Agrobacterium Tumefaciens Strain Research Articles

Enhancing the pathogenicity of Arthrobotrys conoides and A. oligospora against Meloidogyne javanica J2 by transferring of protease (Ac1) gene and evaluation of antagonistic capability of transgenic isolates

Antagonistic fungi are well-known as viable alternatives to chemical control of root-knot nematodes. In this paper, serine protease Ac1 as an important pathogenicity factor was used to enhance the antagonistic activity of Arthrobotrys conoides and A. oligospora against Meloidogyne javanica J2 (second stage juveniles). Ac1 gene was extracted from A. conoides and cloned in pCAMBIA1304 vector. The recombinant plasmid was then transferred to these fungi using two strains of Agrobacterium tumefaciens (LBA4404 and AGL1). Transgenic isolates were confirmed by PCR amplification of Hygromycin B resistance gene (hph) as selectable marker, protease assay using casein substrate and in vitro bioassay. Transferring of Ac1 gene by homologous recombination to A. conoides, increased protease activity. Our results showed that production of protease in both transgenic species was increased compared to wild type. Bioassay results indicated that pathogenicity rates of transformants, in both the number of traps formed and the number of trapped J2, increased compared to the wild type. The results showed that inhibition of root-knot nematode, M. javanica, was markedly increased by transgenic isolates of Arthrobotrys spp. compared to the wild types.

Genotype-independent and enhanced in planta Agrobacterium tumefaciens-mediated genetic transformation of peanut [Arachis hypogaea (L.)

Agrobacterium infection and regeneration of the putatively transformed plant from the explant remains arduous for some crop species like peanut. Henceforth, a competent and reproducible in planta genetic transformation protocol is established for peanut cv. CO7 by standardizing various factors such as pre-culture duration, acetosyringone concentration, duration of co-cultivation, sonication and vacuum infiltration. In the present investigation, Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1301-bar was used for transformation. The two-stage selection was carried out using 4 and 250mgl-1 BASTA® to completely eliminate the chimeric and non-transformed plants. The transgene integration into plant genome was evaluated by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot hybridization. Among the various combinations and concentrations analyzed, highest transformation efficiency was obtained when the 2-day pre-cultured explants were subjected to sonication for 6min and vacuum infiltrated for 3min in Agrobacterium suspension, and co-cultivated on MS medium supplemented with 150µM acetosyringone for 3days. The fidelity of the standardized in planta transformation method was assessed in five peanut cultivars and all the cultivars responded positively with a transformation efficiency ranging from minimum 31.3% (with cv. CO6) to maximum 38.6% (with cv. TMV7). The in planta transformation method optimized in this study could be beneficial to develop superior peanut cultivars with desirable genetic traits.

OPTIMIZATION OF FACTORS AFFECTING THE Agrobacterium tumefaciens- MEDIATED TRANSFORMATION OF Eucalyptus saligna

ABSTRACT This study aimed to evaluate the effect of factors that may affect the genetic transformation of cotiledonary explants of Eucalyptus saligna mediated by EHA105 strain of Agrobacterium tumefaciens. The vector pBI121 carrying gus gene under control of 35S CaMV promoter was used. The effect of the following factors was evaluated: explant pre-culture, use of different antibiotics and presence of acetosyringone (AS) in co-culture media. An antioxidant solution was also used during excision, containing ascorbic acid (250mg.L-1), citric acid (25mg.L-1) and PVP-40 (1g.L-1). Pre-culture of the explants before the co-culture with bacteria was done over a 4-day period in MS culture medium supplemented with 4.4µM BAP and 2.7ìM NAA. After theco-culture period, three concentrations of kanamycin (12.5;25 and 50mg.L-1) combined with 300mg.L-1 Augmentin® in the culture medium were tested The influence of the antibiotic was also evaluated by keeping the explants in a medium containing 50mg.L-1 Km and 300mg.L-1 Augmentin® or 500mg.L-1 cefotaxime. It was concluded that Augmentin® stimulates organogenesis, that a Km concentration of 12.5mg.L-1 allows selection of explants transformed with gus gene and, finally, the addition of AS (50ìM) to the liquid and solid co-culture media has a positive effect on gus gene expression. Moreover, the use of an antioxidant solution during cotyledon excision is dispensable and the pre-culture of the explants has no effect on bud regeneration or gus gene expression. A transformation efficiency of 1.5% was reached.

