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.     

Pulvinus: an ideal explant for plant regeneration in <Emphasis Type="Italic">Caesalpinia bonduc</Emphasis> (L.) Roxb., an important ethnomedicinal woody climber

This study demonstrates the morphogenic potential of pulvinus, an important organ situated at the base of the petiole or rachis of leguminous plants. Plant regeneration via pulvinus-derived calli of Caesalpinia bonduc has been achieved. Organogenic calli have been derived from the explant 45 days after culture on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with 6-benzylaminopurine (BA). Optimum callus induction (100%) occurred when the pulvini were cultured on MS medium fortified with 6 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BA. The highest shoot induction was obtained when the calli were transferred to MS medium supplemented with 5 mg l⁻¹ BA and 1 mg l⁻¹ indole-3-acetic acid (IAA). On this medium, 87% cultures responded with an average number of 4.2 shoots per culture. The maximum root induction from the regenerated shoots was observed on half strength MS medium containing 6 mg l⁻¹ indole-3-butyric acid (IBA). Here 100% shoots rooted with a mean number of 6.3 roots per shoot. The regenerated plantlets were acclimatized and subsequently showed normal growth. This efficient protocol will be helpful for propagating elite clones on a mass scale and could be utilized for genetic transformation study.     

Callus induction and whole plant regeneration in elite Indian maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) inbreds

Callus induction and regeneration ability of five elite maize inbred lines, CM 111, CM 117, CM 124, CM 125 and CM 300 were investigated using 14-day-old immature embryos as explants. Genotype, medium, source of auxin and their concentrations influenced induction of callus. Explants grown on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid at 1 mg l⁻¹ showed the highest frequency of callusing. Among all the media tested, explants grown on N6 medium gave the highest frequency of organogenic callus. Moreover, N6 supplemented with Dicamba promoted higher callus response in terms of both frequency of induction as well as quality, compared to N6 medium with 2,4-D. N6 supplemented with 2 mg l⁻¹ Dicamba induced the highest frequency of organogenic callus. Among the five genotypes tested, CM 124, CM 125, and CM 300 gave the best callus. Explants of both CM 124 and CM 300 incubated on MS medium supplemented with 1 mg l⁻¹ benzyladenine and 0.5 mg l⁻¹ indole acetic acid promoted the highest frequency of shoot induction. Though CM 124 induced higher percentage of shoot formation than CM 300, the mean number of developed shoots per explant was higher for CM 300. The highest frequency of root formation was observed when shoots were grown on MS medium supplemented with 2 mg l⁻¹ naphathalene acetic acid. Percentage of regenerated plants ranged from 54 to 66.     

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.     

Somatic embryogenesis and plant regeneration in two wild cotton species belong to G genome

The present work describes the plant regeneration via somatic embryogenesis in two wild cotton species belonging to G genome: Gossypium nelsonii Fryx and Gossypium australe F Muell. The role of plant hormones and carbohydrates was also evaluated for somatic embryogenesis and somatic embryo development. Normal plants were obtained from G. nelsonii Fryx; abnormal plants and somatic embryos were obtained from G. australe F Muell. The best medium for callus induction for these G genome wild cotton species was MSB₅ supplemented with 0.1 mg L⁻¹ KT and 0.1 mg L⁻¹ 2,4-D. For embryogenic callus proliferation, the best medium used was MSB₅ supplemented with 0.2 mg L⁻¹ KT and 0.5 mg L⁻¹ IBA. The medium MSB₅ supplemented with 0.15 mg L⁻¹ KT and 0.5 mg L⁻¹ NAA was used successfully for root initiation and plant growth. In addition, adding CuSO₄ and AgNO₃ in the callus-inducing and proliferation medium resulted in a number of somatic embryos. Glucose and maltose, the carbon sources in somatic culture, were used for callus induction, but maltose worked even better than glucose for proliferation of embryogenic callus and development of somatic embryos.     

