The effect of conditioned medium on colony formation from ‘black mexican sweet’ corn protoplasts
The effect of conditioned medium on colony formation from ‘black mexican sweet’ corn protoplasts
复制标题
条件培养基对“黑墨西哥甜”玉米原生质体集落形成的影响
DOI：
10.1016/0168-9452(87)90162-2
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发表时间：
1987
期刊：
影响因子：
5.2
通讯作者：
M. Brenner
中科院分区：
文献类型：
--
作者：
D. Somers;P. Birnberg;W. L. Petersen;M. Brenner
‘黑墨西哥甜’玉米(Zea maysL.)悬浮培养原生质体在从黑墨西哥甜(BMS)培养物分离的条件培养基中培养时，菌落形成频率增加。以每毫升5×104个原生质体的集落形成率的增加作为检测条件因子和考察原生质体对条件培养基的反应的生物指标。条件培养液在−14℃下冷冻保存时稳定性较好，在24℃下保存10天仍保持80%的活性，加热至100℃10分钟后失活。条件培养基中活性最强的菌株在悬浮培养的指数生长期得到恢复。在培养12h内向原生质体中加入条件培养液的集落形成率最高，此后9d内不去除。在原生质体中加入条件培养液可提高细胞分裂频率，但不能提高原生质体活力或细胞壁的形成。大部分调节活性是由于被500分子量(MW)截留的超滤膜拒绝的因子所致。苏氨酸、谷氨酸、半胱氨酸和蛋氨酸的混合物(各为0.25μM)和1 mM的Pro可促进集落形成，但水平低于25%的条件培养液。我们的结果表明，BMS培养细胞产生促进和维持BMS原生质体细胞分裂的条件因子，而游离氨基酸不是条件培养液中的活性因子。
Protoplasts from suspension cultures of ‘Black Mexican Sweet’ corn (Zea maysL.) exhibited increased colony formation frequency when cultured in the presence of conditioned medium isolated from Black Mexican Sweet (BMS) cultures. The increase in colony formation frequency of protoplasts plated at 5 × 104protoplasts/ml was used as a bioassay for detecting conditioning factors and to investigate the response of protoplasts to conditioned medium. Conditioned medium was stable when stored frozen at −14°C, retained 80% activity when stored for 10 days at 24°C and was inactivated by heating to 100°C for 10 min. The most active isolates of conditioned medium were recovered during the exponential growth phase of the suspension cultures. Colony formation frequencies were greatest when conditioned medium was added to protoplasts within 12 h of plating and was not removed for 9 days thereafter. Addition of conditioned medium to protoplasts increased cell division frequency and did not increase protoplast viability or cell wall formation above the control cultures. Most of the conditioning activity was due to factors that were rejected by a 500 molecular weight (MW) cutoff ultrafiltration membrane. A mixture of threonine, glutamate, cysteine and methionine (0.25 μM each), which were the only amino acids detected in conditioned medium, and proline (1 mM) increased colony formation but to lower levels than 25% conditioned medium. Our results indicate that BMS culture cells produce conditioning factors that increase and sustain cell division from BMS protoplasts and that free amino acids are not the active factors in conditioned medium.