Progress report for LNE21-430R
Hemp is a new and profitable crop for cannabinoids. Hemp is a diploid, dioecious species, and female plants are desired, since their inflorescences produce the greatest quantity of cannabinoids. When female plants are pollinated, they set seed, which significantly reduces cannabinoid yield. Despite farmers best efforts to remove male plants from their fields, crops remain susceptible to pollen drifting from neighboring farms or feral plants. One way to eliminate seed set is to develop triploid hemp, which produce gametes with imbalanced chromosomes that are non-viable. There are many horticultural examples of triploids that are sterile including watermelon, banana, citrus, and hops. My lab developed triploid hemp plants and it needs to be demonstrated that triploids will not set seed, maintain high cannabinoid content, and that their horticultural production potential is sufficient to allow farmers to successfully grow the crop. This project will compare triploid plants to standard diploid plants both in the greenhouse and the field. In the greenhouse, controlled pollination studies using genetically male pollen will test plant sterility. Plant performance and cannabinoid production will also be evaluated. In the field, horticultural performance, cannabinoid production and ability to resist pollen under real world conditions will be evaluated. Many northeast farmers have completely lost their hemp crop due to inadvertent pollination by plants found in the wild or on nearby farms. If triploid hemp proves to be seedless, then it will enable farmers to grow high cannabinoid yielding hemp without risk of crop loss due to pollination and seed set. In a recent survey of 28 farmers and extension specialists, all respondents were extremely supportive of university research to evaluate the sterility and growth performance of triploid hemp. As part of the process of fully vetting the sterility of triploid hemp, a research farmer will test plants in their annual variety trials. Farmers will participate in annual on-site demonstration events where they may view research plants, receive preliminary research results, and provide feedback on the project.
Evaluate the sterility and growth performance of triploid hemp plants developed through breeding. Greenhouse and field studies will be conducted to confirm that triploid hemp is infertile or exhibits reduced fertility. Sterile triploids would allow northeast farmers to grow hemp without the risk of seed set reducing cannabidiol (CBD) yield or hemp escaping cultivation to the wild. In addition to sterility, triploid hemp will likely possess greater yield and CBD content. Adoption of seedless triploid hemp would improve sustainability and profitability for northeast hemp farmers.
2022 Greenhouse Performance Study
A greenhouse trial was conducted to evaluate the performance of four triploid female genotypes compared to four diploid female genotypes. The triploid genotypes were: ‘Abacus’ x ‘Kentucky Sunshine; ‘Wife’ x ‘Kentucky Sunshine’; ‘Tangerine’ x ‘Kentucky Sunshine’; and ‘Wife’ x ‘Wife’. The diploid genotypes were: ‘Abacus’; ‘Kentucky Sunshine’; ‘Tangerine’; and ‘Wife’. Plants of each genotype were clonally propagated by stem cuttings for this study. Plants were grown in a climate-controlled greenhouse under 18 hr photoperiod (vegetatively) for 2 weeks, after which the photoperiod was adjusted to 12 hr (to induce flowering) for an additional 6 weeks. Plants were grown in trade number 2 nursery containers filled with a peat moss-based potting mix. Plants were top-dressed with 15N– 3.9P–10K controlled-release fertilizer (Osmocote Plus 5- to 6-month formulation) at 30 g per container. Plants received a soluble fertilizer providing 100 ppm nitrogen (N) at every irrigation, which occurred as needed. Study plants were arranged in a randomized complete block design with 4 replications. The experimental unit was a single potted plant. Data was collected per plant for the following parameters: stem caliper (mm), number of shoots, height (cm), total shoot length (cm); cola width (mm); dry weight (g); floral dry weight (g); and percent CBD and THC.
2022 Field Sterility Study
A field study was conducted to evaluate sterility of five triploid female genotypes compared to four diploid female genotypes. Two male diploid genotypes were included to provide pollen. Therefore, a total of 11 genotypes were included in the planting. The female triploid genotypes were: ‘Purple Star’ x ‘Wilhelmina’; ‘Tsunami’ x ‘Wife’; ‘Purple Star’ x ‘Wife’; ‘Wife x ‘Purple Star’; and ‘Kentucky Sunshine’ x ‘Wife’. The diploid female genotypes were: ‘Tsunami’ x ‘Wilhelmina’; ‘Purple Star’ x ‘Wife’; ‘Abacus’ x ‘Wife’; and ‘Wife’ x ‘Wife’. The male genotypes were: ‘Youngsim 10’ x ‘Youngsim 10’ and ‘Purple Star’ x ‘Youngsim 10’. Plants of each female genotype were propagated from seed. Male genotypes were clonally propagated by cuttings from stock plants. Seeds were germinated on 6 May 2022 and seedlings grown in 307-ml containers in a greenhouse under 18-h photoperiod until transplanting to the field on 31 May 2022. The field planting was located at the University of Connecticut Plant Science Research and Education Facility in Storrs, CT (lat. 41.79544°N, long. -72.22836°W). The field soil was a Paxton and Montauk fine sandy loam with 6.7% organic matter and pH 5.9. Plants were grown in rows with 1.8 m spacing on center between rows and 1.2 m spacing on center within rows. The experimental unit was a single plant and plants were arranged as a RCBD with 10 replications (110 plants). There were two blocks per planting row for a total of 5 rows. Plants were fertilized twice, at time of planting and again on 7 Jul 2022, with 10 g of granular fertilizer (All Purpose 10N–4.4P–8.3K; Greenview, Lebanon, PA) per plant each time. Fertilizer was broadcast around the base of the plant by hand. Plants were irrigated by hand as needed throughout the growing season. Weeding was by rototiller between rows and by hand within rows. For each plant 4 terminal colas (10 in in length) were harvested and the flower dry weight and seed weight will be measured. Using these measures, percent seed weight and seed number will be calculated.
