Washington State University: Vegetable Grafting Research Project: April 2010

(This project was completed for my environmental biophysics project. Tables, figures, and some symbols did not come through).

Abstract
Chitosan is a natural compound derived from chitin that activates plant defense response and stomatal closure thereby reducing transpiration. The purpose of this study was to investigate the effect of chitosan on stomatal conductance and survival of grafted Cherokee Purple and Maxifort tomato plants. All plants were self-grafted using the apical grafting technique. Chitosan solution was applied to the foliar surface of treated plants. Effect of chitosan was observed in two growth chambers held at 25C and 37% and 95% relative humidity respectively. Stomatal conductance was measured with a leaf porometer every 24 hours for eight days following grafting. Stomatal conductance was reduced in the initial hours after foliar chitosan treatment in Cherokee Purple but the effect was not sustained. Chitosan-treated Maxifort plants did not show significantly lower stomatal conductance as compared to untreated plants. Results of this study indicate no significant reduction in transpiration in grafted transplants by foliar application of chitosan. Effectiveness of chitosan foliar application in reduction of stomatal conductance and higher graft survival at lower humidity levels remains inconclusive.

Introduction
Chitosan is a natural biodegradable polymer obtained by deacetylation of chitin from crustaceans. Foliar applications of chitosan have been shown to elicit plant defense mechanisms resulting in stomatal closure and reduction in transpiration (Bittelli, Flury et al. 2001; Iriti, Picchi et al. 2009). Grafting vegetables is extensively used in Asia, Europe, Canada, and increasingly in the United States for improved plant vigor and resistance to soil-borne pathogens. During the grafting process, xylem is severed and the scion is under extreme water stress until the xylem tissue of the scion and rootstock heal together. In order to minimize water stress, efforts are made to reduce transpiration for a period of time immediately following grafting (Rivard and Louws 2006). Grafted vegetables are placed in healing chambers with optimal temperatures (20-30C), high relative humidity, between 95-100%, and complete darkness for five days(Grubinger 2007; Zhao 2010). After 5 days, light is slowly increased while humidity and temperature are slowly decreased (Hassell, Memmott et al. 2008). As chitosan is a known antitranspirant, foliar applications of chitosan to newly grafted plants may reduce water stress to scion and subsequently increase graft success. Chitosan could potentially provide a low-cost alternative to climate-controlled healing chambers for both conventional and organic growers with limited space and resources. The purpose of this study was to look at the effect of chitosan on stomatal conductance and survival in grafted plants at suboptimal and optimal humidity levels.

Materials and Methods
Chemicals
One liter of chitosan solution was prepared according to methods used by Bittelli, Flury et al. (2001) with some modification. 1 gram of chitosan (448869; Sigma-Aldrich) was dissolved at a concentration of 1 g L-1 in 100mL of 1% (w/w) lactic acid solution. MilliQ water was added until 0.1% (w/w) lactic acid solution concentration was obtained. A 0.1% lactic acid solution without chitosan was also prepared for control plants. Solutions were neutralized to pH 6.3 with sodium hydroxide (NaOH) using an automatic titrator.

Plant materials
Two varieties of tomato commonly used in grafting research, Maxifort and Cherokee Purple, were chosen for this study. Maxifort (DeRuiters, Inc.) is a commercial rootstock used in production of grafted tomatoes. Cherokee Purple (Territorial Seed Co.) is a popular heirloom tomato variety. Untreated Maxifort seed and organic Cherokee Purple seed were sown on February 22, 2010 in Sunshine Mix #1 in 72 cell trays. A data logger was used to measure temperature and relative humidity between February 22 and April 9, 2010. Average daytime temperature and relative humidity in the greenhouse were 21.6C and 27% respectively. Average nighttime temperature and relative humidity level were 12.8C and 39%.

Grafting and Chemical Application
A total of 76 plants (38 Maxifort and 38 Cherokee Purple) were grafted at 5:00am on April 9, 2010. Prior to grafting, a 10% bleach solution was used to sterilize all surfaces. Grafting was done with sterile double-edged disposable razor blades. Plants were self-grafted using the apical grafting technique, such that the angle of cut on rootstock and scion was identical and contact at graft union optimal. Grafts were held together with silicone grafting clips. Immediately following grafting, treatment plants were sprayed with chitosan solution at a rate of 10 spray-shots on both adaxial and abaxial leaf surface. Control plants were sprayed with 0.1% lactic acid solution at the same rate of application.

40 plants were placed in a growth chamber at 25C with no humidity control. Relative humidity was measured with a capacitance hygrometer every 24 hours. 36 plants were placed in a growth chamber held at 25C and 95% relative humidity (Table 1).