Micropropagation and plant regeneration from embryogenic callus of Miscanthus sinensis

Miscanthus sinensis (Poaceae) is a typical perennial giant grass of East Asia. Due to its high photosynthetic efficiency, low input requirements, and high biomass production, M. sinensis shows outstanding potential as a biofuel feedstock. However, the lack of an efficient tissue culture system may limit its utilization potential. Different explants of M. sinensis were evaluated to develop an efficient tissue culture system. Shoot apices from in vitro-germinated seedling explants were tested for adventitious bud proliferation. The highest level of proliferation (multiplication coefficient 6.69) was obtained when shoot apices were cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg L⁻¹ 6-benzyladenine (BA), 2.0 mg L⁻¹ kinetin, 0.05 mg L⁻¹ α-naphthalene acetic acid (NAA), 3% sucrose, and 0.8% agar. The highest rooting percentage (95.4%) was obtained when adventitious buds were cultured on half-strength MS medium supplemented with 0.2 mg L⁻¹ NAA, 3% sucrose, and 0.8% agar. Significant differences were found in the formation of embryogenic callus among different explant types. The embryogenic callus derived from epicotyls had the highest regeneration capacity when cultured on a medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid, 0.5 mg L⁻¹ BA, and 0.1 mg L⁻¹ thiamine. Under these conditions, the callus induction percentage was 82%.     

Plant regeneration and production of embelin from organogenic and embryogenic callus cultures of <Emphasis Type="Italic">Embelia ribes</Emphasis> Burm. f.—a vulnerable medicinal plant

Embelia ribes, an important vulnerable medicinal liana, was regenerated through organogenesis and embryogenesis using leaf explants. Leaf explants produced organogenic calluses on MS medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l⁻¹ 6-benzylaminopurine. Shoot regeneration was obtained from organogenic calluses on MS medium containing different concentrations of thidiazuron (TDZ) and indole-3-acetic acid (IAA). The frequency of shoot bud organogenesis was highest (23.9 shoots/explant) in MS medium containing 0.5 mg l⁻¹ TDZ and 0.1 mg l⁻¹ IAA. The best result for induction of embryogenic callus was noticed in the combination of 2.0 mg l⁻¹ TDZ and 0.5 mg l⁻¹ 2,4-D. This callus, maintained in the same medium, showed the highest differentiation of embryos (56.5%) after 6 wk of culture. Embryos were transferred to MS medium supplemented with different concentrations of TDZ, and this facilitated conversion of embryos into plants. After 6 wk of subculture, MS medium with 0.05 mg l⁻¹ TDZ favored the highest percentage (52.2%) embryo conversion. As per the present protocol, 52.2% of the embryos underwent conversion, and a mean number of 29.5 shoots per culture was obtained. Shoots developed from both types of calluses were rooted on half-strength MS basal medium supplemented with 1.0 mg l⁻¹ indole-3-butyric acid. HPLC-UV assay demonstrated the highest embelin content (5.33% w/w) in the embryogenic callus cultures. Embelin was isolated from embryogenic callus and was identified using IR and ¹H NMR studies.     

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.     

Efficient and rapid plant regeneration of oil palm zygotic embryos cv. ‘Tenera’ through somatic embryogenesis

An efficient and rapid plant regeneration system through somatic embryogenesis was developed using 13-week-old zygotic embryos of oil palm (Elaeis guineensis Jacq.) cv. ‘Tenera’. Zygotic embryos were cultured on MS and N6 media supplemented with 2.0 mg L⁻¹ picloram, 2,4-D and dicamba. The highest embryogenic callus formation (32%) was observed on N6 medium with 2,4-D after 3 month culture on callus induction medium. Somatic embryos were continuously formed from nodular calli on embryo maturation medium [N6 + 0.1 mg L⁻¹ 2,4-D, 0.16 g L⁻¹ putrescine, 0.5 g L⁻¹ casein amino acids and 2.0 g L⁻¹ activated charcoal(AC)] for 3–5 months. Histological analysis confirmed that embryo development occurred via somatic embryogenesis. For plant regeneration, modified N6 medium (MN6) with AC (0.5 g L⁻¹) without growth regulators, induced both shoot and root formation simultaneously with the highest regeneration rate of 56%. This combined shoot and root induction protocol shortened the culture time to 9–12 months. Furthermore, after acclimatization, more than 85% of transferred plants from our protocol developed successfully in the soil.     

