Dormant Winter Buds Research Articles

Germplasm of Ozark chinquapin (Castanea ozarkensis Ashe) can be cryopreserved by dormant winter buds

Ozark chinquapin (Castanea ozarkensis Ashe) is a forest tree, endemic to the Ozark Mountain region in Eastern United States. Its nutritious nuts were consumed by Native Americans, European settlers, livestock, and wild animals and its wood was an important rot-resistant construction material. Once a significant tree in regional forest communities, the species was nearly eradicated by a chestnut blight caused by Cryphonectria parasitca (Murill) Barr fungus. Some individuals have survived as sprouts from adventitious root buds, but they rarely reach reproductive maturity. While some in situ restoration efforts are underway, the development of a viable ex situ germplasm preservation method is critical to the conservation of this important food-bearing species. Our experiment aimed to develop a cryopreservation method for C. ozarkensis dormant winter buds subjected to eight experimental treatments before desiccation, slow cooling, and storage in liquid nitrogen vapor. The highest post cryogenic viability was 91.2 % for dormant buds pretreated with 0.3 M sucrose for 16 h followed by 0.75 M sucrose for 3 h; this treatment is suggested for cryopreservation of dormant winter buds of Ozark chinquapin germplasm.

Optimized Recovery of Cryostored Dormant Buds of Mulberry Germplasm

A two-step freezing cryoprotocol preceded by desiccation to 15 to 25% moisture content was developed and successfully applied to winter dormant buds of mulberry (different Morus spp.) of a core set comprising 238 accessions studies in our laboratory. The survival and recovery percentage of diverse accessions cryobanked for various periods were tested under in vitro conditions, and several factors were analyzed to determine their role in optimizing the recovery of low-viability accessions. The effect of rates of freezing and thawing (both fast and slow), were tested and recovery compared. Recovery conditions such as dark incubation and rehydration in sterile moist moss grass for different durations after cryopreservation led to a higher survival percentage compared to controls. Two different recovery culture media were compared for their efficiency in survival. On average, the survival under in vitro culture conditions using optimized conditions was high: above 60% in majority of the accessions. Dormant buds showed viability in the range of 25 to 100% with an average of 50.4%. The recovery percentage of winter dormant buds after cryopreservation via slow freezing and slow thawing with rehydration by moist moss grass for 2 h was recorded in the range from 63.3 to 90.9% with an average of 81.05%. Without rehydration, it ranged from 50 to 75% with an average of 60.4%. Regeneration of cryopreserved mulberry germplasm after 6 years of storage indicated no survival loss over different years of storage, and 33-40% of the accessions showed viability above 40%, up to a maximum of 100%. Maximum shoot formation (100%) was obtained from Morus alba. The majority of the accessions were rooted in vitro within 20-25 days of subculture in the auxin rich rooting media, except in wild species M. latifolia and M. laevigata, which took longer (45 to 60 days) for root development. All the rooted plantlets were then transferred to the field and successfully established in a glasshouse.

Cryopreservation of several Ribes species by dormant winter buds

Currant and gooseberry plants (Ribes L.) are native to the temperate regions of the Northern hemisphere through high elevations in Mexico and Central America to the mountainous regions of South America. Annually, the global production is about 838 MT. The fruits are used for cassis, juice preserves, in bakery, dairy products and pharmaceuticals. Some species such as black currant (Ribes nigrum) have antimicrobial, antitumor and immunostimulatory qualities; hence, interest is growing in crop improvement using a diversity of plant genetic resources. Active germplasm collections preserve Ribes cultivars as clonal plantations in the field or under protected cultivation, and species as plants in the field or seeds in cold storage. Long-term preservation of Ribes germplasm may be accomplished by various methods, one of them is cryopreservation of dormant winter buds (DB). This method is reported for preservation of R. nigrum. We have investigated the possibility of applying DBs for cryopreservation of other Ribes species. Eleven accessions in seven species (R. aureum var. aureum, R. aureum var. villosum, R. cereum, R. erythrocarpum, R. mascalerium, R. turbinatum and R. uva-crispa) were cryopreserved over two winter seasons without a pretreatment (standard), with a cryoprotectant (0.75 M sucrose) or a cryoprotectant with an antioxidant (0.75 M sucrose with 0.75 mM vitamin C). A 2-year average viability varied significantly among the accessions processed with the standard method (10 to 87.5%) and among accessions processed with the pretreatments (32.5 to 97.5%); however, the difference between the standard and treatment procedures was not significant. A significant difference in viability between the processing years was observed and there was an interaction between accession, year and treatments for seven accessions processed in three treatments but not in the four accessions processed in the two treatments. Cryopreservation of the broad species in the Ribes germplasm collection may be supported by identifying modifications of the standard cryopreservation procedure.

