Effects of several establishment modes of Miscanthus × giganteus and Miscanthus sinensis on yields and yield trends

Miscanthus is a C4 perennial grass originating from East Asia, the yields of which progressively increase in the first years of growth. Several species for bioenergy have been studied since the mid‐1980s in Europe, in particular (Miscanthus × giganteus [M. × giganteus]), due to its high yields. M. × giganteus is mainly cultivated in France and established from rhizomes. Our study aimed to assess, in field conditions, alternative establishment methods combined with an alternative species, Miscanthus sinensis (M. sinensis). We set up a multi‐environment experimental network. On each trial, we tested two treatments with M. × giganteus, established from rhizomes (G_r‐sd) and from plantlets obtained from rhizomes (G_p‐sd), and two treatments with M. sinensis seedlings transplanted in single (S_p‐sd) and double density (S_p‐dd). ANOVA was performed to compare establishment and regrowth rates across treatments, as well as yields across treatments and site‐years. A logistic model was used to describe yield trends and to compare the maximum yield reached and the rate of yield increase of both species. Results showed that miscanthus establishment from plantlets resulted in higher establishment (between 87% and 92%) and regrowth (between 91% and 94%) rates compared to establishment from rhizomes. Treatments with M. × giganteus obtained higher average yields across site‐years than those with M. sinensis, but more variable yields across site‐years. We showed a strong species effect on yields, yield components (shoot weight, shoot density and shoot number per plant) and light interception (through leaf area index). Lastly, to use M. sinensis established from transplanted plantlets as an alternative to M. × giganteus, research would be required on the breeding of M. sinensis sterile seeds to avoid risks of invasiveness.     

Spatiotemporal patterns of shoots within an isolated <Emphasis Type="Italic">Miscanthus sinensis</Emphasis> patch in the warm-temperate region of Japan

The shoot configuration of each monoclonal patch of phalanx-forming tallgrass, Miscanthus sinensis, is characterized by the formation of a fairy ring, which forms as the result of developing vacant inner areas. One large-sized M.sinensis patch (patch L), observed over a 9-year survey period, underwent lateral expansion in almost all directions as a result of peripheral shoot births. In the year after the shoots in each part of patch L reached a maximum density (D_max), the number of shoots decreased by approximately 20% per year. However, the overall number of within-patch shoots was stable during the survey because the patch area increased at the periphery. Twelve patches (>900cm² in area) with orthotropic shoots were selected to observe the distribution pattern of within-patch shoots, and the patch areas were divided into three parts: the exterior, intermediate and interior areas. In 10 of these 12 patches, shoot densities were lowest in the interior areas and highest in the exterior areas, which led to ring formation. The shoot density of each subarea was inversely related to the age of the subarea. This raises the possibility that in any part of these patches, shoot densities decrease annually from D_max in a similar way.     

Clonal Plasticity in Response to Reciprocal Patchiness of Light and Nutrients in the Stoloniferous Herb Glechoma longituba L.

Interconnected ramets of clonal plants can functionally specialize in the uptake of resources from aboveground and/or underground sources. Ramet pairs of the clonal stoloniferous herb Glechoma Iongltuba L. were grown In spatially heterogeneous environments with complementary availability of light and nutrients. Plasticity with respect to root-shoot ratio, fitness-related traits （biomass, number of ramets and dry weight per ramet）, morphological traits （lamina area, root length） were experimentally examined. The aim was to understand the adaptation of G. Iongltuba to an environment with reciprocal patchiness of light and soil nutrients by plasticity In Its root-shoot ratio and clonal morphology. The results showed that the performance of ramets growing In patches with high light Intensity and low soil nutrients into the adjacent opposite patches was Increased in terms of fitness-related traits. However, the performance of those from patches with low light Intensity and high soil nutrients into the adjacent opposite patches was not changed. The root-shoot ratio and clonal morphology were plastic. Morphological traits such as lamina area and root length were altered In a way that enhanced the capture of light resources and soil nutrients. Apparent reciprocal resource transport between the ramets In an environment of reciprocal patchiness of resources can enhance the growth of ramets with complementary resource deficiencies.     

