Developments Of Novel Cryopreservation Techniques For Blueberry And Apple Adventitious Buds

Cryopreservation has been considered as an ideal means for long-term preservation of plant germplasm.Shoot tips are preferred as explants for cryopreservation,because they are easy to regenerate into plantlets and are genetically stable,compared with other tissues and cells.In shoot tip cryopreservation,excision of shoot tips requires skillful work,and is laborand time-consuming.In addition,excision of shoot tips may cause mechanical injury to and induces browning of the explant.The objective of the present study was to develop novel cryopreservation protocols for shoot tips of blueberry and apple.The main results obtained are summarized as the followings:1.We established a new cryopreservation method for one of the blueberry cultivars,the “Misty”,using small leaf squares bearing adventitious buds.The third and fourth fully-opened leaves next to the shoot terminal bud of ‘Misty' were excised from 4-week old stock shoots,and three transverse cuts were made through the midrib.These prepared leaf explants were cultured for adventitious bud regeneration,with their adaxial surface down,in adventitious bud regeneration medium composed of WPM supplemented with 20?M ZT.The cultures were maintained at 22 ± 2 °C in the dark for 3 days and transferred to light condition.After 21 days of adventitious bud regeneration,small leaf squares(SLSs,2 × 3 mm),each bearing multiple adventitious buds,were cut from the leaf explant,precultured on WPM containing 0.3 M sucrose and maintained at 22 ± 2 °C in the dark for 24 h.Then the precultured SLSs were treated for 30 min with a loading solution composed of WPM containing 1.0 M sucrose and 2 M glycerol,followed by exposure to plant vitrification solution 2(WPM+30% sucrose +15% ethylene glycol +15%DMSO+0.4M sucrose)at 0 °C for 40 min.Each of dehydrated SLS was then transferred onto an aluminum foil and 3?l of PVS2 was dropped until it covered the SLS.After a direct immersion in liquid nitrogen for 1h,cryopreserved SLSs were re-warmed in WPM containing 1.2 M sucrose for 20 min at room temperature.The cultures were then placed at 22 ± 2 °C in the dark for 1 day and transferred to the light conditions for recovery.With this procedure,more than 23 adventitious buds were produced in each leaf explant,and 100% of SLSs were able to survive and resume shoot regrowth,with seven shoots per SLS obtained following cryopreservation.In ‘Misty',the morphology of plantlets regenerated from cryopreserved SLSs was identical to that of the in vitro-derived ones.No polymorphic bands were detected using inter-simple sequence repeat(ISSR)markers and random amplified polymorphic DNA(RAPD)in plantlets recovered from cryopreservation,indicating the genetic stability of the plantlets regenerated from cryopreserved SLSs.The method was then applied for other cultivars ‘Northland' and ‘Bluegold' and 100% of SLSs were able to survive and resume shoot regrowth.2.The third to fifth fully-opened leaves next to the shoot terminal bud of ‘Rui Xue' and ‘Rui Yang' were excised,and three transverse cuts were made through the midrib.These prepared leaf explants were cultured in adventitious bud regeneration medium composed of 2 mg/L TDZ and 1 mg/L IBA.The cultures were placed at 22 ± 2 °C in the dark for 3 weeks and the regeneration rate of adventitious buds of each leaf explants reached 91.7%.As a result,5.9 and 3.9 shoots were obtained from ‘Rui Xue' and ‘Rui Yang' respectively.We established a new cryopreservation method for apple leaf regeneration by small leaf squares(SLSs)from adventitious bud.After 28 days of adventitious bud regeneration,small leaf squares(SLSs,3 × 3 mm),each bearing 1-2 adventitious buds,were cut from the leaf explant,precultured on MS.The cultures were placed at 22 ± 2 °C in the dark for 24 hours and then transferred to the loading solution composed of MS containing 0.8 M sucrose and 2 M glycerol,followed by exposure to plant vitrification solution 2 at 0 °C for 30 min.Each of dehydrated SLS was then transferred onto an aluminum foil and PVS2 was dropped until it covered the SLS for 1h.The cryopreserved SLSs were re-warmed in unloading solution composed of MS containing 1.2 M sucrose for 20 min followed by post-thaw culture MS containing 30g/L sucrose,4g/L agar,8g/L Gellan gum,0.5mg/L 6-BA and 0.5mg/L GA3 for recovery.After the cultures were placed at 22 ± 2 °C in the dark for 3 days and were transferred to the light conditions for recovery for 7 days,a survival rate of 71.1%-81.1% was obtained.To date,regeneration rate has not been obtained.The largest advantage of use of SLSs-bearing adventitious buds for cryopreservation developed in the present study eliminates shoot tip excision.In addition,production efficiency of shoot tips was significantly o improved.This cryopreservation method can be considered the most efficient cryopreservation protocol so far reported.