These researches were conducted to in vitro propagation and evaluation of culture environments, to develop their acclimatization and efficient transferring method, to produce ex vitro gametophytes and to extraction system for bioactive compounds of six moss gametophytes. In vitro cultures were initi...
These researches were conducted to in vitro propagation and evaluation of culture environments, to develop their acclimatization and efficient transferring method, to produce ex vitro gametophytes and to extraction system for bioactive compounds of six moss gametophytes. In vitro cultures were initiated from apical shoot, leaves of gametophytes and capsules of sporophytes with respective concentration of commercial bleach and time combinations. High percentages of survivability were found with 1% NaOCl for 2 minutes sterilization on culture derived from both the gametophytes tips and leaves. Bleach failed to sterilize the capsule of Hypnum plumaeforme and Polytrichum commune. In that case before use of bleach treated with 70% ethanol reduced the contamination and maximum survivability was achieved. Gametophytes were successfully produced by subculture of apical shoots, basal shoots and chopped gametophytes. The numbers of gametophytes per culture flax were greatest using chopped gametophytes culture among all the culture types. Seven types of culture media and four types of total nitrogen and sucrose concentrations were tested for gametophytes production. Knop macro salts and Nitsch and Nitsch trace elements was most convenient in all types of media. Respective light and temperature factors were also evaluated for micropropagation of this moss. Higher sucrose concentrations tended to have a negative effect on gametophytes elongation and gametophytes numbers. Nitrogen deficiency and excessiveness both inhibited growth of gametophytes. Temperature variation caused highly significant changes in number and length of gametophytes and fresh weight. A common optimum seems to be around 2,000-3,000 lux for proper growth of gametophytes. The protonemal gemmae and gametophytes inducing effect of the IAA and kinetin were examined using semi solid media. Gametophytes regeneration was induced from in vitro gametophytes on K+N medium supplemented with 0.01, 0.1, 1 and 10 ?M of IAA and kinetin. High frequency of bud and gemmae were selected then subcultured to same supplemented of IAA and kinetin. Protonema produced gemmae, callus and gametophytic bud. Production of protonemal gemmae varies according to the concentration of IAA and kinetin, whereas low concentrations of growth regulators promoted gemmae formation and bud induction. To acclimatize the regenerated moss, gametophytes was investigated using direct sowing, blending and chopping of in vitro propagules. The direct sowing of in vitro propagules had significant role to regenerate of new gametophytes. The blending process of gametophytes allowed producing significant number of gametophytes for Climacium japonicum and Thuidium tmariscinum moss. Four different types of inoculums (0.5, 1, 2 and 4g) were tested to acclimatize the gametophytes. Higher inoculums were produced more gametophytes but most of them were tiny and reduced growth and development during cultivation. Three level of light shade (50, 70 and 90%) was compared to adapt moss gametophytes from in vitro to in vivo condition. Our results showed that 70% shade was found out very effective and enhanced gametophytes growth for acclimation of moss gametophytes. In order to select a suitable growing media during acclimation of moss gametophytes we have tested the peat and bark soil separately with perlite using three kinds of ratio (3:1, 4:1 and 5:1). Higher amounts of peat and lower amounts of bark with perlite were effective to produce moss during acclimation. Surface and sub irrigation was tested for gametophytes production of mosses during acclimation. The surface irrigation was better than sub irrigation in respect of gametophytes acclimation for most of studied moss species. To produce of ex vitro moss, the propagation of gametophytes was investigated using direct sowing, blending and chopping of propagules. The direct sowing of gametophytes propagules had significant role to regenerate of new gametophytes for most of the species. The blending process of gametophytes allowed to producing significant number of gametophytes for all moss species. Four different types of inoculums (0.5, 1, 2 and 4g) were tested to produce the gametophytes. Robust gametophytes were produced using 0.5g of inoculums for most moss species. Three level of light shade (50, 70 and 90%) was compared to adapt moss gametophytes ex vitro condition. 50-70% shade was found out very effective and enhanced gametophytes growth for production of moss gametophytes. In order to select a suitable growing media during ex vitro condition of moss gametophytes we have tested peat and bark soil separately with perlite using three kinds of ratio (3:1, 4:1 and 5:1). Higher amounts of peat and lower amounts of bark with perlite were effective to produce moss gametophytes. Surface and sub irrigation was tested for gametophytes production of mosses during ex vitro condition. The surface irrigation was also better than sub irrigation in respect of gametophytes growth and development for most of studied moss species. Extraction system for bioactive compounds of six moss gametophytes was developed. Total phenolics, flavonoids contents and radical scavenging of ABTS and DPPH was examined with 80% ethanol, 100% methanol and water extraction. The alcoholic extraction was significantly higher than water as a solvent for all studied species. The methanolic extraction showed optimum contents of phenolics. P. commune exhibited the maximum contents of phenolics. There was no significant changes in flavonoid contents among the solvents except. Climacium japonicum and Thuidium tamariscinum moss. Methanol extraction was performed optimum contents of flavonoid for P. commune moss comparatively others. The methanolic extraction of H. plumarforme was found to be optimum for radical scavenging. The most potent RC50 value was exhibited by 100% methanolic extraction for all species. The significantly highest DPPH RC50 value was observed on H. plumaeforme and followed by L. glaucum moss. The RC50 value of water extract was found comparative lower in DPPH radical scavenging assay. Extraction of antioxidant and other phenolics compound of moss may be important tools in antimicrobial strategies.
