Sphagneticola Calendulacea Research Articles

In vitro tetraploidization for the augmentation of wedelolactone in Sphagneticola calendulacea (L.) Pruski

A practical and reliable method for in vitro tetraploidization of Sphagneticola calendulacea (L.) Pruski [synonym Wedelia chinensis (Osbeck) Merrill] has been established to enhance the production of wedelolactone. Shoot tip and nodal explants from in vitro-grown culture (2n = 50) were exposed to the antimitotic chemical, i.e., colchicine, at various concentrations (0, 0.025, 0.05, 0.1, 0.3, and 0.5%; w/v) for 12, 24, 36, 48, and 60 h. The treated explants were then incubated and proliferated on Murashige and Skoog (MS) medium fortified with 0.2 mg l−1 thidiazuron and 0.05 mg l−1 naphthalene acetic acid, followed by root induction in 1.0 mg l−1 indole-3 acetic acid enriched ½MS medium. Treatment of shoot tips with 0.05% colchicine for 24 h supported the maximum rate of survival (63.33%) of explants as well as tetraploid induction (42.93%). Morphological, stomatal, and cytological characteristics along with the secondary metabolite content of the in vitro tetraploids were compared to that of diploids. The recovered tetraploid plants possessed superior plant height, stem diameter, leaf size, root number, and increased length and width of stomata but decreased stomatal frequency. The tetraploid plants demonstrated twice the chromosome number (2n = 4x = 100) than the diploids as confirmed through cytology, spectrophotometry and flow cytometry. High-performance thin-layer chromatography showed a significant enhancement in the wedelolactone content of tetraploid plants (541.48 µg g−1 of dried sample) in comparison to diploid plants (325.43 µg g−1 of dried sample), signifying the prospective of this technique for the trade value improvement.

Characterization of the complete chloroplast genome sequence of Sphagneticola calendulacea (Asteraceae)

Sphagneticola calendulacea is a valuable medicinal herb. With the spread of a congeneric invasive species Sphagneticola trilobata in South China, the already vulnerable S. calendulacea populations are being threatened further by natural hybridization with the invading species. In this study, we assembled and characterized the complete chloroplast genome of S. calendulacea as a resource for future studies on this species. The chloroplast genome was 151,748 bp in size, with a large single-copy (LSC) region of 83,270 bp, a small single-copy (SSC) region of 18,348 bp, separated by two inverted repeat (IR) regions of 25,065 bp each. A total of 134 genes were predicted. Phylogenetic analysis showed close relationship between S. calendulacea and Eclipta prostrata within the Heliantheae tribe.

Development of transgenic hairy roots and augmentation of secondary metabolites by precursor feeding in Sphagneticola calendulacea (L.) Pruski

Sphagneticola calendulacea (L.) Pruski [synonym Wedelia chinensis (Osbeck) Merrill] is an important medicinal plant that possesses a variety of bioactive secondary metabolites. A stable transgenic hairy root culture has been developed using engineered Agrobacterium rhizogenes strain LBA1334 harboring pCAM:2 × 35S:gusA binary vector, to achieve high yield of secondary metabolites. Putative transgenic hairy roots appeared with highest transformation frequency (66.67%) when leaf explants were immersed in A. rhizogenes suspension for 10 min and co-cultivated for three days. Incorporation of foreign genes was confirmed by initial GUS staining assay followed by polymerase chain reaction analysis of rolA, rolB, rolC and gusA genes. Time course study revealed that the transgenic hairy roots grew rapidly in ½ Murashige and Skoog liquid medium with highest biomass production of 0.61 g per 50 ml dry weight after 28 days of culture. In the precursor feeding experiment, the biomass of hairy roots was further increased to 0.698 g per 50 ml dry weight when 0.5 mM phenylalanine was added to the 10 days old growing culture. High Performance Thin Layer Chromatography showed that the transgenic hairy roots accumulated enhanced level of wedelolactone (422.01 μg g−1 dry weight), which was 1.43 and 1.37 times higher than those of in vivo (294.44 μg g−1 dry weight) and in vitro-grown (308.28 μg g−1 dry weight) plantlets, respectively. Precursor feeding with phenylalanine further augmented the accumulation of wedelolactone to 440.33 μg g−1 dry weight in the transgenic hairy roots. In addition, the content of several important phenolic and flavonoid compounds were increased in the transgenic hairy roots as analyzed by High Performance Liquid Chromatography. The protocol offers a scope to introduce foreign genes and further regulate the production of secondary metabolites.

Conservation, ex vitro direct regeneration, and genetic uniformity assessment of alginate-encapsulated nodal cuttings of Sphagneticola calendulacea (L.) Pruski

