Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

Micropropagation of Aristolochia rotunda L.

An original protocol of micropropagation of Aristolochia rotunda L., an important herb for the survival of an endangered butterfly was developed. The cytokinin 6-benzylaminopurine affected the production of new shoots but had no effect on shoot length. The addition of indole-3-butyric acid (IBA) had a negative effect on shoot length. Best results in terms of rooting percentage and root length were achieved with 1.5 μM IBA. The protocol can be employed to enhance the number of A. rotunda in the environment.     

Genetic fidelity of long-term micropropagated shoot cultures of vanilla (Vanilla planifolia Andrews) as assessed by molecular markers

Occurrence of genetic variants during micropropagation is occasionally encountered when the cultures are maintained in vitro for long period. Therefore, the micropropagated multiple shoots of Vanilla planifolia Andrews developed from axillary bud explants established 10 years ago were used to determine somaclonal variation using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats markers (ISSR). One thousand micro-plants were established in soil of which 95 plantlets (consisting of four phenotypes) along with the mother plant were subjected to genetic analyses using RAPD and ISSR markers. Out of the 45 RAPD and 20 ISSR primers screened, 30 RAPD and 7 ISSR primers showed 317 clear, distinct and reproducible band classes resulting in a total of 30 115 bands. However, no difference was observed in banding patterns of any of the samples for a particular primer, indicating the absence of variation among the micropropagated plants. Our results allow us to conclude that the micropropagation protocol that we have used for in vitro proliferation of vanilla plantlets for the last 10 years might be applicable for the production of clonal plants over a considerable period of time.     

In vitro bulblet production of Brunsvigia undulata from twin-scales

Many South African medicinal plants are over-collected for use in traditional medicines. This necessitates developing methods for increasing production. Micropropagation can be used as an alternative to conventional propagation methods. Twin-scales, cut from large parent bulbs, were cultured on MS medium (Murashige and Skoog, 1962) supplemented with 25 plant growth regulator combinations. Bulblets formed on twin-scales in 24 of the treatments. All explants formed bulblets on plant growth regulator-free medium. The effect of plant growth regulators, activated charcoal, explant orientation, explant origin and photoperiod on bulblet production was investigated. Bulblet formation was greatest when twin-scales were excised from the middle of the parent bulb, placed adaxial side down on plant growth regulator-free medium and kept in a 16 h photoperiod.     

Control of development and valepotriate production by auxins in micropropagated Valeriana glechomifolia

Valeriana glechomifolia is a plant species endemic to southern Brazil that accumulates valepotriates, which are terpene derivatives, in all of its organs. Valepotriates are the presumed sedative generic components of the pharmaceutically used species of Valeriana. The influence of various concentrations of the auxins indole-3-acetic acid, indole-3-butyric acid and -naphthaleneacetic acid on the growth of micropropagated V. glechomifolia was investigated under conditions of transient and continuous exposure. Changes in the development of roots and shoots as well as the production of the valepotriates acevaltrate, valtrate and didrovaltrate (analyzed by high-performance liquid chromatography) were evaluated. The best performance in valepotriate production, growth and survival under ex vitro conditions following plant acclimatization was achieved in the continuous presence of 5.71 M IAA. When cultured in medium containing IAA plants produced stable levels of valepotriates throughout the entire cultivation period.Abbreviations ACE Acevaltrate - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - DW Dry weight - DID Didrovaltrate - NAA -Naphthaleneacetic acid - PVP Polyvinylpyrrolidone - VAL Valtrate     

In vitro plantlet regeneration of<Emphasis Type="Italic"> Schinopsis balansae</Emphasis> (Anacardiaceae)

A protocol for the in vitro regeneration of rooted plants from nodal single bud segments of 10-year-old Schinopsis balansae trees was developed. Nodal segments were harvested from actively growing shoots of plants grown from seeds and maintained in pots under greenhouse conditions, and from epicormic shoots obtained by forced flushing of branches. Culture of nodal segments on nutrient medium containing the mineral salts and vitamins of Murashige and Skoog medium at 1/4 strength (1/4 MS), supplemented with 100 mg l^–1 ascorbic acid, 3% sucrose, and 5–15 M 6-benzyladenine resulted in regeneration of multiple shoots. Rooting of regenerated shoots was observed in 1/4 MS medium with vermiculite as the substrate and supplemented with 7.5 M indolbutyric acid.     

