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LEDs and Their Potential in Somatic Embryogenesis of Panax vietnamensis Ha et Grushv.
Abstract
In recent years, light-emitting diodes (LEDs) have been utilized by many researchers in order to develop an understanding of light-regulated plant growth and development. However, there has been little discussion on using LEDs during in vitro cultures of Panax vietnamensis. This study examines the influence of various LEDs to determine the effectiveness of specific lighting conditions for callus formation and the subsequent development of somatic embryos, plantlet formation, and saponin accumulation. The results showed that growth and development had significant differences, and different lighting conditions were optimal for various stages. Callus cultured under yellow LEDs (Y-LEDs) resulted in the maximum fresh and dry weights (1197, 91.7 mg, respectively) after 3 months of culture. The most effective plantlet formation (11.21 plantlets per explant) from embryogenic calli was obtained when the combination of 60% red LED (R-LED) and 40% blue LED (B-LED) was used. The results of this study also provide additional evidence of LEDs influencing on the accumulation of saponin content. R-LED (20%) in combination with B-LED (80%) gave the highest MR2 content. However, the highest Rg1 and Rb1 contents were found under fluorescent light. The present results highlight the significance of using LEDs for improvement in micropropagation of P. vietnamensis.
... Micropropagation via somatic embryogenesis (SE) provides a useful tool for replacing the traditional method with true-to-type plantlets (Pais 2019). This method has also been successfully performed on Panax ginseng Mayer (Zhang et al. 2014), Panax notoginseng (Shoyama et al. 1997;Qiang et al. 2020), Panax quinquefolius (Qiang et al. 2020), Panax japonicus (You et al. 2007), Panax vietnamensis (Nhut et al. 2012(Nhut et al. , 2017Cuong et al. 2021). The efficiency of SE depends on many factors such as sterilization, plants, explant types, mineral medium, and plant growth regulators (You et al. 2007). ...
... Moreover, it is known that auxin (2,4-D, NAA, IBA) involved in the plant cell division regulation and differentiation processes (Teixeira da Silva and Malabadi 2012), leading to somatic embryogenesis. The regulation of auxin on somatic embryo induction has been reported in many previous studies in P. ginseng, P. vietnamensis, P. ginseng and P. quinquefolius, P. japonicus (Choi 1988;Nhut et al. 2017;Kim et al. 2019;Cuong et al. 2021). Overall, optimized types and concentrations of the auxin requirements generally depended on plants, explants, and genotypes. ...
Micropropagation of Lang Bian ginseng (Panax vietnamensis var. langbianensis), an endemic medicinal plant, via somatic embryogenesis (SE) and rhizome formation were investigated. Rhizome explants disinfected with 0.15% AgNPs for 30 min resulted in the most effective surface disinfection (contamination, necrosis and survival with callus induction of 36.00%, 14.70% and 49.30%, respectively). Moreover, shoot regeneration (45.33%) and the number of shoots (5.2 shoots) from 0.15% AgNPs disinfected- rhizome were significantly higher than those in others after 12 weeks of culture. The leaf was cut transverse thin cell layer (L-tTCL: 1 mm × 5 mm) and petiole cut longitudinally TCL (P-lTCL: 0.5 mm × 10 mm). L-tTCL explant cultured on MS medium supplemented with 7 mg/L NAA gave 100% SE and 32.80 embryos; meanwhile, P-lTCL explants also obtained 100% SE and 51.80 embryos in 1 mg/L 2,4-D treatment. Adventitious root formation and callus induction were also observed in the auxin-supplemented treatments. In addition, 12-week-old secondary somatic embryos, mainly cotyledonary stage, were obtained from the primary somatic embryo cultured on 1/2 MS medium supplemented with 1 mg/L 2,4-D. Plantlets derived from secondary embryos grew well with 63.34% rhizome formation, 1.27 cm in rhizome length and 0.65 cm in rhizome diameter. Meanwhile, plantlets derived from adventitious shoots only induced adventitious roots and callogenesis after 20 weeks of culture. The analysis of saponins by HPLC method showed that twenty-week-old in vitro rhizomes had Rg1, Rd and Rb1.
... Ánh sáng ảnh hưởng lớn đến quá trình tăng trưởng của cây trồng bởi nó rất cần thiết cho quá trình quang hợp [1,2]. Diệp lục của cây trồng hấp thụ ánh sáng trong vùng nhìn thấy gồm: ánh sáng xanh dương (425÷490 nm), đỏ (620÷700 nm) và đỏ xa (700÷740 nm) [1][2][3]. ...
