Source: UNIV OF WISCONSIN submitted to
CRANBERRY COLD HARDINESS IN RELATION TO DORMANCY AND BUD DEVELOPMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1009297
Grant No.
(N/A)
Project No.
WIS01945
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jun 1, 2016
Project End Date
May 31, 2019
Grant Year
(N/A)
Project Director
Atucha, AM, .
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Horticulture
Non Technical Summary
Large changes in cranberry bud hardiness occur both in the fall as plants go into dormancy, and during early spring as they emerge from dormancy and prepare to begin another season of growth and fruit production. However, the timing and exact nature of these changes are not well understood due to the lack of information in physiological processes of dormancy, temperature acclimation and deacclimation, as well as the anatomical changes in and around the terminal bud of the cranberry plant. This lack of understanding contributes to the challenges growers face in decision-making regarding flooding strategies for harvest, cold temperature plant protection throughout the remainder of fall and early winter, establishment of winter ice cover, protection strategies for early spring before visual signs of bud swelling, as well as the resumption of sprinkle irrigation protection. The goal of the present studygoal is for growers to be able to make better informed and efficient flooding protection choices based on an increased understanding of the nature, depth, and timing of changes in the plant's dormancy and cold hardiness status.Cold hardiness of cranberry buds will be evaluated using differential thermal analysis (DTA) in addition to histological work to determine the presence or absence of an ice nucletion barrier. Timing ofterminal bud endodormancy onset and release will be tracked by forcing upright cuttings in a greenhouse hydroponic system. Finally, evaluation of the effect of ABA foliar application on cranberry bud cold hardiness will be conducted during fall, testing different concentrations of foliar ABA concentration (0-800mgL-1), followed by evaluating timing of applications.
Animal Health Component
0%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20311211020100%
Goals / Objectives
The main goal of this project is to gain abetter understating of the physiological changes that occur during fall at the onset of dormancy and early spring at the release of dormancy, and the relation to plant cold hardiness, which will lead to more efficient decisions regarding frost protection management. The objectives of this study are to: 1) Define changes in cranberry terminal bud cold hardiness from fall to spring over the period of plant dormancy, focusing on the transition periods at the onset and emergence from dormancy; 2) Evaluate visible and measurable environmental and plant physiological factors for use as indicators to changes in cranberry bud cold hardiness level; and 3) Evaluate the effect of foliar ABA application on cranberry bud cold hardiness during fall. The results of this study will allow growers to make more informed and assertive decision on when are the critical periods for cold protection, as well as potential management strategies to increase cold hardiness of terminal buds during fall. Ultimately, reducing production cost by savings on fuel and water during these critical periods will be essential for cranberry grower to stay competitive in a market with declining fruit prices.
Project Methods
The outlined research will be conducted in a laboratory setting, as well as on a commercial cranberry marsh, Cranberry Creek Cranberries, located in Necedah WI. Laboratory work will be conducted in the Atucha Lab, Department of Horticulture, Plant Science 273.1) Changes in Cranberry terminal bud cold hardiness will be determine usingdifferential thermal analysis (DTA).'Stevens', 'HyRed', and 'Ben Lear' cranberry uprights will be collected from September to April, targeting 5 critical periods: pre-harvest, post-harvest, pre ice formation for winter protection, after ice-off in early spring, and initial terminal bud swelling.The terminal mixed buds will be mounted on thermoelectric modules, and placed in a Tenney environmental chamber (Thermal Products Solutions, New Columbia, PA). Temperature will be lowered at 4°C h-1 from 1 to -50°C. Differential thermal output will be recorded and mean number of exotherms and median LTE temperatures will be calculated from the number of LTEs and the temperatures at which they occurred for each variety and sampling date.The temperature at which 50% of the buds are killed (LT50) will be determined using Bittenbender and Howell's (1974) variation of Spearman-Kärber equation.2) The presence and nature of an ice nucletion barrier in cranberry buds will be determine