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.     

Effects of sucrose and pH levels on in vitro shoot regeneration from leaf explants of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> and accumulation of bacoside A in regenerated shoots

Brahmi (Bacopa monnieri) is an important medicinal plant mainly used for the treatment of neurological disorders and depression. Recent investigations revealed that bacoside A is major chemical component shown to be responsible for memory facilitating action of brahmi. The current investigation was carried out to assess the potential for increasing biomass and the concentration of bacoside A in the in vitro regenerated shoots by varying sucrose and pH levels of shoot regeneration medium. The leaf explants were cultured on the Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ kinetin (KN) and with varying concentrations of sucrose (0, 1, 2, 3, 4, 5 and 6% at pH 5.8) and pH (4.5, 5.0, 5.5, 6.0 and 6.5 with 2% sucrose) with the objective of verifying the effects of sucrose and pH level on shoot regeneration and to verify the accumulation of bacoside A in the regenerated shoots. The shoot biomass increased (150.50 ± 2.84 shoots per explant, fresh wt 6.31 ± 0.12 g and dry wt 250 ± 5.00 mg) on the medium supplemented with 2% sucrose and pH which was set at 4.5. The results of HPLC analysis indicate that increase in sucrose concentration (0, 1, 2, 3, 4, 5 and 6% at pH 5.8) lead to decrease in the bacoside A content (39.51, 22.43, 13.05, 12.17, 10.73, 9.56 and 8.93 mg g⁻¹ dry wt, respectively) in regenerated shoots. These findings provide evidence that stressful condition of inadequate supply of carbon elevated synthesis of bacoside A in brahmi shoots. However, 2% sucrose is found suitable for biomass accumulation. Therefore, medium supplemented with 2% sucrose and pH set at 4.5 was found suitable for both biomass (6.31 ± 0.12 g fresh wt and 250 ± 5.00 mg dry wt) and bacoside A accumulation (13.09 mg g⁻¹ dry wt).     

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.     

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.     

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.     

Callus induction and plant regeneration in <Emphasis Type="Italic">Dorem ammoniacum</Emphasis> D., an endangered medicinal plant

Dorema ammoniacum D. Don. (Apiaceae), a native medicinal plant in Iran, is classified as a vulnerable species. Root, hypocotyl, and cotyledon segments were cultured on Murashige and Skoog (MS) (1962) medium supplemented with either 2,4-dichlorophenyoxyacetic acid (2,4-D) or naphathalene acetic acid (NAA), at 0–2 mg l⁻¹, alone or in combination with either benzyladenine (BA) or kinetin (KN), at 0–2 mg l⁻¹ for callus induction. The best response (100%) was observed from root segments on MS medium containing 1 mg l⁻¹ NAA and 2 mg l⁻¹ BA. The calli derived from various explants were subcultured on MS medium supplemented with BA (1–4 mg l⁻¹) alone or in combination with NAA or indole-3-butyric acid (IBA), at 0.2 or 0.5 mg l⁻¹ for shoot induction. Calli derived from hypocotyl segments showed significantly higher frequency of plantlet regeneration and number of plantlets than the calli derived from root and cotyledon segments. Therefore, MS medium supplemented with 2 mg l⁻¹ BA and 0.2 mg l⁻¹ IBA produced the highest frequency of shoot regeneration (87.3%) in hypocotyl-derived callus. The optimal medium for rooting contained 2.5 mg l⁻¹ IBA on which 87.03% of the regenerated shoots developed roots with an average number of 5.2 roots per shoots within 30 days. These plantlets were hardened and transferred to the soil. The described method can be successfully employed for the large-scale multiplication and conservation of germplasm this plant.     

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

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

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.     

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.     

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.     

Direct Adventitious Shoot Formation from Apical Shoot Explants of Euphorbia tirucalli

The induction of adventitious buds from apical shoot explants of Euphorbia tirucalli was studied. On average, 10.5 adventitious buds were efficiently induced in a ring on the segment from one apical explant on MS (Murashige and Skoog) medium supplemented with 0.5 mg l⁻¹ thidiazuron and 0.5 mg l⁻¹ benzylaminopurine. The adventitious buds could develop into adventitious shoots during subsequent cultures on hormone-free MS medium. For rooting, shoot clumps were cultured on half-strength MS medium containing 0.2 mg l⁻¹ α-naphthaleneacetic acid or indole-3-butyric acid. All the rooted plants survived establishment in soil within 2 months.     

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.     

Micropropagation of a tropical fruit tree Spondias mangifera Willd. through direct organogenesis

An efficient in vitro propagation is described for Spondias mangifera Willd., a medicinally important tree, using nodal explants obtained from 4-week-old seedlings. The frequency of shoot regeneration from seedling node was affected by various concentrations of BAP and successive transfer of mother explant. MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium supplemented with 1.0 mg l⁻¹ of 6-benzylaminopurine (BAP) was optimal for shoot multiplication. Upon this medium, highest number of shoots (about 10.6) per explants was obtained after fourth subculture of mother explants. Half-strength MS medium containing IAA (1.0 mg l⁻¹) was most effective for rooting of shoots. Regenerated plantlets were successfully acclimatized and transferred into soil with 80–90% survival rate. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the mother plants. This is the first report on micropropagation of S. mangifera, which can be applied for further genetic transformation assays and pharmaceutical purposes.     

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.     

