Shoot induction and regeneration using internodal transverse thin cell layer culture in <Emphasis Type="Italic">Sesamum indicum</Emphasis> L.

An efficient protocol for shoot regeneration was developed for sesame (Sesamum indicum L.) internodes using the transverse thin cell layer (tTCL) culture method. The frequency of shoot regeneration and the number of adventitious buds produced from regenerated shoots depend significantly on explant age, thickness of the tTCL sections, and the phytohormones supplemented to the culture medium. A combination of 6-benzyladenine (2.0 mg l⁻¹) and α-naphthaleneacetic acid (0.5 mg l⁻¹) was found to be the best phytohormone combination for shoot bud induction, with the maximum number of shoots obtained when the tTCL sections were 0.5–1.0 mm thick and derived from 4- to 6-week-old seedlings of sesame. Well-developed shoots were rooted on MS medium without phytohormones, and 80% of the regenerated plantlets were successfully established in soil.     

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 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.     

Transformation of <Emphasis Type="Italic">Liquidambar formosana</Emphasis> L. via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> using a mannose selection system and recovery of salt tolerant lines

Malaxis acuminata is a terrestrial orchid that grows in shady areas of semi-evergreen to shrubby forests. It is highly valued for its medicinal properties as dried pseudo-bulbs are important ingredients of several Ayurvedic preparations. In this study, adventitious shoot buds were induced from internodal explants of M. acuminata grown on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Of the three cytokinins used, TDZ at 3 mg l⁻¹ induced the highest frequency (82%) of organogenic explants. However, all responding explants produced only a single adventitious shoot irrespective of the type and concentration of the cytokinin. Adding 0.5 mg l⁻¹ α naphthaleneacetic acid (NAA) to the medium enhanced adventitious shoot formation. In the presence of 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA, frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant. Prolonged culture or subculture on the same medium did not promote further shoot production. However, transfer of these cultures to MS medium supplemented with 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA and various concentrations of different polyamines (PAs), including spermine, spermidine, and putrescine, significantly increased mean shoot number per explant. The highest frequency of shoot induction (100%) and mean shoot number per explant (14.6) was observed on MS medium with 3 mg l⁻¹ TDZ, 0.5 mg l⁻¹ NAA, and 0.4 mM spermidine. Regenerated shoots were excised and subcultured on an elongation medium consisting of MS medium with 3 mg l⁻¹ BA. Moreover, the highest frequency of rooting (96%) and mean number of roots per shoot (3.3) was observed on MS medium with 4 mg l⁻¹ indole-3-butyric acid (IBA) and 1.5 mg l⁻¹ activated charcoal (AC). Almost 90% of rooted shoots were successfully acclimatized and established ex vitro.     

Somatic embryogenesis and shoot organogenesis in the medicinal plant <Emphasis Type="Italic">Pulsatilla koreana</Emphasis> Nakai

We have developed a system for the in vitro regeneration of pasqueflowers (Pulsatilla koreana Nakai). The system was based on somatic embryogenesis and shoot organogenesis. Over a growth period of 6 weeks, multiple shoots were initiated from leaf, petiole, and pedicel explants on Murashige and Skoog (MS) medium containing 0.5 mg l⁻¹ indole-3-acetic acid (IAA) and zeatin (Zn), kinetin (Kin), or 6-benzyladenine (BA). We achieved 100% of adventitious shoot induced when petiole and pedicel explants were cultured on MS, 0.5–2.0 mg l⁻¹ Zn, and 0.5 mg l⁻¹ IAA. Somatic embryos developed from the explants and generated shoots on MS medium containing 0.25 mg l⁻¹ Zn and 0.5 mg l⁻¹ IAA. Globular and heart-shaped stages of somatic embryos were observed. Histological studies have revealed the stages of development of somatic embryos. For propagation and growth, the regenerated shoots from organogenic or embryogenic calluses were transferred to MS medium containing either (1) 1.5 mg l⁻¹ Zn and 0.05 mg l⁻¹ IAA or (2) 1.0 mg l⁻¹ BA and 0.05 mg l⁻¹ IAA. After the length of the shoots reached 3 cm, the shoots initiated by organogenesis as well as those initiated by somatic embryogenesis were transferred to the root induction medium. After 2 months of culture in half-strength MS with 1.5 mg l⁻¹ α-naphthalene acetic acid (NAA), the rooting ratio was 93%. Finally, the rooted plantlets were acclimatized in a mixture of mountain soil and perlite.     