Agrobacterium tumefaciens-mediated transformation of Dendrobium lasianthera J.J.Sm: An important medicinal orchid.

A protocol for genetic transformation mediated by Agrobacterium tumefaciens and production of transgenic Dendrobium lasianthera has been developed for the first time. The 8-week-old protocorm explants were used as target of transformation with Agrobacterium tumefaciens strain LBA4404 carrying plasmid pG35SKNAT1. Several parameters such as infection period, Agrobacterium density, concentration of acetosyringone, and co-cultivation period were evaluated for the transformation efficiency. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's Multiple Range Test (DMRT) with p < 0.05. Subsequently, KNAT1 gene expression was confirmed by polymerase chain reaction (PCR) analysis. The highest efficiency of transformation (70%) obtained from protocorm explants infected with Agrobacterium culture was at the OD600 concentration of 0.6 for 30 min, and co-cultivated with acetosyringone 100 µM for 5 days. The results of confirmation by PCR analysis show that the KNAT1 gene has been integrated and expressed in the genome of Dendrobium lasianthera transgenic.

Agrobacterium tumefaciens-mediated transformation of Jatropha curcas L. with a polyhydroxyalkanoate gene (phaC)

Polyhydroxybutyrate is a component of bioplastics that is synthesized under the control of enzymes encoded by pha multigenes. The genes are naturally present in Ralstonia eutropha. However, the production of bioplastics in bacteria is inefficient because the bacterial biomass is relatively small compared with plants or fungi. As such, engineering techniques have been developed that enable pha genes to be inserted into plant biomass, and then be expressed in the biomass of the plant to produce polyhydroxybutyrate. The objectives of this study were to transform the tissue of Jatropha curcas using the phaC gene (a pha gene), to regenerate the transformed plant, and to confirm the presence of the inserted genes with PCR. The genetic transformation of J. curcas was mediated by Agrobacterium tumefaciens strain GV3101 containing pARTC by dipping the cotyledon tissue of J. curcas in a suspension of the bacterium for 30 min, followed by cocultivation for 3 d on Murashige and Skoog (MS) medium. The tissue was then placed on a selection medium, i.e. MS medium containing 13.3 µM BAP and 0.05 µM IBA with the addition of 20 mg/L kanamycin. The results showed that 12.35% of the tissue survived and regenerated into a shoot after 1–2 months. Molecular analysis of the transformed tissue was performed using phaC and nptII primers, in order to detect the presence of the phaC and nptII genes. Specific bands were detected at 659 bp and 700 bp, corresponding to the nptII primer and phaC primer, respectively.

Establishment of Miscanthus sinensis with decreased lignin biosynthesis by Agrobacterium–mediated transformation using antisense COMT gene

This study was to determine a transformation system for Miscanthus sinensis, and to optimize factors and conditions required for expression of an antisense caffeic acid O-methyltransferase gene in the M. sinensis (MsCOMT-AS). Transformation of callus derived from seeds and immature inflorescences of M. sinensis was established by using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pMBP1. In order to establish the stable transformation system, several transformation factors such as explant type, strain, co-culture periods, acetosyringone concentration, and selective markers were assessed. In this study, seven putative transgenic plants were obtained from callus transformation and plantlet regeneration. Various tests including PCR analysis and RT-PCR were used to detect the transgenic insert. The transgenic plants were also characterized for their agronomic and morphological characteristics, expression of MsCOMT-AS gene, and variation in lignocellulosic content. Biomass related traits such as plant height, number of leaves, length of leaf, stem diameter, fresh weight, dry weight, and cell size of the control plants were superior to transgenic plants. Total lignin content of transgenic plants was lower than that of the control plant due to reduced caffeic acid O-methyltransferase (COMT) gene expression related to lignin production. Cellulose and hemicellulose content in transgenic plants were not increased. Variation in cellulose and hemicellulose content had no correlation with variation in lignin content of transgenic plants. In conclusion, transgenic M. sinensis was obtained with down-regulated COMT gene. Lignin synthesis was decreased what offers possibility of crop modification for facilitated biofuel production.

