Micropropagation of <Emphasis Type="Italic">Pinus massoniana</Emphasis> and mycorrhiza formation in vitro

A protocol was developed for the micropropagation of Pinus massoniana and mycorrhiza formation on rooted microshoots. Seedling explants were first cultured on Gresshoff and Doy (GD) medium supplemented with 6-benzyladenine (BA) alone or in combination with α-napthaleneacetic acid (NAA) to stimulate the formation of intercotyledonary axillary buds. The frequency of axillary bud induction was up to 97% on medium supplemented with 4.0 mg l⁻¹ BA and 0. 2 mg l⁻¹ NAA, and the average number of buds per explant reached up to 5.5 on medium with 4.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Axillary buds elongated rapidly after being transferred to half-strength GD medium containing activated charcoal (0.1% w/v). Shoot proliferation was achieved by cutting elongated shoots into stem segments and subculturing on GD medium containing 2 mg l⁻¹ BA and 0.2 mg l⁻¹ NAA. Root primordia were induced in 82% of shoots when transferred to half-strength GD medium containing 0.2 mg l⁻¹ NAA. Root elongation was achieved in a hormone-free GD agar medium or a perlite substrate. Rooted plantlets were inoculated with the mycelium of ectomycorrhizal fungus Pisolithus tinctorius and the formation of ectomycorrhiza-like structures was achieved in vitro.     

In vitro propagation of four threatened <Emphasis Type="Italic">Paphiopedilum</Emphasis> species (Orchidaceae)

The effects of seed maturity, media type, carbon source, and organic nutrient additives on seed germination, protocorm development, and plant growth of Paphiopedilum villosum var. densissimum Z. J. Liu et S. C. Chen were investigated. Micropropagation frequency was enhanced through the use of 200-day-old seed, Knudson C (KC) medium, and the presence of both glucose and coconut milk in the medium. The effects of various plant growth regulators on the frequency of shoot organogenesis in four Paphiopedilum species were also investigated. Explants of P. villosum var. densissimum and P. insigne (Lindl.) Stein incubated in the presence of 5 mg l⁻¹ 6-benzyladenine (BA) with 0.5 mg l⁻¹ α-naphthalene acetic acid (NAA) and 0.2 mg l⁻¹ BA with 0.1 mg l⁻¹ NAA, respectively, showed a twofold increase in the frequency of shoot organogenesis. For explants of P. bellatulum (Rchb. f.) Stein and P. armeniacum S. C. Chen et F. Y. Liu, the combination of 5.5 mg l⁻¹ BA with 0.5 mg l⁻¹ NAA and 4 mg l⁻¹ BA with 0.1 mg l⁻¹ NAA, respectively, resulted in the highest frequencies of shoot organogenesis.     

Fast versatile regeneration of <Emphasis Type="Italic">Trifolium alexandrinum</Emphasis> L.

Trifolium alexandrinum L. (Egyptian clover) is one of the most important forage crops in the world. Its regeneration in tissue culture has been described in a few reports but the efficiency, accurate time scales and applicability to various genotypes of the described procedures are uncertain. Therefore their suitability for genetic transformation is unclear. In this study, were report new fast procedures for regeneration of Egyptian clover that are applicable to the regeneration of various genotypes (Mescawi-ahaly, Sakha3 and Sakha4). Shoots were regenerated from intact and wounded cotyledons as well as hypocotyls of Mescawi-ahaly on naphthaleneacetic acid/benzyladenine (NAA/BA) and naphthaleneacetic acid/thidiazuron (NAA/TDZ) media. The highest shoot regeneration frequencies were obtained from intact cotyledons on NAA/BA (0.05 mg l⁻¹ NAA combined with 2.0 mg l⁻¹ BA) and NAA/TDZ (0.05 mg l⁻¹ NAA combined with 1.0 mg l⁻¹ TDZ) media (66.2 and 43.1% respectively) compared to 18.4 and 10.1% for wounded cotyledons on NAA/BA and NAA/TDZ respectively. 21.0% shoot regeneration frequency was observed for hypocotyls on NAA/BA (2.0 mg l⁻¹ NAA combined with 0.5 mg l⁻¹ BA) medium but no regeneration was obtained on NAA/TDZ medium. Rooting of the regenerated shoots was induced on indole butyric acid (IBA: 0.24 mg l⁻¹) or NAA (2.0 mg l⁻¹) media where IBA medium supported significantly higher frequencies of rooting as well as survival of the whole plantlets after transfer to soil. However, the rooting and survival frequencies also depended on the type of explant and the medium used for shoot regeneration. The two cultivars Sakha3 and Sakha4 were regenerated using the culture conditions optimized for Mescawi-ahaly with comparable efficiencies, indicating that the described procedure is not genotype dependent. The time scale of whole plantlet regeneration ranged from 7.5 weeks for intact and wounded cotyledons to 10 weeks for hypocotyl explants.     

