Abstract
Pawpaw (Asimina triloba) is a fruit-bearing tree native to North America, and nurseries have a strong public demand for grafted trees. Most nurseries currently propagate pawpaw cultivars clonally through chip budding and whip-and-tongue grafting, both onto pawpaw seedling rootstock. Flexibility in successful grafting and budding techniques can optimize the type and quality of available scionwood while using the available labor. In an initial experiment, a range of pawpaw selections were subjected to grafting and budding techniques that indicated candidate cultivars and advanced selections for further examination. Therefore, the objective of this study was to determine the optimal method of propagating the pawpaw cultivars, KSU-Atwood™, KSU-Chappell™, and the advanced selection Hi7-1 using chip budding, whip and tongue, T-budding, and a cleft grafting tool. Additional trees, each of KSU-Atwood™ and KSU-Chappell™, were also subjected to green budding in the summer with the current season’s scionwood. Whip and tongue grafting was the most successful method, with a scion survival rate (SSR) of 95.8%, followed by the cleft grafting tool (SSR = 66.7%) and chip budding (SSR = 50.0%), then T-bud grafting (SSR = 25.0%), and lastly green bud grafting (SSR = 0.0%). There were no cultivar differences for the grafting techniques used. The whip-and-tongue method had the highest scion survival rate; however, cleft grafting and chip budding can be effective propagation methods for nurseries if scionwood quality does not support the whip-and-tongue grafting technique. In addition to whip-and-tongue grafting, the cleft grafting tool is a viable option for grafting pawpaw for use by personnel with less grafting experience or skill.
The North American pawpaw (Asimina triloba) is a native fruit tree that can be cultivated for ornamental purposes or commercial production for its unique-tasting fruit (Pomper and Layne 2005). Small commercial orchards of pawpaw are found throughout the Southeastern and Midwestern United States for fresh market sales and processing. The fruit has a tropical-like flavor that can vary greatly between varieties (Duffrin and Pomper 2006). There are over 50 pawpaw cultivars that are commercially available, with many recent releases (Peterson 2003; Pomper and Layne 2005). Kentucky State University (KSU) has released three pawpaw cultivars, KSU-Atwood™ in 2009, KSU-Benson™ in 2016, and KSU-Chappell™ in 2018 with excellent fruit flavor and quality (Pomper et al. 2011, 2020). Many nurseries are currently unable to meet the public demand for pawpaw cultivars.
The seeds of the pawpaw fruit are genetically different from the parent tree, therefore clonal propagation methods such as grafting and budding onto seedling rootstock are important to the nursery industry for propagating cultivars. Most nurseries propagate pawpaw cultivars clonally through chip budding and whip-and-tongue grafting, both using pawpaw seedling rootstock because there are no clonal rootstocks for pawpaw, and no cultivars are grown on their own root system (Geneve et al. 2003; Pomper et al. 2009). Clonal propagation of pawpaw genotypes has been attempted via cuttings, tissue culture, and layering, all with limited success (Geneve et al. 2003). Rooting of stem cuttings has also not been successful, and root cuttings have limited success, in the clonal propagation of pawpaw. Pawpaw has the ability to send out autonomous shoots in tissue culture, but rooting of shoots in tissue culture remains a challenge (Geneve et al. 2003). Therefore, grafting or budding is the standard practice for clonally propagating pawpaw (Geneve et al. 2007).
Few studies have compared the success rates of different grafting or budding techniques for the clonal propagation of pawpaw. Pomper et al. (2009) reported that chip budding of pawpaw rootstock seedlings that retained leaves increased scion budbreak (76%) on rootstocks compared with budding onto rootstocks with all leaves removed (54%). Layne (1996) suggested that T-budding was not an optimal method for the propagation of pawpaw; however, no data have been published in this area. T-bud grafting is named for the “T” shape that is cut into the stem in which the bud is inserted. Inverted T-bud grafting is when the second slit is made at the bottom of the first slit instead of the top, resulting in an upside-down “T” marking in the bark. Inverted T-bud grafting is preferred because it helps prevent water from pooling in the graft union, which can lead to the growth of mold. The interruption in the downward flow of hormones and metabolic products below the bud leads to earlier healing and a stronger union (Stoltz and Strang 2005). Green budding is a propagation technique used in the spring or late summer for pecans and other tree species. Green buds at the base of the current season’s growth are removed in a chip and used in budding rootstock in an effort to increase the propagation period for nurseries.