Complete sequence of the tumor-inducing plasmid pTiChry5 from the hypervirulent Agrobacterium tumefaciens strain Chry5

Agrobacterium tumefaciens strain Chry5 is hypervirulent on many plants including soybean that are poorly transformed by other A. tumefaciens strains. Therefore, it is considered as a preferred vector for genetic transformation of plants. Here we report the complete nucleotide sequence of its chrysopine-type Ti-plasmid pTiChry5. It is comprised of 197,268 bp with an overall GC content of 54.5%. Two T-DNA regions are present and 219 putative protein-coding sequences could be identified in pTiChry5. Roughly one half of the plasmid is highly similar to the agropine-type Ti plasmid pTiBo542, including the virulence genes with an identical virG gene, which is responsible for the supervirulence caused by pTiBo542. The remaining part of pTiChry5 is less related to that of pTiBo542 and embraces the trb operon of conjugation genes, genes involved in the catabolism of Amadori opines and the gene for chrysopine synthase, which replaces the gene for agropine synthase in pTiBo542. With the exception of an insertion of IS869, these Ti plasmids differ completely in the set of transposable elements present, reflecting a different evolutionary history from a common ancestor.

Agrobacterium- Mediated Transformation of Tobacco Plants using Walnut Polyphenol Oxidase Gene

Polyphenol oxidase (PPO) is a well-recognized copper-containing enzyme which can catalyze oxidation of a great variety of phenolic compounds. PPO is found in diverse microorganisms, plants, and animals. To examine the anti-pathogenic role of PPO in walnut (Juglans regia L.), Agrobacterium tumefaciens strain LBA4404 was used to transform tobacco (Nicotiana tobacum L.) explants. Recombinant binary vector pBI121, containing the walnut JrPPO gene and the nptII gene as a selectable marker, was incorporated into A. tumefaciens. MS medium supplemented with 50 mg/l of kanamycin and 200 mg/l of cefotaxime was used as a selection medium. Plantlets were regenerated from putatively transgenic calli and polymerase chain reaction and Southern blot analysis were performed to confirm the integration of JrPPO into the genome. To evaluate the function of PPO in pathogen resistance, transgenic tobacco plants were exposed to Pseudomonas syringae pv. tabaci. According to symptom progress and quantitative analyses, although there was no significant difference in transgenic tobacco, but mean comparison showed that disease severity of transgenic tobacco was less than wild plants. Finally, it may be concluded that PPO has a notable function in the resistance process in walnut, but tobacco transgenic plants might not be a suitable test plant to examine the resistance role of PPO in walnut.

The effects of β-lactam antibiotics and hygromycin B on de novo shoot organogenesis in apple cv. Golden Delicious

Since the genetic transformation of the apple is strongly genotype-dependent and generally inefficient, the evaluation of factors affecting shoot regeneration are crucial for the establishment of a successful transformation process. In this report, we evaluated the effects of the ?-lactam antibiotics meropenem and timentin on in vitro regeneration via de novo shoot organogenesis from leaf explants of apple cv. Golden Delicious, as well as on the growth of the Agrobacterium tumefaciens strain EHA 105, and compared them with the commonly used ?-lactam cefotaxime. Also, we report for the first time the effect of hygromycin B as a selective agent in the domesticated apple, as regards shoot regeneration and shoot multiplication efficiency. We observed that cefotaxime and timentin at concentrations higher than 100 mg L-1 were sufficient to prevent Agrobacterium growth during a two-week period, while meropenem exhibited an inhibitory effect on bacterial growth at all tested concentrations (25-150 mg L-1). Cefotaxime at a concentration of 300 mg L-1 increased the number of regenerated shoots per explant (9.39) in comparison with the control (7.67). In contrast to cefotaxime, meropenem and timentin caused a decrease in shoot regeneration efficiency, but larger and more developed shoots were obtained on meropenem (25-125 mg L-1) after the same period of cultivation. Hygromycin B at a concentration of 5 mg L-1 or higher completely inhibited shoot regeneration and induced explant tissue necrosis. Therefore, the selection procedure with a final concentration of 4 mg L-1 throughout organogenesis and 10 mg L-1 for further shoot growth and multiplication is recommended for an efficient transformation process in apple cv. Golden Delicious.

