Spike preservation: A simple method to preserve pollen viability and in vitro germination in wheat

Spike preservation: A simple method to preserve pollen viability and in vitro germination in wheat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article Spike preservation: A simple method to preserve pollen viability and in vitro germination in wheat Irum Khan, Muhammad Sajjad This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2659708/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Wheat pollen grains have very short longevity and are non-viable after ~30 minute at room temperature and ~60 minutes at 4 ° C. Pollen grain viability can be preserved maximum to ~24 hrs with existing preservation methods. Results Herein, we developed two simple methods-anther preservation and spike preservation- to preserve the pollen grain viability in wheat. The methods were validated using viability and in vitro germination of pollen grains of 50 diverse spring wheat genotypes. Anthers and spikes were collected for anther preservation and spike preservation methods, respectively, and stored at room temperature (22°C) and fridge (4°C) for 0 and 1-week. Pollen viability were assessed using Alexander staining techniques at two storage temperatures 22°C and 4°C. In vitro germination was determined using liquid germination medium at 4°C. After 1 week, the pollen viability and in vitro germination as determined with Spike preservation method were higher as compared with those of anther preservation method at 4°C. In addition, in vitro pollen germination and pollen viability significantly reduced as storage duration increased. After 1-week, the pollen grains preserved with anther preservation method at 4°C failed to germinate. Conclusion The Spike preservation method is effective for preserving the pollen grain viability and in vitro germination in a large panel of wheat genotypes. This new method is instrumental to further our understanding on pollen grain viability and germination. Pollen viability wheat germination spike anther preservation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Wheat ( Triticum aestivum L.) is an allohexaploid (2n = 6x = 42, AABBDD) and obligate self- pollinating species [ 1 , 2 ]. Successful pollination and fertilization in wheat breeding rely on haploid male gametophyte. In flowering plants including wheat, pollen grain is male gametophyte that is shed after the completion of second pollen mitosis. The mature tricellular wheat pollen grain has relatively high moisture contents and is short-lived [ 3 ]. Therefore, pollination is necessary within 30 to 40 min after pollen shedding to achieve successful seed sets [ 4 ] under a wide range of temperate environments [ 1 ]. Terminal heat stress at anthesis stage is an emerging threat to wheat crop in South Asia and some other parts of the world. Heat stress at anthesis stage desiccates pollen grains affecting their size, viability, vigour and potential of making pollen tube to fertilize the female gametophyte [ 5 ]. Pollen viability reflects the performance of pollen grains in terms of stainability, germinability and fertilization ability [ 6 ]. Pollen vigour refers to the speed and rate of germination and pollen tube formation. In wheat, pollen viability is mostly determined by pollen staining methods and within 30 minutes at room temperature or within few hours when stored at 4°C [ 7 , 8 , 9 , 10 , and 11 ]. In vitro pollen germination tests are performed mostly on fresh pollen grains since after shedding pollen grains remain alive for 30–40 minutes at room temperature or few hours when preserved at 4°C [ 12 , 11 ]. High quality crops also depend on the strength of the pollen and it is the most important factor in the breeding program [ 13 ]. In the first 1.5 hours of storage, the viability of creeping bent grass ( Agrostis stolonifera L.) pollen decreased significantly, and after 3 hours, it was completely lost [ 14 ]. Within two hours in a field condition, pollens of maize became non-viable [ 15 ]. The pollen viability is significantly affected by moisture content, temperature and genotypes [ 16 ]. Many crops depend on low temperature and low humidity while wheat crop prefers low temperature and high humidity for pollen viability and longevity [ 17 ]. Adhikari and Campbell [ 17 ] in their study on buckwheat concluded that a high humidity was necessary for pollen longevity and viability. Genetic variations in blooming times among elite parents are one of the main problems with wheat breeding programs. Wheat breeders can overcome variations in flowering periods by preserving pollens from the desired male parents until pollination can take place. In general, wheat breeders believe that wheat pollen has a very limited lifespan and may be a barrier to the economically viable production of hybrid seed [ 4 ]. However, there are few reports on the best storage conditions for wheat pollen grain [ 18 ]. According to Fritz and Lukaszewski [ 4 ], pollination with pollen that had been kept for 45 minutes reduced seed set in spring wheat from 100% to less than 10%. Athwal and Kimber [ 19 ] found no seed set in "Chinese Spring" wheat pollinated only 5 minutes after anther dehiscence. Roemer [ 20 ] reported that pollen stored at low temperatures preserved germination capability better than pollen stored at high temperatures. According to Hoekstra and Bruinsma [ 21 ], higher respiration is the reason for the viability reduction in three-celled pollen. When kept at 0°C–10°C and 80–100% RH, the pollen of various grasses only lasts a few days [ 22 ]. Further, Andronescu [ 23 ] reported that Graminaceous taxa's pollen grains lose viability very quickly, and under ideal storage conditions, successful preservation was thought to only last for around 10 days [ 24 ] However, Li Xun Zhen et al. [ 25 ] preserved rice pollen grains at low temperature for 15 days. For measuring pollen germination vigour and percentage over time, in vitro techniques have been used [ 12 , 11 ]. A genotype can be regarded a good pollinators if its pollen viability is high, and examination of pollen viability and pollen tube formations are crucial criteria for pollen