In vitro propagation of Western Australian Rushes (Restionaceae and related families) by embryo culture. Part 1. In vitro embryo growth

This study outlines the first method for initiating micropropagation of a wide range of wild and commercially significant species of various Southern Hemisphere rushes (Restionaceae) by embryo culture. Most species showed best embryo growth on half-strength Murashige and Skoog basal medium, with the addition of gibberellic acid (1–5 M) and/or zeatin (1–5 M). Results showed there was a high level of variability in seed germinability as indicated by in vitro embryo growth (6–96%), with no apparent link to growth form, generic status or habitat preference. Embryo growth was achieved for several rare and/or restricted taxa (Loxocarya magna ined Lepidobolus contorta Meney & Dixon Hopkinsia anoectocolea (F. Muell.) Cutler, Lepidobolus contorta Meney & Dixon ined). The low germination of whole seeds of most species (average of 10%) indicates that whole seed germination under controlled conditions considerably understates the potential germinability of species of Restionaceae. In this context, embryo culture is useful for assessment of germinability and for initiating stock cultures for micropropagation.Abbreviations 1/2MS half-strength (minerals only) Murashige and Skoog (1962) basal medium - GA₃ gibberellic acid     

Plant regeneration from callus cultures of Piper longum L. by organogenesis

Plant regeneration from callus cultures of Piper longum was achieved through organogenesis. In vitro grown shoots were used as explants for callus induction. Competent callus was initiated around the nodal ring of tissue using Murashige and Skoog medium supplemented with 1.0 mg.l^–1- naphthaleneacetic acid and 0.2 mg.l^–1 N⁶-benzyladenine. Optimum growth regulator concentrations for shoot induction and shoot elongation were found to be 0.5 mg.l^–1 indole-3-acetic acid with 1.5 mg.l^–1 benzyladenine, and 0.1 mg.l^–1 indole-3-acetic acid with 0.2 mg.l^–1 benzyladenine, respectively. Elongated shoots were rooted on half-strength Murashige and Skoog medium having 0.1 mg.l^–1 indole3-acetic acid. The rooted plants were successfully established in soil.Abbreviations BA, N⁶ Benzyladenine - 2, 4-D 2, 4- dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - 2iP 2-isopentenyladenine - Kn Kinetin - MS Murashige and Skoog (1962) - NAA -Naphthaleneacetic acid     

In vitro shoot proliferation and plant regeneration from kurrat (Allium ampeloprasum var. kurrat) seedlings

Shoots were produced from kurrat seedling and mature plant explants cultured in Murashige and Skoog medium (MS) alone or supplemented with 4.4 M benzyladenine (BA). Shoots were also produced from explants through a two-step procedure. Regenerated shoots were induced to form roots on MS medium with 5 g I^-1 activated charcoal. Plants were successfully established in soil.Abbreviations AC activated charcoal - BA benzyladenine - MS Murashige & Skoog's (1962) medium - 2,4-d 2,4-dichlorophenoxyacetic acid     

Micropropagation of mature siberian elm in two steps

A simple and reliable two-step micropropagation system was developed for mature Siberian elm (Ulmus pumila L.). Shoot-tip cultures were initiated and multiplied on a modified Murashige and Skoog medium supplemented with 1 M benzyladenine, B5 vitamins, solidified with 0.22% Gelrite, and subcultured weekly. Proliferated shoots 2–3 cm in length rooted successfully in sterile and non-sterile soil mix at 100% efficiency. Healthy plants were acclimatized to the ambient environment.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine - IBA indole-3-butyric acid - MS Murashige & Skoog medium (1962)     

Somatic embryogenesis from leaf of witloof chicory through suspension culture

A procedure for regeneration of somatic embryogenesis from witloof chicory leaf has been developed. Explants were taken from the distal third part of leaf vein and cultured on Murashige and Skoog medium (MS) containing 100 mg/1 casein hydrolysate 1.3 M 2,4-D, and 1.3 M kinetin. A pale yellowisl nodular callus was formed after 4 weeks which was maintained in the same medium for 8–12 weeks with one change to a fresh medium every 4 weeks. Callus was then suspended in the same medium without agar for 4–6 weeks with one change to a fresh medium every 2 weeks. Embryo-like structures appeared upon transfer to liquid MS containing 1.8 M benzyladenine. Embryo germination was accomplished in half strength MS medium with or without 1 g/l activated charcoal.Abbreviations AC activated charcoal - BA benzyladenine - MS Murashige & Skoog (1962) medium     

