Embryogenic responses of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. callus induced from transgenic hairy roots

Agrobacterium rhizogenes A4M70GUS-mediated transformation of two local breeding lines of sugar beet was obtained using 4-week-old seedlings. Root formation efficiency was 61.54% for SBa genotype and 36.36% for SBb genotype. Five highly proliferated hairy root lines have been established in liquid hormone-free MS medium. Transgenic nature of the hairy root clones was evaluated by GUS assay, PCR and RT-PCR analyses. Hairy root-derived calli were induced using different plant growth regulators (PGRs): auxin, auxin/cytokinin and cytokinin. The best callus induction response was achieved on MS medium containing both 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg/l thidiazuron (TDZ). Globular embryo-like structures were observed in friable callus after its prolonged cultivation on MS medium supplemented with TDZ and giberellic acid (GA₃) at 1 mg/l each, followed by growth on MS medium containing 1% glucose and 0.5 mg/l 2,3,5-triiodobenzoic acid (TIBA). Histological analysis revealed somatic embryos at different stages of development in hairy root-derived callus of sugar beet.     

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Parkia timoriana (DC.) Merr.: a multipurpose tree legume

In vitro regeneration of Parkia timoriana (DC.) Merr. has been achieved using cotyledonary node explants. The ability to produce multiple shoots has been evaluated using semi-solid Murashige and Skoog (MS) basal medium and Gamborg’s B-5 basal medium supplemented with various concentrations of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BA) either in single or in combinations. The explants cultured in MS medium supplemented with combinations of 2.7 μM NAA and 11 μM BA showed the maximum frequency of multiple shoots (96.66%) formation and number of shoots per explants (6.60), respectively. For rooting, full and half strength MS medium supplemented with various concentrations of indole-3-butyric acid (IBA) and NAA were studied and the highest number of root formation was observed in full-strength MS supplemented with 9.8 μM IBA. Using Agrobacterium tumefaciens strain EHA105 pCAMBIA2301 various optimum conditions for efficient transformation were determined by recording the percentage of GUS⁺ explants. Following the optimized conditions, the co-cultured explants were cultured on semi-solid shoot regeneration medium containing MS medium + 2.7 μM NAA + 11 μM BA + 100 mg/l kanamycin + 500 mg/l cefotaxime. After 8 weeks of culture, the regenerated shoots were rooted in rooting medium (RM) containing MS medium + 9.8 μM indole-3-butyric acid (IBA), 3% sucrose, 7.5 mg/l kanamycin and 500 mg/l cefotaxime. Successful transformation was confirmed by histochemical GUS activity of the regenerated shoots, nptII gene PCR analyses of the regenerated kanamycin resistant plantlets and Southern analysis of putative transgenic PCR⁺ plants.     

Highly efficient shoot regeneration of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) using a two-stage culture procedure and assessment of genetic integrity of micropropagated plants by RAPD

A two-stage culture procedure has been developed for highly efficient shoot regeneration from leaf and internode explants of Bacopa monnieri. Adventitious shoot buds were obtained on the shoot induction medium containing Murashige and Skoog’s (MS) basal salt supplemented with 1.5 mg/l thidiazuron and 0.5 mg/l naphthalene acetic acid; these shoot buds were subcultured on the multiplication (second) medium amended with BAP (benzyl amino purine). Multiplication medium containing 0.5 mg/l BAP produced more shoots (135) and longer shoots (7.8 cm) with more nodes (6). Best response of root induction with more number of roots (16.5) and longer roots (8.7 cm) was observed in half strength MS basal medium supplemented with 1.0 mg/l IBA (indole-3-butyric acid) and 0.5 mg/l phloroglucinol. In vitro obtained plants were transferred to the field after hardening with a 100% survival rate. Random amplified polymorphic DNA analysis was carried out using five random primers. The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic integrity of micropropagated plants.     

Effects of explant types and media salt strength on growth and secondary metabolite accumulation in adventitious roots of <Emphasis Type="Italic">Periploca sepium</Emphasis> Bunge

In order to study the capabilities of Periploca sepium adventitious root induction in different types of explants, we selected leaves, roots and stems with or without buds. The growth of adventitious roots and periplocin content in these roots were determined. In order to investigate the suitable media salt strength, we cultured the adventitious roots in different salt strength (0.25, 0.50, 1.0, 1.5, 2.0) of Murashige and Skoog (MS) media supplemented with 1 mg/l indole butyric acid (IBA) and 30 g/l sucrose. The results showed that both leaf and root explants were proven suitable for the adventitious root induction; however, the stems could hardly induce adventitious roots no matter whether the stems had buds or not. Further studies reported that adventitious root proliferation and periplocin production derived from root explants were higher than those derived from leaf explants. So the root explants were the optimum explants for adventitious root induction, growth and periplocin production. The salt strength experiment showed that with the increasing salt strength (1.0–2.0 MS), adventitious root growth decreased significantly, as well as periplocin content in comparison with lower (0.25–0.5 MS) salt strength media.     