evaluation [ 26 ]. On a solid medium raffinose, Cheng and McComb [ 27 ] produced the longest wheat pollen tube. Testing pollen viability and pollen germination are essential for determining the quantity of pollen needed for good pollination. The pollen viability tests are indispensable for hybrid wheat breeding programs and breeding heat tolerant pure line wheat varieties [ 28 ]. Pollen viability is now considered as a key trait for heat tolerance in wheat [ 29 , 5 ]. Nonetheless, performing pollen viability tests to evaluate large number wheat lines are not feasible due to very short life span of pollen grains after their shedding from anther. To overcome this obstacle, we tried two simple and pollen preservation methods namely anther preservation and spike preservation. Pollen grains of a panel of 50 diverse genotypes were used to compare the results of the two preservation methods under two temperature conditions and two storage durations. Materials And Methods Plant materials The pure seed of about 50 diverse spring wheat genotypes including land races, pre-green revolution, post-green revolution and recent cultivars and advanced lines were collected and arranged for three replications. Field experiment The experiment was conducted at National Agricultural research Center (NARC) Islamabad, Pakistan .The seed of about 50 diverse spring wheat cultivars and elite lines were grown in randomized complete block design (RCBD) with three replications. The seeds were sown with wheat planter in 1.2 m x 3m plots, consisting of six rows, 20 cm apart. The standard agronomic practices were used in each experiment to raise good quality crops. Pollen collection Spikes at anthesis stage were collected and kept in plastic bags. The bagged spikes were then transferred to lab. Two preservation methods were used for measuring pollen viability to test which method is robust and fast to extract pollen and maintain pollen viability for long term. Method 1: Anther Preservation Method Spikes with yellow anthers were collected at the flowering stage in order to sample the anthers thereafter, anthers were extracted from sampled spikes by using forceps to open the glume and lemma. Then, the sampled anthers were stored for future use in tightly sealed plastic vials. Method 2: Spikes Preservation method. At anthesis stage, spikes were harvested, collected, and preserved in plastic bags and immediately transported to the lab. Pollen storage The anthers and spikes that were harvested for each genotype of wheat were kept at ambient temperature (22°C) and at (4°C) in the fridge as storage temperatures. After 0 (for fresh, non-stored pollens) and 1-week (for stored pollens) at two storage temperatures (22°Cand 4°C) pollen vitality was assessed. Pollen viability was tested after 0-week (non-stored, fresh pollens) and 1-week (stored pollens) in each storage temperature. Pollen viability To determine the best long-term pollen storage conditions for maintaining high viability of pollen, two different storage temperatures (22°C and 4C°) with two storage durations (0-week and 1-week) were tested. The pollen grains of 50 genotypes collected from both procedures under two different storage conditions were placed on slides with one to two drops of ALEXANDER's solution and then it was covered with coverslips [ 6 ]. Next, a compound microscope (Olympus). was used to observe the level of pollen staining at a 5X magnification. However, in the standard stain, pollen that was fully and darkly stained (magenta-red or red) was viable, pollen that was lightly stained (magenta-red or red) was semi-viable, and pollen that was stained (blue-green, blue, or non-stained having no colour) was non-viable [ 30 ]. Pollen viability was measured as the proportion of stained to total pollen grains, and it was represented as a percentage [ 31 ]. In vitro pollen germination test To examine pollen germination in 50 genotypes of wheat under anther and spike preservation method at 4°C, liquid pollen germination medium (without agar and peptone water) was made by dissolving H3BO3 (0.05g), Ca(NO3)2.4H2O (0.03g), and BK Salts viz (MgSO4.7H2O (0.2g) and KNO3(0.1g)), maltose (19%), and polyethylene glycol (PEG6000, 13%) at 6 pH as described by [ 32 ]. Using a light compound microscope (Olympus BX41 with DP12 camera) pollen grains and germinated pollen grains from a randomly selected microscopic field were counted to calculate in vitro pollen germination. All images were taken at 5x´magnification. When the pollen tube's length exceeded the pollen grain's diameter, the pollen grain was considered germinated [ 31 ]. Results The pollen viability and in vitro pollen germination data of 50 spring wheat lines were determined using two preservation techniques—anther preservation and spike preservation under two storage conditions or temperatures, namely room temperature (22°C) and fridge (4°C), with two storage durations—0 week for fresh pollens and 1 week for stored pollens (Table S1 and Table S2). Non-stored (0 week) pollen was shown to had better viability and in vitro pollen germination rates than stored pollen (1 week). With anther preservation method at room temperature (22°C) maximum pollen viability (100%) was recorded for fresh non-stored pollens (0 week). After 1 week of storage at room temperature (22°C), all genotypes had non-viable (60–100%) pollens except Punjab-76 (Table S1 and Fig. 1). With anther preservation method at 4°C, all genotypes had viable pollens for fresh non-stored pollens (0-week). However, after 1-week of storage in fridge at 4°C, 37 genotypes showed maximum pollen viability (100%) while other had 40%-70% viability. In vitro pollen germination of 50 spring wheat genotypes ranged from (10%-30%) at 4°C for fresh non-stored pollens (0-week). After one week of storage at 4°C, all 50 spring wheat lines did not show any germination. Thus, pollen germination (10%-30%) was observed in all genotypes of wheat only for fresh non- stored (0 week)) as compared with those stored at 4°C for 1-week which did not germinate (Fig. 4 ). With spike preservation method at room temperature (22°C), the highest pollen viability(100%viable) was recorded for fresh