A high-frequency in vitro multiplication,micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multipurpose endangered medicinal shrub

Physiology and Molecular Biology of Plants - An efficient and reproducible in vitro propagation protocol has been established for Cadaba fruticosa (L.) Druce. Surface-sterilized nodal stem segments...     

The Rhodococcus fascians-plant interaction: morphological traits and biotechnological applications

Rhodococcus fascians is a Gram-positive bacterium that infects dicotyledonous and monocotyledonous plants, leading to an alteration in the normal growth process of the host. The disease results from the modulation of the plant hormone balances, and cytokinins are thought to play an important role in the induction of symptoms. Generally, on the aerial parts of the plants, existing meristems were found to be most sensitive to the action of R. fascians, but, depending on the infection procedure, differentiated tissues as well gave rise to shoots. Similarly, in roots not only actively dividing cells, but also cells with a high competence to divide were strongly affected by R. fascians. The observed symptoms, together with the determined hormone levels in infected plant tissue, suggest that auxins and molecules of bacterial origin are also involved in leafy gall formation. The complexity of symptom development is furthermore illustrated by the necessary and continuous presence of the bacteria for symptom persistence. Indeed, elimination of the bacteria from a leafy gall results in the further development of the multiple embryonic buds of which it consists. This interesting characteristic offers novel biotechnological applications: a leafy gall can be used for germplasm storage and for plant propagation. The presented procedure proves to be routinely applicable to a very wide range of plants, encompassing several recalcitrant species. Received: 14 January 1999 / Accepted: 19 June 1999     

Tissue culture for the conservation and mass propagation of <Emphasis Type="Italic">Vriesea reitzii</Emphasis> Leme and Costa,a bromeliad threatened of extinction from the Brazilian Atlantic Forest

The Brazilian Atlantic Forest biome is considered a hotspot of biodiversity. It is estimated that today the remaining primary vegetation covers only 7.5% of its original area. Bromeliad species are important components of this biome. Some of these species are endemic, like the highly endangered Vriesea reitzii. Tissue culture techniques have been often employed for the mass propagation and conservation of threatened bromeliad species. In the present work we describe a procedure for the micropropagation and in vitro conservation of V. reitzii. Seedling explants were cultured on MS and LPm liquid media supplemented with BA, NAA and GA₃. The induction and multiplication of shoots were observed in the MS medium supplemented with NAA (2 μM) and BA (4 μM). The best conditions for maintenance and conservation of shoots were half-strength or MS medium. Shoot elongation was observed in MS medium supplemented with GA₃ (10 μM). MS medium supplemented with NAA (1 μM) and BA (2 μM) enabled an efficient proliferation system. The acclimatization of shoots longer than 2 cm resulted in 100% survival rate.     

Sucrose utilization during potato microtuber growth in bioreactors

 Potato microtubers are used as pathogen-tested in vitro stocks for certified seed potato production. Microtubers grown in a rotating bioreactor grew at a faster rate when the medium was replaced frequently. Although the total microtuber number was not affected, the number of microtubers over 1 g quadrupled when 75% of the medium was replaced every 2 weeks when compared with no medium refreshment. Significantly slower microtuber growth rates resulted when a lower sugar concentration (40 g 1⁻¹ instead of 80 g 1⁻¹) was used or when a mixture of glucose and fructose replaced sucrose. Although high sucrose levels are necessary for optimal microtuber production, the sucrose supplied was rapidly hydrolyzed into glucose and fructose, making the long-term maintenance of desirable sucrose levels difficult. These results indicate that successful strategies to reduce sucrose hydrolysis without inhibiting microtuber growth will improve the efficiency of sucrose utilization in potato microtuber bioreactors. Received: 1 December 1998 / Revision received: 6 May 1999 · Accepted: 19 May 1999