Seed germination and in vitro regeneration of the African medicinal and pesticidal plant, Bobgunnia madagascariensis

Propagation of the medicinal and pesticidal tree, Bobgunnia madagascarensis is difficult due to poor and erratic germination of its seeds and slow growth of its seedlings. This study involved two separate experiments. The first evaluated the effect of pre-sowing treatments and growing medium on ex vitro seed germination and early seedling development. The second experiment involved in vitro germination, shoot initiation and rooting of shoots. Pre-sowing seed treatments involved soaking seeds in cold and hot water for 12 and 24 h and soaking in different concentrations (0, 100, 200, 400 and 800 mg/l) of gibberellic acid for 24 h. Soaking of seeds in cold or hot water for up to 24 h did not achieve more than 45% germination, while seeds treated with gibberellic acid achieved <20% germination rates. On the other hand, in vitro procedures achieved 30 to 70% germination of seeds. Seedling survival of ex-vitro germinated seeds was higher (>76%) when seeds were sown in a growing medium without compost compared with a medium with compost (<43%). All shoot-tips isolated from the in vitro germinated seedlings on B5 media without plant growth regulators continued to grow as a single shoot, while shoot-tips cultured on B5 supplemented with 0.1 mg/l of naphthaleneacetic acid (NAA) and thidiazuron (TDZ) produced two shoots each after four weeks. It was concluded that B. madagascariensis seeds had very low ex vitro germination percentages. Although, in vitro cultures improved seed germination, axillary shoot multiplication and rooting were not satisfactory. Therefore, further studies are needed to develop an optimal in vitro multiplication protocol for B. madagascariensis.Key words: Axillary shoot multiplication, gibberellic acid, in vitro regeneration, seed germination, Swartzia

Plant regeneration in Piper longum L. (Piperaceae) through direct and indirect adventitious shoot development

Methods for large scale multiplication of Piper longum are reported. Shoot tips cultured on Murashige & Skoog's medium supplemented with 0.5 mgl-1 benzyl adenine or kinetin in combination with 0.5 mgl-1 I-naphthalene acetic acid developed multiple shoots at very low frequency. Multiple shoots originated in large numbers, only from nodes of vertically planted explants, while shoots dnveloped de novo from all over the surface, when planted horizontally in the same medium containing 2·5 mgl-1 benzyl adenine. Leaf segments also developed adventitious buds in large numbers in Murashige & Skoog's medium containing 3 mgl-1 benzyl adenine, I mgl-1 kinetin and 10% (v/v) coconut water. Callus induced from leaf and stem explants in Murashige & Skoog's medium with 1-2 mgl-1 2, 4 dichlorophenoxy acetic acid in combination with Imgl-1 benzyl adenine developed shoot buds when transferred to medium containing 3 mgl-1 benzyl adenine. All the shoots were rooted in Murashige & Skoog's basal medium. The isolated plantlets, hardened in vermiculite were transferred to pots containing garden soil and sand. &nbsp

Plant regeneration in Piper longum L. (Piperaceae) through direct and indirect adventitious shoot development

Methods for large scale multiplication of Piper longum are reported. Shoot tips cultured on Murashige & Skoog's medium supplemented with 0.5 mgl-1 benzyl adenine or kinetin in combination with 0.5 mgl-1 I-naphthalene acetic acid developed multiple shoots at very low frequency. Multiple shoots originated in large numbers, only from nodes of vertically planted explants, while shoots dnveloped de novo from all over the surface, when planted horizontally in the same medium containing 2·5 mgl-1 benzyl adenine. Leaf segments also developed adventitious buds in large numbers in Murashige & Skoog's medium containing 3 mgl-1 benzyl adenine, I mgl-1 kinetin and 10% (v/v) coconut water. Callus induced from leaf and stem explants in Murashige & Skoog's medium with 1-2 mgl-1 2, 4 dichlorophenoxy acetic acid in combination with Imgl-1 benzyl adenine developed shoot buds when transferred to medium containing 3 mgl-1 benzyl adenine. All the shoots were rooted in Murashige & Skoog's basal medium. The isolated plantlets, hardened in vermiculite were transferred to pots containing garden soil and sand. &nbsp

Plant regeneration in Piper longum L. (Piperaceae) through direct and indirect adventitious shoot development

