Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line

Key message

An improved Agrobacterium -mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds.

Abstract

This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T₀ plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T₀ plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.     

Slash pine genetic transformation through embryo cocultivation with A. tumefaciens and transgenic plant regeneration

Agrobacterium-mediated genetic transformation has been widely used to generate transgenic plants in angiosperms. However, progress in conifer species has lagged because of the recalcitrant nature of gene transfer. In this study, a transgenic plant regeneration system has been established for slash pine (Pinus elliottii Engelm.) using Agrobacterium-mediated transformation. Among the different Agrobacterium tumefaciens strains (EHA105, GV3101, and LBA4404) tested, the highest frequency (60%) of transient β-glucuronidase-expressing embryos was obtained from Agrobacterium strain GV3101 with over 330 blue spots per embryo. To improve the frequency of transformation, different cocultivation conditions were analyzed. Combination of Agrobacterium density at OD₆₀₀?=?0.9, 50 s sonication of embryos, and the addition of 50 μM acetosyringone produced the highest transformation efficiency, in which 56.2% of embryos formed hygromycin-resistant calli. Transient gene expression was observed in cotyledons and hypocotyls, but transgenic plants were only produced from callus cultures derived from embryonic cotyledons of transformed slash pine. Stable integration of transgenes in the plant genome of slash pine was confirmed by polymerase chain reaction, Southern blot, and Northern blot analyses. Transgenic lines with a single T-DNA copy were produced from Agrobacterium strains EHA105 (80.4%), GV3101 (95.7%), and LBA4404 (66%). These results demonstrated that a stable transformation system has been established in slash pine, and this system could provide an opportunity to transfer economically important genes into slash pine.     

Agrobacterium tumefaciens-mediated transformation of Atropa belladonna

Belladonna or deadly nightshade (Atropa belladonna L.) is an important medicinal plant in the family Solanaceae. It is a model plant for studying plant alkaloid biosynthesis. In this study, a reliable protocol for efficient transformation of A. belladonna using Agrobacterium tumefaciens was developed. Hypocotyl and cotyledon explants were co-cultivated with three opine-type Agrobacterium strains (LBA4404: pBISN1, GV3101: pBISN1, and EHA105: pBISN1). Selection and regeneration of transformed cells were conducted on two regeneration media; RM1 (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium (MS) salts, Gamborg B5 vitamins (Gamborg et al. in Exp Cell Res 50:151–158, 1968), 4.56 μM zeatin, and 2.9 μM indole-3-acetic acid (IAA)] and RM2 (MS salts, B5 vitamins, 4.65 μM kinetin, and 1.14 μM IAA), each containing 100 mg l^?1 kanamycin and 250 mg l^?1 timentin. Both regeneration media and type of explant had significant effects on frequencies of transformation. Using an optimal regeneration medium and regardless of the strain of Agrobacterium used, over 80 % of hypocotyl explants and 60 % of cotyledons developed at least one transformed shoot after 2–3 months of selection. Most transformants exhibited a normal phenotype while growing in the greenhouse. Southern blot analysis confirmed the stable integration of the nptII transgene in T₁ plants.     

Agrobacterium-mediated genetic transformation of selected tropical inbred and hybrid maize (Zea mays L.) lines

The study was carried out to evaluate the amenability of tropical inbred and hybrid maize lines, using Agrobacterium mediated transformation technique. Agrobacterium tumefaciens strains EHA101 harbouring a pTF102 binary vector, EHA101, AGL1, and LBA4404 harbouring pBECK2000.4 plasmid, LBA4404, GV and EHA105 harbouring pCAMBIA2301 plasmid, and AGL1 harbouring the pSB223 plasmid were used. Delivery of transgenes into plant tissues was assessed using transient β-glucuronidase (gus) activity on the 3rd and 4th day of co-cultivation of the infected Immature Zygotic Embryos (IZEs) and embryogenic callus. Transient gus expression was influenced by the co-cultivation period, maize genotype and Agrobacterium strain. The expression was highest after the 3rd day of co-culture compared to the 4th day with intense blue staining was detected for IZEs which were infected with Agrobacterium strains EHA105 harbouring pCAMBIA2301 and EHA101 harbouring pTF102 vector. Putative transformants (T_o) were regenerated from bialaphos resistant callus. Differences were detected on the number of putative transformants regenerated among the maize lines. Polymerase chain reaction (PCR) amplification of Phosphinothricin acetyltransferase (bar) and gus gene confirmed the transfer of the transgenes into the maize cells. Southern blot hybridization confirmed stable integration of gus into PTL02 maize genome and segregation analysis confirmed the inheritance of the gus. A transformation efficiency of 1.4 % was achieved. This transformation system can be used to introduce genes of interest into tropical maize lines for genetic improvement.     

