Final Report for GNE13-064
The purpose of this project was to develop a more cost effective, efficient and sustainable method of propagating new hazelnut cultivars. The specific goal was to improve the bud retention, vegetative growth and long term survival of softwood and semi-hardwood hazelnut cuttings through the use of novel plant growth regulators as well as optimization of propagation techniques including (tissue type, timing of tissue collection, media type, and cultivar/genotype). Despite infrastructure related setbacks, we were able to draw some general conclusions on improved propagation techniques for hazelnut cuttings. Throughout the experiment, cultivars tended to have significant effects on propagation success. Specifically, Rutgers selections H3R11P55, H3R11P52 and OSU 408.040 were all found to have a greater percentage of bud retention than other cultivars tested. Semi-hardwood cuttings of H3R11P55 were as high as 57% and softwood cuttings of H3R11P52, and OSU 408.040 both had nearly 50% bud retention. In addition, basal and canopy stem cuttings across cultivars, were found to result in significantly greater survival rates than apical shoot cuttings. Knowledge of optimal tissue type and genotype on propagation success will be used to continue efforts to develop improved hazelnut propagation techniques in the future.
Hazelnuts are a high-value, low-input crop with great potential for production in the northeastern U.S. Historically, production in this region has been limited by the disease eastern filbert blight (EFB). However, recent breeding efforts at Rutgers University have identified EFB-resistant plants that produce high-quality nuts. Pre-commercialization testing of the new selections is necessary prior to large scale adoption of the crop. This testing requires planting several small-to medium size (1-5 acres) clonal orchards to measure yields and investigate production and harvest techniques. Unfortunately, the challenges of asexual propagation of hazelnuts have limited this phase of development. Hazelnuts were traditionally propagated by layering and grafting. Layering is inefficient and grafting is not a preferable option due to the lack of tested EFB-resistant rootstocks. Recently, hazelnuts have begun to be propagated through micro-propagation, although this technique is not cost-effective when utilized for small- to medium-scale propagation. Rooting stem cuttings would be an ideal system for this setting. However, this method has not been proven to be consistently successful, with the survival of rooted stems being the major limiting factor. Fortunately, recent research has shown the use of the ethylene inhibitor 1-methylcyclopropene and/or gibberellic acid can greatly increase bud retention, continued rooting success and subsequent survival of hazelnut cuttings. We initially proposed a factorial study building upon recent knowledge and use of plant growth regulators to optimize the rooting of hazelnut stem cuttings. As the experiment progressed, several additional propagation techniques (described subsequently) were introduced into the experimental design. A more efficient method of propagation will expedite the establishment of pre-commercialization trials and should reduce the costs of mass producing planting stock, which in turn will reduce the costs of establishing orchards.
The goal of the project was to optimize the rooting and survival of both semi-hardwood and softwood hazelnut stem cuttings, which would present a more efficient and cost-effective method of clonal hazelnut propagation then that currently available. The objectives of the project are listed below followed by the any changes to the objectives, and explanations of the changes.
1. Measure the effectiveness and optimize the ethylene inhibitor (1-MCP) method described in Contessa et al. (2011) for enhancing bud retention of semi-hardwood hazelnut cuttings taken at different dates.
2013 Experiments: In 2013 semi-hardwood cutting experiments yielded poor rooting and subsequent bud retention despite treatments. This was in large part due to infrastructure related issues. It was noted that cuttings that dropped their leaves early (90% of cuttings) due to drought stress did not develop roots, while cuttings that held leaves rooted at a higher percentage.
2014 Experiment: In 2014 AVG (an ethylene blocking compound) was added to the experiment to assess its efficacy in enhancing bud retention, but yielded similar poor results.
2015 Experiment: This objective was not tested in 2015.
2016 Experiment: It was noted in previous years, that bud retention issues seemed to result in part from lack of moisture/humidity in the propagation benches, thus most recently in 2016 semi-hardwood cuttings were propagated in an entirely enclosed growth/humidity chamber with LED lighting.
2. The effectiveness of the foliar formulation of 1-MCP was to be compared with the wettable release (gas) formulation for its efficacy in the prevention of bud abscission and as an aid to survival and subsequent shoot growth of semi-hardwood hazelnut cuttings.
