A reproducible genetic transformation system for cultivated Phaseolus acutifolius (tepary bean) and its use to assess the role of arcelins in resistance to the Mexican bean weevil

A reproducible Agrobacterium tumefaciens-mediated genetic transformation method that delivers fertile and morphologically normal transgenic plants was developed for cultivated tepary bean (Phaseolus acutifolius L. Gray). Factors contributing to higher transformation efficiencies include (1) a low initial concentration of bacteria coupled with a longer cocultivation period with callus, (2) an initial selection of callus on a medium containing low levels of the selectable agent, (3) omission of the selectable agent from the medium during callus differentiation to shoots and (4) the efficient conversion of transgenic shoots into fertile plants. All plants regenerated with this procedure (T₀) were stably transformed, and the introduced foreign genes were inherited in a Mendelian fashion in most of the 33 independent transformants. Integration, stable transmission and high expression levels of the transgenes were observed in the T₁ and/or T₃ progenies of the transgenic lines. The binary transformation vectors contained the -glucuronidase reporter gene, the neomycin phosphotransferase II selectable marker gene and either an arcelin 1 or an arcelin 5 gene. Arcelins are seed proteins that are very abundant in some wild P. vulgaris L. genotypes showing resistance to the storage insect Zabrotes subfasciatus (Boheman) (Coleoptera, Bruchidae). Transgenic beans from two different cultivated P. acutifolius genotypes with high arcelin levels were infested with Z. subfasciatus, but they were only marginally less susceptible to infestation than the non-transgenic P. acutifolius. Hence, the arcelin genes tested here are not major determinants of resistance against Z. subfasciatus.Electronic Supplementary Material Supplementary material is available for this article at     

Agrobacterium-mediated transformation of Phaseolus acutifolius A. Gray

Agrobacterium-mediated transformation of Phaseolus acutifolius A. Gray has been achieved. Regeneration-competent callus, obtained from bud explants of greenhouse-grown plants, was co-cultivated with Agrobacterium tumefaciens C58C1Rif^R(pMP90) harbouring a binary vector with the neomycin phosphotransferase II (nptII) and β-glucuronidase (uidA) marker genes. Transient expression of uidA was detected in five out of six genotypes tested. Transgenic callus lines of three genotypes were established on geneticin-containing medium. Plants were recovered from one line (genotype NI 576). This line had been transformed with a binary plasmid which, in addition to the marker genes, contained a genomic fragment encoding the Phaseolus vulgaris arcelin-5a protein. This seed storage protein presumably confers resistance to the insect Zabrotes subfasciatus, a major pest of P. vulgaris. Integration of foreign DNA was confirmed by molecular analysis. The introduced genes segregated as a single locus. Arcelin-5a was produced at high levels in seeds. The possibility of using P. acutifolius as a `bridging' species to introduce transgenes into the economically more important species P. vulgaris is discussed. Received: 20 July 1996 / Accepted: 23 August 1996     

Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in banana cv. ‘Dwarf Cavendish’ (Musa AAA): effect of spermidine on transformation efficiency

An efficient method has been developed for somatic embryogenesis, plant regeneration and transformation of the important banana cultivar ‘Dwarf Cavendish’ (Musa AAA). A high embryogenic response was obtained in 1.36 % of immature male flower explants. Once embryogenic structures were transferred to liquid medium, embryogenic cell suspensions (ECSs) with high regeneration capacity were obtained. ECSs were incubated under different conditions with Agrobacterium tumefaciens strain EHA101 harboring vector pFAJ3000 that contains pNos-nptII-tOcs and p35S-uidAintron-t35S expression cassettes. The effect of spermidine and infection time on transformation efficiency was examined. The highest efficiency was obtained when ECSs were infected for 6 h, in medium supplemented with 200 μM acetosyringone and 1.0 mM spermidine, with more than 600 independent lines/~50 mg FW of settled cells. Spermidine showed an enhancing effect, increasing significantly the transient Gus expression and the number of transformed embryo colonies and regenerated plants in comparison with the same treatments without this polyamine. This is the first report showing efficient Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in the ‘Dwarf Cavendish’ banana cultivar.     