Growth of clonal modules on <i>Agropyron michnoi</i> in the Songnen Plain of Northeast China

Spatial expansion of clonal plants and growth of their modules are of concern in the field of plant ecology. After measuring a large number of samples, we analyzed the module components and the growth patterns of vegetatively propagated Agropyron michnoi clones in the Songnen Plain on Northeast China. The results showed that the plasticity of clonal growth was large; the coefficients of variation of both extensive areas and the quantitative characters of modules were more than 20%. The numbers of ramets, seedlings, and buds and the cumulative length of the rhizomes showed exponentially and linearly increasing patterns with increases of the area and the total number of modules. The biomass of each module, total number of modules and total biomass showed an allometric growth pattern, which was best described by power functions. For A. michnoi, there was a relatively stable investment to sexual reproduction; it showed a priority for allocating biomass to reproductive ramets, and also to rhizomes and buds formation.     

Ramet phenology and clonal architectures of the clonal sedge <Emphasis Type="Italic">Schoenoplectus americanus</Emphasis> (Pers.) Volk. ex Schinz &; R. Keller

The clonal plant Schoenoplectus americanus shows variable belowground clonal architecture as a result of producing two types of ramets: those with very long rhizomes (long rhizome ramet, LRR) and those with very short ones (short rhizome ramet, SRR). In a previous study we demonstrated that the two types of ramets are functionally specialised. The production of SRRs results in the formation of consolidated clonal patches with densely packed shoots, while the production of LRRs results in a more diffuse network of connected rhizomes with widely spaced shoots. We hypothesised that the two types of ramets would be produced at different times during the growing season because of their functional differences. The production of LRRs throughout the growing season would enable the species to continuously explore new habitats while the production of SRRs early in the growing season would enable the species to occupy and consolidate resources in available open patches. We evaluated this hypothesis through field observations in different communities with S. americanus and indeed found that SRRs were produced early in the growing season while LRRs tended to be produced over an extended period of time. Plants in high-quality environments (i.e. higher light conditions) produced more SRRs, and these were formed early in the growing season. In contrast, plants in low-quality environments produced more LRRs, and these were formed continuously over the growing season. We also observed that the shoot longevity was greater for SRR. In high-quality patches, the production of the lower cost SRRs results in a more rapid occupancy of open spaces; in lower quality patches, the production of LRRs throughout the growing season enables plants to explore the immediate environment for higher quality patches.     

Shoot population dynamics of persisting clones of<Emphasis Type="Italic"> Miscanthus sinensis</Emphasis> in the warm-temperate region of Japan

The population dynamics of annual shoots in four clonal patches of Miscanthus sinensis was surveyed in terms of clone persistence. Over a study period of 3 years, a stable net shoot number was attained through a balanced replacement of old shoots by tillering 2–3 times a year. The birth rate was significantly correlated with the average monthly temperature. This suggests that a warm climate advances the date of tillerings, and shoots become taller as they emerge earlier. Five cohorts were identified, corresponding to shoots that were born in spring, early summer, summer, autumn and early winter. The autumn cohorts were more numerous and had a greater longevity than the other cohorts; they were thus were best suited for overwintering and, consequently, maintaining the population from one generation to the next. The shoots of earlier cohorts grew too tall to survive the cold of winter, which might cause freezing injury, while later cohorts shoots excelled in overwintering because of their shortness. However, if the delayed shoots are very short, they will also have difficulty in surviving the early season of growth. Therefore, M. sinensis shoots have an optimum size for survival.     

