The total seed weight was recorded, and we calculated the total seed production by weighing and averaging five groups of 100 seeds. A t-test was used to compare the differences in inflorescence growth and seed production for treatments using massive, irradiated pollen or non-irradiated pollen against the results from open pollination. This analysis was conducted using the R software. Lastly, seeds from the massive pollination experiment were used for a certation experiment. Two hundred seeds were randomly selected from female plants within the same treatment and were then planted in the greenhouse under the previously described conditions. Plant sex was recorded after anthesis. Finally, the sex ratio observed in both the irradiated massive pollination group and the non-irradiated massive pollination group was subjected to a statistical analysis for comparison against the sex ratio found in the open pollination group using a chi-square test.Two powder types were effective in reducing the seed set in Amaranthus palmeri as shown by our dose-response analysis . Data from the two tested powder types were pooled because there was no difference between the full model and the reduced model , as shown in Appendix 1. This suggests four parameters can be fixed across curves of talc powder and wheat powder without significantly reducing the goodness of fit.
The seed set in both treatments was lower than the seed set from open pollination . The effective pollen share in the mixture was 4.81 , vertical grow racks producing a seed set halfway between the lower limit and upper limit. The ED50,the ratio reducing seed set by 50%, was not estimable because the lower limit of the model was greater than half the maximum response . To minimize seed set and conserve irradiated pollen, a mixture ratio of 25%:75% is recommended; this is the smallest ratio that yielded seed set close to the lower limit while minimizing the amount of irradiated pollen required . This ratio can effectively reduce seed set in A. palmeri while conserving the limited resources of irradiated pollen. The lower seed set observed with pure powder application compared to open pollination can be attributed to the physical barrier created by the powder, which covers the stigma and prevents non-irradiated pollen from fertilizing the ovule and producing seeds . With an increase in the proportion of irradiated pollen in the mixture, there is a decrease in the seed set. This is due to the fact that while the irradiated pollen can germinate on the stigma and produce a pollen tube, it is incapable of fertilizing the egg cell, thereby failing to produce any seeds . When selecting an optimal diluent for pollen application, it is crucial to take into account factors such as non-toxicity and preventing any disruption to pollen-stigma interactions. Artificial supplementary application of pollen using non-toxic diluents like wheat flour and talc powder, has shown positive results in various plants. For instance, in raspberry , talc-diluted pollen is employed to enhance fruit production . Similarly, in Cannabis sativa, the use of cryop reserved pollen mixed with wheat flour yields seeds of comparable number, size, and morphology to those produced with untreated firesh pollen .
However, when wheat flour is combined with irradiated pollen of Palmer amaranth, it tends to clump and degrade pollen flow more than talc powder.The combined effect of sterile pollen application starting time, application firequency and application interval had a statistically significant impact on seed production . Initiating application 7 days after anthesis consistently resulted in the lowest calculated seed production per plant, reduced by about 50% relative to the open pollination as shown in Figure 3. Estimated seed production from single applications of sterile pollen at 14 or 21 days after anthesis did not show a significant difference compared to open pollination . Although not statistically significant, a trend was observed where increasing the number of applications or decreasing the interval between them tended to reduce seed production per plant. Based on these findings, the optimal application strategy for the sterile pollen technique is to begin at 7 days after anthesis and apply three times at 7-day intervals. Due to the indeterminate nature of Palmer amaranth inflorescences , flowers varied in age at the time we applied irradiated and sterile pollen, resulting in within individual variation. Additionally, not all plants within the population flower simultaneously, leading to between-individual variation where a portion of the population may not be exposed to sterile pollen. Those flowering variations among female flowers present challenges in terms of the timing for the application of the sterile pollen technique. Furthermore, the initiation of flowering occurred earlier in males than females under both water stress and control conditions . When applying a pollen-powder mixture to female flowers, our aim is to cover as many stigmas as possible while minimizing the influence of naturally occurring pollen on seed development, considering the earlier flowering of males compared to females.