2013 Experiment: In 2013 the treatments were first performed, and no significant effect on bud retention was observed. The poor results were again attributed in part to the non-optimal growing conditions.
2014 Experiment: In 2014 the experiment was performed in a similar way as 2013, and showed treatment effects.
2015 Experiment: This objective was not tested in 2015.
2016 Experiment: This objective was not tested in 2016.
3. To investigate and optimize the application of ethylene inhibitors and gibberellic acid on bud retention, shoot growth, and overall survival of softwood hazelnut cuttings.
2013 Experiment: This objective was not tested in 2013.
2014 Experiment: In 2014 an experiment was performed where cuttings were treated with different types and combinations of gibberellic acid and 6-BAP to enhance bud retention and shoot growth. All treatments resulted in little root or shoot growth.
2015 Experiment: To improve upon previous propagation attempts, in 2015 two factorial experiments were established to explore several different methods of softwood cutting propagation to find optimal conditions for root initiation and subsequent shoot growth. Since our previous results have shown significant differences in the survival of cuttings among cultivars, 14 cultivars were included in the experimental design to ensure the propagation method was applicable to many hazelnut varieties. Additional factors added to the experimental design were media, rooting hormone concentration, and tissue type.
2016 Experiment: This objective was not explicitly tested in 2016, but a foliar formulation of IBA was used as a treatment to enhance root growth in cuttings.
2013 Materials and Methods:
Semi-hardwood Cutting Experiment (Objectives 1 and 2)
The first round of semi-hardwood hazelnut cuttings were taken in 2013 from the following breeding selections; H3R11P55, H3R11P52, and Oregon State University (OSU) 408.040 and 541.147. Although they were not initially proposed to be included, the OSU selections were added to the study because they are also promising cultivars for production in the northeast and ample stem tissue was available for developing the factorial experiment. The first round of cuttings was taken on September 16 and 17, 2013, and subsequently stuck on one propagation bench in a randomized complete block design. They were given their designated 1-MCP treatments and all were dipped in Hormodin #3 IBA .8% immediately before being stuck into the benches. The second round of cuttings were taken on September 30 and October 1, and also stuck in a randomized complete block design on a separate bench.
Each round of cuttings was kept in their benches for ~ 10 weeks. After ~10 weeks the cuttings were taken out of their benches and analyzed for the number of live buds, dead buds, leaves, roots and individual root length and width.
2014 Materials and Methods:
Softwood Cutting Experiment (Objective 3)
In Mid-June of 2014 softwood cuttings of three genotypes (OSU 408.020, OSU 541.147 and H3R11P52) were collected, dipped in 1000 ppm Dip-N-Grow then stuck in rooting benches. At 2, 4, and 6 weeks after the collection date the cuttings were sprayed with the following treatments: water (control), 125 ppm foliar fertilizer, 50 ppm GA 4,7, 50 ppm GA3 + 10 ppm 6-BAP, 50 ppm GA 4,7 + 10 ppm 6-BAP, 50 ppm GA3. At 8 weeks after collection the cuttings were dug up and assessed for number of live buds, number of roots (expressed as a root rating 1-5), and number of new leaves.
Semi-hardwood Cutting Experiment (Objectives 1 and 2)
At the end of September 2014, semi-hardwood cuttings were taken from two genotypes (‘Ratoli’ and OSU 541.147) and dipped in 500 ppm IBA salt and stuck in rooting benches. Foliar treatments were applied to the cuttings immediately after they were stuck, and gaseous treatments were applied prior to IBA dip and sticking. The rooting hormone was changed from last years experiment because of a much higher rooting success in cuttings treated with 500 ppm IBA (in a small-scale preliminary softwood cutting experiment earlier in the year). The cuttings were monitored closely over an 8 week period for bud retention, and at 8 weeks the cuttings were dug up and the number of live buds, dead buds, young leaves and roots were recorded.
2015 Materials and Methods:
Softwood Cutting Experiment (Objective 3)
In 2015, softwood cutting experiments were established with slightly different treatment structure. The following growing conditions were investigated: growing media, rooting hormone concentrations, and developmental tissue stage. The following is a description of the new treatments and why they were chosen.
