Highly efficient <Emphasis Type="Italic">in vitro</Emphasis> adventitious shoot regeneration of peppermint (<Emphasis Type="Italic">Mentha</Emphasis> x <Emphasis Type="Italic">piperita</Emphasis> L.) using internodal explants

An in vitro regeneration system with a 100% efficiency rate was developed in peppermint [Mentha x piperita] using 5- to 7-mm-long second internode stem segments of 3-wk-old stock plants. Shoots developed at sites of excision on stem fragments either directly from the cells or via primary calluses. The optimal medium for maximum shoot initiation and regeneration contained Murashige and Skoog (MS) salts, B5 vitamins, thidiazuron (TDZ, 11.35 μM), ZT (4.54 μM), 10% coconut water (CW), 20 g l⁻¹ sucrose, 0.75% agar, adjusted to pH 5.8. A frequency of 100% shoot initiation was achieved, with an average of 39 shoots per explant. This regeneration system is highly reproducible. The regenerated plants developed normally and were phenotypically similar to Black Mitcham parents.     

Organogenesis of Phaseolus angularis L.: high efficiency of adventitious shoot regeneration from etiolated seedlings in the presence of N6-benzylaminopurine and thidiazuron

A step-wise procedure for the regeneration of fertile plants by organogenesis from cultures of the economically important Phaseolus angularis L., cultivars: KS-6, KS-7 and KS-8 using etiolated seedlings was established. Pre-culture of 5-day old seedling explants with MS (Murashige and Skoog (1962) Physiol Plant 15:473–493) + B₅-vitamins (Gamborg et al. (1968) Exp Cell Res 50:151–158) liquid medium containing either 5.0 μM TDZ or 5.0 μM BAP under dark condition was essential for organogenesis. Bud growth and shoot multiplication were stimulated by reducing the BAP concentrations from 5.0 to 2.5 μM after 3 weeks. The maximum frequency of shoot induction was 65.2% (33.8 ± 2.54 shoots/explant) in cultivar KS-8 followed by KS-7 34.6% (23.4 ± 1.91 shoots/explant) and KS-6 30.6% (21.2 ± 2.28 shoots/explant). The multiplied buds elongated after transferring to solid MSB₅ medium supplemented with 4.0 μM GA₃, 12.5 μM AgNO₃ and 0.4 μM IBA. Up to 98% rooting efficiency of was obtained when the shoots were pulse-treated with liquid medium containing 4.5 μM IBA for 10 min. The rooted plantlets were transferred to pots in the greenhouse, where they grew, mature, flowered and bared pod normally. The efficient shoot bud induction capability was found to be cultivar dependent. All the three cultivars tested formed multiple shoots. This efficient and rapid regeneration system may also be helpful for Agrobacterium- or particle gun-mediated transformation for this important legume crop.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of Indian Kino (<Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> Roxb.) using Thidiazuron

An efficient protocol was developed for micropropagation of an economically important timber-yielding multipurpose tree, Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes (CNs) derived from 18-d-old axenic seedlings on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.1–10 μM). The highest shoot regeneration frequency (90%) and maximum number (15.2 ± 0.20) of shoots per explant was recorded on MS medium amended with 0.4 μM TDZ. Continuous presence of TDZ inhibited shoot elongation. In the primary medium, TDZ-initiated cultures were transferred to the secondary medium supplemented with another cytokinin, 6-benzyladenine (BA), for shoot growth and elongation. Maximum (90%) shoot elongation with an average shoot length of 5.4 ± 0.06 cm was observed at 5 μM BA. To further enhance the number of shoots per explant, mother tissue was repeatedly subcultured on fresh shoot induction medium after each harvest of newly formed shoots. Thus, by adopting this strategy, an average of 44 shoots per explant could be obtained. About 65% of in vitro regenerated shoots produced a maximum number (4.4 ± 0.2) of roots per shoot by a two-step culture procedure employing pulse treatment and subsequent transfer of treated shoots to a low concentration of 0.2 μM indole-3-butyric acid along with phloroglucinol (3.96 μM). The in vitro-raised plantlets were successfully acclimatized first under culture room conditions, then to greenhouse with 70% survival rate.     