non-stored pollens (0 week). However, after 1 week of storage at room temperature (22°C), 31 genotypes were non-viable (50–100%) and 27 genotypes were semi-viable (10–100%). On the other hand, at 4°C all genotypes were viable (100%) for fresh non-stored pollen (0 week). However, after 1 week of storage in fridge at 4°C all 50 genotypes showed (50–87%) viability (Table 2 and Fig. 2). However, in vitro pollen germination 50 spring wheat genotypes ranged from (30–50%) at 4°C for fresh non-stored pollens (0 week). After 1 week of storage at 4°C in fridge, 50 spring wheat genotypes showed 2–14% pollen germination (Table 2 and Fig. 3). Taken together, pollen grains preserved with the spike preservation method at 4°C had better pollen grain viability and in vitro germination after 1 week (Fig. 4 ). When florets were kept closed and entire spikes were preserved, pollen viability was not rapidly lost since their possibilities of shedding decreased. The spike preservation method is efficient to preserve pollen grain viability in wheat for a longer period of time. Discussion Wheat pollen grain is known to have a relatively high moisture content and a short life span [ 3 ]. Therefore, pollination must be done within 30–40 minutes of pollen shedding in order to produce effective seed sets [ 4 ]. Pollen viability has a significant effect on fertilization [ 33 ], embryonic development [ 34 ] and quality of seed [ 35 ]. Plant pollen viability and longevity vary depending on species and environmental conditions [ 6 ]. The ability of a pollen to retain its viability over time and under different storage temperatures depends on both the pollen's genetic makeup and environmental influences. [ 36 , 37 , 38 ]. Optimum storage conditions for pollen also vary depending on species or cultivar [ 39 ]. There are only a few studies on the short-term storage of spring wheat pollen at various temperatures [ 18 ]. To prevent pollen viability loss, proper storage temperatures are required [ 40 ].Thus the primary goal of this study was to establish the optimum storage temperature range for spring wheat pollen as well as how long wheat pollen can be stored under different storage conditions before losing viability. The results showed the Spike preservation method was effective to preserve pollen viability and germination under two storage temperatures (22°C and 4°C). Many factors can influence the viability of pollen, including pollen handling during collection, flowering maturation stage, and environmental conditions such as air temperatures and moisture contents [ 41 , 42 , and 37 ]. Low temperatures are typically used for the long-term preservation of pollen because they reduce soluble sugar and organic acid consumption due to decreased pollen respiration [ 43 , 44 ]. The findings showed that pollen stored at low temperatures (4°C) had a higher percentage of germination than pollen stored at room temperature (22°C) for one week, and that germination percentages decreased as storage duration increased among all wheat genotypes. The inactivation of essential germination enzymes and substrates may be the root cause of the pollen grains' decreased ability to germinate when kept at room temperature [ 45 ]. The loss in pollen viability during storage is caused by enzymatic activity that reduces the amount of respiratory substrates [ 46 ]. Fresh pollen at 0 week of storage had the highest pollen viability and in vitro pollen germination for all wheat genotypes. It has been reported that storing pollen at lower temperatures and lower humidity levels often increases pollen lifespan [ 47 ]. Our findings demonstrated that wheat genotypes, pollen storage conditions, germination assessment tests, pollen viability, and storage period all affected pollen viability and in vitro germination. The findings of the current study concur with those from other authors [ 48 , 49 , 50 , 47 , 51 , 52 ]. The genetic diversity among the genotypes under study may be the cause of the variations in pollen viability and germination [ 53 ]. Plant pollen viability and longevity vary depending on species and environmental conditions [ 6 ]. There are genotype-specific differences in pollen viability and longevity [ 38 , 54 , and 55 ]. In vitro pollen germination percentages were consistently lower than pollen viability tests for all 50 spring wheat genotypes evaluated. This could be as a result of the effect of various uncontrollable parameters including pollen density, the best growing medium, and the environmental requirements for each genotype [ 56 ]. In accordance with the findings of the current study, Cheng and McComb [ 27 ] found that wheat germination under in vitro conditions of pollen grains was low and variable, with a maximum germination rate of 6.8%. According to Devrnja et al. [ 57 ], trinucleate pollens germinate more quickly than binucleate pollens but have a much shorter lifespan. Additionally, according to Mulcahy and Mulcahy [ 58 ], some species with trinucleate pollen have difficulty developing pollen tubes in vitro . It has been reported that differences in pollen longevity among plant species are caused by differences in pollen desiccation tolerance [ 59 ]. The results of pollen viability and germination tests conducted at 22°C and 4°C revealed that the maximum levels of pollen viability (100%) and germination (2–14%) were found at 4°C by spike preservation method. In agreement with the current findings, D'Souza [ 60 ] observed that wheat pollen viability was increased after storage at 2 ° to 5°C. In the current study, there were no differences in pollen viability for fresh (non-stored) pollens at 22°C under either preservation method. The current study found that storage duration had a significant impact on pollen viability and pollen germination. Under all storage temperatures, the pollen viability and germination rates of all wheat genotypes rapidly declined during the study period. According to the findings, fresh pollen had a high viability at 0 weeks but rapidly declined after 1 weeks of storage using either preservation technique. After a week of pollen storage at 4°C using anther preservation technique, the germination rate of