Source: JUST GREENS, LLC submitted to NRP
STOMATAL CONDUCTANCE TO IMPROVE LEAFY GREEN SHELF LIFE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1022852
Grant No.
2020-33610-31705
Cumulative Award Amt.
$98,893.00
Proposal No.
2020-00514
Multistate No.
(N/A)
Project Start Date
Jul 15, 2020
Project End Date
Dec 14, 2021
Grant Year
2020
Program Code
[8.13]- Plant Production and Protection-Engineering
Recipient Organization
JUST GREENS, LLC
1114 HANSHAW RD
ITHACA,NY 148502744
Performing Department
(N/A)
Non Technical Summary
The long-term goal of this project is to increase the shelf life of leafy green vegetables. Short shelf life of produce is a significant concern for the produce industry and consumers worldwide. We believe the opportunity resides with longer shelf life, which will result in significantly less waste and enhanced revenue along the value chain, improved consumer satisfaction, and increased food safety. Water loss through open stomata is believed to be the main cause of reduced shelf life. However; the importance of stomatal control to prolong shelf life has not been investigated yet. To fill in this gap in knowledge, we propose to develop preharvest water and light management practices to stimulate and maintain stomatal closure after harvest for prolonged shelf life and improved food safety.The objectives are: (1) establish baseline for pre- and postharvest stomatal closure under current growing practices, (2) evaluate duration of preharvest drought that forces stomatal closure, (3) evaluate timing of pre-harvest drought for stomatal closure, (4) evaluate the frequency of preharvest drought on stomatal closure, and (5) evaluate the impacts of dark periods on the stomatal response to drought. Leaf stomatal traits (aperture, conductance) and leaf produce properties (water loss, firmness, turgidity) will be determined for all treatments at the R&8D facility of AeroFarms.After completion of Phase I, we will apply for Phase II to determine how we could translate our findings to commercial production including automation of controls. We expect that longer shelf life will result in significantly less waste and enhanced revenue along the value chain, in addition to improved consumer satisfaction, and increased food safety.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20314301020100%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1430 - Greens and leafy vegetables;

Field Of Science
1020 - Physiology;
Goals / Objectives
The goal of this research by AeroFarms is to develop a crop cultivation and harvesting algorithm and process that enhances the shelf life (and, thus, quality), of leafy greens in a controlled environment agriculture setting. AeroFarms has had numerous discussions with retailers, agricultural experts, and food industry leaders about the need to improve shelf life and increase consumption of nutritious leafy greens. Our objective is to investigate the effect of drought treatment and changes to light spectrum, intensity, and frequency on postharvest shelf life in leafy greens. AeroFarms will conduct targeted R&D on environmental factors pre- and post-harvest with the objective to develop a crop cultivation and harvesting algorithm/process that produces higher quality leafy greens. Improvement of leafy greens shelf life will improve the economics for the industry, increase consumer acceptance, reduce food waste and improve the environmental impact of indoor farming.
Project Methods
Drought and light will be the main variables explored using two leafy greens representing plant families commonly grown indoors.1. Materials and Methods:Plants will be grown in the controlled vertical farm at the AeroFarms R&D facility at 212 Rome Street, Newark, NJ. This growing system consists of multi-story grow towers containing rows of trays. Leafy greens are grown a proprietary cloth medium. Roots penetrate the cloth fiber and absorb water and nutrients from a fertigation mist emitted from nozzles located below the cloth. Short episodes of drought will be applied by completely withholding the misting.2. Measurement of Dependent and Independent Variables In Phase I, several independent and dependent variables will be tracked throughout pre- and postharvest handling. Since postharvest stomatal closure depends on time after harvest, light, and temperature, and varies between species (McLauchlan et al., 2001; Thomson, 2005), a time-series measurement will be necessary to accurately assess the ratio of percentage of closed stomates.We propose to take leaf samples for the different time points and measure dependent variables on them. Stomatal opening will be assessed both microscopically and by measuring stomatal conductance, an indicator of stomatal opening that is used as surrogate for stomatal closure (Monteith et al., 1988).Measurement of the water loss or desiccation response of stomata will be determined according the methods of Aliniaeifard et al. (2014) and Arve et al. (2013). To visualize the stomatal closure the stomatal aperture will be assessed microscopically according the method as described by Scarpeci et al. (2016 and 2017). Photos of leaf sections from each stage will be taken and the state of every stomate in a unit area will be recorded.3. TasksTask 0: PreparationsA level of an AeroFarms aeroponic grow tower will be reconfigured, adding in additional sensors for temperature and humidity at a one per square meter density. Additional automatic valving will be added to allow precise control of the timing of the drought stress.Task-1 Baseline- Pre- and Postharvest Stomata Under Standard Growing ConditionsIn this task, experimental towers will be prepared to grow leafy greens using the standard tray structure and growing conditions. Sets of measurements for dependent variables will be conducted to determine baseline stomatal aperture and conductance under constant light and irrigation, to which plants of Tasks 2-5 will be compared to.Task 2: Drought Duration Before HarvestPlants will be grown to baby leaf size. For the initial days of growing, control plants and test plants will be treated the same. On the harvest day, the plants will receive 4 treatments and drought cycle timing will be recorded. The distance of the roots to the surface of the drip pan below will be recorded. Upon harvest, the stomatal closure will be recorded.Task 3: Timing of Drought Before HarvestThe control will be plants grown with no drought applied. The experimental variable will be the number of days before harvest when the drought stress will be applied.The length of the drought stress will be the best one determined from task 2. The measurement will be of the distance from the roots to the surface of the drip pan.Task 4: Frequency of Drought Before HarvestIn Task 4, we will measure the effect of having multiple occurrences of the drought stress treatment chosen after tasks 2 and 3. In Task 2 we chose a duration, in Task 3 we chose a day for that duration to occur in the growth cycle. In Task 4 these treatments will be the base case, but additional drought treatments will be added on the days previous to the one chosen in Task 3.Task 5: Impact of Dark Period on Stomatal Response to Drought.Stomata should naturally close under darkness. However, commonly used extended photoperiod or continuous lighting can lead to poor stomatal closure (Arve et al., 2013; Islam et al., 2010; Slootweg and Van Meeteren, 1991; van Meeteren and Aliniaeifard, 2016).Task 5 will evaluate the stomatal response to the best scenarios observed previously in Task 4 by exposing plant to different periods of 6 darkness.

