In Vitro Culture And Analysis Of Essential Oil Onstituents With GC-MS In Mellisa Officinali

In this experiment,stems with buds were used as explants for the systematical studies on the tissue culture of stem segment and for further establishing the industrialized in vitro propagation model in Mellisa officinalis;and the systematical research on the vitrification in the culture of Mellisa officinali was also discussed.Leaves,stems and seeds were used as explants for the studies on the callus culture and plant regeneration.At the same time,GC-MS analysis was used for analyzing the difference of the volatile oil components of Mellisa officinalis between the plantlets in vitro and the seeding of potted planting.These studies would establish an effective way on tissue culture of Mellisa officinalis which could provide scientific evidence on industrial production of plantlets and protection of germplasm.The main results were as follows.1.Stem segment culture of Mellisa officinalis.Stems with buds were used as explants for the studies on tissue culture.The results showed that:the best season of taking explants was March.The best disinfection method for stems was 75%ethanol for 30 s +0.1%HgCl₂ 5 min on MS medium.Adventitous buds induction rate was 100.0%on the medium:MS +0.5 mg/L 6-BA + 0.1 mg/L NAA.The most suitable medium for the multiplication of the adventitious buds was MS +1.0 mg/L 6-BA +0.2 mg/L NAA +0.1 mg/L IBA,the multiplication time was 4.6.The most suitable medium for rooting was 1/2 MS +0.5 mg/L IBA,rooting rate reached 100.0%.Substrate with perlite,vermiculite and sand by 1:1:1 was the optimal,the survival rate was 93.3%.2.Establishment of the industrialized in vitro propagation model of Mellisa officinalis. The main technical parameters were as follows:the proliferation coefficient was up to 4.5,the subculture cycle was 25 days,and the subculture time was 12 times every year;the rooting rate was up to 100%,and the survival rate of transplanting was 93.3%,.According to these parameters, a bud could proliferate to 4038000 copies a year theoretically when contamination rate was less than 5%and vitrification rate was less than 5%.3.Studies on the vitrification in the culture of Mellisa officinalis.Stems with buds of normal plantlets were used as explants,compared to different factors:6-BA,temperature,the containers,subculture time,and sucrose,studied on the control measures for the vitrification of Mellisa officinalis in vitro Culture.The results show that:when plantlets on the appropriate cultured medium with 0.5-2.0 mg/L 6-BA,30g/L sucrose and 7g/L agar,the rate of the vitrification could be lowest.When the temperature was 25℃,plantlets sealing with plastic film in glass bottles for 30 d subculture could be an effective control of the vitrification.4.Callus culture of Mellisa officinalis.Seeds,leaves,petiole and stems of Mellisa officinalis were used as explants for the studies on callus culture.The results showed that:the most suitable disinfection method for seed was 75%ethanol for 30 s +0.1%HgCl₂ 5 min in MS medium,and the germination rate was 85.5%.The most suitable medium for callus induction was MS+1.0 mg/L 6-BA+0.1 mg/L 2,4-D,callus induction rate was 97.1%;there were no significant difference of callus induction rate when callus induced from leaves,petiole and stems。The most suitable medium for the multiplication of the Callus was MS +2 mg/L 6-BA +0.01 mg/L NAA +0.2 mg/L 2,4-D,the growth rate reached 76.7%.The most suitable differentiation medium was MS +1.0 mg/L 6-BA +0.1 mg/L NAA +0.1 mg/L 2,4-D,differentiation rate was 3.75%.5.GC-MS analysis of essential oil constituents of Melissa officinalis in vitro culture. Mellisa officinalis in vitro were used as the materials,using GC-MS separated 19 kinds of substances.Main composition were 6-Methyl-5-heptene-2-one,citronellal,neryl alcohol,β-citral, geraniol,1,3,4-Trimethyl-3-cyclohexen-1-carboxaldehyde,α-citral,nerol acetate,butylated hydroxytoluene,viridiflorol and so on.Compared to normal plants of Melissa officinalis there were 11 kind of same material in two samples together,although the contents existenced some differences,all of the main substances neryl alcohol,β-citral,geraniol,α-citral and nerol acetate have great content in the two samples.