Somatic embryogenesis of <Emphasis Type="Italic">Merwilla plumbea</Emphasis> (Lindl.) Speta

A simple and efficient system was developed for rapid somatic embryogenesis from leaf explants of Merwilla plumbea, a traditional but threatened medicinal plant in South Africa. Friable embryogenic callus (FEC) was obtained from leaf explants on embryogenic callus induction medium containing agar-solidified Murashige and Skoog (MS) salts and vitamins, 8.3 μM picloram, 2.3 μM thidiazuron (TDZ) and 20 μM glutamine. FEC was subsequently incubated in embryogenic callus proliferation medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.1 μM picloram for 7 days before it was transferred to liquid somatic embryo medium (SEM_L) containing MS medium supplemented with 0.4 μM picloram and 0.9 μM TDZ. In SEM_L supplemented with 150 mg L⁻¹ haemoglobin, 5.4–35.6 somatic embryos per settled cell volume of 500 mg FEC were obtained. These embryos were at globular to cotyledonary developmental stages. Embryo maturation, germination and plant formation rate was 94.4% following transfer of SEs to half-strength MS medium supplemented with 1.4 μM gibberellic acid. Plantlets transferred into soil acclimatized in the misthouse and established successfully in the greenhouse (100%). This is the first report on induction of Merwilla plumbea somatic embryogenesis. The protocol developed offers controlled vegetative propagation by alleviating extinction threats, ensures germplasm conservation and provides a system for physiological, biochemical, molecular and cellular studies of embryo development.     

Phenylpropanoid production in callus and cell suspension cultures of <Emphasis Type="Italic">Buddleja cordata</Emphasis> Kunth

Plant tissue cultures represent a potential source for producing secondary metabolites. In this work, Buddleja cordata tissue cultures were established in order to produce phenylpropanoids (verbascoside, linarin and hydroxycinnamic acids), as these metabolites are credited with therapeutic properties. Highest callus induction (76.4–84.3%) was obtained in five treatments containing 2,4-Dichlorophenoxyacetic acid (2,4-d: 0.45–9.05 μM) with Kinetin (KIN: 2.32, 4.65 μM), whereas highest root induction (79.6%) corresponded to the α-Naphthaleneacetic acid (9.05 μM) with KIN (2.32 μM) treatment. Verbascoside was the major phenylpropanoid produced in in vitro cultures (root, white and green callus) [66.24–86.26 mg g⁻¹ dry weight (DW)], while linarin and hydroxycinnamic acid production was low (0.95–3.01 mg g⁻¹ DW). Verbascoside and linarin production were improved in cell suspension culture (116 mg g⁻¹ DW and 8.12 mg g⁻¹ DW, respectively).     

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.     

Comparison of in vitro regeneration pathways in <Emphasis Type="Italic">Punica granatum</Emphasis> L.

When cotyledonary explants, excised from in vitro germinated seedlings, of pomegranate (Punica granatum L.) were incubated on solid Murashige and Skoog (1962) medium supplemented with 21 μM naptheleneacetic acid (NAA) and 9 μM 6-benzyladenine (BA), 80% of explants developed callus. A high frequency of shoot organogensis was obtained when explants were incubated on MS medium supplemented with 8 μM BA, 6 μM NAA, and 6 μM giberrellic acid (GA₃). However, adding 24 μM silver nitrate (AgNO₃) to this medium markedly enhanced shoot regeneration frequency (63%) and mean number of shoots per explant (11.26) and length of shoots (2.22 cm). Highest frequency of in vitro rooting, mean number of roots/shoot (4.32), and mean root length (2.71 cm) were obtained when regenerated shoots were transferred to half-strength MS medium supplemented with 0.02% activated charcoal. Well-rooted plantlets were acclimatized, and then transferred to soil medium. Moreover, when zygotic embryos of P. granatum, excised from seeds collected at 16 weeks following full bloom, were incubated on MS medium containing 30 g l⁻¹ sucrose, 15% coconut water, 21 μM NAA, and 9 μM BA, they developed the highest frequency of embryogenic callus, clumps with globular embryos, and mean number of both globular and heart-shaped embryos per callus clump. Subjecting zygotic embryo explants to six-week dark incubation period was essential for embryogenic callus induction, and these were subsequently transferred to 16 h photoperiod for further growth and development of somatic embryos. Germination of somatic embryos was observed when these were transferred to MS medium was supplemented with 60 g l⁻¹ sucrose.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">indica</Emphasis> rice cv. ADT 43

