In vitro plant regeneration through direct organogenesis in Populus deltoides clone G48 from petiole explants

A rapid and efficient protocol is developed for in vitro plantlet regeneration of Populus deltoides clone G48 using petiole explants. The highest frequency of shoot regeneration (74.75%) from petiole was obtained on MS medium supplemented with 0.50?mg/l BAP and 0.20?mg/l IAA. The regenerated shoots started turning brown and necrotic after 10?C15?days in culture. To overcome the browning problem, the explants along with the developing shoot buds were transferred to modified MS medium containing 0.50?mg/l BAP, 0.20?mg/l IAA, 15?mg/l AdS, 0.1% PVP, 100?mg/l casein hydrolysate, 50?mg/l L-glutamine, 250?mg/l (NH₄)₂SO₄ and 0.5% agar. Shoot multiplication and elongation took place on the same medium. Indole-3-acetic acid at 0.10?mg/l was most effective for root regeneration. Using the current protocol, it took 2?months to regenerate plantlets. The in vitro regenerated plantlets were successfully acclimatized and established in greenhouse conditions. This regeneration system using petiole explants provides a foundation for Agrobacterium-mediated genetic transformation of P. deltoides clone G48 for incorporation of various silviculturally important traits.     

Highly efficient production of transgenic Scoparia dulcis L. mediated by Agrobacterium tumefaciens: plant regeneration via shoot organogenesis

Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with β-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on the MS medium supplemented with 1 mg l⁻¹ indole-3-butyric acid and 15 mg l⁻¹ hygromycin. Successful transformation was confirmed by PCR analysis using uidA- and hpt-specific primers and monitored by histochemical assay for β-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at a rate of 54.6% with an average of 3.9 ± 0.39 transgenic plantlets per explant was achieved in the present transformation system. It took only 2–3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient and fast genetic transformation system was developed for S. dulcis using an Agrobacterium-mediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering studies in this medicinally important plant.     

High frequency organogenesis in hypocotyl,cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.     

Development of anAgrobacterium-mediated transformation system for regenerating garland chrysanthemum (Chrysanthemum coronarium L.)

Although efficient shoot regeneration and selection are essential for genetic transformation mediated byAgrobacterium, success has been limited with the garland chrysanthemum (Chrysanthemum coronarium L.). In this study, we developed a useful protocol for shoot regeneration with leaf disk explants. The optimal concentrations of NAA and BA were 0.2 mg L⁻¹ and 0.5 mg L⁻¹, respectively. To optimize the selection system for regenerating plants from genetically transformed tissues, we tested the effects of four antibiotics (kanamycin, hygromycin, carbenicillin, and cefotaxime). Among them, 5 mg L^-1 hygromycin proved adequate as a selectable marker, whereas 500 mg L^-1 carbenicillin was effective in eliminating excessiveAgrobacterium after co-cultivation. Transgenic plants were obtained by first co-culturing garland chrysanthemum leaf disks withA. tumefaciens strain EHA105, which harbors plasmid pRCVII containing the hygromycin resistance (hpt) and β-glucuronidase (GUS) genes. After the transgenic plants were confirmed via Southern analysis, they were rooted in soil and appeared phenotypically normal. Our report is the first to describe the optimum conditions for producing transgenic plants of this species.     

Shoot Regeneration from Cultured Leaf Explants of Lycium barbarumand Agrobacterium-Mediated Transformation1

A method for fast plant regeneration via organogenesis directly from Lycium barbarumleaf explants has been developed. The key factor for shoot regeneration was the presence of benzyladenine (BA) in the medium. NAA could only induce root formation and explant callusing. Murashige and Skoog (MS) medium supplemented with 2 mg/l BA and 0.5 mg/l NAA is the most efficient condition for shoot formation, with up to 92.6% shoot regeneration and no callus formation. All adventitious shoots cultured on MS medium supplemented with 1 mg/l IAA formed an extensive root system. Regenerated plants were morphologically normal and were also proved to be diploid (2n = 24). Using the optimized regeneration system, the genetic transformation of L. barbarumwas carried out mediated by Agrobacterium tumefaciensEHA101(pIG121Hm). 11.8% leaf explants produced kanamycin-resistant shoots after infection by A. tumefaciens.The putative transgenic nature of plants was confirmed by GUS assay and PCR analysis. Expression of the nptIIgene in the regenerated plants was also detected by observing the callus formation by leaf pieces on MS medium containing 0.2 mg/l 2,4-D and 0–100 mg/l kanamycin.     

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.     