Agrobacterium mediated genetic transformation of commercial jute cultivar Corchorus capsularis cv. JRC 321 using shoot tip explants

We have developed a reproducible method of Agrobacterium tumefaciens mediated stable genetic transformation of white jute (Corchorus capsularis cv. JRC 321) utilizing the shoot organogenesis potential of the shoot tip apical meristem. A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 was used in the transformation experiments. The explants were subjected to varying durations of preculture and cocultivation with A. tumefaciens in the presence of acetosyringone in order to optimize the conditions conducive for the highest expression of transgene. A schedule of 1 day preculture of shoot tips followed by 3 days cocultivation was optimized for Agrobacterium mediated stable genetic transformation of C. capsularis cv. JRC 321. The optimized lethal doses of the antibiotic hygromycin B for shoot tips (12 mg/L) and for 5 days old seedlings (14 mg/L) were employed in efficient selection of the transformed tissues. This method of transformation resulted in a mean transformation efficiency of 4.09 %. Stable expression of the intron harbored gusA transgene was observed in mature organs of the transformed plants and their progenies. Genomic integration and inheritance of the hpt transgene was further confirmed by Southern blot analysis. The transformed plants exhibited normal morphology and most of them produced viable progenies, many of which segregated in a 3:1 ratio following Mendelian inheritance for a single dominant locus. However, strong P value support for 3:1 segregation ratio was obtained in case of two lines of independent transformants. Nevertheless, the method of transformation mentioned in this protocol could be effectively implemented in genetic transformation of many other cultivars of jute due to the genotype independent regeneration potential of the shoot tip explants.     

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD₆₀₀ of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.     

Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes

To determine the optimum conditions for Agrobacterium-mediated gene transfer, peach explants including cotyledons, embryonic axes and hypocotyl slices from non-germinated seeds and epicotyl internode slices from germinating seeds were exposed to Agrobacterium-mediated transformation treatments. The GUS (uidA) marker gene was tested using two different A. tumefaciens strains, three plasmids and four promoters [CaMV35s, (Aocs)3AmasPmas (“super-promoter”), mas-CaMV35s, and CAB]. GFP was tested with six A.␣tumefaciens strains, one plasmid (pLC101) and the doubleCaMV35s (dCaMV35s) promoter. The CaMV35s promoter produced more GUS expression than the CAB promoter. A. tumefaciens strains EHA105 and LBA4404 harboring the same plasmid (pBIN19) differed in their effects on GUS expression suggesting an interaction between A. tumefaciens strain and plasmid. A combination of A. tumefaciens EHA105, plasmid pBIN19 and the CaMV35s promoter produced the highest rates of transformation in peach epicotyl internodes (56.8%), cotyledons (52.7%), leaves (20%), and embryonic axes (46.7%) as evaluated by the percentage of explants expressing GUS 14 days after co-cultivation. GFP expression under the control of the dCaMV35s promoter was highest for internode explants but only reached levels of 18–19%. When GFP-containing plasmid pCL101 was combined with each of five A. tumefaciens strains the highest levels of transformation were 20–21% (internode and cotyledons, respectively). When nine peach genotypes were co-cultivated with A. tumefaciens strain EHA105 and GFP-containing plasmid pCL101 the highest levels of transformation were 26–28% (cotyledons and internodes, respectively). While GFP represents a potentially useful transformation marker that allows the non-destructive evaluation of transformation, rates of GFP transformation under the conditions of this study were low. It will be necessary to optimize expression of this marker gene in peach.     

Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration

A reproducible and efficient transformation method was developed for the banana cv. Rasthali (AAB) via Agrobacterium-mediated genetic transformation of suckers. Three-month-old banana suckers were used as explant and three Agrobacterium tumefaciens strains (EHA105, EHA101, and LBA4404) harboring the binary vector pCAMBIA1301 were used in the co-cultivation. The banana suckers were sonicated and vacuum infiltered with each of the three A. tumefaciens strains and co-cultivated in the medium containing different concentrations of acetosyringone for 3 days. The transformed shoots were selected in 30 mg/l hygromycin-containing selection medium and rooted in rooting medium containing 1 mg/l IBA and 30 mg/l hygromycin. The presence and integration of the hpt II and gus genes into the banana genome were confirmed by GUS histochemical assay, polymerase chain reaction, and southern hybridization. Among the different combinations tested, high transformation efficiency (39.4 ± 0.5% GUS positive shoots) was obtained when suckers were sonicated and vacuum infiltered for 6 min with A. tumefaciens EHA105 in presence of 50 μM acetosyringone followed by co-cultivation in 50 μM acetosyringone-containing medium for 3 days. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into banana has been developed and that this transformation system could be useful for future studies on transferring economically important genes into banana.     

