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.     

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.     

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.     

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.     

An efficient plant regeneration system with <Emphasis Type="Italic">in vitro</Emphasis> flavonoid accumulation for <Emphasis Type="Italic">Hylotelephium tatarinowii</Emphasis> (Maxim.) H. Ohba

An efficient micropropagation system for Hylotelephium tatarinowii (Maxim.) H. Ohba, a rare medicinal plant, has been developed. Callus induced from leaf explants placed onto Murashige and Skoog (MS) medium with supplementation of plant growth regulators. When the concentration of 2,4-dicholorophenoxy acetic acid was as high as 2.0 mg l⁻¹ in combination with 0.5 mg l⁻¹ 6-benzylaminopurine (6-BAP), the callus induction rate reached 92.1%. Adventitious shoots were observed on callus exposed to 1.0 mg l⁻¹ 6-BAP, with 81.5% frequency of shoot regeneration after 30 d. Flower buds appeared after subculture. Regenerated shoots could flower normally in vitro. Up to 100% of the regenerated shoots formed complete plantlets on half-strength MS medium without any growth regulator, with an average of 5.9 roots per shoot explant. Quantitative analysis of flavonoids and rutin showed that the phytochemical profile of callus and regenerated plants was similar to that of wild plants.     

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.     

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.     

In vitro hormone-regulated growth and floral induction of <Emphasis Type="Italic">Cuscuta reflexa</Emphasis>: a parasitic angiosperm

The role of different growth regulators in callus induction, shoot regeneration, floral induction and chlorophyll content of the obligatory parasitic plant Cuscuta reflexa has been studied. Callus development was excellent from the nodal part of the shoot explants in modified Murashige and Skoog (MMS) media supplemented with 2 mg L⁻¹ benzyl adenine (MMS1c). Supplementation of 2 mg L⁻¹ naphthalene acetic acid (NAA) along with MMS1c (MMS2c) was responsible for estimable shoot induction and development in callus. 2,4-Dichloro acetic acid (2,4-D) played a crucial role in the floral induction of C. reflexa in vitro. MMS supplemented with 2 mg L⁻¹ NAA and 2 mg L⁻¹ 2,4-D (MMS3b) supported floral induction after shooting in vitro. MMS supplemented with 3 mg L⁻¹ 2,4-D (MMS4a) rapidly induced flower directly from the stem explants without showing any elongation of shoot. MMS1c along with MMS3b (MMS5a) showed callus proliferation followed by shoot elongation and floral induction. In vitro MMS5a grown plants show a sharp increase in the chlorophyll contents. Cytokinin treatment further increases the chlorophyll level of the plant.     

Strategies for overcoming genotypic limitations of in vitro regeneration and determination of genetic components of variability of plant regeneration traits in sorghum

Development of suitable strategy to overcome genotypic limitations of in vitro regeneration in sorghum would help utilize high yielding but poor tissue culture responsive genotypes in genetic manipulation programmes. A factorial experiment was conducted with two explants (immature embryos and inflorescences), eight genotypes (five Sorghum sudanense and three Sorghum bicolor genotypes), three levels of 2,4-D (1 mg l⁻¹, 3 mg l⁻¹, and 5 mg l⁻¹), and two levels of kinetin (0.0 mg l⁻¹ and 0.5 mg l⁻¹). The induced callus was transferred to the regeneration media with factorial combinations of IAA (1.0 mg l⁻¹ and 2.0 mg l⁻¹) and kinetin (0.5 mg l⁻¹ and 1.0 mg l⁻¹). S. sudanense regenerated at significantly higher frequency (38.91%) and produced shoots more intensely (2.2 shoots/callus) than S. bicolor (26.93%, 1.26 shoots/callus). Immature inflorescences regenerated at a much higher frequency (46.48%) and produced significantly more number of shoots (2.71 shoots/callus) than immature embryos (22.35%, 0.99 shoots/callus). Moreover, differences for plant regeneration between genotypes of the same species were minimal when using immature inflorescences. Increase in the 2,4-D concentration in callus induction media exhibited inhibitory effect on callus induction, growth, shoot induction and number of shoots/callus but inclusion of kinetin in callus induction media improved these responses. Use of immature inflorescence explant and inclusion of kinetin in callus induction media could overcome genotypic limitations of plant regeneration to a large extent. The extent of variability, heritability and expected genetic advance was more in plant regeneration traits than in callus induction traits. This indicated that the variability in respect of these attributes in the genotypes may be due to the additive gene action and selection of genotypes for these characters would be rewarding.     

