Influence of <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> on induction of hairy roots and benzylisoquinoline alkaloids production in Persian poppy (<Emphasis Type="Italic">Papaver bracteatum</Emphasis> Lindl.): preliminary report

Persian poppy (Papaver bracteatum Lindl.) is an important medicinal plant and source of the opium alkaloids codeine, morphine and thebaine. Transgenic root cultures of P. bracteatum Lindl. are well-defined model systems to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis. Agrobacterium rhizogenes was able to produce hairy roots on wounded Persian poppy seedlings. Excised shoots from 7-day-old Persian poppy were co-cultivated with the A. rhizogenes strain R15834 carrying the pBI121 binary vector. All media, except for the co-cultivation medium, included 40 mg l⁻¹ paromomycin to select for pBI121 transformants and 200 mg l⁻¹ cefotaxime to eliminate the Agrobacterium. Eight weeks after infection, paromomycin-resistant roots appeared on 45–50% of explants maintained on hormone-free medium. Isolated hairy roots were propagated in liquid medium containing 1.0 mg l⁻¹ 1-naphthaleneacetic acid to promote rapid growth. Also, callus induction and shoot regeneration of transformed Calli in vitro was achieved on B5 medium containing 1.0 mg l⁻¹ 1-naphthaleneacetic acid. Detection of the neomycin phosphotransferase gene and GUS histochemical localization confirmed the integrative transformation of root cultures. This is the first study to illustrate useful protocol to introduce foreign genes into transgenic Persian poppy hairy root cultures using A. rhizogenes strain R15834.     

Agrobacterium tumefaciens-mediated transformation of Lesquerella fendleri L., a potential new oil crop with rich lesquerolic acid

A protocol was developed for regeneration and Agrobacterium-mediated genetic transformation of Lesquerella fendleri. Calli were first induced from hypocotyls and cotyledons on MS plus 0.5 mg l⁻¹ BA, 1 mg l⁻¹ NAA and 1 mg l⁻¹ 2,4-D, then co-cultivated for 2–3 days in darkness on MS supplemented with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹ NAA and 100 μmol l⁻¹As together with Agrobacterium tumefaciens strain EHA105/pCAMBIA1301 that harbored genes for uidA (GUS) and hygromycin resistance. Following co-cultivation, calli transfected by A. tumefaciens were transferred to MS with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹NAA, 500 mg l⁻¹ Cef and 10 mg l⁻¹ hygromycin and cultured for 10 days, then the hygromycin was increased to 20 mg l⁻¹ on the same medium. After 4 weeks the resistant regenerants were transferred to MS with 0.5 mg l⁻¹BA, 0.2 mg l⁻¹ NAA, 500 mg l⁻¹ Cef and 25 mg l⁻¹ hygromycin for further selections. Transgenic plants were confirmed by polymerase chain reaction analysis, GUS histochemical assay and genomic Southern blot hybridization. With this approach, the average regeneration frequency from transfected calli was 22.70%, and the number of regenerated shoots per callus was 6–13. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for improvement of this Lesquerella species.     

Effects of type of explant and age,plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

Plant regeneration was achieved through direct and indirect somatic embryogenesis in Eucalyptus camaldulensis. Callus was induced from mature zygotic embryos and from cotyledon explants collected from 10, 15, 25, and 30-day-old seedlings cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of naphthaleneacetic acid (NAA). Maximum callus induction from mature zygotic embryos was obtained on MS basal medium containing 1 mg l⁻¹ NAA. The frequency of callus development varied based on the age of the cotyledon explants 10-day-old explants giving highest percentage on MS basal medium supplemented with 1 mg l⁻¹ NAA. Callus obtained from mature zygotic embryos gave highest frequency of somatic embryogenesis on MS basal medium containing 0.5 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ NAA. Separate age wise culture of the calli, obtained from cotyledons of different ages cultured separately, revealed high somatic embryogenic potential on callus from 10-day-old cotyledons. Direct somatic embryogenesis too was obtained from hypocotyl explants without an intervening callus phase on MS basal medium containing 0.5 mg l⁻¹ BA. The effects of abscisic acid (ABA), sucrose, and different strengths of MS medium on somatic embryo maturation and germination were also investigated. Number of mature somatic embryos increased with lower concentrations (0–1 mg l⁻¹) of ABA while no significant differences were observed at higher concentrations (2–5 mg l⁻¹) of ABA. Compared to basal medium containing lower concentrations of sucrose (1%), the MS medium supplemented with higher levels of sucrose (4%) showed significantly lower frequency of mature somatic embryos. Basal medium without any dilution gave the highest number of immature embryos. However, the number of mature embryos was high at higher medium dilutions.     

