This study describes a protocol for in vitro propagation of Chonemorpha grandiflora (Roth) S.M. & M.R. Almeida (Apocynaceae) through axillary bud proliferation and rooting. For shoot induction, the combination of 13.3 μM N⁶-benzyladenine (BA) and 2.46 μM indole-3-butyric acid (IBA) in Murashige and Skoog (MS) medium was better than other growth regulators. Liquid medium was superior over solid medium for root formation and growth. IBA was more effective than other auxins for root induction. Addition of silver nitrate in the presence of IBA significantly increased the number, length, and branching of roots. Shoot tips encapsulated in medium containing 0.49 μM IBA and 11.7 μM silver nitrate exhibited 95% conversion to plantlets. This protocol enables the production of 250 plantlets from a single nodal explant within 7 mo. Plantlets showed 90% survival after acclimatization in soil and were morphologically similar to the source plant under field conditions.

This study describes a protocol for the regeneration of complete plantlets of Terminalia arjuna from nodal explants of mature trees. Shoot multiplication from nodal explants was achieved by culturing on Murashige and Skoog (MS) medium containing different concentrations of 6-benzyladenine (BA), thidiazuron or kinetin, or BA in combination with α-naphthaleneacetic acid (NAA). The best shoot multiplication response was obtained from nodal explants grown on modified MS (half-strength major salts and Fe-EDTA) medium containing 4.44 μM BA and 0.53 μM NAA. Seasonal variations significantly affected the proliferation potential of nodal explants and best proliferation was observed from explants collected during April to May. Microshoots were rooted on half-strength MS medium with 4.92 μM IBA. The rooted shoots were acclimatized successfully.

This study reports an efficient and direct shoot bud differentiation and multiple shoot induction from nodal segments of underground stoloniferous rhizomes of Houttuynia cordata Thunb. The frequency of shoot bud regeneration was influenced by the type of cytokinin and concentrations. Among the various concentrations used, benzylaminopurine (BAP, 17.74 μM) or kinetin (Kn, 18.58 μM) was found to be most effective for rapid and maximum shoot bud differentiation. The number of shoots per explant was higher (20.00 ± 2.61) on Murashige and Skoog (MS) medium supplemented with Kn (18.58 μM) compared to BAP and 6-γ,γ-(dimethyl-allylamino)-purine (2iP) during initial 40-d-old culture. Subsequent shoot differentiation and multiplication were achieved in MS medium containing 9.29 μM Kn and 15% (v/v) coconut milk. Elongation and growth of multiple shoots were also obtained on MS medium containing either 2.32 μM Kn or 2.46 μM 2iP alone. The rate of shoot multiplication during subcultures declined with an increase in the size of proliferating shoot cluster. Reducing shoot cluster size to three to four shoots and subculturing together in shoot multiplication medium resulted in a better shoot multiplication and growth, which could be maintained for 2 yr. The elongated shoots (>20 mm) were successfully rooted on MS medium supplemented with 19.60 μM indole-3-butyric acid. Regenerated plants were successfully established in soil and were found to be healthy and uniform. The protocol reported in this study can be used for conservation and utilization of elite clone of H. cordata.

Cotyledonary node and leaf nodal explants excised from 14-d-old in vitro-grown seedlings of Albizia odoratissima were cultured on a Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP), N⁶-(2-isopentenyl) adenine (2-iP) and kinetin, used either solely or in combinations. The highest frequency for shoot regeneration (82.5%), the maximum number of shoots per explant (6.9), and the maximum shoot length (2.55 cm) were obtained from cotyledonary node explants cultured on a MS medium containing 10 μM BAP and 10 μM 2-iP with 30 g l⁻¹ sucrose. Successful rooting was achieved by placing the microshoots on MS medium with 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was best for plant acclimatization, in which 75% of plants survived.

