Search

Main Conclusion: Studies on the tissue culture of apple have allowed for molecular, biotechnological and applied breeding research to advance. In the past 8 years, over 100 papers advancing basic biology, genetic transformation and cryobiology have emerged. Apple (Malus × domestica Borkh.; Rosaceae) is an important fruit crop grown mainly in temperate regions of the world. In vitro tissue culture is a biotechnological technique that has been used to genetically improve cultivars (scions) and rootstocks. This updated review presents a synthesis of findings related to the tissue culture of apple and other Malus spp. between 2010 and 2018. Increasingly complex molecular studies that are examining the apple genome, for example, in a bid to identify the cause of epigenetic mutations and the role of transposable elements in this process would benefit from genetically stable source material, which can be produced in vitro. Several notable or curious in vitro culture methods have been reported to improve shoot regeneration and induce the production of tetraploids in apple cultivars and rootstocks. Existing studies have revealed the molecular mechanism underlying the inhibition of adventitious roots by cytokinin. The use of the plant growth correction factor allows hypothetical shoot production from leaf-derived thin cell layers relative to conventional leaf explants to be determined. This updated review will allow novices and established researchers to advance apple and Malus biotechnology and breeding programs.

Plant cryopreservation has provide secure back-ups of germplasm collections of vegetatively propagated crops. Often, recovery levels vary among laboratories when the same cryogenic procedures are used for the same genotypes. The present study investigated the effects of Apple stem grooving virus (ASGV) on shoot proliferation of in vitro stock cultures and recovery of cryopreserved shoot tips of 'Gala' apple. Results showed that virus infection reduced shoot proliferation of in vitro stock cultures and cell ability to regenerate normal shoots in cryopreserved shoot tips. Virus infection increased total soluble protein, total soluble sugar and free proline levels and altered endogenous levels of indoleacetic acid (IAA) and zeatin riboside (ZR), but induced severe cell membrane damage and caused alternation in mitochondria shape of the in vitro stock shoots. The altered levels of IAA and ZR were most likely to be responsible for the reduced shoot proliferation of in vitro stock culture. Cell damage and alternations in mitochondria shape in ASGV-infected shoot tips were most likely responsible for the reduced cell ability to regenerate normal shoots following cryopreservation. To the best of our knowledge, this is the first study on effects of virus infection on recovery of cryopreserved shoot tips. Results reported here emphasize that healthy in vitro stock cultures should be used for cryopreservation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Apple stem grooving virus (ASGV), a difficult-to-eradicate virus from apple propagative materials, causes serious damage to apple production. The use of virus-free plants has been and is an effective strategy for control of plant viral diseases. This study aimed to eradicate ASGV from virus-infected in-vitro-cultured shoots of four apple cultivars and one rootstock by combining thermotherapy with cryotherapy. In vitro stock shoots infected with ASGV were thermo-treated using an alternating temperature of 36°C (day) and 32°C (night). Shoot tips were excised from the treated stock shoots and subjected to cryotherapy. Results showed that, although thermotherapy did not influence shoot survival rates, it reduced shoot growth and proliferation of in vitro shoots. Shoot regrowth rates decreased while virus eradication frequencies increased in cryo-treated shoot tips as time durations of thermotherapy increased from 0 to 6 weeks. Shoot regrowth and frequency of virus eradication were positively and negatively correlated, respectively, with the size of shoot tips. The protocol established here yielded shoot regrowth rates and virus eradication frequencies of 33 to 76% and 30 to 100%, respectively, in the four apple cultivars and one rootstock. Thermotherapy altered virus distribution patterns, subsequently resulting in production of a larger virus-free area in the thermo-treated shoot tips. Many cells in the top layers of apical dome and some cells in the youngest leaf primordia survived in cryo-treated shoot tips; these cells were most likely free of virus infection. Thus, plants regenerated from the procedure of combining thermotherapy with cryotherapy were free of ASGV, as judged by reverse-transcription polymerase chain reaction. To the best of our knowledge, this is the widest-spectrum technique reported thus far for the production of ASGV-free plants and provides a novel biotechnology for the production of virus-free plants in Malus spp.

Key Message: Cryopreservation provides valuable genes for further breeding of elite cultivars, and cryotherapy improves the production of virus-free plants in Malus spp., thus assisting the sustainable development of the apple industry. Apple (Malus spp.) is one of the most economically important temperate fruit crops. Wild Malus genetic resources and existing cultivars provide valuable genes for breeding new elite cultivars and rootstocks through traditional and biotechnological breeding programs. These valuable genes include those resistant to abiotic factors such as drought and salinity, and to biotic factors such as fungi, bacteria and aphids. Over the last three decades, great progress has been made in apple cryobiology, making Malus one of the most extensively studied plant genera with respect to cryopreservation. Explants such as pollen, seeds, in vivo dormant buds, and in vitro shoot tips have all been successfully cryopreserved, and large Malus cryobanks have been established. Cryotherapy has been used for virus eradication, to obtain virus-free apple plants. Cryopreservation provided valuable genes for further breeding of elite cultivars, and cryotherapy improved the production of virus-free plants in Malus spp., thus assisting the sustainable development of the apple industry. This review provides updated and comprehensive information on the development and progress of apple cryopreservation and cryotherapy. Future research will reveal new applications and uses for apple cryopreservation and cryotherapy.

A droplet-vitrification procedure is described for cryopreservation of Malus shoot tips. Survival patterns, recovery types, histological observations, and genetic integrity were compared for Malus shoot tips cryopreserved using this droplet-vitrification procedure and an encapsulation-dehydration procedure that was previously reported by us. In both procedures, three types of shoot tip recovery were observed following cryopreservation: callus formation without shoot regrowth, leaf formation without shoot regrowth, and shoot regrowth. Three categories of histological observations were also identified in cross-sections of shoot tips recovered after cryopreservation using the two cryogenic procedures. In category 1, almost all of the cells (94-95%) in the apical dome (AD) were damaged or killed and only some cells (30-32%) in the leaf primordia (LPs) survived. In category 2, only a few cells (18-20%) in the AD and some cells (30-31%) in the LPs survived. In category 3, majority of the cells (60-62%) in the AD and some cells (30-33%) in the LPs survived. These data suggest that shoot regrowth is correlated to the presence of a majority of surviving cells in the AD after liquid nitrogen exposure. No polymorphic bands were detected by inter-simple sequence repeats or by random amplified polymorphic DNA assessments, and ploidy levels analyzed by flow cytometry were unchanged when plants recovered after cryoexposure were compared to controls. The droplet-vitrification procedure appears to be robust since seven genotypes representing four Malus species and one hybrid recovered shoots following cryopreservation. Mean shoot regrowth levels of these seven genotypes were 48% in the droplet-vitrification method, which were lower than those (61%) in the encapsulation-dehydration procedure reported in our previous study, suggesting the latter may be preferred for routine cryobanking applications for Malus shoot tips., (Copyright © 2015 Elsevier B.V. All rights reserved.)