Progress report for FNC23-1359
Project Information
All of the cooperators on this farm have had an interest in low-maintenance fruit crops and have grown these crops at our respective farms for many years. We have used traditional orchard layout, forest farming, and silvopasture practices; and for many of us, persimmons have been a crop of increasing interest due to their low input requirements, their impressive disease-resistance, and their reliable cropping where many other fruits fail. Weston and Darren have grown persimmons on a homestead scale and sold the grafted trees for some time now. The historical roots of Cliff England’s operation go back as far as 1960, and Matt Renkoski was a recipient of a SARE grant around 2020 as he launched his persimmon orchard grafted on wild seedlings of Diospyros virginiana. Jamie currently manages a grove of Satsuma oranges in southern Alabama. We’ve all been interested in shifting our farms toward sustainability, and for many of us persimmons have been part of the transition.
Growers throughout North America and in the southern NC-SARE region have realized that persimmons are one of the simplest fruit crops to grow without pesticides and heavy fertilization. Persimmons resist many diseases and increase consumer access to local, nutritious food. As more and more people grow this fruit, the public is acquiring a taste for all types of persimmons. But for many people, non-astringent persimmons (such as ‘Fuyu’) are still considered the most desirable. Unlike astringent cultivars, non-astringent varieties can be shipped and eaten while they are still firm.
Breeders have labored to develop persimmon varieties that produce desirable fruit and also tolerate the winter conditions found throughout the southern NC-SARE region, however their work is far from finished. Cold-hardy hybrids between wild persimmon (Diospyros virginiana) and the oriental persimmon (Diospyros kaki) have been released and eagerly received by growers, but so far there have been no non-astringent hybrid persimmons released to growers in the NC-SARE region. This means that growers cannot easily compete with consumer expectations set by California and other warm regions. As of now, non-astringent persimmons primarily grow in zone 7 and southward, which excludes all of the NC-SARE region. But we would like to see this changed! We are laying groundwork for the potential breeding of a truly non-astringent, cold-hardy hybrid persimmon that can grow in many portions of the NC-SARE region!
There are two different genes that can be used to generate the non-astringent persimmon phenotype in Diospyros kaki, and for this reason we see primary ways that a non-astringent hybrid persimmon could potentially be generated as well. Japanese non-astringent persimmon varieties carry a recessive gene which is responsible for the non-astringent phenotype, while Chinese non-astringent persimmons carry a dominant allele responsible for a similar phenotype. As part of this project, we have laid groundwork for potentially introgressing either one of these genes in the background of a cold-hardy Asian-American hybrid persimmon cultivar.
During the course of this grant, we are grateful to have obtained cuttings of the very rare Taishuu Japanese persimmon variety—said to sell for $30/each in some markets, as well as cuttings of the Chinese Luotian persimmon—a cultivar which carries the Chinese dominant allele for non-astringency. These varieties were difficult to obtain, but they carry the genetics that we need to experiment with the two breeding strategy outlined above. We have also established a small breeding orchard in southern Alabama, where we have planted these cultivars just mentioned, along with others varieties needed for achieving our breeding goals.
Further, as part of this project, we have conducted some additional persimmon research relevant to our goals. We attempted to chemically induce male blooms on persimmon trees that naturally produced female flowers. This is relevant to our work because often male flower production is a limiting factor in persimmon breeding. We have also worked with a lab to test molecular markers that can be used to identify persimmon trees which will 1) be expected to produce male flowers (in addition to females) and 2) have the non-astringent phenotype induced by the Japanese recessive allele. The mixed results of these research projects are noted below.
Project outreach occurred via several channels. Several posts to the “Persimmon World” Facebook group during the commencing year of the project alerted group members of our goals and our efforts. In winter 2023/2024, an article featuring our work and written by Weston Adams, our project coordinator, was published in Pomona, the journal of the North American Plant Explorers. Around the same time, Hannah Walhout reached out to Weston, requesting some details to include in a persimmon article featured by FoodPrint, which was published January 18, 2024. All of these communication channels served to raise awareness about persimmons and our project.
As explained in more detail below, this project took some unexpected turns. But progress was made nonetheless. Preliminary results from our tests indicate that female Diospyros kaki trees may not respond to the chemical method we used to induce male blossoms. Our test specimen tree did not produce male blooms even when treated repeatedly with a silver thiosulfate solution--a treatment that been known to induce male blossoms in otherwise-female plants of other species.