Efficient indirect induction of protocorm-like bodies and shoot proliferation using field-grown axillary buds of a <Emphasis Type="Italic">Lycaste</Emphasis> hybrid

This work presents a rapid and reliable micropropagation method for a Lycaste hybrid using a field-grown axillary bud culture system. Intact buds (2–4 mm in length) were excised from a mature pseudobulb and were cultured in half-strength MS basal medium, which was supplemented with 0.5 mg l⁻¹ benzyladenine (BA), 1.0 mg l⁻¹ thidiazuron (TDZ) and 2% (w/v) sucrose. After 2 months, the calli exhibited vigorous growth and eventually turned green, forming protocorm-like bodies (PLBs) originating in the surface of each callus. The results of this work reveal that the combination of 0.5 mg l⁻¹ BA and 1.0 mg l⁻¹ TDZ treatments was highly effective in indirectly multiplying shoots from callus-PLB mixed explants, which yielded up to 400 shoots in the fourth time subcultures (within 24 weeks). Histological observations showed the apical meristem of adventitious bud is based on a longitudinal section of a callus sample. Histological and scanning electronic microscopy also indicated that PLBs derived from calli could be regarded as organogenesis but not somatic embryogenesis. Shoots with a length of around 2–3 cm generated in vitro were excised and cultured in MS medium supplemented with 0.5 mg l⁻¹ IBA exhibited the best rooting response (78.3%), and an average of 1.8 roots per explant was produced within 4 weeks.     

Androgenesis induction in microspore culture of sweet pepper (Capsicum annuum L.)

Isolated microspore culture experiments were carried out in sweet pepper (Capsicum annuum L.) F₁ hybrid genotypes. In the first experiment, four culture media (W14, B5, MS and NLN) were compared to test their effectiveness in inducing the formation of microspore-derived structures in two genotypes. The experiments revealed the superiority of B5 medium. In the second experiment, the effects of different ratios of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.1, 0.2 and 0.5 mg l⁻¹) and kinetin (0, 0.2 and 0.5 mg l⁻¹) were also investigated in B5 medium with two genotypes. The effect of growth regulators were investigated on the production of microspore-derived calli and embryo-like structures (ELSs), the ratio of the two and plant regeneration (number of regenerated plantlets) in microspore culture. The histological experiments revealed the differences between the microspore-derived ELSs and calli. The most promising results were obtained on the investigated parameters in the presence of 0.1 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ kinetin producing the highest number of plantlets in both genotypes tested. In the response of 11 genotypes, the androgenesis induction was successful in each sweet pepper genotypes tested using the best basic medium and growth regulators combination. In case of 11 genotypes, the number of ELSs ranged from 20 to 100/Petri dish (an average of 48.1 ELS/Petri dish), while the number of green plantlets varied from 0 to 8 plantlets/Petri dish (an average of 1.5 plantlets/Petri dish) depending on the genotype. The spontaneous rediploidization rate obtained was 25% in isolated microspore.     

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.     

<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.     

Impact of colchicine application during callus induction and shoot regeneration on micropropagation and polyploidisation rates in two <Emphasis Type="Italic">Miscanthus</Emphasis> species

Grasses from the genus Miscanthus have several characteristics that make them very favourable crops for efficient, low input, multifunctional and environmentally friendly biomass production. This study is aimed to improve a polyploidisation method to effectively induce polyploids in Miscanthus sinensis and Miscanthus x giganteus. Colchicine was applied for 2, 4 or 7 d in micropropagation systems using inflorescence segments at two different points: during callus induction (313 and 626 μM colchicine) and during shoot regeneration from callus (313 μM colchicine). Among the tested combinations, the most effective (up to 40%) was the 4-d colchicine treatment of a shoot-forming callus cultured 4 d before the experiment on regeneration medium under light conditions. In vitro colchicine treatment during callus induction and during shoot regeneration from callus resulted in no chimeric polyploids as well as a very low number of albinos (2.5%). Additionally, some combinations using colchicine did not significantly reduce the rates of micropropagation effectiveness. The obtained material is promising for the creation of new high-biomass-yielding forms in the Miscanthus genus. In all genotypes tested, chromosome doubling significantly increased pollen stainability. According to preliminary results, induced tetraploids are fertile and useful in hybrid production. Leaves of polyploid forms of two genotypes demonstrated significantly greater width in comparison to the controls.     

