There are several methods to make pollen grains functionally deficient and thereby reduce seed set . The most commonly used is ionizing irradiation with X-rays or gamma rays due to their ease of use, effective penetration, consistent results, and minimal disposal issues . Irradiated pollen grains can be physiologically alive, depending on the irradiation dosage, but are infertile. Irradiated grains can germinate on the stigma and even produce pollen tubes but cannot fertilize egg cells to produce embryos . Further, when sterile pollen grains are deposited on a stigma through artificial pollination, they can interfere with fertile pollen in the process of fertilization and disrupt seed production, as has been shown in apple , pear , citrus , cacao , and melon . The use of sterile pollen to reduce weed seed production is similar to the insect sterile technique , an environmentally-friendly and biologically-based method for controlling insect pest. This technique involves sterilizing male insects by irradiation and subsequently releasing the sterile males to mate with wild females , resulting in infertile eggs and reduced insect pest population sizes. Pollinating the female plants of Palmer amaranth with sterile pollen resulted in 40% reduction in the number of newly formed seeds . However, the sterile pollen technique has been rarely used as a weed control technique but could potentially be effective on dioecious weedy species because female and male flowers are in separate plants and pollen grains can be collected from male plants, sterilized and then released on female plants. The summer annual dioecious weed A. palmeri is one of the most devastating weeds in the US. It was ranked as the worst weed in US corn fields in a survey by the Weed Science Society of America . Furthermore, industrial drying rack it has evolved resistance to nine herbicide classes used and is able to produce up to one million seeds per plant .
This weed is a particularly suitable candidate for exploration of the sterilepollen technique for weed control. Being a dioecious species with separate male and female plants, it relies on cross-pollination for successful seed production. This makes it feasible to collect pollen grains from male plants, sterilize them, and subsequently release them onto female plants. The primary goal of this research was to examine the effectiveness of sterile pollen technique, SPT, as a means of disrupting seed production in A. palmeri. To this end, it was necessary to determine the optimal irradiation dose for pollen sterilization as excessively high doses may kill the pollen entirely, thereby eliminating their preventative effects on fertile pollen, while low doses may allow the treated pollen to maintain its fertility. Accordingly, a broad range of irradiance doses was tested in combination with an extensive array of artificial pollination treatments to fully explore the potential effects of the SPT on seed production in A. palmeri. Our hypothesis is that pollinating with sterile pollen, irradiated at an optimal dose, could reduce seed production in this weed. Furthermore, we speculated that the maximum reduction in seed output could be achieved when pollination with sterile pollen precedes open pollination.Seeds of A. palmeri collected from Kansas were planted in May 2020 into 3-L pots filled with UC Davis potting medium containing in a greenhouse set at a 24/32 ℃ night/day temperature regime and extended photoperiod . Fertilizers were applied as 80 ml of a general-purpose fertilizer solution weekly at 350 ppm N starting from the 2-true leaf plant stage with drip irrigation applied at 65 mL/min for two minutes twice per day. Once plants reached the flowering stage, 50 male and 50 female plants were isolated and grown in separate greenhouses .
Pollen collections were made from male plants by gentle tapping or shaking of the inflorescence. Pollen grains from all male plants were pooled and released onto aluminum foil held beneath the inflorescence. The collected pollen was then sieved through 250-mm mesh to remove large floral materials. Pollen was placed in Petri dishes covered with parafilm and then irradiated immediately with gamma rays from Cesium-137 at six dosages of 0 , 100, 200, 300, 400 and 500 Gray at the UC Davis Center for Health & the Environment . Irradiated and untreated pollen were immediately used for pollen viability tests and hand -pollination experiments as described below.Pollen viability was assessed immediately after irradiation by using a test solution consisting of a 1% concentration of the substrate 2,5-diphenyl tetrazolium bromide in 5% sucrose. The MTT assay measures cellular metabolic activity as an indicator of cell viability and cytotoxicity . In this assay, viable pollen appears dark violet and non-viable pollen did not stain at all . Viability of 100 pollen grains for each dose at each irradiation dose was assessed by analyzing the brightness of the resulting tetrazolium stain using a digital camera and ImageJ software Version 1.46r . Grey values were used to indicate the brightness of a pixel. Because the range for grey values is 0- 255, grey value percentages were calculated by dividing the recorded grey values by 255 and multiplying by 100. Higher grey value percentages indicated lower pollen viability. Theeffects of irradiation dosage on grey value percentages were analyzed using ANOVA with Dunnett’s test.In the 2020 experiment, six lateral inflorescences of similar size from each female plant were selected to receive the following treatments: hand pollination with 1) non-irradiated pollen only, 2) irradiated pollen only, 3) non-irradiated pollen followed by irradiated pollen, 4) irradiated pollen followed by non-irradiated pollen, 5) no pollination, or 6) open pollination .
