• Korean Journal of Plant Resources
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• v.28 no.6
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• pp.697-703
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• 2015

Twenty apple germplasm accessions from the Korean Genebank were successfully cryopreserved using two-step freezing to back up genetic resources maintained by field collections. This study examined the morphological and genetic stability of cryopreserved dormant apple buds that were stored in liquid nitrogen, and then rewarmed and regrown. Whole plants were regenerated directly from dormant buds through budding without an intermediary callus phase. The cryopreserved buds produced high levels of shoot formation (76.2-100%), similar to those of noncryopreserved buds (91.3-100%), with no observed differences between cryopreserved and noncryopreserved materials. Three of the twenty cryopreserved apple germplasm accessions were used to assess morphological and genetic stability. No differences in morphological characteristics including shoot length, leaf shape, leaf width/length ratio, and root length were observed between controls (fresh control and noncryopreserved) and cryopreserved plantlets. The genetic stability of regenerants (before and after cryopreservation) was investigated using inter simple sequence repeat (ISSR) markers. The ISSR markers produced 253 bands using four primers, ISSR 810, SSR 835, ISSR 864, and ISSR 899. These markers showed monomorphic banding patterns and revealed no polymorphism between the mother plant and regenerants before and after cryopreservation, suggesting that cryopreservation using two-step freezing does not affect the genetic stability of apple germplasm. These results show that two-step freezing cryopreservation is a practical method for long-term storage of apple germplasms.

• International Journal of Industrial Entomology and Biomaterials
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• v.47 no.2
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• pp.126-133
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• 2023

Genetic resources of mulberry trees are commonly preserved as trophosomes, which are vulnerable to environmental factors, such as natural disasters, diseases, and pests. This study establishes a basic protocol for ultra-low temperature cryopreservation of mulberry trees using a two-step freezing process. The procedure was established using the "Daeshim" variety and then tested on genetic resources from 24 other mulberry varieties. Samples were first dried to a moisture content of 33-43% in a low-temperature forced-air chamber at -5 ℃, then slowly frozen from -5 ℃ to -20 ℃, and preserved in liquid nitrogen (-196 ℃). To determine the regeneration rate, isolated dormant buds were inoculated into MS basal medium, and grown shoots were grafted onto 1-year-old rootstock via chip budding and then cultured. After freezing in liquid nitrogen, the "Daeshim" variety exhibited a survival and regeneration rate of more than 70% and 50%, respectively. Applying the two-step freezing process to genetic resources from 24 mulberry species yielded average survival and regeneration rates of 85.3% and 75.5%, respectively. Morus alba showed survival and regeneration rates of 100%, confirming the efficacy of the two-step freezing method. These results indicate the high feasibility of ultra-low-temperature cryopreservation through two-step freezing of dormant buds from mulberry genetic resources. Additional research is required into the variations in regeneration rates with freezing period in liquid nitrogen.

• Korean Journal of Plant Resources
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• v.15 no.2
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• pp.89-95
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• 2002

In woody plant germplasms, using prefrozen dormant buds for materials is one way to achieve successful cryopreservation. The protocol of cryopreservation for White birch (Betula platyphylla var. japonica) winter vegetative buds is the following. First, the branches of White birch were collected in January 20, when the vegetative buds were still in a state of quiescence. The winter buds with about 5㎜ of xylem tissue were removed from the branches. They were dehydrated to moisture contents about 44% by air dry treatment. The buds were prefrozen, with the temperature being decreased by 5∼-20$\^{C}$ and then transfered to the LN(liquid nitrogen) maintained below -l96$\^{C}$. After cryopreservation, the vegetative buds were rapidly thawed in a water bath at 40$\pm$5$\^{C}$. In this case, the cell survival rate of samples was about 86%. After sterilization, buds were then cultured on MS medium. These results demonstrate the feasibility for cryopreservation of winter vegetation buds of Betula platyphylla var. japonica.

