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Micropropagation and reintroduction of the endemic Tripleurospermum ziganaense (Asteraceae) to its natural habitat
Abstract
In this study, a rapid and efficient biotechnological method was developed for the critically endangered (CR) endemic species Tripleurospermum ziganaense (Asteraceae) in Turkey to reintroduce it to its natural habitat. Murashige and Skoog basal medium (MS) supplemented with 1.0 mg L−1 gibberellic acid (GA3) was highly effective with 80% germination percentage. MS medium containing 1.0 mg L−1 6-BA and 0.1 mg L−1 IBA was superior for the highest average shoot number of 11.73-fold per explant. The same basal medium strengthened with 1.0 mg.L−1 2iP plus 0.1 mg L−1 IBA was highly favorable for the highest average shoot length of 47.77 mm. In vitro micropropagated plants had similar DNA ploidy levels (x) and chromosome number (2n = 18) compared to mother plants. Rooting success was achieved between 93.33 and 100% in all rooting media. MS medium strengthened with 1.0 mg L−1 IBA was remarkably effective in terms of the highest average root number with 11.71-fold per explant. On the other hand, MS medium fortified with 0.25 mg L−1 NAA was quite conspicuous with an average root length of 124.03 mm per explant. All rooted plantlets were initially acclimatized in climate room conditions and then transferred from optimal greenhouse conditions into a botanical garden with 100% and 90% survival rates, respectively. Afterward, plantlets were reintroduced into experimental plot near their wild population of T. ziganaense, with 90% survival. Flow cytometry and cytology-assisted cytogenetic fidelity assessments of the micropropagated plantlets exhibited ploidy as well as chromosomal stability within the plantlets and with coherence to their mother plant. The preliminary results obtained from the combination of micropropagation with reintroduction efforts offered an excellent opportunity for in situ and ex situ conservation efforts to bolster populations of threatened endemic T. ziganaense. This approach consisting of the components of conservation, propagation, and reintroduction (CPR) may potentially serve as a model for saving and enriching other species at risk.
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Grazielanthus is a monotypic, dioecious and microendemic plant genus of the Brazilian Atlantic Forest. Its only species, Grazielanthus arkeocarpus , is categorized as Critically Endangered on the IUCN Red List and comprises only one small population, in the Poço das Antas Biological Reserve, Rio de Janeiro State. Collaborative activities have been developed since 2013 to implement in situ and ex situ conservation actions for this species. Successful in situ planting has increased the number of individuals in its natural population. Ex situ conservation efforts have resulted in the cultivation of the species in two Brazilian living plant collections, and this will soon increase to three collections.
Helichrysum arenarium, known as sandy everlasting, is a perennial plant species, used in traditional medicine. In Bulgaria, it is protected by the Biodiversity Act and under a special regime of use based on the Medicinal Plants Act. Seeds from five Bulgarian populations were in vitro germinated and seedlings were sub-cultured on five media supplemented with different plant growth regulators (PGRs) and control MS medium free of PGR. The type of cytokinin turned out to be of crucial importance, as the presence of kinetin stimulated formation of rhizomes and direct organogenesis, BAP caused callogenesis, indirect organogenesis and hyperhydricity, and Meta-topolin – necrosis. Best results were obtained on medium with reduced macrosalt and sucrose concentrations as rhizomes gave rise to numerous well-developed and spontaneously rooted plantlets, up to 55 originating from a single seed. Plants were potted in soil mixture, adapted to phytotron conditions, and transferred to the greenhouse for acclimation, then planted on the ex situ collection. All plants flowered during the first summer outdoor. Nuclear DNA amount was measured by flow cytometry to check possible abnormalities of the in vitro multiplied plants. The estimated DNA amount varied in the range 1C = 0.85–0.89 pg. No deviations in the ploidy level were detected. No difference between the studied populations was found. In vitro micropropagation was shown to be an appropriate method for rapid plant multiplication of H. arenarium thus offering the opportunity for its introduction into agriculture.
The present study reports development of an improved in vitro regeneration protocol of Strychnos potatorum L. f. a wild threatened medicinal tree species. The higher rate of morphogenetic plant regeneration was observed in seedlings derived young nodal bud explants when compared to shoot tips explants. Nodal explants cultured on MS medium supplemented with 1.0 mg/L–1 6-benzylaminopurine (BAP) in combination with 0.5 mg/L–1 naphthalene acetic acid (NAA) induced a maximum number of shoots of (10.5 ± 0.53) with a mean shoot height of (5.2 ± 0.36 cm) was obtained. BAP was found more significant in multiple shoot induction when compared to other cytokinins used in this study. In vitro rooting was achieved on half-strength MS medium augmented with different concentrations of auxins. NAA at 0.5 mg/L–1 produced the highest number of healthy roots (8.6 ± 0.70). The rooted plantlets were successfully hardened in the paper cups containing farmyard manure and garden soil in 1:1 ratio and successfully transferred to field conditions. About 80% of the plants survived in the natural habitat. The field-grown plants showed similarity in its growth characteristics to their mother plant. An assessment of clonal fidelity of regenerated plants was undertaken using inter simple sequence repeat (ISSR) markers which revealed that they are monomorphic and more comparable to their mother plants. This efficient reproducible protocol can be useful for the production of true-to-type nature and ex situ conservation of this threatened S. potatorum tree species.
Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America. It is an herbaceous and shrubby plant belonging to the Asteraceae family. Stevia is considered as a natural sweetener and a commercially important plant worldwide. The leaves of S. rebaudiana contain steviol glycosides (SGs) which are highly potent and non-caloric sweeteners. The sweetening property of S. rebaudiana is contributed to the presence of these high potency, calorie free steviol glycosides. SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals. SGs amount in the plant mostly varies from 8% to 10%, and the enhancement of SGs is always in demand. These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories. SGs are almost 300 times sweeter than sucrose. Being used as alternative sugar intensifier the commercial value of this plant in bio-pharmaceutical, food and beverages industries and in international market is increasing day by day. SGs have made stevia an important part of the medicinal world as well as the food and beverage industry, but the limited production of plant material is not fulfilling the higher global market demand. Therefore, researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S. rebaudiana. This review aims to describe the emerging biotechnological strategies and approaches to understand, stimulate and enhance biosynthesis of secondary metabolites in stevia. Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.
This study aimed to develop an efficient micropropagation protocol for Disanthus cercidifolius Maxim., an ornamental shrub. Sprouting buds of two genotypes (‘Trubaʼ and ‘PdSʼ) were used as an initial plant material. For the in vitro propagation experiment, the nodal segments were cultured on a MS medium supplemented with either N⁶-benzyladenine (BA) or zeatin (both at 0.5–3 mg l⁻¹). As a control, MS medium without growth regulators was used. The highest number of shoots per explant (6.95 ± 0.33 in genotype ‘PdSʼ and 7.93 ± 0.41 in genotype ‘Trubaʼ) was achieved on a medium supplemented with 2 mg l⁻¹ BA. Half-strength WPM and half-strength MS medium supplemented with indole-3-butyric acid (IBA) (0.1–1 mg l⁻¹) were tested for in vitro rooting of the shoots. Optimal rooting performance was achieved on a half-strength WPM containing 0.5 mg l⁻¹ IBA in genotype ‘PdSʼ, and 0.1 mg l⁻¹ IBA in genotypes ‘Trubaʼ. The rooted plantlets were transferred ex vitro with 85% survival in genotype ‘PdSʼ and 82.5% in genotype ‘Trubaʼ. Eighteen samples from each genotype were subjected to ISSR analysis and flow cytometry to assess plant genetic fidelity after micropropagation. Fifteen ISSR primers gave rise to monomorphic patterns indicating no detected genetic variation in regenerants. Flow cytometric analysis showed that the ploidy level in all tested in vitro regenerants remained stable. The micropropagation protocol optimized here represents a reliable and efficient method for the large-scale production of plants of the ornamental shrub Disanthus cercidifolius.