Long-term cultured callus and the effect factor of high-frequency plantlet regeneration and somatic embryogenesis maintenance in <Emphasis Type="Italic">Zoysia japonica</Emphasis>

In this study, we have demonstrated that Zoysia japonica callus induced from mature seeds can produce high frequencies of plant regeneration and somatic embryogenesis, even following a prolonged period of subculturing. Initial callus cultures were induced from mature seeds of Japanese lawngrass (Z. japonica Steud.) incubated on a medium containing major N₆ medium salts, minor Murashige and Skoog (MS) medium salts, and modified MS medium organic elements supplemented with 3 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01–0.02 mg L⁻¹ 6-benzyladenine. Compact callus were selected and subcultured monthly on a medium containing 2 mg L⁻¹ 2,4-D, 0.5 mg L⁻¹ kinetin, 500 mg L⁻¹ casein hydrolysate, 500 mg L⁻¹ proline, and 500 mg L⁻¹ myoinositol. Callus maintained in vitro for 18 mo could be induced to regenerate plantlets with a frequency of >90%. By contrast, 36-mo-old callus cultures failed to produce normal shoot regeneration. However, the addition of CuSO₄ to the subculture media maintained >90% regeneration frequencies in such long-term callus cultures. Histological observations revealed that plant regeneration occurred both through somatic embryogenesis and organogenesis pathways. The ability to sustainable regeneration in long-term callus cultures will be valuable to the program of genetic transformation and somaclonal variant selection.     

A rapid system for micropropagation of <Emphasis Type="Italic">Swertia chirata</Emphasis> Buch-Ham. ex Wall.: an endangered medicinal herb via direct somatic embryogenesis

An improved method of direct somatic embryogenesis (SE) was developed in Swertia chirata for the first time using leaves and roots of in vitro-grown young seedlings. In the present study, 2,4-dichlorophenoxyacetic acid (2,4-D) was assessed individually and in combination with other auxins, as well as with cytokinin for its effectiveness to induce somatic embryos. Leaf explants with abaxial side in the medium produced maximum number of somatic embryos. This system omits the callus stage and thus reduces the process of SE in S. chirata by 35–45 days. Embryos at different stages of development were observed. Maturation of heart stage embryos were observed on Murashige and Skoog (MS) medium containing 1 mg L⁻¹ 2,4-D. Upon transfer to the germination medium, they were converted to cotyledonary stage and then plantlets of 33% and 68% of them were converted to cotyledonary stage and then plantlets on MS medium supplemented with 0.05 and 0.1 mg L^-1 GA₃ respectively. The 2,4-D alone at 1.0 or 1.5 mg L⁻¹ was found to be better for embryogenic tissue initiation than 2,4-D in combination with indole-3-acetic acid or α-naphthalene acetic acid. For further embryo development, 2,4-D was combined with cytokinins such as 6-benzylaminopurine (BAP) and kinetin or plant growth regulator free medium or medium with 50% reduced concentration of the same hormone while subculturing. Mean germination and percentage of survival were maximum in the medium containing 1.0 mg L⁻¹ 2,4-D in combination with 0.1 mg L⁻¹ BAP. Regenerated plantlets were morphologically and genetically identical. This method offers a vast scope for the clonal propagation of endangered 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.     