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 plant regeneration in <Emphasis Type="Italic">Dorem ammoniacum</Emphasis> D., an endangered medicinal plant

Dorema ammoniacum D. Don. (Apiaceae), a native medicinal plant in Iran, is classified as a vulnerable species. Root, hypocotyl, and cotyledon segments were cultured on Murashige and Skoog (MS) (1962) medium supplemented with either 2,4-dichlorophenyoxyacetic acid (2,4-D) or naphathalene acetic acid (NAA), at 0–2 mg l⁻¹, alone or in combination with either benzyladenine (BA) or kinetin (KN), at 0–2 mg l⁻¹ for callus induction. The best response (100%) was observed from root segments on MS medium containing 1 mg l⁻¹ NAA and 2 mg l⁻¹ BA. The calli derived from various explants were subcultured on MS medium supplemented with BA (1–4 mg l⁻¹) alone or in combination with NAA or indole-3-butyric acid (IBA), at 0.2 or 0.5 mg l⁻¹ for shoot induction. Calli derived from hypocotyl segments showed significantly higher frequency of plantlet regeneration and number of plantlets than the calli derived from root and cotyledon segments. Therefore, MS medium supplemented with 2 mg l⁻¹ BA and 0.2 mg l⁻¹ IBA produced the highest frequency of shoot regeneration (87.3%) in hypocotyl-derived callus. The optimal medium for rooting contained 2.5 mg l⁻¹ IBA on which 87.03% of the regenerated shoots developed roots with an average number of 5.2 roots per shoots within 30 days. These plantlets were hardened and transferred to the soil. The described method can be successfully employed for the large-scale multiplication and conservation of germplasm this plant.     

Plant regeneration of the mining ecotype <Emphasis Type="Italic">Sedum alfredii</Emphasis> and cadmium hyperaccumulation in regenerated plants

An efficient micropropagation system for mining ecotype Sedum alfredii Hance, a newly identified Zn/Cd hyperaccumulator, was developed. Frequency of callus induction reached up to 70% from leaves incubated on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l⁻¹ 6-benzyladenine (BA), and 83% from internodal stem segments grown on MS medium with 0.1 mg l⁻¹ 2,4-D and 0.1 mg l⁻¹ BA. Callus proliferated rapidly on MS medium containing 0.2 mg l⁻¹ 2,4-D and 0.05 mg l⁻¹ thidiazuron. The highest number of adventitious buds per callus (17.3) and frequency of shoot regeneration (93%) were obtained when calli were grown on MS medium supplemented with 2.0 mg l⁻¹ BA and 0.3 mg l⁻¹ α-naphthalene acetic acid (NAA). Elongation of shoots was achieved when these were incubated on MS medium containing 3.0 mg l⁻¹ gibberellic acid. Induction of roots was highest (21.4 roots per shoot) when shoots were transferred to MS medium containing 2.0 mg l⁻¹ indole 3-butyric acid rather than either indole 3-acetic acid or NAA. When these in vitro plants were acclimatized and transferred to the greenhouse, and grown in hydroponic solutions containing 200 μM cadmium (Cd), they exhibited high efficiency of Cd transport, from roots to shoots, and hyperaccumulation of Cd.     

An <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation system using callus of <Emphasis Type="Italic">Zoysia tenuifolia</Emphasis> Willd. ex Trin

