Failure of sexual reproduction found in micropropagated critically endangered plants prior to reintroduction: a cautionary tale

Micropropagation is a useful technique for ex situ multiplication and restoration of critically endangered plant species, but the sexual reproductive behaviour of micropropagated plants is seldom evaluated prior to reintroduction. We examined the critically endangered species Rulingia sp. ‘Trigwell Bridge’, with only three remaining plants known in the wild, as a model case to examine this issue. Abnormalities in micropropagated plants of this species related to four floral traits (lengths of sepals, petals and anthers and width of anthers). The number of pollen grains per flower of abnormal individuals was lower than in plants with apparently normal flowers (wild types), but not significantly so (P = 0.068). Pollen viability for the abnormal plant (0.87 ± 0.26%) was significantly lower than for the plants exhibiting wild‐type floral morphology (45.42 ± 4.47%). Experimental manipulations were used to examine the mating behaviour of normal and abnormal plants. The results showed that both male and female reproductive failure was linked to individuals exhibiting abnormal flowering attributes. Such aberrant reproductive performance in a micropropagated rare species predicates caution when using micropropagated plants in reintroduction programmes, highlighting the importance of screening for reproductive normality prior to release of micropropagated plants (especially for critically endangered species where reliance on in vitro propagation methods is often a necessity). © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 165 , 278–284.     

Assessing the Tolerance of Castor Bean to Cd and Pb for Phytoremediation Purposes

This study evaluated Cd and Pb accumulation by castor bean (Ricinus communis cv. Guarany) plants grown in nutrient solution, aiming to assess the plant’s ability and tolerance to grow in Cd- and Pb-contaminated solutions for phytoremediation purposes. The plants were grown in individual pots containing Hoagland and Arnon’s nutrient solution with increasing concentrations of Cd and Pb. The production of root and shoot dry matter and their contents of Cd, Pb, Ca, Mg, Cu, Fe, Mn, and Zn were evaluated in order to calculate the translocation and bioaccumulation factors, as well as toxicity of Cd and Pb. Cadmium caused severe symptoms of phytotoxicity in the plant’s root and shoot, but no adverse effect was observed for Pb. Castor bean is an appropriate plant to be used as indicator plant for Cd and tolerante for Pb in contaminated solution and it can be potentially used for phytoremediation of contaminated areas.     

Oviposition specificity and behavior of the watermilfoil specialist Euhrychiopsis lecontei

Eurasian watermilfoil (Myriophyllum spicatum L.) is a nuisance aquatic weed, exotic to North America. The freshwater weevil Euhrychiopsis lecontei (Dietz) is a potential control agent of Eurasian watermilfoil and is a fully submersed aquatic specialist herbivore. Its presumed original host is the native northern watermilfoil (Myriophyllum sibiricum Komarov). We conducted a set of oviposition experiments to reveal first and second oviposition preference of Euhrychiopsis lecontei when presented with seven macrophytes. We tested differences between source (lake) populations of weevils, differences in behavior between weevils reared on the exotic Eurasian watermilfoil and the native northern watermilfoil and between weevils in the presence and absence of their preferred hostplant. Oviposition assays confirmed that E. lecontei is a watermilfoil specialist. Out of the 207 females that laid eggs, only three oviposited on a non-watermilfoil plant, Megalodonta beckii. The weevils' degree of specificity was influenced by the watermilfoil species on which they were reared. Weevils reared on Eurasian watermilfoil tended to oviposit on Eurasian watermilfoil, spent more time on Eurasian watermilfoil than on other plants, and spent more time off plants and took longer to oviposit when Eurasian watermilfoil was removed. Weevils reared on northern watermilfoil did not exhibit a preference for either watermilfoil species in oviposition or in time allocation, although they oviposited on and spent significantly more time on watermilfoils than on other species. Rearing of the two populations on their complementary watermilfoil hostplant resulted in responses typical of the rearing plant, not the original host. These results show that although both weevil populations are watermilfoil specialists, Eurasian-reared weevils prefer Eurasian watermilfoil in general host attraction and oviposition, whereas northern-reared weevils do not. The results support the contention that E. lecontei may be a good biocontrol agent for Eurasian watermilfoil because of its high specificity. The results also suggest that the current host range expansion of the weevil to Eurasian watermilfoil has the potential to become a host shift due to the increased specificity. Herbivory in freshwater systems is not well studied, and the E. lecontei-M. spicatum relationship is a rare example of submersed freshwater specialist herbivore-host-plant interactions.     