Establishment of culturing conditions and assessment of antioxidant activity and somaclonal variation in the adventitious root suspension cultures of <Emphasis Type="Italic">Oplopanax elatus</Emphasis> Nakai

Oplopanax elatus Nakai, a plant traditionally used in folk medicine, is currently in population decline due to uncontrolled harvesting. In the present study, we investigated the factors affecting O. elatus adventitious root production, including hormones (alone or in combination), explant type, basal salt type and strength, sucrose concentration, pH, and temperature. Results revealed that adventitious root formation was optimal with root explants grown on 1/2 Murashige and Skoog (MS) medium containing 0.5 mg L^?1 Indole-3-butyric acid (IBA) (pH 5.8) at 25 °C. Chlorogenic acid concentration was highest in roots propagated in 1/2 MS medium containing 0.5 mg L^?1 IBA; vanillin, another phenolic compound, was also detected in cultures. Liquid media containing 3% sucrose exhibited the highest radical scavenging activity and total phenolic compound contents. X-ray diffraction revealed significant differences in the elemental intensity between adventitious root and field-grown plantlet extracts. Analysis of simple sequence repeats confirmed that adventitious roots regenerated in vitro were genetically similar to their mother plant. Thus, we identified the optimal conditions for proliferation of O. elatus adventitious roots in liquid culture, from which, secondary metabolites, particularly bioactive compounds associated with the medicinal use of this plant, can be mass produced without further population deterioration.     

Micropropagation of the Thai orchid <Emphasis Type="Italic">Grammatophyllum speciosum</Emphasis> blume

Protocorm-like bodies (PLBs) were induced from shoot tips of Grammatophyllum speciosum, a Thai orchid. The highest frequency of PLBs (93%) were observed on explants incubated on 1/2-Murashige and Skoog (MS) liquid medium containing 2% (w/v) sucrose without any plant growth regulators (PGRs). Tests with different carbon sources compared to sucrose revealed that maltose promoted the highest relative growth of G. speciosum PLBs (7-fold increase), while trehalose and sucrose yielded 5-fold and 4-fold increases, respectively. In 1/2 MS liquid medium, addition of 15 mg/l of chitosan promoted a 7-fold increase in PLB growth while 25 mg/l promoted a 4-fold increase. However, the relative growth rate in solid culture was significantly lower than that in liquid culture. In addition, chitosan supplementation in solid medium promoted shoot formation but not rooting. Plantlet regeneration was induced using a combination of NAA and BA supplementation in 1/2 MS solid medium with optimum induction shoot and root formation at 2.0 mg/l NAA and 1.0 mg/l BA. Using this protocol, approximately 8 months was required to obtain a hundred plantlets from one shoot tip. The plantlets showed no changes in ploidy when tested by flow cytometry.     

Morphogenesis and in vitro production of caffeoylquinic and caffeic acids in Baccharis conferta Kunth

We established a protocol for the in vitro propagation of Baccharis conferta Kunth. This plant is used to treat gastrointestinal problems, cramps, pain, respiratory problems, and insect bites. A high rate of shoot multiplication was obtained from nodal segments on Murashige and Skoog (MS) culture medium. The shoots regenerated roots without exogenous plant growth regulators (PGRs). All explants of wild leaves on MS medium containing 5 μM of thidiazuron (TDZ) produced friable callus. An organogenic response was achieved after 3 wk of culture when callus segments were transferred to MS medium containing a combination of plant growth regulators (PGRs): either (i) 5 μM indole butyric acid (IBA) + 5 μM kinetin (KIN) or (ii) 0.5 μM IBA + 1.10 μM benzylaminopurine (BAP). The morphogenetic responses of callus were characterized by scanning electron microscopy. Shoots regenerated from callus and formed roots on MS medium without PGRs. The micropropagated plantlets and the organogenic callus showed similar chemical profiles in HPLC-mass spectrometry analyses. The main compounds present in the cultures were caffeoylquinic acids. Only plantlets contained small amounts of triterpenes (erythrodiol and ursolic acid). These findings will be useful for the micropropagation of this important native resource, and for further studies on its biology.