nearly all wheat genotypes dropped to 0.00%. However, after a week of pollen storage at 4°C resulted in germination (2–14%) using the spike preservation method. Our findings corroborate those of Parzies et al. [ 61 ], who found that in a genotype of barley originating from a semi-arid region, pollen viability decreased upto 90% after anthesis and less than 50% within 24 hours. Our findings supported earlier studies on other species of cereal, which showed that pollen grains from plants like sorghum and maize have extraordinarily short lifespans, with times between a few minutes and several hours [ 15 , 62 ]. Pollen viability and in vitro pollen germination with the spike preservation method was higher than those using anther preservation method in the selected panel of 50 spring wheat genotypes. According to a prior study, Spring wheat pollens preserved using anther preservation technique exhibited germination(1.64%), after 24 hours of storage ,but completely lost viability and revealed (0.00% after 48 and 72 hours) [ 18 ]. Nevertheless, with the Spike preservation method we observed 2–14% germination at 4°C after 1-week of storage. Conclusion Pollen preserved at 4°C with the spike preservation method displayed higher pollen viability and germination when compared will pollen stored at 4°C with anther preservation method. Convincingly, the Spike preservation method works well to preserve pollen grain viability and germination at 4°C after 1-week of storage. The Spike preservation method will be useful for research on artificial pollination, hybrid wheat breeding. The preservation of pollen grains with this new method will also facilitate molecular studies on pollen grain in wheat. Declarations Ethical Approval Not applicable Competing interests The Authors declare no conflict of interest Authors' contributions I.K performed the experiment, analyzed data and wrote the first draft and M.S conceptualized the study, supervised the experiments and improved the manuscript. Funding Not applicable Availability of data and materials Not applicable References Shewry PR. Wheat. 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Mulcahy GB, Mulcahy DL.The effect of supplemented media on the growth in vitro of bi-and trinucleate pollen. Plant Science. 1988;55(3): 213-216. Song J,Tachibana S. Loss of viability of tomato pollen during long-term dry storage is associated with reduced capacity for translating polyamine biosynthetic enzyme genes after rehydration. Journal of Experimental Botany. 2007;58(15-16): 4235-4244. D'SouzA L. Studies on the suitability of wheat as pollen donor for cross pollination, compared with rye, Triticale and Secalotricum. Zeitschrift fur Pflanzenzuchtung. 1970; 63: 246-69. Parzies HK, Schnaithmann F, Geiger HH. Pollen viability of Hordeum spp genotypes with different flowering characteristics. Euphytica.2005; 145: 229-235. Tuinstra MR ,Wedel J. Estimation of pollen viability in grain sorghum.Crop science. 2000;40(4): 968-970. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2659708","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":181398604,"identity":"8b694962-26c7-43b7-923e-411c0ac3a898","order_by":0,"name":"Irum Khan","email":"","orcid":"","institution":"COMSATS University Islamabad","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Irum","middleName":"","lastName":"Khan","suffix":""},{"id":181398606,"identity":"092f77e5-08ef-4b48-9685-8db5e6e4120f","order_by":1,"name":"Muhammad Sajjad","email":"data:image/png;base64,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","orcid":"","institution":"COMSATS University Islamabad","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Sajjad","suffix":""}],"badges":[],"createdAt":"2023-03-06 07:59:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2659708/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2659708/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":34196255,"identity":"0d49ef87-8479-4fc0-a4cf-736d84af0fb7","added_by":"auto","created_at":"2023-03-13 23:07:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":276118,"visible":true,"origin":"","legend":"\u003cp\u003ePollen grains of spring wheat lines with Anther preservation method considered as ; (A) indicates 79% semi-viable at 22°C for fresh pollens; \u003cstrong\u003eblue arrow indicates semi-viable and\u003c/strong\u003e \u003cstrong\u003eblack arrow indicates non-viable \u003c/strong\u003e(B) represents all non-viable pollens after storage of 1 week at 22°C .(C) indicates 5.7 % viability after storage of 1 week at 22°C ; \u003cstrong\u003eblue arrow\u003c/strong\u003e \u003cstrong\u003eindicates semi-viable and black arrow indicates viable\u003c/strong\u003e (D) indicates 2.56% viability after storage of 1-week at 22°C ; \u003cstrong\u003eblack arrow indicates viable and blue arrow indicates non-viable\u003c/strong\u003e. All images were taken under a light compound microscope (OLYMPUS) at 5x magnification.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2659708/v1/d2a21bb793835c1b76a5fd6b.png"},{"id":34196254,"identity":"938c0623-c43d-44f6-b1eb-147def6474df","added_by":"auto","created_at":"2023-03-13 23:07:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":183623,"visible":true,"origin":"","legend":"\u003cp\u003ePollen grains of Spring wheat lines with Spike preservation method. (E) indicates 79.4 % viability after storage of 1 week at 4°C; the\u003cstrong\u003e black arrow indicates viable pollens and the blue arrow indicates semi-viable pollens\u003c/strong\u003e. (F) indicates all viable pollen grains for non-stored fresh pollens at 4°C.(G) represents all semi-viable pollens after storage of 1 week at 22°C.All images were taken under a light compound microscope(OLYMPUS) at 5x magnification.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2659708/v1/f20eb1c682223b0df1063665.png"},{"id":34197036,"identity":"b7b62691-70fc-41ae-b830-81486676f759","added_by":"auto","created_at":"2023-03-13 23:15:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":192222,"visible":true,"origin":"","legend":"\u003cp\u003ePollen germination after storage treatment with spike preservation method at 4°C (H) and (I); \u003cstrong\u003ethe blue arrow indicates no germination and the black arrow indicates germination having pollen tube\u003c/strong\u003e( 5x under light compound microscope).