CN105685072A - 一种植物组培外植体包膜、抗菌、促生剂及使用方法 - Google Patents

一种植物组培外植体包膜、抗菌、促生剂及使用方法 Download PDF

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CN105685072A
CN105685072A CN201610140571.4A CN201610140571A CN105685072A CN 105685072 A CN105685072 A CN 105685072A CN 201610140571 A CN201610140571 A CN 201610140571A CN 105685072 A CN105685072 A CN 105685072A
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樊仲书
王海斌
张玉华
樊昌华
孔祥海
樊君早
陈晓婷
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Longyan Hekang Biotechnology Co Ltd
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Abstract

本发明公开一种植物组培外植体包膜、抗菌、促生剂及使用方法，所述的促生剂中含有按以下质量百分数计的组分：羧化壳聚糖1%~2%、硫脲0.2%~0.4%、抗生素0.005%~0.007%、恶霉灵0.02%~0.03%、乌洛托品0.05%~0.07%、霜霉威盐酸盐0.03%~0.05%、MS+6-BA 0.12%~0.14%、萘乙酸0.14%~0.18%、生长素0.16%~0.18%、矮壮素0.4%~0.6%。通过将植物组培外植体生芽、生根及抗菌所需的物质与壳聚糖混合后在硫脲的作用下形成的混合物易于成膜；外植体在包膜、抗菌、促生剂中浸泡后，在外植体表面形成一层复合膜，该复合膜不仅有利于促进外植体生芽率、生根率，同时有利于保护植物外植体免受微生物侵染，提高成活率。

