Optimization of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transient expression of heterologous genes in spinach

The Agrobacterium-mediated transient assay is a relatively rapid technique and a promising approach for assessing the expression of a gene of interest. Despite the successful application of this transient expression system in several plant species, it is not well understood in spinach. In this study, we analyzed various factors, including infiltration method, Agrobacterium strain and density, and co-infiltration of an RNA silencing suppressor (p19), that affect transient expression following agroinfiltration in spinach. To evaluate the effects of these factors on the transient expression system, we used the β-glucuronidase (GUS) reporter gene construct pB7WG2D as a positive control. The vacuum-based infiltration method was much more effective at GUS gene expression than was the syringe-based infiltration method. Among the three Agrobacterium strains examined (EHA105, LBA4404, and GV2260), infiltration with the GV2260 strain suspension at a final optical cell density (OD₆₀₀) of 1.0 resulted in the highest gene expression. Furthermore, co-expression of suppressor p19 also increased the efficiency and duration of gene expression and protein accumulation. The results indicate that the use of optimized conditions for transient gene expression could be a simple, rapid, and effective tool for functional genomics in spinach.     

Histological study of organogenesis in Cucumis melo L. after genetic transformation: why is it difficult to obtain transgenic plants?

Melon (Cucumis melo L.) is widely considered as a recalcitrant species for genetic transformation. In this study, we developed different regeneration and transformation protocols and we examined the regeneration process at different steps by histological studies. The highest regeneration rate (1.13 ± 0.02 plants per explant) was obtained using cotyledon explants of the ‘Védrantais’ genotype on Murashige and Skoog (MS) medium supplemented with 0.2 mg/l 6-benzylaminopurine (BAP) and 0.2 mg/l dimethylallylaminopurine (2-iP). Agrobacterium tumefaciens-mediated transformations with the uidA reporter gene were realized on cotyledon explants cultivated in these conditions: 70–90% of explants expressed a transient GUS activity during the early stages of regeneration, however, only few transgenic plants were obtained (1.8–4.5% of stable transformation with the GV2260pBI101 strain). These results revealed a low capacity of melon GUS-positive cells to regenerate transgenic plants. To evaluate the influence of the Agrobacterium infection on plant regeneration, histological analyses were conducted on explants 2, 7, 15, and 28 days after co-culture with the GV2260pBI101 strain. Genetic transformation occurred in epidermal and sub-epidermal cells and reached the meristematic structures expressing a high level of GUS activity during 14 days of culture; but after this period, most of the meristematic structures showed premature cell vacuolization and disorganization. This disruption of the GUS-positive meristematic areas could be responsible of the difficulties encountered to regenerate melon plants after genetic transformation.     

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.     

Development of an efficient regeneration and Agrobacterium-mediated transformation system in crab apple (Malus micromalus) using cotyledons as explants

Apple has become a model species for Rosaceae genetic and genomic research, but it is difficult to obtain transgenic apple plants by Agrobacterium-mediated transformation using in vitro leaves as explants. In this study, we developed an efficient regeneration and Agrobacterium-mediated transformation system for crab apple (Malus micromalus) using cotyledons as explants. The proximal cotyledons of M. micromalus, excised from seedlings that emerged from mature embryos cultured for 10–14 d in vitro, were suitable as explants for regeneration and Agrobacterium-mediated transformation. Cotyledon explants were cocultivated for 3 d with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA2301 on regeneration medium. Kanamycin-resistant buds were produced on cotyledon explants cultured on selective regeneration medium containing 20 mg/L kanamycin. Acetosyringone supplemented in the Agrobacterium suspension or in the cocultivation medium slightly enhanced the regeneration of kanamycin-resistant buds. The maximum percentage of explants with kanamycin-resistant buds was 11.7%. The putative transformed plants were confirmed by histochemical analysis of β-glucuronidase activity and the polymerase chain reaction amplification of the neomycin phosphotransferase II gene. This transformation system also enables recovery of nontransformed isogenic controls developed from embryo buds and is therefore suitable for functional genomics studies in apple.     

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.     

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.     

An improved protocol for<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Antirrhinum majus</Emphasis> L.

