of transgenic papaya plants using leaf tissue as explants

Optimization process for de-novo organogenesis and regeneration of transgenic papaya plants using leaf tissue as explants

Melatonin supplementation enhances browning suppression and improves transformation efficiency and regeneration of transgenic rough lemon plants (Citrus × jambhiri).

Enzymatic browning poses a significant challenge that limits in vitro propagation and genetic transformation of plant tissues. This research focuses on investigating how adding antioxidant substances can suppress browning, leading to improved efficiency in transforming plant tissues using Agrobacterium and subsequent plant regeneration from rough lemon (Citrus × jambhiri). When epicotyl segments of rough lemon were exposed to Agrobacterium, they displayed excessive browning and tissue decay. This was notably different from the 'Hamlin' explants, which did not exhibit the same issue. The regeneration process failed completely in rough lemon explants, and they accumulated high levels of total phenolic compounds (TPC) and polyphenol oxidase (PPO), which contribute to browning. To overcome these challenges, several antioxidant and osmoprotectant compounds, including lipoic acid, melatonin, glycine betaine, and proline were added to the tissue culture medium to reduce the oxidation of phenolic compounds and mitigate browning. Treating epicotyl segments with 100 or 200 μM melatonin led to a significant reduction in browning and phenolic compound accumulation. This resulted in enhanced shoot regeneration, increased transformation efficiency, and reduced tissue decay. Importantly, melatonin supplementation effectively lowered the levels of TPC and PPO in the cultured explants. Molecular and physiological analyses also confirmed the successful overexpression of the CcNHX1 transcription factor, which plays a key role in imparting tolerance to salinity stress. This study emphasizes the noteworthy impact of supplementing antioxidants in achieving successful genetic transformation and plant regeneration in rough lemon. These findings provide valuable insights for developing strategies to address enzymatic browning and enhance the effectiveness of plant tissue culture and genetic engineering methods with potential applications across diverse plant species.

An efficient method for hairy root transformation and transgenic plant regeneration of Melia azedarach

An efficient method for hairy root transformation and transgenic plant regeneration of Melia azedarach

Efficient transformation and regeneration of transgenic plants in commercial cultivars of Citrus aurantifolia and Citrus sinensis.

Citrus is one of the major horticultural crops with high economic and nutraceutical value. Despite the fact that conventional research has developed numerous improved varieties, citriculture is still susceptible to various stresses and requires innovative solutions such as genetic engineering. Among all the currently available modern approaches, Agrobacterium-mediated transformation is the most efficient method for introducing desired traits in citrus. However, being a non-host for Agrobacterium, various citrus species, including Citrus aurantifolia and Citrus sinensis, are recalcitrant to this method. The available reports on Agrobacterium-mediated transformation of commercial citrus cultivars show very low transformation efficiency with poor recovery rates of whole transgenic plantlets. Here, we provide an efficient and reliable procedure of Agrobacterium-mediated transformation for both C. aurantifolia and C. sinensis. This protocol depends on providing callus-inducing treatment to explants before and during Agrobacterium co-cultivation, using optimum conditions for shoot regeneration and modifying in-vitro micrografting protocol to combat the loss of transgenic lines. As transgenic citrus shoots are difficult to root, we also developed the ideal conditions for their rooting. Using this protocol, the whole transgenic plantlets of C. aurantifolia and C. sinensis can be developed in about ~ 4months, with transformation efficiency of 30% and 22% for the respective species.

Ticarcillin degradation product thiophene acetic acid is a novel auxin analog that promotes organogenesis in tomato.