Cytokinin-induced organogenesis in Lessertia (Sutherlandia) frutescens L. using hypocotyl and cotyledon explants affects yields of l-canavanine in shoots

A simple protocol for direct shoot organogenesis and plant regeneration in Lessertia frutescens using hypocotyl and cotyledon segments is reported. l-canavanine content in the derived shoots is also quantified. Media containing different concentrations and combinations of the cytokinins kinetin (K) and benzyladenine (BA) were tested for shoot induction potential. The best shoot regeneration rate (83%) was obtained from hypocotyl segments cultured in Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ K; these hypocotyls also produced the largest number of shoots per explant (3.5) and the longest shoots per explant (13.3 mm). The best shoot regeneration rate (46%) using cotyledons as explant material was obtained in MS medium supplemented with 1 mg l⁻¹ K and 1 mg l⁻¹ BA or with 5 mg l⁻¹ K and 0.5 mg l⁻¹ BA. The highest number of cotyledon-derived shoots (1.5) was obtained in MS medium containing 2 mg l⁻¹ K and 0.5 mg l⁻¹ BA, and the longest cotyledon-derived shoots (6.1 mm) were obtained in MS medium containing 1 mg l⁻¹ K and 0.5 mg l⁻¹ BA. Shoots derived from hypocotyls cultured on media containing 1 mg l⁻¹ K contained the highest quantity of l-canavanine (1.42 mg g⁻¹) relative to the control (0.52 mg g⁻¹). Shoots derived from cotyledons cultured on media containing 2 mg l⁻¹ K contained the highest quantity of l-canavanine (2.07 mg g⁻¹) compared to the control. Scanning electron microscopy revealed that shoots regenerated directly from the wounded epidermal tissue, although callus formation was observed in most cultures. Young shoot clusters proliferated into healthy adventitious shoots that were subsequently transferred directly onto rooting medium (MS medium containing 4 mg l⁻¹ indole-3-butyric acid), eliminating the need for an additional multiplication or elongation phase. The in vitro plants were successfully acclimatized in a growth chamber, achieving an 85% survival rate.     

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.     

High Frequency of Shoot Regeneration from Hypocotyls and Stem Segments of Antirrhinum majus (Snapdragon)

A broadly applicable direct shoot regeneration method from hypocotyls and stem explants has been developed for six cultivars of Antirrhinum majus L. In order to establish a stable and high frequency of shoot regeneration system, leaves, hypocotyls and stem explants of six cultivars were tested with 72 combinations of auxin (naphthaleneacetic acid (NAA) or 3-indoleacetic acid (IAA)) and cytokinin (6-benzylaminopurine (BA) or zeatin (Z)). A few adventitious shoots were directly regenerated from hypocotyl segments of cv. Orchid on MS medium with NAA + BA, IAA + BA, NAA + Z and IAA + Z. High frequency of direct shoot regeneration was obtained from hypocotyl segments on MS medium with 0.05, 0.1 or 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z and 0.5 mg l⁻¹ IAA + 2 mg l⁻¹ Z. Finally, stable and high frequency (92–100%) of shoot regeneration with more than 10 adventitious shoots per explant was achieved from the hypocotyls and stem explants of all six cultivars on MS medium with 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z. The shoots emerged directly from the hypocotyls and stem segments 4 weeks after culture initiation.     

Efficient regeneration and antioxidant potential in regenerated tissues of <Emphasis Type="Italic">Piper nigrum</Emphasis> L.

