Anther Culture And Leaves Protoplast Isolation And Culture Of Salvia Miltiorrhiza

Salvia miltiorrhiza Bge, belongs to the Labiatae sage; is a perennial herbaceous plant. Salvia miltiorrhiza radix et rhizome is one of the major and widely used medicinal materials. Currently, there are many researches on the drug action mechanism of S.miltiorrhiza, but the reports about the anther culture and protoplast isolation was little. It is an important way for promoting the germplasm initiative in anther culture and protoplast culture.in the study, the anther of S. miltiorrhiza Bunge and S. miltiorrhiza Bunge f.alba respectively as explant, a series of influence factors of anther culture was studied,such as anther development period, the composition and morphology of culture medium, species and concentration of plant growth regulators(PGR). In addition, callus differentiation into the bud and taking root were optimized, so a complete set of regeneration system for in vitro culture of S. miltiorrhiza anther was established. Moreover,in the study,the leaves of S. miltiorrhiza as material, the enzyme liquid combination, osmotic concentration, incubation time, centrifugal force and protoplast conditions of isolation and purification the culture medium composition were optimized, and the system of protoplast isolation and culture of S. miltiorrhiza was established. This study provides a certain practice guidance for the innovation of the germplasm resources of S. miltiorrhiza, it is conducive to further promote the S. miltiorrhiza varieties breeding for the final quality stabilization of S. miltiorrhiza.The main results were as follows:Before the anther culture, the flower bud was pretreated with 75% alcohol 30 s, followed by 0.1% HgCl2 10 min,then washed with sterilized water for 5 times and kept at 4 oC for 2 d in mononuclear period. The sterilized anthers were incubated 30 d on the MS+2,4-D 0.25 mg/L+6-BA 1.25 mg/L for the callus differentiation; after the callus were formed, the callus were transfered onto the MS medium(MS +6-BA 0.5 mg/l + 0.1 mg/l NAA) to obtain more cluster seedlings and then transfered onto the 1/2 MS medium(1/2 MS +IBA0.2 mg/l) to root differentiation so as to the later transplantingThe young leaves of S. miltiorrhiza were incubated for 8 h at 25 oC 50 rpm, in the enzyme liquid combination(2.0%cellulose enzyme+0.5% segregation-10+0.3% pectinase enzyme R Y- 23+0.6 mol/l mannitol) to obtain the protoplast- enzyme mixture. The protoplast- enzyme mixture was filtrated for 5 min with 300 mesh sieve at 700 rpm for 5 min, repeated for three times, to obtain the purified protoplast. The yield and vitality of protoplast could reached to 8.54×106 /g FW and 85.24% respectivelyBefore the protoplast culture, the density of S. miltiorrhiza protoplast was suspended, then incubated in the suspension of medium(DPD + 0.5 mg/L 2, 4-D+1.0 mg/L 6-BA+0.5 mg/l KT+400 mg/l casein hydrolysate, 0.5 mol/L mannitol, pH=5.8) and adjused to 1×106 /ml, then 3 ml protoplast suspension was incubated in the same components of DPD solid medium in 25 oC constant darkness four days, the recovery of the cell wall could be observed.