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

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

The effect of thidiazuron level on in vitro regeneration type and peroxidase profile in <Emphasis Type="Italic">Eucalyptus microtheca</Emphasis> F. Muell

The green twigs of 1-year-old Eucalyptus microtheca F. Muell seedlings were cultured on modified MS medium, supplemented with α-naphthalene acetic acid (NAA) and kinetin (Kin) hormones at 12 different concentrations. After 4 weeks, the combination of 1 mg l⁻¹ NAA + 1 mg l⁻¹ Kin induced the highest number of axillary shoots. Meanwhile, embryogenic calli were observed in media containing 4 mg l⁻¹ NAA + 0.5 mg l⁻¹ Kin, without any regeneration. The hormone treatments were followed by subculturing the twigs in different levels of thidiazuron (TDZ). The combination of 1 mg l⁻¹ NAA + 1 mg l⁻¹ Kin together with 0.01 mg l⁻¹ TDZ resulted in an increase of direct shoot, while higher amounts of TDZ led to adventitious shoot induction. Somatic embryogenesis was observed in the treatment containing 0.01 mg l⁻¹ TDZ + 4 mg l⁻¹ NAA + 0.5 mg l⁻¹Kin. The peroxidase (POD) band patterns in regenerated plantlets were investigated in order to determine the effect of different levels of TDZ on loci synthesis. A dimer locus, a tetramer locus and two epigenetic bands (a new band for NAA + Kin and the other for TDZ) were observed in the POD profiles. In case of low (0.01 mg l⁻¹ and 0.1 mg l⁻¹) levels of TDZ, one heterozygote allele was disappeared from dimer locus, while at higher TDZ levels, the dimer locus lost its stability and tetramer locus showed a high activity. Thus, POD allele patterns seems to be a feasible marker for different types of regeneration.     

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.     

High-frequency adventitious shoots regeneration from leaf of non-heading Chinese cabbage (<Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis>) cultured in vitro

The objective of the present work was selection of cultivar and suitable medium for regenerating shoots from leaf segments of non-heading Chinese cabbage. We evaluated six types of supplemented media with 2.0, 5.0 and 10.0 mg l⁻¹ 6-BA; 1.0 and 2.0 mg l⁻¹ TDZ; 0.1, 0.3, 0.5, 0.8 and 1.0 mg l⁻¹NAA; 3.0, 5.0 and 7.5 mg l⁻¹AgNO₃; 0.01 mg l⁻¹ 2–4, D and 4.0 mg l⁻¹ KT for shoot regeneration and six cultivars “Sanchidaye”, “Liuchuandasuomian”, “Qingyou 4”, “Liangbaiye”, “AiKang 5” and “Hanxiao F3”, furthermore for root formation three types of supplemented media with 0.2, 0.3, 0.5 mg l⁻¹ NAA, and for survival rate two types of base media: turf + vermiculite + manure (1:2:0.2) and soil + vermiculite (1:2). Culturing leaf segments on MS medium supplemented with 2 mg l⁻¹ TDZ; 0.5 mg l⁻¹ NAA and 7.5 mg l⁻¹ AgNO₃ gave the highest number of shoots per leaf segment (66) while roots were best formed on the medium supplemented with 0.2 mg l⁻¹ NAA. Survival rate was highest (61.6%) in the turf: vermiculite: manure (1:2:0.2) medium. The highest percentage of responding leaf segments, number of shoots per leaf segment, rooting percentage and survival rate were observed in “Liuchuandasuomian”. The plantlets were transferred to the soil and grown into mature plants in pots. These results could be used for preliminary selections of cultivars to transfer disease resistance (Bt) gene through agrobacterium in non-heading Chinese cabbage.     

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.     