Plasticity in R/S ratio, morphology and fitness-related traits in response to reciprocal patchiness of light and nutrients in the stoloniferous herb, Glechoma longituba L

Clonal fragments of the stoloniferous herb Glechoma longituba were subjected to a complementary patchiness of light and soil nutrients including two spatially homogeneous treatments (SR–SR and IP–IP) and two spatially heterogeneous treatments (IP–SR and SR–IP). SR and IP indicate patches (shaded, rich) with low light intensity (shaded, S), high nutrient availability (rich, R) and patches (illuminated, poor) with high light intensity (illuminated, I) and low nutrient availability (poor, P), respectively. Plasticity of the species in root–shoot ratio, fitness-related traits (biomass, number of ramets and dry weight per ramet) and clonal morphological traits (length and specific length of stolon internodes, area and specific area of laminae, length and specific length of petioles) were experimentally examined. The aim is to understand adaptation of G. longituba to the environment with reciprocal patches of light and soil nutrients by plasticities both in root–shoot ratio and in (clonal) morphology. Our experiment revealed performance of the clonal fragments growing from patches with high light intensity and low soil nutrient availability into the adjacent opposite patches was increased in terms of the fitness-related characters. R/S ratio and clonal morphology were plastic. Meanwhile, the capture of light resource from the light-rich patches was enhanced while the capture of soil nutrients from either the nutrient-rich or the nutrient-poor patches was not. Analysis of cost and benefit disclosed positive effects of clonal integration on biomass production of ramets in the patches with low light intensity and high soil nutrient availability. These results suggest an existence of reciprocal translocation of assimilates and nutrients between the interconnected ramets. The reinforced performance of the clonal fragments seems to be related with specialization of clonal morphology in the species.     

Edaphic environment, gall midges, and goldenrod clonal expansion in a mid-successional old-field

During the middle stage of old-field succession, genets of clonal plants vie to take over space from annual and short-lived perennial plants. We studied factors that may influence the relative rates of expansion of Solidago altissima genets in an old-field population attacked by the gall midge Rhopalomyia solidaginis. Genets growing in more clayey soil expanded more slowly, as evidenced by differences in rhizome growth. Edaphic conditions also affected galling frequencies, with genets in more sandy soil having twice as many galls. Gall midges reduced goldenrod stem growth, and stem height was positively correlated with rhizome growth. For a given stem height, galled ramets allocated relatively more biomass to rhizome growth than ungalled ramets. The end result was that galled ramets produced the same number and sizes of rhizomes as ungalled ramets.     

Radiation capture and conversion efficiencies of Miscanthus sacchariflorus,M. sinensis and their naturally occurring hybrid M. × giganteus

Miscanthus is a rhizomatous C4 grass of great interest as a biofuel crop because it has the potential to produce high yields over a wide geographical area with low agricultural inputs on marginal land less suitable for food production. At the moment, a clonal interspecific hybrid Miscanthus × giganteus is the most widely cultivated and studied in Europe and the United States, but breeding programmes are developing newer more productive varieties. Here, we quantified the physiological processes relating to whole season yield in a replicated plot trial in Wales, UK. Light capture and conversion efficiency were parameterized for four carefully selected genotypes (M. sinensis, M. sacchariflorus and Miscanthus × giganteus). Differences in the canopy architecture in mature stands as measured by the extinction coefficient (k) were small (0.55–0.65). Sensitivity analysis on a mathematical model of Miscanthus was performed to quantify the accumulative intercepted photosynthetically active radiation (iPAR) in the growing season using (i) k, (ii) variation in the thermal responses of leaf expansion rate, (iii) base temperature for degree days and (iv) date start of canopy expansion. A 10% increase in k or leaf area per degree day both had a minimal effect on iPAR (3%). Decreasing base temperature from 10 to 9 °C gave an 8% increase in iPAR. If the starting date for canopy expansion was the same as shoot emergence date, then the iPAR increases by 12.5%. In M. × giganteus, the whole season above ground and total (including below ground) radiation‐use efficiency (RUE) ranged from 45% to 37% higher than the noninterspecific hybrid genotypes. The greater yields in the interspecific hybrid M. × giganteus are explained by the higher RUE and not by differences in iPAR or partitioning effects. Studying the mechanisms underlying this complex trait could have wide benefits for both fuel and food production.     