In planta agrobacterium-Mediated genetic transformation in okra {Abelmoschus esculentus (L.) moench}

In Planta methods have been developed in several plant species to overcome the problems associated with their tissue culture-based transformation. In present study, a method for genetic transformation in okra (Abelmoschus esculentus) was established using in planta Agrobacterium-mediated genetic transformation. The embryos of imbibed seeds pricked from meristematic region of plumule were used as explants for transformation. Agrobacterium tumefaciens strain EHA105 was carrying cry1Ac gene against fruit and shoot borer, CaMV 35S as promoter and nptII as plant selectable marker gene. The putative transgenic plants were confirmed by amplifying transgene through polymerase chain reaction.

Screening of Colombian soybean genotypes for Agrobacterium mediated genetic transformation conferring tolerance to Glyphosate

Soybean is a very important crop worldwide due to its multiple uses as raw material for industry and to its high nutritional value. Colombia consumes a large amount of imported soybean because domestic production does not supply demand. There are soybean varieties adapted to the environmental conditions in the Colombian territory, but none of them have been enhanced by genetic engineering to confer competitive advantages compared to imported product. In this research, the Colombian soybean varieties SK7, p29 and Soyica p34 ability to be genetically transformed by Agrobacterium tumefaciens strains AGL0 and EHA105 using a Glyphosate tolerance cassette was tested. It was found that SK7 variety presented a better regeneration performance from the cotyledonary node, and also had the highest transformation frequency with AGL0 strain. The p29 variety was also transformed, but a lower efficiency was registered. It was not possible to transform Soyica p34 variety under the established parameters. This research is an advance towards the construction of a platform to enhance the generic transgenic crops in Colombia.

Genetic engineering of sweet potatoes (Ipomoea batatas) using isopentenyl transferase gene for enhanced drought tolerance

Nawiri SO, Oduor RO, Jalemba AM. 2017. Genetic engineering of sweet potatoes (Ipomoea batatas) using isopentenyl transferase gene for enhanced drought tolerance. Asian J Agric 1: 85-99. Approximately 70% of yield crop reduction worldwide is caused by drought. Due to severe drought which happened many times as a result of climate change, substantial yield deprivation is usual among the major cereals such as maize, wheat, and barley.. Therefore, drought tolerant crops that still yield amidst erratic climatic phenomenon are greatly needed. Due to its capability to produce high yield in a short period, sweet potato is suitable for cultivation in regions with limited or erratic rain water supply where other food crops cannot grow easily. Nevertheless, its sensitivity to water deficit may lead to the adverse crop growth and yield. By conventional hybridization method, sweet potato is tried to be improved, but it gives unsatisfied results due to its high male sterility, sexual incompatibility and hexaploid nature of its genome.The aim of this study, therefore, is to develop new varieties of sweet potato with improved tolerance to water-deficit stress for sustainable production of sweet potato under water-limited conditions. Three sweet potato genotypes: Jewel, Kemb36, and Ksp36 were transformed using isopentenyl transferase gene (IPT) that delays drought-induced senescence via up-regulation of cytokinin biosynthesis, under the control of a waterdeficit responsive and maturation specific promoter (PSARK). The PNOV-IPT gene construct was introduced into sweet potato to evaluate their transformability and regenerability. It is done via Agrobacterium tumefaciens strain EHA101 and the plants subsequently regenerated via somatic embryogenesis. Jewel genotype recorded the highest transformation and regeneration frequency followed by Kemb36 and KSP36. Calli were cultured on media supplemented with various mannose concentrations to evaluate the suitability of mannose as a selectable marker for sweet potato, and it was figured out that 30 g/L concentration was optimal for selection of transformed events. At the time of PCR analysis, Jewel had the highest transformation efficiency followed by Kemb36. At the time for evaluation on drought tolerance under controlled conditions, the sweet potato showed delayed senescence and greater drought tolerance under water deficit conditions in the glasshouse. These plants exhibited better growth, higher yield, higher water status maintenance, higher chlorophyll content, and thus higher photosynthetic rates under reduced water conditions in comparison to wild-type. These results, therefore, indicated that expression of isopentenyl transferase gene in sweet potato significantly improves drought tolerance. Therefore, IPT gene should be used to transform other economically important food crops to delay drought-induced senescence and enhance drought tolerance.

