Genotype independent and efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of the medicinal plant <Emphasis Type="Italic">Withania somnifera</Emphasis> Dunal

Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in traditional and modern medicines as active constituents. A high frequency genotype and chemotype independent Agrobacterium-mediated transformation protocol has been developed for W. somnifera by optimizing several factors which influence T-DNA delivery. Leaf and node explants of Withania chemotype was transformed with A. tumefaciens strain GV3101 harboring pIG121Hm plasmid containing the gusA gene encoding β-glucuronidase (GUS) as a reporter gene and the hptII and the nptII gene as selection markers. Various factors affecting transformation efficiency were optimized; as 2 days preconditioning of explants on MS basal supplemented with TDZ 1 μM, Agrobacterium density at OD₆₀₀ 0.4 with inclusion of 100 μM acetosyringone (As) for 20 min co-inoculation duration with 48 h of co-cultivation period at 22 °C using node explants was found optimal to improved the number of GUS foci per responding explant from 36?±?13.2 to 277.6?±?22.0, as determined by histochemical GUS assay. The PCR and Southern blot results showed the genomic integration of transgene in Withania genome. On average basis 11 T₀ transgenic plants were generated from 100 co-cultivated node explants, representing 10.6 % transformation frequency. Our results demonstrate high frequency, efficient and rapid transformation system for further genetic manipulation in Withania for producing engineered transgenic Withania shoots within very short duration of 3 months.     

Adventitious shoot regeneration and in vitro biosynthesis of steroidal lactones in <Emphasis Type="Italic">Withania coagulans</Emphasis> (Stocks) Dunal

A micropropagation system through leaf explant culture has been developed for Withania coagulans. Shoot bud proliferation occurred through both adventitious and de novo routes depending on the hormonal regime of the culture medium. Green compact nodular organogenic callus developed on Murashige and Skoog (MS) medium supplemented with 2.3 μM kinetin (Kn) and lower levels of 6–benzyladenine (BA) (13.3 μM) while multiple adventitious shoot bud differentiation occurred on medium fortified with 2.3 μM kinetin (Kn) and higher levels of BA (22.2 μM). Shoot buds were transferred to proliferation medium containing 2.2 μM BA, 2.3 μM Kn, and 3.9 μM phloroglucinol (PG) for further growth and development of shoot system. Elongated shoots were rooted using a two-step procedure involving pulse treatment of 7 days in a medium containing 71.6 μM choline chloride (CC) and 3.9 μM PG and then transferred to rooting medium containing ½ MS, 1.2 μM IBA, 3.6 μM PAA, and 14.3 μM CC for 3 weeks. Well-rooted plants were transferred to a greenhouse for hardening and further growth. Random amplification of polymorphic DNA (RAPD) showed monomorphic bands in all the plants thereby confirming clonality of the regenerants. Thin layer chromatography (TLC) showed the presence of withanolides in the regenerated plants. Quantification through reverse-phase HPLC revealed increased concentration of withanolides in the regenerated plants compared to the field-grown mother plant. Accumulation of withaferin A and withanolide A increased up to twofold and that of withanone up to tenfold. Direct regeneration via leaf explants will be useful for Agrobacterium-mediated genetic transformation, and will facilitate pathway manipulation using metabolic engineering for bioactive withanolides.     

Factors affecting genetic transformation and shoot organogenesis of Bacopa monnieri (L.) Wettst

Efficient Agrobacterium tumefaciens mediated T-DNA delivery and subsequent shoot organogenesis has been achieved from Bacopa monnieri. Various factors influenced T-DNA delivery as evident from transient GUS assay. The transient GUS expression was significantly higher (97.7 %) in explants that were pre-cultured before bacterial infection on medium supplemented with 100 μM acetosyringone. Incorporation of acetosyringone into the co-cultivation medium also enhanced transient GUS activity. Explant injury with carborundum paper, co-cultivation period of 2 days and a bacterial density of 0.4 OD₆₀₀ showed higher transient GUS expression. Following co-cultivation, shoot organogenesis was achieved from leaf segments on basal Murashige and Skoog medium containing 58 mM sucrose. Supplementation of antibiotics (cefotaxime or carbenicillin) at > 250 μg/ml into the medium significantly promoted shoot organogenesis from leaf explants (71.5 % in control and > 83.0 % on medium containing 500 μg/ml of carbenicillin or cefotaxime). Stable transformation of regenerated shoots was confirmed on the basis of GUS activity and PCR amplification of DNA fragments specific to reporter gene (uidA) and selection marker gene (nptII). The expression level of nptII gene in independent transgenic lines was studied using quantitative real time-PCR. Stable transformed shoots after rooting were successfully established in the pots.     

Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf midribs as explants in ramie [Boehmeria nivea (L.) Gaud]

In this study, leaf midribs, the elite explants, were used for the first time to develop an efficient regeneration and transformation protocol for ramie [Boehmeria nivea (L.) Gaud.] via Agrobacterium-mediated genetic transformation. Sensitivity of leaf midribs regeneration to kanamycin was evaluated, which showed that 40 mg l^?1 was the optimal concentration needed to create the necessary selection pressure. Factors affecting the ramie transformation efficiency were evaluated, including leaf age, Agrobacterium concentration, length of infection time for the Agrobacterium solution, acetosyringone concentration in the co-cultivation medium, and the co-cultivation period. The midrib explants from 40-day-old in vitro shoots, an Agrobacterium concentration at OD₆₀₀ of 0.6, 10-min immersion in the bacteria solution, an acetosyringone concentration of 50 mg l^?1 in the co-cultivation medium and a 3-day co-cultivation period produced the highest efficiencies of regeneration and transformation. In this study, the average transformation rate was 23.25 %. Polymerase chain reactions using GUS and NPTII gene-specific primers, Southern blot and histochemical GUS staining analyses further confirmed that the transgene was integrated into the ramie genome and expressed in the transgenic ramie. The establishment of this system of Agrobacterium-mediated genetic transformation and regeneration of transgenic plants will be used not only to introduce genes of interest into the ramie genome for the purpose of trait improvement, but also as a common means of testing gene function by enhancing or inhibiting the expression of target genes.     

Stable germ line transformation of a leafy vegetable crop amaranth (Amaranthus tricolor L.) mediated by Agrobacterium tumefaciens

We have optimized a procedure for genetic transformation of a major leafy vegetable crop, Amaranthus tricolor L., using epicotyl explant co-cultivation with Agrobacterium tumefaciens. Two disarmed A. tumefaciens strains EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT harboring the neomycin phosphotransferase II gene (nptII) and the β-glucuronidase gene (gus), were evaluated as vector systems. The former displayed a higher transforming efficiency. Several key factors influencing the transformation events were optimized. The highest percentage of transformed shoots (24.24%) was achieved using hand-pricked epicotyl explants, a 10-min infection period, with 100 μM acetosyringone-pretreated Agrobacterium culture corresponding to OD₆₀₀???0.6 and diluted to 10⁹ cells ml^?1, followed by 4 d co-cultivation in the regeneration medium. Putative transformed explants capable of forming shoots were selected on medium supplemented with 75 μg?ml^?1 kanamycin, and transient as well as stable glucuronidase expression was determined by histochemical analysis. From a total of 48 selected shoot lines derived from independent transformation events with epicotyl explants co-cultivated with EHA 105, 32 showed positive PCR amplification for both the nptII and gus genes. Germ line transformation and transgene stability were evident in progeny of primary transformed plants (T₀). Among T₁ seedlings of 12 selected transgenic plant lines, kanamycin-resistant and kanamycin-sensitive seedlings segregated in a ratio typical of the Mendelian monohybrid pattern (3:1) as verified by the chi-square (χ ²) test. Southern hybridization of genomic DNA from kanamycin-resistant T₁ transgenic segregants to an nptII probe substantiated stable integration of the transgene. Neomycin phosphotransferase (NPTII) activity was detected in leaf protein extracts of selected T₁ transgenic plants, thereby confirming stable expression of the nptII gene.     

Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars

For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacterium-mediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l^?1 citric acid, 100 µM acetosyringone, and 100 mg l^?1 l-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l^?1 hygromycin and the embryos were germinated in basal medium containing 20 mg l^?1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into soybean has been developed.     