‘Collar propagation’ as an alternative propagation method for rhizomatous miscanthus

The demand for perennial nonfood crops, such as miscanthus, is increasing steadily, as fossil resources are replaced by biomass. However, as the establishment of miscanthus is very expensive, its cultivation area in Europe is still small. The most common propagation method for miscanthus is via rhizomes, the harvesting of which is very labour‐intensive. Seed propagation is promising, but not suitable for sterile genotypes. In this study, a new vegetative propagation method, ‘collar propagation’, was tested in field and controlled environment studies. Collars are built at the junction between rhizome and stem. They can be harvested in a less destructive way than rhizomes by pulling out the stems from winter‐dormant miscanthus plants. One genotype of each of the species M. sacchariflorus, M. × giganteus, M. sinensis in combination with three fragment types (collars, rhizomes, collars + rhizomes) were tested for establishment success and plant performance. The performance (e.g. dry matter yield) of collar‐propagated plants was either better than or not significantly different from rhizome‐propagated plants. Pregrown plantlets transplanted into the field showed no significant differences in establishment success between the fragments within a genotype. When directly planted into the field however, the fragment ‘rhizome+collar’ had a significantly better establishment success than the other two. The winter survival rate of the fragment ‘rhizome+collar’ was 70% for M. sacchariflorus and 75% for M. × giganteus. Emergence success from collar‐derived plants was not affected by harvest date (harvested monthly from November to February). This study showed that miscanthus propagation via collars is feasible and a promising alternative to rhizome propagation, as the multiplication rate of collars is comparable to that of rhizome propagation. Collar propagation is the more suitable method for the tested genotypes of the species M. sachariflorus and M. × giganteus, but not for M. sinensis genotypes, which may be better propagated by seeds.     

Growth and fecundity of fertile Miscanthus × giganteus (“PowerCane”) compared to feral and ornamental Miscanthus sinensis in a common garden experiment: Implications for invasion

Perennial grasses are promising candidates for bioenergy crops, but species that can escape cultivation and establish self‐sustaining naturalized populations (feral) may have the potential to become invasive. Fertile Miscanthus × giganteus, known as “PowerCane,” is a new potential biofuel crop. Its parent species are ornamental, non‐native Miscanthus species that establish feral populations and are sometimes invasive in the USA. As a first step toward assessing the potential for “PowerCane” to become invasive, we documented its growth and fecundity relative to one of its parent species (Miscanthus sinensis) in competition with native and invasive grasses in common garden experiments located in Columbus, Ohio and Ames, Iowa, within the targeted range of biofuel cultivation. We conducted a 2‐year experiment to compare growth and reproduction among three Miscanthus biotypes—”PowerCane,” ornamental M. sinensis, and feral M. sinensis—at two locations. Single Miscanthus plants were subjected to competition with a native grass (Panicum virgatum), a weedy grass (Bromus inermis), or no competition. Response variables were aboveground biomass, number of shoots, basal area, and seed set. In Iowa, all Miscanthus plants died after the first winter, which was unusually cold, so no further results are reported from the Iowa site. In Ohio, we found significant differences among biotypes in growth and fecundity, as well as significant effects of competition. Interactions between these treatments were not significant. “PowerCane” performed as well or better than ornamental or feral M. sinensis in vegetative traits, but had much lower seed production, perhaps due to pollen limitation. In general, ornamental M. sinensis performed somewhat better than feral M. sinensis. Our findings suggest that feral populations of “PowerCane” could become established adjacent to biofuel production areas. Fertile Miscanthus × giganteus should be studied further to assess its potential to spread via seed production in large, sexually compatible populations.     

Expansion of <Emphasis Type="Italic">Phragmites australis</Emphasis> (Cav.) Trin. ex Steud. (Common Reed) into <Emphasis Type="Italic">Typha</Emphasis> spp. (Cattail) Wetlands in Northwestern Indiana,USA