Adventitious regeneration in blackberry (<Emphasis Type="Italic">Rubus fruticosus</Emphasis> L.) and assessment of genetic stability in regenerants

Adventitious shoot regeneration from leaves of blackberry cultivar Čačanska Bestrna was examined using 30 different combinations of treatment. Young, fully expanded leaves taken from in vitro proliferating shoots were cultured on Murashige and Skoog (MS) medium containing either N ⁶-benzylaminopurine (BAP) (2.0 mg l⁻¹) or thidiazuron (TDZ) (1.0 and 2.0 mg l⁻¹) alone, or either of them combined with indol-3-butyric acid (IBA), α-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) at different concentrations (0.1, 1.0 and 2.0 mg l⁻¹). Indirect shoot formation was observed in 12 different treatments, though the efficacy varied greatly among types and concentrations of plant growth regulators. TDZ at 1.0 mg l⁻¹, applied either alone or combined with IBA, was significantly more effective than BAP in inducing shoot regeneration. The highest regeneration rate (41.66%) was obtained on medium containing TDZ alone. Cytological, flow cytometry and isozyme analyses were used for screening of genetic stability/instability in regenerants. Cytological study, based on chromosome counts in root tip meristems, and flow cytometry analysis indicated that adventitious shoots of Čačanska Bestrna are tetraploid with 2n = 4x = 28 as well as those derived from axillary buds. However, a study conducted on the peroxidase patterns of the different blackberry regenerating lines showed differences between some lines and control plants (in vivo plants and micropropagated plants). These differences were visible with 3,3′,5,5′-tetramethylbenzidine (TMBZ) as hydrogenous donor for peroxidase detection.     

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.     

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 efficient protocol for micropropagation of lemon (<Emphasis Type="Italic">Citrus limon</Emphasis>) from mature nodal segments

The influence of the basal medium and different plant growth regulators on micropropagation of nodal explants from mature trees of lemon cultivars was investigated. Although the basal medium did not affect any of the variables, explants on DKW medium were greener. Several combinations of 6-benzyladenine (BA) and gibberellic acid (GA) were used to optimise the proliferation phase. The number of shoots was dependent on the BA and GA concentrations and the best results were obtained with 2 mg l⁻¹ BA and 1 or 2 mg l⁻¹ GA. Explants length was shorter with the higher BA concentrations and, in all genotypes, shoot length was greater with 2 mg l⁻¹ GA. The best results for productivity (number of shoots × the average shoot length) were obtained with 2 mg l⁻¹ BA and 2 mg l⁻¹ GA, although explants with chlorosis and narrow leaves were observed. The presence of BA and GA in the proliferation medium was essential for the explant multiplication but GA had a greater influence. The transfer of in vitro shoots to rooting media, containing different concentrations of indole butyric acid (IBA) and indole acetic acid (IAA) produced complete plantlets. Lemon shoots rooted well in all rooting combinations. The highest rooting percentages were obtained on media containing 3 mg l⁻¹ IBA alone or IBA in combination with 1 mg l⁻¹ IAA and on these media the highest numbers of roots were produced. The average root length was affected significantly by the IBA and IAA concentrations. Root length was greater when only 3 mg l⁻¹ IBA was used, and in this rooting medium explants had a better appearance, with greener and larger leaves. The success during the acclimatisation was close to 100% and the plantlets exhibited normal growth in soil under greenhouse conditions.     

In vitro regeneration and morphogenesis studies in common bean

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

Cyclic secondary somatic embryogenesis and efficient plant regeneration in camphor tree (<Emphasis Type="Italic">Cinnamomum camphora</Emphasis> L.)

An efficient protocol for secondary somatic embryogenesis in camphor tree is reported. Secondary somatic embryos (SSEs), initially obtained from the primary embryos of a nascent embryogenic culture in 2002, were proliferated and maintained for more than 4 yr via cyclic secondary somatic embryogenesis. Throughout this period, the embryo populations retained a high level of competence for plant regeneration. SSEs were produced on the surfaces of the cotyledons and radicular ends of maternal somatic embryos (MSEs). Histological observations of the various stages of secondary embryo development revealed four typical stages, namely, globular, heart-shaped, torpedo, and cotyledonary. The process of secondary embryogenesis continued in a cyclic way, with each newly formed embryo producing a subsequent generation of secondary embryos. In order to progress developmentally beyond proliferation cycles, cotyledonary embryos from one of embryogenic lines (L14) were cultured on Murashige and Skoog (MS) medium with 0.1–3.0 mg l⁻¹ abscisic acid (ABA) or 0.05–1.0 mg l⁻¹ thidiazuron (TDZ) in darkness for 2 mo to achieve maturation. Matured embryos were then transferred to MS-based germination medium containing either 0.1 mg l⁻¹ TDZ, 0.2 mg l⁻¹ indole-3-butyric acid (IBA), and 0.5 mg l⁻¹ 6-benzylaminopurine (BA) or 0.1 mg l⁻¹ TDZ and 0.2 mg l⁻¹ IBA and were cultured in light for germination. Over 50% of embryos matured in the presence of 0.5 mg l⁻¹ ABA were able to germinate with shoots and poor root system. Frequencies of embryos germinating normal shoots among different genotypes did not change significantly. A total of 93% of the shoots from the germinated embryos converted to plantlets on half strength MS medium with 0.5 mg l⁻¹ IBA by 3 wk. Plantlets acclimatized successfully to ex vitro conditions and developed as field-grown plants with normal appearance.     

