Cryopreservation Of Seeds:Dormancy And Water Content In Perspective

The project is aimed to shed more light on the key problems faced during seed storage at gene bank standards and cryopreservation.Two important problems were addressed(1)dormancy and(2)high water content.Though seeds with dormancy and high water content are amendable to cryopreservation,often the storage is not easy.Chapter 2 focuses on the cryopreserving dormant seeds of Dodonea viscosa having PY.More specifically,this chapter focuses on a vital question of when to give dormancy breaking during storage,how to break dormancy and storing at liquid nitrogen temperature.Chapter 3 extends this idea on Prunus armeniaca collected from Xinjiang province China.This species is supposed to have double dormancy(but see chapter 3).These species have been chosen because D.viscosa might serve as an important reference for species with PY and have restoration potential.P.armenica is an important cash crop in China and presumably provide an important reference to other Rosaceae family species and other families with hard endocarp(hereafter stone).Chapter 4 and 5 report the freezing behavior of Lactuca sativa an orthodox seeds in their imbibed state and provide insights in to recalcitrant seed storage.In particular,chapter 4 provides some explanations on optimizing seed storage protocols on recalcitrant seeds by focusing on cooling rate.Whereas chapter 5 address the importance and requirement of cryoprotectant.In chapter 2,dormancy and feasibility of cryopreservation were investigated.Seeds of Dodonaea viscosa(Sapindaceae)have a water-impermeable seed coat,i.e.physical dormancy(PY).Although mechanical scarification,dry heat,sulphuric acid and hot water treatment make seeds permeable under laboratory conditions,the mechanisms by which dormancy is alleviated in natural environments have not yet been understood completely.The present investigation aims to understand the pattern of dormancy alleviation in D.viscosa seeds using an artificial burial approach for 2 years.Secondary aim of this chapter was to develop cryopreservation protocols.Freshly collected seeds held in hydrated soil at 10/20°C,15/20°C,15/30°C,20/35°C and 25°C for 32 weeks germinated to less than 15%,irrespective of storage temperature.Dry storage of seeds at15,20,25 and 30°C for 1 year did not break dormancy.Hot water treatment at 80 and90°C for 30 s broke dormancy in 90% of the seeds.On the other hand,burying seeds ata depth of 3–5 cm in the natural environment for 2 years increased germination from 7to 71%.In particular,seeds exhumed after summer in both years showed a significant increase in germination percentage(p < 0.05).However,seeds buried after summer did not germinate to a higher percentage when exhumed prior to summer.It is suggested that a high summer temperature,rising above 60°C in the top soil layer of the tropics,is a likely factor breaking dormancy.Most seeds germinated during burial,which indicates that light is not a cue for germination.Seeds survived both desiccation and liquid nitrogen storage,indicating the orthodox behavior.Applying dormancy breaking before or after storage had no significant difference in final germination percentage after storage,however breaking dormancy at the end of storage is suggested to have increased viability.In chapter 3,I investigated the mechanism of dormancy and cryopreservation in Apricot seeds.Apricot(Prunus armeniaca L.)is an important cash crop cultivated in five continents of the world for its edible fruit.Due to the overexploitation of endemic forest to plant commercial varieties,there is an urgent need to conserve the genetic resources.The techniques to break dormancy and feasibility of cryopreservation were investigated using freshly collected fully mature P.armeniaca seeds(true seeds +endocarp)from Xinjiang province,China.No seeds germinated at the time of collection when incubated at 20/25°C for 5 weeks.The endocarp was permeable to water,thus dormancy was not due to an impermeable coat,i.e physical dormancy.Manual removal of endocarps resulted in germination of 10% of seeds without any other treatment.Gibberellic acid treatment improved germination in 66-83% of the seeds and cold-wet stratification relieved dormancy in 69-80% of seeds.Thus,seeds have non-deep to deep physiological dormancy.Drying to water content of 3.3% Fwb.did not lead to viability loss confirming the orthodox nature.Seeds dried over silica gel(4.1% Fwb.)survived better compared to air dried samples(7.8% Fwb.)following liquid nitrogen storage.Although storing dormancy broken seeds in liquid nitrogen resulted in lesser germination percentage after 6 months of storage compared to the dormancy broken at the end of storage,this effect was not statistically significant(p > 0.05).The results demonstrate seeds of P.armeniaca can be stored in liquid nitrogen for conservation purpose.The main objective of chapter 4 was to extend the information available on seed freezing tolerance mechanism studied in Lactuca sativa(L.)to elucidate whether extracellular freezing plays a pivotal role in seed storage at liquid nitrogen temperature.Differential scanning calorimetry(DSC)and programmable freezer were used to study the effects of cooling rate on the survival of fully hydrated L.sativa at low temperatures.The cooling rates are 60?C h-1 and 3?C h-1 separately.Seeds were cooled to the minimum temperatures of-40°C and-20°C.Germination percentage of seeds was taken as a parameter to evaluate the cryopreservation.Rapidly cooled(60?C h-1)hydrated seeds showed both high temperature exotherm(HTE)and low temperature exotherms(LTE),with mortality correlating with temperatures below the LTE at c.-18?C as a consequence of ice formation in the embryo.In contrast,cooling hydrated seed slowly at 3?C h-1 generated only one exotherm above-10?C,seed viability was unaffected by cooling beyond this exotherm to a temperature minimum of-40°C.Seeds cooled at 3?C h-1to temperatures below-20°C survived subsequent storage at liquid nitrogen for 24 h,although many produced abnormal seedlings.The number of seeds survived and produced normal seedlings increased with decrease in temperature.No statistically significant difference(p > 0.01)observed between seeds cooled to various temperatures and subsequently cryopreserved at temperatures below-20°C to a terminal temperature of-40°C.However,seeds fast cooled to all temperatures failed to tolerate liquid nitrogen.These results demonstrate that the mechanisms used by seeds in natural environment can be successfully transformed to bank seeds using low temperatures.Chapter 5 critically evaluates the importance of cryoprotectant in cryopreservation of imbibed lettuce seeds.Dry seeds of Lactuca sativa(iceberg lettuce)and L.sativa cv.Hance salad that had imbibed water for 40 hours at 4°C(to moisture contents of 68 ±3.9 and 62 ± 2.9%,respectively)followed by imbibition in 15 or 35% dimethyl sulfoxide(DMSO)for eight hours,were cooled to various sub-zero temperatures(>-40°C)at 3 or 60°C h-1 in a programmable freezer.After cooling to the various low temperatures,seeds were either thawed directly in a water bath at 40°C or plunged into liquid nitrogen for one week before thawing at 40°C.Viability was assessed as the number of seeds that germinated at 21°C.The ice formation temperature,which was detected using a thermocouple during cooling(in the range of-12 to-40°C),did not affect subsequent germination following liquid nitrogen storage.The higherconcentration of DMSO was toxic whilst the lower concentration was essential for higher seed survival after both sub-zero and liquid nitrogen storage.Survival of both cultivars was best achieved after pre-cooling to-25 to-40°C.Pre-cooling at 3°C h-1 was effective for iceberg lettuce seeds but not for those of L.sativa Hance salad,implying a cultivar-specific effect.These findings provide important insights into the interaction of water content,pre-cooling,cooling rate and cryoprotectant concentration on seed storage.