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH₄NO₃ and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - BM basal medium - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - Kn kinetin - MS Murashige & Skoog - NAA naphthaleneacetic acid     

In vitro multiplication ofVanilla planifolia using axillary bud explants

A clonal propagation method has been developed for efficient multiplication ofVanilla planifolia. Multiple shoots were developed from axillary bud explants using semi-solid Murashige and Skoog (MS) medium supplemented with N⁶-benzyladenine (BA, 2 mg l^–1) and -naphthaleneacetic acid (NAA, 1 mg l^–1). The multiple shoots were transferred to agitated liquid MS medium with BA at 1 mg l^–1 and NAA at 0.5 mg l^–1 for 2–3 weeks, and subsequently cultured on semi-solid medium. Using this method, an average of 42 shoots were obtained from a single axillary bud explant over a period of 134 days. Use of an intervening liquid medium has been found to enhance multiplication of shoots inV. planifolia.Abbreviations BA N⁶-benzyladenine - DMRT Duncan's multiple-range test - KC Knudson (1946) medium - KCB KC basal medium - Kn kinetin - MS Murashige and Skoog (1962) medium - MSB MS basal medium - 1/2 MSB half-strength MSB - MS-D double-phase MS medium - MS-L liquid MS medium - MS-S semi-solid MS medium - NAA -Naphthaleneacetic acid     

In vitro somatic embryogenesis ofDalbergia sissoo Roxb. —a multipurpose timber-yielding tree

Somatic embryogenesis was achieved in callus cultures dervied from 40-day-old semimature zygotic embryos ofDalbergia sissoo on semi-solid Murashige and Skoog (MS) salts and vitamins supplemented with 0.46–1.16 M kinetin, 6.78–9.04 M 2,4-dichlorophenoxy acetic acid (2,4-D) and 30 g/1 sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to half-strength basal MS medium supplemented with 0.46-1.16 M kinetin and 6.78–9.04 M 2,4-D with 2% (w/v) sucrose. The light-green somatic embryos germinated on half-strength MS salts and vitamins supplemented with 0.5 mg/1 abscisic acid and 2% (w/v) sucrose. The developmental stages of somatic embryogenesis were studied by light and scanning electron microscopy.Abbreviations ABA Abscisic acid - BA 6-benzyladenine - Kn kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) basal medium     

The novel paclitaxel-producing system: establishment of Corylus avellana L. hairy root culture

Hazelnut (Corylus avellana L.), contains a valuable medicinal substance known as Paclitaxel®, which is one of the most effective anticancer drugs. The original plants produce negligible amount of paclitaxel; therefore, tissue culture techniques, especially hairy root culture, could be one of the most practical methods to enhance the amount of paclitaxel. The main goal of this study was to assess the induction of hairy roots in C. avellana. The effects of different strains of Agrobacterium rhizogenes including c58c1pRiA₄, K599, and 15834, and six culture media, MS (Murashige and Skoog), half-strength MS, quarter-strength MS, WPM (woody plant media), half-strength WPM, and quarter-strength WPM, were evaluated. The results showed that the maximum amounts of the rooted explants were obtained with c58c1pRiA4 strain in quarter-strength WPM medium. The investigations of explant type (leafstalk, petiole, lamina, and stem) and different propagation media (quarter-strength WPM, half-strength MS, and half-strength SH ((Schenk and Hildebrandt) medium) showed that the leafstalk was the most optimal explant for hairy root induction, and half-strength SH was the best culture medium for growth of the hairy roots in liquid medium. HPLC analyses confirmed the presence of paclitaxel (3.2 μg g⁻¹ (DW)) in hairy root extracts.

     

Multiplication of the endangered Indian pitcher plant (Nepenthes khasiana) through enhanced axillary branching in vitro

Axenic seedling-derived two- to three-node stem segments of Nepenthes khasiana Hook.f. were successfully cultured on Woody Plant Medium containing 2.2 M benzyladenine to produce a 0.5–1.5 cm axillary shoot from each node in 7–8 weeks. The rapid growth along with the axillary branching of this shoot enabled amassing of 6–12 shoots during subculture. Excised shoots transferred to basal medium or rooted in medium containing 2.7 M naphthaleneacetic acid produced typical pitchers at leaf tips. Rooted plants were established in pots at 90–95% survival rate.Abbreviations AA ascorbic acid - AC activated charcoal - CA citric acid - BA 6-benzyladenine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - KC Knudson-C (1946) basal medium - MS Murashige & Skoog (1962) basal medium - WPM Woody Plant basal medium (Lloyd & McCown 1980)     