Preservation of fruit genetic resources in Germany

The preservation of fruit genetic resources has a long-lasting tradition in Germany. It dates back to the early decades of the 20th century. Ever since, a multitude of cultivars of different fruit crop species is preserved in federal and state governmental institutions as well as in communities and towns, non-governmental organizations, and by private individuals. The German Fruit Genebank has been established as a decentralized network with the aim to coordinate the preservation in the different germplasm collections in Germany to minimize the risk of losing cultivars of fruit species and therefore to maintain a part of the cultural heritage. Currently, the German Fruit Genebank is composed of six species-specific networks consisting of 48 different collections. In addition to the cultivar collections, the Fruit genebank of the Institute of Breeding Research on Fruit Crops, Dresden-Pillnitz includes collections of crop wild relatives of the fruit species important for fruit production in Central Europe. Special emphasis is given on the genera Malus and Fragaria representing 46 and 22 taxa respectively. These accessions are valued for their diverse alleles but also for their specific genotypes. Different cryopreservation protocols were elaborated. For Fragaria a protocol combining vitrification with cold acclimation and for Malus a protocol based on dormant winter buds can now be realistically used to build up a Cryo bank as a duplicate collection. Based on the results provided, a combination of the traditional ex situ conservation approach and cryopreservation approach has a great potential to improve conservation of fruit genetic resources contributing to the National Program for Genetic Resources of Agricultural and Horticultural Crops in Germany.

Salix dormant bud cryotolerance varies by taxon, harvest year, and stem‐segment length

AbstractWillow (Salix L.) species have good qualities for becoming a biomass crop for production of biofuels. They grow on marginal soils, produce high yields of wood in a short period of time, and are easily propagated. Salix is one of the few genera that may be cryopreserved as dormant winter buds (DBs) instead of using tissue culture techniques. The objective of our study was to evaluate selected factors that affect cryotolerance of nine Salix germplasm accessions in the USDA‐ARS National Plant Germplasm System collection. One‐year‐old branches harvested in January over 3 yr were cut into either 6‐ to 7‐ or 10‐cm segments containing at least two DBs. Segments were treated with a slow‐cooling procedure and stored in vapor‐phase liquid N (LNV). Control segments were stored at −5 °C until planting. The LNV‐exposed and the −5 °C‐stored nodal segments were warmed and directly planted in a cold greenhouse. Six weeks later, the material was evaluated for shoot and root development. Segments that developed a shoot were considered viable. Average viability varied among years (0–35.1%) even for the same accession, species (4.2–51.4%), and segment length. Dormant buds on 10‐cm segments had higher average viability (82.2%) than did DBs on 6‐ to 7‐cm segments (43.9%), suggesting higher suitability for cryopreservation. In the material studied, Salix DB viability was correlated with branch diameter, DB density, and the ability to develop shoots and roots prior to cryopreservation.

Molecular analysis of bunch architecture in grapevine

A loose grape cluster is a desired trait in grapevine breeding, since it reduces the abundance and severity of fungal infections. This is the result of better coverage of the grape bunch by antifungal spraying agents and better air exchange within the grape cluster. The reduced exposure to high humidity acts as a physical barrier against pathogens that need high moisture to proliferate, e.g., Botrytis cinerea. The aim of this study was to identify genes that influence bunch architecture and to deduce molecular markers to accelerate the selection process in grapevine breeding. Bunch compactness was characterized by phenotyping of several subtraits. The experiment comprised plants of a mapping population (‘GF.GA-47-42’ × ‘Villard blanc’) and a set of ‘Pinot noir’ clones with loose as well as compact clusters. The latter were sampled from three different wine-growing regions in Germany. A genetic mapping and quantitative trait locus (QTL) analysis approach based on the trait-segregating population of 150 F1 individuals yielded many reproducible QTLs distributed along the genome. Some of them were concentrated in specific genomic regions. Further statistical evaluation revealed eight such QTL clusters to be of major relevance and identified flanking molecular markers for marker-assisted selection. In a transcriptional profiling approach, two loosely and two compactly clustered clones of ‘Pinot noir’ were compared. RNA from dormant winter buds and compound buds harvested during the growing period were used in differential gene expression experiments. RNA sequencing was performed at three different stages of development. Candidate genes found in these experiments were selected and re-analyzed in a more comprehensive set of ‘Pinot noir’ clones from the three different German growing locations. Finally, two genes appeared to be differentially expressed between the two groups of clones over the three locations.