through miscroscopy work.To determine the presence of a suberin based barrier, terminal mixed buds of Ben Lear, Stevens, and HyRed will be collected from September to April, targeting 5 critical periods: pre-harvest, post-harvest, pre ice formation for winter protection, after ice-off in early spring, and initial terminal bud swelling. Buds will be excised from the upright leaving a 1 mm upright section attached. Suberin staining will be performed on fresh section with a 0.1% berberine solution, followed by 30 min incubation in aniline blues, and then viewed with an epifluorescence microscope. Permeability in the base of the bud will be measured using a large fluorescent tracer (Rhodamine green), if the pore size of the axis changes then the tracer will be excluded from the bud. For the tracer treatment, buds will be excised as described above, and the upright portion of the bud will be submerged in the tracer for 12 h to allow for uptake of the dye. Longitudinal sections of the bud will be observed with a microscope.3) Terminal cranberry budendodormancy onset and release will be tracked by forcing upright cuttings in a greenhouse hydroponic system. The extent of growth inhibition will be related to the accumulation of chilling and near-freezing temperature experience. A subset of uprights with formed terminal buds will be collected from Ben Lear, Stevens and HyRed beds. The proximal ends of uprights will be trimmed to around 8-10 cm with razor blade under water. Uprights will be arranged in rows in pink insulation foam holders to suspend at least 3 cm of cut ends in plastic tubs. Water in the plastic tubs will be aerated with aquarium pumps. Tubs will be placed under fluorescent lights set to a 16-hour day at room temperature. At one, two, and three weeks intervals, we will record the number of buds that have swollen and those that have broken. Depth of dormancy will be expressed as days to 50% budbreak (first appearance of green tissue). Buds that do not break will be visually inspected to determine viability and adjust percent budbreak. Uprights will be collected from the field at biweekly intervals starting from when the daily minimum temperature drops below 7°C and continue until beds are flooding for the formation of ice for overwintering protection. Sample collections will resume in the spring when beds are first accessible after ice-off in early spring.4)Leaf anthocyanin development and chlorophyll degradation in fall will be related to eco- and endodormancy status, with the opposite trend in early spring charted in relation to the plant's status in endo- and ecodormancy. To quantify leaf anthocyanin and chlorophyll, upright of Ben Lear, Stevens and HyRed will be collected starting as described in Objective 1 C. Tissue samples will be ground in acetone/Tris buffer solution (80:20 vol:vol, pH=7.8) for chlorophyll measurements and cold methanol/HCL/water (90:1:1, vol:vol:vol) for anthocyanin measurements. The absorbance of the extracted solutions will be measured with a spectrophotometer.5) Evaluation of the effect of ABA applications on cranberry bud cold hardiness during fall will be determine by treating cranberry vines with foliar ABAapplication at 50% fruit set stage at concentrations of 0, 200, 400, 600, and 800 mgL-1 to evaluate ABA phytotoxicity.Phytotoxicity will be evaluated in leaves through visual observation (using a score rating based on the percentage of damaged leaf area) starting 7 days after ABA application and continuing until damage is observed. Fruit damage will be evaluated before harvest, and percentage of undeveloped berries will be recorded. To identify optimum timing of ABA applications, 'Stevens' cranberry vines will be treated with eight foliar treatment as follow: 0 (deionized water) and 400 mgL-1 of ABA applied at 21 days after 50% fruit set; 0 (deionized water) and 400 mgL-1 of ABA applied at 21 days after 50% fruit set 42 days after 50% fruit set; 0 (deionized water) and 400 mgL-1 of ABA applied when 50% of fruit is at veraison, and 0 (deionized water) and 400 mgL-1 of ABA applied after harvest. Freezing tolerance of terminal buds will be determined using DTA on a monthly basis starting in September until beds are flooding for the formation of ice for overwintering protection.