Development of an Agrobacterium-mediated transformation method for pear (Pyrus communis L.) with leaf-section and axillary shoot-meristem explants

We have developed a new Agrobacterium-mediated transformation method for the low-frequency-regenerating pear (Pyrus communis L.) cvs. Silver bell and La France. Leaf sections derived from in vitro shoots were initially used for the transformation procedure. Under optimum transformation conditions, which included culture and selection on 30 mg/l kanamycin (Km) combined with 500 mg/l sulbenicillin, a 3.2% transformation efficiency was obtained for cv. Silver bell, but no transformants of La France were obtained because of the very low regeneration frequency. Axillary shoot meristems were then examined as potential explants for La France. Selection in 5 mg/l Km and 375 mg/l carbenicillin resulted in transformed shoots being produced at an efficiency of 4.8%, and the apparent white Km-sensitive shoots were not formed during a 2-year subculture on micropropagation medium containing 50 mg/l Km. Therefore, transformations using axillary shoot meristems may be an alternative method for pear cultivars recalcitrant to regeneration from leaf sections.     

Direct organogenesis in hop - a prerequisite for an application ofA. tumefaciens-mediated transformation

The regeneration ability of primary explants derived from mericlones of two commercial Bohemian hops was investigated. It was found that these hops are able to regenerate shoots by direct organogenesis on media containing BAP or zeatin at concentrations 0.5–2 mg dm⁻³. The highest regeneration of shoots was achieved from either petioles or internodes at frequencies 21% and 52%, respectively, on the medium containing zeatin (2 mg dm⁻³), while relatively low amount of regenerated shoots (1.3%) was observed for leaf blade explants. On the other hand, more efficient rooting occurred on the leaf blades then on other explants. A similar pattern of regeneration we observed for HLVd-infected mericlones of clone Osvald 31 even though viroid concentration inin vitro cultures was about 8-fold higher than in field-grown plants and was 31.1 pg mg⁻¹ of fresh mass in the average. These results suggest that HLVd infection did not impair organogenesis. We found that high 2,4-D concentration pretreatment (11 mg dm⁻³) did not promote somatic embryogenesis. Although this treatment suppressed direct organogenesis, the inhibition was not complete and in low frequency the shoot regeneration was seen. Sensitivity of hop explants to antibiotics commonly used inAgrobacterium-mediated transformation was assayed. It was found that kanamycin (100–200 mg dm⁻³) suppressed efficiently callogenesis, root formation and shoot proliferation. An estimation of effect of kanamycin (200 mg dm⁻³) and ticarcillin (500 mg dm⁻³) on morphogenesis was performed using regeneration medium. The inhibitory effects observed suggest that these conditions could be used inAgrobacterium transformation/selection system. Communicated by J. TUPY     

A rapid and efficient method for in vitro shoot organogenesis and production of transgenic Bacopa monnieri L. mediated by Agrobacterium tumefaciens

Efficient shoot regeneration and Agrobacterium-mediated genetic transformation systems were developed for Bacopa monnieri L. (Scrophulariaceae), a plant well known for its medicinal properties. Leaf explants were cultured on Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP), and in combination with either indole-3-acetic acid (IAA) or napthalene-3-acetic acid. A combination of BAP (17.80 μM) and IAA (2.28 μM) maximized shoot initiation (85.2 ± 2.43) with greatest shoot length (2.8 ± 0.22), and was obtained directly from leaf explants without an intervening callus phase. Leaf segments from in vitro grown plants were co-cultivated with Agrobacterium tumefaciens LBA4404 harboring pCAMBIA1301 with ?-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes. The co-cultivated explants were transferred to selective shoot induction and elongation medium. The elongated hygromycin-resistant shoots were subsequently rooted on MS medium supplemented with 4.9 μM indole-3-butyric acid and 25 mg/l hygromycin (SSRM). Successful transformation was confirmed by monitoring histochemical GUS activity during shoot elongation and PCR analyses using uidA- and hpt-specific primers. Integration of hpt into the genome of transgenic plants was also verified by Southern blot analysis. The highest transformation efficiency achieved was 70.6%, with an average of 10.4 ± 0.15 transgenic plantlets per explant using the present transformation system. Therefore, these highly efficient and rapid regeneration and transformation systems create significant potential for engineering of B. monnieri with a view to detailed biomolecular analyses or for further enhancement of its medicinal properties.     

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.     

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 tumefaciens-mediated transformation of rutabaga (Brassica napus var. napobrassica) cultivar “American Purple Top Yellow”

An efficient protocol for genetic transformation of rutabaga (Brassica napus var. napobrassica) cultivar ??American Purple Top Yellow?? was developed by optimizing several factors influencing gene delivery and plant regeneration. A two-step regeneration protocol, adapted from canola, was optimal for rutabaga regeneration using hypocotyl explants. Transient expression studies monitored by histochemical ??-glucuronidase (GUS) assays indicated that several factors, including Agrobacterium tumefaciens strain, cocultivation time, and cocultivation medium, affected gene delivery. For stable transformation, precultured hypocotyl explants were cocultivated with Agrobacterium cells on sterilized filter paper overlaid on callus induction medium containing 100???M acetosyringone for 6?d under a 16-h photoperiod. Selection and regeneration of transformed cells were conducted on media containing 50?mg?l^?1 kanamycin and 250?mg?l^?1 Timentin. Using this protocol, GUS- and PCR-positive transformants were obtained from 3.2 to 4.2?% of hypocotyl explants inoculated with each of the three Agrobacterium strains after 3?C5?mo. Most transformants exhibited a normal phenotype. Southern blot analysis confirmed stable integration of the gusA transgene in T₀ plants.     