Rapid,in situ detection of Agrobacterium tumefaciens attachment to leaf tissue

Attachment of the plant pathogen Agrobacterium tumefaciens to host plant cells is an early and necessary step in plant transformation and agroinfiltration processes. However, bacterial attachment behavior is not well understood in complex plant tissues. Here we developed an imaging‐based method to observe and quantify A. tumefaciens attached to leaf tissue in situ. Fluorescent labeling of bacteria with nucleic acid, protein, and vital dyes was investigated as a rapid alternative to generating recombinant strains expressing fluorescent proteins. Syto 16 green fluorescent nucleic acid stain was found to yield the greatest signal intensity in stained bacteria without affecting viability or infectivity. Stained bacteria retained the stain and were detectable over 72 h. To demonstrate in situ detection of attached bacteria, confocal fluorescent microscopy was used to image A. tumefaciens in sections of lettuce leaf tissue following vacuum‐infiltration with labeled bacteria. Bacterial signals were associated with plant cell surfaces, suggesting detection of bacteria attached to plant cells. Bacterial attachment to specific leaf tissues was in agreement with known leaf tissue competencies for transformation with Agrobacterium. Levels of bacteria attached to leaf cells were quantified over time post‐infiltration. Signals from stained bacteria were stable over the first 24 h following infiltration but decreased in intensity as bacteria multiplied in planta. Nucleic acid staining of A. tumefaciens followed by confocal microscopy of infected leaf tissue offers a rapid, in situ method for evaluating attachment of A. tumefaciens' to plant expression hosts and a tool to facilitate management of transient expression processes via agroinfiltration. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

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.     

Development of efficient miniprep transformation methods for Artemisia annua using Agrobacterium tumefaciens and Agrobacterium rhizogenes

Extensive studies have been carried out for the optimization of regeneration and transformation conditions for both Agrobacterium tumefaciens- and Agrobacterium rhizogenes-mediated transformation of the highly medicinal plant Artemisia annua. Most protocols describe laborious transformation procedures requiring no less than 3 mo to obtain transgenic plants. This study reports rapid and efficient protocols for A. tumefaciens- and A. rhizogenes-mediated transformation of A. annua, which were equally effective for transformation of Artemisia dubia. In both transformation procedures, stem explants responded best for maximal production of transformed plants and hairy roots. In the case of A. tumefaciens-mediated transformation, stem explants were pre-cultured for 2 d followed by infection with A. tumefaciens strain LBA4404 for 48 h. A. annua explants showed maximal transformation rate (43.5%) on half-strength Murashige and Skoog medium containing 40 mg/L kanamycin in only 20 d. The same method was tested using a related species A. dubia and resulted in a transformation rate of 41.3%, demonstrating that this protocol is efficient and genotype-independent. In the case of A. rhizogenes-mediated transformation for the production of hairy root cultures, in vitro-grown stem explants were infected with a single colony of A. rhizogenes strain LBA9402 by creating incisions at different places of the stem explants, which resulted in production of hairy roots in only 7 d. The method was tested in both A. annua and A. dubia, which resulted in transformation rates of 90 and 87.5%, respectively. Integration of the transgene and copy number was confirmed by PCR and Southern blot analyses, respectively. The miniprep transformation protocols developed for both A. tumefaciens- and A. rhizogenes-mediated transformation are simple, efficient, and potentially applicable to other species of Artemisia for transfer of pharmaceutically important genes.     

Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation

The effects of timentin on shoot regeneration of tobacco (Nicotiana tabaccum) and Siberian elm (Ulmus pumila L.) and its use for the suppression of Agrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were determined. Timentin is a mixture of ticarcillin and clavulanic acid, and at concentrations of 200–500 mg/l with ratios of ticarcillin:clavulanic acid of 50:1 and 100:1, it had little effect on shoot regeneration of tobacco or Siberian elm. Timentin was as effective in suppressing A. tumefaciens as carbenicillin and cefatoxime at concentrations commonly used in transformation. The disarmed A. tumefaciens strain LBA4404 in infected tobacco leaf tissues was visually undetectable after three subcultures on medium with 500 mg/l of timentin and 250 mg/l carbenicillin. Timentin was stable in solid agar medium and remained effective for at least 70 days, but was unstable when stored as a mixed stock solution or as separate ticarcillin and clavulanic acid stock solutions at –20°C or –80°C freezer for 4 weeks. Timentin may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation. Received: 8 September 1997 / Revision received: 19 November 1997 / Accepted: 2 December 1997     

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.     