An assessment of genetic fidelity of micropropagated plants of Chlorophytum borivilianum using RAPD markers

Rapid micropropagation was achieved in Chlorophytum borivilianum Santapau and Fernandes using shoot base as explants. Multiple shoots were induced on Murashige and Skoog’s (MS) medium supplemented with 3.0 mg dm⁻³ 6-benzylaminopurine, 0.1 mg dm⁻³ 1-naphthaleneacetic acid, 150 mg dm⁻³ adenine sulphates and 3 % saccharose. Rooting was readily achieved upon transferring the shoots onto half strength MS medium supplemented with 0.1 mg dm⁻³ indolebutyric acid and 2 % saccharose. Micropropagated plantlets were hardened in the greenhouse and successfully established in soil. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic stability of the micropropagated plants. Thirty one arbitrary decamers were used to amplify genomic DNA from in vitro and in vivo plant material to assess the genetic stability. All RAPD profile analysis from micropropagated plants was genetically similar to mother plants.     

Random amplified polymorphic DNA (RAPD) markers for genetic analysis in micropropagated plants of Populus deltoides Marsh

RAPD markers were used to assess genetic fidelity of 23 micropropagated plants of a single clone (L34) of Populus deltoides. Eleven arbitrary 10-base primers were successfully used to amplify DNA from in vivo and in vitro material. Of these, 5 distinguished a total of 13 polymorphisms common across 6 micropropagated plants. Apart from these 6 plants, the amplification products were monomorphic across all the micropropagated plants, the mother plant and 4 additional field-grown control plants. Our results show that RAPD markers can be used to gain rapid and precise information about genetic similarities or dissimilarities in micropropagation systems that might not be so easily evident from other commonly used techniques.     

Monitoring of cultivar identity in micropropagated olive plants using RAPD and ISR markers

Randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers were applied to assess the genetic stability of micropropagated olive (Olea europaea L. cv. Maurino) plants regenerated by axillary buds. Initial olive explants, isolated from one donor tree, were multiplied on Murashige and Skoog medium for 12 repeated subcultures. A total of 40 RAPD and 10 ISSR markers resulted in 301 distinct and reproducible band classes showing homogeneous RAPD and ISSR patterns. The amplification products revealed genetic stability among the micropropagated plants and between them and the donor plant. The results demonstrate the genetic stability of nine year old mature micropropagated olive plants cultured in field, and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.     

An Assessment of Genetic Integrity of Micropropagated Plants of Plumbago Zeylanica by RAPD Markers

Clones of Plumbago zeylanica were micropropagated using nodal culture. The application of random amplified polymorphic DNA (RAPD) in assessing the genetic integrity of the micropropagated plants was evaluated by polymerase chain reaction. Twenty arbitrary decamers were used to amplify genomic DNA from in vitro and in vivo plant material to assess the genetic fidelity. All RAPD profiles from micro-propagated plants were monomorphic and similar to those of field grown mother plants. No polymorphism was detected within the micropropagated plants.     

Plant regeneration of <Emphasis Type="Italic">Erigeron breviscapus</Emphasis> (vant.) Hand. Mazz. and its chromatographic fingerprint analysis for quality control