Optimization of various factors affecting Agrobacterium-mediated transformation and regeneration of putative transgenic Malaysian MR219 rice with Bacillus SP 289 endo-β-1,3-1,4-glucanase gene

Sheath blight disease caused by Rhizoctonia solani is the most destructive diseases in rice cultivation. Development of transgenic indica rice MR219 through Agrobacterium tumefaciens strain EHA 105 harbouring the plasmid pCAMBIA 1305.2 with endo-beta-1,3-1,4-glucanase gene from Bacillus SP 289 is one of the strategies to engineer disease resistance. Four optimisation parameters were examined such as Agrobacterium culture cell density (0.1 to 1.0 based on OD600nm), callus immersion time in the Agrobacterium culture (30 to 120 minutes), duration of the subsequent drying time (15 to 120 minutes) and co-cultivation period (1 to 6 days). Hygromycin-resistant embryogenic calli developed after 8 to 10 weeks of transformation. Improved transformation rates were achieved when calli were incubated with an Agrobacterium suspension with a culture density of OD 600nm 0.2 for 30 mins, followed by 90 mins of drying time and co-cultivation for 3 days. PCR analysis of the transformants confirmed the presence of Bacillus SP 289 endo-beta-1,3-1,4-glucanase and hpt genes in the rice genome. The transgenic rice plants obtained in this study will be tested against sheath blight disease.

Targeted expression of insecticidal hybrid SN19 gene in potato leads to enhanced resistance against Colorado potato beetle (Leptinotarsa decemlineata Say) and tomato leafminer (Tuta absoluta Meyrick)

The expression of insecticidal genes must be induced at appropriate time and in sufficient amount to confer protection against targeted pests. However, the increased scientific reports of resistance development in insect pest against insecticidal delta-endotoxins, produced by Bacillus thuringiensis, provide impetus for the development of alternative insect management strategies. The present study was conducted to investigate the importance of targeted expression of a hybrid insecticidal gene (SN19) in potatoes. For this purpose, two plant expression vectors were constructed by cloning hybrid SN19 gene (cry1Ba-domain I–III and cry1Ia-domain II) under the control of a wound-inducible promoter isolated from Asparagus officinalis (AoPR1) and CaMV 35S promoter, and were transferred to Agrobacterium tumefaciens strain EHA 105. Four potato genotypes (Marabel, Innovator, Tokat 10/1 and Tokat 6/24) were transformed with EHA 105 strain harboring pTF101.1 35S–SN19 and pTF101.1 AoPR1–SN19 constructs. Phosphinothricin (PPT) was used at concentration of 1 mg/l for selection of primary transformants. PCR results showed the presence of both introduced SN19 and bar genes in 43 plants out of total 154 putative transgenics. Expression of SN19 protein in primary transformants was confirmed by Western blot assays. The mechanical wounding of transgenic plants exhibited more accumulated levels of SN19 proteins during post wounding period. Leaf biotoxicity assays with Colorado potato beetle (Coleoptera) and tomato leafminer (Lepidoptera) exhibited 100% mortality of the pests in primary transformants. Based on our mortality results with both constructs, we concluded that the potato transgenic lines exhibited targeted expression of insecticidal gene under the control of AoPR1 promoter upon insect wounding with eliminated toxicity of Cry protein and hence can be further used effectively in potato breeding programme.

P.4.d.002 - Evaluating self-knowledge and self-control workshops for patients with anxiety disorders

Pseudomonas aeruginosa is an opportunistic pathogen, enable of causing infections especially in immunocompromised patients. Recently many isolates developed multiple drug resistance, resulting in treatment failure in serious infections. In this study, the effect of tobramycin incorporated with bismuth-ethanedithiol loaded on niosomes on the quorum sensing and biofilm production by P. aeruginosa was evaluated. Thin layer hydration method with cholesterol (30%), Span 40 and Tween 40 were used to make niosomes. The physical properties and particle size of the niosomes were investigated. Micro dilution method was used to determine the Minimum Inhibitory Concentration (MIC) for tobramycin, niosomal tobramycin, bismuth ethanedithiol, niosomal bismuth ethanedithiol, tobramycin incorporated with bismuth-ethanedithiol and niosomal tobramycin incorporated with bismuth-ethanedithiol. Biofilm formation was evaluated using microtiter plate. The effect of different combination on N-acyl homoserine lactone (AHL) production was evaluated in presence of Agrobacterium tumefaciens strain (GV3101). The best combination inhibiting the growth of various strains of P. aeruginosa were niosomal tobramycin and niosomal tobramycin incorporated with bismuth-ethanedithiol which reduced the MIC of tobramycin significantly. Sub-MIC concentration of these compounds reduced the rate of biofilm formation 80% lower than the untreated bacteria, and effectively inhibited the production of AHL molecule. The prepared formulations containing non-ionic surfactants, can kept the drug and gradually release it. Encapsulation of tobramycin in combination with bismuth-ethanedithiol in niosome had the ability to reduce the MIC of tobramycin and effectively inhibiting the biofilm formation. These combinations can be used as an excellent combination for further evaluation for treatment of infections caused by MDR isolates of P. aeruginosa.