Expansion of Phragmites australis (Cav.) Trin. ex Steud. (common reed) into stands of Typha spp. (cattail; Typha australis L. and T. x glauca) is common in the wetlands of northwestern Indiana (USA). To understand this phenomenon better, we investigated the production of shoot sprouts and proportional allocation of biomass as well as a potential role for the water table in the relative dominance of each species. The reduction in sprouts from rhizomes upon vegetative expansion of Phragmites appeared to be the most likely process causing the decline of Typha. The latter had a shoot density of 39/m² in plots without Phragmites, but this dropped to 13 shoots m⁻² in plots that had been invaded by Phramites. Such a decline was likely caused by reduced reserves; e.g., the belowground biomass of Typha decreased from 11.3 g m⁻² without Phragmites to 8.1 g m⁻² with Phragmites. The latter also reduced its belowground biomass but not its shoot density in the presence of Typha. The mean weight of Phragmites shoots was 2.9 g, and nearly all produced inflorescences. Meanwhile, Typha failed to develop spadices despite its shoots having a greater biomass (7 g). This suggests that Phragmites is more efficient than Typha in shoot growth. Springtime flooding appeared to promote the sprout of Typha shoots from shallow rhizomes (≈18 cm below the soil surface), whereas the shoot density of Phragmites showed no correlation with water level in that season. Deep-rooted Phragmites (≈39 cm) occurred on both high and low water-table sites, whereas the shallow-rooted Typha was limited to only the former. Phragmites will likely continue its expansion, by vegetative sprouts from rhizomes, into Typha wetlands.     

Are clonal plants more tolerant to grazing than co-occurring non-clonal plants in inland dunes?

Clonal traits such as clonal integration and storage functions of rhizomes or stolons may provide clonal plants with additional advantages against grazing over non-clonal plants. Here, we hypothesize that clonal species have a larger capacity for compensatory growth than co-occurring non-clonal species. In inland dunes in northern China, individual plants of two rhizomatous clonal species (Bromus ircutensis and Psammochloa villosa) and two non-clonal ones (Artemisia intramongolica and Astragalus melilotoides) were subjected to 0% (control), 50% (moderate) and 90% (heavy) shoot removal. Compared with control, heavy clipping greatly increased the relative growth rate in Bromus and Psammochloa, but decreased that in Artemisia and Astragalus. Heavy clipping affected above-ground dry weight and the number of modules more negatively in Artemisia and Astragalus than in Bromus and Psammochloa. These results support the hypothesis and suggest that clonal species are more tolerant to grazing than co-occurring non-clonal species in inland dunes.     

High Frequency in vitro Multiplication of Centella asiatica: An Important Industrial Medicinal Herb

An efficient protocol was developed for rapid clonal propagation of the important medicinal plant, Centella asiatica (L.) Urban, using shoot tip culture. High frequency bud break (88 %) and multiple shoot formation (16.8 shoots/shoot tip) were induced from a shoot tip segment, which was cultured on MS medium supplemented with 6‐benzylaminopurine (BAP) (17.76 μM) and gibberillic acid (GA₃) (1.44 μM). Half‐strength Murashige and Skoog (MS) medium supplemented with naphthalen acetic acid (NAA) (10.74 μM) induced the maximum (27.66) number of roots. Plantlets with 3–4 fully expanded leaves and well‐developed roots were successfully transferred to potted soil which exhibited a 95 % survival. The protocol enables the harvest of more than 25,000 plantlets within 160 days starting from a single shoot tip explant.     

People,money, and protected areas: the collection of the caterpillar mushroom <Emphasis Type="Italic">Ophiocordyceps sinensis</Emphasis> in the <Emphasis Type="Italic">Baima Xueshan</Emphasis> Nature Reserve,Southwest China

The caterpillar mushroom Ophiocordyceps sinensis (syn. Cordyceps sinensis) is among the most valuable mushrooms in the world, and plays a major role for the local economies in its distribution area on the Tibetan Plateau and adjacent regions. Large proportions of its habitat fall into protected areas, and best practice of sustainable harvest is under discussion, considering both, O. sinensis as a valuable income source for rural poor and protection of its populations and habitat. This study aims for a detailed analysis of O. sinensis collection in a nature reserve in Southwest China. We found that harvesting is unevenly distributed among households and villages, with households who have access to the resource but lack adequate alternatives for income generation such as rewarding wage labor, fertile agricultural fields or harvest of other high value products being most involved. Although collection is de jure forbidden, authorities of the nature reserve apply adaptive management strategies for sustainable resource use. This includes the allocation of collection areas to communities based on their traditional land use strategies and the control of harvesters from outside, triggering self-policing of the resource by the local people. The strategies applied provide a promising model also for other protected areas where the caterpillar mushroom is collected.     

