Sonic-induced cellular vibrations disrupt poricidal cone trichomes to boost tomato floral pollination

doi:10.21203/rs.3.rs-3198339/v1

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Sonic-induced cellular vibrations disrupt poricidal cone trichomes to boost tomato floral pollination

https://doi.org/10.21203/rs.3.rs-3198339/v1

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Artificial pollination methods rely primarily on air-jets, vibrating wands and trellis tapping which can spread pathogens. This problem can be addressed by non-contact sonic techniques that vibrate cells via sound waves yet how frequency and intensity affect pollination, seed set and fruit size remain unclear. Our study systematic characterizes frequency-dependent vibration events on greenhouse-grown tomato flowers comparing them with contact-induced oscillations from a vibrating wand and a mechanical shaker arm. Sonic vibrations in the frequency range from 50 to 10,000 Hz increased pollination, fruit size, weight, and seed set in Sweetelle and Endeavour commercial varieties. Scanning electron microscopy revealed sonication loosened the trichomes joining the poricidal cone lobes that encase the anthers filled with pollen. Ultra-sonic frequencies enlarged fruit size, whereas seed set remained constant thereby challenging the floral cells power-law rheological characteristics in different frequency scales. Our bioacoustics non-contact precision technology can be used to boost tomato floral self-pollination.

Teaser

Leveraging bioacoustics from bees and bats to boost precision pollination of tomato flowers and fruit size in commercial varieties

Biological sciences/Plant sciences/Plant reproduction/Pollination

Biological sciences/Plant sciences/Plant cell biology

Biological sciences/Biological techniques/Biophysical methods

Biological sciences/Biological techniques/Microscopy

Biological sciences/Plant sciences/Plant reproduction/Fruiting

The global area cultivated for pollinator-dependent crops has rapidly increased relative to pollinator-independent crop regions ¹. Agroecosystem activity has contributed to a loss in biodiversity and impacted upon natural pollinators ². The European honeybee (Apis mellifera L.) and northern hemisphere bumblebee (Bombus spp.) are very effective crop pollinators and their services necessitates periodic relocation of hives to dependent crops ^3,4. In more recent times, the expansion of horticultural crops like almonds (Prunus dulcis), blueberries (Vaccinium spp.), and tomatoes (Solanum lycopersicum) requires increased pollinator movements ^5,6. Biosecurity laws in certain countries like Australia prohibit the importation of bumblebee pollinators due to a devastating threat to ecological habitats ⁷. World-wide pollination services have been threatened by the spread of parasitic mite pests such as Varroa destructor, which attack endemic as well as commercial honeybees and broods ⁸. Managing natural pollinators can pose a multifaceted challenge, particularly within protected cropping facilities that are specifically designed to ensure superior quality and consistency of fresh market produce through precise environmental control ^7,9,10. As a result, protected cropping facilities incur a significant labour cost associated with manual-pollination services required for producing fresh-market tomatoes. Precision artificial pollination services have potential to add value by sustaining ecological biodiversity and enhancing horticultural crop production ¹¹.

The demand for automating artificial pollination has prompted the development of electronic wands capable of inducing plant vibrations, as well as the utilization of drones and air jets to create mechanical stimulation of flowers. These methods replicate the natural interactions between plants and insects during foraging, facilitating the dispersion of pollen ^11-13. Artificial vibration devices significantly increase floral self-pollination compared with a no-pollination control, although elicit inferior outcomes compared with bee pollinators that have different capacities to generate buzz-induced vibrations ^13-15. Buzz-pollination encompasses the evolutionary convergence of specialized floral morphologies and pollinator behaviours by which bees use both mechanical and sonic means to vibrate flowers and release pollen ^16,17. Bumblebees produce greater thoracic vibrations than other pollinators, generating capacity to move floral structures as well as induce sonic-vibrations depending upon the bee–flower coupling ^16,18. From the plant perspective, information about floral characteristics important for mediating pollen removal and the effect of non-contact cellular vibration can contribute to our understanding of insect-mediated buzz-pollination.