Hill’s thistle (Cirsium hillii (Canby) Fernald) is a perennial plant endemic to the Great Lakes region of North America. Hill’s thistle is listed as threatened in Ontario and Canada where it is found in globally rare alvar habitats. The main objective of this study was ex-situ conservation of Hill’s thistle using in vitro culture techniques and reintroduction of micropropagated plants back to their natural habitat in Bruce Peninsula National Park, Ontario, Canada. Two out of twenty-nine available seeds were successfully germinated under in vitro condition. An efficient micropropagation protocol was optimized with 100% survival during acclimatization of plantlets in the greenhouse. Three hundred micropropagated plants were reintroduced to twelve different sites within Bruce Peninsula National Park in June and July 2017. Plants were monitored for survival, rosette growth, and flowering on all sites from 2017–2019. After four months of planting, 67 to 99% of the plants were alive in different sites and 90 to 99% of them survived over winter. In the following years, shoot regeneration and flowering were observed on most sites. This study further confirms the benefit of plant tissue culture techniques to ensure revival of Hill’s thistle ecological biodiversity through the reintroduction of micropropagated plants. This approach consisting of the components of conservation, propagation, and reintroduction (CPR) may potentially serve as a model for saving and enriching other species at risk.
A rapid micropropagation protocol was designed to produce Calamintha sylvatica plantlets by using nodal segments as explants for the shoot formation. 6-BA favored the highest shoot formation and biomass yield, whilst kinetin was found superior for the highest shoot length (38.97 ± 2.85 mm) and node numbers (2.89 ± 0.63). Rosmarinic acid was detected as major phenolic acid, ranging from 7.59 mg/100 g to 81.44 mg/100 g. Hexane extracts from natural and in vitro propagated plantlets showed activity only against Staphylococcus aureus ATCC 25923 with MIC values at 6.25 and 3.33 m/mL, respectively while in the latter case, extracts from natural plantlets exerted higher cytotoxic activity than those of micropropagated ones (IC50 values were 83 µg/mL and 98 µg/mL on HeLa cells, respectively). C. sylvatica showed high micropropagation performance and produced remarkable amount of rosmarinic acid in vitro as well as antimicrobial and cytotoxic effect. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********
The study aimed to develop an efficient, rapid, and large-scale in vitro regeneration system for propagation, conservation, and restoration of an endemic and critically endangered herb, Ceropegia mohanramii. The cultures were established using nodal explants on Murashige and Skoog’s (MS) medium supplemented with 6-benzylaminopurine (BAP: 1.0 mg/l). Nodal buds cultured on MS medium supplemented with BAP (2.0 mg/l) along with indole-3-butyric acid (IBA, 0.5 mg/l) resulted with production of maximum number of shoots (17.1 ± 1.2) in hundred percent of the cultures. MS medium supplemented with BAP (2.0 mg/l) along with diverse concentrations of indole-3acetic acid (IAA) promoted the in vitro flowering. In vitro regenerated shoots were transferred to one-half MS medium fortified with singular supplementation of auxins, where IBA (1.5 mg/l) served optimal for production of maximum number of roots (5.7 ± 0.6). In vitro derived plantlets were hardened under controlled conditions in a glasshouse and subsequently transferred to soil. Over 1200 saplings were transplanted to eight different localities of the Western Ghats where over 76% survival is recorded after 1 year of transplantation.
Helichrysum italicum (Roth) G. Don is a Mediterranean vegetal species from the Asteraceae family. The ornamental value of the flowers and the properties of its essential oils contribute to its popularity. The aim of the present study was to compare the effect of different cytokinins on the multiplication of Helichrysum italicum and to develop a reliable protocol for in vitro micropropagation of this medicinal and ornamental plant. Nutrient media based on both MS (Murashige and Skoog, 1962) or DKW (Driver and Kuniyuki, 1984) formulations were used. They were enriched with different cytokinins - BAP, Kinetin, or Zeatin (5μM). The best multiplication rate was achieved on DKW medium, supplemented with 5μM Kinetin. A high percentage of rooting was achieved on the control treatment and the nutrient media, supplemented with IBA.
Different Eryngium species have been used with ornamental, agricultural and medicinal purposes, as a consequence of their chemical constituents. In the southwest Europe the endemic Eryngium viviparum, presents a high risk of extinction and ex situ strategies are high recommended for efficient conservation and re-introduction program. The objective of this study was to satisfy a dual objective: (i) to develop an ex situ conservation strategy through micropropagation and (ii) taking advantage of the extraordinary potential of plant tissue culture, produce a considerable amount of plant material to carry out a preliminary phytochemical study, based on the accumulation of phenolic compounds and their associated antioxidant activity. First a factorial design was conducted in order to study the effect of two cytokinins (6- benzylaminopurine, BAP, and kinetin, KIN), at three levels (0, 1 and 2 mg L⁻¹), on shoot multiplication. Later another factorial design was applied, by using three levels of MS medium salt strength (full, half and quarter- strength) and four sucrose levels (0, 1, 2, and 3%) for improving shoot elongation and rooting. In parallel, a preliminary quantification of total phenolic and flavonoid contents from E. viviparum aerial parts was determined. The simple micropropagation protocol designed allowed obtaining a high rates of shoot multiplication (5.1–5.8 new shoots), rooting (100%) with healthy long roots (3.1–3.5 cm) and plantlet acclimatization (96%). Moderate antioxidant activity was recorded in hydromethanolic extracts from E. viviparum aerial parts. High correlation between total phenolic content and BAP levels in the culture media was found. In conclusion, the micropropagation procedure described here for the endangered E. viviparum can be used as new and very efficient ex situ conservation strategy, and as potential source of antioxidants, conferring an added-value to this plant.
Medicinal and aromatic plants and their refined natural products have
gained global attraction for their therapeutic potential against many human diseases. Nigella sativa is a medicinally important plant, commonly known as Black cumin or Black seed is a dicotyledon plant of the Ranunculaceae family. It is in common use for a longer time in history as preservative and spice and has been extensively utilized by different communities around the globe. Black cumin has been an eminent component of traditional medicine systems like Unani and
Tibb, Ayurveda and Siddha. Its biological activities include ntidiarrheal,
analgesic, antibacterial, liver tonic, diurectic, digestive agent and to treat several skin disorders. Furthermore, the therapeutic properties also include antidiabetic, anticancer, antihypertensive, anti-inflammatory, hepatoprotective, spasmolytic and bronchodialator. This is all because of its miraculous healing power that it has been
ranked as top ranked, among evidence based herbal medicines. The literature supports that the pharmacological activities of Nigella sativa are mainly because of the essential oil and its constituents particularly thymoquinone. The current review is an attempt to present a detailed literature survey regarding chemical composition, phytochemistry, therapeutic potential and biotechnological approaches to enhance
the medicinal potential of this valuable plant.