Tissue culture and Agrobacterium-mediated genetic transformation studies in four commercially important indica rice cultivars

This research was undertaken to find an efficient tissue culture system and Agrobacterium-mediated genetic transformation method for recalcitrant indica rice cultivars. For this, mature seeds of commercially important indica rice varieties, ASD16, ADT43, IR 64, and Pusa Basmati were cultured on MS and N6 medium supplemented with 2 mg l-1 2, 4-D + 30 g l-1 sucrose. The calli grown in N6 medium showed better friability and embryogenic response. Out of the four varieties tested, ASD16 and IR64 showed better callusing and embryogenic capacity as compared to ADT43 and Pusa Basmati. For genetic transformation studies, embryogenic calli of all the cultivars were co-cultivated with the Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCambia 1305.1 with GUS gene. GUS assay was performed for the putative transformed calli and its activity was found to be qualitatively higher in ASD16 and IR64 than the other two varieties. The best responsive ASD16 transformed calli was regenerated and the putative transgenic lines were regenerated. ASD16 transformed calli were confirmed by GUS assay. PCR analysis confirmed the presence of both GUS and HPT genes in ASD16 transgenic lines.

Gene transfer utilizing pollen-tubes of Albuca nelsonii and Tulbaghia violacea

Abstract Developing a tissue culture-independent genetic transformation system would be an interesting technique for gene transfer in valuable medicinal and horticultural plants. Efficient gene delivery (Agrobacterium tumefaciens strain LBA 4404: harbouring PBI121 plasmid) was achieved with Km-resistant pollen grains (pollen tube technique) and were found to be GUS-positive for Albuca nelsonii (31.3%) and Tulbaghia violacea (32.6%). The Km-resistance (95.6% for A. nelsonii and 86.7% for T. violacea) and GUS-positive (100% for A. nelsonii and 97.5% for T. violacea) putative transgenic seedlings in vitro were obtained with 200 mg L-1 Km. The in vitro plants were obtained from leaf explants of putative transgenic seedlings and were confirmed to be Km-resistant and GUS-positive (T. violacea, 73.7% and A. nelsonii, 80.5%). The plants were successfully acclimatized in the greenhouse. We describe a tissue culture-independent gene transfer technique with high efficiency clonal transgenic plant production for A. nelsonii and T. violacea. This can also be applied to biotechnological crop improvement of the same species and potentially to other plants.

A novel in vitro transformation of Lepidium draba L. using rapid direct shoot regeneration.

The present research is carried out to study Lepidium draba gene transformation for the first time, using direct shoot explants. As a prerequisite for gene transformation, the regeneration conditions in L. draba were optimized. We achieved an efficient and reproducible protocol for successful direct shoot regeneration without intervening callus formation. The results indicate that L. draba is the insistent species of Brassicaceae in direct shoot regeneration. Various explants of L. draba were genetically transformed with different strains of Agrobacterium tumefaciens, viz., LBA4404, GV3850, GV3101, and EHA105, using the vector pBI121. Expression of GUS reporter protein was assayed by histochemical staining. In addition, using the PCR method with specific primers proved the integration of GUS gene into the plants. The highest transformation efficiency was achieved with Agrobacterium strain GV3850. Moreover, we found that infected hypocotyl and root explants of L. draba interestingly yielded higher transformation efficiency, so that in hypocotyls on average exceeded 70% of the explants. This study showed that L. draba, in addition to the numerous desirable traits, has a high potential for gene transfer.