The mechanisms by which pollinators contribute to reproduction vary among plant species. While some plants rely on the simple translocation of available pollen to receptive stigmas, others necessitate specific stimuli to activate the male physiology and make pollen available ^19,20. Furthermore, dioecious species need pollen vectors for their gametes to unify, whereas monoecious demand a similar but different role from the pollinator ^21,22. Tomatoes have self-fertile hermaphroditic flowers and unique reproductive strategies that require specific stimuli of the anthers for pollen release ²³. This restriction mechanism is hypothesised to meter pollen expulsion according to the abundance and transfer efficiency of pollinators, with the goal of maximum pollen utilisation ²³. The arrangement of poricidal anthers found in tomato flowers are distinguished between insects on the proxy of their vibration capacities ²⁴. The efficacy of low-level mechanical vibrational frequencies of flowers generated by artificial devices, are like those produced by bees, and can adequately pollinate glasshouse-grown tomatoes ¹¹. Considering the mechanism that releases pollen from the tomato anther, it is unclear whether higher frequency vibrational stimulations can propagate more energy than insect vibrations and achieve superior self-pollination outcome. Fruit size, seed set, and postharvest quality correlates with the extent of successful pollination and fertilization ²⁵.

The precise mechanism by which vibrational energy exerts inertia upon individual pollen grains, allowing their escape from the anther locule, remains a hot topic. A combination of centrifugal, kinetic, and electrostatic forces is thought to be responsible ²⁶. Energy transmission from the pollinator to the floral structures involves physical and acoustic mechanisms ²⁷. Whilst the role of the acoustic component has previously been understated as a by-product of mechanical stimulation, a recent report suggested that it may be involved in energy transmission to the pollen grains ¹⁸. Vibrations may vary according to differentials in amplitudes of displacement, acceleration, and velocity ²⁸. Pollinator taxa will display varying sound generating capabilities ²⁹. Hang and co-authors (2022) put forward a theory that elucidated the response of living cells to stimuli of different frequencies, positing that the behaviour of living cells can be accurately described by a frequency-dependent power-law. In the low-frequency range, living cells exhibit a weak dependence on frequency, meaning their response to the stimulus changes only slightly at this frequency range. At high frequencies, however, living cells exhibit a tendency towards a constant response, indicating that they become less sensitive changing frequencies and their response becomes consistent ³⁰. Plants have specific natural resonant frequencies, at which most of the vibration energy is concentrated when subjected to external stimulations. The natural frequencies depend on the plant’s physical properties (e.g., mass, stiffness, moisture content) such as those influenced by turgor pressure ³¹. Vibrations at the natural frequencies can also more efficiently propagate through their tissues than other non-resonant frequencies ³², therefore non-resonant frequencies normally induce lower acceleration and are hence less effective for pollen expulsion ³³. The force-frequency relationship, duration, and repetition of cell vibration events can lead to transient and/or long-lasting changes in plant growth, yield, and stress acclimation responses ³⁴. What remains unknown is whether the cellular response to contact and non-contact vibrational stimuli is dependent upon the resonant emission frequency and its effect on the success of self-pollination.

The objective of this study was to establish a comprehensive understanding of the influence of floral velocity and amplitude, induced by a diverse range of sonic frequencies ranging from 50 Hz to 10 kHz, on tomato pollination success and the underlying cellular structures involved in pollen release. We hypothesized that sonic vibration frequencies can significantly affect pollination outcomes in terms of fruit size, weight, and seed set and that higher frequency sounds emitted by bats for echolocation can outperform conventional mechanical contact pollination methods. It is worth noting that mechanical, contact-based excitation methods can only generate low frequency vibrations in flowers near the stems’ natural frequencies. By contrast, high frequency vibrations can be induced directly into the flowers by using amplifier operated speakers. To this end, we employed two different methods to trigger floral vibration: an active subwoofer speaker to induce acoustic waves that directly induced vibration in the flowers and a mechanical shaker and electric wand that excited the stem. Two commercial tomato varieties were grown within a protected cropping environment and subjected to a bioacoustics experimental strategy. We quantified floral vibration responses and generated frequency emission patterns induced by three vibration sources. Vibrational frequency ranges were deployed to tomato flowers and a quantified strategy assessed fruit traits (Figure 1). Our development of a non-contact sonication method offers the first step towards engineering a simple, scalable, and robotic method for automated pollination within the tomato cropping industry.