In vitro regeneration of Viola stagnina Kit., endangered in most part of its European distribution range, was successfully obtained based on the newly developed protocol. Adventitious shoots via direct and indirect organogenesis were induced on leaf blade and petiole fragments on solidified Murashige and Skoog (MS) medium supplemented with 0.5 or 1 mg l⁻¹ thidiazuron, respectively. Shoots were rooted on half-strength MS medium with 2% sucrose and 0.5 mg l⁻¹ indole-3-acetic acid (IAA), and plantlets were successfully acclimatized. Sixty-five of the regenerated plants (72% of isolated shoots cultured on rooting medium) survived at the experimental plot conditions. Among recovered via organogenesis plants, individuals ‘true-to-type’ derived from initial plant G1 and also plants genetically distant from initial plants were detected by ISSR markers. In all groups of clones genetic indices (number of genotypes, polymorphic markers, gene diversity, total gene diversity, mean gene diversity) were lower than in natural populations. Regenerated plantlets had the same genome size estimated by flow cytometry as initial material and plants from natural populations. They developed chasmogamous flowers with highly viable pollen grains (over 90%), cleistogamous flowers, and set seeds from both flower types in the first and second seasons cultivated at experimental plots. This is the first report of a successfully developed micropropagation protocol of V. stagnina, and the first detailed genetic analysis of recovered plants with the use of ISSR markers and genome size measurements allowing to discuss the advantageous role of somaclonal variation in ex situ plant conservation with the use of in vitro micropropagation.
The study of genome size variation can contribute valuable information on species relationships as well as correlate to several morphological or ecological features, among others. Here we provide an extensive report on genome sizes on genus Tripleurospermum and its closely related genus Matricaria, which are two typically Mediterranean genera particularly widespread and diverse in Turkey, the origin of most of the populations here studied. We analyse and discuss genome size variation in the first relatively complete molecular phylogenetic framework of Tripleurospermum (based on ITS and ETS ribosomal DNA–rDNA–regions). We find cases of intraspecific genome size variation, which could be taxonomically significant. Genome downsizing is also detected as the typical response to polyploidisation in Tripleurospermum taxa, being most conspicuous at the tetraploid level. Several positive correlations with genome size, including those with pollen and stomatal size or cypsela length, among others, are also found. Remarkably, taxa presenting rhizomes tend to present higher genome sizes, confirming a trend to accumulate nuclear DNA in such species, which could be explained by the nutrient reserves availability in their storage organs, allowing genome expansion, or by the lower rates of sexual reproduction in rhizomatous taxa.
The lantern flowers (genus: Ceropegia L. — Apocynaceae) are among the most fascinating group of flowering
plantswhich comprises of over 200 species of herbs and climbers. Although most of the species have appreciable
importance as a source of edible tubers, ornamental flowers and implications in folk medicine, the conventional
propagation techniques limit the mass propagation and their commercialization. Plant tissue culture and other
biotechnological tools proved their importance in breeding and large-scale propagation of lantern flowers. In
recent past, numerous in vitro regeneration approaches for micropropagation and conservation have been
established for lantern flowers. Molecular markers represent powerful tools for constructing phylogenetics,
taxonomic confirmations and reconsiderations of lantern flowers. Moreover, molecular markers are routinely
used for ascertaining genetic integrity among the micropropagated clones of lantern flowers. This review
provides comprehensive information on the biotechnological interventions for propagation, conservation, and
improvement of lantern flowers. Protocols for various in vitro regeneration approaches, genetic composition,
genetic fidelity analysis and in vitro secondary metabolite production are compared and optimized results
are highlighted.
Silene bolanthoides Quézel, Contandr. & Pamukç. is an endemic species from Kazdagi (Mt. Ida), Canakkale- Balikesir, Turkey. In order to develop an efficient shoot regeneration protocol, the leaf, nodal and internodal explants of S. bolanthoides were cultured on Murashige and Skoog (MS) medium containing benzyladenine (BA) alone or in combination with a-naphthaleneacetic acid (NAA). The highest number of regenerated shoots (5.75±0.1) was obtained from nodal explants that were cultured on MS medium with 8.8 μM BA+0.54 μM NAA. Regenerated shoots were rooted on MS medium without plant growth regulators (PGRs). Rooted plants (2-3 cm) were separately transferred to pots containing a mixture of peat and perlite (3:1 v/v) and acclimatized successfully in a growth chamber. Genetic stability of the propagated plants was assessed by flow cytometry and cytological analysis. Flow cytometry analysis demonstrated that regenerated plants had 2.61±0.01 pg nuclear DNA (2C) and seed-derived plants had on average 2.58±0.02 pg/2C. Cytological analysis showed that the regenerated plants had the same chromosome number as seed-derived plants of S. bolanthoides (2n=24). It was determined that regenerated plants were uniform in chromosome number and had a similar DNA content to the seed-derived ones, indicating that the described efficient shoot regeneration protocol can be applied for ex situ conservation of this species.
An efficient micropropagation protocol was developed to produce Stachys annua plantlets and the extracts obtained from both micropropagated and naturally growing individuals were evaluated for their possible antimicrobial, antioxidant and cytotoxic activities. Mean number of shoot (4.5 ± 0.54 per explant) and node number (4.56 ± 0.5 per explant) as well as biomass yield based on fresh (0.29 ± 0.02 gm per explant) and dry weight (0.029 ± 0.003 gm per explant) were found to be the highest on MS medium with 6-BA, whilst the highest mean shoot length (36.65 ± 1.58 mm) was obtained from MS medium containing 2iP. Hexane extracts from both sources showed activity against Staphylococcus aureus whilst methanol extracts of micropropagated plantlets exerted activity on Pseudomonas aeruginosa. In antioxidant activity assays, the best antioxidant activity was up to IC50 9.41 mg/mL in DPPH and 1409.5 (mg/100 gm trolox equivalent) was found in FRAP. Extracts from natural plantlets showed higher cytotoxic activity micropropagated ones, with IC50 of 0.099 µg/mL and IC50of 0.211 µg/mL on HeLa cells, respectively. Total phenolics ranged from 87.47 to 605.12 in micropropagated samples while 771.46 (mg/100 gm gallic acid equivalent) in natural resources. © 2017, National Institute of Science Communication and Information Resources (NISCAIR). All rights reserved.
Most temperate terrestrial orchids are endangered species. Attempts to produce plantlets from plants of the genus Epipactis by asexual methods have totally failed. This study was conducted using somatic embryogenesis as a rapid vegetative propagation technique for conservation of E. veratrifolia. For these purposes, effects of different types of plant growth regulators (PGRs), different types of explants, light and dark conditions, and the effect of gibberellic acid (GA3) and Paclobutrazol (PBZ) were investigated on somatic embryogenesis induction. Abscisic acid (ABA) pre-treatment effectiveness on inducting somatic embryogenesis and increasing the number of embryos per explant were investigated. Subsequently, 16 media were tested to find the best medium for plant regeneration and shoot and root proliferation. BA (3 mg L⁻¹) resulted in a better response than the other PGRs by supporting the development of 17 embryos per protocorm. PBZ, which resulted in 11 embryos per explant, was better than GA3. FAST medium supplemented with organic substances was recognized as the best medium for plant regeneration and shoot and root proliferation. ABA pre-treatment had positive effect on somatic embryogenesis initiation. This study, for the first time, succeeded in finding a rapid and suitable protocol for propagation and genetic resource conservation of E. veratrifolia. © 2016 The Journal of Horticultural Science & Biotechnology Trust
Calendula maritima is a critically endangered endemic plant of Western Sicily. Besides habitat destruction, the hybridization with the contiguous congener species C. fulgida is a major threat to its conservation. For this reason, seed- based propagation and seed storage are not appropriate for conservation purposes. In the present paper we describe a rapid and prolific In vitro plant regeneration method by direct organogenesis from leaves of C. maritima. Leaf explants were cultured on solid Murashige and Skoog (MS) medium in the presence of several plant growth regulator combinations. The best shoot multiplication rate (2.5 shoots/explant) was obtained on the medium containing 4.4 μM 6-benzylaminopurine in combination with 10 μM ß-naphthoxyacetic acid. Regenerated shoots were successfully rooted on solid MS medium supplemented with several auxins and the best result was obtained with 1.0 μM indole-3-acetic acid (35% of plantlets rooted). Plantlets were thereafter established in the greenhouse (survival frequency 75%) and no phenotypic variations were observed between regenerants and the mother plants.