Transformation of the oilseed crop Camelina sativa by Agrobacterium-mediated floral dip and simple large-scale screening of transformants

Camelina sativa is a promising under-exploited oilseed crop with potential to become a biofuel feedstock. The ability to transform C. sativa would allow for the rapid introduction of novel traits into this emerging crop. We report the development of an Agrobacterium-based floral dip transformation method, requiring no vacuum-infiltration step, with transformation efficiencies up to 0.8%. C. sativa cultivars Ames 26665, ??Calena?? A3U7761, Ames 1043, and ??Celine?? were tested using Agrobacterium tumefaciens strains GV3101, EHA105, and At503. Use of all strains and cultivars resulted in transformed plants; however, GV3101 was the only Agrobacterium strain and Ames 1043 the only C. sativa cultivar to yield transformed plants under all conditions tested. Progeny analysis revealed that in approximately 78% of the transformed plants, the transgene segregated as a single locus. Furthermore, a high-throughput, filter paper-based PCR method was developed to screen marker-free transformed plants. Together, these methods will allow for easier introduction of new genes into this promising oilseed crop.     

Agrobacterium‐mediated transformation of reed (Phragmites communis Trinius) using mature seed‐derived calli

Reed (Phragmites communis) is a potential bioenergy plant. We report on its first Agrobacterium‐mediated transformation using mature seed‐derived calli. The Agrobacterium strains LBA4404, EHA105, and GV3101, each harboring the binary vector pIG121Hm, were used to optimize T‐DNA delivery into the reed genome. Bacterial strain, cocultivation period and acetosyringone concentration significantly influenced the T‐DNA transfer. About 48% transient expression and 3.5% stable transformation were achieved when calli were infected with strain EHA105 for 10 min under 800 mbar negative pressure and cocultivated for 3 days in 200 μm acetosyringone containing medium. Putative transformants were selected in 25 mg l^?1 hygromycin B. PCR, and Southern blot analysis confirmed the presence of the transgenes and their stable integration. Independent transgenic lines contained one to three copies of the transgene. Transgene expression was validated by RT‐PCR and GUS staining of stems and leaves.     

Plant regeneration and genetic transformation of C. canadensis: a non-model plant appropriate for investigation of flower development in Cornus (Cornaceae)

Key message

Efficient Agrobacterium -mediated genetic transformation for investigation of genetic and molecular mechanisms involved in inflorescence architectures in Cornus species.

Abstract

Cornus canadensis is a subshrub species in Cornus, Cornaceae. It has recently become a favored non-model plant species to study genes involved in development and evolution of inflorescence architectures in Cornaceae. Here, we report an effective protocol of plant regeneration and genetic transformation of C. canadensis. We use young inflorescence buds as explants to efficiently induce calli and multiple adventitious shoots on an optimized induction medium consisting of basal MS medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.1 mg/l of 1-naphthaleneacetic acid. On the same medium, primary adventitious shoots can produce a large number of secondary adventitious shoots. Using leaves of 8-week-old secondary shoots as explants, GFP as a reporter gene controlled by 35S promoter and hygromycin B as the selection antibiotic, a standard procedure including pre-culture of explants, infection, co-cultivation, resting and selection has been developed to transform C. canadensis via Agrobacterium strain EHA105-mediated transformation. Under a strict selection condition using 14 mg/l hygromycin B, approximately 5 % explants infected by Agrobacterium produce resistant calli, from which clusters of adventitious shoots are induced. On an optimized rooting medium consisting of basal MS medium supplemented with 0.1 mg/l of indole-3-butyric acid and 7 mg/l hygromycin B, most of the resistant shoots develop adventitious roots to form complete transgenic plantlets, which can grow normally in soil. RT-PCR analysis demonstrates the expression of GFP transgene. Green fluorescence emitted by GFP is observed in transgenic calli, roots and cells of transgenic leaves under both stereo fluorescence microscope and confocal microscope. The success of genetic transformation provides an appropriate platform to investigate the molecular mechanisms by which the various inflorescence forms are developed in Cornus plants.     