An efficient micropropagation system for Erigeron breviscapus (vant.) Hand. Mazz., an important medicinal plant for heart disease, has been developed. Shoot organogenesis occurred from E. breviscapus leaf explants inoculated on a medium supplemented with a combination of plant growth regulators. On average, 17 shoots per leaf explant were produced after 30 days when they were cultured on MS basal salts and vitamin medium containing 5 μM 6-benzylaminopurine (BAP) and 5 μM 1-naphthaleneacetic acid (NAA). All the regenerated shoots formed complete plantlets on a medium containing 2.5–10 μM indole-3-butyric acid (IBA) within 30 days, and 80.2% of the regenerated plantlets survived and grew vigorously in field conditions. Based on the variation in common peaks and the produced amount of the most important bioactive component, scutellarin, a high performance liquid chromatography (HPLC) fingerprinting system was developed for quality control of these micropropagated plants. Chemical constituents in E. breviscapus micropropagated plants varied during plant development from regeneration to maturation, the latter of which showed the most similar phytochemical profile in comparison with mother plants. The regeneration protocol and HPLC fingerprint analysis developed here provided a new approach to quality control of micropropagated plants producing secondary metabolites with significant implications for germplasm conservation.     

If you build it,will they come? Use of restored dunes by beach mice

Restoration of coastal habitat fragmented, degraded, or destroyed by development and climate‐related processes such as sea level rise and storm surge usually involves planting native plants to restore habitat structure, but whether and how restored areas benefit taxa other than plants is rarely reported. Installing restoration plantings is one method used to build habitat such as beach dunes where dunes have been lost, potentially creating habitat for dune‐dependent species. We compared use of natural vegetated dunes, open sand gaps, and restoration plantings (habitat treatment) by Perdido Key beach mice (Peromyscus polionotus trissyllepsis) over 3 years using tracking tubes to assess the value of restoration plantings for beach mice. Tubes were monitored in two seasons (early and mid‐summer), and under new and full moon conditions. Mice used restoration plantings less than natural vegetated dunes but more than open sand gaps, which suggests restoration plantings may facilitate movement of mice across fragmented areas. Both season and moon phase influenced the effect of habitat treatment, interactions which may be attributable to perceived risk associated with movement under a combination of different conditions of ambient light, vegetation cover, and habitat novelty. Our results show restoration plantings provide habitat for movement and foraging, and may ameliorate some consequences of sea level rise and storms for beach mice and potentially other dune‐dependent species into the future.     

Rapid clonal propagation of Saccharum officinarum L. Vars. CO-6907 and CO-86249 and to assess the genetic uniformity through molecular markers

Abstract

An efficient protocol was developed for in vitro clonal propagation of Saccharum officinarum Vars. CO-6907 and CO-86249 through axillary meristem culture. Maximum meristem elongation was achieved on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kn) within 15 days of culture. Multiple shoots were induced from meristems on MS basal medium supplemented with 1.0 mg/L BA, 0.5 mg/L Kn, 0.25 mg/L 1-napthaleneacetic acid (NAA) and 3% (w/v) sucrose. Addition of 0.1–0.25 mg/L gibberellic acid into the multiplication medium found the better shoot elongation. Repeated subculture on multiplication medium induces higher rate of shoot multiplication. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 1.0–2.0 mg/L NAA or indole-3-butyric acid and 6% (w/v) sucrose. While either decreasing or increasing of sucrose concentration in the rooting medium, the percentage of rooting was reduced. Maximum percentage of rooting was achieved on medium having 2.0 mg/L NAA with 6% (w/v) sucrose. About 80% of micropropagated plantlets were hardened in the greenhouse and successfully established in the soil. Random Amplified Polymorphic DNA marker was used to detect the variability among the micropropagated plants developed through in vitro. The results showed that there was no polymorphism among the micropropagated plants. This study will help for propagation of quality planting material of high-yielding variety of sugarcane for commercialization.     