Progress 07/15/21 to 12/14/21

Outputs
Target Audience:The target audience for this project will be the entirety of the research department of Aerofarms to communicate information learned through analyzing experimental data and at select times, specific representatives from the engineering and business development departments to communicate relevant information about the research. Changes/Problems:An extra experiment was added after the Objective 2 results indicated decrease in the leafy green product quality before a significant change in stomatal closure could be measured. The new experiment was termed as Objective 2.1 and focused on a new purpose to pinpoint the exact period of the preharvest drought until which the product quality is maintained. Adding a new experiment was extremely valuable for two reasons: (a) preharvest drought period must end before the product quality is affected that also affects harvest procedure and (2) at this stage of the project defining a suitable drought period regardless of it impact on the stomatal closure is critical for developing strategies for remaining Objectives that usage other means of drought and light conditions to promote stomatal closure. Thus, results from Objective 2.1 will be used to develop strategies for achieving Objective 3. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Complete the current experiment that will provide critical data for designing the experiment. Continue to analyze data and complete remaining experiments for Objective 3, 4, and 5 that are associated with the SBIR Phase I award. In part, the addition of the new experiment that was not planned previously, also delayed the project completion timeline of March 30. This and possibility of a follow up experiment, may casue us to file for an extension. Any extended SBIR Phase I work will be funded internally by the award recipient.

Impacts
What was accomplished under these goals? This phase project focused on defining preharvest drought practices and light conditions that will result in increased postharvest stomatal closure in leafy greens, which will reduce postharvest water losses and maintain product freshness and shelf life. For Objective 1, we have created baseline by quantifying stomatal closure for the two species of leafy greens grown in the current growing method. The results gave basis to strategize method of data collection. For Objective 2, the single event of preharvest drought was evaluated to identify stomatal closure rates resulting from a single drought stress immediately before harvest. Results indicated leaf desiccation before any significant reduction in the indicators of the stomatal closure was determined, which can affect the product quality. Therefore, an extra experiment as Objective 2.1 was designed and currently in progress to identify exact period of preharvest drought until which the product quality is maintained. The results from For the Objective 2.1 will be used to develop strategies for Objective 3 where we define stomatal closure rates as a function of the timing of a single drought stress event before the harvest. The results from the experiments from previous objectives will guide the strategies for next objectives.