An improved micropropagation of <Emphasis Type="Italic">Spilanthes acmella</Emphasis> L. through transverse thin cell layer culture

An efficient and improved in vitro propagation system for Spilanthes acmella L. using transverse thin cell layer (tTCL) culture system was established. The frequency of shoot regeneration from tTCL nodal segments was affected by concentrations of plant growth regulators and orientation of the explant. MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium with 5.0 mg dm⁻³ BAP was optimal for shoot regeneration. Upon this medium, the explant inoculated in the upright orientation exhibited a high frequency of shoot regeneration (about 97%), and the highest number of shoots (31.5) per explant. The intact node (1.0–1.5 cm) cultured on the same medium had significantly lower shoot multiplication ability with only 4.5 shoots per responsive explant. As compared to BAP alone, the combination of BAP and Kin or NAA did not have positive effects on shoot multiplication from tTCL nodal segments. Rooting of shoots was achieved on growth regulator free full-strength MS medium. Plantlets were transplanted into soil with 90–100% survival rate.     

Micropropagation of <Emphasis Type="BoldItalic">Embelia ribes</Emphasis> Burm f. through proliferation of adult plant axillary shoots

Micropropagation of Embelia ribes was achieved through proliferation of axillary shoots obtained from mature plants. Nodal shoot segments, collected March–May, exhibited high-frequency (75%) shoot initiation when cultured on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ) at 1.13 μM and indole-3-butyric acid (IBA) at 0.49 μM. Subculture of sprouted shoots from the original explants on medium containing TDZ (1.13 and 0.45 μM) during the first and second subcultures was found essential for further shoot proliferation, while inhibition of shoot elongation by TDZ could be overcome by transferring shoot cultures onto MS medium containing 6-benzylaminopurine (BAP; 11.10 μM) for the third subculture. Treating the explants with an antioxidant mixture of 568 μM ascorbic acid, 119 μM citric acid, and 307 μM glutathione prior to inoculation, coupled with subculture at 2-wk intervals onto fresh medium, both helped to reduce browning of the explants and facilitated production of five to six shoots/explant. MS medium supplemented with BAP (4.44 μM) and IBA (0.49 μM) induced shoot multiplication, producing five to six shoots/explant with a shoot length of 3 to 4 cm over a 4-wk culture period. Shoots of 3 to 4 cm in length exhibited 100% rooting within 4 wk after transfer to media containing half the nutrient salt concentration of MS medium with 3.69 μM IBA. Ex vitro rooting in the greenhouse from the in vitro shoots treated with 4.93 μM IBA for 30 min exhibited 95% rooting in soilrite™ medium in a 4-wk period. About 85% of micropropagated plants were established successfully in root trainers. Three-month-old, hardened plants could further be successfully established in the field. In 1 yr, by using the above protocol, 3,200 plants could be produced from a single shoot and 2,700 could be established in the field.     

Micropropagation,berberine content and antitumor activity of Jeffersonia dubia (Maxim.) Benth et Hook

An efficient micropropagation protocol was developed for Jeffersonia dubia using sucker explants. High frequency of multiple shoot formation was induced when the sucker explants were cultured on Chu’s (N6) medium with different concentrations of thidiazuron (TDZ) plus 0.54 µM α-naphthaleneacetic acid (NAA). The maximum frequency of shoot formation (96.2 %) was obtained on N6 medium with 2.27 µM TDZ plus 0.54 µM NAA. The highest mean number of shoots per explant (13.6) was obtained in temporary immersion system using an immersion frequency of 30 s every 30 min. The highest frequency of rooting (100 %), number of roots per shoot (5.8), and root length (6.3) was observed in half-strength N6 medium supplemented with 2.69 µM NAA. The regenerated plantlets (30 days old) were successfully acclimatized in the greenhouse with 98 % survival rate. The berberine content and cytotoxicity were higher in in vitro-developed calli and shoots than in leaves of field-grown plants. The greatest content of berberine was found in shoots (1381 μg g⁻¹) followed by calli (1092 μg g⁻¹) and leaves of field-grown plants (92 μg g⁻¹). At 1000 μg mL⁻¹ concentration, growth inhibition rate of berberine, callus, shoot, and leaf (in vivo) extracts were 68.4, 57.1, 54.2, and 17.7 %, respectively.

     

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.     

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

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

Rapid in vitro multiplication and plant regeneration from nodal explants of Andrographis paniculata: a valuable medicinal plant