In vitro regeneration of the important North American oak species <Emphasis Type="Italic">Quercus alba</Emphasis>, <Emphasis Type="Italic">Quercus bicolor</Emphasis> and <Emphasis Type="Italic">Quercus rubra</Emphasis>

North American oak species, with their characteristic strong episodic seasonal shoot growth, are highly problematic for clonal micropropagation, resulting in the inability to achieve a stabilized shoot multiplication stage. The potential for initiating and proliferating shoot cultures derived from Quercus alba, Q. bicolor and Q. rubra explants was investigated, and a micropropagation method for these species was developed. Branch segments from 6 to 7-year-old trees were forced-flushed and the forced shoots were used as source of explants for culture initiation. A consistent shoot multiplication stage was achieved, in 13 of the 15 genotypes established in vitro, although marked differences occurred in explants from different genotypes/species. The control of efficient shoot multiplication involved the culture of decapitated shoots in a stressful horizontal position on cytokinin-containing medium with a sequence of transfers within a 6-week subculture cycle, which was beneficial to overcoming the episodic character of shoot growth. During each subculture cycle, the horizontally placed explants were cultured on media containing 0.2 mg l⁻¹ benzyladenine (BA) for 2 weeks with two successive transfers (2 weeks each) to fresh medium with 0.1 mg l⁻¹ BA, giving a 6-week subculture cycle. The general appearance and vigor of Q. alba and Q. bicolor shoot cultures were improved by the inclusion of both 0.1 mg l⁻¹ BA and 0.5 mg l⁻¹ zeatin in the medium used for the second transfer within the 6-week subculture cycle. Addition of AgNO₃ (3 mg l⁻¹) to the shoot proliferation medium of Q. rubra had a significant positive effect on shoot development pattern by reducing deleterious symptoms, including shoot tip necrosis and early senescence of leaves. The three species showed acceptable in vitro rooting rates by culturing microcuttings in medium containing 25 mg l⁻¹ indolebutyric acid for 48 h with subsequent transfer to auxin-free medium supplemented with 0.4% activated charcoal. Although an initial 5-day dark period generally improved the rooting response, it was detrimental to the quality of regenerated plantlets. However, activated charcoal stimulated not only the rooting frequencies, but it also enhanced plant quality, as evidenced by root, shoot and leaf growth.     

Callus induction,plant regeneration,and long-term maintenance of embryogenic cultures in <Emphasis Type="Italic">Zoysia matrella</Emphasis> [L.] Merr

An efficient protocol of callus induction, plant regeneration and long-term maintenance of embryogenic cultures for manilagrass was developed. Callus induction and embryogenic callus formation were influenced by cytokinins and nodal positions. Murashige and Skoog (MS) medium with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 0.02 mg l⁻¹ kinetin (KT) or 6-benzyladenine (BA) gave the highest frequency for both callus induction and embryogenic callus formation compared with 0.02 mg l⁻¹ thidiazuron (TDZ) or N⁶-(2-isopenteny) adenine (2iP). The frequency of callus induction of different nodes (from the first to the sixth node) varied from 22.5 to 92.1%, and the embryogenic callus formation frequencies ranged from 13.3 to 25.7%. The highest frequencies of callus induction and embryogenic callus formation (92.1 and 25.7%, respectively) were observed in the fourth node group. During subculture on callus induction and maintenance medium, somatic embryos formed on the surface of the embryogenic callus. On regeneration medium, the regeneration rates of embryogenic callus varied from 96.8 to 100% during the 4-year period of subculture. The results also indicate that preservation of manilagrass callus is stable at low-temperature (4°C) over a period of 11 months. No significant differences were found in the activities of superoxide dismutase (SOD), peroxidase (POD) and proline content of the plants regenerated from the 4-year subcultured callus on different regeneration media.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

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.     

In vitro micropropagation of Ruscus aculeatus

We have developed three protocols for the rapid micropropagation of Ruscus aculeatus. The primary explants utilised were immature embryos, aerial buds excised from rhizomes and shoot buds regenerated from organogenic calli. In order to increase the plant regeneration from the primary explants, we used organogenic calli from cladode, stem and rhizome segments. We tested more than 20 culture media for callus induction and shoot regeneration and the best results were obtained when rhizome segments were cultured on Murashige and Skoog medium supplemented with 0.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid and 1 mg dm⁻³ kinetin.     

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.