Efficient regeneration of transgenic plants from explants after transformation is one of the crucial steps in developing genetically modified plants with desirable traits. Identification of novel plant growth regulators and developmental regulators will assist to enhance organogenesis in culture. In this study, we observed enhanced shoot regeneration from tomato cotyledon explants in culture media containing timentin, an antibiotic frequently used to prevent Agrobacterium overgrowth after transformation. Comparative transcriptome analysis of explants grown in the presence and absence of timentin revealed several genes previously reported to play important roles in plant growth and development, including Auxin Response Factors (ARFs), GRF Interacting Factors (GIFs), Flowering Locus T (SP5G), Small auxin up-regulated RNAs (SAUR) etc. Some of the differentially expressed genes were validated by quantitative real-time PCR. We showed that ticarcillin, the main component of timentin, degrades into thiophene acetic acid (TAA) over time. TAA was detected in plant tissue grown in media containing timentin. Our results showed that TAA is indeed a plant growth regulator that promotes root organogenesis from tomato cotyledons in a manner similar to the well-known auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). In combination with the cytokinin 6-benzylaminopurine (BAP), TAA was shown to promote shoot organogenesis from tomato cotyledon in a concentration-dependent manner. To the best of our knowledge, the present study reports for the first time demonstrating the function of TAA as a growth regulator in a plant species. Our work will pave the way for future studies involving different combinations of TAA with other plant hormones which may play an important role in in vitro organogenesis of recalcitrant species. Moreover, the differentially expressed genes and long noncoding RNAs identified in our transcriptome studies may serve as contender genes for studying molecular mechanisms of shoot organogenesis.

Regeneration of Transgenic Ficus lyrata via Indirect Somatic Embryogenesis and Isolation of Variants for Development of New Cultivars

Ficus lyrata is a popular ornamental foliage plant with unique violin- or guitar-shaped green leaves. In our previous study, a grapevine gene VvMybA1 was introduced into F. lyrata via Agrobacterium-mediated transformation, which resulted in the availability of purple-leaved plants. Since VvMybA1 is a transcription factor, the regeneration of transgenic purple-leaved plants might potentially produce variants with multicolored leaves. The objective of this study was to establish a method for regenerating purple-leaved F. lyrata and determine if variants with different coloration or variegation could be isolated from regenerated populations. Leaf explants derived from a completely purple-leaved transgenic plant were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 6-benzyladenine (BA) and α-naphthalene acetic acid (NAA). Callogenesis occurred in leaf explants, and a subculture of callus-borne explants on the same medium resulted in callus proliferation and the occurrence of somatic embryos. Somatic embryos were more effectively induced from callus pieces cultured on MS medium supplemented with 8.88 μM of BA and 0.27 μM of NAA. More than 30 embryos were induced per callus piece, and the embryos matured and converted to plantlets. MS medium supplemented with 4.92 μM of indolyl-3-butanoic acid (IBA) greatly improved root development. Plantlets were transplanted into soilless substrate and grown in a shaded greenhouse for morphological evaluation. Nine variants with different degrees of coloration and variegation were isolated from the regenerated populations. Our results suggest that the regeneration of transgenic plants that harbor a transcription factor, such as VvMybA1, could be an additional way of isolating novel variants for the development of new cultivars of ornamental plants.

Commercial scale genetic transformation of mature seed embryo explants in maize.

A novel, efficient maize genetic transformation system was developed using Agrobacterium-mediated transformation of embryo explants from mature seeds. Seeds from field grown plants were sterilized and crushed to isolate embryo explants consisting of the coleoptile, leaf primordia, and shoot apical meristem which were then purified from the ground seed bulk preparation. The infection of relevant tissues of seed embryo explants (SEEs) by Agrobacterium was improved by the centrifugation of the explants. Transgenic plants were obtained by multiple bud induction on high cytokinin media, followed by plant regeneration on hormone-free medium. Three different selectable markers (cp4 epsps, aadA, and nptII) were successfully used for producing transgenic plants. Stable integration of transgenes in the maize genome was demonstrated by molecular analyses and germline transmission of the inserted transgenes to the next generation was confirmed by pollen segregation and progeny analysis. Phenotypic evidence for chimeric transgenic tissue was frequently observed in initial experiments but was significantly reduced by including a second bud induction step with optimized cytokinin concentration. Additional improvements, including culturing explants at an elevated temperature during bud induction led to the development of a revolutionary system for efficient transgenic plant production and genome editing. To our knowledge, this is the first report of successful transgenic plant regeneration through Agrobacterium-mediated transformation of maize mature SEEs. This system starts with mature seed that can be produced in large volumes and the SEEs explants are storable. It has significant advantages in terms of scalability and flexibility over methods that rely on immature explants.