Evaluation of factors influencing Agrobacterium-mediated spinach transformation and transformant selection by EGFP fluorescence under low-selective pressure

We developed an efficient Agrobacterium-mediated transformation protocol for spinach (Spinacia oleracea L.) that uses root-derived callus. Evaluation of this protocol was based on the systematic evaluation of factors that influence transformation efficiency. Four of the five factors that were tested significantly affected the transformation efficiency, including spinach cultivar, Agrobacterium tumefaciens strain and density, and the duration of co-cultivation. Transgenic spinach plants were generated based on optimized conditions, consisting of callus explants of the cultivar Gyeowoonae, A. tumefaciens strain EHA105 with OD₆₀₀ of 0.2, a co-cultivation period of 4 d, and 100 μM acetosyringone supplemented in the inoculation and co-cultivation media. After co-cultivation with A. tumefaciens, explants were cultured in low-selective and then non-selective conditions to enhance the growth of putative transgenic explants. Visualization of the fluorescent marker, enhanced green fluorescent protein (EGFP), was used to select transgenic explants at several stages, including callus, somatic cotyledonary embryo, and plantlet. The best results for fluorescence visualization screening were obtained at the somatic cotyledonary embryo stage. On average, 24.96?±?6.05% of the initial calli regenerated shoots that exhibited EGFP fluorescence. The putative transgenic plants were subjected to β-glucuronidase (GUS)-staining assay, phosphinothricin acetyltransferase (PAT) strip test, and molecular analyses to assess the transgene incorporation into plant genome and its expression. All EGFP-positive plants tested were confirmed to be transgenic by GUS-staining assay, PAT strip test, and molecular analyses. The transformation system described in this study could be a practical and powerful technique for functional genetic analysis and genetic modification of spinach.     

Factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Hybanthus</Emphasis><Emphasis Type="Italic">enneaspermus</Emphasis> (L.) F. Muell.

Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD₆₀₀, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.     

Optimization of regeneration and Agrobacterium-mediated transformation of immature cotyledons of chickpea (Cicer arietinum L.)

Immature cotyledons collected at different time intervals from four genotypes of chickpea (C 235, BG 256, P 362 and P 372) were cultured adaxially on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine, thidiazuron, kinetin, zeatin and dimethylallylaminopurine (2-iP), either alone or in combination with indole-3-acetic acid (IAA) or α-napthoxyacetic acid (α-NOA) for dedifferentiation and regeneration of adventitious shoots. Morphogenesis was achieved with explants cultured adaxially on MS medium with 13.68 μM zeatin, 24.6 μM 2-iP, 0.29 μM IAA and 0.27 μM α-NOA. Explants prepared from pods of 21 days after pollination, responded favourably to plant growth regulator treatment in shoot differentiation. Histological studies of the regenerating explants, revealed the initiation of meristematic activity in the sub-epidermal region during the onset of morphogenesis, which can be correlated with elevated activity of cytokinin oxidase-dehydrogenase, for cytokinin metabolism. The regenerated shoots were efficiently rooted in MS medium supplemented with 2.46 μM indole-3-butyric acid and acclimatized under culture room and glasshouse conditions for normal plant development leading to 76–80 % survival of the rooted plantlets. The immature cotyledon explants were used for Agrobacterium-mediated transformation with critical manipulation of cultural conditions like age of explant, O.D. of Agrobacterium suspension, concentration of acetosyringone, duration of sonication and co-cultivation for successful genetic transformation and expression of the reporter gene uidA (GUS). Integration of transgene was confirmed by molecular analysis. Transformation frequency up to 2.08 % was achieved in chickpea, suggesting the feasibility of using immature cotyledon explants for Agrobacterium-mediated transformation.     

Agrobacterium-mediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp. indica cv. ADT 43)

A simple and efficient protocol for the Agrobacterium-mediated transformation of an agronomically useful abiotic sensitive popular indica rice cv. ADT 43 has been developed. Initiation of calli were best achieved from the leaf bases of 4 days old rice seedlings on LS medium supplemented with 2.5 mg/L 2,4-D and 1.0 mg/L thiamine-HCl. Rice calli immersed in Agrobacterium suspension (strain EHA 105, OD₆₀₀ = 0.8) were co-cultured on LS30-AsPC medium for 2 days at 25 ± 2 °C in the dark. Based on GUS expression analysis, 10 min co-cultivation time with 100 μM acetosyringone was found optimum for the delivery of gus gene. Calli were proved to be very sensitive to Agrobacterium infection and we found that the level of necrotic response can be minimized after co-cultivation with 30% LS, 10 g/L PVP, 10% coconut water and 250 mg/L timentin which improved the final transformation efficiency to 9.33%. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgene in the progeny (T₁) of these plants. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants (T₀ and T₁).     