A field study to evaluate sterility of four triploid genotypes and one diploid genotype (control) was installed in June 2021. However, this planting perished because of extreme rainfall (that more than doubled the historic average) and flooding in summer 2021 in Connecticut and other parts of the northeast due to four storms that that came up the East Coast.
A greenhouse trial was conducted to evaluate the sterility of two triploid female genotypes of the strain Wife compared to a diploid female genotype of the strain Wife (control). Plants of each genotype were clonally propagated by stem cuttings for this study. Plants were grown in a climate-controlled greenhouse under 18 hr photoperiod (vegetatively) for 2 weeks, after which the photoperiod was adjusted to 12 hr (to induce flowering) for an additional 9 weeks. Plants were grown in trade number 2 nursery containers filled with a peat moss-based potting mix. Plants were top-dressed with 15N– 3.9P–10K controlled-release fertilizer (Osmocote Plus 5- to 6-month formulation) at 30 g per container. Plants received a soluble fertilizer providing 100 ppm nitrogen (N) at every irrigation, which occurred as needed. At peak flowering plants were hand pollinated (at 12 shoots per plant) with pollen from genetically male cannabis plants. A paint brush was used to deliver a heavy sprinkling of pollen to each shoot. The male plants were cultivated in the same greenhouse section as the female study plants. Since the male plants produced copious quantities of pollen, it is reasonable to assume that in addition to hand pollination, natural pollination occurred to a great extent. Study plants were arranged in a randomized complete block design with 11 replications. The experimental unit was a single potted plant. At the end of the study, plants were harvested and all seed collected per plant. For the triploid genotypes seed was hand counted. For the diploid control plants the total seed per plant was calculated using the weight of a quantified subsample of seed. The number of viable (brown), unviable (white/tan), and irregular shaped unviable seed was counted for every triploid plant and for a subsample of seed per diploid plant. Unviable seeds were hollow inside, which was confirmed by cutting the seeds open. Seed caliper was measured for 10 viable seeds per plant. Percent CBD and THC based on HPLC analysis was determined. Seed germination percent was measured for 15 viable seeds collected per study genotype. Flow cytometry analysis was conducted for 20 seed collected from triploid plants to determine their ploidy.
2022 Greenhouse Performance Study
Triploids and diploids performed similarly for most measured parameters however, statistical analysis has not yet been completed for this study. Triploids appears to have slightly greater stem caliper and terminal cola width. The triploid ‘Wife’ x ‘Wife’ had less dry weight and floral dry weight compared to the other triploid and diploid genotypes, which may be due to its slightly inbred status. Future studies will compare triploid and diploid hybrid genotypes developed from the same parental cultivar crosses. If triploid genotypes continue to demonstrate reduced fertility and similar performance to diploids, then they could be a desirable option for growing areas with high pollen drift and risk of seed set, which reduces yield.
2022 Field Sterility Study
The field study was a very successful experiment. Even rainfall in spring facilitated plant establishment. There were no unusual weather events that compromised the study as occurred in 2021. Data collection and analysis is in process for the 2022 field study.
2022 Research Farmer Trial
Two triploid genotypes were provided as seed to Heather Darby, Extension Professor, University of Vermont (UVM) for includion in hemp research trials at UVM. Dr. Darby is named in this project as a research farmer and advisory group member. The triploid genotypes provided were reported to be highly uniform in performance, but the data is still being analyzed.
2021 Greenhouse Sterility Study
Diploid plants produced 3000 seed per plant and triploid plants produced 100 seed per plant. Of the seeds produced, 89% were viable for diploids and only 9% were viable for triploids. Of the seeds produced by triploid genotypes, 52% to 60% were irregular in shape and unviable, while only 2% were irregular for the diploid control. Triploid seed germinated at only 13% to 53% and out of 20 seed tested by flow cytometry, 19 were diploid and one was triploid. All seed tested from diploids were diploid. We can conclude that the triploid genotypes evaluated had significantly reduced fertility and are highly resistant to pollen, since they produced only 0.3% viable seed compared to the diploid control.
Education & Outreach Activities and Participation Summary
I provided a presentation about this project for the 2022 Industrial Hemp Conference hosted by the University of Vermont on March 15, 2022. A meeting to share information about the project with stakeholders is in planning for March 2023.
On December 9, 2021, a virtual field day was conducted, which included two separate educational presentations. After each presentation there was a lengthy and dynamic discussion with attendees about the project and results of the research to date. There were 20 attendees of which 15 were farmers or in industry, and 5 were extension educators. The advisory group for this project, consisting of 3 growers and 3 extension educators, attended this event and participated in the discussions.
I provided a presentation about this project for the 2nd Annual Hemp Science & Technology Virtual Symposium, which aired on December 1, 2021. There were 360 registered for the event and 99 attended the session live. The session was made available to registrants to watch for one year.
After the virtual field day on December 9, 2021, an online survey was disseminated to attendees to gauge learning outcomes. The survey was anonymous and did not distinguish farmers from extension educators. There were 20 attendees and 9 of them responded to the survey. The key areas in which farmers reported changes in knowledge were: (1) what is triploid hemp, (2) how is triploid hemp seed produced, and (3) triploid hemp sterility in the greenhouse. For each key area, respondents were asked to rate their level of understanding before and after the meeting on a scale of 0 to 5 with 0 = no understanding and 5 = great understanding. Respondents' knowledge of key area 1 increased from 3.6 before the event to 4.9 after the event. For key areas 2 and 3, respondents' knowledge increased form 3.0 before the event to 4.8 after the event.