Methods for large scale multiplication of Piper longum are reported. Shoot tips cultured on Murashige & Skoog's medium supplemented with 0.5 mgl-1 benzyl adenine or kinetin in combination with 0.5 mgl-1 I-naphthalene acetic acid developed multiple shoots at very low frequency. Multiple shoots originated in large numbers, only from nodes of vertically planted explants, while shoots dnveloped de novo from all over the surface, when planted horizontally in the same medium containing 2·5 mgl-1 benzyl adenine. Leaf segments also developed adventitious buds in large numbers in Murashige & Skoog's medium containing 3 mgl-1 benzyl adenine, I mgl-1 kinetin and 10% (v/v) coconut water. Callus induced from leaf and stem explants in Murashige & Skoog's medium with 1-2 mgl-1 2, 4 dichlorophenoxy acetic acid in combination with Imgl-1 benzyl adenine developed shoot buds when transferred to medium containing 3 mgl-1 benzyl adenine. All the shoots were rooted in Murashige & Skoog's basal medium. The isolated plantlets, hardened in vermiculite were transferred to pots containing garden soil and sand. &nbsp

Fungal community composition at the last remaining wild site of yellow early marsh orchid (Dactylorhiza incarnata ssp. ochroleuca)

The yellow early marsh orchid (Dactylorhiza incarnata ssp. ochroleuca) is a critically endangered terrestrial orchid in Britain. Previous attempts to translocate symbiotic seedlings to a site near the last remaining wild site demonstrated some success, with a 10% survival rate despite adverse weather conditions over a two-year period. However, to facilitate future reintroduction efforts or conservation translocations, a more comprehensive understanding of the fungal microbiome and abiotic soil characteristics at the final remaining wild site is required. Obtaining comprehensive information on both the fungal community and soil nutrient composition from wild sites has significant benefits and may prove critical for the success of future conservation translocations involving threatened orchids. This preliminary study, conducted at the last remaining wild site, revealed a significant correlation between the relative abundance of the orchid mycorrhizal fungal order Cantharellales and the concentrations of nitrate and phosphate in the soil. Another orchid mycorrhizal fungal group, Sebacinales, was found to be distributed extensively throughout the site. The composition of fungal communities across the entire site, orchid-hosting and non-orchid-hosting soils is discussed in relation to reinforcing the current population and preventing the extinction of this orchid

Recovery of mycorrhizal fungi from wild collected protocorms of Madagascan endemic orchidAerangis ellisii(BS Williams) Schltr. and their use in seed germination in vitro

Orchid mycorrhizal fungi (OMF) are critical for seed germination and maintaining natural populations of orchids, yet the degree of specificity of most orchids to their mycorrhizal associates remains unknown. Many orchids are at risk of extinction, whether generalists or specialists, but orchid species of narrow fungal specificity are arguably under increased threat due to their requirement for specific fungal symbionts. This study characterises the fungi associated with Aerangis ellisii, a lithophytic orchid from a site in the Central Highlands of Madagascar. Culturable OMF isolated from spontaneous protocorms of this species from the wild were used for seed germination. In vitro germination and seedling development of A. ellisii were achieved with fungi derived from A. ellisii and an isolate from a different Aerangis species 30 km away. The significance of these findings and their importance to conservation strategies for this species and other Aerangis spp. is discussed. These results have important implications for the conservation of A. ellisii populations in Madagascar

Fungal diversity of selected habitat specific Cynorkis species (Orchidaceae) in the central highlands of Madagascar

About 90% of Cynorkis species are endemic to the biodiversity hotspot of Madagascar. This terrestrial habitat-specific genus received little study for fungal diversity to support conservation. We evaluated the diversity of culturable fungi of 11 species and soil characteristics from six sites spanning a >40 km radius in and along the region’s inselbergs. Peloton-forming fungi were grown in vitro from root/protocorm slices and positively identified using DNA sequencing. The fungal diversity was then correlated with soil pH, NO3-N, P, and K. All species harbored either putative mycorrhizal associates in the Rhizoctonia complex or Hypocreales fungi. Tulasnella Operational Taxonomic Units (OTUs) were most prevalent in all soil types while Serendipita OTUs were found in species inhabiting granite/rock outcrops in moist soil (seepage areas). Most Cynorkis species were present in soil with low NO3-N and P levels with diversity of mycorrhizal fungi inversely correlated to NO3-N levels. Of the different orchid life stages sampled, only one species (Cynorkis fastigiata) yielded putative mycorrhizal fungi from juvenile stages. As diversity of mycorrhizal fungi of Cynorkis spp. was negatively correlated with NO3-N, and majority of the studied taxa were found in soils with low NO3-N and P contents, reintroduction studies must include analysis of N and P in detail. For the first time, we showed that the assemblage of culturable fungi in the roots of habitat-specific species of Cynorkis (Orchidaceae) are intimately tied to specific soil characteristics