High-frequency direct shoot regeneration from <Emphasis Type="Italic">Drymaria cordata</Emphasis> Willd. leaves

An efficient and reproducible procedure is described for direct shoot regeneration in Drymaria cordata Willd. using leaf explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine. The regeneration frequency varied with the plant growth regulator concentrations, orientation of the explants, and the carbon source and basal salts present in the regeneration medium. The highest mean number of shoots per explant (10.65 ± 1.03) was recorded on MS plates containing 3% sucrose and 0.8% agar supplemented with 0.1 mg/l NAA and 1.0 mg/l BAP. Shoot buds were induced in the basal parts of the leaf explants. Concentrations of NAA exceeding 1 mg/l suppressed shoot regeneration. Explants bearing the entire lamina and petiole were much more responsive than those having only the lamina. The plantlets that regenerated from the leaf explants were rooted successively on MS medium alone or in combination with indole butyric acid (IBA). The highest mean number of root organogenesis, with 25.67 ± 3.68 roots per leaf segment, was obtained in the presence of 1 mg/l IBA. Histological investigations of the regenerating shoots showed that the shoot buds had emerged from epidermal cells without callus formation. More than 90% of the in vitro-propagated plants survived when transferred to a greenhouse for acclimatization. Thus, this optimized regeneration system may be used for rapid shoot proliferation and genetic transformation.     

An efficient protocol for Agrobacterium-mediated genetic transformation of Antirrhinum majus

Snapdragon (Antirrhinum majus L.) is a popular ornamental and model plant species, and the recently released reference genome could greatly boost its utilization in fundamental research. However, the lack of an efficient genetic transformation system is still a major limiting factor for its full application in genetic and molecular studies. In this study, a simple method for quick regeneration and efficient Agrobacterium-mediated transformation of snapdragon was developed. Cotyledon petiole and hypocotyl explants derived from two-week-old seedlings were cultured on MS media supplemented with 2 mg/L zeatin (ZT), 0.2 mg/L 1-naphthaleneacetic acid (NAA), and 2 mg/L AgNO₃, and adventitious shoots were regenerated through organogenesis with an average regeneration of 48.00% and 41.33%, respectively. By contrast, the regeneration frequency was only 22.67% for cotyledon petiole and 25.67% for hypocotyl explants in the absence of AgNO₃. Moreover, the application of AgNO₃ promoted indirect shoot organogenesis, while direct shoot organogenesis occurred in the absence of AgNO₃ from both hypocotyl or cotyledon petiole explants. Agrobacterium-mediated genetic transformation systems were developed with this high-efficient regeneration system. The transformation efficiency has been improved from 0 to 1% through the direct shoot organogenesis to 3 to 4% via the indirect shoot organogenesis. This efficient regeneration and genetic transformation method could be important for future use of snapdragon as a model plant to address some fundamental questions which are hard to be solved by using other model plant species, and to accelerate the breeding process through CRISPR/Cas9 genome editing.

     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

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.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.     

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.     

Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium tumefaciens — mediated gene transfer

Summary A transformation system that allows regeneration of transgenic pea plants from calli selected for antibiotic resistance was developed. Explants from axenic shoot cultures and seedling epicotyls were cocultivated with nononcogenic Agrobacterium tumefaciens strains, and transformed callus could be selected on callus-inducing media containing either 15 mg/l hygromycin or 75 mg/l kanamycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on hygromycin-resistant calli, but not on the calli selected for kanamycin resistance. Regenerated shoots could subsequently be rooted and transferred into the greenhouse. In addition, the effects of different callus-inducing and growth media on organogenesis were investigated. The transformation of the calli and regenerated plants was confirmed by DNA analysis.     

Age and orientation of the cotyledonary leaf explants determine the efficiency of de novo plant regeneration and Agrobacterium tumefaciens-mediated transformation in Jatropha curcas L.

Effects of age and orientation of the explant on callus induction and de novo shoot regeneration from cotyledonary leaf segments of Jatropha curcas were studied. The callus induction and shoot regeneration capacity of cotyledonary leaf segments were found significantly related to the age of the explants and their orientation in culture medium. The youngest explant, derived from the cotyledonary leaf of germinated seed induced the highest regeneration response as compared to one- and two-week-old explants. A gradient response with age of the explant was observed in percentage of callus induction, shoot regeneration from callus and the number of shoots per regenerating callus. The explants cultured with their abaxial side in medium showed significantly higher regeneration response. The youngest explant was found to be most amenable to Agrobacterium-mediated transformation as compared to older explants. The fact that callus induced from the edges of the explant followed by de novo shoot induction, and strong transient gus expression observed in the edges of the explant are significant for routine Agrobacterium-mediated transformation and generation of stable transgenic plants in J. curcas.