A rapid and efficient method for the large-scale propagation of a highly valuable medicinal plant, Andrographis paniculata Nees, through in vitro culture of nodal explants obtained from 15-d-old aseptic seedling has been developed. High frequency direct shoot proliferation was induced in nodal explants cultured on Murashige and Skoog’s medium supplemented with 6-benzylaminopurine (BAP). Amongst the various cytokinins tested (BAP, kinetin, thidiazuron and 2-isopentyl adenine), BAP proved to be the most effective. The shoot forming capacity of the nodal explants was influenced by the BAP concentration (1–12.5 μM), and the optimal response was observed at 10 μM BAP, which induced an average of 34 shoots in 94% of the cultures within 4 wk. Significant differences were recorded in terms of average number of shoots per explant (8.6–34.1) among the different concentrations of BAP investigated. Concentrations of all cytokinins tested reach a level that can be considered above the optimum level, as marked by a reduced frequency of shoot proliferation. The multiple shoots obtained on various concentrations of BAP failed to elongate even after transfer to hormone-free MS medium. Elongation of the induced shoots was achieved on MS basal medium supplemented with 1.0 μM GA₃ within 2 wk. A proliferating shoot culture was established by repeatedly subculturing the original nodal explants on shoot multiplication medium after each harvest of the newly formed shoots. The explants retained their morphogenic potential even after three harvests. Therefore, in 90 d, about 60–70 shoots were obtained from a single nodal explant and the nodal explants from primary shoots further regenerated equivalent number of shoots, depicting their high frequency regeneration potential in A. paniculata. Rooting was best induced in 94% of shoots cultured on MS medium supplemented with 2.5 μM indole-3-butyric acid (IBA), within a wk. The plantlets were successfully transferred to soil after hardening with a 92% survival rate. The system is rapid: the initiation of shoot buds to the transplanting of regenerants to soil is completed in 8–9 wk.     

Pretreatment in thidiazuron improves the in vitro shoot induction from leaves in Curculigo orchioides Gaertn., an endangered medicinal plant

Leaf regeneration via direct induction of adventitious shoots obtained from an endangered medicinal plant, Curculigo orchioides Gaertn. by pretreating with thidiazuron. C. orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Direct inoculation of leaf pieces on MS medium supplemented with various concentrations of BAP (2–8 μM) or TDZ (2–8 μM) alone or in combination with NAA (0.5 and 1.0 μM) produced low shoot induction both in terms of % response and number of shoots per explant. Hence, leaf explants were pretreated with 15, 25 or 50 μM thidiazuron (TDZ), for 6, 24 or 48 h with the aim of improving shoot regeneration from cultured explants. After pretreatment, explants were transferred to an agar solidified MS medium that was supplemented with BAP (4 μM), TDZ (6 μM), BAP (4 μM) + NAA (1.0 μM), TDZ (6 μM) + NAA (0.5 μM). Control explants were incubated directly on the medium without any pretreatment. The pretreatment of explants with 15 μM TDZ for 24 h significantly promoted the formation of adventitious shoots and the maximum response was observed on MS medium supplemented with 6 μM TDZ. In this medium, 96 % cultures responded with an average number of 16.2 adventitious shoots per explant. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when TDZ pretreated leaves were cultured onto MS medium supplemented with BAP or TDZ alone or in combination with NAA. The rooted plantlets were successfully transplanted to soil with 90% success. The present investigation indicated the stimulatory role of TDZ pretreatment in regulating shoot regeneration from leaf explants of C. orchioides.     

Liquid culture for efficient micropropagation of Wasabia Japonica (MIQ.) matsumura

Summary An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoort tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N⁶-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3±0.9 and 15.0±0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8±1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4±0.6 shoots per explant) or 10.0 μM kinetin (10.9±0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055±77 mg and 34.2±1.0 mm, respectively) were significantly higher than those derived from full-strength MS semisolid medium (437.6±17.3 mg and 15.4±0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after heardening.     

Micropropagation of Sesbania rostrata from the Cotyledonary Node

Multiple shoots were induced from the cotyledonary nodes derived from seedling of Sesbania rostrata on Nitsch (1969; N) medium supplemented with various concentrations of benzyladenine (BA). 1 mg dm⁻³ BA proved to be the best, eliciting 5.8 ± 1.0 shoots per explant in 100 % cultures. The elongation of shoots was best at 2.0 mg dm⁻³ BA. The shoot proliferation capacity increased to 7.5 shoots per explant following transfer of explants to the fresh shoot multiplication medium (MS + 1.0 mg dm⁻³ BA), after an initial incubation of 30 d. To further enhance number of shoots per explant an alternative strategy of cultivation of mother explant on fresh shoot multiplication medium after excision of shoots was adopted. Following the repeated harvesting of shoots an average of 33 shoots per explant could be obtained. The in vitro regenerated shoots produced roots when transferred to half-strength MS medium supplemented with 3 % sucrose and 1 mg dm⁻³ IBA. The developed plantlets were planted in the soil and transferred to the field after an acclimatization period of 3 – 4 months. These plants produced flowers and fruits in the field and exhibited the development of prominent and more organized stem nodules as compared to the in vivo raised plants of the same age. This revised version was published online in July 2006 with corrections to the Cover Date.     