... Ánh sáng ảnh hưởng lớn đến quá trình tăng trưởng của cây trồng bởi nó rất cần thiết cho quá trình quang hợp [1,2]. Diệp lục của cây trồng hấp thụ ánh sáng trong vùng nhìn thấy gồm: ánh sáng xanh dương (425÷490 nm), đỏ (620÷700 nm) và đỏ xa (700÷740 nm) [1][2][3]. α-Al 2 O 3 có nhiều tính chất ưu việt như: tính chất cơ học tốt, ổn định về mặt hoá học và không độc hại. Đặc biệt, α-Al 2 O 3 dễ dàng pha tạp với các kim loại chuyển tiếp và nguyên tố đất hiếm. ...
Red-emitting α-Al2O3:Mn4+ fluorescent powders were prepared by co-precipitation method and sintered at1000 to 1500oC in the air. Effects of sintering temperature and Mn4+ concentration on the optical properties of the materials were investigated. The X-ray diffraction (XRD) showed that the crystallinity of Al2O3 belonged to hexagonal with R3C space group. The excitation spectrum consisted of two broad intense bands centered at 399 and 568 nm and was assigned to 4A2g(F)→4T1g(F) and 4A2g(F)→4T2g(F) transitions, respectively. Under the excitation wavelengths of 399 and 568 nm, the α-Al2O3:0.1% Mn4+ phosphors emitted efficient red light at 694 nm caused by 2Eg→4A2g transition. A red LED is made by coating Al2O3:0.1% Mn4+ powder on a violet chip located in the deep-red light region. These results showed that α-Al2O3:Mn4+has the potential to produce red LED lightings for plants.
... (Fraser, Harvey, 1986) or root tissue (Liu et al., 1998) and leaf explant (Lan et al., 2007) of Actinidia kolomikta, etc. In addition, SEMs was influenced by many factors such as plant growth regulator (Grzybkowska et al., 2018), explant (Pencik et al., 2015), light (Nhut et al., 2017), etc. Besides, thin cell layer (TCL) technique also successfully applied in SEMs on many different crops ; however, the effect of TCL technique on SEMs of A. chinensis has not been specifically studied. ...
Somatic embryogenesis (SEMs) is an effective micropropagation method that has been successfully applied to many different plant species; however, little is known about the SE in Actinidia chinensis Planch. - A species with high economic value. In this study, the efficiency of SEMs from leaf main vein and petiole of A. chinensis (seedlings germinated from seeds sterilized with 200 ppm silver nanoparticles for 7 min) via thin cell layer (TCL) technique was performed and compared with that of whole leaf main vein and whole petiole explants. The leaf main vein (mv - 1 mm × 10 mm in size) and leaf petiole (p - 1 mm × 10 mm in size) explants of in vitro A. chinensis were cut through transverse and longitudinal TCL (mv-tTCL and mv-lTCL for mv explants or p-tTCL and p-lTCL for p explants) with different sizes (½, ¼, ¹/6, ¹/8 of mv-lTCL or p-lTCL and ½, ¹/3, ¼ of mv-tTCL or p-tTCL, respectively). Somatic embryo (SE) was recorded in the wounds of mv and p explants after 8 weeks of culture. For mv explants which cultured on MS medium supplemented with 0.02 mg/L NAA, 0.5 mg/L TDZ, 30 g/L sucrose and 8 g/L agar – MSE medium, the ½ mv-tTCL explant (mv was cut into 2 tTCL explants) gave the highest SEMs (98.67%) and number of somatic embryos per explant (10.66 embryos); meanwhile, p explants cultured on MSE medium, the ½ p-tTCL explant (p was cut into 2 tTCL explants) obtained the highest number of somatic embryos per explant (8.66 embryos) after 8 weeks of culture. In addition, the growth correction factor of SEMs was optimal at ½ mv-tTCL explant (21.04) or ¹/6 p-lTCL (p was cut into 6 lTCL explants) and ¹/8 p-lTCL (p was cut into 8 lTCL explants) (31.36 and 32.00). The current study has shown that the procedure of leaf main vein and petiole TCL derived- SEMs of A. chinensis is the optimal one. Large amounts of somatic embryo induction were observed from mv-TCL and p-TCL with full shapes of the globular, heart, torpedo and cotyledon. The shoot regeneration from somatic embryo with the cotyledon shape was optimal on medium containing 1.0 mg/L BA in terms of total number of shoots, shoot height and number of shoots. In addition, plantlets derived from shoot cultured on the MS medium supplemented with 0.4 mg/L NAA and planted in plastic pots with the combination of humus, coconut fiber and biofertilizers substrates (with ratio of 2:3:1 in weight) showed higher acclimatization and growth than those from other treatments in the greenhouse.