• International Journal of Industrial Entomology and Biomaterials
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• v.15 no.1
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• pp.23-33
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• 2007

A simple and reliable cryo technique using desiccation and slow freezing of winter dormant buds was employed for 238 core collection of mulberry germplasm collected from diverse geographical regions and maintained under tropical conditions in the ex situ field gene bank to develop long-term biodiversity conservation for ensuring sustainable utilization of these valuable resources. Desiccation and freezing tolerance of bud grafts and excised shoot apices in the axillary buds of different Morus species under in vivo and in vitro condition indicated species-specific variation and most of the wild Morus species were found sensitive. In vitro regeneration and cryopreservation($-196^{\circ}C$) protocols using differentiated bud meristem like axillary winter dormant buds were worked out for a wide range of Morus species, land races, wild and cultivated varieties. Successful cryopreservation of mulberry winter dormant buds of different accessions belonging to M. indica, M. alba, M. latifolia, M. cathayana, M. laevigata, M. nigra, M. australis, M. bombycis, M. sinensis, M multicaulis and M. rotundiloba was achieved. Among wild species Morus tiliaefolia, and M. serrata showed moderate recovery after cryopreservation. Survival rates did not alter after three years of cryopreservation of different Morus species. ISSR markers were used to ascertain the genetic stability of cryopreserved mulberry, which showed no difference detected among the plantlets regenerated from frozen apices in comparison to the non-frozen material.

• Journal of Plant Biotechnology
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• v.44 no.4
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• pp.379-387
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• 2017

Various sizes (0.2 ~ 1.2 mm) and developmental stages (referred to as Stage 1 ~ 3) of apical and lateral meristems were excised, together or separately, directly from dormant buds of apple 'Hongro'. They were mixed infected by Apple scar skin viroid (ASSVd), Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV), which are major viruses attacking apples. A total of 31 callus lines (> 10 mm in diameter) were obtained by culturing the explants on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 3.0 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-butyric acid (IBA), and they were subjected to RT-PCR analysis for virus detection. A high rate of virus elimination (expressed as the percentage of calli that did not amplify during RT-PCR, i.e., RT-PCR negative calli per total number of calli obtained) was achieved for ACLSV (100%), ASSVd (93.7%), and ASPV (93.7%), whereas it was only 25.8% for ASGV. ASPV was detected in the presence of 2 ~ 3 bracts. Simultaneous virus elimination of ASSVd, ASPV, ACLSV, and ASGV occurred during the meristem culture, in which the early stages of the dormant buds (Stage 1) were used, because ASGV was mostly eliminated during that stage. The results of the present study will be valuable for the production of virus-free apple trees.

• The Korean Journal of Ecology
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• v.14 no.3
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• pp.305-316
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• 1991

Growth hadit and demograph in a mature population of polygonatum and polygonatum humile in kanghwa island were studied for two growing seasons. Shoots of two species emerged early spring from the growing apices of the underdground rhizomes which had persisted for up to 1~5 year.after flowering, the ramets produced two rhizome buds at the shoot base. Bacause of the apical dominance in the rhizome system, a new rhizome was developed from only one bud, an actual bud,and the other latent buds were suppressed sothat remained dormant. The latent dud produced a new rhizome only when the actual dud was severed by the herbivores or by the physical obstacles. Therefore, the ramet number is not increased by the new rhizome from the latent bud. however, new ramets dould sometimes grow from latent buds which had been produced more than a year ago. Production of these ramets was main means increasing the ramet numbew and widening the potential zone of exploitation. Changes in size class of each ramet were noyiceable after a tear in small size-classes. Small ramets replaced themselves with larger-sized ramet, while large ramets with similar-sized or smallar-sized ramets. ramet numbers were average 0.82and 1.14 times of those fromthe previous year inp. involucratum and p. humile, though there was between-site variation. Almost all the ramets in the quadrats were alive during the growing season. when the entire rhizome systems were excavater next spring, there were many rhizomesegments without shoots, especially in p. involucratum. therefore, the drcrease of ramet number in p. involucratum in probably due to the climatic factors of winter.

• Journal of Ginseng Research
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• v.40 no.4
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• pp.409-414
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• 2016

Background: The low survival rate of in vitro regenerated Panax ginseng plantlets after transfer to soil is the main obstacle for their successful micropropagation and molecular breeding. In most cases, young plantlets converted from somatic embryos are transferred to soil. Methods: In vitro thickened taproots, which were produced after prolonged culture of ginseng plantlets, were transferred to soil. Results: Taproot thickening of plantlets occurred near hypocotyl and primary roots. Elevated concentration of sucrose in the medium stimulated the root thickening of plantlets. Senescence of shoots occurred following the prolonged culture of plantlets. Once the leaves of plantlets senesced, the buds on taproots developed a dormant tendency. Gibberellic acid treatment was required for dormancy breaking of the buds. Analysis of endogenous abscisic acid revealed that the content of abscisic acid in taproots with senescent shoots was comparatively higher than that of taproots with green shoots. Thickened taproots were transferred to soil, followed by exposure to gibberellic acid or a cold temperature of $2^{\circ}C$ for 4 mo. Cold treatment of roots at $2^{\circ}C$ for 4 mo resulted in bud sprouting in 84% of roots. Spraying of 100 mg/L gibberellic acid also induced the bud sprouting in 81% roots. Conclusion: Soil transfer of dormant taproots of P. ginseng has advantages since they do not require an acclimatization procedure, humidity control of plants, and photoautotrophic growth, and a high soil survival rate was attained.