A reproducible and highly efficient protocol for Agrobacterium tumefaciens-mediated transformation of indica rice (Oryza sativa L. subsp. indica cv. ADT 43) was established. Prior to transformation, embryogenic callus were induced from mature seeds incubated on Linsmaier and Skoog (LS) medium supplemented with 2.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ thiamine-HCl. Callus, intact mature seeds, and other in vitro derived explants (leaf bases, leaf blades, coleoptiles, and root-tips) were immersed in a bacterial suspension culture of A. tumefaciens strain EHA 105, OD600 of 0.8, and co-cultivated on LS medium for 2 days in the dark at 25 ± 2°C. Based on GUS expression analysis, 10 min incubation time of explants on a co-cultivation medium containing 100 μM acetosyringone was optimum. Following β-glucuronidase (GUS) assay and polymerase chain reaction (PCR) analysis, transformants were identified. Stable integration of the transgene was confirmed in four putatively transformed T₀ plants by Southern blot analysis. The copy number of the transgene in these lines, one to two, was then determined. Among the observations made, necrosis of co-cultivated explants was a problem, as well as sensitivity of callus to Agrobacterium infection. Levels of necrosis could be minimized following co-cultivation of explants in a medium consisting of 30% LS and containing 10 g l⁻¹ (14), polyvinyl pyrrolidone, 10% coconut water, and 250 mg l⁻¹ timentin (15:1). This latter medium also increased the final transformation efficiency to 15.33%.     

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.     

High-frequency shoot regeneration from leaf explants through organogenesis in bitter melon (<Emphasis Type="Italic">Momordica charantia</Emphasis> L.)

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature leaf explants of Momordica charantia, a very important vegetable and medicinal plant. Calluses were induced from immature leaf explants excised from in vitro (15-day-old seedlings) mature leaf explants of vivo plants (45 days old). The explants were grown on Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l⁻¹ sucrose, 2.2 g l⁻¹ Gelrite, and 7.7 μM naphthalene acetic acid (NAA) with 2.2 μM thidiazuron (TDZ). Regeneration of adventitious shoots from callus (30–40 shoots per explant) was achieved on MS medium containing 5.5 μM TDZ, 2.2 μM NAA, and 3.3 μM silver nitrate (AgNO₃). The shoots (1.0 cm length) were excised from callus and elongated in MS medium fortified with 3.5 μM gibberellic acid (GA₃). The elongated shoots were rooted in MS medium supplemented with 4.0 μM indole 3-butyric acid (IBA). Rooted plants were acclimatized in the greenhouse and subsequently established in soil with a survival rate of 90%. This protocol yielded an average of 40 plants per leaf explant with a culture period of 98 days.     

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.     

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.     

<Emphasis Type="Italic">Bridelia stipularis</Emphasis>: a new source for anthocyanin production in vitro

The present study prospects Bridelia stipularis (L.) Blume as a new source of anthocyanins through leaf and internode explants-derived callus cultures. Murashige and Skoog (MS) medium fortified with 21.48 μM α-naphthaleneacetic acid was superior for callus growth. Of the different regimes, the anthocyanin production relied on synergic effects of plant growth regulators, pH, light, and carbon source. The calluses incubated in light on MS medium with 4% glucose containing 2.22 μM N⁶-benzyladenine (BA) and 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) at pH 3.5 yielded the highest amount (a mean of 0.42 mg g⁻¹ callus) of anthocyanins. Subsequent cultures of the calluses on the above medium yielded a stable production of anthocyanins. Medium containing glucose was superior to that with sucrose for anthocyanin formation. Kinetin was inhibitory to anthocyanin accumulation. Suspension cultures of MS medium containing 2.26 μM 2,4-D and 2.22 μM BA at pH 5.0 started excretion of anthocyanins into the medium on reaching to pH 4.4–4.6.     