Effects of silver nanoparticles (AgNPs) on somatic embryogenesis and plantlets with rhizome of Panax vietnamensis were presented in this study. The highest number of somatic embryos (140) was obtained on MS medium containing 1 mg/L 2.4-D, 0.5 mg/L NAA, 0.2 mg/L Kin and 1.6 mg/L AgNPs. Plantlets with rhizome growth enhanced on SH medium supplemented with 1 mg/L NAA and 1.2 mg/L AgNPs and limited the ethylene gas content in the culture flasks. A low concentration of ethylene gas (0.92 ppm) stimulated plantlets with rhizome growth and development, and provided positive effect on the formation and quality of plantlets. The rhizome size of the plantlets in 1.2 mg/L AgNPs treatment was larger than those in the control without AgNPs. Plantlets acclimatized on the soilless substrate were higher survival rate (93.65%) as compared to the control (44.44%) after one year in the greenhouse. In particular, the rhizome saponin content was doubled compared with that in the planlets without AgNPs after 4-year planting in the greenhouse. These results may be scaled up for micropropagation of this important medicinal plant.
In recent years, LED (light-emitting diode) has been the subject of research within the field of plant growth and development. However, there has been little discussion about using LED in vitro cultures of Panax vietnamensis, one of the important medicinal plants belonging to the Panax genus. This study examines the influence of various LED lamps on callus growth and plant formation of P. vietnamensis. Results show significant differences in growth and development, as various light conditions were suitable for different stages. Callus of 70 mg in fresh weight cultured under yellow LEDs resulted in growth of 1197 mg in fresh weight and 91.7 mg of dry weight, within a period of three months. The most effective plant formation was obtained when embryogenic calli were cultured under the combination of 60% red LED and 40% blue LED with an average of 11.21 plantlets per explant; the shoot clump fresh weight and dry weight were of 1147 and 127 mg, respectively, and the average plant height was 3.1 cm. It was also shown that this light condition was the most efficient for P. vietnamensis in vitro plant growth and development. This study provided additional evidence regarding the influence of different LEDs on ginsenoside production applying high-performance liquid chromatography (HPLC) analysis with photo-diode array (PDA) detection at ultraviolet (UV) wavelength 203 nm. The highest MR2 content was recorded when plants maintained under 20% red LED combined with 80% blue LED. However, the highest Rg1 and Rb1 content was found under fluorescent light. The results presented might provide new strategies using LEDs for adequate micropropagation protocols of P. vietnamensis.
Callus culture was initiated from expiants of mature root tissues of ginseng (Panax ginseng C.A. Meyer) on MS medium enriched with 2,4-D. The ageing callus produced numerous embryoids in this medium. Reculture of these embryoids in media (1/2 MS or B5) supplemented with benzyladenine and gibberellic acid resulted in profuse plantlet regeneration.
Almost all Panax spp. (family Araliaceae) have been used in folk medicine. The most famous variety is P. ginseng, which was recorded in Chinese Materia Medica 2000 years ago. Ginseng is one of the most important Chinese medicines used in tonics to combat stress and cancer, disturbances of the central nervous system, and hypothersmia, and for radio-protection. It contains many dammarane and oleanane saponins of which the biological activity has been studied widely. P. pseudoginseng var.notoginseng is cultivated in Kumming province in China. It is also an important Chinese crude drug used as an astringent and tonic. P. japonicus grows mainly in Japan and China, and is used as a stomachic and a hair-growing tonic in Japan.
Panax vietnamensis Ha et Grush. has been interested and researched from 1973. Application of biotechnology to increase root biomass of P. vietnamensis is studying in Vietnam, whereas, industrial-scale culture of multiple adventitious roots of P. ginseng has succeeded by bioreactor system in South Korea. In previous study, we had determined optimal medium for callus-regenerated adventitious roots of P. vietnamensis on MS medium (Murashige and Skoog,1962) supplemented with 3 mg/l NAA, 30 g/l sucrose and 8 g/l agar. In this study, we investigated optimal medium for multiplication of adventitious roots and primary application of bioreactor system to multiplication of adventitious roots of P. vietnamensis provide material for saponin isolation. Results had shown optimal medium for multiplication of adventitious roots is SH medium (Schenk and Hildebrandt, 1972) supplemented with 3 mg/l NAA, 30 g/l sucrose, 8 g/l agar. Increase of adventitious roots biomass after 8 weeks culture were achieved 6.05 times on SH medium supplemented with 3 mg/l NAA, 30 g/l sucrose by 3-litre balloon-type bubble bioreactor (Korea).