Harmonic flower responses to stem vibrations are induced by an electric wand

An accelerometer, charge amplifier and data acquisition (DAQ) apparatus were setup to identify the main operating frequency of the electric vibrating wand (Fig. 2A). The frequency produced by the commercial tuning wand peaked at 40 Hz (Fig. 2B). The velocities of three floral organs (anther cone, petal, and petal-cone junction) during stem electric vibrations were assessed using a laser vibrometer setup in the glasshouse (Fig. 2C). The flower vibrated at an average frequency of approximately 55 Hz, resulting in a vibration velocity of around 7.4 mm/s. Four additional harmonic frequencies (integer multiples of the fundamental frequency value caused by resonance of the vibrating flower) of 110, 163, 275, and 493 Hz were identified (Fig. 2D). Fourier transformation of the vibrational signal in time revealed spectral components indicative of a consistent flower velocity during a 3 s interval (Fig. 2E). The floral vibration frequency remained consistent across the three floral components, spanning from 51 Hz to 55 Hz. Among them, the anther cone exhibited the highest velocity (~26 mm/s), while the petal displayed the lowest velocity (~7 mm/s). The vibration velocity of the petal-floral axis junction (~20 mm/s), fell between the velocities observed for the anther cone and petal (Fig. 2F). Therefore, electric vibrations to the plant stem below the truss induced the strongest and yet consistent vibrational response to the poricidal cone in a harmonic manner ranging from 55 to 493 Hz.

Flower vibrational response spectrum to a mechanical shaking arm

The frequency and vibration response of tomato flowers to a mechanical shaking arm designed to stimulate stem movement between 40 to 1000 kHz was assessed (Fig. 3A). Firstly, the mechanical arm output velocity was quantified relative to sliding range of input frequency up to 150 Hz. Two vibrational peaks were observed. One ranged from 40-50 Hz and a second stronger signal from 80-100 Hz, after which the arm velocity began to decline (Fig. 3B). A comparison between the electric wand and mechanical arm stimulating a tomato stem, revealed that the floral vibrational response at 40 Hz of mechanical arm movement was twice (~14 mm/s) that of the tuning fork (~7 mm/s) (Fig. 3C). The vibration velocity of the flower was highest when the stem was vibrated at 50 Hz by the mechanical shaker arm (~40 mm/s), and rapidly declined to 80 Hz after which it gradually weakened unable to stimulate flower movement (Fig. 3C).

Sonication induces floral vibrational bandwidth responses with higher acceleration

The floral response was measured during non-contact vibrations induced by a subwoofer speaker operating between 30 to 10,000 Hz (Fig. 3D). The highest floral velocity was observed at ~60 Hz (3460 µm/s) and declined rapidly revealing two smaller peaks around 180 Hz (~291 µm/s) and 700 Hz (~229 µm/s) before stabilizing between 1 and 10 kHz at 47 to 18 µm/s respectively (Fig. 3E). The acceleration of floral vibration was similar at 50, 700, and 10,000 Hz (130, 103 and 115 µm/s², respectively) (Fig. 3F). Sonic vibrations at 1000 Hz induced the highest sound pressure level (SPL) of ~94 dB and steadily decreased to ~50 dB at 10,000 Hz (Fig. 3G). The vibrational bandwidth response to a subwoofer treatment of three sequential flowers on a single truss was quantified using a laser vibrometer. Within a bandwidth range of 55 - 60 Hz, the three flowers showed a similar peak amplitude velocity of 8 mm/s (Fig. 3H). Within the frequency range of 840 to 1000 Hz, the peak amplitude flower velocity of the three flowers reached its maximum at 880 Hz (~800 µm/s) (Fig. 3I). Therefore, the floral velocity was highest at lower sonic frequency bandwidth (50-60 Hz) and there were two other distinct peaks of high acceleration around 840-1000 Hz and 10,000 Hz.