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience for this project are fruit growers, in particular cranberry growers, and the scientific community. Changes/Problems:Objective 3 "Evaluate the effect of foliar ABA application on cranberry bud cold hardiness during fall" will not be completed. The main reason being the difficulty ofdetermining cold hardiness of cranberry buds through controlled freezing test and visual evaluation under a microscope. When we proposed to evaluate the effect of ABA on cold hardiness of cranberry buds, we were hoping DTA would be a reliable way to evaluate cold hardiness of cranberry buds. However, through the work done inobjective 1, we discovered that DTA could not measure cold hardiness in cranberry buds, as cranberry buds do not deep supercool. DTA is a relatively high throughput method to evaluate cold hardiness, compared with visual evaluation, which would have made the evaluation of a large number of samples needed to determine theeffect of ABA treatment on cold hardiness of buds possible. However, with visual evaluation of buds there are a very limited number of samples that can be evaluated in a reasonable amount of time after the controlled freezing test is performed. In the spring of 2018, we started a new study with the objective of evaluating theresponses of cranberry buds to temperatures during ecodormancy.During spring of 2018,0.2 m2sections of cranberry sods (including roots, stems, and uprights) were collected from the field and placed in three controlled environment treatments (10, 15, 20°C) with a 16-hour photoperiod. Cranberry upright phenology was evaluated weekly in all treatments and in the field until 100% bloom was achieved. The data collected will be used to calculate a developmental index for ecodormancy. What opportunities for training and professional development has the project provided?One student is being trained on this project. How have the results been disseminated to communities of interest?1/25/2018 Oral presentation on results from the project delivered at the Wisconsin Cranberry School. Stevens Point, WI 8/6/2018 Oral presentation at scientific conference.Villouta, C., Workmaster, B.A., Bolivar-Medina, J., and Atucha, A. 2018. Mechanisms of freezing stress survival in cranberry dormant buds. 11th International Plant Cold Hardiness Seminar: Importance of Cold Hardiness in a Warming Climate. August 5-10, Madison WI. What do you plan to do during the next reporting period to accomplish the goals?1) Dormancy study data collection will continue through winter and spring 2019. 2) Workto determine presence or absence of ice barrier will continue during 2019. 3) Controlled freezing test to evaluate cold hardiness of cranberry buds will continue during fall 2018 and sprig 2019. In addition, we will conduct regrowth evaluation of sampled subjected to controlled freezing test, to determine the severity of the damage observed.

Impacts
What was accomplished under these goals? Goal 1. Define changes in cranberry terminal bud cold hardiness from fall to spring over the period of plant dormancy, focusing on the transition periods at the onset and emergencefrom dormancy. Goal 1.a. Determine if differential thermal analysis (DTA) can reliably estimate cranberry bud cold hardiness:This sub objective was completed in 2017. G0oal 1.b.Determine the presence or absence of an ice nucleation barrier:Cranberry terminal budswere sampled for histochemical evaluation, during fall 2018. Buds were fixed inglutaraldehydeandembeddedin resin.Cutted sectionswere stained with toluidine blue and rat monoclonal antibodies JIM5 against de-methyl-esterified HG and JIM7 against methyl-esterified HG. Sectionswill be evaluated underfluorescent and light microscopy to determine the presence of an ice barrier. During fall 2017 and spring 2018, cranberry terminal buds were sampled and a controlled freezing test was performed to visualize freezing events of the plant material. A FLIR 655SC video thermo camera recorded the samples during the control freezing test. Results from the video showed two distinctive frozen events; the first one from the base of the upright up through the stem and stopping at the bud axis, and the second one at lower temperature showing the freezing of the bud itself. This suggests the presence of an ice barrier at zone were the bud connects to the stem. Goal 1.c. Determine timing of terminal budendodormancyonset and release:During fall 2017, 0.2 m2sections of cranberry sods (including roots, stems, and uprights) were collected from the field the first week of November, and placed in three controlled environment treatments with short photoperiod (8 hours): a control treatment at 25°C, a freezing treatment of constant -3°C, and chilling treatment of constant 3°C.Samples in freezing and chilling environments were moved to forcing conditions (25°Cand 16 hr photoperiod) after reaching 700, 1000, 1500 and 2000 hours of exposure to freezing or chilling conditions.The rate of bud break was evaluated at a two day-interval. Due to the freezing condition in the -3°C treatments, cranberry samples died and no data was collected for this treatment. 