Efficient Agrobacterium -mediated transformation of Antirrhinum majus L. was achieved via indirect shoot organogenesis from hypocotyl explants of seedlings. Stable transformants were obtained by inoculating explants with A. tumefaciens strain GV2260 harboring the binary vector pBIGFP121, which contains the neomycin phosphotransferase gene (NPT II) as a selectable marker and the gene for the Green Fluorescent Protein (GFP) as a visual marker. Putative transformants were identified by selection for kanamycin resistance and by examining the shoots using fluorescence microscopy. PCR and Southern analyses confirmed integration of the GFP gene into the genomes of the transformants. The transformants had a morphologically normal phenotype. The transgene was shown to be inherited in a Mendelian manner. This improved method requires only a small number of seeds for explant preparation, and three changes of medium; the overall transformation efficiency achieved, based on the recovery of transformed plants after 4–5 months of culture, reached 8–9%. This success rate makes the protocol very useful for producing transgenic A. majus plants.Communicated by G. Jürgens     

Agrobacterium tumefaciens-mediated transformation of Elatior Begonia (Begonia×hiemalis Fotsch)

We report a method for Agrobacterium-mediated transformation of Elatior Begonia (Begonia×hiemalis Fotsch). Young leaf discs were infected with Agrobacterium tumefaciens strains AGL0 and LBA4404. Each strain has a binary vector plasmid, pIG121Hm that includes the β-glucuronidase (GUS) gene with an intron as a reporter gene, and both the neomycin phosphotransferase II and the hygromycin phosphotransferase genes as selection markers. Explants were cultured on modified MS medium supplemented with 1.0 mg/l BA, 0.5 mg/l IAA, 300 mg/l ticarcillin, and either 100 mg/l kanamycin and 5 mg/l hygromycin, or 300 mg/l kanamycin for selection and regeneration. Out of 500 explants infected with AGL0, 16 plantlets were regenerated, and out of 628 explants infected with LBA4404, two plantlets were regenerated after 4 months of culture. Transformation was confirmed by Southern blot analysis of the GUS gene and by histochemical assays of GUS activity in plant tissues. Ten in vitro transgenic plants were obtained from AGL0 infected explants only.     

Developing a rapid and highly efficient cowpea regeneration,transformation and genome editing system using embryonic axis explants

Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium-mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium-mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas-mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies.     

Agrobacterium-mediated transformation of cauliflower: optimization of protocol and development of Bt-transgenic cauliflower

A number of factors that are known to influence genetic transformation were evaluated to optimizeAgrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized intoAgrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the syntheticcryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae.     

Efficient Agrobacterium-mediated genetic transformation method using hypocotyl explants of radish (Raphanus sativus L.)

To investigate the gene function of radish (Raphanus sativus L.), several attempts have been made to generate genetically transformed radish. However, no efficient and relatively simple method for the genetic transformation of radish has been developed to date. In this study, we established an Agrobacterium-mediated genetic transformation method using the hypocotyl-derived explants of radish cultivar “Pirabikku”. Primarily based on the Brassica transformation procedure, we optimized it for radish transformation. Using this system, the transformation efficiency of radish hypocotyl explants by Agrobacterium tumefaciens strain GV3101 harboring pIG121-Hm was 13.3%. The copy number of transfer DNA integrated into the genome was either one or two in the four independent transgenic plants. Two of the four plants exhibited male sterility and did not produce self-pollinated seeds. Examination of the expression of the β-glucuronidase (GUS) gene in T₁ plants from fertile T₀ plants showed that the GUS genes were inherited. The improvement in the genetic transformation in this study might pave the way for accelerated molecular breeding and genetic analysis of radish.     

An efficient method for transient gene expression in monocots applied to modify the Brachypodium distachyon cell wall

Background

Agrobacterium-mediated transformation is widely used to produce insertions into plant genomes. There are a number of well-developed Agrobacterium-mediated transformation methods for dicotyledonous plants, but there are few for monocotyledonous plants.

Methods

Three hydrolase genes were transiently expressed in Brachypodium distachyon plants using specially designed vectors that express the gene product of interest and target it to the plant cell wall. Expression of functional hydrolases in genotyped plants was confirmed using western blotting, activity assays, cell wall compositional analysis and digestibility tests.

Key Results

An efficient, new, Agrobacterium-mediated approach was developed for transient gene expression in the grass B. distachyon, using co-cultivation of mature seeds with bacterial cells. This method allows transformed tissues to be obtained rapidly, within 3–4 weeks after co-cultivation. Also, the plants carried transgenic tissue and maintained transgenic protein expression throughout plant maturation. The efficiency of transformation was estimated at around 5 % of initially co-cultivated seeds. Application of this approach to express three Aspergillus nidulans hydrolases in the Brachypodium cell wall successfully confirmed its utility and resulted in the expected expression of active microbial proteins and alterations of cell wall composition. Cell wall modifications caused by expression of A. nidulans α-arabinofuranosidase and α-galactosidase increased the biodegradability of plant biomass.

Conclusions

This newly developed approach is a quick and efficient technique for expressing genes of interest in Brachypodium plants, which express the gene product throughout development. In the future, this could be used for broad functional genomics studies of monocots and for biotechnological applications, such as plant biomass modification for biofuel 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.     