Predicting the Effectiveness of <Emphasis Type="Italic">Phragmites</Emphasis> Control Measures using a Rhizome Growth Potential Bioassay

In the last century, Phragmites australis (common reed) has expanded from a minor component of the mid-Atlantic tidal wetlands to a dominant species in many locations. Expansion of Phragmites results in decreased plant diversity and alterations to the tidal characteristics of the marsh, resulting in decreased wetland value. Management efforts have used a variety of strategies in an attempt to control its expansion. We tested a greenhouse bioassay that provided insight into the rhizome vitality of six herbicide-treated sites in the Alloway Creek Watershed, NJ well in advance of the growing season. At three sites, rhizomes were exhumed and classified by depth (0–25 cm and 25–75 cm) and appearance (color and firmness). Concurrently, the same protocol was followed, but conducted on an areal basis at three additional sites. Material was grown in sand under greenhouse conditions void of nutrient supplements for 70 days, after which shoots were removed and the rhizomes replanted for 30 days. Effectiveness of control strategies was quantified by examining rhizome color, vitality, and shoot densities in the field. Color was indicative of quality of rhizome reserves. Less than 0.2% of the firm, brown rhizomes produced shoots upon initial planting and none produced shoots upon replanting, whereas 50.9% of white rhizomes produced shoots on initial planting. Rhizome vitality was quantified by examining shoot emergence and the morphology of the shoots. Coupling rhizome vitality with observed field densities resulted in a predictive capability, and shoot density and biomass predictions were compared to field measurements in July 2001. We tested and accurately predicted the relative shoot densities and shoot biomass of the three sites for which we collected rhizome material on an areal basis. The result is a rapid, valuable, and cost-effective monitoring tool that can quickly quantify the effects of past control methods and predict future growth potential.     

Effects of lead contamination on the clonal propagative ability of Phragmites australis (common reed) grown in wet and dry environments

Clonal propagation is important for the survival and maintenance of the common reed Phragmites australis. Pot culture experiments were conducted to investigate the effects of lead (Pb) concentration (0, 500, 1500, 3000, 4500 mg·kg^?1) and water stress on the clonal reproductive ability of this species. The Pb concentration found in plant organs, in decreasing order, was roots >shoots >rhizomes. There was a negative relationship between the growth of clonal propagative modules (excluding axillary shoot buds) and Pb concentrations, which caused a decrease in biomass, rhizome growth and number of axillary and apical rhizome buds. Daughter axillary shoots exhibited a tolerance strategy, with no significant change in their number; the axillary and apical rhizome buds, daughter apical rhizome shoots and rhizomes exhibited compensatory growth during the late stage of Pb (excluding 4500 mg·kg^?1) treatment in a wet environment. Pb applications above 500 mg·kg^?1 reduced these parameters significantly in the drought treatment, except for the number of axillary shoot buds, which did not change. Our results indicate that clonal propagative resistance to Pb contamination can occur via tolerance strategies, compensatory growth and a Pb allocation strategy, enabling these reeds to maintain population stability in wet environments. However, clonal modular growth and reproductive ability were inhibited significantly by the interaction between drought and Pb, which would cause a decline in P. australis populations in a dry environment. Lead concentrations of 4500 and 500 mg·kg^?1 in soils might meet or exceed the Pb tolerance threshold of clonally propagated reeds in wet and dry environments, respectively.     

Organic matter economy of Euphorbia adenochlora with special reference to its life cycle

The dry matter economy of Euphorbia adenochlora Morra et Decne was investigated in a moist tall grassland in the Tajimagahara nature reserve, which protects a riverside grassland on the Arakawa river in Saitama Prefecture, central Japan. Euphorbia adenochlora is a typical short clonal species in the flood plain meadows, but is listed as an endangered species. It starts to grow in late February, and grows fast in early April by using one-third to about half of the dry matter of the rhizomes, which are composed of annual segments up to 8–10 years of age. During the period of fastest growth in early April, the bulk density (g dry weight cm^–3) of the rhizomes attained the minimum value. By the end of May, it reached its maximum. When ¹³CO₂ was fed to the shoots by means of photosynthesis, the concentration of ¹³C-containing photosynthates was highest in the new rhizome segment; the older the segment the lower the concentration of ¹³C. Shoot-clipping of E. adenochlora in late April, when bulk density was near the minimum, greatly depressed shoot growth in the following year. At the beginning of June, all of the aerial shoots of E. adenochlora died out with the growth of tall grasses. These results suggest that E. adenochlora has a life cycle as a spring ephemeral.     