In vitro-stored plant germplasm is usually evaluated by visual analysis of the plant based on subjective characters. To reduce the variability in these evaluations, we developed a digital-image evaluation system for in vitro-stored plantlets. This study compares the standard visual evaluation system with a digital analysis system to determine if digital analysis can effectively quantify the health of diverse Humulus germplasm. Eight cultivars of Humulus lupulus L. were stored on standard Murashige and Skoog (MS) medium with iron alone (EDTA chelated) and on MS iron with 100 or 200 mg l⁻¹ sequestrene 138 iron (EDDHA chelated). Digital images of the upper two nodes of each plantlet were evaluated for red, green, blue, green/red ratio, and modified normalized difference vegetation index (MNDVI=R−G/R G). Evaluation of each plantlet for MNDVI values showed consistent significant differences for all treatments only at the upper node. Significant differences for visual and the MNDVI values among the three iron treatments were observed at the upper node of most of the eight hop cultivars. Regression analysis of the upper node MNDVI values vs. whole-plant visual ratings showed positive correlations for most cultivars. Effects of iron treatments on storage duration were also analyzed for both visual and digital systems. There were significant differences among MNDVI values for plantlets stored on medium with standard MS iron alone (EDTA chelated) and with the addition of sequestrene 138 iron. In general, the MNDVI value of the upper node correlated well with visual ratings and could be used to determine the health of in vitro stored hops.

This study describes a protocol for plant regeneration from cultured seedling explants of Arctium lappa. Hypocotyls and cotyledons of A. lappa were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid and 0.5–2.0 mg l⁻¹ benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0–2.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 0.5–2.0 mg l⁻¹ BA. These regenerated shoots were rooted on MS medium with 1.0 mg l⁻¹ indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mg l⁻¹ NAA. The regenerated plants acclimatized in soil were normal morphologically and in growth characters. They flowered and set seeds in the following year after acclimatization.

This study reports a method for high-frequency shoot organogenesis and plant establishment of Potentilla potaninii Wolf. Hypocotyl and cotyledon explants of P. potaninii were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of benzyladenine (BA) and α-naphthaleneacetic acid (NAA) to induce adventitious shoot formation for micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants grown on MS medium supplemented with 5.0 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA. The regenerated shoots rooted most efficiently on half-strength MS medium supplemented with 1.0 mg l⁻¹ NAA and 0.5 mg l⁻¹ indole-3-acetic acid or indole-3-butyric acid. The acclimatized plants with normal morphology and growth characters flowered and set seeds in the following year.

This study reports a protocol for germination of Givotia rottleriformis (var. Tel. Thella Poniki) using zygotic embryo culture. A 100% germination was obtained by culturing the embryos on Murashige and Skoog medium containing 30 g l⁻¹ sucrose. A sucrose concentration lower or higher than 30 g l⁻¹ resulted in lower germination or promoted callus formation. The seedling growth was promoted by the addition of 100 mg l⁻¹ tyrosine in the medium. Seedlings germinated in the presence of 0.2–0.4 mg l⁻¹ α-naphthaleneacetic acid and 0.3–0.5 mg l⁻¹ indole-3-butyric acid were abnormal, showing a slender stem with slender roots or forming callus with stout roots. Germination also affected embryo orientation in culture; placing embryos upright on the medium was most beneficial for germination. The in vitro-germinated seedlings were acclimatized in soil under shady conditions with a survival rate of 60–70%. These plants were phenotypically normal, healthy, and similar to donor plants. This protocol will be useful for overcoming seed dormancy and for rapid multiplication and conservation of G. rottleriformis using zygotic embryo culture.

Experiments were carried out with Taxus baccata cell lines showing different paclitaxel-producing capacities (between 1.74 and 19.91 mg l⁻¹) when growing in a selected product-formation medium that specifically stimulated the production of taxane to the detriment of cell growth. Through mixing low-, medial- and high-producing lines, it could be observed that paclitaxel productivity in the resulting mixed lines was clearly higher than the mean productivity of the individual lines before mixing. This suggests that culture components generated by high-producing individual lines within the population might induce paclitaxel production. Although the accumulation of paclitaxel and baccatin III was higher when 100 μM methyl jasmonate was added to the subcultures of the mixed lines, the results indicate that exogenously applied methyl jasmonate was not the first factor to stimulate taxane production. The possible effects of methyl jasmonate elicitation and paclitaxel accumulation on cell viability are also considered.