<Emphasis Type="Italic">Crambe tataria</Emphasis>: actions for ex situ conservation

In vitro micropropagation by direct organogenesis and somatic embryogenesis via callus was developed for Crambe tataria (Brassicaceae). C. tataria is an endemic species of the Pontic-Pannonic region, but it is also present in Italy, where it is localized in Friuli on a characteristic grassland formation, called “magredi”. C. tataria is regarded as an endangered species. Leaf and root explants were subjected to plant regulator treatments, which invoked different morphogenic responses. Leaf explants produced more callus than root explants and a higher amount of callus was obtained with 1 mg l⁻¹ 2,4-D in combination with 2 mg l⁻¹ Kin. Somatic embryogenesis was obtained in calli maintained in a delayed subculture regime on media containing BAP in combination with NAA. Root explants cultured with BAP combined with NAA developed adventitious rosette shoots. Shoots rooted on half-strength MS media, and the number of roots per plantlet and their length were heavily dependent on sucrose content. The in vitro regenerated plantlets were acclimatized ex vitro and a mean of 50% of the plantlets survived and showed a true-to-type growth habit. This study describes the development of two in vitro micropropagation protocols, via direct organogenesis and via embryogenesis from callus, that are the basis for the application of in vitro tools for the establishment of basal collections with representative genetic diversity and for the long-term storage of plant genetic material.     

Plant regeneration via somatic embryogenesis of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> in temporary immersion system (TIS)

The present study describes a protocol for plant regeneration via somatic embryogenesis in temporary immersion system (TIS) for Camptotheca acuminata. Somatic embryos were induced by culturing hypocotyl segments from 14-day-old in vitro grown C. acuminata seedlings in TIS. Hypocotyl segments were placed in culture vessels modified with a mechanical device to support the fixation of explants. Cultures were maintained under a 16 h photoperiod with a light intensity of 60 μmol m⁻² s⁻¹ PPF at 25 ± 1°C. After 16 weeks of incubation embryogenic calli were formed above the edge of the mechanical device in the basal Murashige and Skoog (MS) medium containing 35 g l⁻¹ sucrose and without hormonal supplementation. For plantlet regeneration, somatic embryos at cotyledonary stage were cultured in three different concentrations of 6-benzylamino-purine (0.5, 1.0 and 1.5 mg l⁻¹ BAP) and in plant growth regulator (PGR) free medium. In general, 0.5 mg l⁻¹ BAP was found to be the most effective concentration for growth and development of Camptotheca embryos in TIS. Conversion of somatic embryos into plantlets was also successfully achieved on sterile substrates moistened with 0.5 mg l⁻¹ BAP. Plantlets derived from cotyledonary embryos were rooted in vitro with 0.5 mg l⁻¹ indole-3-butyric acid (IBA) before transfer to ex vitro conditions.     

Somatic embryo proliferation, maturation and germination in Catharanthus roseus

In the present study an efficient somatic embryogenesis method has been developed in Catharanthus roseus. Friable embryogenic callus was induced from hypocotyl of in vitro germinated seeds on Murashige and Skoog basal nutrient media supplemented with various auxins particularly 2,4-D (1.0 mg l⁻¹). However, only NAA (1.0 mg l⁻¹) produced somatic embryos in cultures. Embryo proliferation was even high on the same medium added with BAP. Cotyledonary somatic embryo germinated and converted into plantlets in BAP (0.5 mg l⁻¹) added medium following a treatment with gibberellic acid (1.0 mg l⁻¹) for maturation. Carbon sources and concentrations had a marked influence on maturation process. Plantlet conversion was better achieved when embryos were matured on 3% fructose or 3–6% maltose. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as raw material, genetic modification to embryo precursor cell may improve alkaloid yield further.     