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

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

Micropropagation of <Emphasis Type="Italic">Pueraria tuberosa</Emphasis> (Roxb. Ex Willd.) and determination of puerarin content in different tissues

Pueraria tuberosa, a medicinally important leguminous plant, yielding various isoflavanones including puerarin, is threatened, thus requiring conservation. In this study, fresh shoot sprouts of P. tuberosa, produced by tubers, were used as explants for in vitro micropropagation. Surface-sterilized nodal shoots were incubated on Murashige and Skoog (MS) medium supplemented with 8.88 μM benzyladenine (BA), 50 mg l⁻¹ ascorbic acid, and 25 mg l⁻¹ of each of citric acid and adenine sulphate. Cut ends of nodal stem segments rapidly turned brown, and cultures failed to establish. When 100 mg l⁻¹ ascorbic acid (ABA) and 25.0 mg l⁻¹ polyvinyl pyrrolidone (PVP) were added to the medium, explants remained healthy, and cultures were established. Bud-breaking of nodal stem explants resulted in multiple shoot formation. Shoots proliferated (35–40 shoots per culture vessel) on MS medium as described above, but supplemented with 4.44 μM BA and 0.57 μM indole acetic acid (IAA) and additives. After 4–5 passages, proliferating shoots exhibited tip-browning and decline in growth and multiplication. However, when shoots were transferred to fresh shoot proliferation medium supplemented with 2.32 μM kinetin (Kn), sustained growth and high rate of shoot proliferation (50–60 shoots per culture vessel) was observed. Shoots rooted when transferred to medium consisting of half- strength MS medium with 9.84 μM indole butyric acid (IBA) and 0.02% activated charcoal. Alternatively, individual shoots were pulsed with 984.0 μM IBA and transferred to glass bottles containing sterile and moistened soilrite. These shoots rooted ex-vitro and were acclimatized in the greenhouse. Plants were then analyzed for puerarin content using HPLC, and leaves showed maximum accumulation of purerarin.     

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

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

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of mature <Emphasis Type="Italic">Prunus serotina</Emphasis> (black cherry) and regeneration of transgenic shoots

A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced for 12 h with 200 μM acetosyringone for vir gene induction before leaf explant inoculation. Explants were co-cultured for 3 days, and then cultured on woody plant medium supplemented with 9.08 μM thidiazuron, 1.07 μM napthaleneacetic acid, 60 μM silver thiosulphate, 3% sucrose, plus 200 mg l⁻¹ timentin in darkness for 3 weeks. Regenerating shoots were selected 27 days after initial co-culture, on Murashige and Skoog medium with 3% sucrose, 8.88 μM 6-benzylaminopurine, 0.49 μM indole-3-butyric acid, 0.29 μM gibberellic acid, 200 mg l⁻¹ timentin, and 30 mg l⁻¹ kanamycin for five subcultures. After 5–6 months of selection, transformation efficiencies were determined, based on polymerase chain reaction (PCR) analysis of individual putative transformed shoots relative to the initial number of leaf explants tested. The transformation efficiency was 1.2%. Southern blot analysis of three out of four PCR-positive shoots confirmed the presence of the neomycin phosphotransferase and AG genes. Transgenic shoots were rooted (37.5%), but some shoot tips and leaves deteriorated or died, making acclimatization of rooted transgenic plants difficult. This transformation, regeneration, and rooting protocol for developing transgenic black cherry will continue to be evaluated in future experiments, in order to optimize the system for several mature black cherry genotypes.     