High-efficiency gene transfer to recalcitrant plants by Agrobacterium tumefaciens

 Agrobacterium tumefaciens efficiently transforms most plants. A few dicotyledonous plants and most monocotyledonous plants are, however, recalcitrant to A. tumefaciens infection. We investigated whether the constitutive synthesis of a high level of the T-strand DNA intermediate can improve the transformation efficiency of plants. We previously described a mutation in the vir gene regulator virG, virGN54D, that allows constitutive expression of the vir genes. We also described the isolation of a mutant plasmid that is present at a significantly high level in A. tumefaciens. The two mutations were combined to produce an A. tumefaciens strain that synthesizes a high level of T-strand DNA in an inducer-independent manner. DNA transfer efficiency of the mutant was measured by monitoring β-glucuronidase (GUS) expression in a transient transfer assay. A significant increase in the efficiency of DNA transfer to both rice and soybean was observed with the double mutant. The presence of virGN54D had a major positive effect on transformation efficiency. Received: 4 August 2000 / Revision received: 9 October 2000 / Accepted: 12 October 2000     

Agrobacterium tumefaciens-mediated transformation of a taxol-producing endophytic fungus, Cladosporium cladosporioides MD2

In this study, an Agrobacteriurn tumefaciens-mediated transformation (ATMT) protocol was successfully developed for the genetic transformation of a taxol-producing fungus, Cladosporium cladosporioides MD2, and the co-cultivation conditions affecting the transformation efficiency were optimized. The optimal transformation conditions were that 1 ml of C. cladosporioides MD2 spore suspension (10⁸ spores/ml) was mixed with an equal volume of A. tumefaciens cultures, which contained 400 μl of A. tumefaciens LBA4404 (OD₆₆₀ ≈ 0.6) and 600 μl LB medium that were used to make up difference in volume, and the mix cultures were supplemented with 300 μM acetosyringone (AS) and co-cultivated at 26°C and 50 rpm for 48 h. Stable transformants were obtained through analysis of the mitotic stability of inserted T-DNA and the presence of hygromycin resistance gene (hpt II). This study laid a fine groundwork for development of transgenic C. cladosporioides MD2 strains.     

Susceptibility to Agrobacterium tumefaciens and cotyledonary node transformation in short-season soybean

 Short-season adapted soybean [Glycine max (L.) Merrill] genotypes (maturity group 0 and 00) were susceptible to Agrobacterium tumefaciens in tumor-formation assays with A. tumefaciens strains A281, C58 and ACH5. The response was bacterial-strain and plant-cultivar dependent. In vitro Agrobacterium-mediated transformation of cotyledonary node explants of these genotypes with A. tumefaciens EHA105/pBI121 was inefficient but resulted in a transgenic AC Colibri plant carrying a linked insertion of the neomycin phosphotransferase and β-glucuronidase (gus) transgenes. The transgenes were transmitted to the progeny and stable gus expression was detected in the T₇ generation. The low rate of recovery of transgenic plants from the co-cultured cotyledonary explants was attributed to inefficient transformation of regenerable cells, and/or poor selection or survival of such cells and not to poor susceptibility to Agrobacterium, since, depending on the cultivar, explants were transformed at a rate of 27–92%, but transformation events were usually restricted to non-regenerable callus. Received: 8 January 1998 / Revision received: 30 June 1999 / Accepted: 12 July 1999     

Factors influencing regeneration and Agrobacterium tumefaciens-mediated transformation of common bean (Phaseolus vulgaris L.)

