Tissue culture of Hypericum brasiliense Choisy: Shoot multiplication and callus induction

Hypericum brasiliense, a non-domesticated plant has been shown to have useful medicinal properties. This plant has not been cultivated so a protocol for mass propagation based on selection of superior clones was developed and a protocol established for the culture of callus cells that could be used for in vitro metabolite production. A micropropagation method based on amplification of nodal buds was developed, by selection, from ten seedling clones that were examined for growth rate, multiplication rate and rooting. The effect of various basal media, growth regulator types and concentrations were examined for optimal callus induction. Optimal callus induction occured on either Murashige and Skoog's or Gamborg's media supplemented with 1 to 2 mg l^–1 of 2,4-dichlorophenoxyacetic acid.Abbreviations B5 Gamborg's medium - 2,4-Dscd 2,4-dichlorophenoxyacetic acid - IAA indolacetic acid - MS Murashige & Skoog's medium - NAA naphtaleneacetic acid     

Micropropagation of Capparis decidua (Forsk.) Edgew. — a tree of arid horticulture

A method for micropropagation of mature trees of Capparis decidua was developed. Multiple shoots were obtained from nodal explants on Murashige and Skoog's (1962) medium+0.1mgl^–1 NAA+5.0mgl^–1BAP+additives (50mgl^–1 ascorbic acid and25 mgl^–1 each of adenine sulphate, L-arginine and citric acid) at 28 ± 2°C, 12 h/dphotoperiod and 35–40 mol m^-2s^–1 photon flux density. The shoots were multiplied by (i) subculture of nodal shoot segments onto MS +0.1 mgl^-–1 IAA+1.0mgl^–1 BAPH+additives, and (ii) repeated transfer of original explant onto MS+ 0.1mgl^–1 IAA+mg l^–1 BAP+additives, at intervals of 3 weeks. Sixty to 70% of the shoots rooted when pulse treated with 100 mg l^–1 IBA in half strength MS liquid medium for 4h, and then transferred onto hormone-free half-strength agar-gelled MS basal saltmedium. Incubation in dark at 33 ± 2°C for 6d favoured root induction. In vitro hardened plants were transferred to pots.Abbreviations IAA Indole-3-aceticacid - IBA Indole-3-butyric acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - 2-ip Isopentenyl adenine - B₅ Gamborg et al. (1968) medium - MS Murashige and Skoog's (1962) medium - WP Woody plant medium (Lloyd and McCown 1981)     

Simple strategy for the in vitro conservation of Ipsea malabarica an endemic and endangered orchid of the Western Ghats of Kerala,India

A simple protocol was developed for plantlet regeneration from seedling leaf segments of pigeonpea cultivar ICPL 93115 through shoot organogenesis. Ten-day-old seedlings aseptically grown on MS medium were used for furnishing leaf segments. Initial incubation for 5 days in dark at 25±2 °C followed by transfer to 10/14-h light / dark cycle (12.1 mol photons m^–2 s^–1) favoured regeneration. The decisive role of 6-benzyl adenine at different concentrations was established on shoot organogenesis. Murashige and Skoog's (MS) (1962) medium fortified with BA of 5.0 mg l^–1 was optimum for shoot regeneration and MS + BA of 1.0 mg l^–1 for shoot elongation, while MS + IAA (1.0 mg l^–1) + kinetin (0.1 mg l^–1) showed good results for rooting.     

