樟科等树种种子超低温保存机理与技术研究
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摘要
顽拗性林木种子超低温保存的研究，延长种子的贮藏寿命具有重要的理论和实际意义。本研究针对樟科5种树种种子及乳源木莲种子，进行超低温保存，测定保存前后的种子生理生化特性，如脱氢酶、电导率、α—淀粉酶、MDA和脯氨酸等，从而研究种子超低温保存机理。同时探讨出各树种超低温保存的最适含水量、冷解冻技术和防冻剂预处理技术，为林业生产提供科学的理论依据。
1．超低温保存后20％含水率的肉桂离体胚其脱氢酶和α—淀粉酶以快冻快解方式活力最高，电导率值最低，表明其膜系统最完整；MDA以缓冻快解值最低，表明其膜脂过氧化程度低，膜伤害最小；20％含水率的脯氨酸以缓冻快解方式处理最高，脱水耐性能很好地得到诱导；20％含水率的肉桂种子的电导率以缓冻快解的膜结构完整性最好。在添加防冻剂后，离体胚的最适贮藏含水率提高到25％，且比没有预处理的保存效果更好。不同防冻剂种类和浓度的保存效果不同。
2．15％含水率的樟树离体胚以快冻快解方式其各生理指标均表明所受的低温伤害最小，活力最好，添加防冻剂后虽然没有提高最适贮藏含水率，但远比没有预处理的保存效果更好。
3．10％含水率的山苍子离体胚其脱氢酶和α—淀粉酶以快冻快解方式处理活性最高，MDA最低，膜结构完整性最好，而脯氨酸则以缓冻快解方式处理最高，脱水耐性能很好地得到诱导。防冻剂处理后，最适贮藏含水率提高到15％，以缓冻缓解效果最好。
4．10％含水率的建润楠离体胚的脱氢酶和α—淀粉酶以快冻快解方式处理活性最高；电导率和MDA以快冻快解方式处理最低，表明其膜伤害最小；脯氨酸含量在15％含水率时含量最高。添加防冻剂后，离体胚的最适贮藏含水率提高到15％，且比没有预处理的保存效果更好。
5．檫树种子的离体胚在15％含水率缓冻缓解脱氢酶含量最高，种子活力最高；而其MDA含量最少，表明其膜脂过氧化程度最低；离体胚在15％含水率快冻快解方式处理其电导率最低，α—淀粉酶活性最高，脯氨酸含量最大。添加防冻剂后以缓冻缓解效果最好。
6．乳源木莲种子以15％含水率快冻快解方式处理电导率值最低；离体胚在10％含水率缓冻快解时脱氢酶含量最高；快冻快解方式处理α—淀粉酶和脯氨酸含量最高。添加防冻剂后种子15％含水率快冻快解方式处理电导率值最低；离体胚在15％含水率快冻快解时脱氢酶含量最高；10％含水率缓冻快解时α—淀粉酶活性最高；15％含水率缓冻快解时MDA含量最低，而脯氨酸含量最高。
总之，超低温保存林木种子，含水量是保存成败的关键因素之一，适度的脱水有助于提高贮藏成功率。不同的树种的最适贮藏含水率不同，但对大多数顽拗性种子超低温保存而言15％含水率的种子和离体胚是最佳的。添加防冻剂后最适贮藏含水率可提高到20％左右，且贮藏效果很好，接近于对照。
The study on cryopreservation mechanism of recalcitrant seeds germplasm of tree species has very important theoretics and practic sense ,it can improve the seeds vigor and prolong the span of seeds conservation, which of six species were study on this article .such as Cinnamomum cassia,Cinnamomum caphora(L.),Litseacubeba,Machilus oreophila,Manglietia yuyuanensis Law.This study dealt with cryopreservation mchanism based on analysis of various rules of physiology and biochemistry characteristic, including dehydrogenase , a -amylase activity ,relative conductivity, MDA and proline content .Meanwhile the optimal technique of cryopreservation, including optimal moisture content (MC), freezing technique, thawing technique and pre-treatment technique of cryoprotectants,were drew out to serve for forest production. The results showed as follows.