Antibiosis and bmyB Gene Presence As Prevalent Traits for the Selection of Efficient Bacillus Biocontrol Agents against Crown Gall Disease.

This study aimed to improve the screening method for the selection of Bacillus biocontrol agents against crown gall disease. The relationship between the strain biocontrol ability and their in vitro studied traits was investigated to identify the most important factors to be considered for the selection of effective biocontrol agents. In fact, previous selection procedure relying only on in vitro antibacterial activity was shown to be not suitable in some cases. A direct plant-protection strategy was performed to screen the 32 Bacillus biocontrol agent candidates. Moreover, potential in vitro biocontrol traits were investigated including biofilm formation, motility, hemolytic activity, detection of lipopeptide biosynthetic genes (sfp, ituC and bmyB) and production of antibacterial compounds. The obtained results indicated high correlations of the efficiency of the biocontrol with the reduction of gall weight (p = 0.000) and the antibacterial activity in vitro (p = 0.000). Moreover, there was strong correlations of the efficiency of the biocontrol (p = 0.004) and the reduction in gall weight (p = 0.000) with the presence of the bmyB gene. This gene directs the synthesis of the lipopeptide bacillomycin belonging to the iturinic family of lipopeptides. These results were also confirmed by the two-way hierarchical cluster analysis and the correspondence analysis showing the relatedness of these four variables. According to the obtained results a new screening procedure of Bacillus biocontrol agents against crown gall disease could be advanced consisting on two step selection procedure. The first consists on selecting strains with high antibacterial activity in vitro or those harbouring the bmyB gene. Further selection has to be performed on tomato plants in vivo. Moreover, based on the results of the biocontrol assay, five potent strains exhibiting high biocontrol abilities were selected. They were identified as Bacillus subtilis or Bacillus amyloliquefaciens. These strains were found to produce either surfactin or surfactin and iturin lipopeptides. In conclusion, our study presented a new and effective method to evaluate the biocontrol ability of antagonistic Bacillus strains against crown gall disease that could increase the efficiency of screening method of biocontrol agents. Besides, the selected strains could be used as novel biocontrol agents against pathogenic Agrobacterium tumefaciens strains.

AGROBACTERIUM-MEDIATED TRANSFORMATION OF TOMATO (LYCOPERSICON ESCULENTUM Mill.) USING A SYNTHETIC Cry1Ab GENE FOR ENHANCED RESISTANCE AGAINST TUTA ABSOLUTA (Meyrick)

The effect of different plant hormones on in vitro regeneration of tomato (Lycopersicon esculentum Mill.) two cultivars Castle Rock and Super strain B, obtained from hypocotyls and cotyledons of cultured in vitro seedlings was examined. The optimal concentrations of plant growth regulators were the MS medium containing 3 mg L-1 of zeatin and 0.1 mg L-1 of NAA of cotyledon and hypocotyl explants of Super strain B cultivar. While, the best regeneration medium was the MS medium containing 1 mg L-1 ZEA, 0.5 mg L-1 BAP and 0.1 mg L-1 IAA of cotyledon and hypocotyl explants of Castle Rock cultivar. Agrobacterium‐mediated genetic transformation system has been expanded of tomato cultivar Super strain B. Transformation efficiency of hypocotyl explants was studied with Agrobacterium tumefaciens strain GV3101 harboring binary vector pICBV19, containing gus and bar genes. Transformed tomato shoots were obtained from hypocotyls explants on MS medium contained with 3 mg L-1 ZEA and 0.1 mg L-1 NAA. The gene expression of gus and bar genes was tested in the putatively transformed tomato plantlets through GUS histochemical and leaf painting assays. The plasmid pBI121 containing an insect resistance gene (cry1Ab) and kanamycin gene (nptII) as a plant selectable marker were introduced into these explants. Putatively transformed plantlets were examined by PCR and southern blot analysis. The expression of cry1Ab gene was tested through insect bioassay. For insect bioassay, experiments were conducted to determine the mortality percentage of Cry1Ab toxin protein against Tuta absoluta (Meyrick). The results indicated that Cry1Ab toxin protein expressed in transformed tomato plants showed 100% instars larval mortality was obtained after feeding for 4–5 days.