Biochemical profile of callus cultures of <Emphasis Type="Italic">Pogostemon cablin</Emphasis> (Blanco) Benth

Patchouli is an aromatic shrub of commercial interest because its essential oil is rich in patchoulol. This study aimed to evaluate the effect of growth regulators on callus production, analyze the essential oil production in calli and evaluate metabolic differences between callus, in vitro grown-plantlets and greenhouse-grown plants in three different accessions of patchouli. Calli were induced from leaf explants on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 6-benzyladenine (BA). The largest size calli from different accessions were obtained in the presence of the two plant growth regulators (PGRs). For accession POG014, presence of 0.022 mg l⁻¹ 2,4-D plus 0.022 mg l⁻¹ BA were optimum. For accession POG021, presence of 0.110 mg l⁻¹ 2,4-D plus 0.022 mg l⁻¹ of BA induced the largest callus, whereas for accession POG002, 0.022 mg l⁻¹ 2,4-D and 0.225 mg l⁻¹ BA, as well as 0.11 mg l⁻¹ 2,4-D and 0.022 mg l⁻¹ BA promoted the development of largest callus. Among all accessions, peroxidase activity was highest in organogenic calli of accession POG014, whereas, polyphenol oxidase activity was highest in in vitro-grown plantlets of accession POG021. Biochemical variables differed significantly among the treatments, with the exception of total sugar levels. The highest concentrations of total sugars were observed in the calli and in vitro-grown plantlets of POG014 and POG021. Essential oils were not detected in callus tissues.     

Effects of plant growth regulators and <Emphasis Type="SmallCaps">l</Emphasis>-glutamic acid on shoot organogenesis in the halophyte <Emphasis Type="Italic">Leymus chinensis</Emphasis> (Trin.)

The halophyte Leymus chinensis (Trin.) is a perennial rhizome grass (tribe Gramineae) that is widely distributed in China, Mongolia and Siberia, where it is produced as a forage product. In this report, we establish a highly reproducible plant regeneration system through somatic embryogenesis. Two explants, mature seeds and leaf base segments were used; these parts displayed different responses to combinations of growth factors that affect embryogenic callus induction, callus type optimization and plant regeneration. The highest callus induction frequency was obtained on Murashige and Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of 5.0 mg l⁻¹ l-glutamic acid. The inclusion of 5.0 mg l⁻¹ l-glutamic acid was found to significantly promote primary callus induction, embryogenic callus formation and callus status improvement. Subculturing on maintenance medium for 1–2 months before plant regeneration was found to be essential for the optimization of callus type and the maturation of embryogenic callus. Callus relative water content and growth rate were simultaneously investigated during callus maintenance, and found to possibly be related to callus type. Shoots were differentiated from the embryogenic callus on the optimal medium with MS salts containing 0.2–0.5 mg l⁻¹ α-naphthalene acetic acid (NAA), 2.0 mg l⁻¹ kinetin (Kn) and 2.0 g l⁻¹ casamino acids in 71.0 and 69.2% of wild-type (WT) and Jisheng No.1 (JS) plants, respectively. Plant regeneration was variable depending on NAA levels, and the addition of casamino acids stimulated the maturation of embryogenic callus and plant regeneration. Transferring callus with shoots onto half-strength MS medium resulted in rooting within 1 week. The growth of regenerated plants was also surveyed in the field. This is the first report of plant regeneration through somatic embryogenesis from mature seeds and leaf base segments of L. chinensis.     

Response of in vitro strawberry to antibiotics

By identifying antibiotics that had the least phytotoxic effects on explants during genetic transformation, we evaluated the effect of various antibiotics on callus induction and morphogenesis from leaf explants and in vitro growth of Fragaria × ananassa Duch. cv. Toyonaka. Results showed that kanamycin (Kan) significantly inhibited callus induction, bud differentiation and root morphogenesis while carbenicillin (Carb), cefotaxime (Cef) and an equal concentration of Cef and Carb up to 500 mg L⁻¹ had no significant effects on callus induction and shoot growth. Kan, even at 2.5 mg L⁻¹, significantly inhibited callus induction, shoot regeneration and root formation, while no shoots regenerated at concentrations above 15 mg L⁻¹. Rooting was completely inhibited in the presence of 50 mg L⁻¹ Kan. Cef had negative effects on shoot regeneration from leaf explants and in vitro growth of strawberry. Compared to Cef, Carb at ≤300 mg L⁻¹ significantly promoted shoot and root organogenesis. However, an equal concentration of Carb plus Cef could alleviate the negative effect of Cef on strawberry. Results from relative electrolyte leakage, root and antioxidant activities, O₂^·− production rate, H₂O₂, proline and MDA contents showed that Kan, Cef and Carb caused electrolyte leakage and triggered active enzymatic processes and metabolism. This offers a possible mechanism for the inhibition or stimulation of strawberry growth caused by these antibiotics.     

