Studies On The Technique Of The Fusion And Culture Of Protoplast In Allium Sativum L.

This reseaech was conduectd at Horticultural experiment station of Shandong Agricultural University and Ministry of Education Key laboratory of Horticultural Crop Biology from 2008-2010. This study discussed the factors that affected callus inducement of garlic bulb and acquired embryogenic callus. The cell suspension culture was established using embryogenic callus. On that base protoplasts were isolated from cell suspension. The factors influencing protoplasts isolation and purification were studied, and protoplasts culture and protoplasts fusion was performed initial. The main results were described as follows:Media with different levels of 2,4-D and 6-BA gave significant differences in the induction of calluses, 2,4-D was found to be the key factor for callus inductionIn and subculture, MS+2,4-D2mg/L+6-BA1mg/L was the best inducing medium for producing callus, and MS+NAA1mg/L+6-BA0.1mg /L was suitable for pullulating, while the medium without hormone was befitting for taking root. The high growth hormone was suitable to induce the garlic callus, but not for its differentiation.The factors infected the culture of garlic callus were as followed: (1)explant type: Callus induction from the bottom bulb, the middle bulb, and the top bulb was investigated. The best medium for inducing embryonic callus from the bottom bulb and middle bulb was MS+2,4-D2mg/L+6-BA0.5mg/L. MS+2,4-D1.5mg/L was favorable to callus induction from the top bulb, and the induction rate was 78.3%. (2) garlic breed: We used Sulian garlic, Cangshan garlic and Jinxiang garlic in this experiment, and the results showed that the Cangshan garlic had the highest rate of callus, which was 78.12% and obviously higher than the other breeds. (3) type of medium: through the three kinds of medium the MS medium had highest rate of callus taking, and had obvious difference with B5 medium and 1/2MS medium. B5 had the rate as 68.09%, 1/2MS had 62.50%, while MS medium had 82.35% induction rate.Somatic embyrogenic suspension culture of embryonic callus of garlic were established. It was showed that there were no significant differences in the time needed to establish suspension culture among different garlic breed. Change in cell number followed an"S"distribution. Cell fresh and dry weights during suspension culture indicated that cell growth rates were quickest during the earliest stages of culture, peaked on day 16 and then decreased.Protoplasts were isolated from cell suspensions, embryogenic calli and leaves of in vitro-grown plantlets. The conditions of isolation and purification protoplasts of garlic were studied. The results showed that suspension cells which were subcultured 5 days were the best initiation material for protoplast isolation, embryogenic calli the second, and the yield and viability of protoplasts from leaves of in vitro-grown plantlets the lowest. The optimal enzyme solution for protoplasts isolation from suspension cells was 2.0% Cellulase Onozuka R-10+0.2%Pectolgase Y-23+0.55mol/L mannitol+5mM MES +10mMCacl₂(pH5.8). High yield and viability protoplasts were obtained when incubated on constant-temperature shaker with 90rpm at (25±1)℃for 5 hours and centrifugation with 1000r/min for 5min.Protoplasts culture was conducted: The best cultured method was shallow liquid culture, and the cultured medium was 1/2MS+1mg/L2.4-D+0.5mg/L 6- BA+0.1%casein hydrolysate+1%glucose+0.55mol/L mannitol, and the optimal culture density was 1×10⁵/mL. The highest division and cell colony frequency were 7.4% and 0.57% respectively. The first division was observed in 4⁵ days.In this work, protoplasts isolated from cell suspensions of Cangshan garlic as a donor were irradiated by UV at the intensity of 380uW/cm² for 30s and were fused with those of Sulian garlic as a recipient by PEG method. The initial protoplast fusing was observed.