Growing Media: Oasis rooting cubes were compared to standard propagation media (a mixture of 1 part ProMix to 1 part pearlite). Oasis cubes are synthetic polyurethane based foam that contain micronutrients and are purported to allow for better aeration than standard peat based media. In addition to potentially higher rooting success, foam cubes are easier to handle and preparte and their use can lessen damage to roots when transplanting. My decision to include foam rooting cubes in the experimental design was based in part on a previous study that showed foam cubes was based in part on a previous study that showed foam cubes increase cutting success in semi-hardwood peach cuttings (Avery and Beyl, 1991).
Hormone concentrations: In a small unpublished study we noted that Dip-N-Grow rooting hormone (a mixture of IBA and NAA) was most effective in inducing root growth in hazelnuts. In our softwood cutting experiments we used Dip-N-Grow at two rates a 1:20 dilution (500 ppm IBA+ 250 ppm NAA) and a 1:15 dilution (750 ppm IBA + 375 ppm NAA). These rates were similar to suggested (Dip-N-Grow) rooting concentrations for semi-hardwood cuttings.
Tissue Type: There are four main tissue types to use as cuttings for propagation in hazelnuts, canopy shoot cutting tissue, which consists of stem cuttings and apical tip cuttings, and sucker or basal hazelnut tissue, which also consist of stem cuttings and apical tip cuttings. Both apical and stem shoot tissue, along with stem basal cutting tissue were used in the factorial treatment design. Tip cuttings from basal tissue were not utilized due to the short internode length.
Experiment 1: The experiment was set up as a 14x2x2x2 factorial design. Cuttings were taken in June of 2015 from the following 14 hazelnut cultivars, which are considered the first factor in the design (cultivars: Jefferson, and Yamhill; Oregon State University breeding selections: OSU 859.050, OSU 859.053 and OSU 859.086; Rutgers University breeding selections: CR11P10, CR12P35, CR4P43, H3R12P62, H3R4P23, CR11P7, CR6P56, H3R7P25, and CR7P58). The following factors were also included: developmental tissue type (canopy shoot stem tissue or shoot apical tissue), rooting hormone treatment (Dip-N-Grow rooting treatment at either a 1:15 dilution or 1:20 dilution), and media type (Oasis rooting cubes or 1:1 Promix to pearlite packed in a propagation bench).
Experiment 2: The experiment was set up as a 10x3x2x2 factorial design. Cuttings were taken in June of 2015 from the following 10 hazelnut cultivars, which are considered the first factor in the design (Jefferson and Yamhill; Oregon State University breeding selections: OSU859.050, and OSU 859.086; Rutgers University breeding selections: CR11P10, CR4P43, CR11P7, CR6P56, H3R7P25, and CR7P58). The following factors were also included: developmental tissue type (shoot stem, shoot apical and basal stem tissue), rooting hormone treatment (Dip-N-Grow rooting treatment at either a 1:15 dilution or 1:20 dilution), media type (oasis rooting cubes or a 1:1 promix: pearlite media mix in rootmaker flats (11 cu in. cells)).
The following rating scale was used to assess the health of each cutting: 0= dead, 1= dead tip but the remainder of the cutting is alive, 2= buds are retained and the overall health of the cutting is good, and 3= bud break has occurred and the cutting is growing new shoots/leaves. This scale was used to rate the cuttings at 3, 8 and 11 weeks after being stuck in the benches. The data was recorded then analyzed using SAS 9.4.
Avery, J. D. and Beyl, C. B. 1991. Propagation of Peach Cuttings Using Foam Cubes. Hortscience. 26(9).
2016 Materials and Methods:
Semi-hard Cutting Experiment refocus of the objectives- The main objective was to find a better growing system to prolong the longevity of cuttings through the enhancement of bud retention and root growth.
The 2016 experimental design which implemented some successful treatments from previous years is shown below.
The 2016 rooting experiment was smaller than previous years, due to space limitations in a 4’ x 4’ grow tent. This grow tent was obtained from a fellow researcher and was used in an attempt to optimize growing conditions for hazelnut cuttings. The grow tent was equipped with an LED lighting system (12 hour day-length automated timer), fan and 2 humidifiers, although manual daily watering was still necessary to maintain moisture in the chamber.
On July 27, 2016 cuttings were taken from 4, 6 year old mother plants/varieties, which included; Yamhill, breeding selection 541.147, Theta, and a Rutgers breeding selection.