Assessment of genetic stability of two micropropagated wild olive species using flow cytometry and microsatellite markers

Micropropagated plants from two wild-olive species, Olea maderensis and O. europaea ssp. europaea var. sylvestris were screened for genetic stability. O. maderensis shoots were elongated/multiplied on OMG medium with zeatin (9.12 μM), and rooted on 1/2 OMG with NAA (3.22 μM). O. europaea var. sylvestris shoots were elongated/multiplied on OM medium with zeatin, and rooting was optimal after a hormonal shock (IBA 100 μM) followed by transfer to the same medium without growth regulators. In both species, acclimatization was successful and plants looked normal and morphologically identical to the donor field trees. Genetic variability was assessed at several stages of the micropropagation process using flow cytometry (FCM) and nuclear microsatellites (SSR). No changes in ploidy level were found among micropropagated plants, though small deviations, putatively due to the negative effects of cytosolic compounds on propidium iodide staining, between these and field plants were observed. In SSRs analyses, ten SSR markers were able to distinguish between genotypes. No mutations were found in these tested SSR loci among the donor tree and micropropagated plants, suggesting, for the tested markers, genetic uniformity throughout the process. The FCM and SSR results obtained do not exclude the occurrence of other changes in the nuclear genome but, considering the morphological stability of micropropagated plants, indicate that both protocols are suitable and efficient for large scale, true-to-type micropropagation of these two wild olive species.     

Using Macrophytes in Urban Stream Rehabilitation: A Cautionary Tale

Native macrophytes were transplanted into a small urban stream as part of a rehabilitation program, that also meandered the previously channeled stream, naturalized stream banks, and planted native riparian vegetation. Transplanted macrophytes minimized spread of introduced macrophytes and were viewed beneficially by residents, as was the stream rehabilitation. We transplanted the native macrophyte Myriophyllum triphyllum into five larger streams dominated by exotic macrophytes—some of which were weeded prior to transplanting—to see whether Myriophyllum could prevent regrowth of weeded plants. Transplanted Myriophyllum plants were washed away in two streams, reflecting high shear stresses there. Myriophyllum cover in the other streams decreased as weeded plants regrew. Our attempt at eliminating exotic macrophytes in patches in large streams was unsuccessful. Furthermore, council authorities weeded other experimental sections following complaints from residents of excess macrophyte growth. This problem highlighted conflicting multiple values placed on urban streams by managers and the public. A repeat survey of residents living near the original rehabilitated stream showed that many respondents were now critical of excessive plant growth—both in‐stream and riparian. A recurring comment made concerned the apparent lack of maintenance to the stream, giving it an untidy appearance. Difficulties with propagating and transplanting native macrophytes into larger streams, coupled with a negative perception of native vegetation (both in‐stream and riparian) if it looks unmanaged, suggest that planting macrophytes or riparian plants as part of urban stream rehabilitation programs may be more problematic than realized.     

Determination of genetic stability of long-term micropropagated plantlets of Platanus acerifolia using ISSR markers

Inter-simple sequence repeat (ISSR) markers were used to assess the genetic stability of long-term micropropagated plantlets of London plane tree (Platanus acerifolia Willd.). Twenty micropropagated plantlets were chosen from a clonal collection of shoots that originated from a single mother shoot. This clonal collection had been maintained under in vitro culture conditions for at least 8 years, as achieved by axillary branch multiplication. Out of 38 ISSR primers screened, 16 primers were found to produce clear reproducible bands resulting in a total of 103 distinct bands with an average of 6.44 scorable bands per primer. Of these 103 bands, 86 were monomorphic across all 20 of the plants tested and 17 showed polymorphisms (16.5 % polymorphism). Based on the ISSR band data, similarity indices between the plantlets ranged from 0.92 to 1.00. These similarity indices were used to construct an UPGMA dendrogram and demonstrated that all 20 micropropagated plants grouped together in one major cluster with a similarity level of 91 %. A total of 1771 scorable bands were obtained from the full combination of primers and plantlets and only 51 (2.88 %) were polymorphic across the plantlets which indicates that this micropropagated line of P. acerifolia is genetically stable.     

