木薯茎尖小滴玻璃化法超低温保存技术优化

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Abstract： Cassava （Manihot esculenta Crantz），as an important food and economic crop，has significant implications for the conservation of its germplasm resources.Currently，the droplet-vitrification method is the most straightforward and widely applied technique for cryopreservation of shoot tips，but systematic optimization of this method for cassava shoottips has not been conducted.Therefore，this study first optimized the key steps in the droplet-vitrification cryopreservation proces for cassava shoot tips.This study found that adding a 4-hour sucrose preculture before treatment with loading solution，combined with ice-cold protection using PVS2 plant vitrification solution for 50 to 60 minutes，significantly improved the cryopreservation effect. However，the applicability test results for fifteen cassava varieties were unsatisfactory. Consequently，this study proceeded to optimize the regeneration medium after cryopreservation of shoot tips and found that removing 1- naphthaleneacetic acid（NAA）and adding a lower concentration of 6-benzylaminopurine（6-BA） in the regeneration medium could prevent the formation of calus from the shoot tips after frezing，thereby improving theregeneration rate. Ultimately，an optimized regeneration medium，designated as RMB2，was developed： （20 GA3+4.4g/L MS+30g/L Sucrose +7g/L Agar. The regeneration rates of cassava varieties COL777 and SC8002 after cryopreservation and culture on RMB2 were 86% and 69% ， respectively.Testing of seven varieties on RMB2 revealed that four varieties achieved shoottip regeneration rates above 30% after cryopreservation. Overall， 80% of the cassava varieties tested achieved regeneration rates above 30% after cryopreservation using the optimized droplet-vitrification method，making this method more widely applicable across different genotypes.Thecryopreservation system established in this study，based on the dropletvitrification method，is simple and efcient，which can effectively promote the cryopreservationof cassava germplasm resources and better safeguard them.

Key words： cassava； droplet-vitrification；cryopreservation；cryoprotectants；preculture；plant growthregulators

植物种质资源是生物多样性的关键组分，对维持生态稳定及推动农业育种工作意义重大。（剩余19287字）