Gas exchange measurement as a non-destructive viability assay for frozen-thawed, winter-dormant apple buds

Low temperature studies with winter-dormant buds are severely limited by the lack of a rapid, non-destructive assay for their viability. Investigations involving the winter harvest of ecodormant buds of woody subjects, including cryopreservation, are restricted if viability cannot be assessed until dormancy is broken. If post-treatment grafting indicates low survival of the harvested population then further collection and study has to be delayed until the next winter season. This study trials the use of a portable gas exchange system able to discriminate between live and dead buds rapidly, with the assay confirmed as non-destructive by subsequent micropropagation. Active respiration was recorded for 85% of a winter-dormant Malus domestica buds population that showed 91% viability when grafted (n = 45). Lethally stressed material gave no false positive results. When micropropagated after respiratory measurement, a population viability of 76% was recorded. There was a significant, positive correlation between respiration and fresh weight for buds of mass >10 mg, from a population with a mean fresh weight of 17 mg.

Cryopreservation of fruit germplasm

Most temperate fruit species are genetically heterozygous and vegetatively propagated. Active collections of fruit genetic resources in Germany are generally maintained in the field, e.g., as potted plants for Fragaria and as trees for pome and stone fruit species. The plant material in active collections should be kept in duplicate to ensure security in case of disease or environmental disaster. The aim of this study was to develop an efficient complementary conservation strategy for fruit genetic resources. Although costly, fruit tree cultivars can be duplicated as field collections at a second site within the framework of the German Fruit Genebank, which is a decentralized fruit-specific network. Wild species accessions, particularly those of the genera Malus spp. (apple) and Fragaria spp. (strawberry) as well as strawberry cultivars, can also be duplicated by means of cryopreservation. In the current study, long-term cryopreservation was initiated for 194 Fragaria genotypes. A protocol combining vitrification with cold acclimation was effective and highly reproducible, with an average regrowth rate of 86%. In Malus, a general cryopreservation protocol based on dormant winter buds was adopted. Based on the results provided in this study, a combination of traditional ex situ conservation and cryopreservation can greatly improve the stability and security of fruit germplasm conservation.

Pre-budburst temperature influences the inner and outer arm morphology, phenology, flower number, fruitset, TSS accumulation and variability ofVitis viniferaL. Sauvignon Blanc bunches

Background and Aims: Inflorescence morphology, flower formation and subsequent fruit development of grapevines are influenced by genetic, environmental and cultural practices. While the effect of temperature on inflorescence primordia number per bud is documented, its effect during dormancy and budburst (BB) on floral development is less clear. In our study, winter dormant buds were passively heated for different periods of time from mid-winter (July) to BB (October). Methods and Results: Individual canes were heated pre-BB for different periods in polyethylene tunnels. Heating during winter dormancy had no effect on the number or position of inflorescences on the shoot, or on the type of structure occurring at the outer arm position of the inflorescence. Heating buds for either July or August to BB advanced the date of BB and the start date of flowering by 12–14 and 14 days, respectively, compared with that of the unheated Control treatment. There was a significant (P < 0.05) negative correlation between the mean temperature 12 days pre-BB and flower number per shoot. The time between 50% flowering and the fruit TSS reaching 14°Brix was not influenced by winter heating or the date of flowering; the difference in this time interval, however, between the inner and outer arm components increased as shoot node number increased. Conclusions: Flower number per inflorescence had the greatest influence on the proportion of fruitset. The date of flowering of the outer arm was later at higher inflorescence positions on the shoot and later than that of the inner arm. Likewise, the outer arms of bunches higher up the shoot took longer from flowering to accumulate a fruit TSS of 14°Brix. Significance of the Study: Much of the within-vine variability in berry composition at harvest can be attributed to the position of bunches on the shoot and the presence of an outer arm. The outer arm on apical bunches took significantly longer to progress from flowering to a fruit TSS of 14°Brix, suggesting that removal would significantly reduce within-vine fruit variability.