Zoysia tenuifolia Willd. ex Trin. is one of the most popularly cultivated turfgrass. This is the first report of successful plant regeneration and genetic transformation protocols for Z. tenuifolia using Agrobacterium tumefaciens. Initial calli was induced from stem nodes incubated on a Murashige and Skoog (1962) (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg l⁻¹ 6-benzyladenine (BA), with a frequency of 53%. Compact calli were selected and subcultured monthly on the fresh medium. Sixty-nine percent of the calli could be induced to regenerate plantlets when the calli incubated on a MS medium supplemented with 0.2 mg l⁻¹ BA under darkness. For genetic transformation, calli 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 (gus-int) as a reporter gene. Following co-cultivation, about 12% of the callus explants produced hygromycin resistant calli on MS medium supplemented with 2 mg l⁻¹ 2,4-D, 1 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, 500 mg l⁻¹ cefotaxime after 8 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 0.2 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 46% of the resistant calli differentiated into shoots. Finally, all the resistant shoots were rooted on 1/2 MS media supplemented with 50 mg l⁻¹ hygromycin, 250 mg l⁻¹ cefotaxime. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. About 5% of the total inoculated callus explants produced transgenic plants after approximately 5 months. The procedure described will be useful for both, the introduction of desired genes into Z. tenuifolia and the molecular analysis of gene function.     

In vitro root culture of <Emphasis Type="Italic">Ocimum sanctum</Emphasis> L. and evaluation of its free radical scavenging activity

Young leaf explants of Ocimum sanctum L. incubated on solidified Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ 1-naphthaleneacetic acid (NAA) and 0.2 mg l⁻¹ kinetin (Kn) developed rhizogenic callus. When these were subcultured onto MS medium supplemented with 1.5 mg l⁻¹ 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.5 mg l⁻¹ NAA, friable rhizogenic callus was observed. Upon transfer of this friable callus onto liquid MS medium containing 4 mg l⁻¹ NAA and 1.3 mg l⁻¹ 6-benzyladnine (BA) under continuous agitation at 90 rpm and 16 h photoperiod, roots with an optimum dry weight of 1,460 mg l⁻¹ were obtained. An ethyl acetate extract of these roots exhibited 1, 1–diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity.     

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.     

Plant growth regulators,adenine sulfate and carbohydrates regulate organogenesis and in vitro flowering of <Emphasis Type="Italic">Anethum graveolens</Emphasis>

In vitro regeneration protocol for Anethum graveolens (Apiaceae) was developed using leaf explants. MS basal medium used in experiments was augmented with various hormones for caulogenic and rhizogenic response. The optimum callus induction (100%) was obtained by leaf explants on MS media fortified with BA (0.5 mg l⁻¹) singly and in combination with NAA (0.1 and 0.2 mg l⁻¹). BA at 0.5 mg l⁻¹, KN at 1.0 mg l⁻¹ and NAA at 0.1 mg l⁻¹ induced highest number of multiple shoots (10.0 ± 0.25) per explant and they also showed in vitro flowering within 3 weeks of culture. Influence of adenine sulfate on regeneration frequency of callus was evaluated. The highest frequency of rooting (100%) with 6.0 ± 0.25 roots per explants was obtained in one-fourth strength MS medium supplemented with 1/4 MS + IBA 0.5 mg l⁻¹ within 4 weeks of transfer to the rooting medium. In vitro flowering (35%) was obtained with MS fortified with BA alone and also in combination with KN and NAA (5.3 ± 0.42 flowers per explants). In vitro flowering response was tested with different carbohydrates (fructose, glucose, mannose and sorbitol) and optimized. Hardening was successfully attained under controlled conditions inside the plant tissue culture room. The proposed method could effectively be applied for the conservation and clonal propagation to meet the pharmaceutical demands of this medicinally important species.     

Shoot regeneration and free-radical scavenging activity in <Emphasis Type="Italic">Silybum marianum</Emphasis> L.

The morphogenic potential and free-radical scavenging activity of the medicinal plant, Silybum marianum L. (milk thistle) were investigated. Callus development and shoot organogenesis were induced from leaf explants of wild-grown plants incubated on media supplemented with different plant growth regulators (PGRs). The highest frequency of callus induction was observed on explants incubated on Murashige and Skoog (MS) medium supplemented with 5.0 mg l⁻¹ 6-benzyladenine (BA) after 20 days of culture. Subsequent transfer of callogenic explants onto MS medium supplemented with 2.0 mg l⁻¹ gibberellic acid (GA₃) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) resulted in 25.5 ± 2.0 shoots per culture flask after 30 days following culture. Moreover, when shoots were transferred to an elongation medium, the longest shoots were observed on MS medium supplemented with 0.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, and these shoots were rooted on a PGR-free MS basal medium. Assay of antioxidant activity of in vitro and in vivo grown tissues revealed that significantly higher antioxidant activity was observed in callus than all other regenerated tissues and wild-grown plants.     

<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.     

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.     