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2659708/v1/34fb5ec2493003340e8090a4.png"},{"id":34196258,"identity":"c6134bfa-04a7-4e51-8989-5e30a94dbf97","added_by":"auto","created_at":"2023-03-13 23:07:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":125351,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e)Pollen viability of 50 spring wheat genotypes after storage at 4°C and 1-week durations under two preservation methods. (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e pollen germination of 50 spring wheat genotypes after storage at 4°C and 1-week durations with the two preservation methods.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2659708/v1/623f4ac581ad7b0ea20bb09e.png"},{"id":34197037,"identity":"b675e0df-8127-420c-8c81-1cc11d842d6b","added_by":"auto","created_at":"2023-03-13 23:15:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1241799,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2659708/v1/12e6867f-0325-48fe-a357-baf1bfe8aba7.pdf"},{"id":34196257,"identity":"222a1c4d-dccd-4559-9db6-d71dccfc7498","added_by":"auto","created_at":"2023-03-13 23:07:12","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20702,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarytablesBMCPlantMethods.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2659708/v1/c97b182044bb26de300876a3.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Spike preservation: A simple method to preserve pollen viability and in vitro germination in wheat","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) is an allohexaploid (2n\u0026thinsp;=\u0026thinsp;6x\u0026thinsp;=\u0026thinsp;42, AABBDD) and obligate self- pollinating species [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Successful pollination and fertilization in wheat breeding rely on haploid male gametophyte. In flowering plants including wheat, pollen grain is male gametophyte that is shed after the completion of second pollen mitosis. The mature tricellular wheat pollen grain has relatively high moisture contents and is short-lived [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, pollination is necessary within 30 to 40 min after pollen shedding to achieve successful seed sets [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] under a wide range of temperate environments [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Terminal heat stress at anthesis stage is an emerging threat to wheat crop in South Asia and some other parts of the world. Heat stress at anthesis stage desiccates pollen grains affecting their size, viability, vigour and potential of making pollen tube to fertilize the female gametophyte [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Pollen viability reflects the performance of pollen grains in terms of stainability, germinability and fertilization ability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Pollen vigour refers to the speed and rate of germination and pollen tube formation.\u003c/p\u003e \u003cp\u003eIn wheat, pollen viability is mostly determined by pollen staining methods and within 30 minutes at room temperature or within few hours when stored at 4\u0026deg;C [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, and \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cem\u003eIn vitro\u003c/em\u003e pollen germination tests are performed mostly on fresh pollen grains since after shedding pollen grains remain alive for 30\u0026ndash;40 minutes at room temperature or few hours when preserved at 4\u0026deg;C [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. High quality crops also depend on the strength of the pollen and it is the most important factor in the breeding program [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the first 1.5 hours of storage, the viability of creeping bent grass (\u003cem\u003eAgrostis stolonifera\u003c/em\u003e L.) pollen decreased significantly, and after 3 hours, it was completely lost [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Within two hours in a field condition, pollens of maize became non-viable [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The pollen viability is significantly affected by moisture content, temperature and genotypes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Many crops depend on low temperature and low humidity while wheat crop prefers low temperature and high humidity for pollen viability and longevity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Adhikari and Campbell [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] in their study on buckwheat concluded that a high humidity was necessary for pollen longevity and viability. Genetic variations in blooming times among elite parents are one of the main problems with wheat breeding programs. Wheat breeders can overcome variations in flowering periods by preserving pollens from the desired male parents until pollination can take place. In general, wheat breeders believe that wheat pollen has a very limited lifespan and may be a barrier to the economically viable production of hybrid seed [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, there are few reports on the best storage conditions for wheat pollen grain [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. According to Fritz and Lukaszewski [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], pollination with pollen that had been kept for 45 minutes reduced seed set in spring wheat from 100% to less than 10%. Athwal and Kimber [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] found no seed set in \"Chinese Spring\" wheat pollinated only 5 minutes after anther dehiscence. Roemer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] reported that pollen stored at low temperatures preserved germination capability better than pollen stored at high temperatures. According to Hoekstra and Bruinsma [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], higher respiration is the reason for the viability reduction in three-celled pollen. When kept at 0\u0026deg;C\u0026ndash;10\u0026deg;C