A study of Arabidopsis found a 50% reduction in seed set when pollen was irradiated at 400 Gy and less than 10% seed set with pollen irradiated at 800 Gy . The effects of different irradiation doses on seed set varied depending on the sequence of pollination treatments applied . The seed set from pollination with irradiated pollen after non-irradiated pollen with bagging was higher than pollination with non-irradiated pollen after irradiated pollen with bagging . This finding shows that pollination with irradiated pollen is most effective in reducing seed set when it is applied before any fertile and fully functional pollen reaching the stigma. Furthermore, applying irradiated pollen before non-irradiated pollen across all irradiation doses significantly reduced seed set compared to open pollination , even though it may not completely disrupt double fertilization under some doses. Irradiated but non-viable pollen may act as a physical barrier covering the stigma and preventing viable non-irradiated pollen from fertilizing ovules and producing seeds. The additional treatments in 2021 which combined different sequences of pollinating with irradiated pollen and open pollination produced lower seed set than open pollination . However, vertical grow room design the timing of the pollination with irradiated pollen is critical as it can greatly affect the reduction of the seed set.
Seed set on inflorescences that received open pollination after irradiated pollen was lower than that of the inflorescences pollinated with irradiated pollen after open pollination for all irradiation doses . This suggests that irradiated pollen has a preventive effect on subsequently arriving, naturally occurring pollen when it has initial access to the stigma and that it successfully disrupts fertilization twice in two synergid-celled plants.Introducing irradiated pollen for pollination prior to the arrival of naturally occurring pollen on the stigmas of female plants can potentially create a temporal advantage, enhancing the efficiency of SPT. However, determining the optimal timing for dispersing irradiated pollen remains challenging. Due to the indeterminate nature of A. palmeri inflorescences , flowers will be at various ages when irradiated pollen is applied . Furthermore, not all plants will flower simultaneously; consequently, a proportion of plants in a population will not have flowers exposed to sterile irradiated pollen when it is applied . Because of within- and between-individual variations in the timing of flowering, multiple bouts of pollination with irradiated pollen are necessary to achieve desirable level of reduction in seed output. Even with multiple applications, there remain the questions about how many applications should be made and what time intervals.
To address these questions a thorough analysis of the flowering phenology of A. palmeri is required. Our previous study has shown that male plants of A. palmeri enter the flowering stage earlier than females but anthesis happens earlier in females than males . The earlier anthesis in female plants of A. palmeri can make the SPT more effective. Early pollination with irradiated pollen, such as at the first occurrence of female anthesis, can block or interfere with these receptive stigmas and prevent fertilization with fertile pollen from naturally occurring males.Under field conditions, pollen viability was reduced within 30 min of anthesis and approached non-viability at 240 min following anthesis under field conditions . As non-irradiated pollen moves from male plants to female plants, its viability may rapidly decrease due to long inter-plant distances and heat stress experienced during summer. The sensitivity of pollen to temperatures has been established through studies in tomato , maize , and soybean . High temperatures can lead to increased respiration and metabolism, water loss, and a rapid decrease in vitality, while low temperatures can slow down metabolism and respiration, reducing the rate of pollen viability loss . Even if the pollen lands on a compatible stigma, it may have reduced viability and vigor and be unable to germinate. Thus, developing a mechanism to preserve irradiated pollen viability and implement the sterile pollen technique over short distances to the target with high efficiency will be crucial for success of SPT.
In conclusion, we tested the possibility of using sterile irradiated pollen as means of disrupting seed production in A. palmeri in a similar way to the Insect Sterile Technique . Results demonstrated that an irradiation dose of 300 Gy seems to be the most effective in reducing seed set in A. palmeri. Furthermore, we observed that the greatest reduction in seed set was achieved when irradiated pollen was introduced to the stigma through artificial pollination prior to open pollination. It appears that irradiated pollen exerts a preventive effect on naturally occurring pollen that arrives later. Although the focus of this project was a single weed species, the method can be extended to the control of seed production for multiple weed species simultaneously where sterile pollen from multiple weed species can be mixed and released in a single application. The sterile pollen technique could be particularly helpful for managing herbicide-resistant weeds which have withstood in-season control and have reached a reproductive stage.Weeds can cause considerable damage to agricultural production . While herbicides and tillage have been the primary tools used in controlling weeds in modern agricultural systems, over-reliance on these tactics has resulted in negative impacts on both the environment and crop productivity . The breeding system of a weed species plays a critical role in its ability to invade and establish in new environments . However, research on reducing seed production in weedy species through exploitation of breeding system limitations has received little attention. Palmer amaranth , a dioecious weed species with male and female reproductive organs on separate plants, is a highly aggressive and invasive weed species. It is ranked as the worst weed in US corn fields in a survey conducted by the Weed Science Society of America and is now a serious threat to agricultural production systems in over 40 countries . Dioecy enforces outcrossing, which minimizes inbreeding depression and increases genetic variation within populations through pollen dispersal by male plants . Studies have shown that the long-distance dispersal of pollen in Amaranthus palmeri has facilitated the transfer of herbicide resistance genes to susceptible female plants, resulting in offspring with acquired herbicide resistance . However, successful fertilization in dioecious species relies on proximity and synchronization of male and female flowers. These limitations present an opportunity for the development of novel management strategies for A. palmeri. One possible strategy for controlling dioecious weed species is to induce pollen sterility, whereby a plant becomes incapable of producing viable seeds. At certain doses, irradiated pollen retains physiological viability and can germinate on the stigma to produce a pollen tube but is genetically inactive and cannot fertilize the egg cell to form seeds . The effectiveness of the sterile pollen technique in managing Palmer amaranth has been shown experimentally, as results showed 300 Gy is the most effective irradiation dose to balance induced sterility and mating competitiveness . This dosage significantly reduced seed set by at least 50% when compared to open pollination. This method is similar to the sterile insect technique, clone rack which utilizes eco-friendly and species-specific methods to decrease mosquito populations by releasing a large number of sterile male mosquitoes into the environment to mate with females . This study aimed to explore how to increase the effectiveness of sterile pollen application to reduce seed production in Palmer amaranth. Under field conditions, it can be challenging to uniformly apply small volumes of pollen to stigmas.