With many nurseries unable to meet the public demand for pawpaw cultivars, methods that increase the success of scion viability after grafting would be advantageous to producers. Nurseries usually have a range of scionwood quality available after scionwood collection, with some wood being better suited to budding than grafting. Having a range of successful grafting and budding techniques could optimize both the type and quality of scionwood available, as well as the capabilities of available labor. A comparison of the success rate of clonal propagation methods for pawpaw cultivars such as chip budding, whip-and-tongue, T-budding, and cleft grafting with a grafting tool, would assist commercial nurseries and homeowners in the propagation of cultivars. Therefore, the objective of this study was to determine the optimal method among four grafting or budding techniques for the pawpaw cultivars KSU-Atwood™, KSU-Chappell™, and advanced selection Hi7-1.
Materials and methods
Plant material
In all grafting and budding studies, the rootstock trees were grown from seed that was extracted from the fruit of a mix of pawpaw cultivars and included ‘Sunflower’ and ‘Susquehanna’, which were grown in orchards at the KSU Harold R. Benson Research and Demonstration Farm (Frankfort, KY, USA). The seed was stratified at 5 °C for at least 3 months in moist peatmoss and sown to a depth of 3 cm into 4-L Treepots (Stuewe and Sons, Corvallis, OR, USA) containing ProMix–BX (Premier Horticulture, Red Hill, PA, USA) with Osmocote (14–14–14) 3- to 4-month release of 2.22 kg·m−3 and placed in the greenhouse (Pomper et al. 2002, 2009). Seedling germination rates were ∼90%. The seedlings were allowed to grow for 8 months and then overwintered in a greenhouse at 4 °C.
Grafting and budding experiment
For an initial experiment in Mar 2016, dormant scionwood of 11 pawpaw cultivars and advanced selections, including KSU-Atwood™, KSU-Chappell™, Hi1-4, G5-23, Hi7-1, G9-109, G9-111, Hy3-120, G4-25, G6-120, and KSU-Benson™, was collected, placed in plastic storage bags, and stored under refrigeration (4 °C) until used. Three grafting or budding techniques were implemented using dormant scionwood and actively growing 2-year-old pawpaw rootstock. Chip budding and whip-and-tongue grafts were performed using a grafting knife (Victorinox, Ibach, Switzerland), and cleft grafts were performed with a Topgrafter grafting tool (Raggett Industries, Grisborne, New Zealand). Buds or graft unions were then wrapped with ParaFilm® (Bemis Company Inc., Oshkosh, WI, USA) to prevent moisture loss and prevent desiccation of the scion. There were nine trees grafted or budded for each cultivar, with three trees for each grafting method for each cultivar. Trees were grown under greenhouse conditions, and scion survival rate was evaluated in Oct 2016 for grafting or budding success (Pomper et al. 2002, 2009).
In Mar 2018, scionwood for the pawpaw selections, KSU-Atwood™, KSU-Chappell™, and the advanced selection Hi7-1 was collected after the bud chilling requirement was satisfied for use in the experiments and stored in plastic bags at 4 °C until used. The rootstock seedlings in this study were allowed to grow for 8 months and then overwintered in a greenhouse at 4 °C. The rootstock seedlings continued to grow in the greenhouse the second year and were then overwintered. The rootstock seedlings were then allowed to leaf out again and were used in the grafting and budding experiments in May 2018. Therefore, a 2-year-old, actively growing rootstock was used for the grafting and budding experiments. Four grafting or budding methods (chip budding, whip-and-tongue, T-budding, and cleft grafting with a grafting tool) were implemented with rootstock and scion material from three pawpaw genotypes (KSU-Atwood™, KSU-Chappell™, and the advanced selection Hi7-1). The grafting/budding experiment was conducted over 3 days in May–June 2018 with each grafting technique conducted each day to create a two-way randomized complete design. There were 12 combinations of scionwood and grafting/budding methods with eight replications, for a total of 96 grafted trees. Eight potted 2-year-old trees of each cultivar were grafted using each grafting method at the KSU H.R. Benson Research and Demonstration Farm and grown in greenhouses (Pomper et al. 2002, 2009). All four grafting techniques used dormant scionwood and actively growing rootstock. Chip budding and whip-and-tongue grafts were performed using a grafting knife, T-budding, and cleft grafts were performed with a Topgrafter grafting tool. Buds or graft unions were then wrapped with ParaFilm® to prevent moisture loss and prevent desiccation of the scion. The chip buds were cut to 30 to 60 cm in height above the graft union, leaving at least six functional leaves to provide energy for the development of the scion bud. For T-budding on pawpaw, the bud was inverted and placed under the slipping bark. Plants were labeled by variety for the graft and type of graft used. After the scion began to grow, all competing buds were rubbed off weekly starting on 7 Jun 2018, to decrease apical dominance. On 26 Jul 2018, when the scions were at least 30 cm long, the remaining rootstock leaves were removed, and the stock was cut back to 20 to 25 mm above the union. Shadecloth, cooling systems, irrigation, and fertilization regimes were used to keep greenhouse growing conditions optimal for plant growth (Pomper et al. 2002, 2009). Budbreak was evaluated every 2 weeks starting 14 Jun 2018, and the number of buds broken and number of leaves growing on the scion were recorded. Midseason growth was measured and recorded on 12 Jul 2018.