and 80\u0026ndash;100% RH, the pollen of various grasses only lasts a few days [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Further, Andronescu [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] reported that Graminaceous taxa's pollen grains lose viability very quickly, and under ideal storage conditions, successful preservation was thought to only last for around 10 days [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] However, Li Xun Zhen et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] preserved rice pollen grains at low temperature for 15 days. For measuring pollen germination vigour and percentage over time, \u003cem\u003ein vitro\u003c/em\u003e techniques have been used [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A genotype can be regarded a good pollinators if its pollen viability is high, and examination of pollen viability and pollen tube formations are crucial criteria for pollen evaluation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. On a solid medium raffinose, Cheng and McComb [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] produced the longest wheat pollen tube.\u003c/p\u003e \u003cp\u003eTesting pollen viability and pollen germination are essential for determining the quantity of pollen needed for good pollination. The pollen viability tests are indispensable for hybrid wheat breeding programs and breeding heat tolerant pure line wheat varieties [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Pollen viability is now considered as a key trait for heat tolerance in wheat [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Nonetheless, performing pollen viability tests to evaluate large number wheat lines are not feasible due to very short life span of pollen grains after their shedding from anther. To overcome this obstacle, we tried two simple and pollen preservation methods namely anther preservation and spike preservation. Pollen grains of a panel of 50 diverse genotypes were used to compare the results of the two preservation methods under two temperature conditions and two storage durations.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eThe pure seed of about 50 diverse spring wheat genotypes including land races, pre-green revolution, post-green revolution and recent cultivars and advanced lines were collected and arranged for three replications.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eField experiment\u003c/strong\u003e \u003cp\u003eThe experiment was conducted at National Agricultural research Center (NARC) Islamabad, Pakistan .The seed of about 50 diverse spring wheat cultivars and elite lines were grown in randomized complete block design (RCBD) with three replications. The seeds were sown with wheat planter in 1.2 m x 3m plots, consisting of six rows, 20 cm apart. The standard agronomic practices were used in each experiment to raise good quality crops.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePollen collection\u003c/strong\u003e \u003cp\u003eSpikes at anthesis stage were collected and kept in plastic bags. The bagged spikes were then transferred to lab. Two preservation methods were used for measuring pollen viability to test which method is robust and fast to extract pollen and maintain pollen viability for long term.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethod 1: Anther Preservation Method\u003c/h3\u003e\n\u003cp\u003eSpikes with yellow anthers were collected at the flowering stage in order to sample the anthers thereafter, anthers were extracted from sampled spikes by using forceps to open the glume and lemma. Then, the sampled anthers were stored for future use in tightly sealed plastic vials.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethod 2: Spikes Preservation method.\u003c/b\u003e At anthesis stage, spikes were harvested, collected, and preserved in plastic bags and immediately transported to the lab.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePollen storage\u003c/strong\u003e \u003cp\u003eThe anthers and spikes that were harvested for each genotype of wheat were kept at ambient temperature (22\u0026deg;C) and at (4\u0026deg;C) in the fridge as storage temperatures. After 0 (for fresh, non-stored pollens) and 1-week (for stored pollens) at two storage temperatures (22\u0026deg;Cand 4\u0026deg;C) pollen vitality was assessed. Pollen viability was tested after 0-week (non-stored, fresh pollens) and 1-week (stored pollens) in each storage temperature.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePollen viability\u003c/strong\u003e \u003cp\u003eTo determine the best long-term pollen storage conditions for maintaining high viability of pollen, two different storage temperatures (22\u0026deg;C and 4C\u0026deg;) with two storage durations (0-week and 1-week) were tested. The pollen grains of 50 genotypes collected from both procedures under two different storage conditions were placed on slides with one to two drops of ALEXANDER's solution and then it was covered with coverslips [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Next, a compound microscope (Olympus). was used to observe the level of pollen staining at a 5X magnification. However, in the standard stain, pollen that was fully and darkly stained (magenta-red or red) was viable, pollen that was lightly stained (magenta-red or red) was semi-viable, and pollen that was stained (blue-green, blue, or non-stained having no colour) was non-viable [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Pollen viability was measured as the proportion of stained to total pollen grains, and it was represented as a percentage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e pollen germination test\u003c/strong\u003e \u003cp\u003eTo examine pollen germination in 50 genotypes of wheat under anther and spike preservation method at 4\u0026deg;C, liquid pollen germination medium (without agar and peptone water) was made by dissolving H3BO3 (0.05g), Ca(NO3)2.4H2O (0.03g), and BK Salts viz (MgSO4.7H2O (0.2g) and KNO3(0.1g)), maltose (19%), and polyethylene glycol (PEG6000, 13%) at 6 pH as described by [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Using