This study reports the analysis of secondary metabolites of gentiopicrin, swertiamarin, and sweroside in shoot and root cultures of yellow wort (Blackstonia perfoliata), which were initiated from seeds, grown on Murashige and Skoog (MS) medium. Shoot cultures of B. perfoliata were inoculated with suspension of Agrobacterium rhizogenes strain A4M70GUS and hairy roots appeared at the infected sites after 3 wk of inoculation. Tips of adventitious roots of B. perfoliata were grown on hormone-free MS medium and three clones of the transformed roots regenerated shoots spontaneously. Gentiopicrin, swertiamarin, and sweroside were detected in both roots and shoots of B. perfoliata in vitro and in vivo, but gentiopicrin was found to be the major compound. The concentration of growth regulator in the medium affected the production of secoiridoids in B. perfoliata in vitro, where the level of gentiopicrin was higher in plants grown in the presence of indole-3-butyric acid, but the presence of 6-benzylaminopurine was inhibitory to secoiridoid production.

Three barley tissue-specific promoters (Dhn12, Itr1, and Ltp1) were transcriptionally fused to the C1 and/or the B-peru genes involved in anthocyanin biosynthesis. Transient expression of these constructs was examined in different wheat tissues. Constructs with tissue-specific promoters were only active in embryos. A green fluorescent protein (GFP) driven by the actin promoter was used as an internal standard to monitor the effectiveness of each bombardment. Further, normalization of the transient expression assay using the GFP reference significantly reduced the variability between separate bombardments, and the intensity of anthocyanin pigmentation quantified by image analysis allowed for a rapid and accurate evaluation of different promoters. Compared to CaMV35S promoter, tissue-specific promoters were more effective in directing anthocyanin production, specifically in wheat embryos, and the C1 gene was more effective than B-peru. Among the tissue-specific promoters, the Ltp1 was superior to Itr1 and Dhn12 in combination with C1 and/or B-peru gene(s). Analysis of nucleotide sequences of all three tissue-specific promoters revealed the presence of G-Box, E-box, and RY elements, which might trigger embryo-specific expression in wheat.

This study reports an Agrobacterium-mediated transformation of green-colored cotton (Gossypium hirsutum L.). A tissue culture procedure was optimized to induce callus formation from hypocotyl explants and subsequent differentiation into the embryogenic type. Callus formation could be induced by growing explants on Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Among the four genotypes studied, embryogenic calli and plant regeneration were observed only in var. G9803. Agrobacterium-mediated transformation of G9803 with the fiber-specific expansin gene GhExp1 was achieved based on the establishment of these tissue culture methods. A total of 32 individual regenerants resistant to kanamycin were generated within 7 mo., with a transformation frequency of 17.8%. Transformation was confirmed by Southern blot analysis and RT-PCR. These results represent the first step towards genetic manipulation of the colors and fiber quality of green-colored cottons by biotechnology.

We report an improved procedure of in vitro propagation of Scrophularia yoshimurae – a medicinally important plant species indigenous to Taiwan. Induction of maximum shoot buds (22.75 per explant) was obtained with shoot tip explant cultured on Murashige and Skoog medium supplemented with 1.0 mg l⁻¹ benzyladenine (BA) and 0.2 mg l⁻¹ α-naphthaleneacetic acid and gelrite using dispense paper (DP) for ventilation closure of culture vessels. The type of gelling agents (agar and Gelrite) affected both quantity and quality of the shoots induced. Using aluminum foil for ventilation closure resulted in a higher number of hyperhydric shoots. Hyperhydricity was reduced by culturing shoots on a medium devoid of plant growth regulators in conjunction with the use of DP. Plantlet growth in vessels using DP was healthier and all plantlets survived after being transplanted to soil.