Publications


    Progress 07/15/20 to 12/14/21

    Outputs
    Target Audience:This report will be aimed at a technically or scientifically literate person with no assumed knowledge of commercial leafygreenhorticulture.The target audience of the efforts of our project will be other members of the commercial horticulture field, who will find outabout our research through patent filings and later outreach. Changes/Problems:An extra experiment was added after the Experiment 2 results indicated decrease in the leafy green product quality before a significant change in stomatal closure could be measured. With this discovery, an experiment was added to investigate the preharvest drought period that must end before the product quality is affected that also affects harvest procedure and to accurately define a suitable drought period regardless of it impact on the stomatal closure, which is critical for developing strategies for remaining experiments. What opportunities for training and professional development has the project provided?This work provided the opportunity for Jr. Scientists and research associates to practice their skills on relevant topics including machine vision, phenotyping and microbial quantification. It also served as a topic for presentations and seminars at departmental and corporate levels How have the results been disseminated to communities of interest?The results have been disseminated internally. Weekly results are presented at the R&D departmental meeting. Longer term results have been presented to the CEO, COO, and other senior leadership at meetings such as our AeroCafegroup meeting, in which the most impactful ways to improve the quality of our leafy greens. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? Experiment 1 created the baseline of the stomatal and other measurements and helped to standardize the data collection procedures under the control growth conditions. Stomatal conductance (SC), water potential (WP), and stomatal pore area (SPA) were all parameters that were collected at specified times (t= -60, 0, 30, 60, 120, 180, 1440 minutes) relative to leaf excision. The postharvest SC did not change significantly between T30 and T180 for arugula but was significantly higher at T180 vs T30 for red romaine. A decline in SC is generally expected after the leafy greens has been harvested. However, red romaine showed an increased SC at T180 versus T30. The postharvest leaf WP did not differ significantly between T30 and T180 for both arugula and red romaine. For stomatal pore area, there was no significant effect of time (from T=30 through T=180) on SPA for arugula. However, there was a significant effect of time for red romaine, especially between T30 and T180. The linear regression relationship among SC, WP and SPA was not significant in both species. The SPA measurements were omitted from further experiments due to extremely large variability among samples. Experiment 2 was devised to find the adequate length of a single pre-harvest drought event that will have the greatest effect on postharvest leaf SC and WP.. Experimental data (SC and WP) was collected for harvested leaves at specific times (T= 60, 180, 300, 420 minutes) relative to irrigation cut off immediately after leaf excision. Experiment 2.1 was created to re-examine the appropriate length of a single drought event immediately before harvest with an emphasis on sampling location, Experimental data (SC and WP) was collected for harvested leaves with this new sampling procedure at specific times (T= 120, 150, 180, 210, 240, 270/300, 420 minutes) relative to irrigation cut off immediately after excision. There was a significant difference in mean SC between the edge and middle sampling locations for Arugula at specific times T=180, T=240, and T=330. The only significant differences between locations across all time treatments was between Control at T=120 and Drought at T=240. There were however no significant differences across times or locations for Red Romaine. The imposition of drought showed a significant effect between locations at T=180 with a p-value of 0.0018. Otherwise, no other significant differences between locations and time-treatments were found for WP. The goal of experiment 3 was to find whether the identified drought duration immediately before harvest in Exp. 2.1 can be also applied days (1, 2 and 3 days before harvest as well as immediately before harvest, 0-day treatment) before the harvest as a single event that will result in decreased stomatal conductance. The goal of experiment 4 was to examine the effect that the frequency (in 1, 2, 3, and 4 consecutive days before harvest) of a specified drought period(s) have on stomatal conductance and water potential.. There was no significant (p-values > 0.05) regression relationship between the quantity of drought stresses as determined by the frequency of drought and the percent decrease of the stomatal conductance across these treatments. The goal of experiment 5 was to find whether pre-harvest periods of darkness with or without compounded drought will decrease stomatal conductance. Shelf life for both varieties and product attributes like moisture in the packaging were also examined in a preliminary trial.