Endeavour flowers exhibit a weak power-law dependence towards sonic-induced self-pollination

Endeavour self-pollination efficiency to vibrational stimulation by an electric wand, mechanical shaker arm (40 and 80 Hz) and sonication (50, 180, 250, 900, and 10,000 Hz) was assessed by quantifying seed set (number of seeds/fruit, and seed weight/fruit), fruit size (fruit height and width) and weight relative to the untreated controls (Fig. 4). The number of seeds formed per tomato fruit reflects the pollination efficiency. All floral vibration frequencies, regardless of the treatment device, significantly enhanced seed number per fruit by ~1.9- to 2.1-fold (~94-110 %) relative to the untreated controls (Fig. 4A). Floral vibration treatments substantially increased seed weight per fruit by 1.9- to 2.5-fold (94-143 %) compared with the control (Fig. 4B). There were no significant differences in seed set among the three pollination treatments revealing that the lower frequencies (40-50 Hz) were equally effective as the higher frequencies (10,000 Hz) in triggering self-pollination (Fig. 4A-B).

Cellular vibration enhanced the overall fruit size relative to the untreated control, yet the phenotypic variations depended on the specific vibrational treatment and frequency applied (Fig. 4C). Lower vibrational frequencies generated by the electric wand (40 Hz), and subwoofer (50 Hz) significantly enhanced fruit weight (72-82 %), height (17 %), and width (20-22 %) (Fig. 4D-F). Mechanical arm vibrations at 40 Hz significantly increased fruit weight (88 %) and height (38 %) yet the width was only marginally larger compared with the control. At 80 Hz, the mechanical arm enhanced height (20 %) but the width (18 %) and overall weight were only marginally trending greater compared to the control. (Fig. 4D-F). There was an overall linear trend whereby the fruit size increased with sonic frequency such that the distribution of fruit parameter plot ranges was notably higher between 250 and 10,000 Hz compared with lower frequencies (Fig. 4C-F). Fruit height was significantly enhanced by sonic vibrations at 250 Hz (29 %), 900 Hz (31 %) and 10,000 Hz (38 %) (Fig. 4E). The fruit width (33-49 %) and weight (118-188%) were substantially higher at 10,000 Hz (Fig. 4D and 4F). A cross-section of the tomato fruits revealed that sonication at higher frequencies enhanced the mesocarp thickness without altering seed set in comparison with the electric vibrating wand (Fig. 4G). Therefore, the highest sonic frequency of 10,000 Hz unexpectedly hyper-induced fruit height, width, weight, and mesocarp thickness revealing a robust frequency-dependent power-law cell behavior observed in response to infra- to ultra-sonication induced floral vibrations.

Sweetelle flowers exhibit a weak power-law dependence towards sonic-induced self-pollination

The pollination efficiency of cell vibrational frequencies induced by infra- (250 Hz) and ultra-sonication (10,000 Hz), mechanical shaking arm (40, 80 Hz), and electric vibrating wand (40 Hz) were next evaluated using Sweetelle flowers (Fig. 5). The seed set and seed weight per fruit were similar across all treatments, while exhibiting a remarkable increase of 3.7-6-fold (268-497%) and 2.8-4.4-fold (182-333%), respectively, in comparison with the untreated control (Fig. 5A-B). The lower 40 Hz of mechanical shaking arm vibration did not change the fruit height, width and weight. However, sonic frequencies (250 and 10,000 Hz), the 40 Hz vibrating wand, and 80 Hz of mechanical arm movement significantly enhanced fruit weight, height, and width, by 1.9-2.1fold (94 -109 %), 1.2-1.3 (24-30 %) and 1.3-1.4 (33-42%), respectively compared with the control (Fig. 5C-F). Collectively, these findings illustrate that infra- and ultra-sonication can trigger self-pollination in Sweetelle and enhance fruit size effectively similar to that of the gold standard electric vibrating wand.