This study reports the direct regeneration of flower buds from cultured sepal segments of Sinningia speciosa Hiern. Two types of floral bud regeneration were observed: regeneration of floral buds only (designed as B^F) and regeneration of both floral and vegetative buds (designed as B^{F V}). The capacity of B^F regeneration was closely related to the location of sepal segments and the concentration of exogenous gibberellin (GA₃) and cytokinin in the medium. On the medium containing 1.0 mg l⁻¹ GA₃, the addition of 6-benzyladenine (BA) significantly increased the frequency of total flower bud (B^F B^{F V}) formation, with the frequency up to 91.5% in the presence of 0.4 mg l⁻¹ BA. On the medium containing 0.1 mg l⁻¹ BA, the addition of GA₃ also increased the frequency of total flower bud regeneration, with the frequency up to 74.3% in the presence of 1.0 mg l⁻¹ GA₃, but no further increase in regeneration was observed when the GA₃ concentration was higher than 1.0 mg l⁻¹. The capacity of B^F regeneration from different locations of sepal segments was differential. The adaxial part of sepal segments gave rise to the highest frequency of 56.7 and 84.3% for B^F and B^F B^{F V}, respectively.

A rapid and one-step protocol for direct regeneration of shoots from cumin embryo explants has been developed. Embryo explants with shoot meristems were cultured on shoot regeneration medium for 15–22 d. After embryo culture, shoots were regenerated from the area adjacent to the region between the cotyledons and embryo axis within 2 wk, without any intermediate callus phase. Shoot proliferation and elongation were achieved on shoot regeneration medium without subculture. Among the different combinations of 6-benzylaminopurine, α-naphthaleneacetic acid (NAA), and indole-3-acetic acid (IAA) tested, 0.8 mg l⁻¹ (4.3 μM) NAA in combination with 0.3 mg l⁻¹ (1.71 μM) IAA in the B5 medium resulted in the most efficient direct shoot regeneration. No significant difference was detected for the number of regenerated explants when different heterogeneous endemic varieties were compared. This plant regeneration procedure was applicable to different cumin genotypes and regenerated plants were phenotypically normal.

Using the system for genetic transformation and transgenic plant regeneration via somatic embryogenesis (SE) of Lycium barbarum established in this laboratory, this study reports the optimization of the factors affecting the efficiency of transformation, including pre-culture period, leaf explant source, use of acetosyringone, strains and density of Agrobacterium, and temperature of co-cultivation. The optimized transformation protocol for L. barbarum included pre-culture of leaf explants from 3-wk-old seedlings for 3 d on the medium for callus induction followed by inoculation with Agrobacterium strain EHA101 (pIG121Hm), co-cultivation for 3 d at 24°C, and transfer to the selection regeneration medium with 50 mg l⁻¹ kanamycin (Kan). Using this protocol, 65% L. barbarum explants gave rise to Kan-resistant and GUS-positive calli. In addition, the expression of introduced transgene (nptII) in clonal progeny was verified by formation of calli and somatic embryos from leaf segments of nine transgenic plants grown on the Kan-containing medium. All explants formed calli at 50 mg l⁻¹ Kan and seven out of nine transgenic plants were found to possess callus-forming capacity even at 100 mg l⁻¹ Kan. These calli also possessed higher SE potential on SE medium supplemented with 25 mg l⁻¹ Kan.

This study investigated factors affecting the production of somatic embryos in Blighia sapida (ackee). Explants obtained from fully expanded leaves or cotyledons of immature zygotic embryos excised from brown (BSCZE) or green seeds (GSCZE) were cultured on Murashige and Skoog medium supplemented with 9, 18 and 36 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 or 22.1 μM benzylaminopurine (BAP) or 0.2–19.9 μM thidiazuron (TDZ). Leaf explants grown on media supplemented with the different combinations of 2,4-D and BAP formed callus, but they were non-embryogenic, while explants were not responsive on TDZ-supplemented media. GSCZE explants grown in the presence of 2,4-D/BAP combinations of 9/4.4, 18/4.4 or 36/4.4 μM formed non-embryogenic callus profusely, but explants gave rise to organized globular protuberances (GPs) and non-embryogenic callus on media containing TDZ, with the best concentration at 0.4 μM. BSCZE explants grown on TDZ-supplemented media also formed callus, but no GPs were detected. When GPs were cultured on media containing TDZ and abscisic acid they (ABA), gave rise to the highest number of somatic embryos. The medium was also beneficial for the development of somatic embryos from the globular to cotyledonary stage.