In vitro regeneration and morphogenesis studies in common bean

An efficient protocol for high frequency in vitro regeneration of multiple shoots and somatic embryos from the embryonic axis of common bean (Phaseolus vulgaris) was developed. Ten common bean cultivars representing a wide range of diversity among current commercial market classes were used for in vitro regeneration evaluation in our study. These cultivars were tested on 63 different media formulations consisting of combinations of cytokinins, namely benzyladenine (BA) and thidiazuron (TDZ) at concentration levels of 0.0, 1.0, 2.5, 5.0 and 10.0 mg l⁻¹ and auxin, namely naphthalene acetic acid (NAA) and indole-3-acetic acid (IAA) at concentration levels of 0.0, 0.05, 0.1 and 1.0 mg l⁻¹. P. vulgaris cv. Olathe pinto bean performed the best producing over 20 multiple shoots per explant while cv. Condor black bean was the poorest with nine multiple shoots per explant. The optimum media for regeneration of multiple shoots was 4.4 mg l⁻¹ Murashige and Skoog (MS) containing 2.5 mg l⁻¹ BA and 0.1 mg l⁻¹ IAA supplemented with 30 mg l⁻¹ silver nitrate. Adventitious shoots and somatic embryos were regenerated on 4.4 mg l⁻¹ MS medium containing 1 mg l⁻¹ TDZ and 0.05 mg l⁻¹ NAA supplemented with 30 mg l⁻¹ silver nitrate or activated charcoal. Efficient and effective rooting of plantlets was achieved by dipping the cut end base of in vitro regenerated shoots in 1.0 mg l⁻¹ indole-3-butyric acid (IBA) solution and culturing on media containing 4.4 mg l⁻¹ MS supplemented by 0.1 mg l⁻¹ IAA, NAA or IBA.     

Cryoconservation and regeneration of axillary shoot meristems of <Emphasis Type="Italic">Indigofera tinctoria</Emphasis> (L.) by encapsulation–dehydration technique

In vitro axillary shoot proliferation was achieved from single-node explants of Indigofera tinctoria on a well-defined medium, Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ N ⁶-benzyl adenine (BA) and 0.1 mg l⁻¹ indole-3-acetic acid. Axillary shoot meristems from cultures derived from up to three subcultures were used in the encapsulation–dehydration technique. Preconditioned, calcium alginate-encapsulated, and precultured axillary shoot meristems were subjected to different lengths of desiccation in a laminar flow cabinet. Maximum survival and regeneration rates of 56.7% and 62.2%, respectively, were obtained in half-strength (half the macro- and micronutrients and full-strength vitamins) MS medium supplemented with 0.5 mg l⁻¹ gibberellic acid and 0.2 mg l⁻¹ BA after 4 h of desiccation, during which the moisture content was reduced to 16.0%. According to the analysis of six random amplified polymorphic DNA markers, plantlets derived from cultures initiated with cryopreserved plant material were genetically identical to those derived from nonfrozen (control) tissues.     

Production of monoterpenoids and aroma compounds from cell suspension cultures of <Emphasis Type="Italic">Camellia sinensis</Emphasis>

Cell suspension cultures of Camellia sinensis were established in 250 ml shake flasks. Flasks contained 50 ml liquid medium of either Murashige and Skoog (MS), N/5 MS or Heller medium containing different levels of 6-benzyladenine (BA) (0.05–2 mg l⁻¹), 2,4-dichlorophenoxyacetic acid (2,4-D) (1–10 mg l⁻¹), and sucrose (10–50 g l⁻¹). Moreover, the pH of the medium was varied from 5.2–6.2. In addition, cultures were subjected to light irradiation as well as to complete darkness. Following optimization of aroma and terpenoid extraction methods, cell cultures were analyzed for the volatile compounds using GC/MS. A total of 43 compounds were identified using the micro SDE apparatus. Among the major monoterpenoids obtained were α-terpineol and nerol. Moreover, other high aroma-value compounds, including 2-ethyl hexanol, benzyl alcohol, benzene acetaldehyde, nonanal and phenylethylalcohol were also detected. The highest levels of these compounds were obtained from cell suspension cultures grown in MS medium containing 5 mg l⁻¹ 2,4-D, 1 mg l⁻¹ BA and 30 g l⁻¹ sucrose at pH of 5.8 with incubation in complete darkness.     