Light was excluded for 7 days. Stomatal conductance for each plant was measured with a steady state leaf porometer (Decagon SC-1) at 3 hours and 8 hours after grafting. Subsequent stomatal conductance measurements were collected every 24 hours over the next 7 days. As tomatoes are hypostomatous, stomatal conductance was measured only on the abaxial leaf surface. Plants were misted with 10 spray shots of water every 24 hours. On Day 8, plants were returned to the greenhouse for 2 hours with harvest crates over them to diffuse direct solar radiation. Plants were returned to greenhouse for 4 hours on Day 9. On Day 10, plants were permanently moved from the growth chamber to greenhouse.

Statistical Analysis
Statistical analysis was performed at a 95% confidence level (=0.05) using Minitab 15 (Minitab, Inc.). Two-sample t-tests were performed on Maxifort and Cherokee Purple varieties at each time interval with treatments consisting of chitosan-treated plants and untreated plants. An F-test was conducted to determine equality of variance between two samples thereby determining the appropriate t-test: pooled t-test or Welch’s t-test. Although sample size was small (n=9-10), a two-proportion comparison was conducted for percent survival of chitosan-treated plants versus untreated plants for each day of data collection. Pearson correlation coefficients were calculated for stomatal conductance and time after grafting. Correlation coefficients were also determined for standard deviation of stomatal conductance and time after grafting. Analysis was specific to variety and no comparisons were made between Cherokee Purple and Maxifort varieties.

Results
37% Relative Humidity
Although the growth chamber did not have humidity control, temperature was maintained at a constant 25C and thus the growth chamber maintained a nearly constant 37% relative humidity. Stomatal conductance decreased between 3 hours and 8 hours after grafting. Leaves were too wilted at 29 hours to accurately measure stomatal conductance using the porometer, so no further readings were taken.

At three days, all plants had wilted. At four days however, some scions began to regain turgor pressure. After eight days, there was a significant difference between percent survival of chitosan-treated and untreated Cherokee Purple plants (P0.05). At full acclimation on Day 10, chitosan-treated Cherokee Purple plants had 30% higher survival than untreated plants for both humidity levels. There was no significant difference in percent survival between chitosan-treated and untreated Maxifort plants.

95% Relative Humidity
On Day 10, there was no significant difference in survival of chitosan-treated and untreated plants healed in the 95% hr growth chamber for either Maxifort or Cherokee Purple. Maxifort plants had 90% survival on Day 10 regardless of treatment. On Day 5 and Day 6, chitosan-treated plants had significantly higher percent survival than untreated plants (P0.01). However, at completion of the experiment on Day 10, there was no significant difference between survival between Cherokee Purple treatment groups with 80% survival of chitosan-treated plants and 50% survival of untreated plants (Fig. 1).

Initially, it was hypothesized that standard deviation of stomatal conductance would increase over time, as xylem tissue of some grafts healed together while scion and rootstock of other plants remained disconnected. However, only chitosan-treated Cherokee Purple plants showed a significant correlation between standard deviation of stomatal conductance and time after grafting (P0.05).

Cherokee Purple plants that were treated with chitosan had significantly lower stomatal conductance than untreated plants 3 hours after grafting (Fig. 2). At Day 1 and Day 6 stomatal conductance for chitosan-treated plants were however significantly higher than untreated plants (P0.05).

There was no significant difference between chitosan-treated Maxifort grafts and untreated grafts at any time during the healing process (Fig. 3).

Discussion
Numerous studies have documented the antitranspirant effect of foliarly applied chitosan (Lee, Choi et al. 1999; Bittelli, Flury et al. 2001; Iriti, Picchi et al. 2009). However in this study, chitosan-treated plants did not have significantly lower stomatal conductance than untreated plants during the seven days after grafting for either tomato variety. Although stomatal conductance of chitosan-treated Cherokee Purple plants was significantly lower than untreated plants three hours after grafting, this effect was not sustained and Maxifort plants showed no significant reduction in stomatal conductance. The insignificant effect of chitosan in this study may have been due to the lack of light during the healing process, as stomata typically close in dark conditions. Also, the process of grafting possibly could have induced physiological stress responses in the plant that superseded the chitosan-induced defense response.

37% relative humidity is much lower than current research recommendations for post-graft healing, and the decrease in stomatal conductance between 3 hours and 8 hours and complete wilting at 29 hours was therefore expected (Rivard and Louws 2006; Zhao 2010). It was expected that, due to wilting, all grafts would fail. The survival of 50-60% of chitosan-treated grafts and 20-40% of untreated grafts at suboptimal humidity was unexpected. Although the greater percent survival of chitosan-treated Cherokee Purple grafts was statistically significant, the number of plants per treatment was very small. Thus, efficacy of chitosan foliar application in reduction of stomatal conductance and higher survival rates of grafts at suboptimal humidity levels remains inconclusive.