Cuttings were taken and stuck in Promix HP, in Rootmaker® flats. A total of 100 stem cuttings were taken per variety, and organized into the following factorial treatment structure. Note treatments were minimal in this experiment, since it was a trial run of the new grow tent system.
- 50 cuttings of each variety were placed on heating pads (~75 degrees) and 50 were not heated. Bottom heat has been shown to increase rooting in cuttings of some perennial crops, and has been used in previous years of this project.
- Half of the cuttings receiving bottom heat and half of the non-heated cuttings across all cultivars received a foliar spray treatment of 300 ppm IBA, the remaining cuttings were sprayed with water. Both the IBA and water treatments were sprayed till run off, both 1 day after sticking cuttings and then again 2 days after sticking cuttings. IBA was used because it has been shown to enhance rooting when used as a foliar treatment on cuttings of some perennial species. (Kroin, 2008)
Kroin, J. 2008. Propagate Plants from Cuttings Using Dry-Dip Rooting Powders and Water Based Rooting Solutions. Combined Proceedings International Plant Propagators Society Volume 58. 360-372.
Throughout the 2013 experiment there were significant mechanical issues with the misting system that were beyond our control, and as a result parts of the benches were dry for extended periods of time. These periods of dryness caused premature leaf drop in many of the cuttings (especially the second round). Due to the very low overall rooting percentage (<10%), which we suspect is due to misting issues, all rooted cuttings were saved and none were destructively sampled.
Preliminary, although possibly confounded results indicate that there is no significant treatment effect on bud retention (cutting survival) based on the various treatments they were exposed to. However, there was a significant cultivar effect on bud retention. The results indicate that OSU 541.147 has a significantly lower percentage of bud retention in all treatments (8.2% for the first round of cuttings, and 3.8% for the second round of cuttings (P,.0001). The remaining cultivars all showed significantly higher (but similar bud retention rates than OSU 541.147. The first round of cuttings showed an overall 7% survival rate, and the second round of cuttings showed a 1% survival rate. Despite the low survival rate, there seems to be a weak correlation between the number of leaves retained after 10 weeks and the number of roots formed after 10 weeks (the correlation coefficient was r=.5, where 0 indicates the variables are not correlated and 1 shows a high level of correlation). Given this correlation, drought factors beyond our control (causing early leaf drop), could explain the poor root growth and subsequent cutting survival.
Very few new leaves had grown on any of the cuttings after 8 weeks, but a number of cuttings did show live buds and significant root growth. The treatment effects on bud retention and rooting can be visualized in Supplementary Figures 1 and 2 respectively. The water, foliar fertilizer, and GA4,7+ 6-BAP treatment did not result in significantly different bud retention but all three showed significantly higher bud retention than any other treatment.
Although all cuttings received the same rooting compound application prior to sticking, the root growth was negatively affected by application of the foliar products in this study. There was a significantly lower root rating in cuttings treated with any compound except for foliar fertilizer which had a similar rooting response to the cuttings treated with water.
There was also a significant difference in genotype response where H3R11P52 had significantly higher bud retention than OSU 541.147 and higher rooting response than either of the other cultivars.
There was no significant genotype by treatment interaction in bud retention or rooting in this experiment.
After 8 weeks many cuttings were still alive but none of the cuttings showed root growth. The cuttings showed various degrees of live bud retention though. Equally significant high bud retention was observed in cuttings treated with three different compounds; control cuttings treated with water in the gas chamber, 500 ppb 1-MCP in the gas chamber, and 1.5 ppm 1-MCP liquid. The highest average percentage of live buds was 82% found in the cuttings placed with water in the gas chamber (Supplementary Figure 3).
There was a significantly higher percentage of bud retention for ‘Ratoli’ (85%) than OSU 541.147 (52%).
There was no significant genotype by treatment interaction on live bud retention in this experiment.
Up until 11 weeks after sticking the softwood cuttings for both experiments, most were healthy. After 11 weeks in the benches though, the majority of cuttings across all cultivars died. This can be visualized in histograms of both experiments over time (Supplemental Figures 4-9). We hypothesized that the sudden death of many cuttings could have been due to overheating of the greenhouse in August 2015.