Enhanced regeneration of haploid plantlets from microspores of <Emphasis Type="Italic">Brassica napus</Emphasis> L. using bleomycin,PCIB, and phytohormones

The effects of three periods of incubation (10, 20 and 30 min) at different levels of bleomycin (0, 0.1, 0.2, 0.3, 0.4 and 0.5 μg ml⁻¹), as well as three periods of exposure (12, 24 and 48 h) to different levels of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), including 1, 2, 3, 4 and 5 mg l⁻¹, on microspore embryogenesis of rapeseed cv. ‘Amica’ were investigated. Microspore embryogenesis was significantly enhanced following 20 min treatment with 0.2 μg ml⁻¹ bleomycin compared with untreated cultures. Highest embryo yield (163 embryos Petri dish⁻¹) was observed with 24 h treatment of 4 mg l⁻¹ PCIB. The highest percentage of secondary embryogenesis was observed on B₅ medium containing 0.15 mg l⁻¹ of gibberellic acid (GA₃) and 0.2 mg l⁻¹ 6-benzyladenine (BA) in 4–6 mm microspore-derived embryos (MDEs). Most callus formed on B₅ medium containing 0.15 mg l⁻¹ GA₃, 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ indole-3-acetic acid (IAA) when 4–6 mm embryos were used. Regeneration was highest on B₅ medium containing 0.05 mg l⁻¹ GA₃ or 0.1 mg l⁻¹ BA and 0.2 mg l⁻¹ IAA with 2–4 mm embryos. Microspore embryogenesis and plant regeneration could be improved by both bleomycin and PCIB when the appropriate MDE length and phytohormone level were selected.     

An efficient regeneration system via direct and indirect organogenesis for the medicinal plant <Emphasis Type="Italic">Dysosma versipellis</Emphasis> (Hance) M. Cheng and its potential as a podophyllotoxin source

Dysosma versipellis (Hance) M. Cheng is an endangered plant due to overharvesting for the extraction of podophyllotoxin. Thus, the in vitro technique is valuable for the propagation of this species. When the explants of rhizome buds were cultured on Murashige and Skoog’s (MS) medium with 6-benzyladenine (BA) (1.0 mg l⁻¹), gibberellic acid (GA₃) (0.5 mg l⁻¹) and zeatin (Zea) (0.5 mg l⁻¹), multiple buds were regenerated directly on the explants without callusing within 6 weeks. Callus was induced from the leaf segment cultures on MS basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5 mg l⁻¹) and BA (0.2 mg l⁻¹) within 4 weeks. The adventitious buds were differentiated when the calli were subcultured on MS medium supplemented with BA (1.0 mg l⁻¹) and thidiazuron (TDZ) (0.2 mg l⁻¹) within 6 weeks. The adventitious buds obtained from callus and the rhizome-buds rooted with a frequency of 100% on half strength MS medium fortified with indole-3-butyric acid (IBA) 0.5 mg l⁻¹ and activated charcoal (AC) 0.5 g l⁻¹ for 4 weeks. The rooted shoots were successfully transplanted from a mixture of vermiculite:soil (1:1 v/v) to the field with a survival rate of 85%. Podophyllotoxin production in calli, cultured rhizomes, rhizomes of transplanting plants from the garden and rhizomes in the wild field was confirmed by high-performance liquid chromatography (HPLC) analysis. Our results suggest that calli, cultured rhizomes and rhizomes of transplanting plants would be the potential sources of podophyllotoxin.     

An efficient in vitro propagation protocol for <Emphasis Type="Italic">Pinguicula lusitanica</Emphasis>, a rare insectivorous plant

In this study, an efficient protocol was developed for in vitro propagation of Pinguicula lusitanica L., a rare insectivorous plant with pharmacological value and limited reproductive capacity. The effects of two concentrations (0.1 and 0.5 mg l⁻¹) of a range of plant growth regulators, including cytokinins (BA, KIN, and ZEA) and auxins (IAA, IBA, and NAA), and three concentrations of MS medium macronutrients (total, 1/2 and 1/4MS) on proliferation and rooting, were investigated. P. lusitanica shoots showed abundant proliferation and rooting capacity, both of which were significantly influenced by MS medium strength. The use of 1/2MS supplemented with 0.5 mg l⁻¹ BA or KIN ensure a 29-fold rate of proliferation. Best rooting frequency and higher root number and length were attained in 1/4MS medium containing 0.2 mg l⁻¹ IAA. Sixty percent of the plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development. These plantlets can be used to replenish declining populations in the wild and/or for the extraction of bioactive compounds, reducing pressure on wild stocks.