Agrobacterium tumefaciens mediated genetic transformation of Tripterygium wilfordii and its application to enhance the accumulation of triptolide

Triptolide, an important representative active compound of Tripterygium wilfordii, is hopeful to be developed into modern drug. However, the content of triptolide in natural plants is very low, which seriously restricts its development in the pharmaceutical industry. As a perennial woody vine, research on the genetic transformation of T. wilfordii is challenging and slow, which limits the process of improving triptolide content through biotechnology. In this study, the concentrations and combinations of growth regulators for T. wilfordii callus induction and regeneration were firstly optimized. The 2,4-dichlorophenoxyacetic acid (1 mg/L) was efficient to induce callus formation from leaf explants, while 2.0 mg/L 6-benzylaminopurine with 0.1 mg/L α-naphthaleneacetic acid had the best effect on regeneration from callus. Subsequently, the effect of different concentrations of screening agent (kanamycin or hygromycin B) on callus induction rate was determined. Then, Agrobacterium tumefaciens mediated genetic transformation was established and validated by two different plant expression vectors, pCambia1301 and pJCV53, corresponding to β-glucuronidase histochemical assay and monomeric red fluorescent protein fluorescence detection, respectively. It took 3–4 months from A. tumefaciens infection to complete transgenic plant regeneration. Based on this transformation system of T. wilfordii, the overexpression of miltiradiene synthase gene (TwMS) was performed. Real-time quantitative PCR analysis indicated that the expression levels of TwMS were highly up regulated in the transgenic plants compared with control line. The detection of high-performance liquid chromatography showed that overexpression of TwMS resulted in a 1.20–1.51-fold increase of triptolide in the transgenic lines. These results indicated that this efficient A. tumefaciens mediated genetic transformation provides a useful tool for future T. wilfordii genetic engineering studies to enhance the accumulation of triptolide in this economically important medicinal plant.

Genome-wide Characterization of the JmjC Domain-Containing Histone Demethylase Gene Family Reveals GhJMJ24 and GhJMJ49 Involving in Somatic Embryogenesis Process in Cotton.

The Jumonji C (JmjC) domain-containing protein family, an important family of histone demethylase in plants, can directly reverse histone methylation and play important roles in various growth and development processes. In the present study, 51 JmjC genes (GhJMJs) were identified by genome-wide analysis in upland cotton (Gossypium hirsutum), which can be categorized into six distinct groups by phylogenetic analysis. Extensive syntenic relationship events were found between G. hirsutum and Theobroma cacao. We have further explored the putative molecular regulatory mechanisms of the JmjC gene family in cotton. GhJMJ24 and GhJMJ49 were both preferentially expressed in embryogenic callus compared to nonembryogenic callus in cotton tissue culture, which might be regulated by transcription factors and microRNAs to some extent. Further experiments indicated that GhJMJ24 and GhJMJ49 might interact with SUVH4, SUVH6, DDM1, CMT3, and CMT1 in the nucleus, potentially in association with demethylation of H3K9me2. Taken together, our results provide a foundation for future research on the biological functions of GhJMJ genes in cotton, especially in somatic embryogenesis in cotton tissue culture, which is crucial for the regeneration of transgenic plants.

Production of Recombinant Proteins in Transgenic Tobacco Plants.

Nicotiana tabacum (the tobacco plant ) has numerous advantages for molecular farming, including rapid growth, large biomass and the possibility of both cross- and self-fertilization. In addition, genetic transformation and tissue culture protocols for regeneration of transgenic plants are well-established. Here, we describe the production of transgenic tobacco using Agrobacterium tumefaciens and the analysis of recombinant proteins, either in crude plant extracts or after purification, by enzyme-linked immunosorbent assays, sodium dodecyl sulfate polyacrylamide gel electrophoresis with western blotting and surface plasmon resonance.