Influence of tissue culture conditions on apogamy in Dryopteris affinis sp. affinis

The naturally-occurring apogamy of some ferns can be modified by culture conditions and growth regulators. Gametophytes of the apogamic fern Dryopteris affinis sp. affinis L., were cultured on Murashige and Skoog (MS) basal medium. Changes in concentration of MS medium components, sucrose, agar and different pH values were tested. The addition of benzyladenine (4.43 M) and naphthalene acetic acid (0.53 M) enhanced sporophyte proliferation on the gametophytes. After one month in culture, the gametophytes formed callus with a high morphogenic capacity. Culture of calli on medium without growth regulators yielded about 10,000 sporophytes per 1 g fresh weight of callus. This pattern of differentiation slowed with time to a point where only gametophyte regeneration was observed.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - F.W. fresh weight - MS Murashige & Skoog medium - NAA 1-naphthalene acetic acid - SE standard error     

Regeneration of the tropical legume Aeschynomene sensitiva Sw. from root explants

Regeneration of Aeschynomene sensitiva Sw. after callogenesis was obtained from small (2–5 mm long) root explants of 30-day-old seedlings aseptically cultivated on Murashige and Skoog medium supplemented with various concentrations of growth regulators. After 4 weeks, the best results were observed with 0.54 M -naphthaleneacetic acid and 2.22 M benzyladenine. On this medium, the rate of regeneration depended on seedling age and agar concentration. The highest number of shoots per explant was obtained with small cuttings from 30-day-old seedlings grown on a medium containing 8 g l^–1 of agar. Regeneration success was also dependent on explant size. When longer explants (7–20 mm) were cut from the main root, direct regeneration was obtained in two weeks. These cuttings also generated shoots through callogenesis in four weeks but always in lower quantities than with direct regeneration, whatever the seedling age. here also, the best regeneration was obtained with cuttings from 30-day-old seedlings maintained on a medium with 8 g l^–1 of agar. Regenerants were rooted on growth-regulator-free Murashige and Skoog medium and then acclimatized in a greenhouse. A better survival to transplantation was observed when plantlets were inoculated with the photosynthetic Bradyrhizobium strain ORS 278. Stem and root nodules developed on the inoculated plantlets and were able to fix nitrogen.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA - naphthaleneacetic acid - MS Murashige & Skoog (1962) medium     

Plant regeneration in vitro of South Pacific taro (Colocasia esculenta var. esculenta cv. Akalomamale,Aracea)

Summary Axillary bud expiants from South Pacific (Solomon Islands) taro, Colocasia esculenta var. esculenta cv. Akalomamale (Araceae) cultured on a modified Murashige-Skoog medium containing 1 mg NAA 1^–1 and TE formed callus and produced multiple plantlets. Explants died if NAA was present at levels lower than 0.1 mg 1^–1. BA was not required and may have been inhibitory. Plantlets developed faster and became larger following transfer to a hormone-free medium two weeks after the start of culture. Fully grown plants were established in a potting mix and are growing well in a greenhouse.Abbreviations BA benzyladenine - BM basal medium - Ca Colocasia esculenta var. antiquorum - Ce Colocasia esculenta var. esculenta - Ck cytokinin(s) - CW coconut water - HSMSM half strength Murashige Skoog macroelements - HSMS half strength Murashige and Skoog medium - IM initial medium(ia) - MS Murashige and Skoog medium - NAA naphthaleneacetic acid - SM second medium - TE taro corm extract - UCI University of California, Irvine     

Rapid in vitro multiplication of jujube through mature stem explants

Stem explants obtained from a mature tree of Ziziphus mauritiana Lamk were grown on modified Murashige and Skoog medium containing 3800 mg l^-1 potassium nitrate, 2475 mg l^-1 ammonium nitrate, 11 M benzyladenine and 0.5 M indole-3-acetic acid. During successive subcultures 15–20 shoots per inoculum were produced. Rooting was induced by pretreatment with 50 M indolebutyric acid or 1-naphthaleneacetic acid for 24 h followed by transfer to auxin-free White's medium. Plantlets grew well in a soil and vermiculite mixture.Abbreviations IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

Production of ajmalicine and ajmaline in hairy root cultures of Rauvolfia micrantha Hook f., a rare and endemic medicinal plant

Hairy roots of Rauvolfia micrantha were induced from hypocotyl explants of 2–3 weeks old aseptic seedlings using Agrobacterium rhizogenes ATCC 15834. Hairy roots grown in half-strength Murashige & Skoog (MS) medium with 0.2 mg indole 3-butyric acid l^–1 and 0.1 mg -naphthaleneacetic acid l^–1 produced more ajmaline (0.01 mg g^–1 dry wt) and ajmalicine (0.006 mg g^–1 dry wt) than roots grown in auxin-free medium. Ajmaline (0.003 mg g^–1 dry wt) and ajmalicine (0.0007 mg g^–1 dry wt) were also produced in normal root cultures. This is the first report of production of ajmaline and ajmalicine in hairy root cultures of Rauvolfia micrantha.     