Direct shoot organogenesis from hypocotyl explants of Jatropha curcas L. an important bioenergy feedstock

Multiple shoots were induced on hypocotyl segments reared from in vitro germinated seedlings of Jatropha curcas L., using Murashige and Skoog (MS) medium supplemented with N⁶‐benzyl adenine (BA) and kinetin (kin) either alone or in combination with indole‐3‐acetic acid (IAA). The combined treatment of 7.05 μm kin and 1.425 μm IAA resulted in maximum shoot production with an average shoot bud initiation of 12.1 per explant. The regenerated microshoots were transferred to root induction medium containing half‐strength MS salts supplemented with either indole‐3‐butyric acid (IBA) or IAA. Rooting of microshoots was best achieved on half‐strength MS medium supplemented with IBA (9.8 μm ). Rooted plantlets were subsequently acclimatized under green house condition and the plantlets showed 70% survival.     

In vitro propagation of cashew from young trees

Summary Regeneration of cashew (Anacardium occidentale L.) from shoot explants of young grafts of mature tree origin is described. Establishment of shoot cultures was affected by season of collection, source, and type of explant. Explants from young grafts established better than those collected from field trees, and nodal cuttings regenerated better than shoot tips. Maximum percentage bud break and minimum contamination was noticed when shoots were collected in dry months (January to May). Pre-conditioning of stock plants by hormonal spray with 6-benzyladenine (BA) and gibberellic acid (GA₃) and brief presoaking of shoots in BA had no significant effect on culture establishment. MS (Murashige and Skoog, 1962) medium with half-strength major nutrients, 2.74 mM l-glutamine, 87.6 mM sucrose, and 2.25 gl⁻¹ phytagel was ideal for culture initiation. Inclusion of 0.1% polyvinylpyrrolidone (PVP-360) in the media reduced phenolic exudation. Solidified media was superior to liquid medium. Sucrose/glucose as energy source was found essential in the medium and had significant effect on percentage bud break and shoot development. A repeatable axillary shoot-bud induction was obtained on the above basal medium containing thidiazuron (TDZ) alone and in combination with BA. TDZ at 0.45 μM was best for axillary shoot-bud proliferation (4.5 buds per shoot) with maximum response (100%). Bud elongation could be stimulated in multiple shoots on medium containing 116.8 mM sucrose. In vitro rooting on auxin media and pulsing microshoots in 10 mM naphthalenaacetic acid (NAA) was ineffective. Rooting inability was, however, overcome by a micrografting procedure.     

High frequency adventitious shoot induction and plant regeneration from leaves of statice

An efficient regeneration system for large-scale propagation of statice (Limonium altaica cv. Emille) was developed using leaves from mature plants. Leaf segments (5×5 mm sections) were cultured on Murashige and Skoog's medium supplemented with N⁶-benzyladenine (BA) and thidiazuron (TDZ) individually and in combination with indole-3-acetic acid (IAA) and α-naphthaleneacetic acid (NAA). Prolific direct adventitious shoot regeneration occurred on most of the media. The best response in terms of frequency of shoot regeneration (99.5%) and number of shoots per explant (112 shoots per explant) was observed on medium supplemented with 2.85 μM IAA and 1.14 μM TDZ. The shoots rooted easily on half strength MS medium and MS medium with indole-3-butyric acid. In vitro propagated plants could be transferred to soil with survival rates of more than 95%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of a <Emphasis Type="Italic">Casuarina</Emphasis> hybrid (<Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> L. × <Emphasis Type="Italic">Casuarina glauca</Emphasis> Sieber ex Spreng) following facilitated seed germination

A suitable protocol for micropropagation of Casuarina hybrid, Casuarina equisetifolia L. × Casuarina glauca Sieber ex Spreng (C. e. × C. g.), was developed. When seeds without seed coats were cultured on 4 germination media, the optimal seed germination percentage (91%) was obtained on 0.8% agar solidified water medium. Shoot multiplication was achieved by culturing 2-cm long epicotyls, excised from germinated seedlings, on MS (Murashige and Skoog 1962) basal medium supplemented with BA (6-benzylaminopurine) at 4.4, 8.8, 17.8 and 35.6 μM. The greatest percentage of axillary bud sproutings (87.5%), mean number of sprouts per explant (3.8), and shoot length (3.2 cm) were achieved on MS medium supplemented with 17.8 μM BA. MS medium supplemented with 4 different concentrations of IBA (indole-3-butyric acid) (4.3, 8.7, 13.0 and 17.4 μM) were used for rooting of in vitro grown shoots. The highest rooting percentage (65.6%), mean number of roots per explant (2.5) and mean length of roots per explant (1.6 cm) was achieved at 13.0 μM IBA. Rooted shoots grew well after transfer to a substrate of peat and pinebark (7:3) in the greenhouse.     