Comparative Review of Plant Transformation Techniques

Plant transformation is now an important biotechnological tool in plant biology and a practical tool for transgenic plant development. There are many verified methods for stable introduction of novel genes into the nuclear genomes of diverse plant species. As a result, gene transfer and regeneration of transgenic plants are no longer the factors limiting the development and application of practical transformation systems for many plant species. However, the desire for higher transformation efficiency has stimulated work on not only improving various existing methods but also in inventing novel methods. Different methods of transferring the gene into plant cells have been developed and continuous efforts have been made to increase its efficiency. Both direct and indirect methods of gene transfer have their own merits and demerits. Efforts have been made continuously to eliminate drawbacks and to develop an easy and eco-friendly method to transfer foreign genes. Many methods of genetic transformation have been proposed and tried in the laboratories, but most of them result to transient expressions. However, transformation work based on particle bombardment with DNA coated micro projectiles and Agrobacterium mediated transformation have proved to be promising in producing stable transgenic plants from a range of plant species.

Transformation of immature wheat germ (Triticum aestivum L.) by particle bombardment

The aim of the research was to create an effective method for producing transgenic wheat plants suitable for a wide range of promising varieties, both spring and winter crops. The plant material was cultivated at temperatures ranging from 4 to 25°C, either in the dark or in the light, with a 16-hour photoperiod (16/8 - day / night). Osram L36/77 FLUORA and F36W/33 Cool White lamps were used for lighting. The composition of all nutrient media included macroand micro-salts, vitamins B5, phytohormones and carbohydrates. The pH of the medium was adjusted to 5.8 before autoclaving. The medium was sterilized in an autoclave at pressure of 1.2 atmospheres for 15 minutes. An effective method of regeneration of transgenic wheat plants for ballistic transformation has been developed. Plants obtained by this method are phenotypically normal and fully fertile. The transgenic insertion of the target gene is transmitted to the offspring in accordance with Mendel’s laws. The transformation efficiency was high for all the studied varieties and ranged from 1.4 to 7.8%.

CRISPR/Cas9 mediated targeted mutagenesis of LIGULELESS-1 in sorghum provides a rapidly scorable phenotype by altering leaf inclination angle.

Sorghum (Sorghum bicolor L. Moench) is one of the world's most cultivated cereal crops. Biotechnology approaches have great potential to complement traditional crop improvement. Earlier studies in rice and maize revealed that LIGULELESS-1 (LG1) is responsible for formation of the ligule and auricle, which determine the leaf inclination angle. However, generation and analysis of lg1 mutants in sorghum has so far not been described. Here, we describe CRISPR/Cas9 mediated targeted mutagenesis of LG1 in sorghum and phenotypic changes in mono- and bi-allelic lg1 mutants. Genome editing reagents were co-delivered to sorghum (var. Tx430) with the nptII selectable marker via particle bombardment of immature embryos followed by regeneration of transgenic plants. Sanger sequencing confirmed a single nucleotide insertion in the sgRNA LG1 target site. Monoallelic edited plantlets displayed more upright leaves in tissue culture and after transfer to soil when compared to wild type. T1 progeny plants with biallelic lg1 mutation lacked ligules entirely and displayed a more severe reduction in leaf inclination angle than monoallelic mutants. Transgene-free lg1 mutants devoid of the genome editing vector were also recovered in the segregating T1 generation. Targeted mutagenesis of LG1 provides a rapidly scorable phenotype in tissue culture and will facilitate optimization of genome editing protocols. Altering leaf inclination angle also has the potential to elevate yield in high-density plantings.

Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods

The combined Agrobacterium- and biolistic-mediated methods of cotton transformation provide a straightforward and highly efficient protocol for obtaining transgenic cotton. Cotton (Gossypium spp.) is the most important crop for natural textile fiber production worldwide. Nonetheless, one of the main challenges in cotton production are the losses resulting from insect pests, pathogens, and abiotic stresses. One effective way to solve these issues is to use genetically modified (GM) varieties. Herein, we describe an improved protocol for straightforward and cost-effective genetic transformation of cotton embryo axes, merging biolistics and Agrobacterium. The experimental steps include (1) Agrobacterium preparation, (2) seed sterilization, (3) cotton embryo excision, (4) lesion of shoot-cells by tungsten bombardment, (5) Agrobacterium-mediated transformation, (6) embryo co-culture, (7) regeneration and selection of transgenic plants in vitro, and (8) molecular characterization of plants. Due to the high regenerative power of the embryonic axis and the exceptional ability of the meristem cells for plant regeneration through organogenesis in vitro, this protocol can be performed in approximately 4-10weeks, with an average plant regeneration of about 5.5% (± 0.53) and final average transformation efficiency of 60% (± 0.55). The transgene was stably inherited, and most transgenic plants hold a single copy of the transgene, as desirable and expected in Agrobacterium-mediated transformation. Additionally, the transgene was stably expressed over generations, and transgenic proteins could be detected at high levels in the T2 generation of GM cotton plants. The T2 progeny showed no phenotypic or productivity disparity compared to wild-type plants. Collectively, the use of cotton embryo axes and the enhanced DNA-delivery system by combining particle bombardment and Agrobacterium infection enabled efficient transgenic plant recovery, overcoming usual limitations associated with the recalcitrance of several cotton genotypes subjected to somatic embryogenesis. The improved approach states this method's success for cotton genetic modification, allowing us to obtain GM cotton plants carrying traits, which are of fundamental relevance for the advancement of global agribusiness.

A novel and rapid method for Agrobacterium-mediated production of stably transformed Cannabis sativa L. plants

The development of genetically transformed plants is an elusive landmark in Cannabis sativa L. breeding. Despite its economic interest, at present, protocols for producing transgenic C. sativa plants are scarce. We studied the ability of hypocotyl, cotyledon and meristem explants from six C. sativa hemp varieties for transgenic plant regeneration. For this, we firstly evaluated in vitro regeneration rates of hypocotyls cultured in medium without plant growth regulators, and cotyledons cultured in medium supplemented with 0.4 mg L−1 of thidiazuron (TDZ) and 0.2 mg L−1 of α-naphthaleneacetic (NAA). Subsequently, the effect of different kanamycin concentrations (50, 100, 200, 500 and 750 mg L−1) on hypocotyl regeneration rate was determined. Finally, we assessed transformation rates after hypocotyl, cotyledon and meristem co-culture with Agrobacterium tumefaciens strain LBA4404 carrying the binary plasmid pBIN19 containing the β-glucuronidase (uidA) reporter gene and the kanamycin resistance neomycin phosphotransferase (nptII) genes. Plant transformation was validated through in vitro culture of regenerating shoots in kanamycin-containing selective regeneration medium, by GUS histochemical assay for uidA expression, and by PCR amplification of uidA and nptII genes. Our results showed that hypocotyls reached a higher regeneration rate (53.3 %) than cotyledons (18.1 %) without Agrobacterium co-culture. On the other hand, 100 mg L−1 kanamycin proved to be the best concentration in terms of regeneration rate (63.3 %) and spontaneous rooting rate of hypocotyl regenerating shoots (12.2 %), which displayed a 7.1 % of albinism rate. After co-culture with A. tumefaciens and subsequent culture in antibiotic-containing selective regeneration medium, hypocotyl was the best explant type achieving 23.1 % of regeneration rate, which contrasts with the 1.0 % regeneration rate detected for cotyledons. Transgenic plants were obtained from all explant types evaluated. Although there were significant differences among varieties evaluated, hypocotyls proved to be superior to already-developed meristems, reaching a transformation rate of 5.0 % and 0.8 % respectively. Despite the extremely low regeneration rate of cotyledons after A. tumefaciens co-culture, all cotyledon-derived regenerating shoots analyzed were successfully transformed. Our hormone-free protocol doubles the transformation rate of regenerating shoots, also producing transgenic plants three times faster than other already published protocols. This has relevant implications for C. sativa breeding, enabling not only genetic transformation, but also the use of new plant breeding techniques such as targeted genome editing by using CRISPR/Cas systems. This may foster the development of C. sativa varieties with specific biochemical profiles, or tolerant to biotic and abiotic stresses among others.