Through our experiments, the optimal application strategy we identified above allows us to achieve minimal seed production while using a reduced amount of irradiated pollen. The time of flower opening in Palmer amaranth marks the onset of a period in which pollen will be released from male flowers and when pollination, fertilization, and seed production occur in female flowers. Therefore, the timing of flower opening in females plays a significant role in determining the appropriate initiation and interval for application of the sterile pollen technique. In Palmer amaranth, flowers on the same plant have a continuous opening sequence, with varying opening times among the flowers. Flower opening behavior in Palmer amaranth can be influenced by factors such as the time of day and the position of the flower within the inflorescence . Based on our observations, it appears that flowers situated in the middle lower part of the inflorescence tend to open first. In many species, including Palmer amaranth, flower opening occurs in the morning, correlated with an increase in temperature and light intensity, and with a decrease in ambient humidity . The majority of plant species, as indicated by studies utilizing the GloPL Dataset , the Konstanz Breeding System Dataset , and the Stellenbosch Breeding System Dataset , demonstrate pollination firequency plays a critical role in determining seed production . The reduced seed-set rate in early-flowering plants was associated with pollen limitation, asobserved in species like Peucedanum multivittatum and Rhododendron aureum . While the failure of pollen tubes to enter ovules is a common cause of reduced seed production, it is not the sole factor contributing to low fertility, as post fertilization ovule abortion has been observed to decrease fertility in alfalfa . Additionally, reports indicate that embryo abortion can also lead to reduced seed production in various plant species, including red clover and garden pea , as well as in plant families other than the Fabaceae .Regarding massive pollination with non-irradiated and fertile pollen in Palmer amaranth, there were no statistically significant differences in plant height, branch number, inflorescence length, dry weight, seed weight, and seed production per plant compared to open pollination . This indicates our assumed tradeoff between inflorescence outgrowth and fertilization rate is not significant. These results suggest that when plants receive a substantial amount of sterile pollen, the presumed trade-off between inflorescence growth and fertilization rate is not significant either. The activity and development of apical and lateral buds, as well as fruits, are controlled by light, temperature, hormone, carbohydrate, and nutrient signaling . These signals enable communication between the shoot apex and lateral sinks , ensuring the plant’s architecture and reproductive capacity align with available resources . In annual plants, growing tables the suppression of inflorescence growth due to fruit load typically occurs at the late stage of inflorescence development, referred to as the end of the flowering transition . For instance, in Arabidopsis , during this phase, the inhibition of inflorescence shoot growth by fruit load is regulated by auxin and carbohydrate signaling . Our findings suggest that the rate of fertilization has a minor effect on inflorescence outgrowth in Palmer amaranth, likely because the development of fruit in Palmer amaranth, which is a thin membranous structure known as an utricle, has relatively low costs. Pollen limitation has two aspects: quality limitation and quantity limitation. Quality limitation refers to the reduced effectiveness of pollination due to the inferior quality of pollen. In Palmer amaranth, irradiated pollen under 300 Gy doses is genetically inactive and cannot fertilize the egg cell to form seeds . In addition, much literature on inbreeding depression has shown that pollen quality effects associated with both self fertilization and mating between related plants can also reduce seed production .
This reduction is likely because embryos homozygous for deleterious alleles die during development. On the other hand, traditional pollen limitation is typically associated with plants receiving an insufficient quantity of pollen grains to fertilize all their ovules . Extensive reviews show that supplemental pollination often increases , and rarely decreases , seed or fruit production. Regarding sex ratio in offspring after massive pollination in Palmer amaranth, results from massive pollination with irradiated and non-irradiated consistently showed the sex ratio in the progeny population is female dominant as predicted by certation theory . It is 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 small proportion of ovules available to male-determining gametes as was found in Silene alba and Rumexspecies . Several other mechanisms have been proposed to account for female bias. In species with sex chromosomes where males are heterogametic, Y-chromosome degeneration may lead to female-biased populations . This is due to sex viability differences and sex-chromosomal genotype performance during pollination and fertilization. Studies on Rumex nivalis, a species with heteromorphic sex chromosomes , show that both certation and gender-based mortality contribute to female biased sex ratios . Research using sex-specific markers across different life stages revealed that female bias starts in pollen and intensifies from seeds to flowering. Environmental factors, like proximity of females to males, affect these ratios ; females nearer to males capture more pollen, resulting in more female biased ratios. Experiments confirm that higher pollen loads intensify this bias , supporting Correns’ certation hypothesis that larger pollen loads increase gametophytic competition, favoring fertilization by female-determining pollen tubes. In conclusion, we investigated how to improve the efficiency of the sterile pollen technique for reducing seed production in A. palmeri under greenhouse conditions. The optimal formulation is to utilize a mixture of 25% irradiated pollen to 75% talc powder by volume, which enhances pollen distribution by improving flow and uniformity. Furthermore, the efficiency of the sterile pollen technique is affected by variations in the timing of female flower opening and interference from naturally-occurring pollen. These factors make it challenging to further enhance the technique’s efficiency. However, through our investigations, we identified the optimal sterile pollen application strategy: initiating application 7 days after anthesis and repeating it three times at 7-day intervals. This strategy allows us to achieve reduction in seed production while also minimizing the amount of irradiated pollen required. Lastly, we found massive pollination of irradiated pollen or non-irradiated pollen did not have an effect oninflorescence growth, but it did affect sex ratio in the progeny population, resulting in slightly female-biased progeny as predicted by certation theory.The transition to agriculture in humans ~10,000 y ago is often cited as the key innovation that led to large and complex human civilizations . Yet, insects have practiced farming on a much longer timescale. For example, fungus-growing ants have farmed specific “cultivar” fungi for ~50 My . Fungus-growing ants include over 250 known species, the most complex of which, Atta leaf-cutting ants, create colonies that rival large cities in population size . Fungus-growing ants obligately rely on their cultivar fungus to digest otherwise inaccessible plant nutrients. In underground fungus gardens, the cultivar fungus breaks down recalcitrant organic material provided by the ants and in return produces specialized structures that the ants consume, making the cultivar fungus a valuable resource for the ants .