Green budding experiment
In a separate experiment, 20 additional trees, 10 each of KSU-Atwood™ and KSU-Chappell™, were chip bud grafted, or green budded, on 10 Aug 2018, as the new growth had begun to harden off, but the bark was still slipping. Greenbud chip buds were inverted to promote scion survival. The seventh and final data collection of the season happened on 6 Sep 2018, after terminal buds had set and final growth was measured. Initial budbreak and survival rates for all 126 trees were recorded on 9 May 2019. The number of leaves as well as the number of buds that broke on the previous year’s growth was recorded on 23 May 2019.
Statistical analysis
Statistical analysis was conducted using CoStat Statistical software (CoHort Software, Monterey, CA, USA) and subjected to analysis of variance and Fisher’s least significant difference means separation. Treatment means were then separated based on a significance level of P < 0.05 to determine correlations and statistical differences between different grafting methods and scion varieties.
Results and discussion
In the initial 2016 experiment, Hi7-1, KSU-Chappell™, Hi1-4, KSU-Atwood™, G6-120, G9-111, G4-25, G5-23, Hy3-120, and G9-109 were successfully propagated with three grafting and budding methods; however, KSU-Benson™ was not successfully propagated with any methods used, possibly due to scionwood condition (Table 1). All three methods had an average scion survival rate >30%. The experiment provided evidence that all three grafting and budding methods could be used to propagate pawpaw, with whip-and-tongue method resulting in a higher success rate among the majority of cultivars. There was a significant difference among selections for overall average scion survival (Table 1). On the basis of the range of scion survival (50% to 17%) in this 2016 experiment, the selections KSU-Atwood™, KSU-Chappell™, and the advanced selection Hi7-1 were chosen for an expanded study of grafting and budding techniques with scion survival in 2018.
Pawpaw scion survival rate for three grafting or budding methods.
In 2018, five grafting and budding methods were examined. There was no interaction between the main effects: the grafting methods and the cultivars. There was no significant main effect for the cultivar; however, there was a significant main effect (P = 0.0002) for the grafting or budding methods used. Success or scion survival rate was defined as a living graft at the time of data collection at the end of the 4-month evaluation period (June–September). The whip-and-tongue grafting method (95.8%) had a significantly greater scion survival rate than the cleft graft with grafting tool method (66.6%), chip budding (54.1%), and T-budding techniques (37.5%) (Table 2). Similarly, the cleft graft grafting tool method was found to have a significantly higher scion survival rate than T-budding (Table 2). Even after overwintering, the whip-and-tongue grafting method (95.8%) maintained a significantly greater success rate than the rest. While the cleft graft with grafting tool method (66.6%) maintained the same scion survival rate, the survival rate for chip budding (50.0%), and T-budding techniques (25.0%) dropped after overwintering (Table 2). Both the cleft graft with grafting tool method and chip budding methods had significantly higher scion survival rates than T-budding after overwintering. At the end of the evaluation period, the whip-and-tongue method was the most successful method in terms of scion survival rate, and T-budding was the least successful (Table 2). There was no significant difference in scion survival rate (P = 0.3776) or the leaf number produced by scions (P = 0.7317) for each cultivar (Table 3). Similarly, there was no significant difference in leaf number (P = 0.0898) or scion length (P = 0.714) produced for all grafting and budding techniques (Table 3). No difference (P = 0.9258) in scion length was observed for the tested pawpaw cultivars (Table 3). For green budding, no green buds broke after overwintering for either cultivar. There was sufficient replication for there to be a P value of 0.0002 for scion survival rate for the grafting or budding method. Pomper et al. (2009), with larger replication, reported that there were trends for ‘Susquehanna’ buds to have a better success rate than ‘Sunflower’ buds and for chip buds on K8-2 rootstock to have a higher success rate than those on Sunflower rootstock; however, these cultivar trends were also not significant.