Our study was undertaken to better understand how to increase the success rates of recovery plantings of a rare hemiparasite, golden paintbrush (Castilleja levisecta-Orobanchaceae). This species is endemic to western Washington and Oregon, USA, and southwestern British Columbia, Canada. Over 5000 golden paintbrush plants were outplanted as plugs in 2007 at six different native prairie sites that were considered to be suitable habitat, based on general evaluations of vegetation and soil conditions. Outplantings were installed at regular intervals along transects up to 1 km long to include a range of conditions occurring at each site. All plantings were re-examined five years later. The patchy distribution of surviving plugs and new recruits within each reintroduction site suggested success is strongly influenced by microsite characteristics. Indicator species analysis of taxa growing in microsites around outplanted golden paintbrush identified species that were positively or negatively associated with paintbrush survival. Species such as Festuca roemeri, Eriophyllum lanatum, and Viola adunca were strong indicators at some sites; non-natives such as Hypochaeris radicata and Teesdalia nudicaulis tended to be frequent negative indicators. Overall, higher richness of native perennial forbs was strongly correlated with both survival and flowering of golden paintbrush, a pattern that may reflect interactions of this hemiparasite with the immediately surrounding plant community. Topographic position also influenced outcomes, with greater survival occurring on mounds and in swales, where soils generally were deeper. Our findings suggest that assessments of site suitability based on vegetation alone, and coarser, site-level assessments that do not characterize heterogeneity at the microsite scale, may not be strong predictors of restoration success over the longer term and in sites with variability in vegetation and soils. By identifying suitable microsites to focus rare species plantings, survival and efficiency may be significantly enhanced.
A rapid shoot multiplication protocol was established for the endangered cactus Mammillaria mathildae to reintroduce it to its natural habitat. In vitro-germinated seedlings were used as the source of explants. Three explant sources (apical, lateral, and basal excised from in vitro-germinated seedlings) were tested. Shoot multiplication was induced in Murashige and Skoog (MS) medium supplemented with different 6-benzylaminopurine/indole-3-acetic acid (BA/IAA) combinations (0, 22.19, 44.39 and 0, 1.43,2.85, 5.71, respectively). Explants developed abundant callus in the presence of any BA/IAA concentration, whereas hormone-free media produced 0.59 ± 0.11 new shoots (with a 41% callus development) from basal explants. Apical and lateral explants produced 1.14 ± 0.07 and 4.09 ± 0.13 new shoots, respectively, without callus generation. Plantlets originating from lateral explants developed a vigorous rooting system after 2 months growing on MS medium supplemented with 30 g L-1 sucrose. Under greenhouse conditions, 98% of micropropagated M. mathildae survived. Plantlets were reintroduced in an experimental plot near to Juriquilla's wild population of M. mathildae; over 52% of the outplanted M. mathildae lot declined after 5 months. Water availability wasassociated with the decline of outplanted populations during the first month (43%).
The existing literature on plant translocations focuses on post-translocation outcome while still overlooking issues related to the preparation phases. Yet, plant translocation programmes face significant pre-translocation challenges. In the present study, we want to share our pre-transplant experience on four rare plant species (Arnica montana, Campanula glomerata, Dianthus deltoides, and Helichrysum arenarium), highlighting aspects we need to focus on while planning plant translocations. We emphasise some issues that need to be overcome before any translocation is undertaken during the four steps of translocation preparation, i.e. the selection and profiling of the target species, the seed collection, the development of propagation protocols and the assessment of plant fitness of the populations used as seed source. We discuss the implications of our results for designing translocation protocols. Our findings on A. montana show that if local seed sources are constrained to small remnant populations, seed quality may be poor. Preliminary tests using different kinds of growing medium provided valuable information for optimizing plant propagation protocols. Although it is attractive to establish propagation protocols using seeds obtained via Index Seminum (to avoid wasting collected source seeds), the results obtained were not always reproducible on the seeds collected in the wild source populations. Differences in pre-translocation plant fitness were also detected between seed source populations, which might reflect genetic diversity and maternal effects. As the translocated plants should capture as much genetic diversity as possible to ensure a high adaptive potential and improve establishment success, multisource reintroductions can be recommended.
Centaurea cineraria subsp. circae is an endemic plant with a distribution area limited to Circeo mountain (Lazio, Italy), whose population was estimated in a very low number of individuals. The aim of this work is to investigate ex situ conservation strategies such as achene collection and in vitro plant propagation, that will permit to carry out restoration programs. The test carried out on the achenes demonstrated that only 5.5% of them were morphologically healthy. Seed germination tests showed that seeds do not display dormancy and that germination does not require pre-treatments. The higher germination rate (67.5%) was observed under a photoperiod of 12 h/12 h (light/dark) and temperature regime +20 °C/+10 °C. The in vitro studies demonstrated that micropropagation, acclimatization and the transfer outdoors of C. cineraria subsp. circae are not particularly difficult: 74% of shoot explants in MS medium added with 0.5 mg/L benzylaminopurine (BAP) and 2 mg/L kinetin (Kin) formed multiple shoots; 100% of shoots rooted in MS medium added with 0.5 mg/L IBA and over 90% survived the acclimatization phase. After been transferred outdoors the totality of in vitro propagated plants bloomed and appeared morphologically indistinguishable from wild plants. Preliminary chemical analyses showed a similar profile for in vitro propagated and wild plants.
Abstract: This study was designed to micropropagate Vaccinium myrtillus L., a naturally growing bilberry, in the Turkish flora. In order to determine the most effective basal medium, lateral buds were initially selected as explants, which were then individually cultured in Murashige and Skoog medium, Anderson’s rhododendron medium, and McCown’s woody plant medium (WPM) supplemented with 1.0 mg L–1 zeatin in combination with either 0.1 mg L–1 indole-3-butyric acid (IBA) or 0.1 mg L–1 α-naphthalene acetic acid. WPM, supplemented with zeatin and IBA, was found to be the most effective basal medium tested. As shoot regeneration ability highly depends on the medium and cytokinin concentration, WPM basal media containing various concentrations (0.5, 1.0, and 2.0 mg L–1) of 3 different cytokinins, namely zeatin, thidiazuron, and N6-[2-isopentenyl] adenine, were employed together with IBA (0.1 mg L–1) and were compared with basal media containing none of the growth regulators. In terms of shoot multiplication, 2.0 mg L–1 zeatin was found to be superior to the other tested growth regulators when combined with 0.1 mg L–1 IBA. WPM was also used as a basal medium for rooting and was supplemented with different concentrations of IBA (0.25–2.0 mg L–1) with or without activated charcoal (AC). WPM supplemented with 0.5/1.0 mg L–1 IBA/AC was the best culture medium for rooting with 60% root formation.