Cryopreservation of apple (Malusspp.) winter buds

One of the practical approaches for long-term conservation of apple garmplasm is the storage of winter buds in liquid nitrogen. The dormant buds were cryopreserved using two-step freezing method. On dehydration at-20°C, moisture content (MC%) of buds ranged from 37 to 47% after 28 days. On silica gel, the MC% was in the range of 24 and 34% after 4 h desiccation. The cryopreserved buds were rewarmed and patch budded and grafted on the rootstocks in the field. Accessions desiccated on silica gel showed post-thaw recovery. The cryopreserved buds on patch-budding showed 0–10% recovery, while buds on grafting showed higher (up to 25%) regeneration. No natural acclimation at sub-zero temperature can be one reason for low post-thaw recovery across the accessions growing in Indian fields. Winter dormant buds of 35 accessions of Malus spp. were cryobanked.

EXTENDING THE DORMANT BUD CRYOPRESERVATION METHOD TO NEW TREE SPECIES

In cryopreservation of germplasm, using dormant winter buds (DB) as source plant material is economically favorable over tissue culture options. Although the DB cryopreservation method has been known for many years, the approach is feasible only for cryopreserving a select number of temperate tree species. The original method developed for Malus (apple) DB, requires desiccation of stem segments (to 25-30% moisture content), slow cooling (to -30°C), storage in liquid nitrogen vapor (LNV) and viability testing by grafting. We investigated the possibility of using this method for cryopreservation of DB of Juglans regia, J. cinerea, Prunus dulcis, P. persica, Salix exigua and S. triandra germplasm. The post LNV viability of P. dulcis, P. persica and S. triandra DB was very low. Dormant buds of J. cinerea harvested in December were viable in a higher percent than buds harvested in January. The fraction of viable Salix DB on 10 cm branch segments was significantly higher (30 and 80%) than on 6 cm long segments (0 and 45%; P<0.05); this indicated that the longer segments might be more suitable for cryopreservation of the two Salix species. For Juglans regia, the viability after LNV exposure was evaluated by grafting and forced bud break under high relative humidity conditions and the percent of viable buds was similar for both methods; hence testing under mist might be a valid indication of viability. The application of the Malus DB cryopreservation method might also be applicable to preservation of almond, peach and English walnut however studies on factors enhancing post LNV viability are needed.

Melatonin enhances the recovery of cryopreserved shoot tips of American elm (Ulmus americana L.)

Climate change and global migrations of people and goods have exposed trees to new diseases and abiotic challenges that threaten the survival of species. In vitro germplasm storage via cryopreservation is an effective tool to ensure conservation of tree species, but plant cells and tissues are exposed to multiple stresses during the cryopreservation process. The current study was designed to evaluate the potential of melatonin to improve survival through the process of cryopreservation. Shoot tips of in vitro-grown plantlets and dormant winter buds of American elm were successfully cryopreserved in liquid nitrogen (LN) at -196°C under controlled environmental conditions following melatonin treatment and cold acclimation with either vitrification or encapsulation–vitrification protocols. Explants had optimal regrowth following cryopreservation when treated with the plant vitrification solution#2 (PVS2) for 10 min. Supplementation of both preculture and regrowth media with melatonin significantly enhanced regrowth of frozen shoots compared with the untreated control (P < 0.05). Approximately 80–100% of shoot explants grew under optimized conditions using melatonin-enriched media. Shoot tips of dormant winter buds consistently produced nearly 100% regrowth with both techniques. The main steps of the optimized protocol are14-day cold-acclimated cultures exposed to preculture medium with 0.1–0.5 lM melatonin for 24 hr, application of PVS2 for 10 min, rapid cooling in LN, rapid rewarming, removal of cryoprotectants, and recovery on a medium supplemented with 0.1–0.5 lM melatonin. Our results demonstrate the usefulness of the antioxidant melatonin for long-term storage of naturally resistant elm germplasm.