Studies on genetic transformation of Theobroma cacao L.: evaluation of different polyamines and antibiotics on somatic embryogenesis and the efficiency of uidA gene transfer by Agrobacterium tumefaciens

In order to develop a more efficient genetic transformation system for cacao somatic embryos, the effects of polyamines and β-lactam antibiotics on somatic embryogenesis, hygromycin as selective agent, and different factors affecting uidA gene transfer have been evaluated. The polyamines putrescine, spermidine, and spermine significantly improved secondary somatic embryogenesis in cacao. Spermine at 1,000 μM provided the best responses, increasing 6.7× the percentage of embryogenic callus and 2.5× the average number of embryos per embryogenic callus. The β-lactam antibiotics timentin and meropenem, used for Agrobacterium tumefaciens counter-selection, had a non-detrimental effect on secondary somatic embryogenesis, depending on their concentration, whereas the commonly used β-lactam cefotaxime inhibited it, irrespective of the tested concentration. Hygromycin showed a strong inhibitory effect on secondary somatic embryogenesis of cacao, impairing completely the embryo production at 20 mg l⁻¹. Following the criterion of GUS activity, the best conditions for T-DNA transfer into cotyledon explants from primary somatic embryos of cacao were a sonication of the explants for 100 s, a 20-min incubation period in Agrobacterium solution, an Agrobacterium concentration of 1.0 (OD₆₀₀), and cocultivation of the explants on tobacco feeder layers. These findings will have important implications for studies on functional genomics of cacao.     

Somatic embryogenesis and plant regeneration in date palm <Emphasis Type="Italic">Phœnix dactylifera</Emphasis> L., cv. Boufeggous is significantly improved by fine chopping and partial desiccation of embryogenic callus

Plant regeneration through somatic embryogenesis from young leaf explants (5–10 mm long) adjacent to the apex of 5–6 year old offshoots of Tunisian date palm (Phœnix dactylifera L.), cultivar Boufeggous was successfully achieved. Factors affecting embryogenic callus initiation, including plant growth regulators and explant size, were investigated. The highest induction frequencies of embryogenic calli occurred after 6–7 months on MS medium supplemented with 10 mg l⁻¹ 2,4-D and 0.3 mg l⁻¹ activated charcoal. The subculture of these calli onto maintenance medium resulted in the formation of proembryos. Fine chopping and partial desiccation (6 and 12 h) of embryogenic calli with proembryos prior to transfer to MS medium supplemented with 1 mg l⁻¹ ABA stimulated the rapid maturation of somatic embryos. Maturated somatic embryo yield per 0.5 g FW of embryogenic callus was 51 embryos with an average maturation time of 55 days. This was increased to 422 with finely chopped callus, and 124 and 306 embryos following 6 and 12 h desiccation treatments, respectively. The average time to maturation for these 3 treatments was 35, 43 and 38 days, respectively. Subsequent substitution of ABA in MS medium with 1 mg l⁻¹ NAA resulted in the germination and conversion of 81% of the somatic embryos into plantlets with normal roots and shoots. The growth of regenerated somatic plants was also monitored in the field.     

Genetic transformation and overexpression of a rice <Emphasis Type="Italic">Hd3a</Emphasis> induces early flowering in <Emphasis Type="Italic">Saussurea involucrata</Emphasis> Kar. et Kir. ex Maxim

Saussurea involucrata is a valuable traditional Chinese medicinal herb. This is the first report of a successful genetic transformation protocol for S. involucrata using Agrobacterium tumefaciens. Leaf explants were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301, which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following co-cultivation, about 23.7% of the explants produced hygromycin-resistant calli on MS basal medium (Murashige and Skoog in Physiol Plant 15: 473–497, 1962) supplemented with 1 mg l⁻¹ benzyladenine (BA), 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 20 mg l⁻¹ hygromycin, and 500 mg l⁻¹ cefotaxime. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.1 mg l⁻¹ NAA, 0.25 mg l⁻¹ gibberellic acid (GA3), 20 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 67.5% of the resistant calli differentiated into shoots. Finally, 80% of the hygromycin-resistant shoots rooted on MS media supplemented with 0.2 mg l⁻¹ NAA, 20 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime. The transgenic nature of the transformants was demonstrated by detection of β-glucuronidase activity in the primary transformants and by Southern blot hybridization analysis. About 16% of the total inoculated leaf explants produced transgenic plants after approximately 5 months. Using this optimized transformation system, a rice ortholog of the Arabidopsis FLOWERING LOCUS T gene, Hd3a, was transferred into S. involucrata. Introduction of this gene caused an early-flowering phenotype in S. involucrata.     