Epigenetic instability in genetically stable micropropagated plants of <Emphasis Type="Italic">Gardenia jasminoides</Emphasis> Ellis

Gardenia jasminoides Ellis is an evergreen tropical plant and favorite to gardeners throughout the world. Several studies have documented that in vitro micropropagation can be used for clonal propagation of G. jasminoides Ellis, the efficiency remained low. In addition, no information is available on the genetic and epigenetic fidelity of the micropropagated plants. Here, we report on a simplified protocol for high efficient micropropagation of G. jasminoides Ellis cv. “Kinberly” based on enhanced branching of shoot-tips as explants. The protocol consisted of sequential use of three media, namely, bud-induction, elongation and root-induction. By using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation sensitive amplified polymorphism (MSAP), we analyzed the genetic and DNA methylation pattern stability of 23 morphologically normal plants randomly taken from a sub-population (>100) of micropropagated plants originated from a single shoot-tip. We found that of >1,000 scored AFLP bands across the 23 micropropagated plants, no incident of genetic variation was detected. In contrast, of 750 scored MSAP bands, moderate but clear alteration in several DNA methylation patterns occurred in the majority of the 23 micropropagated plants. The changed methylation patterns involved both CG and CHG sites representing either hyper- or hypo-methylation, which occurred without altering the total methylation levels partly due to concomitant hyper- and hypo-methylation alterations. Our results indicated that epigenetic instability in the form of DNA methylation patterns can be susceptible to the in vitro micropropagation process for G. jasminoides Ellis, and needs to be taken into account in the process of large-scale commercial propagation of this plant.     

Vegetative propagation of adult Eucalyptus grandis X urophylla and comparison of growth between micropropagated plantlets and rooted cuttings

Methods for the production of micropropagated plantlets and rooted cuttings were developed and used to vegetatively multiply adult Eucalyptus grandis X urophylla. Rooting success was less than 5% when cuttings excised from twigs of 3-year-old trees were used. The rooted cuttings were grown in the greenhouse as explant- or cutting-donors and maintained at a height of 30 to 100 cm by trimming back periodically. Good rooting success (95%) of cuttings was obtained for epicormic shoots produced from donor plants after trimming 5 times. Explants of both apical and axillary buds taken from the donor plants produced multiple shoots when cultured in vitro. In vitro multiple shoot production was optimal on MS medium containing 0.1 mg/l BA and 0.01 mg/l NAA averaging 13.7 shoots per explant in a 40-day culture period. Shoot elongation was accelerated on a modified MS medium containing half strength potassium nitrate and sucrose. Elongated shoots excised at approximately 1.5 cm in length were successfully rooted on media with NAA or IBA concentrations ranging from 0.1 to 10 mg/l. Root formation was optimal on medium consisting of full strength MS basal macro elements and vitamins, half strength micro elements, 1% sucrose and supplemented with 0.3 mg/l IBA. In the field test, no significant differences were found in tree height and DBH between micropropagated plantlets and rooted cuttings at 1 and 3 years old, with the exception at 2 years old. A considerable difference arose between the 2 types of vegetative propagules in physiological response to flowering, caused by dissimilar degrees of rejuvenation.Abbreviations BA Benzyl-Aminopurine - NAA Naphthalene Acetic Acid - IBA Indole-3-Butyric Acid - MS medium Murashige and Skoog's medium - DBH Diameter at Breast Height     

Effects of thidiazuron on micropropagation of rose

Summary Rose (Rose hybrida L.) plants were micropropagated by axillary shoot proliferation method. Maximum number of microshoots per shoot tip explant were obtained on MS medium supplemented with 5 to 10&#x00B5;M thidiazuron (TDZ). The microshoots formed rooted plants on MS hormone-free medium. No difference in the rooting of microshoots produced on medium containing TDZ or N⁶-benzyladenine was observed. The regenerated plants were successfully transplanted to the field and appeared similar to the parent plant in morphologic features.     

Effect of ectomycorrhizal fungi on survival and growth of micropropagated plants and seedlings of Castanea sativa mill.