2. Evaluateenvironmentally and plant physiological factors for use as indicators to changes in cranberry bud cold hardiness level:During fall 2017 and spring of 2018, dormant cranberry buds were collected to assess cold hardiness level through a controlled freezing test. Buds from the controlled freezing test were dissected for freezing damage assessment using a dissection microscope. Buds were cut longitudinally with a double edge razor blade and immediately observed for damage distribution. Freezing damage was assessed by the evaluation of multiple bud structures: Bud scales, bud axis, lower stem, shoot apical meristem, and flower primordia. Damage for each structure was evaluated by severity according to the proportion of oxidative browning area and water soaking appearance by a discrete scale with four levels of damages, from 0 to 3, being 0 no damage and 3 complete darkened area. Changes in location of the damage and intensity was correlated to temperature data collected in the field, as well as day length during each sample date.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Villouta, C., Workmaster, B.A., Bolivar-Medina, J., and Atucha, A. 2018. Mechanisms of freezing stress survival in cranberry dormant buds. 11th International Plant Cold Hardiness Seminar: Importance of Cold Hardiness in a Warming Climate. August 5-10, Madison WI.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Atucha, A., Villouta, C., and Workmaster, B.A. 2018. Understanding Patterns of Cold Damage In Buds Using Control Freezing Test. Proceedings Wisconsin. Cranberry School. January 24-25. Stevens Point, WI.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience: The target audience for this project are fruit growers, in particular cranberry growers, and the scientific community. Changes/Problems:Objective 1"Define changes in cranberry terminal bud cold hardiness from fall to spring over the period of plant dormancy, focusing on the transition periods at the onset and emergence from dormancy", is divided into 3 subobjectives: 1.1Determine if differential thermal analysis (DTA) can reliably estimate cranberry bud cold hardiness. Completed 1.2Determine the presence or absence of an ice nucleation barrier. This subobjective will be modified to use fresh tissue samples instead of bud sections embedded in resin. 1.3Determine timing of terminal bud endodormancy onset and release. This subobjective will be modified to determine cilling hour requirement for dormancy fufillment, as well as base temperature for growth in the spring after fufillment of chilling. Objective 1.1 has been completed, and we have determined that DTA can not estimate cranberry bud cold hardiness. However, this will affect objective 2 "evaluate environmental and plant physiological factors for use as indicators to changes in cranberry bud cold hardiness level", because we can not evaluate cranberry bud cold hardiness. To fufill objective 2, we collected and analyzed leaf samples for changes in pigment concentration during fall 2016 and spring 2017, however we will not be able to correlate this information with changes in cold hardiness in buds because the results from the DTA analysis we performed as we were sampling for the pigments, did not provide any information on the degree of cold hardiness of the buds. We will correlate changes in pigment development and degradation of cranberry leaves duringfall 2016 and spring 2017 with the temperature data we collected during this period. Objective 3"Evaluate the effect of foliar ABA application on cranberry bud cold hardiness during fall". We will have to modify this objective because we can not evaluate cold hardiness of buds with DTA. We will do visual evaluation of cold damage in buds after controlled freezing tests in a glycol bath. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Conference presentation:Villouta, C., B. Workmaster, J. Bolivar-Medina, and A. Atucha. 2017. Exploring Cranberry Cold Hardiness Using Differential Thermal Analysis. North America Cranberry Researcher and Extension Workers Conference. August 27-30. Plymouth, MA. Non-referred publication:Atucha, A (2017). Identifying frost damage in cranberry buds. Cranberry Crop Management Journal 30 (4): 5. 8/9/2017 Oral presentationon results from the projectdelivered at the Wisconsin Cranberry Summer Field day.Warrens, WI 4/11/2017Oral presentationon results from the projectdelivered at the Wisconsin Cranberry Early Season Grower Workshop. Tomah and Wisconsin Rapids, WI. 1/19/2017Oral presentationon results from the projectdelivered at the Wisconsin Cranberry School. Stevens Point, WI What do you plan to do during the next reporting period to accomplish the goals?1) Dormancy study data collection will continue through winter and spring 2018. 2) Cranberry anatomical study to determine presence or absence of ice barrier on fresh tissue samples will continue during 2018. 3) Evaluate foliar ABA application on bud hardiness.