Callus induction and high-efficiency plant regeneration via somatic embryogenesis in <Emphasis Type="Italic">Papaver nudicaule</Emphasis> L., an ornamental medicinal plant

We describe culture conditions for a high-efficiency in vitro regeneration system of Papaver nudicaule through somatic embryogenesis and secondary somatic embryogenesis. The embryogenic callus induction rate was highest when petiole explants were cultured on Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 0.1 mg l⁻¹ 6-benzyladenine (BA) (36.7%). When transferred to plant growth regulator (PGR)-free medium, 430 somatic embryos formed asynchronously from 90 mg of embryogenic callus in each 100-ml flask. Early-stage somatic embryos were transferred to MS medium containing 1.0 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA to germinate at high frequency (97.6%). One-third-strength MS medium with 1.0% sucrose and 1.0 mg l⁻¹ GA₃ had the highest frequency of plantlet conversion from somatic embryos (91.2%). Over 90% of regenerated plantlets were successfully acclimated in the greenhouse. Secondary somatic embryos were frequently induced directly when the excised hypocotyls of the primary somatic embryos were cultured on MS medium without PGRs. Sucrose concentration significantly affected the induction of secondary embryos. The highest induction rate (89.5) and number of secondary somatic embryos per explant (9.3) were obtained by 1% sucrose. Most secondary embryos (87.2–94.3%) developed into the cotyledonary stage on induction medium. All cotyledonary secondary embryos were converted into plantlets both in liquid and on semisolid 1/3-strength MS medium with 1.0% sucrose.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation and regeneration of transgenic plants using leaf segments as explants in Valencia sweet orange

In this study, attempts were made to develop a protocol for regeneration of transgenic plants via Agrobacterium tumefaciens-mediated transformation of leaf segments from ‘Valencia’ sweet orange (Citrus sinensis L. Osbeck) using gfp (green fluorescence protein) as a vital marker. Sensitivity of the leaf segments regeneration to kanamycin was evaluated, which showed that 50 mg l⁻¹ was the best among the tested concentrations. In addition, factors affecting the frequency of transient gfp expression were optimized, including leaf age, Agrobacterium concentration, infection time, and co-cultivation period. Adventitious shoots regenerated on medium containing Murashige and Tucker basal medium plus 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.5 mg l⁻¹ 6-benzyladenine (BA) and 0.5 mg l⁻¹ kinetin (KT). The leaf segments from 3-month-old in vitro seedlings, Agrobacterium concentration at OD₆₀₀ of 0.6, 10-min immersion, and co-cultivation for 3 days yielded the highest frequency of transient gfp expression, shoots regeneration response and transformation efficiency. By applying these optimized parameters we recovered independent transformed plants at the transformation efficiency of 23.33% on selection medium (MT salts augmented with 0.5 mg l⁻¹ BA, 0.5 mg l⁻¹ KT, 0.1 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 250 mg l⁻¹ cefotaxime). Expression of gfp in the leaf segments and regenerated shoots was confirmed using fluorescence microscope. Polymerase chain reaction (PCR) analysis using gfp and nptII gene-specific primers further confirmed the integration of the transgene in the independent transgenic plants. The transformation methodology described here may pave the way for generating transgenic plants using leaf segments as explants.     

In vitro screening of rice genotypes for drought tolerance using polyethylene glycol

The aim of the present investigation was to study in vitro somatic embryogenesis and to screen calli for drought tolerance using mature embryos as explants. Mature embryos of three aromatic (Pusa Basmati 1, Pant Sugandh Dhan 17, Taraori Basmati) and one non-aromatic (Narendra 359) indica rice (Oryza sativa L.) varieties were used for developing callus on Murashige and Skoog medium supplemented with 2, 4-dichlorophenoxy acetic acid (2, 4-D) (2.0 mg l⁻¹ for Narendra 359 and 2.5 mg l⁻¹ for Pusa Basmati 1, Taraori Basmati and Pant Sugandh Dhan 17). Screening of calli was done by sub-culturing calli for 15 days on Murashige and Skoog (MS) basal medium supplemented with different concentrations of polyethylene glycol (PEG)-6000 as chemical drought inducer. Callus volume decreased and total proline content was found to be increased significantly with increase in PEG concentration. Narendra 359 showed best response in terms of callus growth at 70 g l⁻¹ of PEG. The highest percentage somatic embryogenesis among selected calli was observed in Pusa Basmati 1 and the lowest in Pant Sugandh Dhan 17. Excellent shooting and rooting (94%) was observed in MS + 0.1 mg l⁻¹ naphthalene acetic acid (NAA) and MS + 2.0 mg l⁻¹ 2, 4-D. Regenerated plants were successfully acclimatized with 98% efficiency in greenhouse and grown under pot conditions up to maturity. It was observed that PEG treated somaclones accumulated more proline, chlorophyll content and developed more tiller and height than normal somaclones. Ten random amplified polymorphic DNA (RAPD) primers were used to amplify genomic DNA of somaclones of different varieties. Level of genetic polymorphism existing among these somaclones indicates that these markers can be used in breeding program for improving varieties through in vitro techniques.     