Sonic vibrations disruption of poricidal anther cone trichomes

The mechanism by which sonic vibrations might trigger pollen release was investigated by comparing anther cone structures from unpollinated vs pollinated (40 Hz electric wand and 10,000 Hz sonication) Sweetelle flowers using scanning electron microscopy (SEM). In untreated control flowers, the anther cones were connected by a mesh of interlocked trichome hairs along the edges, tightly joining individual anthers along the central stylar channel (Fig. 6A-C). These trichomes are distributed across the back, front, inside and around the sides of the anther cone (Fig. 6A). Flowers exposed to sonic vibrations (Fig. 6G-I) or the electric vibrating wand (Fig. 6D-F) displayed unentwined trichomes (Fig. 6E,H) that appear loosely packed (Fig. 6D, G) correlating with a change in trichome structure compared with the untreated control (compare Fig. 6C, F, I). The trichomes of vibration treated flowers (both acoustic and mechanical) exhibited a greater visual separation (Fig. 6, E, H). The disentanglement and loosening of the trichome mesh network likely contributed to the enhanced transmission of cell vibrations within the anthers and imposed centrifugal forces to the pollen grains within the anther locules leading to release for successful self-pollination.

Plant perception of sound was once controversial. Sound compression waves force plant cells to vibrate and plant mechano-sensory receptors could perceive the stimuli, triggering a signal transduction cascade that regulates nuclear gene expression and chemical processes ^35,36. We engineered new devices to investigate the frequency-dependent power-law behaviour of floral living cells from two commercial tomato varieties using contact-based mechanical (electric wand and mechanical shaker arm) and precision non-contact sonication (subwoofer speaker) vibration-inducing methods (Fig. 1). Empirical studies suggest that mechanical characteristics of vibrations transmitted from insects or artificial mechanical stimulation can vibrate floral anther cells to release pollen and trigger pollination ³³. The floral vibrations produced by the vibrating wand, mechanical shaker arm and sonication are described with the same basic parameters to those of an oscillatory movement (frequency, velocity, and acceleration) ³⁷. Sound wave vibrations affect cell behavioural characteristics across orders of magnitude with lower and higher frequencies proposed to be frequency-dependent and constant, respectively ³⁰. In nature, sound vibrations from a buzzing bee (~1kHz) induce sweeter nectar production in Oenothera drummondii flowers to increase cross pollination ³⁸. Infra (low) and ultra (high) sonic frequencies can repel whiteflies (300 Hz) and moths (10 kHz) from tomato and strawberry crops, respectively ^39,40. The right balance of bioacoustics can provide benefits for plant cell culture, postharvest fruit storage, and crop production systems ^35,36,41. Therefore, we herein interrogated the relationship between the frequency of vibration with pollination success and fruit size to understand power law behaviour of floral cells.