Clonal multiplication of Gardenia jasminoides Ellis through axillary bud culture

An efficient clonal multiplication system was developed for in vitro propagation of crocin — producing Gardenia jasminoides Ellis plants. Murashige and Skoog's (MS) medium containing 6-benzylaminopurine (BAP 1 mg l^–1) and indole-3-butyric acid (IBA 1 mg l^–1) resulted in multiple shoot initiation at the rate of 21 shoots per explant in 60 d of culture. Transfer of the microshoots into liquid MS medium supplemented with BAP (5 mg l^–1) with two subcultures of 15 d duration in the same medium resulted in 400 ± 25 shoots per explant. Efficient rooting was achieved in MS medium supplemented with -naphthaleneacetic acid (5 mg l^–1). The in vitro raised plants were hardened in a greenhouse and transplanted to the field successfully. The method described will be useful for rapid multiplication of Gardenia for commercial exploitation.Abbreviations MS Murashige and Skoog (1962) medium - BAP 6-benzylaminopurine - Kn kinetin - 2ip 6-(,-dimethylallylamino)purine - NAA -naphthalene- acetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid     

Somatic embryogenesis and plant regeneration in callus culture derived from immature seeds and mature zygotic embryos of Dysosma pleiantha (Hance) Woodson

Callus was induced on the wounded immature seeds and mature zygotic embryos of Dysosma pleiantha (Hance) Woodson (Berberidaceae) on a medium based on Murashige and Skoog's (1962) formula supplemented with 1 mg/l 2,4-dichlorophenoxy-acetic acid (2,4-D). Spontaneous embryoid formation occurred on the media containing low concentrations of 2,4-D (0.1–0.5 mg/l). These embryoids germinated in either MS or B5 medium containing 1 mg/l N⁶-benzyladenine and 1 mg/l gibberellic acid. The regenerated plantlets were successfully transferred to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - GA₃ gibberellic acid - MS medium Murashige and Skoog's (1962) medium     

In vitro propagation of Populus x wilsocarpa — a hybrid of ornamental value

Populus x wilsocarpa, a hybrid of important ornamental value, cannot be seed-propagated, nor grafted, since a compatible rootstock has not been identified. A micropropagation protocol consisting of a series of steps was therefore developed to facilitate the commercial production of this species. The technique involved the transfer of swelling buds to a growth initiation medium with the following composition: N6 macronutrients, MS micronutrients and vitamins supplemented with 0.5 mg l^-1 BAP. The best buds were from dormant twigs, stored at 0–2°C and then forced to burst prior to culture initiation. Shoot multiplication was on a basal WPM medium including 0.1 mg l^-1 BAP and 0.001 mg l^-1 NAA. Shoot elongation and rooting was also on a basal WPM medium supplemented with 1.0 mg l^-1 GA₃ followed by a transfer to a peat-perlite mix in the greenhouse.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellic acid (GA₃) - MS Murashige and Skoog [17] - NAA naphthaleneacetic acid - N6 medium [Chu et al., 7] - WPM woody plant medium [16]     

High frequency plantlets regeneration from seedling explants of Prosopis tamarugo

Callus cultures of Prosopis tamarugo Phil (Leguminosae, Sub family-Mimosoideae) were established from hypocotyls and cotyledons on MS medium supplemented with NAA (2.0 mg l^-1) and BAP (0.2 mg l^-1). Regeneration through various juvenile explants was obtained on hormone-free and high cytokinin containing Murashige and Skoog's medium. Multiple shoot buds formation was observed from the embryonic axis on MS medium incorporated with BAP (5.0 mg l^-1)). Elongation of shoot buds was observed on subsequent transfer to MS medium with BAP (1.0–2.5 mg l^-1) or without BAP. Explants containing apical meristem showed higher number of shoot formation at an early period. De novo shoot buds formation through callus morphogenesis was observed at the base of differentiated shoots on high cytokinin containing medium. All the manipulations of salt strength of MS, nitrogen, carbon, ascorbic acid and polyamines failed to induce organogenesis in isolated callus. In vitro produced shoots were rooted on MS medium supplemented with IBA or NAA singly or in combination.Abbreviations HC high cytokinin (BAP 5.0 mg l^-1) - BAP 6-benzyl amino purine - IBA indole-3-butyric acid - HF hormone free - NAA I-naphthalene acetic acid - MS Murashige & Skoog     