Each inflorescence was meticulously dusted with 1 ml of pollen, ensuring even and gentle distribution using a paintbrush. Thereafter, the inflorescence was immediately enclosed in a paper bag with the exception of inflorescences receiving the open pollination treatment. Hand pollination was conducted through a one-time application. About 6 weeks after pollination, inflorescences were harvested. Flower and seed numbers were measured on the above mentioned six inflorescences for each of five plants at each irradiation dosage. For each replicate, six 1-cm sections of plant branches were dissected and measured for flower and seed numbers. Two categories of seeds were identified and recorded: abnormal seeds and normal full seeds. Seed set was calculated by using the number of viable and full seeds divided by the number of flowers and expressed as percentage. To more closely simulate field conditions, this experiment was repeated in 2021 with three additional treatments: 1) hand pollination with irradiated pollen followed by open pollination , 2) open pollination for two weeks followed by hand pollination with irradiated pollen , and 3) open pollination for two weeks followed by hand pollination with irradiated pollen with no bagging, i.e., open pollination. These treatments began simultaneously when nine lateral inflorescences of similar size from five female plants reached full anthesis. As with 2020 experiment, the hand pollination was performed as a single, one-time application. Data from each year of study was analyzed separately because hand -pollination treatments differed slightly across the two years of experiment. Prior to ANOVA, in order to reduce heteroscedasticity of the residuals, seed set values were transformed using a square root transformation. Two factors, irradiation dose and pollination treatment, were firstly combined into a single factor and a one-way ANOVA was performed on seed set measurements by using aov functions in R . To better explore the interaction between the two factors, drying rack for cannabis the non-crossed treatments were removed to obtain a full-factorial design for a two-way ANOVA. The two-way factorial ANOVA was conducted using lm function followed by slicing each level of irradiation doses, with SLICE function in sasLM package , to perform the F-test for the effect of hand-pollination on seed set at each level of irradiation dose. Lastly, seed set data was back-transformed using the re_grid function in the emmeans package and confidence intervals were constructed using confint function in R .Pollen irradiated at the lowest dose exhibited the lowest grey value percentage while pollen irradiated at 500 Gy had the highest grey value .
The mean grey value of pollen irradiated at 500 Gy was significantly different from the other doses, which indicates this highest irradiation dose reduced pollen viability to a greater degree than the other doses . Under this high irradiation dose, pollen will likely be unable to produce a pollen tube and disrupt the process of double fertilization since it has lost its viability as determined by MTT staining.The viability of pollen is affected by factors such as genotype, pollen maturity, growth media composition , and environmental variables such as air temperature and humidity . Gamma ray irradiation can decrease water content in pollen, reducing the ability to transfer carbohydrate reserves, leading to changes in the cytoplasmic water, abnormal meiosis, irregular gamete formation, and ultimately decreased viability, which has been supported in studies on apples , pumpkins and winter squash , and citrus . The effect of radiation dose on pollen viability is species dependent. In some species irradiation effect is limited. For example, melon pollen can tolerate gammairradiation doses up to 3,600 Gy whereas in winter squash a 300 Gy dose reduced pollen viability by almost 80% . We found significantly reduced viability of A. palmeri pollen irradiated at 500 Gy compared with non-irradiated pollen, which indicates that seed production in this weed is sensitive to ionizing irradiation. However, our goal in the practice of irradiation is not the complete loss of viability. For effective implementation of SPT, it is essential to have semi-functional pollen that can outcompete and displace wild pollen while remaining incapable of fertilizing the ovule. Understanding the sexual reproduction process is important to gain insight about how to increase the competitiveness of irradiated pollen. When the pollen tube enters the female reproductive tissue, intensive communication occurs between the pollen tube and one synergid cell. After the contact of pollen tube and synergid cell, the receptive synergid degenerates . Following release of the two sperm cells from the pollen tube, they interact and fuse with the egg cell nucleus and the central cell nuclei, forming the major seed components embryo and endosperm, respectively. Any of the steps involved in double fertilization or a subsequent event could trigger the block of attraction of multiple pollen tubes to a single ovule . If fusion fails, one synergid can persist and continue to attract multiple pollen tubes until fertile sperm are delivered or the synergid senesces. The recovery of fertilization is limited to the second pollen tube, indicating that there is no third chance for fertilization in two synergid celled plants. The optimal irradiation dosage to sterilize pollen should maintain the function of the vegetative cell but induce failure in cell fusion. If the irradiated pollen can disrupt fertilization twice, there is no third chance for this ovule to produce a seed , thereby reducing overall seed production.Both in 2020 and 2021, the combined effect of irradiation dose and application treatment had a significantly different effect on seed set . Additionally, the effect of different irradiation doses, application treatments, and their interaction on seed set was significant in both years . Female plants that received no pollination did not produce seed in either year so this treatment will not be discussed further in the results. However, in contrast to this observation, a study has proposed apomixis as a potential mechanism for seed production in isolated female plants . In both years, regardless of the irradiation dose, all pollination treatments involving irradiated pollen consistently resulted in lower seed sets compared to open pollination . The mean seed set obtained from pollination treatments involving irradiated pollen never exceeded 35% and decreased to nearly 0% when using only irradiated pollen at doses of 300, 400, and 500 Gy . Seed set decreased with increasing irradiation dose up to 300 Gy in all pollination treatments with irradiated pollen. However, there was an increase in seed set beyond the 300 Gy dose when pollination with irradiated pollen followed by hand pollination with non-irradiated pollen. This suggests that pollen irradiated at 100 Gy and 200 Gy maintained some ability to fertilize egg cells and produce seeds, while pollen irradiated at the higher doses of 300 Gy to 500 Gy were functionally deficient and unable to complete sexual reproduction. The 300 Gy dose seems to be the optimal dose for disrupting seed production inA. palmeri as it produced the lowest seed set when interfering with non-irradiated pollen . Irradiation of pollen has also decreased seed production and seed set in other species.