Litz R E, Grosser J W
Tropical Research and Education Center, University of Florida, 18905 SW 280 St, Homestead FL 33031-3314, USA Fax: +1 305 246 7003 e-mail:
Citrus Research and Education Center, University of Florida, 700 Experiment Station Rd, Lake Alfred FL 33850-2299, USA, , , , , , US.
Plant Cell Rep. 1998 Dec;18(3-4):235-242. doi: 10.1007/s002990050563.
Protoplasts were isolated from embryogenic suspension cultures derived from avocado (Persea americana Mill.) zygotic embryos and nucellus in an enzyme digestion solution consisting of 1% cellulase Onozuka RS, 1% Macerase R10, 0.2% Pectolyase Y-23, 0.7 M mannitol. 24.5 mM CaCl, 0.92 mM NaHPO and 6.25 2-[N-morpholino]ethanesulfonic acid (1.5 ml) mixed with 0.7 M MS8P (2.5 ml). MS8P medium consisted of Murashige and Skoog salts without NHNO, 1 mg l thiamine HCl, 100 mg l myo-inositol, 3.1 g l glutamine and 8P organic addenda. Medium osmolarity was adjusted with 0.15 M sucrose and 0-0.55 M mannitol. Protoplast yields of 3.5×10 protoplasts g were obtained. Growth and development of the protoplasts were significantly affected by osmolarity, nitrogen source, plating density and culture medium dilution. Under optimum conditions, proembryos developed directly from embryogenic protoplasts and subsequently into somatic embryos. Optimum conditions for somatic embryo development included the culture of protoplasts at a density of 0.8-1.6×10 ml in 0.4 M MS8P for 2-3 weeks, followed by subculture in 0.15 M MS8P at a diluted density of 20-40× for 1 month in darkness to obtain somatic embryos. Mature somatic embryos were recovered on semisolid medium; however, a low frequency of plantlet recovery (≤1%) from protoplast-derived somatic embryos was observed.
从鳄梨（Persea americana Mill.）合子胚和珠心组织的胚性悬浮培养物中分离原生质体，酶解液由1%纤维素酶Onozuka RS、1%离析酶R10、0.2%果胶酶Y-23、0.7M甘露醇、24.5mM氯化钙、0.92mM磷酸氢二钠和6.25 2-[N-吗啉代]乙磺酸（1.5ml）与0.7M MS8P（2.5ml）混合而成。MS8P培养基由不含硝酸铵的Murashige和Skoog盐、1mg/L盐酸硫胺素、100mg/L肌醇、3.1g/L谷氨酰胺和8P有机添加物组成。培养基渗透压用0.15M蔗糖和0-0.55M甘露醇调节。原生质体产量达到3.5×10个原生质体/g。原生质体的生长和发育受到渗透压、氮源、接种密度和培养基稀释度的显著影响。在最佳条件下，胚性原生质体直接发育成原胚，随后发育成体细胞胚。体细胞胚发育的最佳条件包括将原生质体以0.8-1.6×10个/ml的密度接种在0.4M MS8P中培养2-3周，然后在0.15M MS8P中以20-40×的稀释密度在黑暗中传代培养1个月以获得体细胞胚。成熟的体细胞胚在半固体培养基上再生；然而，观察到从原生质体来源的体细胞胚中再生植株的频率较低（≤1%）。