The effect of polyamines on the efficiency of multiplication and rooting of <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal and content of some withanolides in obtained plants

An efficient mass multiplication protocol was developed for Withania somnifera (L.) Dunal from nodal explants of field-grown plants on Murashige and Skoog medium (MS) supplemented with 6-benzyladenine (BA) [1.5 mg L^−l], indole-3-acetic acid (IAA) [0.3 mg L^−l] and with the addition of polyamine, spermidine (20 mg L^−l) (shoot multiplication medium). A total of 46.4 shoots were obtained from nodal explants and they were elongated in the same medium in a culture duration of 6 weeks. The elongated shoots produced roots in MS medium fortified with putrescine (20 mg L^−l) after 4 weeks, and all the rooted plants were successfully hardened and acclimatized with a survival rate of 100%. An average of 276 shoots (46 × 6) was produced when at least six nodal explants obtained from each of the 46 in vitro grown shoots were cultured by microcutting method in the same shoot multiplication medium. On an average, 12,696 plants could be produced from all the shoots (276 × 46) by microcuttings in a period of 7 months. HPLC revealed a significant increase in the quantities of withanolide A, withanolide B, withaferin A and withanone in the leaves, stems, and roots of in vitro regenerated plants compared to the field-grown parent plants. Ploidy analysis using flow cytometry revealed genetic stability of in vitro regenerated plants. This protocol will be useful for scale-up production of withanolides on commercial scale.     

Callus induction and high-efficiency plant regeneration via somatic embryogenesis in <Emphasis Type="Italic">Papaver nudicaule</Emphasis> L., an ornamental medicinal plant

We describe culture conditions for a high-efficiency in vitro regeneration system of Papaver nudicaule through somatic embryogenesis and secondary somatic embryogenesis. The embryogenic callus induction rate was highest when petiole explants were cultured on Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 0.1 mg l⁻¹ 6-benzyladenine (BA) (36.7%). When transferred to plant growth regulator (PGR)-free medium, 430 somatic embryos formed asynchronously from 90 mg of embryogenic callus in each 100-ml flask. Early-stage somatic embryos were transferred to MS medium containing 1.0 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA to germinate at high frequency (97.6%). One-third-strength MS medium with 1.0% sucrose and 1.0 mg l⁻¹ GA₃ had the highest frequency of plantlet conversion from somatic embryos (91.2%). Over 90% of regenerated plantlets were successfully acclimated in the greenhouse. Secondary somatic embryos were frequently induced directly when the excised hypocotyls of the primary somatic embryos were cultured on MS medium without PGRs. Sucrose concentration significantly affected the induction of secondary embryos. The highest induction rate (89.5) and number of secondary somatic embryos per explant (9.3) were obtained by 1% sucrose. Most secondary embryos (87.2–94.3%) developed into the cotyledonary stage on induction medium. All cotyledonary secondary embryos were converted into plantlets both in liquid and on semisolid 1/3-strength MS medium with 1.0% sucrose.     

Micropropagation of <Emphasis Type="Italic">Pinus massoniana</Emphasis> and mycorrhiza formation in vitro

A protocol was developed for the micropropagation of Pinus massoniana and mycorrhiza formation on rooted microshoots. Seedling explants were first cultured on Gresshoff and Doy (GD) medium supplemented with 6-benzyladenine (BA) alone or in combination with α-napthaleneacetic acid (NAA) to stimulate the formation of intercotyledonary axillary buds. The frequency of axillary bud induction was up to 97% on medium supplemented with 4.0 mg l⁻¹ BA and 0. 2 mg l⁻¹ NAA, and the average number of buds per explant reached up to 5.5 on medium with 4.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Axillary buds elongated rapidly after being transferred to half-strength GD medium containing activated charcoal (0.1% w/v). Shoot proliferation was achieved by cutting elongated shoots into stem segments and subculturing on GD medium containing 2 mg l⁻¹ BA and 0.2 mg l⁻¹ NAA. Root primordia were induced in 82% of shoots when transferred to half-strength GD medium containing 0.2 mg l⁻¹ NAA. Root elongation was achieved in a hormone-free GD agar medium or a perlite substrate. Rooted plantlets were inoculated with the mycelium of ectomycorrhizal fungus Pisolithus tinctorius and the formation of ectomycorrhiza-like structures was achieved in vitro.     

An <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation system using callus of <Emphasis Type="Italic">Zoysia tenuifolia</Emphasis> Willd. ex Trin

Zoysia tenuifolia Willd. ex Trin. is one of the most popularly cultivated turfgrass. This is the first report of successful plant regeneration and genetic transformation protocols for Z. tenuifolia using Agrobacterium tumefaciens. Initial calli was induced from stem nodes incubated on a Murashige and Skoog (1962) (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg l⁻¹ 6-benzyladenine (BA), with a frequency of 53%. Compact calli were selected and subcultured monthly on the fresh medium. Sixty-nine percent of the calli could be induced to regenerate plantlets when the calli incubated on a MS medium supplemented with 0.2 mg l⁻¹ BA under darkness. For genetic transformation, calli were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, about 12% of the callus explants produced hygromycin resistant calli on MS medium supplemented with 2 mg l⁻¹ 2,4-D, 1 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, 500 mg l⁻¹ cefotaxime after 8 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 0.2 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 46% of the resistant calli differentiated into shoots. Finally, all the resistant shoots were rooted on 1/2 MS media supplemented with 50 mg l⁻¹ hygromycin, 250 mg l⁻¹ cefotaxime. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. About 5% of the total inoculated callus explants produced transgenic plants after approximately 5 months. The procedure described will be useful for both, the introduction of desired genes into Z. tenuifolia and the molecular analysis of gene function.