Half-embryo cocultivation technique for estimating the susceptibility of pea (Pisum sativum L.) and lentil (Lens culinaris Medik.) cultivars toAgrobacterium tumefaciens

Langitudinally sliced embryonic axes from pea and lentil mature seeds cocultivated withA. tumefaciens carrying agus reporter gene in its T-DNA provided a convenient means to evaluate the efficiency of gene transfer to tissues in different cultivars and cocultivation conditions. Use of this technique demonstrated wide variation in susceptibility toAgrobacterium among several pea and lentil commercial genotypes.     

Patterns of transformation intensity on flax hypocotyls inoculated with Agrobacterium tumefaciens

Summary Cellular transformation intensities on flax (Linum usitatissimum) hypocotyl explants using disarmed Agrobacterium tumefaciens were investigated through various preculture durations, cocultivation durations and removal of epidermis. The expression of an intron-containing -glucuronidase (GUS) gene driven by CaMV 35S promoter served as a reporter for determination of transformed tissues on hypocotyls. The binary plasmid p35SGUSINT in octopine-type Agrobacterium strain GV2260 was used as the vector system. A prolonged cocultivation duration (5–7 days) resulted in a much higher transformation staining intensity (frequency * tissue area) than 2- or 3-day-cocultivation duration on hypocotyls variously precultured prior to inoculation. A high staining intensity on the two cut ends was obtained from nonprecultured hypocotyls. A reduction in intensity on the upper cut end of hypocotyls was observed with preculture times greater than 6 days. Peeled hypocotyls with a post-peeling preculture of 2 or 3 days had a high proportion of superficial area covered by transformed tissues after a 7 day-cocultivation duration. These results will help to improve the efficiency of recovery of transgenic plants by increasing the proportion of transformation in the regenerable tissues.     

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.     

Agrobacterium-mediated Japonica rice transformation: a procedure assisted by an antinecrotic treatment

An Agrobacterium-mediated transformation protocol for Japonica rice (cv. R321), using conventional genetic vectors and explants pretreated with antinecrotic compounds is presented. We evaluated the effect of two compounds with known antioxidant activity (ascorbic acid and cysteine) and silver nitrate on the viability of stem sections taken from in vitro rice plantlets, and on their interaction with Agrobacterium tumefaciens (At 2260) containing a shuttle vector bearing the gusand bar genes. After co-culture, calli formed on the callus-induction medium were supplemented with phosphinotricin and cefotaxime; putative transgenic plants were recovered on the regeneration medium after three months. All recovered plants were challenged with the herbicide BASTA under greenhouse conditions, and some resistant individuals were analyzed using PCR and a histochemical GUS test. Southern blot analysis of several R₁ transgenic plants indicated the presence of at least two intact bar gene copies per genome. Inheritance of the bar gene at the R₂ generation was confirmed. Antinecrotic pretreatment of the explants provides an adequate environment for the interaction of A. tumefacienswith the plant cells, thus allowing satisfactory transformation performance without the need of super-binary vectors and hyperinfective A. tumefaciens strains. This revised version was published online in June 2006 with corrections to the Cover Date.     

农杆菌介导的番茄遗传转化的研究进展

番茄(Lycopersicon esculentum)传转化体系对其功能基因的研究和基因工程育种有重要影响,对农杆菌(Agrobacterium tumefaciens)介导的番茄遗传转化的研究进展进行了综述,主要包括影响番茄遗传转化效率的几个因素,如番茄的基因型、外植体状态、预培养和侵染过程、分化培养基中的激素和抗生...     

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.     

Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation of Picrorhiza kurroa: an endangered medicinal herb of the alpine Himalayas

Picrorhiza kurroa Royle ex. Benth. is a medicinal herb of immense therapeutic value with restricted geographic distribution. Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation was developed for this plant. Multiple shoot bud induction was achieved from leaf explants cultured in Gamborg??s B₅ medium containing 3?% (w/v) sucrose, 3?mg/l kinetin and 1?mg/l indole-3-butyric acid. More than 90?% of leaf explants formed shoot buds leading to whole plant regeneration. An Agrobacterium-mediated genetic transformation protocol was developed using A. tumefaciens strain GV3101 harboring binary vector pCAMBIA1302 containing the green fluorescent protein and hygromycin phosphotransferase genes. Leaf explants precultured for 2?d were the most suitable for co-cultivation with Agrobacterium and transformation efficiency was enhanced with 200???M acetosyringone. Putative transformants were selected using media containing 15?mg/l hygromycin. Transformation was verified by detection of the green fluorescent protein using fluorescence microscopy and by polymerase chain reaction. Approximately 56?% of the explants were transformed with an average of 3.4?±?0.4 transgenic plantlets per explant. An efficient regeneration and transformation protocol thus developed enabling a fresh perspective of metabolic engineering in P. kurroa using an Agrobacterium-mediated transformation. This is the first report of direct organogenesis from leaf explants and genetic transformation of P. kurroa.