Somatic embryogenesis and in vitro flowering of 3 species of bamboo

Plant regeneration via somatic embryogenesis was achieved in callus cultures derived from nodal explants of in vitro grown seedlings and excised mature zygotic embryos of three bamboo species on Murashige and Skoog's (MS) basal medium supplemented with 0.5 mg/l kinetin (Kn), 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 10 mg/l adenine sulphate (Ads) and 3% (w/v) sucrose incubated in the light or in the dark. Somatic embryos germinated (95–98%) into normal plants and were transferred to soil with 95% success. In vitro flowering was induced on shoots developed from nodal explants taken from somatic embryo regenerated plants of Bambusa vulgaris, Dendrocalamus giganteus and Dendrocalamus strictus on half-strength MS basal medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA), 0.5 mg/l Ads, 0.5 mg/l gibberellic acid (GA₃) and 3% sucrose.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - Ads adenine sulphate - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - GA₃ gibberellic acid     

Micropropagation of wild beet (Beta maritima) from inflorescence pieces

In order to investigate the regeneration of wild beet (Beta maritima) from inflorescence pieces, the effects of growth regulator, genotype, explant source and stage of plant development on adventitious shoot formation and rooting in vitro and subsequent transplanting in the glasshouse were tested. Inflorescence tips produced more adventitious shoots than sub-apical segments and the best micropropagation was achieved on a Murashige and Skoog (MS) medium supplemented with 1.0 mg l^–1 BAP. Addition of auxin was not beneficial. The induction rate of adventitious shoots was genotype-dependent and influenced by the stage of plant development. Adventitious shoots were produced from the base of the flower buds, i.e. from the receptacle, not from axils or stalks and only a few buds on inflorescence tip explants produced adventitious shoots. Rooting was increased by using a MS medium with 3% sucrose supplemented with 1.0 mg l^–1 NAA. There was no variation in leaf morphology of the transplants. This work shows that inflorescence tips can be used successfully as explants for in vitro multiplication of sugar beet and wild beet.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

In vitro micropropagation of elite rosewood (Dalbergia latifolia Roxb.)

Induction of single and multiple shoots was obtained from nodal expiants of 60–80 year-old elite trees of rosewood on Murashige and Skoog's basal medium supplemented with 6-benzylaminopurine (1.0 mg 1^-1) and -Naphthalene acetic acid (0.05 mg 1^-1) or indole acetic acid (0.5 mg 1^-1). Multiplication of shoots was obtained on MS (reduced major elements) or Woody Plant Medium supplemented with 6-benzylaminopurine (1.0 mg 1^-1) and kinetin (0.5–1.0 mg 1^-1). Excised shoots were rooted on half-strength MS with IBA (2.0 mg 1^-1) to obtain complete plantlets. The regenerated plantlets have been acclimatized and successfully transferred to the soil.Abbreviations MS Murashige and Skoog's (1962) medium - B5 Gamborg (1968) medium - WPM Woody plant medium, Lloyd and McCown (1981) medium - NAA -naphthalene acetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzyl aminopurine - KIN kinetin - PVP polyvinyl pyrrolidone - CH casein hydrolysate - ADS adenine sulphate - L-Gl L-glutamine - L-Arg L-arginine - L-Asp L-asparagine - PG phloroglucinol     

One step shoot tip multiplication and rooting of Digitalis thapsi L.

Shoot tips from seedlings of Digitalis thapsi L. were cultured on Murashige and Skoog's medium and the effect of various auxins (2,4-D, NAA and IAA) were analyzed alone or in combination with cytokinis (BA and kinetin). Shoot multiplication and direct rooting of the new shoots were obtained after four weeks of culture in MS medium without hormones, but callus formation and the appearance of abnormal phenotypes were frequent. The addition of auxins to the cultures prevented the formation of callus but not the appearance of variant phenotypes. Both drawbacks could be avoided by combination of NAA or IAA with BA or kinetin. The best results for shoot multiplication and direct rooting were obtained with 0.5 mg l^-1 NAA and 0.1 or 0.5 mg l^-1 kinetin.Abbreviations BA 6-benciladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - NAA naphtalene acetic acid - MS Murashige and Skoog     

Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl

Summary Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l^–1 2,4-D and 2 g l^–1 casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1–3×10⁷/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l^–1 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×10⁵/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l^–1 each of NAA and BA for 2 months followed by 0.01 mg l^–1 NAA and 5 mg l^–1 BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - f. wt fresh weight - MES 2-(N-morpholino)-ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PE plating efficiency     

Micropropagation of Spilanthes acmella Murr.

