Cryopreservation Of Apple With Virus Infection, And Assessments Of Shoot Tip Cryotechniques For Virus Eradication And Preservation In Apple

Cryopreservation is currently considered an ideal means for the long-term conservation of plant germplasm.Often,recovery levels vary among laboratories when the same cryogenic procedures are used for the same genotypes.Attempts to explore the causes of this phenomenon will undoubtedly benefit much wider applications of cryoprotocols in different laboratories.Virus diseases have for a long time threatened the sustainable development of the apple industry.Availability of highly efficient approaches for production of virus-free plants is important to combat virus diseases.Apple stem grooving virus(ASGV)is a difficult-to-eradicate virus.Establishment of efficient eradication method for this virus benefits the sustainable apple production.Availability of plant virus is a prerequisite in all types of virus-related basic and applied studies.Virus is an obligate parasite that colonizes and replicates only in the living cells of the hosts.Long-term preservation of the virus is difficult.Development of efficient and reliable strategies for long-term preservation of plant virus would largely assist these studies.The objectives of the present study were:(1)to assess effects of ASGV infection on recovery of apple‘Gala’shoot tips following cryopreservation,and causes for abnormal shoot regrowth in cryopreserved virus-infected shoot tips were explored;(2)to develop a procedure combining thermotherapy with cryotherapy for efficient eradication of ASGV from the infected shoot tips.Mechanism as to why combining thermotherapy with cryotherapy can efficiently eradicate ASGV was studied,(3)to establish shoot tip cryopreservation for conservation of ASGV and test efficiency of this method for the virus cryopreservation.The main results obtained are listed as followings:1.In vitro shoots of apple‘Gala’single-infected with ASGV and virus-free shoots were used in the present study.The shoot tips were cryopreserved by droplet-vitrification and post-thaw cultured for recovery.Results showed there were no significant differences in survival and regrowth levels between these two types of shoot tips.However,recovery of the virus-free shoot tips following cryopreservation was much faster than the virus-infected ones,and all of the virus-free shoot tips regenerated into normal shoots.The virus-infected shoot tips following cryopreservation regenerated into three types of regrowth:normal shoots,rosette shoots and rosette shoots with abnormal leaves,accounting for 45%,32%and 23%of the total regrowth,respectively.After 4 weeks of subculture on proliferation medium,the virus-infected in vitro shoots produced 7.2 shoots per explant,significantly higher than 5.1 of the virus-free shoots.The number of shoots≥1.0 cm and<1.0cm,≥0.5 cm/explant were 2.9 and 2.2,respectively,with no shoots shorter than 0.5cm produced in the virus-free shoots.The ASGV-infected explant produced 1.8(≥1.0 cm),2.8(<1.0 cm,≥0.5 cm)and 2.6(<0.5 cm)shoots,accounting for 25%,38.9%and 36.1%of the total shoots.Levels of IAA and ZR in the virus-free shoots were 15.9μg/g and 14.3μg/g,significantly higher than those(12.4 and 11.2μg/g)in the virus-infected ones.Levels of total soluble sugar,protein and free proline in the virus-infected shoots were 11.1 mg/g,8.7 mg/g,and 17.2μg/g,which were 16%,30%and28%higher than those in the virus-free ones,while relative electrolyte leakage in the latter was 8.1%,much lower than 9.8%in the former.Histological observations showed that surviving cells in cryopreserved virus-free shoot tips distributed in the first 8 layers of apical dome(AD)and leaf primordia(LPs)1-4 and their survival percentages were about62%and 34%.Surviving cells in cryopreserved virus-infected shoot tips were found in in the first 5 layers in the AD and LPs 1-4,and their survival percentages were about 46%and30%.Cellular ultrastructural observations revealed ultrastructure of cells in the AD were similar between virus-free and virus-infected shoots,but mitochondria in the latter were more elongated than the former.As a result,the reduced levels of IAA and ZR may cause reduced height and