    Publications


      Progress 07/15/20 to 07/14/21

      Outputs
      Target Audience:The target audience for this project will be the entirety of the research department of Aerofarms to communicate information learned through analyzing experimental data and at select times, specific representatives from the engineering and business development departments to communicate relevant information about the Changes/Problems:An extra experiment was added after the Objective 2 results indicated decrease in the leafy green product quality before a significant change in stomatal closure could be measured. The new experiment was termed as Objective 2.1 and focused on a new purpose to pinpoint the exact period of the preharvest drought until which the product quality is maintained. Adding a new experiment was extremely valuable for two reasons: (a) preharvest drought period must end before the product quality is affected that also affects harvest procedure and (2) at this stage of the project defining a suitable drought period regardless of it impact on the stomatal closure is critical for developing strategies for remaining Objectives that usage other means of drought and light conditions to promote stomatal closure. Thus, results from Objective 2.1 will be used to develop strategies for achieving Objective 3. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Complete the current experiment that will provide critical data for designing the experiment. Continue to analyze data and complete remaining experiments for Objective 3, 4, and 5 that are associated with the SBIR Phase I award. In part, the addition of the new experiment that was not planned previously, also delayed the project completion timeline of March 30. This and possibility of a follow up experiment, may casue us to file for an extension. Any extended SBIR Phase I work will be funded internally by the award recipient.

      Impacts
      What was accomplished under these goals? This phase project focused on defining preharvest drought practices and light conditions that will result in increased postharvest stomatal closure in leafy greens, which will reduce postharvest water losses and maintain product freshness and shelf life. For Objective 1, we have created baseline by quantifying stomatal closure for the two species of leafy greens grown in the current growing method. The results gave basis to strategize method of data collection. For Objective 2, the single event of preharvest drought was evaluated to identify stomatal closure rates resulting from a single drought stress immediately before harvest. Results indicated leaf desiccation before any significant reduction in the indicators of the stomatal closure was determined, which can affect the product quality. Therefore, an extra experiment as Objective 2.1 was designed and currently in progress to identify exact period of preharvest drought until which the product quality is maintained. The results from For the Objective 2.1 will be used to develop strategies for Objective 3 where we define stomatal closure rates as a function of the timing of a single drought stress event before the harvest. The results from the experiments from previous objectives will guide the strategies for next objectives

      Publications


        Progress 07/15/20 to 03/14/21

        Outputs
        Target Audience: Nothing Reported Changes/Problems:An extra experiment was added after the Objective 2 results indicated decrease in the leafy green product quality before a significant change in stomatal closure could be measured. The new experiment was termed as Objective 2.1 and focused on a new purpose to pinpoint the exact period of the preharvest drought until which the product quality is maintained. Adding a new experiment was extremely valuable for two reasons: (a) preharvest drought period must end before the product quality is affected that also affects harvest procedure and (2) at this stage of the project defining a suitable drought period regardless of it impact on the stomatal closure is critical for developing strategies for remaining Objectives that usage other means of drought and light conditions to promote stomatal closure. Thus, results from Objective 2.1 will be used to develop strategies for achieving Objective 3. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Complete the current experiment that will provide critical data for designing the experiment. Continue to analyze data and complete remaining experiments for Objective 3, 4, and 5 that are associated with the SBIR Phase I award. In part, the addition of the new experiment that was not planned previously, also delayed the project completion timeline of March 14, 2021. This and possibility of a follow up experiment, may cause us to file for an extension. Any extended SBIR Phase I work will be funded internally by the award recipient.

        Impacts
        What was accomplished under these goals? This phase of the project focused on defining preharvest drought practices and light conditions that will result in increased postharvest stomatal closure in leafy greens leading to reduced postharvest water losses and maintenance of product freshness and shelf life. For Objective 1, we have created a baseline by quantifying stomatal closure for two species of leafy greens grown using the current growing method. The results gave basis to the best method of data collection. For Objective 2, a single event of preharvest drought was evaluated to identify stomatal closure rates resulting from a single drought stress immediately before harvest. Results indicated leaf desiccation before any significant change in the indicators of the stomatal closure was determined. Without any change in the water status indicators an extra experiment (Objective 2.1) was designed and is currently in progress to identify exact period of preharvest drought until which the product quality is maintained via stomatal closure. The results from Objective 2.1 will be used to develop strategies for Objective 3 where we define stomatal closure rates as a function of the timing of a single drought stress event before the harvest. The results from the experiments from previous objectives will guide the strategies for next objectives.

        Publications