     

Podophyllotoxin production via cell and adventitious root cultures of <Emphasis Type="Italic">Podophyllum peltatum</Emphasis>

Podophyllum peltatum is an important medicinal plant that produces podophyllotoxin (PTOX) with anti-cancer properties. We established the embryogenic cell and adventitious root culture systems in P. peltatum and analyzed PTOX production. For the growth of embryogenic cell clumps in shake flask culture, the most efficient concentration of 2,4-dichloroacetic acid (2,4-D) was 6.78 μM, and the growth of embryogenic cell clumps was 15.9-fold increased in Murashige and Skoog MS liquid medium with 6.78 μM 2,4-D after 3 wk of culture. To induce adventitious roots, half-strength MS medium showed the best results for adventitious root induction compared to full strength MS medium and MS medium lacking NH₄NO₃. Optimal indole-3-butyric acid concentration for adventitious root formation was 14.78 μM. In liquid medium, the frequency of adventitious root formation from root segments was 87.7% and the number of laterally formed adventitious roots was 22.3 per segment. PTOX production in embryogenic cells and adventitious roots was confirmed by liquid chromatography and electrospray ionization–tandem mass spectrometry analysis. High-performance liquid chromatography analysis revealed that adventitious roots contained higher PTOX than embryogenic cell clumps. Elicitor treatment (20 μM methyl jasmonate) strongly enhanced the production of PTOX in both embryogenic cell clumps and adventitious roots. This observation suggests that both embryogenic cell and adventitious root culture can be adopted to produce PTOX.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Acacia crassicarpa</Emphasis> via organogenesis

A protocol was developed for Agroacterium-mediated genetic transformation of Acacia crassicarpa via organogenesis by using in vitro phyllode (leaf) as the explant. Phyllode (leaf) explants were co-cultured with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pBI101 (harboring antisense Pt4CL1 with respect to the Pt4CL1P promoter). The selection for transgenic shoots was performed through two consecutive steps on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators and antibiotics in the following order: 0.5 mg/l thidiazuron (TDZ), 0.5 mg/l α-naphthaleneacetic acid (NAA), 300 mg/l carbenicillin (Car) and 20 mg/l kanamycin (Km) for 10 days; 0.1 mg/l TDZ, 200 mg/l Car and 20 mg/l Km for 60 days; 0.5 mg/l indole-3-butyric acid (IBA), 100 mg/l Car and 20 mg/l Km 50 days. 21.7% of nodules produced multiple adventitious shoot buds, of which 27.7% survived in initial selection. The shoot buds were subjected to repeated selection on MS medium supplemented with 0.1 mg/l TDZ, 200 mg/l Car and 20 mg/l Km for 60 days. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.5 mg/l IBA, 100 mg/l Car 20 mg/l Km 50 days. Genomic PCR analysis confirmed the incorporation of the antisense Pt4CL1 with respect to the Pt4CL1P promoter fragment into the host genome.     

Regeneration of plants from <Emphasis Type="Italic">Fraxinus americana</Emphasis> hypocotyls and cotyledons

A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5, respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting (78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to the emerald ash borer.     

Influence of auxins,cytokinins, and nitrogen on production of rutin from callus and adventitious roots of the white mulberry tree (<Emphasis Type="Italic">Morus</Emphasis><Emphasis Type="Italic">alba</Emphasis> L.)

Rutin is an economically valuable flavone compound with anticancer activity, dietary effects, and anti-aging activity. In this study, callus and adventitious roots were induced from three Morus (mulberry) species. Among the three mulberry species tested for rutin production, roots of the Sugye (M. alba L.) had the highest levels (242.2 μg/g fresh tissue) of rutin. In addition, the mature leaves of this type of tree promoted higher levels of rutin compared to those of young leaves or those undergoing senescence. Adding auxins such as indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) not only enhanced the development of callus and adventitious roots but also increased the protein and rutin contents. In contrast, adding cytokinins such as 6-benzyladenine (BA) and kinetin (KN) retarded callus and adventitious root development as well as the protein and rutin contents. Callus in suspension culture in the presence of IAA produced more rutin than that in the absence of IAA. However, rutin secretion into a medium was greater in the absence of IAA. Different ammonium/nitrate (AM/NI) ratios in a root suspension culture also greatly affected rutin production and its secretion into a liquid medium. As a result, the highest level of rutin was produced when adventitious roots were grown in a 34/66 AM/NI full-strength standard MS medium containing 5 mg/l IAA.     

