Establishment Of Tissue Culture And Plant Regeneration System And Selection Of Conditions For Onion Protoplast Isolation

Onion (Allium cepa.L) is fourth largest vegetable crop in the world, and it is one of the great demanded vegetable plants in the world. In China,it has a wide range of cultivation.Onion originated from central Asia. It has 5,000 years of history. Onion is a biennial cross-pollinated plant.it is highly heterogenous.It has small flower structure. After 2-3 generations will lead to low breeding efficiency,even inbreeding.Different organs were used as explants to establish a regeneration system.we used callus as a material for protoplast isolation,which obtained from experiment, and we obtained high yield of protoplasts.Many factors affected protoplast yield and vitality.In this experiment,we used the bulbs and flower buds as explants, and we seted up onion direct regeneration system,and then we investigated many factors which affected the protoplast and vitality, such as centrifugal force and enzymatic solution.And the result are as follows:1, The establishment of aseptic seedlings of regeneration system of onion:the best medium for callus induction is MS+2,4-D 1.Omg/L+6-BA1.0mg/L, the rates is more than 90%; The best medium for bud is MS+NAA0.5mg/L+6-BA1.0mg/L, bud differentiation rate is 81.67%.2,The establishment of bud culture system regeneration of onion flower:the best callus induction medium is MS+2,4-D2mg/L+6-BA4mg/L, induction rate of bud is 38.33%, the average callus induction rate is 63.83%, direct shoot induction rate is the rate is 77.88%.3, Mannitol were used as osmotic stabilizer to determine the osmotic pressure of onion callus,the best value is 0.6mol/L.4,The establishment of callus protoplasts fast separation system of onion:(1) The best enzyme solution is 0.6 M mannitol+0.1% MES+2.0% Cellulase Onozuka R-10+0.1% PectolyaseY-23+0.5% Macerozyme R-10+CPW solution, pH 5.8, at 25℃and dark conditions for 5-6h. Centrifuging the protoplast is at 600 rpm /min, the callus which cultured 12d is the best condition.(2)The medium for protoplast is B5+1mg/L6-BA+1mg/LNAA+0.1%CH+0.2mol/L glucose+0.2mol/L sucrose+0.6mol/L mannitol.We just got some results that the best culture method is solid-liquid culture and lamellar float culture for onion protoplast, till now, we have just found the first division.