1.After cryopreservation, the dehydrogenase and a -amylase activity of 20% moisture content Cinnamomum cassia embroy was the highest by fast-freezing-quick-thawing method and the relatively conductivity was the lowest. MDA content was very low by mild-freezing-quick-thawing method ;Desiccation-tolerance of exised embryo 20% moisture content was well induced and its proline content accumulated more than any one of other.The vigor are maintained better than other treatment by using method of mild-freezing-quick-thawing.the relative conductivity was lowest by mild-freezing-quick-thawing method. With pre-treatment of cryoprotectants solution, optimal moisture content of exised embryo enhanced to 25%moisture content level, and the conservation effectiveness was better than that of no adding cryoprotectants treatment,which depended on the type and concentration of cryoprotectants.
2.A11 the physiology and biochemistry characteristic index show that 15%moisture content Cinnamomum caphora embroy was adapt to cryopreservation by fast-freezing-quick-thawing mothod. With pre-treatment of cryoprotectants solution,the result was more better than with none.
3.The dehydrogenased and a-amylase activity of 10% moisture content embroy were the highest and MDA content was the lowest by fast-freezing-quick-thawing method. Tts proline content was the highest by mild-freezing-quick-thawing method.With the cryoprotectants the optimal conservation was 15% moisture content and mild freezing mild thawing treament.
4.The optimal treament of machilus oreophila emproy were combination of 10% moisture content and fast-freezing-quick-thawing,which dehydrogenase and a-amylase activity were all high .Relative conductivity and MDA were low and membrane repair ability of embroy is strong with fast-freezing-quick-thawing ,meanwhile the proline content was the lowest of 15% moisture content. With pre-treatment of cryoprotectants solution and mild-freezing-quick-thawing,optimal
MC of ecised embryo enhanced to 15% moisture content level,which effectness is better than that of none.
5.The optimal treatment of Sassafras tsumu embroy is 15% moisture content using mild-freezing-mild-thawing,the dehydrogenase activity was the highest and MDA content was the lowest.The relative conductivity is the lowest, After using the cryoprotectants and mild-freezing-mild-thawing the effectness is the best.
6.Manglietia yuynanensis seed of 15% moisture content through fast-freezing-quick-thawing treatment.The dehydrogenase activity of 10% moisture content embroy with mild-freezing-quick-thawing, while the a-amylase activity and the proline content were the highest with fast-freezing-quick-thawing method.With pretreatment the relative conductivity of 15% moisture content seed through fast-freezing-quick-thawing treatment was the lowest ;the dehydrogenase activity of 15% moisture content embroy with fast-freezing-quick-thawing, while the a-amylase activity of 10% moisture and the proline content of 15% moisture content were the highest with mild-freezing-quick-thawing method.
7.In a whole ,as for the cryopreservation of seed germplasm, especial of recalcitrant seed of tree species,,the key of success is to control moisture c
1．超低温保存后20％含水率的肉桂离体胚其脱氢酶和α—淀粉酶以快冻快解方式活力最高，电导率值最低，表明其膜系统最完整；MDA以缓冻快解值最低，表明其膜脂过氧化程度低，膜伤害最小；20％含水率的脯氨酸以缓冻快解方式处理最高，脱水耐性能很好地得到诱导；20％含水率的肉桂种子的电导率以缓冻快解的膜结构完整性最好。在添加防冻剂后，离体胚的最适贮藏含水率提高到25％，且比没有预处理的保存效果更好。不同防冻剂种类和浓度的保存效果不同。
2．15％含水率的樟树离体胚以快冻快解方式其各生理指标均表明所受的低温伤害最小，活力最好，添加防冻剂后虽然没有提高最适贮藏含水率，但远比没有预处理的保存效果更好。
3．10％含水率的山苍子离体胚其脱氢酶和α—淀粉酶以快冻快解方式处理活性最高，MDA最低，膜结构完整性最好，而脯氨酸则以缓冻快解方式处理最高，脱水耐性能很好地得到诱导。防冻剂处理后，最适贮藏含水率提高到15％，以缓冻缓解效果最好。
4．10％含水率的建润楠离体胚的脱氢酶和α—淀粉酶以快冻快解方式处理活性最高；电导率和MDA以快冻快解方式处理最低，表明其膜伤害最小；脯氨酸含量在15％含水率时含量最高。添加防冻剂后，离体胚的最适贮藏含水率提高到15％，且比没有预处理的保存效果更好。
5．檫树种子的离体胚在15％含水率缓冻缓解脱氢酶含量最高，种子活力最高；而其MDA含量最少，表明其膜脂过氧化程度最低；离体胚在15％含水率快冻快解方式处理其电导率最低，α—淀粉酶活性最高，脯氨酸含量最大。添加防冻剂后以缓冻缓解效果最好。
6．乳源木莲种子以15％含水率快冻快解方式处理电导率值最低；离体胚在10％含水率缓冻快解时脱氢酶含量最高；快冻快解方式处理α—淀粉酶和脯氨酸含量最高。添加防冻剂后种子15％含水率快冻快解方式处理电导率值最低；离体胚在15％含水率快冻快解时脱氢酶含量最高；10％含水率缓冻快解时α—淀粉酶活性最高；15％含水率缓冻快解时MDA含量最低，而脯氨酸含量最高。
总之，超低温保存林木种子，含水量是保存成败的关键因素之一，适度的脱水有助于提高贮藏成功率。不同的树种的最适贮藏含水率不同，但对大多数顽拗性种子超低温保存而言15％含水率的种子和离体胚是最佳的。添加防冻剂后最适贮藏含水率可提高到20％左右，且贮藏效果很好，接近于对照。