We found that the peak amplitude velocity of floral vibrations induced by sonication was highest at lower sonic frequencies, decreased at higher frequencies, and acceleration was almost similar at both higher and lower sonic frequencies (Fig. 3E-F). The vibration velocity of the flower at 40 Hz for the mechanical shaker arm was about twice that of the tuning fork (Fig. 3B). Vibrations at high frequencies (450–1000 Hz) were reported to release more pollen than low frequency vibrations (100–400 Hz) ²⁰. It has been shown however, that floral vibrations transmitted to the anthers by bumblebees at lower frequencies (240–405 Hz) had only a modest effect on pollen ejection ⁴². It has also been found that larger bees capable of generating higher frequencies release more pollen compared with smaller bees ⁴³, suggesting that there may be additional effects of pollinator-specific buzzing that affect pollen removal ⁴⁴. The variation in frequency may not be important to affect pollen emission ^33,42,45. In contrast, duration and peak velocity amplitude are positively associated with pollen ejection during artificial floral vibration ⁴². Contrarily, another study found a positive relationship between frequency and pollen release when expressing amplitude as displacement and velocity. However, when acceleration is used as a proxy for buzz amplitude, the relationship between frequency and pollen release becomes negative ⁴⁵. No significant differences were observed between the three pollination treatments regarding seed set in either tomato variety (Fig. 4 and 5). It seems likely that pollen ejection is equally influenced by multiple cell vibration characteristics, regardless of frequency, and hence one variable may not be sufficient to describe the energy and force transmitted to the anther and hence underpin the success of pollen release. Our findings implicate a constant low power-law cell behavior leading to frequency-independent pollen dispersal and efficient self-pollination.

Sonication and mechanical-induced cell vibrations revealed differences in fruit size (fruit height and width) and weight between the two varieties (Fig. 4 and 5). Sonic frequencies ranging from 50 Hz to 10 kHz linearly increased the size and weight of Endeavour but not Sweetelle tomato fruits. The high frequency-dependent power-law cell behaviour exhibited by Endeavour flowers exposed to 10 kHz ultrasonication could implicate a genetic basis by which frequency-dependent vibrations to floral cell gametes trigger single parent heterosis to enhance fruit vigour and boost tomato fruit size and weight (Fig. 4). This could resemble cross-pollination induced hybrid vigour in the offspring from genetically distinct parents. Plant breeding heterosis is widely used to boost crop vigour, enhance growth, flowering, seed set, and fruit size ⁴⁶. Empirical studies showed that Bombus huntii-mediated pollination significantly enhanced fruit weight of two indeterminate tomato varieties Favorita and Sungold ⁴⁷, and increased fruit production by bumblebee pollination in a series of determinate and indeterminate tomato varieties ⁴⁸. Mechanical pollination using a vibrating wand also increased tomato weight by 35.9% compared with an untreated control, while the non-buzzing honeybee (A. mellifera) improved pollination and yield by only 28.3%, revealing that a sonic buzz can impact upon fruit size ⁴⁹. However, the greatest reported increase in fruit weight (74.5%) compared with an untreated control was attributed to B. terrestris that can floral buzz at 250-300 Hz frequency ⁴⁹. Similarly, B. terrestris-mediated pollination was reported to significantly increase marketable fruit quality when compared with mechanical vibration and auxin treatments ^50,51. The energy transmitted from a pollinator to the floral structure could result from the combined interactive effect of both non-contact acoustic and contact-based mechanical cell vibrations ²⁷. The vibrations generated by the bee thoracic muscles are transmitted to the flower via direct physical contact with the anther ^52,53. Our findings support this notion as the electric wand and mechanical shaking arm both operate by physically contacting the plant to induce floral vibrations. The non-contact sonication evidence provides insights into the capacity of low-frequency-dependent precision movement of air particles, reminiscent of the vibrations produced by a buzzing bee, to induce significant floral cell vibrations. This phenomenon has been observed to effectively trigger self-pollination in two commercially cultivated tomato varieties. The enhanced size of Endeavour fruits exposed to increasing sonication frequency highlight that acoustic and substrate-borne components from a buzzing bee are likely both essential to promote crop vigour and yield.