Somatic embryogenesis and plant regeneration from leaf and petiole explants of <Emphasis Type="Italic">Campanula punctata</Emphasis> Lam. var. <Emphasis Type="Italic">rubriflora</Emphasis> Makino

A simple and efficient protocol was developed for somatic embryogenesis from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino. Somatic embryos (SE) were obtained with greater frequency from petiole explants than from leaf explants when cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L⁻¹ 6-benzyladenine (BA). On this medium, a mean number of 19.5 and 31.2 SE were developed per leaf and petiole explants, respectively. Embryos were induced both light and dark conditions but culturing the explants 2 weeks in the dark followed by 3 weeks under light resulted in high frequency of embryo formation. Globular embryos germinated best on MS medium supplemented with 0.3% (w/v) activated charcoal (AC) and 1.0 mg L⁻¹ GA₃. The germinated plantlets grew further on MS medium containing 0.3% AC. Plantlets were successfully acclimatized in the greenhouse with 94% survival rate. This is the first report on induction of somatic embryogenesis in this genus and also has implications for genetic transformation, and mass clonal propagation.     

High-frequency somatic embryogenesis from germinated zygotic embryos of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and evaluation of the effects of medium strength,sucrose, GA<Subscript>3</Subscript>, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l⁻¹ 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA₃), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA₃ negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l⁻¹ BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.     

The effects of genotype,inflorescence developmental stage and induction medium on callus induction and plant regeneration in two <Emphasis Type="Italic">Miscanthus</Emphasis> species

Several grass species of the genus Miscanthus are considered to be outstanding candidates for a sustainable production of biomass to generate renewable energy. The purpose of this study was to investigate the effects of genotype, the developmental stage of the explant donor inflorescence and the induction medium on the success rate of micropropagation. The experiments were conducted on three genotypes of M. sinensis and one of M. x giganteus. Explants from the youngest inflorescences (0.1–2.5 cm in length) showed a significantly higher callus induction rate than those from more developed inflorescences (2.6–5 cm in length). In addition, cultures initiated from explants from the youngest inflorescences showed significantly the highest rates of callus regeneration and the highest shoot regeneration rate. Three out of the four genotypes tested showed the best shoot regeneration from calli initiated from the youngest inflorescences when cultured on the Murashige and Skoog basal medium (MS) with 5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg l⁻¹ 6-benzyladenine (BA). The percentages of calli from those genotypes showing regeneration ranged from 45 to 76.7%, and the corresponding shoot regeneration rates ranged from 1.85 to 6.33 shoots/callus. This demonstrates that, with some adjustments, efficient micropropagation of Miscanthus sp. is feasible.     

High frequency plant regeneration system for <Emphasis Type="Italic">Nymphoides coreana</Emphasis> via somatic embryogenesis from zygotic embryo-derived embryogenic cell suspension cultures

Culture conditions were established for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Nymphoides coreana. Zygotic embryos formed pale-yellow globular structures and calluses at a frequency of 85.6% when cultured on half-strength Murashige and Skoog (MS) medium supplemented with 0.3 mg l⁻¹ of 2,4-D. However, the frequency of pale-yellow globular structures and white callus formation decreased slightly with an increasing concentration of 2,4-D up to 10 mg l⁻¹ with the frequency rate falling to 16.7%. Cell suspension cultures were established from zygotic embryo-derived calluses using half-strength MS medium supplemented with 0.3 mg l⁻¹ of 2,4-D. Upon plating onto half-strength MS basal medium, over 92.3% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted into potting soil and achieved full growth to an adult plant in a growth chamber. The high frequency plant regeneration system for Nymphoides coreana established in this study will be useful for genetic manipulation and cryopreservation of this 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.