无菌马尾松种子超低温保存技术研究

Cryopreservation of sterile Pinus massoniana seeds

关键词:  马尾松种子, 超低温保存, 干燥, 含水量, 安全含水量范围, 发芽率

Key words:  Pinus massoniana seeds, cryopreservation, desiccation, water content, range of safe water content, germination percentage

摘要:

该研究利用液氮将不同含水量的无菌马尾松种子(27.4%、24.6%、22.7%、16.8%、15.8%、10.7%、7.5%、6.1%、4.8%、3.2%)进行超低温保存。结果表明:(1)在3.2%～6.1%含水量范围内,经液氮冷冻保存后的发芽率随着含水量升高而逐渐升高,在含水量6.1%时,发芽率达到最大值(91.33%); 当含水量大于6.1%时,发芽率逐渐下降,尤其是当含水量大于15.8%时,发芽率迅速下降; 在3.2%～7.5%含水量范围内冻存后发芽率在80%以上。(2)冷冻、化冻方式影响超低温保存效果。种子无需低温预冷,直接投入液氮保存(快速冷冻)效果最好; 室温空气化冻(缓慢化冻)较42 ℃水浴化冻发芽率高,且后者容易发生种皮炸裂现象。(3)种皮对马尾松种子超低温保存过程起到保护作用,使其免受机械损伤,去掉外种皮的种子冻存后发芽率显著下降,且容易出现形态不正常的幼苗。(4)超低温保存对马尾松种子萌发具有一定“刺激”作用,在最适含水量6.1%时,经超低温保存后种子的发芽率显著大于对照种子。总之,含水量、冷冻方法、化冻方法、种皮结构显著影响超低温保存效果,马尾松种子超低温保存的最优方法是将种子含水量控制在6.1%,直接投入液氮快速冷冻后室温空气缓慢化冻,冻后发芽率可在90%以上。马尾松种子超低温保存技术体系的建立,为其种质资源的长期保存提供了技术支持。

Abstract:

To explore long term preservation of Pinus massoniana seeds through cryopreservation technology, aseptic seeds of P. massoniana with different water contents(WC)(27.4%, 24.6%, 22.7%, 16.8%, 15.8%, 10.7%, 7.5%, 6.1%, 4.8%, 3.2%)were cryopreserved in liquid nitrogen. Our results showed that:(1)The germination of cryopreserved seeds increased when the seed WC elevated from 3.2% to 6.1%, reaching the highest rate of 91.33% at the optimum WC of 6.1%; However, seed germination decreased as WC further increased from 6.1% to 27.4%, in particular, with a rapid decline in germination of seeds with 15.8% or higher WC; There is a wide range of safe WC, within 3.2%-7.5%, the average germination rate reached above 80%.(2)In addition to WC, freezing and thawing methods also affected cryopreservation. Rapid freezing by directly immersing seeds into liquid nitrogen is better than pre-freezing at -20 ℃ prior to liquid nitrogen freezing; a slow air-thawing at room temperature gave a better germination result compared with a rapid thawing in a 42 ℃ water bath that caused seed episperm cracking.(3)The episperm also played an important role, which protected the seeds from the mechanical damage. The germination rates without episperm were significantly lower than those with episperm, and the abnormal seedlings were more observed when the episperms were removing.(4)At the optimum WC of 6.1%, cryopreserved seeds germinated better than those non-cryopreserved(control), indicating that cryopreservation may stimulate seed germination of P. massoniana. Collectively, our results showed that water content, freezing and thawing methods as well as the episperm structure can significantly affect cryopreservation of P. massoniana seeds; and the best method for cryopreservation of P. massoniana seeds is to directly freeze seeds with 6.1% WC using liquid nitrogen followed by a slow air-thawing at room temperature, by which the germination can reach above 90%. Results from this study may provide a rational for a long-term storage of P. massoniana seeds through cryopreservation.