Cloning and Characterization of WOX4 Gene from Vitis vinifera L. Involved in Stem Cell Regulation

Abstract Wuschel-related homeobox (WOX) genes play essential, specific, and sometimes redundant roles in plant embryo development, shoot and root meristem maintenance, and plant development. Though much information was quickly gained with members of the WOX gene family of Arabidopsis, monocotyledonous crops, and gymnospermous conifers, little is known about perennial woody plants. In this study, we isolated the first WOX gene family member from grape (Vitis vinifera L. cv. Cabernet Sauvignon), and named it VvWOX4 based on its characteristic domains and phylogenetic analysis. The identity of VvWOX4 was validated by MALDI-TOF MS and Western blot with polyclonal antibody against Arabidopsis thaliana Wuschel. Functional analysis showed that VvWOX4 markedly increased shoot primordia structures when overexpressed under CaMV 35S promoter in tobacco. A different expression pattern was found for VvVOX4 compared with AtWUCHEL and its expression was detected in unique organs of grapevines. Besides the expression in the vegetative shoot apical meristem (SAM) of grape shoot tips, VvWOX4 is expressed in dormant winter buds, inflorescence, young leaves, and tendril tips, but not in root tips. In young leaves, the expression of VvWOX4 is strongly upregulated by wounding, and also by plant growth regulators such as 2 mg L −1 2,4-D, 1 mg L −1 NAA and 1 mg L −1 BAP treatments, while downregulation was monitored by 1 mg L −1 IBA treatment, and there was no response to 0.5 mg L −1 GA3 treatment. Together, our results revealed the first member of grape WOX gene family and indicated different roles and regulation of VvWOX4 in the perennial woody crop grapevine.

Effect of Geographical Location, Year, and Cultivar on Survival of Malus sp. Dormant Buds Stored in Vapors of Liquid Nitrogen

Clonal woody crop germplasm collections often originate and are grown in distinct geographical locations. Because the degree of cold-hardiness is known to be a factor in the successful use of dormant bud cryopreservation for Malus, it was suggested that material from relatively warmer climates would not respond to cryopreservation as well as material from colder environments. To test this hypothesis, the effect of growing provenance on cryosurvival of dormant buds from three Malus (apple) cultivars grown in three locations (Geneva, NY; Davis, CA; and Corvallis, OR) was tested in 3 consecutive years. Dormant winter buds were harvested at the three locations, cryopreserved, and bud viability was tested by grafting. The collective 3-year mean viability for cryopreserved dormant apple buds for the three locations ranged from 63% to 81% of the buds surviving with the highest survival from the Corvallis site; however, the Geneva twigs were exposed to the lowest preharvest temperature. These results suggest that the temperature at the growing location may not hinder application of the dormant bud cryopreservation method with Malus to the extent previously speculated.

22. Critical steps in cryopreservation of dormant winter buds collected under relatively mild winter conditions

22. Critical steps in cryopreservation of dormant winter buds collected under relatively mild winter conditions

Mulberry biodiversity conservation through cryopreservation

A 238 mulberry germplasm accession collection from diverse regions maintained under tropical conditions was identified from an ex situ field gene bank. The purpose was to prioritize the in vitro conservation and cryopreservation to develop long-term biodiversity conservation for ensuring sustainable utilization of these valuable resources. Reliable cryo techniques using desiccation and slow freezing of winter-dormant buds were used. Storage potential of bud grafts of different Morus species at −1.5°C for 90 d indicated species-specific variation, and most of the wild species were found sensitive. In vitro regeneration and cryopreservation (−196°C) protocols using differentiated bud meristems, like axillary winter-dormant buds, were worked out for a wide range of landraces, wild, and cultivated varieties of Morus. Buds maintained under subtropical location are also amenable for cryopreservation. Successful cryopreservation of winter-dormant buds belonging to Morus indica, Morus alba, Morus latifolia, Morus cathayana, Morus laevigata, Morus nigra, Morus australis, Morus bombycis, Morus sinensis, Morus multicaulis, and Morus rotundiloba was achieved. Among wild species, Morus tiliaefolia and Morus serrata showed moderate recovery after cryopreservation. Survival rates did not alter after 3 yr of cryopreservation. Inter-simple sequence repeat markers were used to ascertain the genetic stability of cryopreserved mulberry germplasm accessions, which showed no difference detected among the plantlets regenerated from frozen apices in comparison to the nonfrozen material.