A simple and efficient method for obtaining transgenic soybean callus tissues

In the present study, a simple and efficient method for obtaining transgenic callus tissues of soybean [Glycine max (L.) Merr.] was developed based on Agrobacterium-mediated transformation. Hypocotyl segments of soybean were used as the starting material. Several factors such as soybean genotype, Agrobacterium concentration, inoculation time, co-cultivation period and addition of antioxidants in co-cultivation medium affecting the transformation efficiency were examined. The explants were cultured on callus induction medium containing 0.5 mg L^?1 6-benzylaminopurine and 2.0 mg L^?1, 2,4-Dichlorophenoxyacetic acid for callus induction. Callus tissues were induced at both the acropetal and basipetal ends. CaMV35S::GUS and CaMV35S::GFP transgenic callus tissues were obtained using the optimized protocol. The average transformation efficiency reached up to 87.7 % based on GUS detection. From inoculation with Agrobacterium to obtaining transgenic soybean callus will take about 3 weeks. In order to validate this method for gene function investigation, GVG::GmSARK transgenic soybean callus tissues were obtained and their senescence-associated phenotypes were assessed. To our knowledge, this is the first report using hypocotyl segments as starting materials to obtain transgenic callus, and this system provides a method for high-throughput screening of functional genes of interest in transformed soybean callus.     

Sonication-assisted efficient Agrobacterium-mediated genetic transformation of the multipurpose woody desert shrub Leptadenia pyrotechnica

The desert shrub Leptadenia pyrotechnica (Forssk.) Decne (Asclepiadaceae) is an important multipurpose woody species of tropical and sub-tropical arid regions. The shrub’s excellent pharmacological properties, importance in desert afforestation and role in sand dune fixation has been elaborately studied in recent years which make it a potential candidate for genetic manipulation in the global warming scenario. We have developed an Agrobacterium-mediated transformation protocol for L. pyrotechnica using hypocotyl explants from 5 days old seedlings. The reliability of the protocol has been tested by transforming the species with gus and gfp reporter genes separately. Hypocotyl explants were sonicated for 40 s, infected with Agrobacterium suspension of OD₆₀₀ 0.5, co-cultivated in the dark for 2 days at 26 °C in presence of 200 μM acetosyringone. Transgenic plants were obtained after 20–22 weeks at a frequency of 14 and 11 % for gus and gfp reporter genes respectively. Transgenic plants were confirmed by PCR and Southern blots. Expression of the two reporter genes has been tested in different stages of transgenic plant development. No phenotypic differences between the wild-type and transgenic plants were noted. This method will be very helpful to introduce alien genes-of-interest for various biotechnological applications.     

Efficient somatic embryogenesis and Agrobacterium-mediated transformation of pothos (Epipremnum aureum) ‘Jade’

Leaf and petiole explants of monocotyledonous pothos (Epipremnum aureum) ‘Jade’ were cultured on Murashige and Skoog basal medium supplemented with N-(2-chloro-4-pyridl)-N′-phenylurea (CPPU) or N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA). Somatic embryos appeared directly from explants after 4–8 weeks of culture; 9.1 μM TDZ with 1.1 μM NAA induced 61.1 % leaf discs and 94.4 % of petiole segments to produce plantlets through embryo conversion. Using this established regeneration method and an enhanced green fluorescent protein (GFP) gene (egfp) as a reporter marker, an Agrobacterium-mediated transformation procedure was developed. Leaf discs and petiole segments were inoculated with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pLC902 that contains novel bi-directional duplex promoters driving the egfp gene and hygromycin phosphotransferase gene (hpt), respectively. The explants were co-cultivated with strain EHA105 for 3, 5, and 7 days, respectively prior to selective culture with 25 mg l^?1 hygromycin. A 5-day co-cultivation led to 100 % of leaf discs to show transient GFP expression and 23.8 % of the discs to produce stable GFP-expressing somatic embryos. A 7-day co-cultivation of petiole explants resulted in the corresponding responses at 100 and 14.3 %, respectively. A total of 237 transgenic plants were obtained, and GFP fluorescence was observed in all plant organs. Regular PCR and quantitative real-time PCR analyses confirmed the presence of 1 or 2 copies of the egfp gene in analyzed plants. The highly efficient regeneration and transformation systems established in this study may enable genetic improvement of this vegetatively propagated species through biotechnological means.     