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.     

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.     

Development of an efficient tissue culture protocol for callus formation and plant regeneration of wetland species <Emphasis Type="Italic">Juncus effusus</Emphasis> L.

Wetland species mat rush (Juncus effusus L.) is an important economic plant, but no information is available regarding plant regeneration, callus induction, and its proliferation from in vitro seed grown plantlets. The present study investigates the effects of growth regulator combinations and medium innovation on tissue culture system of five mat rush varieties. Addition of N6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in Murashige and Skoog (MS) medium showed significantly positive effect on callus proliferation, plant regeneration, and its multiplication compared to the medium devoid of BA. The highest callus induction frequency (80.95%, 90.48%, 75.40%, 70.83%, and 83.33%) was observed in MS medium containing 0.5 mg L⁻¹ (2.2 μM) BA in Yinlin-1, Nonglin-4, Gangshan, Taicao, and Taiwan green, respectively. Various growth regulator combinations with successive subculture (medium replacement) were found essential to develop organogenic calluses and to regenerate shoots. The combination of 0.1 mg L⁻¹ BA (0.4 μM) and 2 mg L⁻¹ 2,4-D (9.0 μM) in MS medium was found best for callus proliferation for all the varieties under trial. The plant regeneration required two steps involving successive medium replacements as well as optimal hormonal balances. Successful plant regeneration (over 70%) was observed only by transferring the organogenic callus from regeneration medium I [MS medium containing 0.5 mg L⁻¹ BA (2. μM) and 1.0 mg L⁻¹ kinetin (KT; 4.6 μM)] to the regeneration medium II [MS medium containing 0.5 mg L⁻¹ BA (2.2 μM), 1.0 mg L⁻¹ KT (4.6 μM) and 3.0 mg L⁻¹ indoleacetic acid (IAA; 17.1 μM)]. Our results confirmed the importance of the ratio of auxin (IAA) to cytokinin (BA and KT) in the manipulation of shoot regeneration in J. effusus L. The maximum plant survival frequency and multiplication rates (90.97% and 5.40 and 94.23% and 8.25) were recorded in the presence of 0.5 mg L⁻¹ BA (2.2 μM) in the 1/2 MS multiplication medium for the varieties of Nonglin-4 and Taicao, respectively. About 100% survival rate was also observed for all the varieties in soil conditions. The efficient plant regeneration system developed here will be helpful for rapid micropropagation and further genetic improvement in J. effusus L.     

High frequency shoot organogenesis and somatic embryogenesis in juvenile and adult tissues of seabuckthorn (<Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> L.)

Seabuckthorn (Hippophae rhamnoides) is a multipurpose small tree with unique berries of high nutritional and pharmaceutical values. A clonally propagated plant originating from a 20-year-old tree of H. r. rhamnoides × mongolica hybrid cultivar Julia and seedling offspring of this cultivar were investigated regarding induction of shoot organogenesis in leaf explants and in roots of intact seedlings, and induction of direct somatic embryogenesis in explants from shoot tissue. The highest percentage of leaf explants showing shoot organogenesis was achieved (juvenile explants, 65%; adult explants, 75%) when incubated in Murashige and Skoog (MS) medium supplemented with either 4.5 μM of the phenylurea cytokinin thidiazuron (TDZ) or 2.25 μM TDZ plus 2.2 μM 6-benzyladenine (BA), for juvenile and adult explants, respectively, both supplemented with 0.53 μM α-naphthaleneacetic acid (NAA). Juvenile explants developed on average 18 shoots per explant in the MS medium supplemented with 4.5 μM TDZ, a four fold increase over those incubated on the medium supplemented with 2.25 μM TDZ and 2.2 μM BA. Adult leaf explants grown on medium containing 2.25 μM TDZ and 2.2 μM BA medium produced 12 shoots per explant, while those grown on medium containing 4.5 μM TDZ produced 5 shoots per explant. Shoot organogenesis was observed in roots of intact seedlings pre-cultured on plain medium lacking nutrients (PM) or woody plant medium (WPM) salts and then grown on WPM salts supplemented with 4.4 μM BA, 0.29 μM gibberrelic acid (GA3), and 57.0 μM indoleacetic acid (IAA). The number of shoots formed on each seedling root system was ten fold higher when the pre-culture was in WPM medium indicating a promoting effect of mineral nutrients in the pre-culture medium. Somatic embryogenesis was induced in both juvenile and adult leaf explants in 65 and 78% of the explants, respectively, in MS-based medium supplemented with 2.0 μM N-(2-Chloro-4-pyridyl)-N ¹-phenylurea (CPPU), 0.53 μM NAA and varying concentrations of BA. There was an interaction effect between MS salt strength and BA concentration. The most effective medium for inducing somatic embryogenesis in juvenile explants contained half strength MS salts and 2.2 μM BA and full strength MS salts and 13.2 μM BA for adult explants.     