Key words: Micropropagation, Vaccinium myrtillus, indole-3-butyric acid, N6-[2-isopentenyl] adenine, thidiazuron, zeatin
Viola uliginosa Besser is a European violet having its main distribution range in the Baltic Sea region. Today it is considered endangered and threatened. Species of Violaceae from different genera and sections are known
to produce cyclotides, cyclic polypeptides of much interest due to their medicinal properties and chemical structure. The present study introduced a rare species of violet (V. uliginosa) to in vitro culture for biodiversity protection and as a model for cyclotide biosynthesis research in the Violaceae. Leaf and petiole fragments were cultured on MS medium solidified with agar and supplemented with different concentrations of plant growth regulators: TDZ, KIN and 2,4-D. Direct and indirect (via callus) organogenesis was induced on MS supplemented with TDZ (0.5 or 1 mg l-1) or with equal concentrations (2 mg l-1) of KIN and 2,4-D, followed by callus transfer on 1 mg l-1 TDZ. Shoots were rooted on MS with 2 % sucrose and 0.5 mg l-1 IBA and acclimatized. AFLP marker polymorphism was low but flow cytometry revealed that a large share of the obtained regenerants were tetraploid (2C = 4x = 2.7–2.8 pg), unlike the maternal diploid plants (2C = 2x = 1.4 pg). Eleven different cyclotides were distinguished in the aerial parts of maternal plants. Cyclotide production was significantly higher in tetraploid than in diploid plants regenerated in vitro.
The aim of the work was direct regeneration of shoots from meristems, as well as adventitious shoot production from leaves for regeneration and effective production of highbush blueberry Vaccinium corymbosum L. and lingonberry Vaccinium vitis-idaea L. The results showed that the shoot proliferation intensity of individual cultivars differed. Therefore, optimisation of cytokinin type and concentration for each cultivar was necessary. For meristem regeneration, improved multiplication was achieved on medium with zeatin in comparison with N 6 -Δ 2 -isopentenyl adenine. Thidiazuron was effective in adventitious shoot regeneration from leaf tissue of highbush blueberry, and both thidiazuron and zeatin for lingonberry leaf tissue. Microshoot rooting was achieved on Anderson culture medium supplemented with indole-3-butyric acid or directly in peat after dipping of shoots into indole-3-butyric acid solution under ex vitro conditions.
Germplasm storage of Phyllanthus fraternus by using synseed technology has been optimized. Synseeds were prepared from nodal segments taken from in vitro-grown plantlets. An encapsulation matrix of 3 % sodium alginate and 100 mM calcium chloride with polymerization duration up to 15 min was found most suitable for synseed formation. Maximum plantlet conversion (92.5 ± 2.5 %) was obtained on a growth regulator-free ½-strength solid Murashige and Skoog (MS) medium. Multiple shoot proliferation was optimum on a ½ MS medium containing 0.5 mg/l 6-benzylaminopurine (BAP). Shoots were subjected to rooting on MS media containing 1 mg/l α-naphthaleneacetic acid (NAA) and acclimatized successfully. Encapsulated nodal segments can be stored for up to 90 days with a survival frequency of 47.33 %. The clonal fidelity of synseed-derived plantlets was also assessed and compared with that of the mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeat analysis. No changes in molecular profiles were observed among the synseed-derived plantlets and mother plant, which confirms the genetic stability of regenerates. This synseed production protocol could be useful for in vitro multiplication, short-term storage, and exchange of germplasm of this important antiviral and hepatoprotective plant.
Stevia rebaudiana (Bert.) is an emerging sugar alternative and anti-diabetic plant in Pakistan. That is why people did not know the exact time of propagation. The main objective of the present study was to establish feasible propagation methods for healthy biomass production. In present study, seed germination, stem cuttings and micropropagation was investigated for higher productivity. Fresh seeds showed better germination (25.51-40%) but lost viability after a few days of storage. In order to improve the germination percentage, seeds were irradiated with 2.5, 5.0, 7.5 and 10 Gy gamma doses. But gamma irradiation did not show any significant change in seed germination. A great variation in survival of stem cutting was observed in each month of 2012. October and November were founded the most suitable months for stem cutting survival (60%). In order to enhance survival, stem cuttings were also dipped in different plant growth regulators (PGRs) solution. Only indole butyric acid (IBA; 1000ppm) treated cutting showed a higher survival (33%) than control (11.1%). Furthermore, simple and feasible indirect regeneration system was established from leaf explants. Best callus induction (84.6%) was observed on MS-medium augmented with 6-benzyladenine (BA) and 2, 4-dichlorophenoxyacetic acid (2, 4-D; 2.0 mg l-1). For the first time, we obtained the highest number of shoots (106) on medium containing BA (1.5 mg l-1) and gibberellic acid (GA3; 0.5 mg l-1). Plantlets were successfully acclimatized in plastic pots. The current results preferred micropropagation (85%) over seed germination (25.51-40%) and stem cutting (60%).
The plant Dianthus morisianus Vals. (Caryophyllaceae) is endemic to Sardinia. The Autonomous Region of Sardinia funded a conservation project for this species because it is one of the most threatened plant on the island. The project comprises in situ and ex situ research and experimental projects, such as the construction of protective fences and reintroduction. Juvenile plants, germinated from 200 seeds collected over 2 years and propagated without horticultural treatment, were reintroduced in November 2010. The surviving 113 plants were reintroduced 150 m from the natural population and were monitored monthly. Two years later the survival rate was > 95%, and the fruit yield per plant was higher than that recorded in the natural population. This research emphasizes the importance of identifying an appropriate microhabitat for plant reintroduction. The use of juvenile plants aided the success of the reintroduction and reduced the mortality rate; the knowledge of the species biology, in particular the critical stage of their life cycle, is a crucial factor in plant reintroduction.
Reintroduction of native species has become increasingly important in conservation worldwide for recovery of rare species and restoration purposes. However, few studies have reported the outcome of reintroduction efforts in plant species. Using data from the literature combined with a questionnaire survey, this paper analyses 249 plant species reintroductions worldwide by assessing the methods used and the results obtained from these reintroduction experiments. The objectives were: (1) to examine how successful plant species reintroductions have been so far in establishing or significantly augmenting viable, self-sustaining populations in nature; (2) to determine the conditions under which we might expect plant species reintroductions to be most successful; (3) to make the results of this survey available for future plant reintroduction trials. Results indicate that survival, flowering and fruiting rates of reintroduced plants are generally quite low (on average 52%, 19% and 16%, respectively). Furthermore, our results show a success rate decline in individual experiments with time. Survival rates reported in the literature are also much higher (78% on average) than those mentioned by survey participants (33% on average). We identified various parameters that positively influence plant reintroduction outcomes, e.g., working in protected sites, using seedlings, increasing the number of reintroduced individuals, mixing material from diverse populations, using transplants from stable source populations, site preparation or management effort and knowledge of the genetic variation of the target species. This study also revealed shortcomings of common experimental designs that greatly limit the interpretation of plant reintroduction studies: (1) insufficient monitoring following reintroduction (usually ceasing after 4 years); (2) inadequate documentation, which is especially acute for reintroductions that are regarded as failures; (3) lack of understanding of the underlying reasons for decline in existing plant populations; (4) overly optimistic evaluation of success based on short-term results; and (5) poorly defined success criteria for reintroduction projects. We therefore conclude that the value of plant reintroductions as a conservation tool could be improved by: (1) an increased focus on species biology; (2) using a higher number of transplants (preferring seedlings rather than seeds); (3) taking better account of seed production and recruitment when assessing the success of reintroductions; (4) a consistent long-term monitoring after reintroduction.
