Graduate Thesis Or Dissertation
 

Cryopreservation of in vitro-grown apical meristems

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  • Alternating temperature (22°C / -1°C) pretreatments were effective for increasing cold hardiness and recovery of cryopreserved meristems. Combining low temperature pretreatment with abscisic acid (ABA) or sucrose treatments reduced the time required for optimal cold hardiness of meristems. Desiccation tolerance was directly correlated with increased cold hardiness and recovery of cryopreserved meristems. In v/fro-grown pear (Pyrus L.) plantlets were cold acclimated for up to 16 weeks, then shoot tips were either cryopreserved by slow freezing or tested for cold hardiness. Cold acclimation (CA) consisted of alternating temperatures or constant low temperature. Both alternating and constant low temperature CA significantly improved the cold hardiness and cryopreservation regrowth of pear meristems compared with non-acclimated controls. Alternating temperature treatment was significantly better than constant low temperature treatment. Photoperiod and the length of thermoperiod had little or no effect on cold hardiness and cryopreservation regrowth. Some genotypes required only one week alternating temperature treatment for high regrowth but others needed more than 10 weeks. Pretreatment of shoots with ABA slightly increased cryopreserved meristem regrowth and shoot cold hardiness. However, ABA significantly shortened the CA requirement for high cryopreservation regrowth. The optimal pretreatment for recovery of cryopreserved meristems was 3-wk on medium with 50 μM ABA followed by 2-wk CA. Alternating temperature pretreatments were necessary for successful cryopreservation of Rubus meristems. Shoot tip survival of cryopreserved R parvifolius L. increased greatly after 3-wk CA while R caesius L. required 6 or more weeks. Histological studies showed that all cryopreserved R parvifolius shoot tips continued to grow and regenerated directly from the meristematic domes. Alternating-temperature pretreatments significantly increased cryopreserved meristem regrowth of five genotypes each of Lolium and Zoysia grasses, and also significantly increased the dehydration tolerance of both grasses. Lolium genotypes responded to slow freezing and encapsulation-dehydration with 60-100% regrowth, but produced less than 15% regrowth following vitrification. Zoysia responded with greater than 60% regrowth following encapsulation-dehydration when dehydrated to less than 22% water, but did not respond well to other methods.
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