The organogenic potential and antioxidant potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper) were investigated. Callus induction and shoot regeneration were induced from leaf explants of potted plants cultured on MS medium supplemented with different plant growth regulators. The best callogenic response was observed on explants cultured for 30 days on MS medium supplemented with either 0.5 or 1.5 mg l⁻¹ 6-benzyladenine (BA) + 1.0 mg l⁻¹ α-naphthaleneacetic acid. Subsequent transfer of the callogenic explants onto MS medium supplemented with 1.5 mg l⁻¹ BA + 1.0 mg l⁻¹ gibberellic acid (GA₃) achieved 85% shoot organogenesis after 30 days of culture. The maximum number (7.2) of shoots/explant was recorded for explants cultured in MS medium supplemented with 1.0 mg l⁻¹ BA. Following the transfer of shoots to an elongation medium, the longest shoots (5.4 cm) were observed on MS medium supplemented with 1.0 mg l⁻¹ BA + 1.0 mg l⁻¹ GA₃. The elongated shoots were rooted on MS medium supplemented with different concentrations of indole butyric acid. An assay of the antioxidant potential of the in vitro-grown tissues revealed that the antioxidant activity of the regenerated shoots was significantly higher than that of callus and the regenerated plantlets.     

Regeneration and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of mature woody species <Emphasis Type="Italic">Photinia</Emphasis> × <Emphasis Type="Italic">fraseri</Emphasis> “Red Robin”

An efficient regeneration protocol for genetic transformation was developed from the leaves of an 11-year-old Phtinia × fraseri “Red Robin” tree. A high frequency of adventitious buds (88.63 ± 1.38%) and the highest maximum mean number of adventitious buds per explant (4.65 ± 0.48) were obtained in light conditions on Murashige–Skoog (MS) medium containing 2 mg l⁻¹ benzyladenine (BA) and 0.2 mg l⁻¹ α-Naphthaleneacetic acid (NAA). After preculturing for 6 days, over 95% of the shoots successfully rooted on 1/2 MS medium supplemented with 0.3 mg l⁻¹ indole-3-butyric acid (IBA) within 3 weeks. For genetic transformation, two crucial parameters (30 mg l⁻¹ kanamycin for selection and 30-min suspension time) were optimized. Taken together, a reliable transformation system with an efficiency of more than 5% was established. This genetic transformation protocol can be utilized for further genetic manipulation of the Photinia tree.     

Micropropagation with a novel pattern of adventitious rooting in American sweetgum (Liquidambar styraciflua L.)

Liquidambar styraciflua L. has great potential not only as an ornamental, but also for its commercial importance in pulp and paper production or biomass energy. This study was designed to evaluate the influence of plant growth regulators on adventitious shoot multiplication from shoot tips, and in vitro adventitious rooting. The morphogenic capacity of intact leaves grown in vitro was also assayed for adventitious shoot formation and aerial root development. The highest shoot multiplication rate of 5.9 shoots per explant was achieved with 0.7 mg l⁻¹ 6-benzylaminopurine plus 0.01 mg l⁻¹ indole-3-butyric acid. Thidiazuron, alone or in combination with 6-benzylaminopurine, did not significantly support higher shoot multiplication rates. The organogenic ability of the in vitro grown leaves was significantly lower and slower in comparison with shoot tips. Microshoots rooted readily after transfer to a half-strength woody plant medium supplemented with 0.5–0.7 mg l⁻¹ 1-naphthaleneacetic acid, and were then successfully acclimatised to an ex vitro environment. A novel pattern of adventitious rooting was observed from the aerial parts of microshoots which were not in contact with the medium, including the parenchyma cells of the leaf blades as well as stem nodes and internodes. The regenerated plants established in soil did not show any detectable morphological variation.     

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.     

Cryopreservation of embryogenic cell suspensions of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> L. (G) Don.

The present study describes the potential of in vitro grown adventitious roots of Hypericum perforatum L. commonly known as St. John’s wort at low nutrient and auxin levels in the liquid medium for micropropagation. Roots were regenerated from shoot-derived callus on MS medium containing 4.0 mg l⁻¹ Indole-3 acetic acid (IAA). IAA and Indole-3 butyric acid (IBA) were equally effective for the induction of roots from shoot cultures. Half strength MS medium containing 1.0 mg l⁻¹ IAA was most found suitable for culturing roots in liquid medium. A total biomass of 4.13 ± 0.67 g comprising 226 ± 34.4 shoots and shoot buds along with roots was obtained per culture starting with 200 mg roots inoculum. Pretreatment with kinetin (2.0 mg l⁻¹) enhanced the shoot multiplication. Shoots proliferated profusely from excised roots in static liquid medium supported with glass bead matrix. Growtek^™ vessel was found suitable and cost effective system for high throughput plantlet production. In vitro grown roots regardless of their source of origin were an excellent and easy to handle source of explant for aseptic production of plantlets without loosing the morphogenetic potential over the generations. The plants exhibited 84–99% similarity among themselves through RAPD. The in vitro shoots produced can either be multiplied or rooted perpetually, and alternatively they can also be explored for the in vitro production of hypericin and hyperforin.     