Optimization of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of shoot tip explants of green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek)

Shoot tip explants prepared from seedlings of ML-267 genotype of green gram were inoculated on MSB5 medium supplemented with BAP (0–20 μM) individually or in combination with minimal concentration of auxins (NAA/IAA/IBA) for adventitious shoots formation. BAP alone without auxins was observed to be efficient in multiple shoot induction and optimum shoot proliferation was achieved on MSB5 medium containing 10 μM BAP with 100?% shoot induction frequency. 3-day-old explants gave best shoot multiplication response and the mean shoot number decreased significantly in 4-day and 5-day-old explants. The induced shoots rooted profusely on ½ MSB5?+?2.46 µM IBA and about 90?% of the plantlets survived after acclimatization and set seed normally. Shoot tip explants infected with A.tumefaciens (LBA4404) harboring pCAMBIA 2301?+?AnnBj1 recombinant vector. Various factors which influence the competence of transformation were optimized based on the frequency of transient GUS expression in shoot tip explants. Optimum levels of transient GUS expression were recorded at pre-culture of explants for 2 days, infection for 10 min with Agro-culture of 0.8 OD and co-cultivation for 3 days on co-cultivation medium containing 100 µM acetosyringone in dark at 23?°C. Putative transformed shoots were produced on selection medium (shoot inductionmedium with100 mg/l kanamycin and 250 mg/l cefotaxim). PCR analysis confirmed the presence of AnnBj1, nptII, and uidA genes in T₀ plants. Stable GUS activity was detected in flowers of T₀ plants and leaves of T₁ plants. PCR analysis of T₁ progeny revealed AnnBj1 gene segregated following a Mendelian segregation pattern.     

A system for the transformation and regeneration of the recretohalophyte Limonium bicolor

Limonium bicolor, a typical recretohalophyte, has a specialized salt-secreting structure in the epidermis called the salt gland and plays a significant role in improving saline land. Understanding the molecular mechanisms of salt secretion and salt gland development requires an efficient L. bicolor transformation system, which is described in this report. Leaf explants were incubated with Agrobacterium tumefaciens strain EHA105 harboring the plasmid pTCK303 containing the β-glucuronidase gene (GUS) as the transgene reporter and the hygromycin B resistance gene as a selectable marker. Up to 96.9% of leaves were induced to regenerate shoots on an Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzyladenine and 1.1 μM α-naphthaleneacetic acid; roots were induced on the MS medium containing 2.5 μM indole-3-butyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of L. bicolor. Pre-cultivated explants (2 d old) were incubated with Agrobacterium (0.6–0.7 at OD₆₀₀) in a shaking culture for 20 min; the explants and bacterium were co-cultivated for 4 d in the dark before the explants were transferred to a selection medium containing 8 mg/L hygromycin B and 600 mg/L piperacillin sodium (added to prevent continued Agrobacterium growth). Histochemical assays and PCR to detect the GUS gene showed that transformation frequency was 4.43%. Quantitative PCR and Northern blotting further verified the integration and presence of the GUS gene in L. bicolor. This is the first report of an Agrobacterium-based transformation system for L. bicolor. The system will facilitate a research on the identity and function of genes involved in salt gland development and salt secretion.     

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental × Trumpet and Longiflorum × Asiatic hybrids were maintained on a medium with 8.3 μM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 μM PIC and 0.044 μM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximum.     

High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA^® (4 mg l^?1) containing MS medium followed by BASTA^® foliar spray on 15-day-old black gram plants (35 mg l^?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.     

The development of herbicide-resistant maize: stable Agrobacterium-mediated transformation of maize using explants of type II embryogenic calli

One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for 4 × 14 days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter–bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with 4 mg l^?1 phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the R₁ generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.     

Highly efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic cell suspensions of <Emphasis Type="Italic">Musa acuminata</Emphasis> cv. Mas (AA) via a liquid co-cultivation system

A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0–490 transgenic plants per 0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at 100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar, and was at least 1 month faster for regenerating transgenic plants.