Multiple shoots of Spilanthes acmella Murr. were induced from hypocotyl segments obtained from 1-week-old seedlings on Murashige and Skoog's (MS) medium containing benzyladenine (BA), isopentenyl adenine, and naphthaleneacetic acid (NAA). High frequency shoot proliferation (95 %) and maximum number of shoots per explant (10 ± 0.6) were recorded with 0.5 mg dm^–3 BA in combination with 0.1 mg dm^–3 NAA. A proliferation was achieved by repeatedly subculturing the nodal segments on shoot multiplication medium. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing indole-3-butyric acid (1.0 mg dm^–3). 95 % of the plantlets were successfully acclimatized and established in soil. Transplanted plantlets showed normal flowering without any morphological variation.     

Regeneration of haploid plants from isolated microspores of asparagus (Asparagus officinalis L.)

High percentages of micro-calli and micro-derived embryos were produced from isolated asparagus microspores at late uninucleate stage on MS liquid medium supplemented with 1.0 mg l^–1 2,4-D and 0.5 mg l^–1 BA. Two types of calli, namely compact callus (CC) and loose callus (LC), were found. Plantlets were regenerated via organogenesis, when these calli were transferred onto MS solid medium supplemented with 1.0 mg l^–1 BA and 0.2 mg l^–1 IBA 6 weeks. Embryos were produced from liquid cultured microspores, or from solid cultured micro-calli. The frequencies of haploid plant production from organogenesis and embryogenesis were compared. Effects of plant growth regulators on callus production, plantlet regeneration, and haploid plant production were tested. The combination of BA 1.0 mg l^–1 and IBA 0.2 mg l^–1 resulted the highest precentage of haploid plant production (7.7% from CC, 4.3% from LC).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA 3-indolybutyric acid - BA 6-binzyladinine - NAA naphtalene acetic acid - MS Murashige and Skoog     

Effects of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. f.) Merrill.

A procedure for rapid in vitro multiplication of Tylophora indica (Burm. f.) Merrill., an important indigenous medicinal plant, has been developed. Addition of ascorbic acid was essential to induce sprouting of axillary buds. Optimum multiplication was observed on MS medium containing 6-benzylamino purine (5.0 mg l^–1), -naphathalene-acetic acid (0.5 mg l^–1) and ascorbic acid (100 mg l^–1). Rooting of in vitro produced shoots was readily achieved with indole-3-acetic acid alone (1.0 mg l^–1) in MS. The plantlets thus obtained were successfully transferred to pots in large numbers which grew normally.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - Kn kinetin - MS Murashige & Skoog media - NAA -naphthalene acetic acid     

Micropropagation of Morus laevigata Wall. from mature trees

Multiple shoots were obtained from nodal explants of 10-year-old tree of Morus laevigata on Murashige and Skoog's medium supplemented with different concentrations (0.5–5.0 mg.l^–1) of benzyladenine (BA). Nodal segments taken from in vitro proliferated shoots gave further multiple shoots when cultured on the same basal medium containing 2.5 mg.l^–1 BA. Repeated subculture resulted in rapid shoot multiplication at the average rate of 6-fold per subculture. In vitro raised shoots rooted on MS medium containing 0.1 mg. l^–1 each of 3-indolebutyric acid (ISA) and -naphthaleneacetic acid (NAA). The regenerated plantlets were successfully established in soil under field conditions after a few days of indoor acclimatization.     