Oberprieler, C., Himmelreich, S. & Vogt, R.: A new subtribal classification of the tribe Anthemideae (Compositae). – Willdenowia 37: 89-114. – ISSN 0511-9618; © 2007 BGBM Berlin-Dahlem. doi:10.3372/wi.37.37104 (available via http://dx.doi.org/) A new subtribal classification of the Compositae-Anthemideae is presented based on phylogenetic re-constructions for sequence information of the internal transcribed spacer (ITS) region of the nuclear ri-bosomal DNA (nrDNA) for 103 of the 111 accepted genera of the tribe. Results of the present analyses are compared with results from phylogenetic analyses based on cpDNA ndhF sequence variation and discussed in conjunction with morphological, anatomical, cytological, embryological and phytoche-mical evidence. As a result, 14 subtribes are circumscribed and described in detail, with information provided concerning the generic members and the geographical distribution of these entities. Four subtribes (i.e. Osmitopsidinae, Phymasperminae, Pentziinae and Leucanthemopsidinae) are described as new to science, for a further subtribe a new name (Glebionidinae, replacing the illegitimate Chry-santheminae) is validated.
Present study reports meta-Topolin (mT)-induced in vitro mass propagation of Gerbera jamesonii Bolus ex Hooker f. (a cut-flower with high demand in the floriculture industry), subsequent acclimatization, and cyto-genetic fidelity assessment of the plantlets. Different cytokinins (and cytokinin-like plant growth regulators) i.e., 6-benzyladenine, kinetin, mT, thidiazuron and zeatin in variable doses (0.5–1.5 mg L−1) were supplemented in the basal Murashige and Skoog (MS) medium and comparative growth performance of the inoculated shoot tip explants were studied. Maximum number of de novo shoots (∼14) and leaves (∼40) were obtained in 1.5 mg L−1 mT. Earliest (∼9 days) root induction from shoot with maximum number (∼8) and length (∼6 cm) of roots were recorded in MS medium fortified with 1.5 mg L−1 indole-3-acetic acid. All the micropropagated plantlets (100%) were acclimatized in sand, soil, cow urine and tea leaves extract (1:1:1:1; v v−1) followed by sand-soil (1:1; v v−1) mixture. Histological studies on shoots, roots, and leaves along with leaf gas exchange analysis revealed a stimulated growth, cellular development, photosynthetic and transpiration activities accompanied by significant levels of photosynthetic pigments in plantlets during acclimatization. The flow cytometry-, cytology-, as well as Start Codon Targeted (SCoT) Polymorphism and inter simple sequence repeats (ISSR) marker-assisted fidelity assessments of the micropropagated plantlets exhibited ploidy as well as chromosomal stability, and monomorphic banding patterns confirmed no polymorphism within the plantlets and with their mother plant. The developed protocol should be of immense commercial importance to facilitate large-scale production of true-to-type planting materials of gerbera.
A rapid and efficient in vitro micropropagation system was developed to conserve Tripleurospermum fissurale (Sosn.) E.Hossain (Asteraceae), a rare endemic species for Turkey. Murashige and Skoog basal medium (MS) supplemented with 4.7 µM KIN was found to be the most appropriate basal medium with a 60% germination rate. MS medium supplemented with 4.9 µM 2iP and 0.5 µM IBA gave the highest shoot length of 54.3 ± 3.53 mm. Furthermore, 4.4 µM 6-BA combined with 0.5 µM IBA was superior for the highest shoot number with 3.4 ± 0.49 after 4-wk culture. Cytogenetic analyses indicated that all propagated plants have the same DNA ploidy level (x) and chromosome number (2n = 18) compared with the mother plants. After 4 wk, the rooting percentage achieved 100% in all tested rooting media. MS medium supplemented with 2.7 µM NAA favored the highest root number, root length, and secondary root number with 3.44 ± 0.5, 170.1 ± 4.91 mm, and 26.1 ± 1.65, respectively. Rooted plantlets were initially acclimatized and then transferred in greenhouse conditions. This method could be evaluated for ex situ conservation of rare endemic and endangered plant species.
A unique one-step in vitro propagation protocol via concurrent shoot-root development was established in banana cv. Grand Nain. 6-benzyladenine, kinetin, meta-Topolin, thidiazuron (TDZ), and zeatin (0.5—1.5 mg L ⁻ ¹) individually supplemented in Murashige and Skoog medium were tested for multiple shoot-root regeneration from shoot tip explants. The earliest fresh shoot (∼7 days) and root (∼5 days) initiation, highest number of shoots (∼21) and roots (∼16) with maximum root elongation (16.5 cm) were recorded in 0.5 mg L ⁻ ¹ TDZ. For all intents and purposes, TDZ (0.5 mg L ⁻ ¹) was recorded to be the optimum formulation in terms of the highest simultaneous shoot and root formation that was corroborated further by matrix plot analysis. On the other hand, network plot analysis and principal component analysis illustrated the association between plant growth regulators based on their effect on growth attributes under study. Histological studies on shoots, roots, and leaves revealed a consistent growth and development of plantlets under in vitro condition. Cent percent plantlets were survived during acclimatization, and performed best in cocopeat in comparison to other substrata (vermiculite, perlite, vermicompost-soil, soil-sand, and sand only). The cytology-, flow cytometry- and ISSR primer-assisted fidelity assessments of the in vitro-regenerants exhibited numerical chromosomal as well as ploidy stability and monomorphic banding patterns without any polymorphism within the in vitro-regeneranted plantlets and with their mother plant. Based on the comprehensive information developed in this study, this protocol should be of immense importance to facilitate single-step large-scale production of true-to-type planting materials and to explore the possible comparative physico-chemical roles of the plant growth regulators used in this study.
Tripleurospermum insularum (Asteraceae) is a critically endangered (CR) insular endemic species in Turkey and is facing high risk of extinction. Here, a rapid and efficient in vitro propagation protocol using nodal segments obtained from seedling shoots cultured on Murashige and Skoog (MS) basal medium supplemented with different plant growth regulators (PRGs) was developed to conserve T. insularum. Besides, the cytogenetic fidelity of propagated plants was tested with DNA ploidy level using flow cytometry (FCM) as well as chromosome counting. The highest shoot number and length of shoot per explant were achieved in MS medium containing 4.6 µM zeatin (ZEA) and 0.5 µM indole-3-acetic acid (IAA). No variation in DNA ploidy level (2x) and somatic chromosome number (2n = 18) of all propagated plants were observed. In vitro rooting of shoots was achieved at 100% efficiency in the medium supplemented with 2.9 µM IAA. The rooted plantlets were transferred ex vitro with 74% survival. This is the first report of a successfully developed micropropagation protocol for ex situ conservation of T. insularum.