Impacts
What was accomplished under these goals? We collected dormant cranberry buds on a weekly basis starting the third week of October 2016 until the second week of May 2017, and performed DTA on each sampling date. Although high and low temperature exotherms (LTE) were detected during DTA analysis from buds collected during spring of 2016, results were inconsistent and the number of LTEs recorded did not reflect accurately the number of buds tested. In addition, temperature values of LTEs recorded during this period did not change, even when buds in the field experienced significant changes in temperature. Based on these results, during the spring of 2017, in addition to DTA analysis a second subset of samples was evaluated using a control-freezing test to compare the two methodologies. Results from the comparison between DTA and control freezing test suggest that the LTEs detected by the DTA system might not necessarily reflect the freezing of super cooled tissue within cranberry buds. To further test this hypothesis, during fall 2017 biweekly samples of cranberry buds were collected from the field and cold hardiness of buds was determine using the DTA set up and a set of copper-constant thermocouple attached to individual buds to monitor tissue temperature. Simultaneously, a second set of buds was evaluated through a control freezing test, using a glycol bath and submitting the samples to the same range of temperatures as in the DTA. No LTEs were recorded through the DTA system or through the copper-constant thermocouple, however, significant damage to buds was observed through visual evaluation of buds tested in the control freezing test. The data collected in these studies suggest that cranberry buds may not undergo deep super cooling, thus DTA is not a suitable methodology to estimate cold hardiness of dormant cranberry buds. Terminal buds were sampled for histochemical evaluation, from fall 2015 until spring 2016 at five different stages: Pre-harvest, Post-harvest, Pre-ice, Post-ice and Bud Swell. The samples were classified according to upright nature (i.e. vegetative or reproductive) and bud size. Buds were fixed in glutaraldehyde and embedded in resin. Cut sections were stained with fluorescent dye aniline blue, ruthenium red, and phloroglucinol for callose, pectin, and lignin identification, respectively. Sections were evaluated under fluorescent and light microscopy. No presence of ice barrier was identified in any of the samples evaluated, and there were no differences in tissue composition throughout the different sampling dates evaluated. However, we observed the presence of voids in bud scales, in particular after the sampling of buds during the Pre-ice sampling date. During fall 2017, the same stains were used in fresh bud section. However, no ice barriers were identified as in the fixed sections. During fall 2016, sets of cranberry uprights were collected from the field at three different chilling hours accumulation: 350, 550 and 650 hours. At each sampling date, subsets of uprights were placed in: control in forcing conditions at 25°C; -5 °C constant temperature (freezing) for 700, 1000, 1500 and 2000 hours, and 5 °C constant temperature (chilling) for 700, 1000, 1500 and 2000 hours. Sets in freezing and chilling environments were moved to forcing conditions after reaching 700, 1000, 1500 and 2000 hours of exposure to freezing or chilling conditions. The rate of bud break was evaluated at a two day-interval. After 700 hours of exposure to chilling temperatures, we observed a maximum average of 53% of bud break increasing to 80% with 1000 hours, 95% in 1500 hours, and 90% in 2000 of chilling. After 700 hours of exposure to freezing temperatures, we observed a maximum average of 40% of bud break increasing to 47% with 1000 hours, 59% in 1500 hours, and 54% in 2000 of freezing.During fall 2017, 0.1 square meters area of cranberry vines and roots, refer to as sods, were sampled from the field once the vines had accumulated 350 chilling units. A subset of sods was placed a -3 °C constant temperature (freezing) for 700, 1000, 1500 and 2000 hours, while a second subset was placed at 3 °C constant temperature (chilling) for 700, 1000, 1500 and 2000 hours. This study is currently underway. During fall 2016 and spring 2017, temperature data loggers were placed in the field to monitor hourly temperature experienced at canopy and ground level. Starting a week after harvest, weekly