Strain specific Agrobacterium-mediated genetic transformation of Bacopa monnieri

Agrobacterium-mediated genetic transformation is the most preferred strategy utilized for plant genetic transformation. The present study was carried out to analyze the influence of three different strains of Agrobacterium tumefaciens on genetic transformation of Bacopa monnieri (L.) Pennell. In the present study, B. monnieri was genetically transformed with three different strains of A. tumefaciens viz. LBA4404, EHA105 and GV3101 harbouring expression vector pCAMBIA2301 containing β-glucuronidase (GUS) as a reporter gene. The putative transformants were analyzed by PCR method using transgene specific primers. Expression and presence of GUS reporter protein were analyzed by histochemical staining assay and quantitative analysis of GUS enzyme was done using fluorometric assay. No statistically significant difference in transformation efficiency was found for all the three strains. Interestingly, Gus expression was variable with LBA4404 plants showing highest GUS activity.     

Genetic transformation of selected mature cork oak (Quercus suber L.) trees

A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and -glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil.Abbreviations BA N⁶-Benzyladenine - GUS -Glucuronidase - MSSH Expression-proliferation medium - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase gene - uidA -Glucuronidase gene     

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.     

Transformation of miniature potted rose (Rosa hybrida cv. Linda) with P SAG12 -ipt gene delays leaf senescence and enhances resistance to exogenous ethylene

Key message

The P _SAG12 -ipt gene was transferred to miniature rose, as the first woody species, resulting in increased ethylene resistance due to specific up-regulation of the ipt gene under senescence promoting conditions.

Abstract

Transgenic plants of Rosa hybrida ‘Linda’ were obtained via transformation with Agrobacterium tumefaciens strain harboring the binary vector pSG529(+) containing the P _SAG12 -ipt construct. A. tumefaciens strains AGL1, GV3850 and LBA4404 (containing P_35S-INTGUS gene) were used for transformation of embryogenic callus, but transgenic shoots were obtained only when AGL1 was applied. The highest transformation frequency was 10 % and it was achieved when half MS medium was used for the dilution of overnight culture of Agrobacterium. Southern blot confirmed integration of 1–6 copies of the nptII gene into the rose genome in the tested lines. Four transgenic lines were obtained which were morphologically true-to-type and indistinguishable from Wt shoots while they were in in vitro cultures. Adventitious root induction was more difficult in transgenic shoots compared to the Wt shoots, however, one of the transgenic lines (line 6) was rooted and subsequently analyzed phenotypically. The ipt expression levels were determined in this line after exposure to exogenous ethylene (3.5 μl l^?1) and/or darkness. Darkness resulted in twofold up-regulation of ipt expression, whereas darkness combined with ethylene caused eightfold up-regulation in line 6 compared to Wt plants. The transgenic line had significantly higher content of chlorophyll at the end of the treatment period compared to Wt plants.     

Agrobacterium tumefaciens-mediated in planta seed transformation strategy in sugarcane

Key message

An efficient, reproducible and genotype-independent in planta transformation has been standardized for sugarcane using seed as explant.

Abstract

Transgenic sugarcane production through Agrobacterium infection followed by in vitro regeneration is a time-consuming process and highly genotype dependent. To obtain more number of transformed sugarcane plants in a relatively short duration, sugarcane seeds were infected with Agrobacterium tumefaciens EHA 105 harboring pCAMBIA 1304-bar and transformed plants were successfully established without undergoing in vitro regeneration. Various factors affecting sugarcane seed transformation were optimized, including pre-culture duration, acetosyringone concentration, surfactants, co-cultivation, sonication and vacuum infiltration duration. The transformed sugarcane plants were selected against BASTA^® and screened by GUS and GFP visual assay, PCR and Southern hybridization. Among the different combinations and concentrations tested, when 12-h pre-cultured seeds were sonicated for 10 min and 3 min vacuum infiltered in 100 µM acetosyringone and 0.1 % Silwett L-77 containing Agrobacterium suspension and co-cultivated for 72-h showed highest transformation efficiency. The amenability of the standardized protocol was tested on five genotypes. It was found that all the tested genotypes responded favorably, though CoC671 proved to be the best responding cultivar with 45.4 % transformation efficiency. The developed protocol is cost-effective, efficient and genotype independent without involvement of any tissue culture procedure and can generate a relatively large number of transgenic plants in approximately 2 months.     