Know the breeding system of endemic plants is important to design conservation strategies. Translocations are actions to improve the survival prospects of the species, but nowadays there are only a few studies that analyse their success and make a comparison between translocation and the natural populations. Dianthus morisianus is a threatened narrow endemic plant species growing on sand dunes in SW Sardinia (Italy). The objective of this study was to assess the breeding system, the presence of inbreeding depression and pollen limitation, as well as the success of the plant translocation. All these results were compared with those from the single natural population. The breeding system was tested through five pollination treatments and the reproductive success was analysed by the fruit set, seed set, seed weight, germination and mortality rate. The translocated population behaved like the natural one on fruit and seed formation. Autonomous self-pollination was lower than the other treatments regarding fruit set and seed/ovule ratio in the two studied populations. The species is self-compatible and presents partial self-fertility. The selfing rate was higher in the translocated population and the inbreeding depression presented low values for the natural population, while the translocated population presented negative values. Neither of the populations suffered pollen limitation. The species did not present reproductive problems and it is pollinator dependent. Moreover, the translocated population demonstrated high success after five years, as an increase of the population area and new recruited plants was observed; the offspring were able to flower, fruiting and reproduce. This translocation success increases the survival prospects of the species.
We established a protocol for the in vitro propagation of Baccharis conferta Kunth. This plant is used to treat gastrointestinal problems, cramps, pain, respiratory problems, and insect bites. A high rate of shoot multiplication was obtained from nodal segments on Murashige and Skoog (MS) culture medium. The shoots regenerated roots without exogenous plant growth regulators (PGRs). All explants of wild leaves on MS medium containing 5 μM of thidiazuron (TDZ) produced friable callus. An organogenic response was achieved after 3 wk of culture when callus segments were transferred to MS medium containing a combination of plant growth regulators (PGRs): either (i) 5 μM indole butyric acid (IBA) + 5 μM kinetin (KIN) or (ii) 0.5 μM IBA + 1.10 μM benzylaminopurine (BAP). The morphogenetic responses of callus were characterized by scanning electron microscopy. Shoots regenerated from callus and formed roots on MS medium without PGRs. The micropropagated plantlets and the organogenic callus showed similar chemical profiles in HPLC-mass spectrometry analyses. The main compounds present in the cultures were caffeoylquinic acids. Only plantlets contained small amounts of triterpenes (erythrodiol and ursolic acid). These findings will be useful for the micropropagation of this important native resource, and for further studies on its biology.
Golden paintbrush (Castilleja levisecta Greenm.) is a hemiparasitic herbaceous perennial native to the Pacific Northwest of North America and is considered critically imperilled with only 11 populations remaining in the wild. The main objective of this study was to develop ex situ and in situ conservation through micropropagation and field plantings. In vitro cultures were initiated using nodal explants from two plants raised from seeds collected from a natural population. Shoots were then multiplied on Murashige and Skoog basal medium with 2.0 μmol L⁻¹ 6-benzylaminopurine (BA), 3.0 μmol L⁻¹ kinetin (Kn), 2.2 g L⁻¹ phytagel, and 3% sucrose. Explant position on source plants, culture vessel design, and application of different plant growth regulator levels for BA, Kn, and thiadiazuron (TDZ) were tested to optimize micropropagation protocols. Clones from the plants showed differences in plant height and number of nodes in response to various BA and TDZ concentrations. In vitro shoots were successfully rooted under ex vitro conditions using commercial rooting powder (0.8% indole-3-butyric acid) with an average of ∼17 roots per shoot and acclimatized in the greenhouse with 100% survival rate. Two-month-old plants were transferred to a Parks Canada restoration site at Fort Rodd Hill, Victoria, BC, with 7.5% survival. The use of micropropagation in combination with reintroduction efforts offers an excellent opportunity for conserving endangered plant biodiversity in vitro and facilitating in situ conservation efforts by providing plants for reintroduction.
To obtain benefits of in vitro plant micropropagation, successful acclimatization to ex vitro atmosphere is always required. However, a huge number of in vitro-produced plants cannot survive during ex vitro acclimatization. This study aims to analyze the components that contribute to poor water conservation capacity of in vitro plants after exposure to ambient environment during ex vitro acclimation. Micropropagated shoots grown in vitro (in vitro plants) or after acclimation to greenhouse conditions (greenhouse plants) were microscopically and biochemically analyzed for differences in morphological and biochemical components. Furthermore, dynamic responses of leaves to ex vitro condition were gravimetrically analyzed during 1.5 h desiccation. Compared with greenhouse plants, thinner leaves together with bigger stomata with larger pore area, higher stomatal and epidermal densities were observed in in vitro plants. Plants cultured in vitro kept very high transpiration rate despite of dramatic decrease in leaf RWC during desiccation. Contribution of stomata was more pronounced than the role of cuticule in leaf water loss. Higher concentrations of proline and glycine betaine were observed in the leaves of in vitro plants. Higher (less negative) osmotic potential (ψs) in the leaves of in vitro plants was concurrent with lower levels of potassium and calcium in their leaves. In conclusion, higher compatible solute level in the leaf of in vitro plant does not contribute to water conservation during ex vitro acclimation and low foliar ion levels in in vitro plants can be due to low transpiration rate in plants as a result of in vitro production.
Ex situ conservation of Bulgarian endemic plant Achillea thracica Velen. was achieved by successful in vitro cultivation of mono-nodal segments on MS-B5 medium supplemented with 1.0 mg/L BA for 20 days and subsequent transferring of regenerated plants on hormone free basal MS-B5 medium for root development and accumulation of leaf biomass. In vitro multiplicated plants were successfully acclimated in a growth chamber with 100% survival. GC-MS analysis of the essential oils resulted in the identification of 30, 10 and 28 compounds in in situ grown, in vitro cultivated and ex vitro adapted plants, respectively, constituting 77.7%, 99.9% and 84.1% of the total oils. The wider variety of compounds was found in the essential oils of in situ and ex vitro adapted plants where santolina alcohol, β-eudesmol, 1,8-cineole, germacrene D, α-cadinol and artemisia alcohol were the principal components comprising 68.7% and 69.3 of the oil, respectively. In vitro cultivated plants consist of mainly 1,8-cineole, germacrene D and artemisia alcohol representing 87% of the oil. Different growth conditions affect the composition of essential oils, suggesting their possible involvement in the process of adaptation and surviving in changing environmental conditions.
Tripleurospermum insularum Inceer & Hayırlıoglu-Ayaz sp. nova (Asteraceae, Anthemideae) is described and illustrated. It grows in open places and on rocky slopes in Gökçeada, one of the Aegean Islands. The chromosome number is 2n = 2x = 18. The diagnostic morphological characters that distinguish it from morphologically similar species are discussed and a conservation status is suggested.
The recovery potential of endangered species is limited by the high prevalence of human-modified habitats, while effective in situ conservation strategies to identify and restore disturbed habitat within species ranges are lacking. Our goal was to determine the impact of human disturbance on the endangered endemic Barrens willow (Salix jejuna) to provide science-based protocols for future restoration of disturbed habitats; a key component of conservation and recovery plans for many rare plant species. Our study examined differences in substrate (e.g., % total plant cover, % species cover, substrate type) and vegetation in naturally- (via frost activity) vs human-disturbed limestone barrens (Newfoundland, Canada), across the entire species range of the endangered Barrens willow. There were distinct differences in substrate conditions and vegetation community structure between naturally- and human-disturbed limestone barrens habitat throughout the narrow range of this endemic willow. Human-disturbed sites are more homogeneous and differ significantly from the naturally-disturbed sites having a much coarser substrate (30% more gravel) with less fine grained sands, less exposed bedrock, decreased soil moisture, increased nitrogen content, and reduced phosphorus content. Substrate differences can inhibit return to the natural freeze-thaw disturbance regime of the limestone barrens, negatively affecting long-term persistence of this, and other rare plants. The structure of associated vegetation (specifically woody species presence) negatively affected willow abundance but was not linked to disturbance type. Human-disturbed sites are potential candidates for endangered plant recovery habitat if natural ecosystem processes, vegetation community structure, and habitat heterogeneity are restored, thereby supporting the establishment of long term viable populations.