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.     

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.     

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.     

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

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

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.     

High frequency direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood (Populus deltoides)

Simple, reproducible, high frequency, improved plant regeneration protocol in Eastern Cottonwood (Populus deltoides) clones, WIMCO199 and L34, has been reported. Initially, aseptic cultures established from axillary buds of nodal segments from mature plus trees on MS liquid medium supplemented with 0.25 mg l⁻¹ KIN and 0.25 mg l⁻¹ IAA. Nodal and internodal segments were found to be extra-prolific over shoot apices during course of aseptic culture establishment, while 0.25 mg l⁻¹ KIN concentration played a stimulatory role in high frequency plant regeneration. Diverse explants, such as various leaf segments, internodes, and roots from in vitro raised cultures, were employed. Direct plant regeneration was at high frequency of 92% in internodes, 88% in leaf segments, and 43% in root segments. This led to the formation of multiple shoot clusters on established culture media with rapid proliferation rates. Many-fold enhanced shoot elongation and growth of the clusters could be achieved on liquid MS medium supplemented with borosilicate glass beads, which offer physical support for proliferating shoots leading to faster growth in comparison to semi-solid agar or direct liquid medium. SEM examination of initial cultures confirmed direct plant regeneration events without intervening calli. In vitro regenerated plants induced roots on half-strength MS medium with 0.15 mg l⁻¹ IAA. Rooted 5- to 6-week-old in vitro regenerated plants were transferred into a transgenic greenhouse in pots containing 1:1 mixture of vermicompost and soil at 27 ± 2°C for hardening and acclimatization. 14- to 15-week-old well-established hardened plants were transplanted to the field and grown to maturity. The mature in vitro raised poplar trees exhibited a high survival rate of 85%; 4-year-old healthy trees attained an average height of 8 m and an average trunk diameter of 25 cm and have performed well under field conditions. The regeneration protocol presented here will be very useful for undertaking genetic manipulation, providing a value addition to Eastern Cottonwood propagation in future.     

In vitro regeneration of brahmi shoots using semisolid and liquid cultures and quantitative analysis of bacoside A

The major objectives of this study were to investigate an efficient rapid protocol for mass propagation of adventitious shoots of brahmi using semisolid and liquid cultures; and to assess the amount of bacoside A accumulated in the regenerated shoots. Leaf explants were grown in vitro on Murashige and Skoog semisolid medium supplemented with 0.5, 1.0, 1.5, 2.0 and 2.5 mg l⁻¹ 6-benzyladenine or kinetin (KN) or thidiazuron (TDZ) for 4 weeks. Adventitious shoots developed from leaf explants on all cytokinin supplemented media. After 4 weeks of incubation, leaf explants were split into two batches and one set was subcultured on semisolid medium and another set in liquid medium containing same concentration of cytokinins where they have come from. Highest rate of shoot regeneration was observed for explants cultured on medium with 2 mg l⁻¹ KN. The fresh and dry weight of shoots was also highest with this treatment. Liquid cultures were found suitable for proliferation of shoots (155.6 shoots per explant) and they also favored highest biomass accumulation (8.60 g fresh and 0.35 g dry biomass). The bacoside A contents were determined in shoots using HPLC. Analysis revealed that, the contents were highest with shoots regenerated on medium supplemented with 2 mg l⁻¹ KN. The amount of bacoside A was highest in the shoots regenerated in liquid medium (11.92 mg g⁻¹ DW) and it was 2.2-fold higher compared to shoots grown on semisolid cultures.     

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.     

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.