The study on cryopreservation mechanism of recalcitrant seeds germplasm of tree species has very important theoretics and practic sense ,it can improve the seeds vigor and prolong the span of seeds conservation, which of six species were study on this article .such as Cinnamomum cassia,Cinnamomum caphora(L.),Litseacubeba,Machilus oreophila,Manglietia yuyuanensis Law.This study dealt with cryopreservation mchanism based on analysis of various rules of physiology and biochemistry characteristic, including dehydrogenase , a -amylase activity ,relative conductivity, MDA and proline content .Meanwhile the optimal technique of cryopreservation, including optimal moisture content (MC), freezing technique, thawing technique and pre-treatment technique of cryoprotectants,were drew out to serve for forest production. The results showed as follows.
1.After cryopreservation, the dehydrogenase and a -amylase activity of 20% moisture content Cinnamomum cassia embroy was the highest by fast-freezing-quick-thawing method and the relatively conductivity was the lowest. MDA content was very low by mild-freezing-quick-thawing method ;Desiccation-tolerance of exised embryo 20% moisture content was well induced and its proline content accumulated more than any one of other.The vigor are maintained better than other treatment by using method of mild-freezing-quick-thawing.the relative conductivity was lowest by mild-freezing-quick-thawing method. With pre-treatment of cryoprotectants solution, optimal moisture content of exised embryo enhanced to 25%moisture content level, and the conservation effectiveness was better than that of no adding cryoprotectants treatment,which depended on the type and concentration of cryoprotectants.
2.A11 the physiology and biochemistry characteristic index show that 15%moisture content Cinnamomum caphora embroy was adapt to cryopreservation by fast-freezing-quick-thawing mothod. With pre-treatment of cryoprotectants solution,the result was more better than with none.
3.The dehydrogenased and a-amylase activity of 10% moisture content embroy were the highest and MDA content was the lowest by fast-freezing-quick-thawing method. Tts proline content was the highest by mild-freezing-quick-thawing method.With the cryoprotectants the optimal conservation was 15% moisture content and mild freezing mild thawing treament.
4.The optimal treament of machilus oreophila emproy were combination of 10% moisture content and fast-freezing-quick-thawing,which dehydrogenase and a-amylase activity were all high .Relative conductivity and MDA were low and membrane repair ability of embroy is strong with fast-freezing-quick-thawing ,meanwhile the proline content was the lowest of 15% moisture content. With pre-treatment of cryoprotectants solution and mild-freezing-quick-thawing,optimal
MC of ecised embryo enhanced to 15% moisture content level,which effectness is better than that of none.
5.The optimal treatment of Sassafras tsumu embroy is 15% moisture content using mild-freezing-mild-thawing,the dehydrogenase activity was the highest and MDA content was the lowest.The relative conductivity is the lowest, After using the cryoprotectants and mild-freezing-mild-thawing the effectness is the best.
6.Manglietia yuynanensis seed of 15% moisture content through fast-freezing-quick-thawing treatment.The dehydrogenase activity of 10% moisture content embroy with mild-freezing-quick-thawing, while the a-amylase activity and the proline content were the highest with fast-freezing-quick-thawing method.With pretreatment the relative conductivity of 15% moisture content seed through fast-freezing-quick-thawing treatment was the lowest ;the dehydrogenase activity of 15% moisture content embroy with fast-freezing-quick-thawing, while the a-amylase activity of 10% moisture and the proline content of 15% moisture content were the highest with mild-freezing-quick-thawing method.
7.In a whole ,as for the cryopreservation of seed germplasm, especial of recalcitrant seed of tree species,,the key of success is to control moisture c
引文
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