Organogenesis and embryogenesis inHelianthus tuberosus and in the interspecific hybridHelianthus annuus ×Helianthus tuberosus

A method of plant regeneration from cotyledons ofHelianthus tuberosus, Helianthus annuus ×Helianthus tuberosus and for the backcross of the interspecific hybrids onH. annuus was developed. Induction of somatic embryogenesis and plantlet regeneration from anther culture of the interspecific hybridsH. annuus ×H. tuberosus is reported.Cotyledons were cultured on Murashige and Skoog basal medium (MS) supplemented with indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin) or N⁶-benzylaminopurine (BAP). Shoot regeneration occurred on most of the media tested, but the best results were obtained on media with a high concentration of cytokinins (BAP or kinetin: 4 mg l^–1) and lower concentration of auxin (IAA: 0.5–1 mg l^–1).Embryogenic callus and adventitious buds were initiated from only two anthers of the hybridH. annuus ×H. tuberosus cultured on the MS medium containing BAP (0.2 mg l^–1) and 1-naphtalenacetic acid (NAA: 0.1 mg l^–1). Prolonged culture of these embryogenic calli and buds on the original medium with successive subculture on MS basal medium without growth regulators resulted in embryo formation and shoot differentiation. The plantlets, after rooting, were established in soil.     

High frequency production of haploid embryos in asparagus anther culture

Summary A method for obtaining a high frequency of haploid asparagus embryos through anther culture was developed. Flowers collected from plants in the field in July, August and September 1990, for the genotype G203, were stored at 5°C for 24 h. Anthers were placed on Murashige and Skoog medium (MS) containing 500 mg l ^–1 casein hydrolysate, 800 mg l^–1 glutamine, 2 mg l ^–1 NAA, 1 mg l ^–1 BA and 5 % sucrose at 32 °C in the dark for three to four weeks to induce calli. Calli were then grown at 25 °C with a 16 h photoperiod for three to four weeks. Developing embryos and calli were transferred to embryo maturation medium, MS containing 6% sucrose, 0.1 mg l ^–1 NAA, 0.1 mg l ^–1 kinetin and 0.65 mg l ^–1 ancymidol, for four weeks. More than 50% of the recovered mature embryos germinated on MS containing l mg l ^–1 GA₃. Anthers with microspores at the late-uninucleate stage had the highest frequency of total and embryogenic calli formation, 40% and 15%, respectively. Each embryogenic callus usually produced 10–15 embryos. Aproximately 75 plants per 100 anthers cultured were recovered: 76% haploid, 22% diploid and 2% triploid. High temperature was critical for the induction of embryogenic callus.Abbreviations NAA naphthaleneacetic acid - BA 6-benzylaminopurine - MS Murashige and Skoog (1962)     

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

Micropropagation of Limonium cavanillesii Erben, a threatened statice, from inflorescence stems

Micropropagation of Limonium cavanillesii Erben, a threatened and endemic statice species from Valencia Community (Eastern Spain), was successfully achieved using inflorescence stem pieces as initial explants. Segments 20 mm long from basal parts of immature inflorescences and with axillary buds were cut, sterilised and established in vitro. Shoots obtained from indifferentiated buds were sectioned and then transferred to Murashige and Skoog (MS) medium with 2 mg l^–1 kinetin to provide a plant stock.Shoot multiplication was achieved on MS medium with different cytokinins. The best results for shoot formation were obtained with 2–5 mg l^–1kinetin, 5 mg l^–1 6---dimethylallylaminopurine or 0.1 mg l^–1 6-benzylaminopurine, without significant differences between them. High shoot rooting (80–85%) was obtained within four weeks with indolebutyric acid or indoleacetic acid (0.1 or 0.5 mg l^–1), and also on medium without plant growth regulators. Plant survival to hardened greenhouse conditions was 90% four weeks after plantlet removal from in vitro conditions.This protocol for micropropagation of Limonium cavanillesii is very useful for conservation purposes of endangered statice species, because by using inflorescence stem as initial material it is easier to establish aseptic cultures while preserving the mother plant.