In vitro plant regeneration from seedling explants of Stereospermum personatum D.C.: a medicinal tree

Padar (Stereospermum personatum, family Bignoniaceae) is a well-known medicinal tree. Its complete regeneration occurred through shoot bud culture in vitro. The seeds germinated sequentially on plastic trays and polyethylene bags for 21 days served as explants source. Nodal segments from the seedlings were established on MS medium supplemented with 4.44 μM BA, in which 86.6% nodes showed shoot bud elongation. Then, nodal segments from the developed shoots were cultured on MS medium with several BA concentrations; best shoot multiplication was obtained with 0.44 μM BA. In a second experiment where PVP was added to proliferation medium, nodal segments from developed shoots produced maximum 2.78 shoots per node. The nodal segments showed shoot multiplication up to seventh subculture on. Finally, shoots were rooted on MS medium with 2.46 μM IBA. The plants transferred to net pots containing coco-peat were acclimatized in green house, where more than 80% plants survived and grew normally.     

Isolation,callus formation and plantlet regeneration from mesophyll protoplasts of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. f.) Merrill: an important medicinal plant

Protoplast culture and plant regeneration of an important medicinal plant Tylophora indica were achieved through callus regeneration. Protoplasts were isolated from leaf mesophyll cells and cultured at a density of 5 × 10⁵ protoplasts per gram fresh weight, which is required for the highest frequency of protoplast division (33.7%) and plating efficiency (9.3%). The first division was observed 2 d after plating and the second division after 4 d. Culture medium consists of Murashige and Skoog (MS) liquid medium with 4 μM 2,4-D, 0.4 M mannitol and 3% (w/v) sucrose with pH adjusted to 5.8. After 45 d of culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. The protoplast-derived microcalli were visible to the naked eye within 60 d of culture and reached a size of 0.2–0.4 mm in diameter after 90 d. Calli of 0.2–0.4-mm size were transferred to MS medium supplemented with 2,4-D (4 μM), 3% (w/v) sucrose and 0.8% (w/v) agar, formed friable organogenic calli (7-8 mm size) after 8 wk under incubation in normal light period supplemented with 200 μmol m⁻² S⁻¹ of day light fluorescent illumination. The calli were transferred to MS medium supplemented with thidiazuron (TDZ) (1–7 μM) and naphthalene acetic acid (NAA) (0.2–0.4 μM) for regeneration. The calli developed shoot buds after 3–4 wk, and the frequencies of calli-forming shoots varied from 5% to 44%. Optimum shoot regeneration occurred on MS medium supplemented with 5 μM TDZ and 0.4 μM NAA. On this medium, 44% cultures responded with an average number of 12 shoots per callus. Whole plants were recovered following rooting of shoots in 1/2 MS medium supplemented with 3 μM indole 3-butyric acid.     

Multiple shoot induction and callus regeneration in Sarcostemma brevistigma Wight & Arnott, a rare medicinal plant

An efficient micropropagation protocol based on multiple shoot induction and callus regeneration has been standardized in Sarcostemma brevistigma, a rare medicinal plant. The nodal cuttings were cultured on MS medium supplemented with BA (0.5–8 μM) or Kn (0.5–8 μM) alone or in combination with NAA (0.5–1.5 μM). Maximum multiple shoot induction was observed on MS medium supplemented with 4 μM BA. On this medium, 100% cultures responded with an average number of 11.3 shoots per explant. However, the average shoot length was limited to only 0.9 cm on this medium. The addition of 1 μM NAA along with 4 μM BA gave rise to an average number of 10.9 shoots with an average shoot length of 1.8 cm. Luxuriantly growing callus was obtained on MS medium supplemented with BA (5 μM) and 2,4-D (2 μM). The callus was subcultured on MS medium supplemented with BA (2–15 μM) or Kn (2–15 μM) alone or in combination with NAA (0.5–2 μM) for shoot organogenesis. Optimum callus regeneration was obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. On this medium, 100% cultures responded with an average number of 13.4 shoots per culture. The shoots obtained via multiple shoot induction and organogenesis were rooted on half-strength MS medium supplemented with NAA (1–7 μM) or IBA (1–7 μM). IBA was better than NAA in terms of both the percentage of cultures that responded and the average number of roots per explant. The rooted shoots were successfully transplanted to soil with 86% success. This standardized protocol will help to conserve this rare medicinal plant.