Evaluation of key factors influencing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of somatic embryos of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.)

Key factors influencing the efficiency of transformation of embryogenic cultures, induced from immature zygotic embryos, of avocado cv. ‘Duke 7’ were evaluated. Initially, the sensitivity of somatic embryos to the antibiotics kanamycin, used for selection, carbenicillin, cefotaxime and timentin, all used for elimination of Agrobacterium cells, were evaluated. Isolated globular somatic embryos were more sensitive to kanamycin than embryogenic masses, and 25 mg l⁻¹ kanamycin completely restricted callus proliferation. Cefotaxime at 500 mg l⁻¹ partially inhibited proliferation of embryogenic cultures, while both carbenicillin and timentin did not affect callus growth. For genetic transformation, somatic embryos were infected with A. tumefaciens containing the pBINUbiGUSint plasmid. After 2 days, the embryos were transferred to selection medium supplemented with 50 mg l⁻¹ kanamycin and 250 mg l⁻¹ timentin for 2 months. Then, kanamycin level was increased to 100 mg l⁻¹ for two additional months. The A. tumefaciens strain AGL1 yielded higher transformation rates, 6%, than EHA105 or LBA4404, 1.2%. The percentage of kanamycin resistant calli obtained was significantly influenced by the embryogenic line used as source of explants. Genetic transformation was confirmed by PCR and Southern blot analysis. A significant improvement in the germination rate was obtained when transgenic embryos were cultured in liquid MS medium with 4.44 μM BA and 2.89 μM GA₃ for 3 days in a roller drum and later transferred to the same medium gelled with 7 g l⁻¹ agar. Plants from five independent transgenic lines were acclimated and grown in the greenhouse, being phenotipically similar to control plants.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

An elite protocol for accelerated quality-cloning in <Emphasis Type="Italic">Gerbera jamesonii</Emphasis> Bolus cv. Sciella

A novel, efficient, and simple protocol was developed on in vitro mass propagation and acclimatization of Gerbera jamesonii Bolus cv. Sciella, an ornamental plant with attractive flowers. Shoot tip was used as the primary explant for in vitro establishment in which Murashige and Skoog (MS) medium supplemented with a low level of NAA (0.5 mg l⁻¹) and BAP (1.5 mg l⁻¹) promoted earliest axillary bud initiation within 5 d in 91.6% of the inoculants. Five axillary buds were initiated from a single explant within 13 d after inoculation. A very high rate of shoot multiplication (14 shoots per inoculated axillary bud) and proliferation was achieved when MS medium was fortified with a relatively higher level of BAP (2 mg l⁻¹) and 60 mg l⁻¹ ADS within 27 d of multiple shoot culture. A maximum number of well-developed roots per plant was observed in MS medium with 0.5 mg l⁻¹ IAA in the next 26 d. In the easy low-cost acclimatization process of 20 d, a combination of sand, soil, cow urine, and tea leaves extract (1:1:1:1; v/v) ensured 95% survival rate. Sixty-one well-acclimatized plants were obtained from a single shoot tip within 86 d. The sustained multiple shoot culture for 15 mo paved the way toward the conservation of genetic resources as well as beneficial economics. The clonal fidelity study of micropropagated and sustained cultured clones using ISSR primers ensured the continuous supply of quality propagules retaining genetic uniformity. The in vitro-generated plants performed better over conventionally propagated plants in the field condition.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

Asparagus racemosus cell cultures: a source for enhanced production of shatavarins and sarsapogenin