The mechanism by which pollen ejection is induced by sonication and/or mechanical vibrations has remained unclear. Our examination of buzz-pollinated floral anther cone structures showed that sonic vibrations and the vibrating wand loosened the interlocking trichomes of adjacent anthers of the poricidal cone. The loosened trichomes may enhance the transmission of vibrations within the anthers by increasing elasticity to open the cone walls and allow pollen expulsion (Fig. 6). It is thought that the acceleration of the anther tip during floral vibrations generates centrifugal forces on the pollen grains which causes them to be expelled from of the apical pore ³³. Elastic collisions against the internal walls of the vibrating anther are also thought to transfer kinetic energy to the pollen grains, which causes expulsion ^28,54. As the energy increases, the particles begin to move and escape through the apical pore. Pollen release is a function of anther geometry, velocity and the frequency of the vibrations transmitted from the bee or artificial pollination to the anthers. In addition to these mechanical effects, electrostatic interactions between the pollen grains and the anther walls may also play a significant role in expelling pollen from anthers ⁵⁴. The summation of centrifugal forces, kinetic energy and electrostatic interactions generated by floral vibrations may jointly contribute to the expulsion of pollen grains. The trichome hairs are key structural features that prevent pollen dispersal when interlocked, and low-power frequency dependent cell vibrations can unentwine their mesh network to allow pollen dispersal.

Modern technology and advanced research methods that automate precision pollination devices would be particularly valuable for the protected cropping industry and in particular high-tech glasshouses ¹⁰. Importation of commercially reared bumblebees is not permitted in countries like Australia due to their potential in becoming an agronomic pest and threat to the ecological environment ⁷. Handheld mechanical pollination devices are the best suitable alternative to natural bee pollination for tomatoes grown in protected cropping environments; however, the labour costs can be prohibitive to large-scale industry applications ¹¹. Contactless precision sonication technology that simulates a buzzing bee could be automated to promote effective self-pollination, reduce labour costs, and depending upon the variety potentially enlarge fruit size to boost production.

Plant material

Seeds from two commercial tomato cultivars, Sweetelle and Endeavour RZ, were obtained from Syngenta (Australia) and Rijk Zwaan (Australia) respectively, via Perfection Fresh (Australia Pty Ltd. Homebush, Australia). Seeds were sown in Seed and Cutting Premium Potting Mix (Scotts Osmocote) in 30-cell plastic trays covered with transparent film to maintain an optimum germination temperature of 27 °C and humidity. At the 2-leaf stage, seedlings were transplanted to 50 L pots (10 pots per variety) containing Tomato, Vegetable & Herb Premium Potting Mix (Scotts Osmocote) and grown in a glasshouse located at Western Sydney University in Richmond, New South Wales (33.6000^oS, 150.7500^oE). Plants were maintained at 25 °C during the day and 18 °C at night. Plants were supplied with a commercial granular fertiliser (Aquasol; NPK 23: 3.95: 14) every 2 weeks and watered every other day to maintain soil moisture. Secondary shoots and trusses were pruned weekly as per best horticultural practices ⁵⁵.

Vibrational frequency quantification using a laser vibrometer

To capture plant vibrational velocity responses to mechanical and sonic vibrations, we utilised a helium-neon laser head coupled with a PSV-500 scanning vibrometer (Polytech). The desired frequency range was generated between 50 Hz to 10 kHz using a high-frequency signal generator connected to an amplifier and a data acquisition (DAQ) unit (Polytech). The vibrating wand (Vibri tomato pollinator Vario 12v) and mechanical shaker arm were placed on the stem below the truss to trigger floral vibrations. The sonic vibrations were induced by exciting a high-power subwoofer speaker placed 5 cm from the truss (Fig. 1). Measurements were performed in triplicate per flower. Data were captured both in time and frequency domain using a Fast Fourier Transform (FFT) algorithm. The equivalent FFT is plotted for the time series data to compare the vibrational signals (velocity in mm/s) within the frequency domain. The frequency values corresponded to the highest velocity signals identified from floral vibrations. Data were stored and processed using the DAQ and DIAdem NI software, respectively. The intensity test was conducted to find the sound pressure level of the subwoofer using a decibel meter (Decibel-X pro sound meter).