Thidiazuron: an efficient plant growth regulator for enhancing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Efficient shoot regeneration and Agrobacterium-mediated genetic transformation systems were developed for Petunia hybrida cv. Mitchell. Leaf explants of petunia were cultured on Murashige and Skoog (MS) medium with different concentrations of thidiazuron (TDZ) without auxin. The highest frequency of shoot regeneration (52.1%) and mean number of shoots per explant (4.1) were obtained on medium containing 2 mg l^?1 TDZ. Leaf explants inoculated with Agrobacterium tumefaciens strain EHA101/pIG121Hm harboring ß-glucuronidase (uidA) and hygromycin resistance genes developed putative transformant shoots. The highest frequency of shoot regeneration (22.5%) and mean number of transformant shoots per explant (2.4) were obtained on a selection medium consisting of the above described regeneration medium and containing 25 mg l^?1 hygromycin as the selection agent. Approximately 95% of putative transformant shoots expressed the uidA gene following histochemical ß-glucuronidase (GUS) assay. These were confirmed to be transgenic by PCR analysis and Southern blot hybridization.     

A simple <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation method for rapid transgene expression in <Emphasis Type="Italic">Medicago truncatula</Emphasis> root hairs

Medicago truncatula is widely used as a model legume for symbiotic and pathogenic microbial interaction studies. Although a number of Agrobacterium-mediated transformation methods have been developed for M. truncatula, a rapid root transformation system was not yet available for this model plant. Here, we describe an easy method for rapid transgene expression in root hairs of M. truncatula, using young seedlings co-cultivated with the disarmed hypervirulent A. tumefaciens strain AGL1. This method leads to efficient expression of various GUS and fluorescent reporters in M. truncatula root hairs. We showed that transgene expression is detected as soon as 2 days following co-culture, in root hairs of a particular responsive zone lying 0.5–2 cm behind the root tip. This method can be used with a variety of M. truncatula genotypes, and is particularly useful for rapid investigation of the sub-cellular localization of fluorescent fusion proteins. Moreover, combining distinct Agrobacterium strains during the initial co-culture step efficiently generates co-transformed root hairs, suitable for co-localization of different fluorescent fusion proteins in the same cell.     

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.     

Effect of <Emphasis Type="Italic">Agrobacterium</Emphasis> strain and plasmid copy number on transformation frequency,event quality and usable event quality in an elite maize cultivar

Key message

Improving Agrobacterium -mediated transformation frequency and event quality by increasing binary plasmid copy number and appropriate strain selection is reported in an elite maize cultivar.

Abstract

Agrobacterium-mediated maize transformation is a well-established method for gene testing and for introducing useful traits in a commercial biotech product pipeline. To develop a highly efficient maize transformation system, we investigated the effect of two Agrobacterium tumefaciens strains and three different binary plasmid origins of replication (ORI) on transformation frequency, vector backbone insertion, single copy event frequency (percentage of events which are single copy for all transgenes), quality event frequency (percentage of single copy events with no vector backbone insertions among all events generated; QE) and usable event quality frequency (transformation frequency times QE frequency; UE) in an elite maize cultivar PHR03. Agrobacterium strain AGL0 gave a higher transformation frequency, but a reduced QE frequency than LBA4404 due to a higher number of vector backbone insertions. Higher binary plasmid copy number positively correlated with transformation frequency and usable event recovery. The above findings can be exploited to develop high-throughput transformation protocols, improve the quality of transgenic events in maize and other plants.

     

Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants

Genetic transformation is one of the most widely used technique in crop improvement. However, most of the binary vectors used in this technique, especially cloning based, contain antibiotic genes as selection marker that raise serious consumer and environmental concerns; moreover, they could be transferred to non-target hosts with deleterious effects. Therefore, the goal of this study was reconstruction of the widely used pBI121 binary vector by substituting the harmful antibiotic selection marker gene with a less-harmful selection marker, Basta (herbicide resistance gene). The generated vectors were designated as pBI121NB and pBI121CB, in which Basta gene was expressed under the control of Nos or CaMV 35S promoter, respectively. The successful integration of the new inserts into both the vectors was confirmed by PCR, restriction digestion and sequencing. Both these vectors were used in transforming Arabidopsis, Egyptian wheat and barley varieties using LBA4404 and GV3101 Agrobacterium strains. The surfactant Tween-20 resulted in an efficient transformation and the number of Arabidopsis transformants was about 6–9 %. Soaked seeds of wheat and barley were transformed with Agrobacterium to introduce the bacteria to the growing shoot apices. The percentage of transgenic lines was around 16–17 and 14–15 % for wheat and barley, respectively. The quantitative studies presented in this work showed that both LBA4404 and GV3101 strains were suitable for transforming Egyptian wheat and barley.