Despite the death of many cuttings after 11 weeks in the propagation benches, we were able to report positive effects on rooting due to other treatments. In terms of the developmental stage of tissue that is best suited for softwood cutting propagation, stem cuttings off of hazelnut canopy shoots yielded a greater number of healthy hazelnut cuttings than when apical tips of the same shoots were used. Although, in the second experiment basal (sucker) cuttings were included and yielded a greater percentage of successful cuttings than stem cuttings of shoots across cultivars. Rooting media also showed positive, albeit variable, results from cultivar to cultivar, where the foam cubes generally lead to equal or greater cutting health/success than the 1:1 promix to perlite media in either large propagation benches or rootmaker flats. The hormone concentrations used had similar effects on all cultivars, and we were unable to discern improvements in rooting due to one hormone concentration over another.
A primary limitation to the successful propagation of our hazelnut cuttings in experiments on both softwood and semi-hardwood hazelnuts throughout the course of the grant was the propagation bench system, which provided inconsistent temperature , moisture, and lighting for the cuttings.
In the final year of the project a self-contained LED growth chamber propagation system was obtained, and will be used in future hazelnut propagation experiments. The results from this years trial run with the growth tent/chamber system are described below.
After 2 months (8 weeks), nearly all of the buds were retained on all of the cuttings across cultivars and treatments. Less than three cuttings per variety across all treatments showed a small sign of callous, and no rooting was observed. It is hypothesized that there was an issue of inconsistent (manual) watering, coupled with troubleshooting the fan speed and timing, which created stress on the cuttings early in the experiment. Thus no significant treatment effect on growth/survival of the cuttings was observed. (Data not shown) Despite this result after a trial run with the growth chamber, we are developing a better method for its use with hazelnut cuttings in the future. The results of this experiment confirmed results of previous years experiments in that hazelnut stem cuttings are very sensitive to environmental stress, but the self-contained growth chamber system has the potential to be tweaked to prevent environmental stress issues in the future.
· Results from the 2013 experiment showed there is a significant correlation between semi-hardwood cutting success (indicated through bud retention) and genotype.
· Our preliminary results suggest that treatment of cuttings with 1-MCP has no effect on bud retention and ultimately no effect on overall cutting success.
· Hazelnut cutting were shown to be particularly drought intolerant despite treatment with 1-MCP (as evidence by the low survival rate of the cuttings <10%).
· Leaf retention also appears to be correlated to rooting success, which further corroborates how critical it is for hazelnut cuttings to retain leaves (avoid drought stress) to help induce root growth.
· Results corroborated 2013 findings that genotype has a significant effect on semi-hardwood cutting success, and further showed it maintains a significant effect on softwood cuttings. It also indicates the challenge in finding a method of propagation that leads to success over a large range of cultivars.
· Results from the 2015 experiment showed that stem cuttings off of basal branches lead to greater propagation success than canopy stem or canopy tip cuttings.
· Preliminary results indicate that small scale LED growth chambers could be a viable small scale propagation option for farmers in the future.
Education & Outreach Activities and Participation Summary
A significant portion of this project has been devoted to initially trialing many propagation methods, and has provided us with solid findings/groundwork for further experiments. To date a portion of the results from this project have been presented as a poster at the 2013 NEASHS Conference.
In addition the work will be presented at a Rutgers University hazelnut field day in July 2017.
This research will also be presented as a poster at the Rutgers University Turfgrass association meeting in January of 2017.
Lastly, the results will be written into a non-peer reviewed article for ‘The Nutshell’ which is a publication through the Northern Nut Growers association.
Additional media outlets will also be sought out and used to relay the results to a larger group of hazelnut growers and propagators in the north eastern US.
We did not collect economic analysis data for this project.
Development of an efficient method to root cuttings will greatly impact the ability to propagate hazelnuts at a low cost. This is a particularly important component of the goal to establish hazelnut production in the north eastern united states, where an industry is on the cusp of being established. Given our goals of developing an efficient protocol for propagation by cuttings, we feel once it is presented to farmers it will be easily and readily implemented.
Areas needing additional study
There is still much work to be done in terms of optimizing a hazelnut propagation technique. Our foremost goal in the future will be to address the susceptibility of hazelnuts to environmental stresses early in their establishment. Thus, we will continue to investigate a closed LED growth chamber system. In addition, we will continue to focus on assessing novel plant growth regulators in maximizing the propagation success of hazelnuts.