Floral pollination treatments

Control plants were grown in a separate glasshouse bay to reduce cross-contamination from sonic stimulation events and determine background self-pollination efficacy. Airflow was minimized and plants were spaced to avoid direct contact with the air venting system. Prior to any vibrational stimulus, flowers from Endeavour and Sweetelle trusses were trimmed leaving only 4-6 unopened flowers. For sonic treatments, a signal generator, and a linear digital switching (LDS) amplifier without attenuator connected to a 10 cm diameter subwoofer speaker (Vibe Blackair 1300 Watts and root mean square (RMS) power of 450 watts) had a frequency response of 30 to 30,000 Hz and a sensitivity of 88 dB. Flowers from the Endeavour cultivar were exposed to single sonic frequencies of 50, 180, 250, 900 and 10,000 Hz for 5 min. Flowers from the Sweetelle cultivar were exposed to frequencies of 250 and 10,000 Hz for 5 min. Artificial floral vibrations using a vibrating wand (40 Hz; Vibri tomato pollinator Vario 12v) or an aluminum rod arm attached to a permanent magnet shaker unit mounted on a strong metal frame (operated within a 40-1,000 Hz range when connected to the LDS amplifier) were performed for 5 seconds by stimulating the plant stem directly below the truss (Fig. 3A). A Charge Accelerometer (4393), charge amplifier, and DAQ (DIAdem NI software) were used to identify the optimal frequency of the electric tomato pollinator probe (Fig. 2A). Frequency and acceleration responses of the flower were assessed using a laser vibrometer within the glasshouse bays.

Postharvest traits

The efficacy of pollination treatments was assessed by counting the number of seeds per fruit, total seed weight per fruit (mg), fruit height (cm), fruit width (cm) and fruit weight (g) at the mature ripe stage of horticultural maturity.

Scanning electron microscopy

Tomato flowers were dehydrated progressing through an ethanol series of 70, 90 and 100 % for 30 minutes. Dehydrated samples were then critical-point dried (Leica EM CPD300), mounted onto Scanning Electron Microscopy (SEM) stubs using double-sided conductive carbon tabs, and gold coated using a Leica EM SCF SCD005 cool sputtering device. Samples were examined using a Phenom, XL Desktop SEM under high vacuum at an accelerating voltage of 15 kV. Images were collected using a secondary imagery detector (Everhart-Thornley design) and microanalysis detector (Amptek SSD C1 EDS supported by Phenom Pro-suite software).

Statistical analysis

Statistical significance P<0.05 was calculated by performing a one-way Analysis of Variance (ANOVA) followed by a posthoc Tukey’s test on all data sets using Sigma Plot 14.0 software.

Acknowledgments: We thank Richard Wuhrer and Daniel Fanna from Western Sydney University (WSU) for assistance with the Scanning Electron Microscopy at the Advanced Materials Characterisation Facility. We thank Troy Topp and Naresh Singh from Perfection Fresh (Australia Pty Ltd. Homebush, Australia) for their valuable discussions and insights into pollination strategies utilised in the Australian tomato protected-cropping industry. We also thank Nigel Warwick and Paul Kristiansen from the University of New England for co-supervision of Angus Dingley’s honours research.

Funding: This research article has been prepared using funding provided by the Future Food Systems Cooperative Research Centre (CRC) and Perfection Fresh (P2-010) awarded to Christopher Ian Cazzonelli (WSU) and Chun-H Wang (UNSW). This grant also supported Sidra Anwar’s PhD scholarship extension.

Author contributions:

Conceptualization: CIC, CHW

Methodology: TV, SA, H, LG, WL

Figure Preparation: SA, H

Investigation – data analysis: SA, TV

Supervision: SA by CIC, AD by BMS and CIC, TV by CHW

Writing—original draft: SA, AD

Writing—review & editing: CIC

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data are available in the main text or the supplementary materials. The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Sonic-induced cellular vibrations disrupt poricidal cone trichomes to boost tomato floral pollination

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