Further, our molecular marker results were also mixed. On the positive side, we have successfully confirmed molecular markers which can be used to identify trees of Diospyros kaki that will have the male-flowering trait. These molecular markers can be useful to other persimmon breeders in the United States, because they can help to identify trees with this trait while in the seedling stage. However, we were unable to confirm any markers linked to the non-astringent phenotype. More research would be required to confirm molecular markers effective for this trait.
As hybrid persimmon breeding continues to progress, and as varieties with high-quality fruit and cold-hardiness are developed, I expect that commercial plantings of persimmons will be planted farther and farther north. And I expect that the environmental, social, and economic impact from these orchards will be noteworthy.
Persimmon breeders must focus their efforts on developing a non-astringent hybrid persimmon variety that can withstand temperatures commonly found in the southern portions of the NC-SARE region. Doing so would make an easy-to-grow and nutritious food item available to growers throughout the southern NC-SARE region and in other parts of North America. I have been working with a few others on a plan to tackle this problem.
I will describe below the genetics behind our methods, but in short, we have determined two possible paths to obtaining a non-astringent hybrid persimmon. First, we could attempt to harness the Japanese PCNA non-astringency allele found in Fuyu and many other Japanese non-astringent persimmon varieties. Second, we could bring in the dominant non-astringency allele originating from China and observe the phenotype that this produces in the hybrid.
The first method involves backcrossing the 50/50 Diospyros kaki/Diospyros virginiana hybrid persimmon known as Mikkusu (aka JT-02) to a non-astringent cultivar such as Hana Fuyu. Mikkusu is a cross between a Japanese non-astringent persimmon and a wild American persimmon. The Japanese non-astringent trait that we are discussing is inherited recessively, so we can reasonably assume the non-astringent parent of Mikkusu carried 6 copies of the allele in its hexaploidy genome. Traditionally, researchers have resorted to carefully-planned backcrosses in order to generate new non-astringent Japanese persimmons, and in the case of hybrid persimmons, this first backcross we are working on is similar. It fits the following backcross form:
(American persimmon x non-astringent Japanese persimmon) X non-astringent Japanese persimmon
The consensus among many well-studied persimmon enthusiasts is that this genetic combination could potentially generate a new cold-hardy, non-astringent hybrid cultivar of persimmon suited to parts of the NC-SARE region where non-astringent persimmons are currently very difficult to grow. Replicating this cross as many times as possible will greatly enhance our chances of generating the desired variety or varieties.
But there’s another genetic combination that enthusiasts have been discussing. This is the cross that utilizes the dominant non-astringent trait discovered in China relatively recently. The phenotype (non-astringency of underripe fruit) is similar, but in this case its controlled by a dominant gene. It’s a cross that we have chosen to incorporate into this breeding project only recently, largely because we’ve been able to secure germplasm of the rare Chinese non-astringent persimmon type.
Being a dominant allele, the form of this cross is much simpler. Most likely, we will utilize a hybrid male (75% Diospyros virginiana and 25% Diospyros kaki) crossed to the Chinese non-astringent female that we have been able to obtain. The hope in this cross is that the dominant non-astringent gene from the Chinese line will have a similar effect on the hybrid phenotype as it does on the Chinese D. kaki phenotype.
According to our original grant proposal, we were planning to conduct hundreds of pollinations during the course of this grant. However, due to the difficulty we faced in obtaining the appropriate pollen for our crosses, and due to the resources we were consuming in trying to conduct these crosses, we have adjusted our approach slightly and we have proposed a shift in strategy and a slight broadening of our goals.
Instead of the complicated (and costly) hand pollination trips which even at their best can only yield a limited number of seeds, we have focused on a new approach.
1) First, we are establishing a small research orchard in southern Alabama that is designed specifically for the production of the hybrid seed described in our proposal. It would be ideal if this orchard could be located in the NC-SARE region, but since we are working with cold-sensitive trees, our Missouri climate would certainly kill critical trees in the research planting. For this reason, we must plant a satellite orchard down south with the goal of breeding trees that can be grown in the NC-SARE region. This model has been used before by persimmon breeders such as Cliff England and Jerry Lehman, and it has proven to be a viable breeding strategy. Large number of seed can often be generated through natural insect pollination rather than through expensive and labor-intensive hand pollination. The breeding planting of persimmons is planted on a farm managed by the rare-fruit enthusiast Jamie Whitaker who is willing to cooperate with us in breeding goals. By investing funds in a small research planting, we will be able to potentially generate many more hybrid seeds for the same financial input than we would be by traveling around and tediously performing each cross by hand.
2) Second, we have also invested a portion of our remaining funds into research related to our breeding goals.