Secondary buds in Scots pine trees infested with Gremmeniella abietina

In winter 2000–2001, there was a serious outbreak of Gremmeniella abietina Morelet in southeastern Norway. During the outbreak, we noted that injured Scots pine trees (Pinus sylvestris L.) developed secondary buds in response to the fungus attack, and we decided to study the relationship between injury, appearance of secondary buds and recovery of the trees thereafter. For this purpose, 143 trees from 10 to 50 years of age were chosen and grouped into crown density classes. Injury was assessed in detail, and buds were counted before bud burst in the spring of 2002. In addition, a subset of 15 trees was followed through the summer of 2002 to assess recovery. All injured trees developed secondary buds, with a clear overweight of dormant winter buds in proportion to interfoliar buds. Healthy control trees did not develop secondary buds at all. The secondary buds appeared predominantly on the injured parts of the tree; interfoliar buds in particular developed just beneath the damaged tissue. Most of the secondary buds died during the winter of 2001–2002, mainly because the fungus continued to spread after the first outbreak. Many of the remaining buds developed shoots with abnormal growth during the summer. Secondary buds may help trees to recover from Gremmeniella attacks, but this strategy may fail when the fungus continues to grow and injure the newly formed buds and shoots.

Long-term conservation of Diospyros germplasm using dormant buds by a prefreezing method

Cryopreservation of Diospyros at-150°C followed by a prefreezing treatment using dormant winter buds was investigated. Twig sections bearing one bud each were first partly dehydrated at room temperature for 3 h. Then they were subjected to a successive prefreezing at-5, -10, -15, -20 and-30°C at 24 h intervals, and finally preserved at-150°C in a deep freezer. Thawing in the air at 25°C gave the highest rate (about 70%) of in vitro shoot formation from cryopreserved buds. Shoot regeneration by bud grafting was not successful. Dormant buds appear to be a suitable material for long-term storage of some Diospyros species.

Micropropagation of Chokecherry and Pincherry cultivars

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of chokecherry (Prunus virginiana L.), `Garrington', and pincherry (P. pensylvanica L.f), `Mary Liss' and `Jumping Pound', were examined using various combinations of growth regulators. Dormant winter buds were used as explants. MSMO medium supplemented with 0.49 μM IBA and either 4.44 or 8.87 μM BA was found to be optimal for culture initiation of both species and cultivars. GA3 (28.89 μM) significantly reduced (p=0.0001) the number of successfully established cultures. BA concentrations 8.87–12.82 μM gave optimal shoot proliferation in chokecherry and 4.44 μM BA in both cultivars of pincherry. Auxin treatments were required for ex vitro rooting of approximately 10 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (84%) was obtained with IBA/NAA (9.80/2.69 μM). A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%) mixture, was also effective (75%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.

Elevated CO2 ameliorates birch response to high temperature and frost stress: implications for modeling climate-induced geographic range shifts.

Despite predictions that both atmospheric CO2 concentrations and air temperature will rise together, very limited data are currently available to assess the possible interactive effects of these two global change factors on temperate forest tree species. Using yellow birch (Betula alleghaniensis) as a model species, we studied how elevated CO2 (800 vs. 400 μl l-1) influences seedling growth and physiological responses to a 5°C increase in summer air temperatures (31/26 vs. 26/21°C day/night), and how both elevated CO2 and air temperature during the growing season influence seedling ability to survive freezing stress during the winter dormant season. Our results show that while increased temperature decreases seedling growth, temperature-induced growth reductions are significantly lower at elevated CO2 concentrations (43% vs. 73%). The amelioration of high-temperature stress was related to CO2-induced reductions in both whole-shoot dark respiration and transpiration. Our results also show that increased summer air temperature, and to a lesser degree CO2 concentration, make dormant winter buds less susceptible to freezing stress. We show the relevance of these results to models used to predict how climate change will influence future forest species distribution and productivity, without considering the direct or interactive effects of CO2.