increased proliferation in the ASGV-infected shoots.Virus infection-induced cell membrane damage and alterations in the mitochondria shape may cause decreases in cell tolerance to freezing and subsequently results in the reduced level of normal shoot regrowth in cryopreserved shoot tips.2.ASGV-infected‘Gala’was used in this study for establishment of a protocol combining thermotherapy with droplet-vitrification cryotherapy to eradicate ASGV.In vitro stock‘Gala’shoots infected with ASGV were thermo-treated using an alternating temperature of 36 ~oC(day)and 32 ~oC(night).After 4 weeks of thermotherapy,shoot tips in the size of 1.5 mm with 3-4 primordia were excised and subjected to shoot tip culture or cryotherapy.Regenerated plants were transferred to the greenhouse and grown for 10months before the detection of viruses by RT-PCR.Results showed thermotherapy did not affect shoot survival levels but influenced proliferation and shoot length of in vitro shoots.The number of shoots per explant was 4.7 following thermotherapy and 2.5 without thermotherapy,but the length of shoots was 2.5 cm in the former and 3.5 cm in the latter.Regrowth levels of cryopreserved shoot tips were reduced with prolonged duration of thermotherapy,while the virus-free frequencies were improved.Shoot regrowth levels of cryopreserved shoot tips decreased from 55%to 22%and virus eradication frequencies increased from 23%to 100%as thermotherapy durations increased from 2 weeks to 6weeks The optimized protocol was tested in 3 apple cultivars and 1 rootstock,and produced virus eradication frequencies of 100%for‘Fuji’,40%for‘Nongguo 25’,30%for‘Ruixue’and 83%for‘M9’,respectively.Immunohistological localization of ASGV in the shoot tips found that the virus widely distributed across the AD and the youngest LPs in shoot tips of both‘Gala’and‘Ruixue’.The virus-free areas within shoot tips were enlarged with prolonged duration of thermotherapy.In‘Gala’,no viral signals were detected in the AD and the LPs 1-5 after 4 weeks of thermotherapy.However,in‘Ruixue’,only the AD and LPs 1-3 were found to be virus-free after 4 weeks of thermotherapy.Following cryotherapy,surviving cells were found in the AD and LPs 1-3 of both‘Gala’and‘Ruixue’and much more surviving cells were found in the LP 4 of‘Ruixue’than those were from‘Gala’.The variations in the virus distribution and cell surviving patterns between‘Gala’and‘Ruixue’provide experimental explanations to varying virus-free frequencies among genotypes obtained in the present study.3.In vitro shoots of apple‘Gala’single-infected with ASGV were used in this study.Droplet-vitrification and encapsulation-dehydration methods were applied to cryopreserve shoot tips of the virus-infected shoots.Shoot regrowth levels of 62-67%were obtained in cryopreserved shoot tips and 100%of plants recovered after cryopreservation were virus-infected.The virus-infected shoots derived from cryopreservation were subcultured for proliferation.Results showed although shoot proliferation and virus concentration were reduced in the virus-infected shoots after 8 weeks of subculture,these two parameters reached the comparable levels as in the non-cryopreserved virus-infected stock shoots(control)after 16 weeks of subculture.Cryopreserved ASGV was efficiently transmitted to virus-free apple via in vitro micrografting and the infection rate was 100%after 21 days of grafting.Cryopreserved ASGV was also efficiently transmitted to Nicotiana benthamiana by mechanical inoculation.Virus was detected by RT-PCR from leaves of N.benthamiana that had been inoculated for 21 days.Gene sequencing in three fragments of ASGV genome including those coding coat protein and movement protein showed that cryopreserved ASGV shared 99.87%nucleotide identities with ASGV in the virus-infected in vitro shootsThe results obtained from the present study provided experimental proof and emphasized the necessity to using virus-free material for apple cryopreservation.Thermotherapy combining cryotherapy provided a new means for eradication of difficult-to-eradicate viruses like ASGV that can invade the AD.Virus cryopreservation opened a new avenue for safe,efficient and long-term preservation of plant obligate pathogens.