A systematic study was carried out to optimize regeneration and Agrobacterium tumefaciens-mediated transformation of four common bean (Phaseolus vulgaris L.) cultivars; Red Hawk, Matterhorn, Merlot, and Zorro, representing red kidney, great northern, small red, and black bean commercial classes, respectively. Regeneration capacity of leaf explants, stem sections, and embryo axes were evaluated on 30 media each containing Murashige and Skoog (MS) medium and different combinations of plant growth regulators. For stem sections and leaf explants, none of the media enabled plant regeneration from any of the four cultivars tested, indicating the recalcitrance of bean regeneration from these tissues. In contrast, several media enabled multiple shoot production from embryo axis explants, although optimal regeneration media was genotype-dependent. Under optimal regeneration conditions, multiple shoots, 2.3–10.8 on average for each embryogenic explant, were induced from embryo axis explants at frequencies of 93 % for ‘Merlot’, 80 % for ‘Matterhorn’, 73 % for ‘Red Hawk’, and 67 % for ‘Zorro’. Transient expression studies monitored by an intron-interrupted gusA on explants transformed with A. tumefaciens strains GV3101, LBA4404, and EHA105 indicated that all three A. tumefaciens strains tested were efficient in gene delivery. Gene delivery depended on parameters including strain of A. tumefaciens, co-cultivation time, explant type, and bean genotype. Agroinfiltration also enhanced gene delivery. Kanamycin-resistant and GUS-positive calluses were induced from leaf, stem, and embryo axis explants. Chimeric transformants were obtained from embryo axis explants and showed partial GUS-staining. Lack of efficient regeneration from non-meristem containing tissues is the main limitation for stable transformation of common bean.     

Analysis of a <Emphasis Type="Italic">LEA</Emphasis> gene promoter via <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the desiccation tolerant plant <Emphasis Type="Italic">Lindernia brevidens</Emphasis>

An Agrobacterium tumefaciens-based transformation procedure was developed for the desiccation tolerant species Lindernia brevidens. Leaf explants were infected with A. tumefaciens strain GV3101 harbouring a binary vector that carried the hygromycin resistance gene and an eGFP reporter gene under the control of a native dehydration responsive LEA promoter (Lb2745pro). PCR analysis of the selected hygromycin-resistant plants revealed that the transformation rates were high (14/14) and seeds were obtained from 13/14 of the transgenic lines. A combination of RNA gel blot and microscopic analyses demonstrated that eGFP expression was induced upon dehydration and ABA treatment. Comparison with existing procedures used to transform the well studied resurrection plant and close relative, Craterostigma plantagineum, revealed that the transformation process is both rapid and leads to the production of viable seed thus making L. brevidens a candidate species for functional genomics approaches to determine the genetic basis of desiccation tolerance.     

Optimization of a <Emphasis Type="Italic">Bacopa monnieri</Emphasis>-based genetic transformation model for testing the expression efficiency of pathway gene constructs of medicinal crops

A fast regenerating Agrobacterium tumefaciens-mediated transformation protocol for Bacopa monnieri (L.) Wettst. was developed as a model system for heterologous expression of terpenoid indole alkaloid pathway genes from Catharanthus roseus (L.) G. Don. The direct regeneration of shoots from leaf explants co-cultured with A. tumefaciens resulted in the integration of a tryptophan decarboxylase (tdc) and strictosidine synthase (str) cassette (<hpt-<Tdc2-<Str-gus>) in the regenerated progeny. The highest transformation efficiency (83.88%) was achieved when leaf explants were infected on the adaxial laminar surface by manual pricking with 48- to 72-h-old suspensions (OD₆₀₀ = 0.5–0.6) of A. tumefaciens strain LBA1119 (carrying the binary vector pMOG22). The heterologous expression of tryptophan decarboxylase and strictosidine synthase genes that are otherwise not present in B. monnieri plants was confirmed through semi-quantitative PCR and metabolite quantification assays. The entire protocol duration from co-cultivation through regeneration of transgenic plants to their establishment in the glass house took 40–45 d. The developed B. monnieri model can be used to test expression cassettes carrying genes for plant secondary metabolic pathway engineering, especially those genes that are expressed in differentiated cell, tissue, or organs.     

Exploration on the vacuum infiltration transformation of pakchoi

Agrobacterium tumefaciens mediated vacuum infiltration transformation in planta has been established in pakchoi, a kind of Chinese cabbage, but the transformation frequency in harvested seeds has varied in the range of 0.5 to 3.0 × 10⁻⁴ over several years and is much lower than the transformation frequency in Arabidopsis thaliana. To understand that, the distribution and vitality changes of A. tumefaciens in plant tissues were examined. Results revealed that there was a majority of A. tumefaciens in the flower compared with that in the stem and in the leaf at all times after infiltration. As fact of transformants in the upper part of the treated plant (T₀) stalk and fact of the survival of A. tumefaciens in the plant were proved, possibilities of optimizing the transformation conditions to increase the transformation frequency in pakchoi was discussed.