Advances and Perspectives in Tissue Culture and Genetic Engineering of Cannabis.

For a long time, Cannabis sativa has been used for therapeutic and industrial purposes. Due to its increasing demand in medicine, recreation, and industry, there is a dire need to apply new biotechnological tools to introduce new genotypes with desirable traits and enhanced secondary metabolite production. Micropropagation, conservation, cell suspension culture, hairy root culture, polyploidy manipulation, and Agrobacterium-mediated gene transformation have been studied and used in cannabis. However, some obstacles such as the low rate of transgenic plant regeneration and low efficiency of secondary metabolite production in hairy root culture and cell suspension culture have restricted the application of these approaches in cannabis. In the current review, in vitro culture and genetic engineering methods in cannabis along with other promising techniques such as morphogenic genes, new computational approaches, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR/Cas9-equipped Agrobacterium-mediated genome editing, and hairy root culture, that can help improve gene transformation and plant regeneration, as well as enhance secondary metabolite production, have been highlighted and discussed.

ЭКСПРЕССИЯ ПОВЕРХНОСТНОГО АНТИГЕНА BRUCELLA ABORTUS ОМР16 В РАСТЕНИИ NICOTIANA BENTHAMIANA

Brucellosis is one of the most contagious and infectious diseases with high incidence rates of cattle and humans in Kazakhstan. Using modern biotechnology techniques to develop vaccines that are reliable and affordable for farmers is an alternative solution to the problem. Plant viruses are often used as a vector for obtaining the expression of antigens of the pathogen. The grape virus A (BAB) is widely used among plant viruses. Brucella membrane proteins are the main objects of this research for futher development of vaccines or diagnostic texts against brucellosis, Membrane proteins (OMPs) are cell specific surface antigens that are immunogenic. OMPs are ideal candidates for the production of recombinant brucellosis vaccines. The object of the study was the outer membrane protein (Omp16), which plays an important role in the suppression of TNF-α production in macrophages. In this study, molecular cloning and analysis of the expression of the Omp16 gene, which was used to express the recombinant protein in plants, was carried out. We selected brucella from the vaccine strain of Brucella abortus 19, and the plant Nicotiana benthamiana, as the subjects for our research, since they widely used for the production of recombinant proteins, and they both appropriate for molecular genetic research. A viral vector was constructed to express the brucellosis antigen Omp16 in Nicotiana benthamiana plants. Nineteen explants were used for the regeneration of transgenic plants. As a result of this studies, the introduced gene of Omp16 was under the subgenomic promoter control of the ORF4 and was successfully expressed while maintaining the efficiency of expression in transgenic plants. The efficiency of viral vectors was evaluated at the level of transcription during expression of the protein Omp16 with viral proteins. The entire leaf blade was infiltrated; the density of Agrobacteria was 0.7. We were able to obtained transgenic plants Nicotiana benthamiana carrying the gene of capsid protein BAB, and the expression of the membrane antigen Omp16 in the viral vector was achieved by replacing the ORF4 with the Omp16 gene. The development of transgenic plants was carried out using agrobacterial transformation.

Direct Germline Transformation of Cotton Meristem Explants With No Selection.