We have tested genetic markers that could be used by other persimmon breeders to conduct marker-assisted selection of hybrid persimmon seedlings in the future. Genetic markers are used by plant breeders to identify at the molecular level plants that have specific and desirable phenotypic traits. Breeders can work with labs to screen young seedlings for these markers and discard the seedlings that clearly do not meet breeding objectives. Genetic markers could greatly speed up the breeding and development process with this crop.
With that in mind, I will briefly outline the genetic markers we have sought to optimize. Breeders know that when seedling hybrid persimmon trees are grown out in an orchard, a very high percentage of the trees will end up being males. These trees take up space in the orchard and consume resources, but they never produce fruit. Further, since this project is focused on developing a non-astringent hybrid, we not only want female trees; we also want female trees that carry the non-astringent trait. A review of the literature reveals that genetic markers have been developed for both of these traits.
Blasco et al. (2020) published on some genetic markers that have been proven to reliably identify astringent phenotypes of Asian persimmon (Diospyros kaki) and also to identify specimens that will produce at least some male flowers. The authors did not study whether these markers could be used in Diospyros virginiana or in persimmon hybrids, but if they can be shown to function well in hybrid genomes, the benefit to hybrid persimmon breeding would be very significant. Some of our funds have gone to test these markers and confirm that they give the expected results using known Diospyros kaki phenotypes. We worked with CPS Labs in Pleasant Grove, CA to run these tests.
If the genetic markers which have been used in Diospyros kaki are also applicable in hybrid persimmons, the breeding process could be greatly accelerated.
As additional branch of our research, it should be noted that we completed a test treatment of a female Asian non-astringent persimmon (Diospyros kaki) with silver thiosulfate, the ethylene-blocking compound that has been used in some crops to generate male blossoms on female plants. We treated the tree in early summer (at the time when we expected next years primordial fruit buds to be forming) and also in the spring using a rate that has been proven to work on cucumbers. More research would be needed for conclusive results, but our preliminary tests indicate that silver thiosulfate may not have the same effect on female persimmon trees as it has on female plants of other species. We did not obtain any male blooms on our tree, and we chose to not spend more resources on this part of our research.
This project continues to be an excellent learning experience, and it has also helped to raise awareness of the need for cold-hardy persimmons with high fruit quality. We've acknowledged that this project in its entirety goes beyond the scope of a 2 or 3-year grant, but this phase of the project is allowing us to lay the groundwork for likely future breeding successes.
Cooperators
- - Technical Advisor (Educator and Researcher)
- (Researcher)
- (Researcher)
Research
Our project began in earnest spring of 2023. And although we learned some good things, the activities of the spring caused us to shift our strategy slightly, as has already been described.
Hours were spent searching for sources for pollen, and in the end, we ended up utilizing pollen from the USDA persimmon repository in Davis, CA. A small portable freezer was purchased, and the project coordinator drove through multiple states to conduct hand pollinations at different farms in Missouri, Kentucky, and Indiana during the spring of 2023.
The spring pollinations were done with great care. In order to prevent contamination from foreign pollen, the project coordinator was careful to bag unopened flowers with empty tea bags prior to pollination so that insects would not have access to them. Since persimmon blooms open before dawn, he frequently bagged any candidate blossoms in the evening in preparation for the next day. One or two mornings found him up early in the dark hours of the morning bagging blooms before pollinators had become active.
The pollen was carefully separated from the dried anthers. Using an organza jewelers’ bag and petri dish helped to make very efficient use of the small quantity of pollen we had. Each bloom that had been bagged the night before was carefully inspected to determine which ones had opened and become receptive to pollen. These flowers were then unbagged, stripped of petals, and dipped carefully into the petri dish containing the target pollen. The pollen quickly adhered to the tacky surface of the stigma. Blooms were then promptly re-bagged. By dipping the stigmas directly into the petri dish (and skipping the paint brush method of pollen application), we were able to get many, many crosses from a small quantity of pollen. Each cross was tagged with flagging tape and labeled to document the donor pollen parent. In total, we conducted upwards of 180 controlled crosses amongst the 3 participating farms this past spring. But alas, we were to be (mildly) disappointed. When we returned to these pollinated fruits in the fall, we discovered that most of them were devoid of seeds—they had produced parthenocarpic fruits!
Our project coordinator is employed at Baker Creek Heirloom Seeds where he conducts hand pollination on a variety of crops. Based on this fact, and also based on the information gathered from Cliff England and others, it is believed that our pollination technique was correct. Also, based on communication with Ram Viswanathan who has obtained many seeds from Mikkussu x Diospyros kaki by open polliation, we have significant reason to believe that the cross we were attempting is a compatible cross. This leaves the likelihood that the pollen we received from California was no longer viable by the time we performed the pollinations. And it also caused us to rethink the effectiveness of mail-ordering pollen and traveling around to conduct hand-pollination!