Improved <Emphasis Type="Italic">in vitro</Emphasis> micropropagation method with adventitious corms and roots for endangered saffron

The objective of this study was to investigate development of an efficient in vitro tissue culture system for saffron (Crocus sativus L.) complete with roots and corms. In indirect organogenesis, Murashige and Skoog (MS) media with 3% (w/v) sucrose, 100 mg L⁻¹ ascorbic acid, and the combination of 0.25 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg L⁻¹ 6-benzylaminopurine (BAP) were best for callus initiation and growth while 1.5 mg L⁻¹ BAP was excellent for high rate of adventitious shoot formation. 1 mg L⁻¹ indole-3-butyric acid (IBA) was more preferable for adventitious corm and root initiation as well as growth. Overall, 64% rooting and 33% corm production rates were achieved in indirect organogenesis. In direct organogenesis, MS medium supplemented with 3 % sucrose, 100 mg L⁻¹ ascorbic acid and 1 mg L⁻¹ BAP was optimum for shoot growth. While 1 mg L⁻¹ IBA was best for adventitious corm formation, 2 mg L⁻¹ IBA promoted adventitious root initiation and growth. Overall, 36% and 57% of explants had corm and contractile root, respectively. The high rates suggest that efficient tissue culture system could be achieved for mass propagation and ex situ conservation of threatened saffron genetic resources.     

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.     

Large-scale somatic embryogenesis and regeneration of <Emphasis Type="Italic">Panax notoginseng</Emphasis>

An efficient system for inducing somatic embryogenesis in Panax notoginseng was established using shaker flasks and bioreactor cultures; furthermore, regenerated plantlets were successfully transferred to ex vitro soil conditions. Embryogenic callus was induced from segments of adventitious roots incubated on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 5 weeks of culturing. The highest frequency (100%) of somatic embryogenesis, with a mean of 32.7 somatic embryos per callus, was obtained on embryogenic callus incubated on a medium containing 0.5 mg/L 2,4-D. To scale-up somatic embryo formation, 10 g (~1.65 × 10⁴) of early globular-stage somatic embryos were incubated in a 3 L airlift bioreactor containing 1.5 L 1/2 MS medium without plant growth regulators (PGRs) for a period of 4 weeks; these globular-stage somatic embryos then developed into cotyledonary embryos. When maintained on PGR-free medium, the cotyledonary embryos developed roots but did not develop shoots. However, when they were treated with gibberellic acid (GA₃), they continued to germinate and transformed into plantlets after 2 weeks of culture. Plantlets with well-developed shoots and roots were transferred to an autoclaved vermiculite and perlite mixture, acclimatized for a period of 3 months and successfully transferred to forest mountain soil. Following overwintering, these plants produced new growth.     

Optimisation of plantlet regeneration from leaf and nodal derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andrews

An indirect in vitro plant regeneration protocol for Vanilla planifolia has been established. Juvenile leaf and nodal segments from V. planifolia were used as explants to initiate callus. Nodal explants showed better callus initiation than juvenile leaf explants, with 35.0% of explants forming callus when cultured on Murashige and Skoog (MS) basal medium supplemented with 2.0 mg/l 1-naphthylacetic acid (NAA) and 1.0 mg/l 6-benzyladenine (BA). Almost 10.0% of juvenile leaf explants were induced to form callus on the MS basal medium containing 2.0 mg/l NAA and 2.0 mg/l BA, whereas no callus formed in the presence of any concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and BA. After 8 weeks, callus generated was transferred to MS basal medium containing 1.0 mg/l BA and 0.5 mg/l NAA. A mean number of 4.2 shoots per callus was produced on this medium, with a mean length of 3.8 cm after 8 weeks of culture. Roots formed on 88.3% of plantlets when they were cultured on MS medium supplemented with 1.0 mg/l NAA, with a mean length of 4.4 cm after 4 weeks of culture. Of the rooted plantlets, 90.0% survived acclimatisation and were making new growth after 4 weeks.     