• Horticultural Science & Technology
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• v.31 no.1
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• pp.38-49
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• 2013

Prohexadione-calcium (Pro-Ca), ethephon and transient water stress were evaluated in a factorial design, as potential inhibitors of early-season shoot growth of high density orchard management of apple (Malus domestica Borkh.) trees. In the experiment, water stress was imposed to one-half of the 7-year-old 'Golden Delicious'/M.9 apple trees in each of 5 blocks, by stopping irrigation for 3 weeks between 35 and 56 days after full bloom (AFB). Within each whole unit, the following Pro-Ca and ethephon treatments were randomly allocated at $2{\times}2$ factorial: a) 0 or 250 $mg{\cdot}L^{-1}$ a.i. Pro-Ca applied at 28 days AFB and b) 0 or 300 $mg{\cdot}L^{-1}$ a.i. ethephon applied twice (35 and 71 days AFB). All trees were hand thinned to king flowers prior to treatments. Vegetative shoot growth was markedly reduced by Pro-Ca, with its effect being obvious within 14 days after application, while ethephon and water stress treatments were less effective. Pro-Ca had no effect on fruit set and yield but slightly increased fruit size. Ethephon substantially reduced the fruit size and yield but had no effect on fruit set. Water stress reduced fruit set, fruit size and yield. With regard to fruit quality, Pro-Ca did not influence fruit shape, flesh firmness and soluble solids contents (SSC) but slightly reduced titratable acidity. Ethephon had no effect on fruit shape but increased firmness, SSC and acidity, while water stress did not influence these fruit quality attributes. Dry weight of dormant spur buds was reduced by both Pro-Ca and water stress, while increased by ethephon. The larger dormant buds led to the larger spur flowers at the tight cluster stage the following spring. Return flowering was promoted only by ethephon, especially on previous season's shoots. There were no significant interactions between Pro-Ca and ethephon or water stress on most variables observed in this study.

• Journal of Sericultural and Entomological Science
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• v.30 no.2
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• pp.75-83
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• 1988

These experiments were carried out to define the rest period of mulberry by treating growth regulators in Taegu, Korea. Results obtained were as follows: It was recognized that the depth of rest in Taegu, Korea, was not deeper than that in Tokyo and Kagoshima, Japan. The rest of mulberry was begun at the end of September, subsequently became deeper through the first October into the late October and then turned gradually into quiescence by the beginning of November. Buds sprayed by gibberellic acid ($GA_3$) 10ppm and urea 0.5% were promoted to sprout, while naphthalene acetic acid (NAA) 0.02% inhibited strongly bud sprouting and abscisic acid (ABA) 20ppm had no effect on the rest of mulberry. Gibberellic acid 10ppm enhanced the rate of green color of bud after incubation for 10 days at $30^{\circ}C$. By the portion of mulberry stems, the depth of rest was different that the middle buds were less dormant than those lower. The optimal time required for the mulberry winter bud break is 15 days incubation at $30^{\circ}C$ as treated with $GA_3$.

• Journal of Plant Biotechnology
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• v.44 no.4
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• pp.401-408
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• 2017

Herein, we report the meristem-tip culture from dormant buds of grape 'Kyoho' single-infected with Grapevine fleck virus (GFkV), which is phloem-limited and transmitted by graft inoculation. We produced GFkV-free shoots without thermo- or chemotherapy using meristem-tip explants approximately 0.3 mm (73 explants) and 0.8 mm long (five explants) including shoot apical meristem, 2-5 leaf primordia, and 1-4 uncommitted primordia from dormant buds of the infected woody cuttings (stored at $4^{\circ}C$). Explants were cultured on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 3.0 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-butyric acid (IBA). After 16 weeks of culture, shoot (10-mm long) regeneration frequency achieved from 0.3-mm explants was 4.1% and that obtained from 0.8-mm explants was 40.0%. Virus-free efficiency (expressed as the percentage of RT-PCR negative shoots regenerated) from 0.3- and 0.8-mm explants was 100% and 50%, respectively. Following in vitro multiplication, RT-PCR assays revealed identical results to assays of the first regenerated shoots. Our new methodological approach could be applied for eliminating other viruses in grapevines, as well as for producing virus-free plants in many other deciduous tree species, including fruit trees.