Dry particulates used as pollen diluents can improve the flow and uniformity of pollen distribution . Thus, the first objective was to determine an ideal dry diluent at the most effective mixed ratio for large scale application. Furthermore, due to the indeterminate nature of Palmer amaranth inflorescences , flowers are likely to be at various ages when irradiated and sterile pollen is applied . In addition, not all plants will flower simultaneously, meaning that a proportion of the population will not be exposed to sterile pollen with a single pollination event . These within individual and between-individual variations in flowering pattern make it challenging to maximize the efficiency of this technique. Therefore, our second objective was to identify the optimal combination of starting time, firequency, and number of sterile pollen applications to minimize seed production of Palmer amaranth. The aim is to apply the sterile pollen technique with the ideal timing, firequency, and number of applications to result in the minimum possible seed production. To better understand the ecological implications of sterile pollen technique, we further investigated the effects of massive sterile pollen application. Maximalre productive success depends on the timing of flowering and on balancing the number of seeds produced with resources allocated to individual seeds . The production of large inflorescences itself can be costly as it may deplete resources that could otherwise be allocated to other plant organs . A scientific unknown is whether there exists a trade-off between inflorescence growth and fertilization rate. If a trade-off exists, massive pollination with sterile pollen may have the additional benefit of reducing inflorescence growth. Consequently, our next objective was to investigate the effect of massive pollination on inflorescence growth and seed output. We hypothesized that inflorescence growth and total seed output should be reduced by artificial massive pollination in Palmer amaranth. Massive pollination may also influence sex ratio due to certation, a prezygotic mechanism of sex determination hypothesized to originate from the competition between a female-determining gamete and a male-determining gamete . As a result, when a heavy load of pollen is dusted on female flowers, the female-determining gamete would rapidly reach and sire more than half the ovules and leave a smaller proportion of ovules available to male-determining gametes as was found in Rumex species . We hypothesized that massive pollination would change the sex ratio in the progeny population resulting in a female-biased progeny as predicted by certation theory.Seeds of Amaranthus palmeri were planted into 3-L pots filled with UC Davis potting medium containing a ratio of 1 sand:1 redwood sawdust:1 peat in a greenhouse set at 24/32 C 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 2-true leaves with drip irrigation for two minutes and twice per day. Seedlings were thinned to one plant per pot . Once plants reached the flowering stage, 100 male and 100 female plants were grown in the same greenhouses to simulate mixed population conditions in the field. Pollen was collected by gently tapping or shaking the male inflorescence, causing the pollen grains to be released onto aluminum foil placed beneath the inflorescence. The collected pollen was then sieved through a 250-mm mesh opening to remove large floral materials and stored in polyvinyl containers at 95 to 100% relative humidity until needed. To sterilize the pollen, firesh and mature pollen was placed in Petri dishes with parafilm and irradiated with gamma-rays from Cesium-137 at the UC Davis Center for Health & the Environment . The irradiation was delivered at a dosage of 300 Grey , which was determined to be the most effective irradiation dosage in previous tests . Two types of diluent powders, wheat flour and talc powder, were evaluated for their effectiveness in diluting the sterilized pollen. Previous work has shown that these compounds do not change the biological properties of pollen and can be mixed and applied with pollen uniformly . The sterilized pollen was mixed with each powder at six v/v ratios of 0/100 , 5/95, 10/90, 25/75, 50/50, or 100/0 . The pollen-powder mixture was then uniformly applied to the inflorescence, each about 18 cm long, of receptive female plants using the same amount of pollen-diluent mixture. Each treatment had three replications. For each replicate, five 1 cm sections of treated inflorescences were dissected and analyzed to determine the number of flowers, normal full seeds and abnormal seeds . Seed set was calculated by using the number of viable seeds divided by the number of flowers and expressed as percentage. The optimal pollen-powder mixture was determined based on the formulation that resulted in the lowest seed set.Once seeds reached maturity, we assessed various aspects of inflorescence outgrowth, including plant height, the number of branches per plant, length of the main inflorescence, total length of all inflorescences, and dry weight. Following these measurements, the plants were crushed and sieved to collect the seeds.