Description

一种植物组培外植体包膜、抗菌、促生剂及使用方法
技术领域
本发明涉及一种包膜、抗菌、促生剂，具体涉及一种植物组培外植体包膜、抗菌、促生剂及使用方法。
背景技术
植物组培技术中，外植体污染的控制是植物组织培养成功的关键难点之一。外植体所携带微生物包含外植体内部及表面的细菌或真菌等微生物，在外植体接种过程中常规的消毒手段只能除去外植体表面的微生物而无法根除外植体内部的微生物，在培养过程中容易导致外植体内部的微生物滋生进而污染了植物组培苗，降低成活率。因此，建立一种植物组培外植体的抗菌试剂，同时有利于提高外植体的生芽、生根率具有重要的意义，本案由此产生。
发明内容
本发明的目的在于针对现有技术的不足，提供一种植物组培外植体包膜、抗菌、促生剂及使用方法。通过将植物组培外植体于本发明的制剂中浸泡后，有利于降低植物组培外植体的污染率，提高外植体的生芽与生根率，有效提高经济植物组培的经济效益。
为了达成上述目的，本发明采用的主要技术方案如下：
一种植物组培外植体包膜、抗菌、促生剂，所述的促生剂中含有按以下质量百分数计的组分：羧化壳聚糖1%~2%、硫脲0.2%~0.4%、抗生素0.005%~0.007%、恶霉灵0.02%~0.03%、乌洛托品0.05%~0.07%、霜霉威盐酸盐0.03%~0.05%、MS+6-BA0.12%~0.14%、萘乙酸（NAA）0.14%~0.18%、生长素（IAA）0.16%~0.18%、矮壮素（CCC）0.4%~0.6%。
进一步的，所述促生剂由按以下质量百分数计的组分组成：羧化壳聚糖1%~2%，硫脲0.2%~0.4%，抗生素0.005%~0.007%、恶霉灵0.02%~0.03%、乌洛托品0.05%~0.07%、霜霉威盐酸盐0.03%~0.05%、氯化锌0.02%~0.04%，MS+6-BA0.12%~0.14%、萘乙酸0.14%~0.18%、生长素0.16%~0.18%、矮壮素0.4%~0.6%，维生素B10.06%~0.08%、维生素B20.06%~0.08%、维生素B60.05%~0.07%、维生素B90.04%~0.05%、维生素B120.03%~0.04%，余量为蒸馏水；所有组分质量百分数之和为100%。
所述的抗生素为两性霉素B和青霉素按照质量比1:1复配的混合物。
一种制备如上所述的植物组培外植体包膜、抗菌、促生剂的方法：将羧化壳聚糖加入蒸馏水中，搅拌60min，加入硫脲，搅拌30min，将溶液置于灭菌锅中灭菌，待溶液冷却至40-50℃；在无菌条件下加入抗生素、恶霉灵、乌洛托品、霜霉威盐酸盐、氯化锌，搅拌30min，加入MS+6-BA、萘乙酸、生长素、矮壮素，搅拌30min；然后在无菌条件下，加入维生素B1、维生素B2、维生素B6、维生素B9、维生素B12，搅拌60min，即可制得一种植物组培外植体包膜、抗菌、促生剂。
一种如上所述的植物组培外植体包膜、抗菌、促生剂的使用方法：将外植体表面用无菌水冲洗3次，冲洗后的外植体于促生剂中浸泡2min~3min，超净台中晾置5min，于促生剂中再浸泡1min~2min，超净台中晾置5min，最后于促生剂中再浸泡1min~2min，超净台中晾置15min，即接种至培养基中培养。
本发明的有益效果在于：
本发明将植物组培外植体生芽、生根及抗菌所需的物质与壳聚糖混合后在硫脲的作用下形成的混合物易于成膜；外植体在包膜、抗菌、促生剂中浸泡后，在外植体表面形成一层复合膜，该复合膜不仅有利于促进外植体生芽率、生根率，同时有利于保护植物外植体免受微生物侵染，提高成活率。
具体实施方式
为充分公开本发明的一种植物组培外植体包膜、抗菌、促生剂的制备与使用方法，以下结合实施实例加以说明。
实施例一
将10g羧化壳聚糖加入1L蒸馏水中，搅拌60min，加入2g硫脲，搅拌30min，将溶液置于灭菌锅中灭菌，待溶液冷却至45℃左右；在无菌条件下，加入50mg抗生素、200mg恶霉灵、500mg乌洛托品、300mg霜霉威盐酸盐、200mg氯化锌，搅拌30min；在无菌条件下，加入1.2gMS+6-BA、1.4gNAA、1.6gIAA、4gCCC，搅拌30min；在无菌条件下，加入600mg维生素B1、600mg维生素B2、500mg维生素B6、400mg维生素B9、300mg维生素B12，搅拌60min，制得一种植物组培外植体包膜、抗菌、促生剂。
选择常规的植物组织外植体，将外植体表面用无菌水冲洗3次，将冲洗后的外植体，置于配置的一种植物组培外植体包膜、抗菌、促生剂中浸泡3min，于超净台中晾置5min，于一种植物组培外植体包膜、抗菌、促生剂中浸泡2min，于超净台中晾置5min，于一种植物组培外植体包膜、抗菌、促生剂中浸泡2min，于超净台中晾置15min，将外植体接种至常规的植物组培培养基中，按照常规的植物组培条件进行培养，即可获得大量的健壮芽苗。
将植物外植体于本发明的制剂中浸泡处理后与常规未经处理的外植体共同接种至常规培养基中培养后，经本发明处理的外植体污染率为0.02%，未经处理的外植体污染率为6.85%；生芽率与生根率，本发明处理的外植体比未经处理的外植体提高了3.2倍和3.7倍。
实施例二
将15g羧化壳聚糖加入1L蒸馏水中，搅拌60min，加入3g硫脲，搅拌30min，将溶液置于灭菌锅中灭菌，待溶液冷却至45℃左右；在无菌条件下，加入60mg抗生素、250mg恶霉灵、600mg乌洛托品、400mg霜霉威盐酸盐、300mg氯化锌，搅拌30min；在无菌条件下，加入1.3gMS+6-BA、1.6gNAA、1.7gIAA、5gCCC，搅拌30min；在无菌条件下，加入700mg维生素B1、700mg维生素B2、600mg维生素B6、450mg维生素B9、350mg维生素B12，搅拌60min，制得一种植物组培外植体包膜、抗菌、促生剂。
选择常规的植物组织外植体，将外植体表面用无菌水冲洗3次，将冲洗后的外植体，置于配置的一种植物组培外植体包膜、抗菌、促生剂中浸泡2.5min，于超净台中晾置5min，于一种植物组培外植体包膜、抗菌、促生剂中浸泡1.5min，于超净台中晾置5min，于一种植物组培外植体包膜、抗菌、促生剂中浸泡1.5min，于超净台中晾置15min，将外植体接种至常规的植物组培培养基中，按照常规的植物组培条件进行培养，即可获得大量的健壮芽苗。
将植物外植体于本发明的制剂中浸泡处理后与常规未经处理的外植体共同接种至常规培养基中培养后，经本发明处理的外植体污染率为0.01%，未经处理的外植体污染率为6.21%；生芽率与生根率，本发明处理的外植体比未经处理的外植体提高了3.4倍和3.5倍。
实施例三
将20g羧化壳聚糖加入1L蒸馏水中，搅拌60min，加入4g硫脲，搅拌30min，将溶液置于灭菌锅中灭菌，待溶液冷却至45℃左右；在无菌条件下，加入70mg抗生素、300mg恶霉灵、700mg乌洛托品、500mg霜霉威盐酸盐、400mg氯化锌，搅拌30min；在无菌条件下，加入1.4gMS+6-BA、1.8gNAA、1.8gIAA、6gCCC，搅拌30min；在无菌条件下，加入800mg维生素B1、800mg维生素B2、700mg维生素B6、500mg维生素B9、400mg维生素B12，搅拌60min，制得一种植物组培外植体包膜、抗菌、促生剂。
选择常规的植物组织外植体，将外植体表面用无菌水冲洗3次，将冲洗后的外植体，置于配置的一种植物组培外植体包膜、抗菌、促生剂中浸泡2min，于超净台中晾置5min，于一种植物组培外植体包膜、抗菌、促生剂中浸泡1min，于超净台中晾置5min，于一种植物组培外植体包膜、抗菌、促生剂中浸泡1min，于超净台中晾置15min，将外植体接种至常规的植物组培培养基中，按照常规的植物组培条件进行培养，即可获得大量的健壮芽苗。
将植物外植体于本发明的制剂中浸泡处理后与常规未经处理的外植体共同接种至常规培养基中培养后，经本发明处理的外植体污染率为0.01%，未经处理的外植体污染率为6.32%；生芽率与生根率，本发明处理的外植体比未经处理的外植体提高了3.1倍和3.3倍。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。