Plant regeneration from excised hypocotyl explants of <Emphasis Type="Italic">Platanus acerifolia</Emphasis> willd

Summary A method of plant regeneration from hypocotyl segments of Platanus acerifolia Willd, has been developed. Hypocotyl slices were cultured on Murashige and Skoog (MS) basal medium supplemented with a range of combinations of cytokinins [6-benzyladenine (BA) or kinetin] and auxins [indole-3-butyric acid (IBA), indole-3-acetic acid, α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid] for adventitious shoot induetion. The highest regeneration frequency was obtained with MS medium containing 2.0 mg l⁻¹ (8.88 μM) BA and 0.5 mg l⁻¹ (2.46 μM) IBA. Adventitious buds and shoots were differentiated from hypocotyl-derived cellus or directly from the wounded sites within 4–8 wk. The regenerated shoots were elongated and proliferated efficiently on multiplication medium. Complete plantlets were transplanted to the soil and grew normally in the greenhouse after root formation on rooting medium for 4–6 wk.     

A rapid in vitro propagation of red sanders (<Emphasis Type="Italic">Pterocarpus santalinus</Emphasis> L.) using shoot tip explants

An efficient protocol has been developed for the in vitro propagation of Pterocarpus santalinus L. using shoot tip explants which is a valuable woody medicinal plant. Various parts of this plant are pharmaceutically used for the treatment of different diseases. Multiple shoots were induced from shoot tip explants derived from 20 days old in vivo germinated seedlings on 1:1 ratio of sand and soil after treating with gibberellic acid (GA₃). The highest frequency for shoot regeneration (83.3%) with maximum number of shoot buds (11) per explant was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l of 6-benzylaminopurine (BAP) along with 0.1 mg/l of thidiazuron (TDZ) after 45 days of culture. A proliferating shoot culture was established by repeatedly subculturing the original shoot tip explants on fresh medium after each harvest of the newly formed shoots. Sixty percent of the shoots produced roots were transferred to rooting medium containing MS salts and 0.1 mg/l indole-3-butyric acid (IBA) after 30 days. About 73.33% of the in vitro raised plantlets were established successfully in earthen pots. Random amplified polymorphic DNA (RAPD)-based DNA fingerprinting profiles were generated for the first time using shoot tip explants of this species and confirmed that there was no genetic variability. This protocol might be helpful for the mass multiplication of P. santalinus in the future.     

Direct shoot bud organogenesis and plant regeneration from pre-plasmolysed leaf explants in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

A protocol for high-frequency shoot bud regeneration from the leaves of Catharanthus roseus is reported here for the first time. A 60-min pre-plasmolytic treatment of leaf explants in a cell protoplast washing medium containing 13% (w/v) mannitol followed by their plating on a half-strength Murashige and Skoog (MS) medium supplemented with 7.0 mg/l 6-benzyladenine (BA) and 3.0 mg/l α-naphthaleneacetic acid (NAA) resulted in the de novo induction and development of adventitious shoot buds in more than 75% of explants. Histological observations revealed a direct origin of these shoot buds from hypodermal tissue around the mid-rib. The rooting in the regenerated shoots was obtained in the presence of 3.0 mg/l indole-3-butyric acid (IBA) and the rooted plants could be successfully established in soil with a 70% rate of success. The relevance of the developed protocol in terpenoid indole alkaloids pathway engineering at the whole-plant level in C. roseus is discussed.     

In vitro plantlet regeneration of “dwarf” Indian olive (Elaeocarpus robustus Roxb.): a fruit plant of Bangladesh

A plantlet regeneration protocol was developed on pot-grown mature plants of Elaeocarpus robustus Roxb. cv. Dwarf from nodal and leaf explants. The best yield of adventitious shoots was achieved from the leaf-derived calli in a modified MS (MMS₁, half strength of major salts, full strength of minor salts, and vitamins) medium containing 4.0 μM BA + 4.0 μM Kn + 0.5 μM NAA + 15% coconut water (CW). The shoot multiplication rate was amplified about twofold per culture after the addition of 15% CW to the medium. The rate of shoot multiplication reached maximum at the 5th subculture, and it maintained this rate throughout the 3 subsequent subcultures. The best rooting in vitro was investigated by subculturing the microcuttings in an MMS₂ (half strength of both major salts and minor salts and full strength of vitamins) medium containing 1.0 μM IBA in the dark for one initial week at 30°C, followed by subculturing them in a plant-growth regulator (PGR)-free medium in the light. The plantlets raised in vitro were successfully established under ex vitro conditions.     

Production of bialaphos-resistant Nierembergia repens by electroporation

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient β-glucuronidase (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and 100 μF in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1–2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.