Instead of continuing this approach, we’ve invested in a small research planting in southern Alabama during the second half of our project, as already described. Our hope is that primarily via open-pollination, we can easily obtain hundreds (or thousands) of the seeds that we were laboring so hard to get through hand-pollination. It’s a slower, long-term breeding plan, but with more potential we believe. The genetics of the cross we are attempting have not changed, except that we chose to incorporate the dominant PCNA gene as a second prong to our breeding approach. This prong of the project is possible because we obtained germplasm of the Chinese PCNA persimmon genotype.
As stated, we have completed some silver thiosulfate treatment experiments with the aim of inducing male blooms on otherwise-female trees. Inducing male blooms would have helped us with our challenge of obtaining pollen. Silver-thiosulfate is a compound that has been used to induce male blooms on female trees in other plant species, and we were hopeful that it might do the same in persimmon. As we performed this work, we did reference the University of Wisconsin (n.d.) publication on inducing male blooms in female cucumbers. Our applications were primarily conducted in the late spring, when we supposed that floral primordia for the next growing season were initiating. We sprayed once every 3 or 4 days for a few weeks. While we did not obtain male blossoms on our trial tree, more research and consistent treatment methodologies would be required to make definitive statements about the effect of silver thiosulfate on Diospyros kaki.
Finally in the last stage of our research, we sought to optimize molecular markers that could potentially be used in the breeding of hybrid persimmons. As stated above, Blasco et al. (2020) published on some genetic markers that have been proven to reliably identify astringent phenotypes of Asian persimmon (Diospyros kaki) and also to identify specimens that will produce at least some male flowers. We worked with CPS Labs in Pleasant Grove, CA to optimize these markers. We cannot take credit for discovering the markers; we did not discover them. Our goal was to simply to confirm that they operate as expected using the protocol and equipment of CPS Labs, and using confirmed phenotypes that we provided to CPS labs. Our rational was that once we have confirmed that CPS labs can use these markers effectively on Diospyros kaki, persimmon breeders can take the next step and test the same markers on hybrid plant material.
We tested two sets of markers: one was for the Japanese PCNA trait, and the other was for the male-flowering trait. Through the NPGS Persimmon Repository in California, we obtained four known positive controls and four known negative controls for each of the two traits we were studying. Leaf tissue of these controls were submitted to CPS labs, along with the molecular markers published by Blasco et al. CPS labs ran the molecular markers on our plant material and provided us results, which are discussed below.
Blasco, M., Gil-Munoz, F., Naval, M. del Mar, Badenes, M. L. (2020). Molecular Assisted Selection for Pollination-Constant and Non-Astringent Type without Male Flowers in Spanish Germplasm for Persimmon Breeding. Agronomy2020, 10, 1172; doi:10.3390/agronomy10081172
University of Wisconsis (n.d.). Silver Thiosulfate 6mM Ag(S2O3)2. https://haveylab.horticulture.wisc.edu/wp-content/uploads/sites/66/2016/05/Silver-Thiosulfate-.pdf
In spite of setbacks, this project has already turned out some useful information and has facilitated networking of persimmon enthusiasts! The difficulty we experience in getting seed set prompted us to dig into the compatibility of the crosses we are conducting. We did not obtain material quantities of seed from our initial 2023 pollination efforts, but upon further investigation Ram Viswanathan was able to confirm for us that the Mikkussu F1 hybrid is indeed cross compatible with Diospyros kaki. Previous work has already shown that F1 hybrids between D. virginiana and D. kaki are backcross-compatible with their American parent, so it’s helpful to confirm that these hybrids—and this genotype in particular—can also be compatible with their oriental parent. All of this led us to believe that we were working with sub-optimal pollen in the spring of 2023.
The genetic basis of our project is solid, but the logistics of creating the cross have been challenging. This is why we’ve adjusted our approach in favor of establishing a small planting of persimmons designed specifically for breeding. But as can be imagined with tree-breeding, the far-reaching effects of a project like this extend well beyond the grant timeframe.
As explained above, our preliminary tests of silver thiosulfate treatment on female Diospyros kaki did not yield any male blossoms. Perhaps treatment protocols could be optimized for better results, however such optimization would require more research.
The results of our molecular marker optimization were mixed, but interesting nonetheless. As described above, we tested two sets of molecular markers on known phenotypes obtained from the NPGS Persimmon repository.