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Local knowledge about natural resources is becoming increasingly important in defining strategies and actions for conservation of medicinal plants. This study is trying to display the threatened status of medicinal plants of Ajloun heights region; identify the most important factors affecting the plants in their natural habitats. Previous studies summarized the presence of 46 medicinal plant species grown in the study region are still in use in traditional medicine for the treatment of various diseases needed much effort in terms of conservation. The non endangered species (N) are consisting of 31 species; the vulnerable ones (VU) are 5; the endangered medicinal plants (EN) are five species; they are: Alchemilla vulgaris L., Crocus hyemalis Boiss. and Blanche, Pistacia palaestina Boiss., Rubia tinctorum L. and Salvia triloba L.f.; while the critically endangered species (CE) are four species, they are: Eryngium creticum Lam., Majorana syriaca (L.) Raf., Mandragora autumnalis Bertol. and Matricaria aurea Sch. Bip. well-known safe medicinal plants such as Achillea falcata, Matricaria aurea, Majorana syriaca, Allium sativum and Allium cepa. The use of moderately unsafe or toxic plants was noted to be practiced by practitioners and herbalists rather than the locals. Some widely distributed toxic plants include Ecballium elaterium A. Rich., Euphorbia hierosolymitana Boiss., Mandragora autumnalis Bertol., and Citrullus colocynthis. (L.) Schrad. need further care in treatment. Deforestation, agriculture, mining, industrial plantation, timber extracting and wildfires are the most dangerous factors causing the forest loss in Ajlun. It is highly recommended for enactment of an act for the establishment of the traditional medicinal council, which is tasked with the responsibility for the registration of all traditional medicinal practitioners in the country to organize all the activities, is very essential.
Achillea millefolium belonging to the Astreaceae family is an endangered medicinal plant of Jordan and of its neighboring countries. As an alternative to seed propagation, an efficient micropropagation of A. millefolium and its subsequent rooting were developed as an option for its in vitro conservation. A maximum of 5.9 shoots per microshoot were obtained on Murashige and Skoog agar medium supplemented with 0.9 mg L-1 of 6- Benzyl Amino Purine (BAP). The effect of different types and concentrations of auxins were tested, i.e. IBA (Indole-3-Butyric-Acid), IAA (Indole-3-Acetic-Acid) or Naphthalene Acetic Acid (NAA). Maximum root number (20.8 roots ex-plant-1) was obtained from media containing 1.2 mg L-1 of IBA. A survival of 70% was obtained when rooted explants were acclimatized in vivo in equal portions of perlite and peat soil. In vitro, A. millefolium shoots were successfully stored for up to 32 weeks on MS medium supplemented with different concentrations of either sucrose, glucose or fructose, at 24±2°C. After 32 weeks past, 88.6% of the shoots survived on the medium supplemented with 3% sucrose. Moreover, 85.3% of the shoots were able to re-grow when stored under light conditions. Cryopreservation through vitrification was successfully achieved (80% re-growth) when shoot tips precultured on a medium supplemented with 0.4M sorbitol and 0.1M sucrose for 1 day, followed by loading shoot tips with concentrated plant vitrification solution 2 (PVS2) for 20 minutes, then being dehydrated with PVS2 for 60 minutes at 0°C prior to storage in Liquid Nitrogen (LN).
A micropropagation protocol was developed for the conservation of critically endangered Serbian perennial Nepeta rtanjensis (Lamiaceae). Rooted shoots were obtained from one-node stem segments and shoot tips on a half-strength Murashige and Skoog (MS) medium without growth regulators. The best pH of the medium for axillary buds induction and for rooting of shoots was found to be at 7 and/or 7.2 respectively. The addition of cytokinins to the culture medium did not significantly stimulated auxiliary bud production as compared to the control. On the contrary, on media supplemented with high cytokinin concentrations, only dwarf shoots with rudimentary roots were obtained. All tested concentrations of 6-benzylaminopyrine (BAP) and kinetine (Kn) in combination with 0.1 mg l -1 indole-3-acetic acid (IAA) negatively affected the elongation and rooting of shoots. Plants micropropagated on hormone free medium and rooted in vitro were successfully acclimatized in greenhouse and in open field conditions. The result of successful acclimatization was the production of more than 7000 plantlets with normal sexually reproduction. They flowered, fruited and produced seeds which exhibited 47% germination. The survival rate of plants that were transferred to the open field for the acclimatization and exposed to the winter chill was 99%. The reintroduction of N. rtanjensis occurred in May 2004. One thousand plantlets were planted within the historic range of this plant species. The survival rate was also 99%.
In the present study, the germination characteristics of three endemic species from Turkey, Tripleurospermum pichleri (Boiss.) Bornm., Cirsium leucopsis D.C and Senecio olympicus Boiss. (Asteraceae), were investigated. Germination was studied for fresh seeds, for seeds subjected to short-time chilling (15 days, moist +4°C), to GA3 (100, 150 and 250 ppm) and a combination of chilling and GA3; in all cases seeds were incubated either at 20/10°C day/night with light daytime or at 20°C in darkness with daily short-time dim light (DSDL). In C. leucopsis seeds, all GA3 treatments enhanced the final germination percentages. The mean germination time (MGT) of C. leucopsis was lower under DSDL than with photoperiod. The chilling treatment with GA3 in DSDL significantly increased germination in S. olympicus seeds (from 45 to 87%). Germination increased to 55% in T. pichleri by chilling under photoperiod compared with 32% by chilling followed by DSDL. In conclusion, these three co-existing endemic Asteraceae species have different germination behaviours; something that should be taken into account for ex situ propa-gation. However, an efficient way to germinate all species is to use 250 ppm GA3 and 20/10°C with photoperiod.
Chromosome number and morphology in 14 taxa belonging to 19 populations of Tripleurospermum Sch. Bip. were studied using karyological and numerical taxonomical techniques. Data on chromosome measurements were analysed using cluster analysis. Chromosome numbers of these taxa are 2n = 2x = 18, 4x = 36 and 5x = 42–48. Seven records are new, two are not consistent with previous counts, and the remainder confirm the very limited previous data (one to three records). A new ploidy level (pentaploidy) is reported for the first time for the genus. Some correlations between ploidy levels and morphological characters are noted and several systematic and evolutionary aspects of the genus are discussed in the light of karyological data. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 146, 427–438.
A new species of Tripleurospermum Sch.Bip., Tripleurospermum ziganaense Inceer & Hayırlıoglu-Ayaz (Asteraceae, Anthemideae), is described and illustrated. The species grows in open places, on rocky slopes and on roadsides in north-east Anatolia, Turkey. The diagnostic morphological characters that distinguish it from closely related taxa are discussed, and its conservation status is indicated. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 696–700.