Claims (5)

1.一种植物组培外植体包膜、抗菌、促生剂，其特征在于：所述的促生剂中含有按以下质量百分数计的组分：羧化壳聚糖1%~2%、硫脲0.2%~0.4%、抗生素0.005%~0.007%、恶霉灵0.02%~0.03%、乌洛托品0.05%~0.07%、霜霉威盐酸盐0.03%~0.05%、MS+6-BA0.12%~0.14%、萘乙酸0.14%~0.18%、生长素0.16%~0.18%、矮壮素0.4%~0.6%。
2.根据权利要求1所述的植物组培外植体包膜、抗菌、促生剂，其特征在于：所述促生剂由按以下质量百分数计的组分组成：羧化壳聚糖1%~2%，硫脲0.2%~0.4%，抗生素0.005%~0.007%、恶霉灵0.02%~0.03%、乌洛托品0.05%~0.07%、霜霉威盐酸盐0.03%~0.05%、氯化锌0.02%~0.04%，MS+6-BA0.12%~0.14%、萘乙酸0.14%~0.18%、生长素0.16%~0.18%、矮壮素0.4%~0.6%，维生素B10.06%~0.08%、维生素B20.06%~0.08%、维生素B60.05%~0.07%、维生素B90.04%~0.05%、维生素B120.03%~0.04%，余量为蒸馏水；所有组分质量百分数之和为100%。
3.根据权利要求1或2所述的植物组培外植体包膜、抗菌、促生剂，其特征在于：所述的抗生素为两性霉素B和青霉素按照质量比1:1复配的混合物。
4.一种制备如权利要求2所述的植物组培外植体包膜、抗菌、促生剂的方法，其特征在于：将羧化壳聚糖加入蒸馏水中，搅拌60min，加入硫脲，搅拌30min，将溶液置于灭菌锅中灭菌，待溶液冷却至40-50℃；在无菌条件下加入抗生素、恶霉灵、乌洛托品、霜霉威盐酸盐、氯化锌，搅拌30min，加入MS+6-BA、萘乙酸、生长素、矮壮素，搅拌30min；然后在无菌条件下，加入维生素B1、维生素B2、维生素B6、维生素B9、维生素B12，搅拌60min，即可制得一种植物组培外植体包膜、抗菌、促生剂。
5.一种如权利要求1或2所述的植物组培外植体包膜、抗菌、促生剂的使用方法，其特征在于：将外植体表面用无菌水冲洗3次，冲洗后的外植体于促生剂中浸泡2min~3min，超净台中晾置5min，于促生剂试剂中再浸泡1min~2min，超净台中晾置5min，最后于促生剂中再浸泡1min~2min，超净台中晾置15min，即接种至培养基中培养。
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