a light compound microscope (Olympus BX41 with DP12 camera) pollen grains and germinated pollen grains from a randomly selected microscopic field were counted to calculate \u003cem\u003ein vitro\u003c/em\u003e pollen germination. All images were taken at 5x\u0026acute;magnification. When the pollen tube's length exceeded the pollen grain's diameter, the pollen grain was considered germinated [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe pollen viability and \u003cem\u003ein vitro\u003c/em\u003e pollen germination data of 50 spring wheat lines were determined using two preservation techniques\u0026mdash;anther preservation and spike preservation under two storage conditions or temperatures, namely room temperature (22\u0026deg;C) and fridge (4\u0026deg;C), with two storage durations\u0026mdash;0 week for fresh pollens and 1 week for stored pollens (Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e and Table S2). Non-stored (0 week) pollen was shown to had better viability and \u003cem\u003ein vitro\u003c/em\u003e pollen germination rates than stored pollen (1 week).\u003c/p\u003e\n\u003cp\u003eWith anther preservation method at room temperature (22\u0026deg;C) maximum pollen viability (100%) was recorded for fresh non-stored pollens (0 week). After 1 week of storage at room temperature (22\u0026deg;C), all genotypes had non-viable (60\u0026ndash;100%) pollens except Punjab-76 (Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig.\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eWith anther preservation method at 4\u0026deg;C, all genotypes had viable pollens for fresh non-stored pollens (0-week). However, after 1-week of storage in fridge at 4\u0026deg;C, 37 genotypes showed maximum pollen viability (100%) while other had 40%-70% viability. \u003cem\u003eIn vitro\u003c/em\u003e pollen germination of 50 spring wheat genotypes ranged from (10%-30%) at 4\u0026deg;C for fresh non-stored pollens (0-week). After one week of storage at 4\u0026deg;C, all 50 spring wheat lines did not show any germination. Thus, pollen germination (10%-30%) was observed in all genotypes of wheat only for fresh non- stored (0 week)) as compared with those stored at 4\u0026deg;C for 1-week which did not germinate (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eWith spike preservation method at room temperature (22\u0026deg;C), the highest pollen viability(100%viable) was recorded for fresh non-stored pollens (0 week). However, after 1 week of storage at room temperature (22\u0026deg;C), 31 genotypes were non-viable (50\u0026ndash;100%) and 27 genotypes were semi-viable (10\u0026ndash;100%). On the other hand, at 4\u0026deg;C all genotypes were viable (100%) for fresh non-stored pollen (0 week). However, after 1 week of storage in fridge at 4\u0026deg;C all 50 genotypes showed (50\u0026ndash;87%) viability (Table\u0026nbsp;2 and Fig.\u0026nbsp;2). However, \u003cem\u003ein vitro\u003c/em\u003e pollen germination 50 spring wheat genotypes ranged from (30\u0026ndash;50%) at 4\u0026deg;C for fresh non-stored pollens (0 week). After 1 week of storage at 4\u0026deg;C in fridge, 50 spring wheat genotypes showed 2\u0026ndash;14% pollen germination (Table\u0026nbsp;2 and Fig.\u0026nbsp;3).\u003c/p\u003e\n\u003cp\u003eTaken together, pollen grains preserved with the spike preservation method at 4\u0026deg;C had better pollen grain viability and \u003cem\u003ein vitro\u003c/em\u003e germination after 1 week (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). When florets were kept closed and entire spikes were preserved, pollen viability was not rapidly lost since their possibilities of shedding decreased. The spike preservation method is efficient to preserve pollen grain viability in wheat for a longer period of time.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWheat pollen grain is known to have a relatively high moisture content and a short life span [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, pollination must be done within 30\u0026ndash;40 minutes of pollen shedding in order to produce effective seed sets [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Pollen viability has a significant effect on fertilization [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], embryonic development [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and quality of seed [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Plant pollen viability and longevity vary depending on species and environmental conditions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The ability of a pollen to retain its viability over time and under different storage temperatures depends on both the pollen's genetic makeup and environmental influences. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Optimum storage conditions for pollen also vary depending on species or cultivar [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. There are only a few studies on the short-term storage of spring wheat pollen at various temperatures [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To prevent pollen viability loss, proper storage temperatures are required [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].Thus the primary goal of this study was to establish the optimum storage temperature range for spring wheat pollen as well as how long wheat pollen can be stored under different storage conditions before losing viability.