Pawpaw grafting or budding scion survival rate and growth characteristics.
Grafting and budding scion survival rate and growth characteristics by pawpaw cultivars.
There have been few studies comparing grafting or budding techniques for the clonal propagation of pawpaw. Although many nurseries currently propagate pawpaw cultivars through chip budding and whip-and-tongue grafting on seedling rootstock, having information on a range of successful grafting and budding techniques could optimize both the type and quality of scion wood available, as well as the capabilities of available labor. Scionwood collection from cultivars can often result in varying quality with poor vegetative bud development. Having multiple successful budding and grafting methods will maximize the scionwood available for use by nurseries.
Commercial propagation of pawpaw is currently effective only through grafting and budding techniques (Geneve et al. 2003). In the 2018 study, the whip-and-tongue grafting method (95.8%) had significantly greater scion survival rate than the cleft grafting tool, chip budding, or T-budding techniques. Pomper et al. (2009) had a higher scion survival rate of 87% when retaining six to eight supporting rootstock leaves at budding before leaf removal at 6 weeks; however, when leaves were removed at budding, these authors reported a significant reduction in scion survival rate to 55%. In this current 2018 study, the supporting leaves were removed from the rootstock after ∼6 weeks and chip bud viability was only 54%. The lower scion survival rate than Pomper et al. (2009) could be due to the trees in this current study being larger 2-year-old trees with more root containment stress growing in containers. There may also have been some greater mismatch of cambial areas with the larger trees in this current study, although the budding was done at a higher level on the plant stem to match the diameter of the scion to the stem. This may have also resulted in more competing buds breaking underneath the bud union and interfering with apical dominance of the developing bud and reducing nutrients reaching the bud union. Additionally, a study on grafting height in Dashehari mangoes (Magnifera indica) found that grafts had a higher success rate when grafted at 75 cm than at 100 cm on seedling rootstock (Kumar et al. 2000). The scion buds used in the current study may also have been of lower quality due to difficulties in obtaining large scionwood with plump buds after poor growing seasons or because of other unknown factors that affect scionwood quality year to year. However, these factors apparently did not affect the other forms of grafting and the success rate of those methods. Zenginbal (2007) examined the effects of whip-and-tongue grafting and chip budding in field conditions using two male (Hayward and Bruno) and two female (Matua and Tomuri) cultivars as scions and concluded that whip-and-tongue grafts were more successful than chip budding in kiwifruit. The pawpaw trees in this current study were also grafted a few weeks earlier than by Pomper et al. (2009) in Kentucky; therefore, differences in environmental conditions in the greenhouse such as temperatures, light intensity, and humidity levels influence scion survival rate in this current study. These data suggest that there are many factors that can affect scion survival rate with budding that are not potentially a concern with the whip-and-tongue grafting method. In addition to whip-and-tongue grafting, the cleft grafting tool used in this study had a high scion survival of 66.7%, making this method a viable option for use by personnel with less grafting experience or skill. Additional studies to optimize scion survival rate under greenhouse conditions could be undertaken to optimize the success of specific grafting or budding techniques.
Layne (1996) suggested that T-budding was not an optimal method for the propagation of pawpaws. The inverted T-bud interruption in the downward flow of hormones and metabolic products below the bud can lead to earlier healing and a stronger union (Stoltz and Strang 2005). The T-bud grafting was not an effective method for the propagation of pawpaw in this study. Green budding was also unsuccessful in this current study. Green bud grafting and T-bud grafting overall were not effective methods to propagate pawpaw in this study.
Conclusion
The whip-and-tongue grafting method had a significantly greater scion survival rate than the cleft grafting with a grafting tool, chip budding, or T-budding techniques for the clonal propagation of pawpaw cultivars onto seedling rootstock. In addition to the whip-and-tongue grafting, the cleft grafting tool used in this study had a high scion survival rate, making this method a viable option for use by personnel with less grafting experience or skill. Chip budding can be a successful propagation method with pawpaw, but some additional environmental and plant material factors may be of greater concerns with this method. Whip-and-tongue was the most successful technique for the clonal propagation of pawpaw cultivars for nurseries.
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