A well-organized procedure was established for the conservation and distribution of Sphagneticola calendulacea (L.) Pruski [synonym Wedelia chinensis (Osbeck) Merrill] for the first time, using alginate-encapsulated nodal segments (NSs) as synthetic seeds. The ideal beads were obtained through a combination of 2.5% sodium alginate and 75 mM calcium chloride with 84.40 ± 2.20% rate of shoot emergence. The maximum regeneration (88.84 ± 2.24%) from synthetic seeds was achieved on liquid 1/2Murashige and Skoog (MS) medium in comparison to its other formulations. Furthermore, superior frequency (91.09 ± 2.24%) of complete plantlet (having both shoots and roots) formation was achieved when synthetic seeds were cultured on liquid 1/2MS (1.5% sucrose) fortified with 1.0 mg L−1 N6-benzyladenine plus 0.25 mg L−1 α-naphthalene acetic acid. Synthetic seeds could be effectively stored at low temperature (8 °C) up to 90 days with a survival rate of 52.38 ± 3.06%, whereas higher temperature (25 °C) did not support regeneration after 75 days of storage. The plantlets were successfully acclimatized to natural conditions with ~ 89% survival frequency. To by-pass the time-consuming in vitro culture step after encapsulation, synthetic seeds were directly regrown into complete plantlets ex vitro on sand, soil, and vermicompost (1:1:1; w/w). Regeneration rate of 42.22 ± 2.22% was attained when NSs were pretreated on 1/2MS medium containing 4.0 mg L−1 indole-3-acetic acid for 24 h in dark, prior to encapsulation. The random amplified polymorphic DNA and intersimple sequence repeat fingerprinting profiles demonstrated genetic uniformity amongst the regenerated plantlets, in vitro mother plant, as well as in vivo wild plant.

Factors influencing large-scale micropropagation of Sphagneticola calendulacea (L.) Pruski and clonality assessment using RAPD and ISSR markers

Sphagneticola calendulacea (L.) Pruski (synonym Wedelia chinensis (Osbeck) Merrill) has great significance in traditional systems of medicine and is popularly known for its hepatoprotective properties. A comprehensive micropropagation technique for its conservation and season-independent, large-scale production was established. Initial culture was established in different basal media [Murashige and Skoog (MS), Schenk and Hildebrandt (SH), Gamborg’s (B5), and Woody Plant medium (WPM)] following a non-toxic, simple, and effective sterilization of ex vitro shoot tip (ST) and nodal segment (NS) explants. MS medium favored the highest rate (∼87.27%) of multiple (∼2.03) shoot bud initiation from NS explants that was further multiplied with supplementation of thidiazuron (TDZ), kinetin, N6-(Δ2-isopentenyl) adenine, or zeatin in combination with indole-3-acetic acid (IAA) or α-naphthaleneacetic acid (NAA). MS medium containing TDZ (0.2 mg L−1) and NAA (0.05 mg L−1) resulted in the maximum (∼98.82%) response with the highest number (∼33.12) and length (∼6.04 cm) of multiple shoots. The maximum number (∼12.18) and length (∼6.24 cm) of healthy roots per plant was achieved on ½ MS medium plus 1 mg L−1 IAA. In a simple acclimatization process of 6 wk, the survival rate of plantlets cultured in a combination of soil, sand, and vermicompost (1:1:1; v/v/v) was 82.00%. DNA fingerprinting of the in vitro-regenerated plantlets via analysis of random amplification of polymorphic DNA and intersimple sequence repeat markers showed clonal fidelity of the plantlets with the mother plant.

Responses of the Hybrid between Sphagneticola trilobata and Sphagneticola calendulacea to Low Temperature and Weak Light Characteristic in South China.

Hybridization between exotic and native species is of great interest to evolutionary biologists and ecologists because it usually shows a quick evolution of species and invasiveness. It has been reported that such hybridization frequently increases the adaptation and aggressiveness of the new hybrids. A hybrid between invasive Sphagneticola trilobata and its native congener S. calendulacea was recently found in subtropical China. S. calendulacea has a significantly higher tolerance to low temperature and weak light stress than S. trilobata, and its range includes both tropical and temperate regions. This study examined how the tolerance of the new hybrid to low temperature and weak light conditions (LTWL), expanded its geographical range. All changes of phenotype, gas exchange parameters, chlorophyll fluorescence parameters, contents of malonaldehyde (MDA) and activity of superoxide dismutase (SOD) and peroxidase (POD) indicated that hybridization slightly catalyzed the tolerance of the hybrid to LTWL condition and the responses of the hybrid were more similar with their invasive parent. The results demonstrate that the current hybrid populations may not expand their geographical distribution ranges in a short period, but the distribution of the backcrossed generations is still uncertain. The threat of the hybrid to its native parent in subtropical region should be concerned.

A hybrid of the invasive plant Sphagneticola trilobata has similar competitive ability but different response to nitrogen deposition compared to parent

AbstractHybridization between introduced and related native species has been suggested to be a key driving force of evolutionary processes in invasions that may be accelerated by increasing nitrogen (N) deposition. We carried out two experiments to compare the competitive ability of a newly‐reported putative hybrid (Sphagneticola calendulacea × Sphagneticola trilobata) to that of its invasive paternal parent and investigate its response to different N additions. Growth of the hybrid increased by 39.62 ± 15.69 % when grown with the native parent but showed no significant changes when grown with the invasive parent. Growth of invasive S. trilobata showed no significant changes when grown with the other two taxa. The hybrid and invasive parent inhibited the growth of the native parent by 33.27 ± 15.26 % and 78.56 ± 3.13 %, respectively. Low level N addition (50 kg N ha−1) enhanced the growth of both the hybrid and native parent, whereas medium and high levels of N additions (150, 300 kg N ha−1) had no effects on hybrid growth but decreased native parent growth. However, all N treatments had no significant effects on the growth of the invasive parent. Our results suggest that the hybrid is an equal competitor to its invasive parent and is more susceptible to N deposition. Therefore, this hybrid could be a threat to native diversity but may decline more than the invasive parent under high N conditions.