We are pleased to report that the molecular markers reported by Blasco et al. for the identification of male-flowering genotypes have functioned as expected in our tests run through CPS Labs. The male-flowering genotypes that we submitted returned the expected result when screened with the appropriate marker. This means that the markers published by Blasco et al. can likely be used by persimmon breeders in the United States to screen for this trait in Diospyros kaki, in cooperation with CPS Labs. Determining whether these markers will also work in hybrids will require more research.
The markers published by Blasco et al. for identification of the PCNA phenotype did not work as expected in our test cycles. We submitted known phenotypes to CPS Labs, however they were unable to establish a clear correlation between the phenotypes we submitted, and the markers that Blasco et al. associated with this trait. The molecular identification of this trait appears to be more complicated, and at this time we cannot recommend plant breeders use these markers until further confirmation of their effectiveness is obtained.
Blasco, M., Gil-Munoz, F., Naval, M. del Mar, Badenes, M. L. (2020). Molecular Assisted Selection for Pollination-Constant and Non-Astringent Type without Male Flowers in Spanish Germplasm for Persimmon Breeding.Agronomy2020, 10, 1172; doi:10.3390/agronomy10081172
Educational & Outreach Activities
Participation summary:
Outreach has occurred via a few different methods.
- Facebook updates posted to the "Persimmon World" Facebook group helped to keep other enthusiasts aware of project developments as we conducted pollinations. The posts helped to increased publicity for our project and generated some discussion surrounding our activities.
- In the fall of 2023, I wrote a project update which was published in the winter 2024 issue of Pomona, the journal of North American Fruit Explorers. Pomona is distributed to around 900 passionate fruit growers, so this article gave many North-American rare fruit enthusiasts a peak into the work we have been doing!
- Our project received some bonus publicity fall 2023 when Hannah Walhout from FoodPrint contacted me to conduct a brief email interview! She intended to run a feature on persimmons, and she wanted to hear a few words from the ranks of persimmon breeders! Here feature on persimmons, which includes a reference to the project we are undertaking, can be viewed here: https://foodprint.org/blog/eat-seasonally-this-winter-with-persimmons/
- Out of necessity we called a zoom meeting on December 3, 2023 to discuss the challenges we were having obtaining hybrid seed! The meeting was attended by a total of 7 persimmon growers who were either directly involved in this project, or interested in the work we were doing. The meeting helped me formulate a theory as to what was going wrong, and it allowed us to discuss project goals with other persimmon enthusiasts.
Learning Outcomes
We have taken multiple new pieces of knowledge away from this project. Although they may seem minor, they are tangible and directly applicable to our work.
We've confirmed that the F1 persimmon interspecific hybrid Mikkusu has the potential to backcross successfully to both of its parents. The project coordinator knew at the onset of this project that F1 hybrdis had the potential to backcross to their American parent, but it was not until my conversation with Ram Viswanathan that I confirmed backcross compatibility to their oriental parent.
We’ve also confirmed that the published molecular markers used in our tests and associated with the male-flowering trait in Diospyros kaki apparently function as expected when applied by CPS Labs to Asian persimmon varieties. This information may be helpful to other persimmon breeders in the United States.
Finally, we’ve learned that Diospyros kaki may not respond to silver thiosulfate applications in the way that other plant species have been known to respond—or that if it does respond, it would require different application rates, different application frequencies, or perhaps treatment at another time of the year.
Our learning outcomes have confirmed which varieties we should planted in our new persimmon breeding planting. Two of the prominent varieties we have planted are Mikkusu and Taishuu. The former is planted as a seed parent, and during the course of this grant we have confirmed that it can serve as a seed parent when pollinated by D. kaki. The latter is planted as a pollen parent. It is a Japanese PCNA variety, so it will fulfill the backcross criteria of our Japanese PCNA breeding strategy, and this variety is also known to produce frequent male flowers. Since we have not achieve chemical induction of male flowers, it is important to have a variety that produces these from a genetic basis.
This project is a long-term project indeed. We believe that our strategy is sound, but it takes much time to implement. Additionally, we believe that folks like ourselves—and other fruit tree breeders—need to be open to unexpected outcomes as they work. This is a subject that has come up in discussions about this project. Whether or not we obtain the fruit we are aiming for—a PCNA cold-hardy hybrid variety—we are still likely to find many amazing cultivars in our breeding work. These varieties may not fully meet our criteria, yet they are still valuable new cultivars that are worthy of further propagation and distribution. Focusing on our goals, while keeping our eyes open for the unexpected winner is an important balance in any breeding program.