 Four ectomycorrhizal fungi (Amanita muscaria, Laccaria laccata, Piloderma croceum and Pisolithus tinctorius) were used to produce mycorrhiza on seedlings and micropropagated plants of Castanea sativa in vitro. Pisolithus tinctorius was most effective in colonizing roots of both micropropagated plants and seedlings. A. muscaria and L. laccata only colonized a few feeder roots of some plants and Piloderma croceum did not form mycorrhizas. Mycorrhization of micropropagated plants increased survival and growth during weaning. Accepted: 27 February 1996     

Detection of genetic variation among micropropagated tea [Camellia sinensis (L.) O. Kuntze] by RAPD analysis

Summary Randomly amplified polymorphic DNA (RAPD) techniques were applied to assess genetic instability among micropropagated tea [Camellia sinensis (L.) O. Kuntze] eultivar ‘T-78’. Out of 49 random 10-mer primers, 11 generated polymorphism in four out of 17 micropropagated plants and one mother plant. A total of 221 bands, ranging from 525 bp to 2.5 kb, were produced by the 49 primers. Twenty-four were polymorphic for those four plants. However, the remaining bands were monomorphic among all plants. Polymorphism among those four plants showed an identifical banding pattern suggesting the occurrence of a single mutation. Our results demonstrated that RAPD can be used successfully to determine the genetic instability among micropropagated plants which otherwise were morphologically indistinguishable.     

Micropropagation of <Emphasis Type="Italic">Sapindus trifoliatus</Emphasis> L. and assessment of genetic fidelity of micropropagated plants using RAPD analysis

An efficient in vitro propagation system has been developed for rapid micropropagation of Soapnut (Sapindus trifoliatus Linn.), a medicinally and economically important tree from nodal (axillary bud) segments of seedlings. The frequency of shoot regeneration from seedling node explant was influenced by the age of the seedlings, growth regulators and successive transfer of the mother explant. Explants from 4-week-old seedlings yielded the maximum shoot regeneration frequency (97.22%) on full-strength MS medium supplemented with 1.0 mg l⁻¹ of 6-benzylaminopurine (BAP). After harvesting the newly formed shoots, the mother explants transferred to same medium subsequently produced a maximum of 5.16 shoots per explant after third passage. Further improvement in the morphogenic response occurred when the nodal explants excised from in vitro regenerated shoots were employed, and 6.89 shoots per explant were obtained on the same medium after the third subculture. Optimal rooting (91.67%) was obtained by placing the micro-shoots in liquid MS medium with 1.0 mg l⁻¹ IBA for 24 h and then transferring to the agar solidified MS medium devoid of IBA. The micropropagated shoots with well-developed roots were acclimatized and successfully transplanted to soil with 90% survival rate. Genetic stability of the regenerated plants was assessed using random amplified polymorphic DNA (RAPD). The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic integrity of micropropagated plants. This is the first report of an efficient protocol for regeneration of S. trifoliatus through organogenesis, which can be applied for further genetic transformation assays and pharmaceutical purposes.     

A study on the characteristics of urban ecosystems and plans for the environment and ecosystem in Gangnam-gu,Seoul, Korea

The aim of this study is to establish a plan for the environment and ecosystem by studying the characteristics of urban ecosystems, involving one of Seouls districts for environment-friendly urban management. Biotope type in Gangnam-gu was classified into six large groups: forest, planted area, grassland, stream and wetland, cultivated land and urbanization area. Then the six groups were again divided into 22 small groups on the basis of composition of species, naturalness, diversity of stratification and scarcity. Filicales and Disporum smilacinum communities, moisture-loving native plants which are well worth conserving, were distributed widely throughout the northern slope of Mt. Daemosan, an outer mountain in Gangnam-gu. In contrast, Ageratina altissima, one of the naturalized plants in Seoul, was found to spread over Mt. Maebongsan and the Cheongdam neighborhood park, residual mountain-type parks located along the northern part of the Yangjaecheon stream. The southern part of the stream was found to be used as a route for woodpeckers and small wild birds, since the districts residual mountain type park was connected to large outer forest whose naturalness was in good condition. Under the plan for ecosystem conservation and restoration, conservation and restoration areas with highly valuable conservation biotopes were selected. Under the plan for ecosystem networking, a natural corridor and a green corridor were designed to connect greenspaces for the migration of woodpeckers. The plan for enlarging the greenspaces has a short-, mid- and long-term plan for areas that are need to secured greenspaces. The plan was set after comparing greenspaces in each block within the district.