samples of leaves from 'Stevens' cultivar were collected until ice was made in the beds for winter protection, and sampling resumed in the spring, after ice was melted, and continued until bud break. During the fall period, chlorophyll b concentration remained constant, while chlorophyll a concentration decreased from 10.61 mg/g FW at the beginning of the sample period in mid-October, to 4.55 mg/g FW in late December. During spring, Chlorophyll a, increased from 2.07 mg/g FW at the first sampling date in early March, to a maximum of 4.67 mg/g FW during the last sampling date in late May. Anthocyanins concentration during fall increased from 0.37 in relative absorbance value at 535nm in mid-October, to 1.06 in late December. During spring, the anthocyanin concentration decreased its relative absorbance at 535nm value in leaves from 1.26 in early March, to 0.22 in late May.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Villouta, C., B. Workmaster, J. Bolivar-Medina, and A. Atucha. 2017. Exploring Cranberry Cold Hardiness Using Differential Thermal Analysis. North America Cranberry Researcher and Extension Workers Conference. August 27-30. Plymouth, MA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Workmaster, B.A., Villouta, C. and Atucha, A. 2017. Cranberry Plant Freezing is SuperCool. Proceedings Wisconsin Cranberry School. January 19. Stevens Point, WI.


Progress 06/01/16 to 09/30/16

Outputs
Target Audience:The target audience for this project are fruit growers, in cranberry growers and the scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?04/28/16 An update on the project was presented to the cranberry growers at the Cranberry Spring Field Day. 11/11/16 An update on the project was presented to the Wisconsin Cranberry Board research committee. What do you plan to do during the next reporting period to accomplish the goals?1) Sampling of cranberry buds to evaluate cold hardiness will continue during the winter and spring of 2017, and resume during fall 2017. 2) Dormancy study data collection will continue through the winter and spring of 2017, and resume during fall 2017. 3) Cranberry bud anatomical study to determine presence or absence of an ice nucleation barrier will continue during 2017.

Impacts
What was accomplished under these goals? To evaluate cold hardiness of cranberry buds during fall and spring, we designed and constructed a custom system to electronically control a temperature test chamber. This system allows us to perform real-time controlled freezing of cranberry buds and document the temperatures at which cranberry buds freeze at given date by using differential thermal analysis (DTA). To evaluate if DTA could reliably estimate cranberry bud cold hardiness, we sampled buds on a weekly basis starting in October, and performed DTA. We were able to record high and low temperature exotherms (LTE) and calculated the temperature at which 50% of the buds were killed (LT50) for all sampling dates. To validate the results obtained through the DTA, after the appearance of LTEs samples were removed from the chamber, incubated at room temperature, and inspected under a dissecting microscope for blackened tissue. Data collection in this experiment is still underway. To determine the presence or absence of an ice nucleation barrier between the bud and the bud axis in cranberries, tissue samples were collected at 5 critical periods: pre-harvest, post-harvest, pre-ice formation for winter protection, after ice-off in early spring, and initial terminal bud swelling. The samples were fixed, sectioned, and stained for further analysis with a microscope. Data collection in this experiment is still underway. During fall of 2016, cranberry uprights were collected to determine timing of terminal bud endodormancy onset and release. Samples were collected based on chilling unit (0-7°C) accumulation in the field (350; 550; 650) and subjected to three treatments: forcing at 25 °C; accumulation of chilling units in a cold chamber at 5 °C followed by forcing at 25°C; accumulation of freezing units at -3°C followed by forcing at 25 °C. Data collection in this experiment is still underway. During 2016, we installed a temperature data logger and installed them at canopy-level to calculate chilling unit accumulation and growing degree-days. Leaf anthocyanin development and chlorophyll degradation have been quantified for all sampling dates starting October 2016.

Publications