These researches were conducted to in vitro propagation and evaluation of culture environments, to develop their acclimatization and efficient transferring method, to produce ex vitro gametophytes and to extraction system for bioactive compounds of six moss gametophytes. In vitro cultures were initiated from apical shoot, leaves of gametophytes and capsules of sporophytes with respective concentration of commercial bleach and time combinations. High percentages of survivability were found with 1% NaOCl for 2 minutes sterilization on culture derived from both the gametophytes tips and leaves. Bleach failed to sterilize the capsule of Hypnum plumaeforme and Polytrichum commune. In that case before use of bleach treated with 70% ethanol reduced the contamination and maximum survivability was achieved. Gametophytes were successfully produced by subculture of apical shoots, basal shoots and chopped gametophytes. The numbers of gametophytes per culture flax were greatest using chopped gametophytes culture among all the culture types. Seven types of culture media and four types of total nitrogen and sucrose concentrations were tested for gametophytes production. Knop macro salts and Nitsch and Nitsch trace elements was most convenient in all types of media. Respective light and temperature factors were also evaluated for micropropagation of this moss. Higher sucrose concentrations tended to have a negative effect on gametophytes elongation and gametophytes numbers. Nitrogen deficiency and excessiveness both inhibited growth of gametophytes. Temperature variation caused highly significant changes in number and length of gametophytes and fresh weight. A common optimum seems to be around 2,000-3,000 lux for proper growth of gametophytes. The protonemal gemmae and gametophytes inducing effect of the IAA and kinetin were examined using semi solid media. Gametophytes regeneration was induced from in vitro gametophytes on K+N medium supplemented with 0.01, 0.1, 1 and 10 ?M of IAA and kinetin. High frequency of bud and gemmae were selected then subcultured to same supplemented of IAA and kinetin. Protonema produced gemmae, callus and gametophytic bud. Production of protonemal gemmae varies according to the concentration of IAA and kinetin, whereas low concentrations of growth regulators promoted gemmae formation and bud induction. To acclimatize the regenerated moss, gametophytes was investigated using direct sowing, blending and chopping of in vitro propagules. The direct sowing of in vitro propagules had significant role to regenerate of new gametophytes. The blending process of gametophytes allowed producing significant number of gametophytes for Climacium japonicum and Thuidium tmariscinum moss. Four different types of inoculums (0.5, 1, 2 and 4g) were tested to acclimatize the gametophytes. Higher inoculums were produced more gametophytes but most of them were tiny and reduced growth and development during cultivation. Three level of light shade (50, 70 and 90%) was compared to adapt moss gametophytes from in vitro to in vivo condition. Our results showed that 70% shade was found out very effective and enhanced gametophytes growth for acclimation of moss gametophytes. In order to select a suitable growing media during acclimation of moss gametophytes we have tested the peat and bark soil separately with perlite using three kinds of ratio (3:1, 4:1 and 5:1). Higher amounts of peat and lower amounts of bark with perlite were effective to produce moss during acclimation. Surface and sub irrigation was tested for gametophytes production of mosses during acclimation. The surface irrigation was better than sub irrigation in respect of gametophytes acclimation for most of studied moss species. To produce of ex vitro moss, the propagation of gametophytes was investigated using direct sowing, blending and chopping of propagules. The direct sowing of gametophytes propagules had significant role to regenerate of new gametophytes for most of the species. The blending process of gametophytes allowed to producing significant number of gametophytes for all moss species. Four different types of inoculums (0.5, 1, 2 and 4g) were tested to produce the gametophytes. Robust gametophytes were produced using 0.5g of inoculums for most moss species. Three level of light shade (50, 70 and 90%) was compared to adapt moss gametophytes ex vitro condition. 50-70% shade was found out very effective and enhanced gametophytes growth for production of moss gametophytes. In order to select a suitable growing media during ex vitro condition of moss gametophytes we have tested peat and bark soil separately with perlite using three kinds of ratio (3:1, 4:1 and 5:1). Higher amounts of peat and lower amounts of bark with perlite were effective to produce moss gametophytes. Surface and sub irrigation was tested for gametophytes production of mosses during ex vitro condition. The surface irrigation was also better than sub irrigation in respect of gametophytes growth and development for most of studied moss species. Extraction system for bioactive compounds of six moss gametophytes was developed. Total phenolics, flavonoids contents and radical scavenging of ABTS and DPPH was examined with 80% ethanol, 100% methanol and water extraction. The alcoholic extraction was significantly higher than water as a solvent for all studied species. The methanolic extraction showed optimum contents of phenolics. P. commune exhibited the maximum contents of phenolics. There was no significant changes in flavonoid contents among the solvents except. Climacium japonicum and Thuidium tamariscinum moss. Methanol extraction was performed optimum contents of flavonoid for P. commune moss comparatively others. The methanolic extraction of H. plumarforme was found to be optimum for radical scavenging. The most potent RC50 value was exhibited by 100% methanolic extraction for all species. The significantly highest DPPH RC50 value was observed on H. plumaeforme and followed by L. glaucum moss. The RC50 value of water extract was found comparative lower in DPPH radical scavenging assay. Extraction of antioxidant and other phenolics compound of moss may be important tools in antimicrobial strategies.
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