The efficacy of three auxins, viz. 2,4-0, NAA and dicamba, were compared for the induction of somatic embryogenesis in American ginseng (Panax quinquefolium L.). Somatic embryos (SEs) formed on ginseng cotyledonary, zygotic embryo and shoot explants after 8 weeks of induction by the auxin stimuli. Significantly more somatic embryos were induced by culture of any of the ginseng explants on media supplemented with 2,4-0 than any other auxin treatment. Shoots derived from somatic embryos had the greatest regenerative potential and zygotic embryos the least. Explants generated from green (unstratified) seeds gave similar or higher frequency of embryogenesis as the explants derived from stratified seeds. Histological and SEM studies confirmed that the regenerimts were somatic embryos. Somatic embryos germinated and developed into normal plants in months. About of plantlets from second generation SEs formed flowers within 10 weeks, particularly on media supplemented with The development of a regeneration system for ginseng through somatic embryogenesis is a necessary first step for mass propagation and genetic improvement of American ginseng.
Metabolome analysis was carried out to evaluate the effects of light-emitting diode (LED) spectra on the metabolic processes of ginseng (Panax ginseng C. A. Mayer) adventitious roots. In total, 35 hydrophilic and 11 lipophilic metabolites were identified in ginseng roots irradiated with red (630 nm), blue (465 nm) LED light or fluorescent lamp (FL) light, by gas chromatography-time-of-flight mass spectrometry. Principal components analysis results using the 46 metabolites showed differentiation between the metabolomes of ginseng roots irradiated with red LED, blue LED, and FL light, indicating that metabolic changes were occurred in the ginseng roots by light spectral quality. The corresponding loading indicated that LED light-irradiated ginseng roots had higher sucrose and lower amino acids compared to FL-irradiated ginseng roots. The quantitative results revealed that ginseng roots irradiated with blue LED light had higher concentrations of α-tocopherol and β-amyrin, as well as phenolic acids compared with FL-irradiated ginseng roots. This is the first study to determine the comprehensive metabolic changes in response to LED light in ginseng adventitious roots and to demonstrate the utility of the metabolite profiling approach employed in this study for detecting environmental effects on the plant metabolome.
Light irradiation had remarkable effects on the callus growth of Cistanche deserticola and biosynthesis of phenylethanoid glycosides (PeG). After 30 days of culture, the callus biomass and production of PeG reached 15.5 g dry weight (DW)/l and 1.7 g/l, respectively, at its light saturation point (24 μmol/m2 s). The callus cultured under blue light at 435 nm gave both the highest biomass (18.4 g DW/l) and production of PeG (2.4 g/l), which were 19 and 41% higher than those obtained under white light.
The composition of a medium is described that proved useful to culture callus of a variety of monocotyledonous and dicotyledonous plants. Growth on the medium was often better than on some other excellent media. In addition to supporting rapid cell growth, a soft, friable type of colony growth was often obtained. This type of loose, friable cell growth facilitated work with single cells and the enzymatic removal of cell walls in related studies. A high level of auxin-type growth-regulating substances (AGRS) generally favored cell cultures of monocotyledonous plants, while low levels of cytokinin were essential for most dicotyledonous cell cultures. Some cultures of dicotyledonous plant cells adapted to a cytokinin-free medium containing a high level of 2,4-dichlorophenoxyacetic acid (2,4-D) or p-chlorophenoxyacetic acid (pCPA). The preferred AGRS were 2,4-D and pCPA. Citrate, succinate, and 2(N-morpholino) ethane sulfonic acid (MES) were effective medium buffers, but phosphate alone seemed adequate to buffer the medium at pH 5.8–5.9.
After a series of experiments on photoperiodicity and light intensity under daylight supplied by an ordinary fluorescent lamp in cultivations using a flask and a roux bottle, it was found that irradiation at 27.2 W/m2 for the whole period was effective for anthocyanin production by a suspended culture of Perilla frutescens (shiso). A high amount of anthocyanin pigments, 3.0 g/L, was obtained in a bubble column bioreactor after 10 days of cultivation at an aeration rate of 0.1 vvm with light irradiation at 27.2 W/m2, while 2 g/L was obtained at 13.6 W/m2 and very little at 54.4 W/m2. A high amount of anthocyanin pigments, 2.9 g/L, was also produced using an aerated and agitated bioreactor at an agitation speed of 130 rpm, an aeration rate of 0.1 vvm and light irradiation intensity of 27.2 W/m2. The amount of anthocyanin produced was more than twice that without light irradiation, Keeping the other cultivation conditions the same. The results obtained also showed that the amount of anthocyanin pigment accumulated in a shake flask could be rather well reproduced in bioreactors for both aerated culture, and aerated and agitated culture, by improving the conditions of light irradiation, which conspicuously affects metabolite formation.