Effects of sodium nitroprusside on callus induction and shoot regeneration in micropropagated Dioscorea opposita

The effects of sodium nitroprusside (SNP) on callus induction and shoot regeneration of Dioscorea opposite Thunb. have been studied. Application of 40 μM of SNP depresses accumulation of H₂O₂ in tuber explants of Dioscorea opposita markedly. Supplementation of 40 μM of SNP to the Murashige and Skoog medium with combinations of benzylaminopurine (3 mg dm⁻³) and naphthaleneacetic acid (0.5 mg dm⁻³) reduces the browning of explants and increases the frequency of callus induction from tuber explants significantly. The regeneration frequency of adventitious shoot shows a significant increase in the presence of SNP. Further analysis indicates that treatment with 40 μM of SNP results in significant decreases in catalase and peroxidase activity, while increasing the activity of superoxide dismutase. Supplementation with 40 μM of SNP also promotes the accumulation of non-enzymic antioxidants, including proline and glutathione. The effects on callus induction and shoot regeneration promoted by SNP were reversed by the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)- 4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide. These results indicate that the exogenously applied NO-donor SNP alleviates browning of tuber explants by reducing H₂O₂ accumulation, thereby promoting a higher in vitro proliferation frequency of D. opposita.     

Production of psoralen by in vitro regenerated plants from callus cultures of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L.

Psoralea corylifolia is an attractive, endangered annual producing various bioactive compounds of medical importance. This plant contributes to Indian pharmaceutical and cosmetic industries for the production of commercial medicines, Ayurvedic skin care ointments and soap. The influence of various plant growth regulators (PGRs) and additives on high-frequency rapid adventitious shoot regeneration from transverse thin cell layer (tTCL) hypocotyl explants of P. corylifolia was investigated. Organogenic callus was obtained in tTCL hypocotyl explants on Murashige and Skoog (1962) medium supplemented with 15 μM naphthaleneacetic acid (NAA) and 3 μM benzylaminopurine (BA). The highest adventitious shoot regeneration (107.5 shoots per explant) was achieved in culture when transferred to half-strength solid MS medium. The regenerated shoots were rooted and the plantlets successfully acclimatized in moistened (1/8-MS basal salt solution with 3 μM indole-3-butyric acid (IBA), 1 μM 2-isopentenyladenine (2iP) and 100 mg l⁻¹ Bavistin (BVN)); garden soil, farmyard soil and sand (2:1:1, v/v/v). The acclimatized plants produced flowers in the growth chamber. When planted in the field these plants set viable seed. The psoralen content in different tissues of ex vitro and naturally-grown plants was determined by high-performance liquid chromatography (HPLC). The highest psoralen content was recorded in seeds from naturally-grown (6.48 μg g⁻¹ DW) and ex vitro plants (6.46 μg g⁻¹ DW). This system can be used for rapid mass propagation of P. corylifolia, for conservation strategies, and to produce phytomedicines.