Regeneration of transgenic plants without selectable markers can facilitate the development and commercialization of trait stacking products. A wide range of strategies have been developed to eliminate selectable markers to produce marker-free transgenic plants. The most widely used marker free approach is probably the Agrobacterium-based 2 T-DNA strategy where the gene-of-interest (GOI) and selectable marker gene are delivered from independent T-DNAs (Darbani et al., 2007). The selectable marker gene is segregated away from the GOI in subsequent generations. However, the efficiency of this 2 T-DNA system is much less than the traditional 1 T-DNA system due to the inefficiency of T-DNA co-transformation and high rate of con-integration between the GOI and selectable marker gene T-DNAs. In contrast, no selection transformation utilizes a single T-DNA carrying the GOI and thus eliminates the need to remove the selectable marker insert and potentially provides a viable alternative marker-free system. In this study, we reported the successful regeneration of transgenic cotton plants through Agrobacterium inoculation of seed meristem explants without the use of selective agents. Regeneration of putative transgenic plants were identified by GUS histo-chemical assay. The germline transmission of transgene to progeny was determined by segregation of pollen grains, immature embryos and T1 plants by GUS expression. The results were further confirmed by Southern analyses. The marker-free transformation frequency in this no selection system was similar to current meristem transformation system with selection (0.2%–0.7%). The strategy for further improvement of this system and its implication in improving cotton transformation pipeline and in developing transgene-free genome editing technology is discussed.

Endogenous auxin determines the pattern of adventitious shoot formation on internodal segments of ipecac.

Endogenous auxin determines the pattern of adventitious shoot formation. Auxin produced in the dominant shoot is transported to the internodal segment and suppresses growth of other shoots. Adventitious shoot formation is required for the propagation of economically important crops and for the regeneration of transgenic plants. In most plant species, phytohormones are added to culture medium to induce adventitious shoots. In ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), however, adventitious shoots can be formed without phytohormone treatment. Thus, ipecac culture allows us to investigate the effects of endogenous phytohormones during adventitious shoot formation. In phytohormone-free culture, adventitious shoots were formed on the apical region of the internodal segments, and a high concentration of IAA was detected in the basal region. To explore the relationship between endogenous auxin and adventitious shoot formation, we evaluated the effects of auxin transport inhibitors, auxin antagonists, and auxin biosynthesis inhibitors on adventitious shoot formation in ipecac. Auxin antagonists and biosynthesis inhibitors strongly suppressed adventitious shoot formation, which was restored by exogenously applied auxin. Auxin biosynthesis and transport inhibitors significantly decreased the IAA level in the basal region and shifted the positions of adventitious shoot formation from the apical region to the middle region of the segments. These data indicate that auxin determines the positions of the shoots formed on internodal segments of ipecac. Only one of the shoots formed grew vigorously; this phenomenon is similar to apical dominance. When the largest shoot was cut off, other shoots started to grow. Naphthalene-1-acetic acid treatment of the cut surface suppressed shoot growth, indicating that auxin produced in the dominant shoot is transported to the internodal segment and suppresses growth of other shoots.

Regeneration of transgenic plants by Agrobacterium-mediated transformation of Quercus ilex L. somatic embryos with the gene CsTL1

Here we describe for the first time a protocol for the genetic transformation of the holm oak Quercus ilex. Holm oak populations are seriously affected by a disorder known as oak decline syndrome. However, tolerant plants can be obtained by overexpression of pathogenesis-related (PR) proteins. The aim of the present study was to define a protocol for transforming somatic embryos (SEs) of holm oak with the gene CsTL1 encoding a chestnut thaumatin-like protein (PR protein). Lethal concentrations of kanamycin (kan) were determined in previous studies, in which SE formation was found to be inhibited by kan concentrations of 100 mg/L and above. Genetic transformation was only achieved when target explants were pre-cultured for one or 2 weeks, with a transformation efficiency of 2%. Transformation was also affected by genotype and was only successful in 2 of the 3 embryogenic lines evaluated. A total of 11 transformed lines (10 corresponding to line Q8 and 1 to line Q10–16) were obtained, all of which were maintained by secondary embryogenesis on proliferation medium (Schenk and Hildebrandt medium lacking plant growth regulators). The transgenic embryogenic lines were successfully cryopreserved by a vitrification-based procedure, thus enabling the lines to be preserved while tolerance tests were performed. The presence of CsTL1 in genomic DNA was confirmed by PCR, and expression of the gene was determined by qPCR. CsTL1 expression was up to 5.93 times higher in transgenic lines than in the corresponding untransformed line. Transgenic plants were produced from transformed SEs, with embryo conversion rates ranging from 2.8 to 66.7%.