95% relative humidity is optimal for healing grafted transplants and the high percent survival of both Cherokee Purple and Maxifort support this recommended humidity level (Rivard and Louws 2006; Zhao 2010). There was no significant difference between percent survival of chitosan-treated plants and untreated plants at this level. Based on this small sample size, it appears that chitosan foliar application does not increase survival of grafted transplants healed at optimum relative humidity.

Stomatal conductance data at both humidity levels exhibited a high amount of variance. This is likely due to small sample sizes, physiological responses of scion and rootstocks to grafting, and inconsistencies of leaf porometer readings. A larger sample size would be recommended for future research. In addition, measuring stomatal conductance with the leaf porometer may have injured the graft union through slight agitation or movement of the scion away from the rootstock.

Conclusion
Results from this study do not conclusively demonstrate chitosan as an effective antitranspirant for newly grafted plants. Initial reduction in transpiration was not sustained beyond three hours after grafting for both suboptimal and optimal relative humidity levels. Chitosan-treated plants did show higher percent survival for all varieties and humidity levels. However, percent survival of chitosan-treated transplants was statistically significantly higher only in Cherokee Purple grafts at 37% relative humidity. The mixed results of this study are insufficient to conclude with confidence that chitosan is an effective foliar treatment for reducing water stress during the healing process in grafted transplants.

REFERENCES
Bittelli, M., M. Flury, et al. (2001). "Reduction of transpiration through foliar application of chitosan. ." Agricultural and Forest Meteorology 107: 167-175.

Grubinger, V. (2007, 1/07). "Grafting greenhouse tomatoes." from http://www.uvm.edu/vtvegandberry/factsheets/graftingGHtomato.html.

Hassell, R. L., F. Memmott, et al. (2008). "Grafting Methods for Watermelon Production." HortScience 43(6): 1677-1679.

Iriti, M., V. Picchi, et al. (2009). "Chitosan antitranspirant activity is due to abscisic acid-dependent stomatal closure." Environmental & Experimental Botany 66: 493-500.

Lee, S., H. Choi, et al. (1999). "Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis." Plant Physiology 121: 147-152.

Rivard, C. and F. J. Louws (2006). "Grafting for disease resistance in heirloom tomatoes." North Carolina Cooperative Extension Service.

Zhao, X. (2010). Personal Communication. 4/24/2010.

During the past five months, I have been the teaching assistant for Plant Propagation (HORT 251) here at WSU. I prep labs, write, administer, and grade quizzes, grade lab reports, and entertain the students. On a wonderfully sunny day this week, we took the class out to the Tukey Orchard.

The R.B. Tukey Horticulture Orchard is just off campus and was established for research and education purposes. Surplus fruit is sold to the public and there are u-pick cherries, apples, pears, and peaches (in mild years). Many of the trees are over 30 years old. Aging trees begin to slow in productivity and decrease in yield. The orchard houses lots of varieties. Many varieties were experimental and never gained momentum while other varieties have simply fallen out of favor over the decades. The solution to an aging unfashionable orchard: top-grafting!

Top-grafting is a grafting technique used to revitalize old established orchards or replacing a variety that may be waning in popularity with a rising star (such as 'Pink Lady' or 'Honeycrisp).

In the spring, the established tree's branches are cut back close to where they arise from the trunk. It is important to top graft in the spring, as the bark 'slips' away from the inner wood due to cambium growth. The scion wood's cambium must come into contact and heal with the rootstock's cambium. Slipping the bark away from the cambium, allows you to securely place the scion cambium in contact with the rootstock cambium.

Scion on left has been slipped between bark and cambium. Scion on right has been sealed with grafters wax to prevent it from drying out.

The scion material are new shoots that are about the thickness of a pencil. The lower ends must be cut in a wedge shape to maximize exposure of the cambium and to slip securely in between the bark and inner wood of the rootstock. Cutting the scion in the perfect wedge is a lot more difficult than it initially appears and requires confidence around very sharp knives.

A vertical cut is made about 3/4" down the bark of the rootstock, the bark is gently pried away from the cambium, and the scion is pushed down in between the bark in inner part of the rootstock.

Scion is nailed in place to secure.

The base of the scion is then covered with grafters wax to prevent drying out. The scion is pruned back to 3-4 buds to promote graft healing rather than vegetative growth. All wound surfaces are painted with seal and heal to prevent water loss and disease infection. About 3-4 scions are grafted into each main branch of the rootstock.