In vitro high frequency plant regeneration from hypocotyl and root segments of spinach by organogenesis

An efficient protocol for spinach (Spinacia oleracea L.) plant regeneration from hypocotyl and root segments was established. When the sub-apical hypocotyl and tip-free root segments were cultured on Murashige & Skoog (1962)-based medium containing high concentrations of indole-3-acetic acid (85.62 M) and gibberellic acid (100 M), more than 75% and 90% of the hypocotyl and root explants, respectively, formed shoots. After elongation, more than 92% of the shoots rooted on medium supplemented with 2.85–5.71 M of indole-3-acetic acid. More than 70% of rooted plantlets survived in soil and were fertile. Significant interactions between growth regulator combinations, explant types and environmental conditions on shoot initiation, development and rooting were discussed.Abbreviations BA benzyladenine - BM Murashige & Skoog basal medium - B5 Gamborg et al. medium (1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - 2ip isopentenyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige & Skoog medium (1962) - NAA naphthaleneacetic acid - HS hypocotyl segments - RSS root segments of seedlings - RSV foot segments of in vitro plantlets     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

An unusual root tip formation in hairy root culture of Hyoscyamus muticus

Summary Hairy root cultures of Hyoscyamus muticus were established using Agrobacterium rhizogenes ATCC 15834. In one out of 8 clones established, an unusual root tip formation was observed after transfer of cultures from half-strength Murashige and Skoog (1962) to White's medium (1939). This phenomenon was associated with the production of a fine brownish cell suspension culture. Hairy root development resumed after transfer of the root tips from White to half-strength Murashige and Skoog medium. After plating the isolated brownish cells on hormone-free half-strength Murashige and Skoog or White solid medium, callus proliferation was observed, and then redifferentiation of hairy roots occurred. The polymerase chain reaction analysis of the H. muticus hairy root (clone Z2) revealed that only the tl region of the T-DNA was integrated. The growth and the production of five tropane alkaloids by this clone were examined.Abbreviations PCR Polymerase Chain Reaction - MS medium Murashige and Skoog Medium - 1/2 MS medium half-strength MS medium - WP medium Woody Plant medium - RC medium Root Culture medium - WH medium White medium - HPLC High Performance Liquid Chromatography - wt. weight     

A three media transfer system for direct somatic embryogenesis from leaves ofSenecio x hybridus Hyl. (Asteraceae)

Bisected leaves were cultured on semi-solid Murashige & Skoog medium amended with 13.5 M 2,4-D and 4.5 M BA for 7–14 days, transferred to 0.5% activated charcoal medium (without growth regulators) for three days, then transferred to MS basal medium. Control explants remained on initiation medium for comparison to transferred explants. Twenty-eight days after initiation of cultures, explants exposed to 11–14 days on induction medium yielded the largest number and most developmentally advanced embryos. Mature, white somatic embryos from transferred explants were visible after 35 days. Conversely, somatic embryos on control explants were not morphologically mature until 2–4 months. Mature embryos from transferred explants germinated without a resting stage, whereas embryos from control explants appeared quiescent. Histological examination confirmed embryo anatomy, however embryos, regardless of treatment, had abnormal cotyledons and regenerated plants had multiple stems.Abbreviations AC activated charcoal - BA 6-benzylaminopurine - CrAF III chromium trioxide, acetic acid and formaldehyde - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog medium - NAA 1-naphthalenacetic acid     

In vitro organogenesis in Dalbergia retusa (Papilonaceae)

Plants were obtained via organogenesis from hypocotyl explants of Dalbergia retusa from in vitro germinated seedlings. Adventitious bud induction was achieved on Murashige and Skoog medium containing five BA (benzyladenine) concentrations. The best BA concentration for budding induction and budding development was 8.8 microM. Shoot rooting was obtained on half-strength modified MS basal medium, supplemented with 20 g x l(-1) of sucrose and five concentrations of indole-3-butyric acid (IBA). The highest number of shoot rooting was obtained with 19.7 microM IBA but the highest average number of roots for plantlet was achieved with 24.6 microM IBA. Plants were transferred to greenhouse conditions.