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.     

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.     

In vitro regeneration from cotyledon explants in figleaf gourd (<Emphasis Type="Italic">Cucurbita ficifolia</Emphasis> Bouché), a rootstock for Cucurbitaceae

An efficient plant regeneration system has been developed for figleaf gourd (Cucurbita ficifolia Bouché), which is exclusively used as a rootstock for cucumber. The protocol is based on results obtained from a series of culture experiments involving different parts of the cotyledons and various media. The culture of cotyledon explants was critical for the enhancement of shoot regeneration frequency. The lower parts of the cotyledon excised at the plumule base were found to display a markedly enhanced production of adventitious shoots compared to other cotyledon regions. Culture in silver nitrate-supplemented Murashige and Skoog (MS) medium was not beneficial for shoot regeneration and suppressed root regeneration. Efficient shoot regeneration was obtained on MS medium containing 1.0 mg l⁻¹ zeatin and 0.1 mg l⁻¹ indole-3-acetic acid. Regenerated shoots successfully elongated and rooted in medium containing 0.1 mg l⁻¹ 1-naphthaleneacetic acid after 10–15 days of subculturing. The plantlets were satisfactorily acclimatized in a greenhouse and grew into normal plants without any morphological alterations.     

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.     

In vitro plantlet regeneration system from rhizomes and mannose-binding lectin analysis of <Emphasis Type="Italic">Polygonatum cyrtonema</Emphasis> Hua.

Polygonatum cyrtonema Hua. lectins (PCLs) were extracted from plantlets regenerated from rhizome explants of P. cyrtonema. Rhizome explants demonstrated a high frequency of callus induction (72.5%) and adventitious shoots differentiation (83.7%) on Murashige Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid and 1.0 mg l⁻¹ 6-benzyladenine. The adventitious shoots could root readily on 1/2 MS medium + 0.5 mg l⁻¹ α-naphthaleneacetic acid and regenerate plantlets with a survival rate of 75.0%. Regenerated rhizomes were freeze-dried, macerated and prepared for total RNAs and proteins extraction. The PCL gene was cloned and its expression level was measured by RT-PCR. Western blot using a lectin-specific antibody revealed a similar amount in regenerated rhizomes compared to wild rhizomes, Furthermore, lectin derived from regenerated rhizomes retained its ability to haemagglutinate rabbit blood cells.     

An elite protocol for accelerated quality-cloning in <Emphasis Type="Italic">Gerbera jamesonii</Emphasis> Bolus cv. Sciella

A novel, efficient, and simple protocol was developed on in vitro mass propagation and acclimatization of Gerbera jamesonii Bolus cv. Sciella, an ornamental plant with attractive flowers. Shoot tip was used as the primary explant for in vitro establishment in which Murashige and Skoog (MS) medium supplemented with a low level of NAA (0.5 mg l⁻¹) and BAP (1.5 mg l⁻¹) promoted earliest axillary bud initiation within 5 d in 91.6% of the inoculants. Five axillary buds were initiated from a single explant within 13 d after inoculation. A very high rate of shoot multiplication (14 shoots per inoculated axillary bud) and proliferation was achieved when MS medium was fortified with a relatively higher level of BAP (2 mg l⁻¹) and 60 mg l⁻¹ ADS within 27 d of multiple shoot culture. A maximum number of well-developed roots per plant was observed in MS medium with 0.5 mg l⁻¹ IAA in the next 26 d. In the easy low-cost acclimatization process of 20 d, a combination of sand, soil, cow urine, and tea leaves extract (1:1:1:1; v/v) ensured 95% survival rate. Sixty-one well-acclimatized plants were obtained from a single shoot tip within 86 d. The sustained multiple shoot culture for 15 mo paved the way toward the conservation of genetic resources as well as beneficial economics. The clonal fidelity study of micropropagated and sustained cultured clones using ISSR primers ensured the continuous supply of quality propagules retaining genetic uniformity. The in vitro-generated plants performed better over conventionally propagated plants in the field condition.     

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.