Differential Responses of <i>NHX1</i> and <i>SOS1</i> Gene Expressions to Salinity in two <i>Miscanthus sinensis</i> Anderss. Accessions with Different Salt Tolerance

The lignocellulosic crop Miscanthus spp. has been identified as a good candidate for biomass production. The responses of Miscanthus sinensis Anderss. to salinity were studied to satisfy the needs for high yields in marginal areas and to avoid competition with food production. The results indicated that the relative advantages of the tolerant accession over the sensitive one under saline conditions were associated with restricted Na⁺ accumulation in shoots. Seedlings of two accessions (salt-tolerant ‘JM0119’ and salt-sensitive ‘JM0099’) were subjected to 0 (control), 100, 200, and 300 mM NaCl stress to better understand the salt-induced biochemical responses of genes involved in Na⁺ accumulation in M. sinensis. The adaptation responses of genes encoding for Na⁺ /H⁺ antiporters, NHX1 and SOS1 to NaCl stress were examined in JM0119 and JM0099.The cDNA sequences of genes examined were highly conserved among the relatives of M. sinensis based on the sequencing on approximate 600 bp-long cDNA fragments obtained from degenerate PCR. These salt-induced variations of gene expression investigated by quantitative real-time PCR provided evidences for insights of the molecular mechanisms of salt tolerance in M. sinensis. The expression of NHX1 was up-regulated by salt stress in JM0119 shoot and root tissues. However, it was hardly affected in JM0099 shoot tissue except for a significant increase at the 100 mM salt treatment, and it was salt-suppressed in the JM0099 root tissue. In the root tissue, the expression of SOS1 was induced by the high salt treatment in JM0119 but repressed by all salt treatments in JM0099. Thus, the remarkably higher expression of NHX1 and SOS1 were associated with the resistance to Na⁺ toxicity by regulation of the Na⁺ influx, efflux, and sequestration under different salt conditions.     

Clonal propagation,encapsulation and reintroduction of <Emphasis Type="Italic">Ipsea malabarica</Emphasis> (Reichb. f.) J. D. Hook., an endangered orchid

Summary Rapid clonal propagation and encapsulation of in vitro-formed bulbs of Ipsea malabarica (Reichb. f.) J. D. Hook., an endemic and endangered orchid of the Western Ghats of Kerala, and its reintroduction to the natural habitat were accomplished. Rhizome segments of Ipsea cultured on half-strength Murashige and Skoog (MS) medium supplemented with 6.97 μM kinetin induced the highest number of shoots, at the rate of four shoots per explant within 50 d. Transfer of the isolated shoots increased the rate of shoot multiplication to more than 10 shoots. Subsequent culture enhanced the number of shoots. No decline of shoot multiplication was observed up to the 10th subculture. Shoots developed bulbs during culture which developed into rhizomes. Sucrose at 6–8% reduced the time for the development of bulbs and rhizomes. Roots were developed from the base of the developed shoots as well as from the bulbs. Isolation and culture of bulbs also developed 5–10 shoots within 50 d. Encapsulated in vitro-formed bulbs cultured either on hormone-free halfstrength MS or 6.97 μM kinetin-supplemented medium facilitated 100% conversion. As a step to conservation in situ, 50 plantlets were reintroduced into their natural habitat, i.e. at Vellarimala (at 1300 m height) of the Western Ghats of Kerala, and flowered normally. Development of more than 40 000 plantlets starting from a single explant is possible within 250 d. This threatened endemic orchid stands to benefit greatly from the established protocol and will hopefully curtail the threat of extinction.