Asparagus racemosus is an important monocot medicinal plant that is in great demand for its steroidal saponins called shatavarins. This study was initiated to optimize the conditions for production of shatavarins in cell cultures of A. racemosus in a modified Murashige and Skoog (MS) medium supplemented with six different combinations of growth regulators. Biomass accumulation was correlated with saponin production over a 30-d culture cycle. Biomass and saponin accumulation patterns were dependent on combinations of growth regulators and the pH of the medium. Maximum levels of saponin and biomass accumulation were recorded on day 25 of the culture cycle within a pH range of 3.4 to 5.6. Total saponin produced by the in vitro cultures was 20-fold higher than amounts produced by cultivated plants. Saponin accumulation was not a biomass-associated phenomenon; cultures which showed the highest biomass accumulation were not the highest saponin accumulators. Maximum biomass (28.30 ± 0.29 g l⁻¹) and maximum levels of shatavarin IV(11.48 ± 0.61 mg g⁻¹) accumulation was found using a medium containing 2.0 mg l⁻¹ 2,4-D, 2 g l⁻¹ casein hydrolysate and 0.005% pectinase. The highest levels of sarsapogenin, secreted and intracellular (4.02 ± 0.09 mg g⁻¹), accumulated using a medium containing 1.0 mg l⁻¹ NAA, 1.0 mg l⁻¹ 2,4-D, 0.5 mg l⁻¹ BAP, 2 g l⁻¹ casein hydrolysate and 0.005% pectinase, after 25 d. Shatavarins were secreted into the medium and can be isolated easily for further purification.     

Somatic embryogenesis and shoot organogenesis in the medicinal plant <Emphasis Type="Italic">Pulsatilla koreana</Emphasis> Nakai

We have developed a system for the in vitro regeneration of pasqueflowers (Pulsatilla koreana Nakai). The system was based on somatic embryogenesis and shoot organogenesis. Over a growth period of 6 weeks, multiple shoots were initiated from leaf, petiole, and pedicel explants on Murashige and Skoog (MS) medium containing 0.5 mg l⁻¹ indole-3-acetic acid (IAA) and zeatin (Zn), kinetin (Kin), or 6-benzyladenine (BA). We achieved 100% of adventitious shoot induced when petiole and pedicel explants were cultured on MS, 0.5–2.0 mg l⁻¹ Zn, and 0.5 mg l⁻¹ IAA. Somatic embryos developed from the explants and generated shoots on MS medium containing 0.25 mg l⁻¹ Zn and 0.5 mg l⁻¹ IAA. Globular and heart-shaped stages of somatic embryos were observed. Histological studies have revealed the stages of development of somatic embryos. For propagation and growth, the regenerated shoots from organogenic or embryogenic calluses were transferred to MS medium containing either (1) 1.5 mg l⁻¹ Zn and 0.05 mg l⁻¹ IAA or (2) 1.0 mg l⁻¹ BA and 0.05 mg l⁻¹ IAA. After the length of the shoots reached 3 cm, the shoots initiated by organogenesis as well as those initiated by somatic embryogenesis were transferred to the root induction medium. After 2 months of culture in half-strength MS with 1.5 mg l⁻¹ α-naphthalene acetic acid (NAA), the rooting ratio was 93%. Finally, the rooted plantlets were acclimatized in a mixture of mountain soil and perlite.     

High-efficiency somatic embryogenesis and plant regeneration in California poppy, Eschscholzia californica Cham.

 The development of a rapid protocol for high-efficiency somatic embryogenesis and plant regeneration from seed-derived embryogenic callus cultures of California poppy (Eschscholzia californica Cham.) is reported. The optimized procedure required less than 13 weeks from the initiation of seed cultures to the recovery of plantlets and involved the sequential transfer of cultures onto solid Murashige and Skoog basal medium containing three different combinations of growth regulators. All steps were performed at 25  °C. Friable primary callus was induced from seeds of E. californica cultured on medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid. The primary callus was transferred to medium containing 1.0 mg l⁻¹ 1-naphthaleneacetic acid and 0.5 mg l⁻¹ 6-benzylaminopurine to establish embryogenic callus and promote somatic embryogenesis. Regenerated plantlets were recovered after the conversion of somatic embryos on medium containing 0.05 mg l⁻¹ 6-benzylaminopurine and showed normal development. Embryogenic callus was induced at a frequency of 85%, an average of 45 somatic embryos were produced per callus, 90% of the somatic embryos converted, and about 70% of the plantlets were recovered in soil. The growth rate of somatic embryo-derived shoots could be increased by gibberellic acid treatment, but the resulting plantlets were hyperhydritic. Received: 14 February 1999 / Revision received: 27 April 1999 / Accepted: 14 May 1999     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of mint with <Emphasis Type="Italic">E.</Emphasis> <Emphasis Type="Italic">coli</Emphasis> glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l⁻¹ BAP, 0.2 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 125 mg l⁻¹ cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l⁻¹ NAA and 50 mg l⁻¹ kanamycin. The presence and expression of transgenes in transgenics (T₀) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications. Akhilesh Kumar and Amrita Chakraborty contributed equally.