\u003c/p\u003e \u003cp\u003eThe results showed the Spike preservation method was effective to preserve pollen viability and germination under two storage temperatures (22\u0026deg;C and 4\u0026deg;C). Many factors can influence the viability of pollen, including pollen handling during collection, flowering maturation stage, and environmental conditions such as air temperatures and moisture contents [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, and \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Low temperatures are typically used for the long-term preservation of pollen because they reduce soluble sugar and organic acid consumption due to decreased pollen respiration [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The findings showed that pollen stored at low temperatures (4\u0026deg;C) had a higher percentage of germination than pollen stored at room temperature (22\u0026deg;C) for one week, and that germination percentages decreased as storage duration increased among all wheat genotypes. The inactivation of essential germination enzymes and substrates may be the root cause of the pollen grains' decreased ability to germinate when kept at room temperature [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The loss in pollen viability during storage is caused by enzymatic activity that reduces the amount of respiratory substrates [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFresh pollen at 0 week of storage had the highest pollen viability and \u003cem\u003ein vitro\u003c/em\u003e pollen germination for all wheat genotypes. It has been reported that storing pollen at lower temperatures and lower humidity levels often increases pollen lifespan [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Our findings demonstrated that wheat genotypes, pollen storage conditions, germination assessment tests, pollen viability, and storage period all affected pollen viability and \u003cem\u003ein vitro\u003c/em\u003e germination. The findings of the current study concur with those from other authors [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The genetic diversity among the genotypes under study may be the cause of the variations in pollen viability and germination [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Plant pollen viability and longevity vary depending on species and environmental conditions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. There are genotype-specific differences in pollen viability and longevity [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, and \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. \u003cem\u003eIn vitro\u003c/em\u003e pollen germination percentages were consistently lower than pollen viability tests for all 50 spring wheat genotypes evaluated. This could be as a result of the effect of various uncontrollable parameters including pollen density, the best growing medium, and the environmental requirements for each genotype [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In accordance with the findings of the current study, Cheng and McComb [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] found that wheat germination under in vitro conditions of pollen grains was low and variable, with a maximum germination rate of 6.8%. According to Devrnja et al. [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], trinucleate pollens germinate more quickly than binucleate pollens but have a much shorter lifespan. Additionally, according to Mulcahy and Mulcahy [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], some species with trinucleate pollen have difficulty developing pollen tubes \u003cem\u003ein vitro\u003c/em\u003e. It has been reported that differences in pollen longevity among plant species are caused by differences in pollen desiccation tolerance [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of pollen viability and germination tests conducted at 22\u0026deg;C and 4\u0026deg;C revealed that the maximum levels of pollen viability (100%) and germination (2\u0026ndash;14%) were found at 4\u0026deg;C by spike preservation method. In agreement with the current findings, D'Souza [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] observed that wheat pollen viability was increased after storage at 2 \u0026deg; to 5\u0026deg;C. In the current study, there were no differences in pollen viability for fresh (non-stored) pollens at 22\u0026deg;C under either preservation method. The current study found that storage duration had a significant impact on pollen viability and pollen germination. Under all storage temperatures, the pollen viability and germination rates of all wheat genotypes rapidly declined during the study period. According to the findings, fresh pollen had a high viability at 0 weeks but rapidly declined after 1 weeks of storage using either preservation technique. After a week of pollen storage at 4\u0026deg;C using anther preservation technique, the germination rate of nearly all wheat genotypes dropped to 0.00%. However, after a week of pollen storage at 4\u0026deg;C resulted in germination (2\u0026ndash;14%) using the spike preservation method. Our findings corroborate those of Parzies et al. [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], who found that in a genotype of barley originating from a semi-arid region, pollen viability decreased upto 90% after anthesis and less than 50% within 24 hours. Our findings supported earlier studies on other species of cereal, which showed that pollen grains from plants like sorghum and maize have extraordinarily short lifespans, with times between a few minutes and several hours [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePollen viability and \u003cem\u003ein vitro\u003c/em\u003e pollen germination with the spike preservation method was higher than those using anther preservation method in the selected panel of 50 spring wheat genotypes. According to a prior study, Spring wheat pollens preserved using anther preservation technique exhibited germination(1.64%), after 24 hours of storage ,but completely lost viability and revealed (0.00% after 48 and 72 hours) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Nevertheless, with the Spike preservation method we observed 2\u0026ndash;14% germination at 4\u0026deg;C after 1-week of storage.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003ePollen preserved at 4\u0026deg;C with the spike preservation method displayed higher pollen viability and germination when compared will pollen stored at 4\u0026deg;C with anther preservation method. Convincingly, the Spike preservation method works well to preserve pollen grain viability and germination at 4\u0026deg;C after 1-week of storage. The Spike preservation method will be useful for research on artificial pollination, hybrid wheat breeding. The preservation of pollen grains with this new method will also facilitate molecular studies on pollen grain in wheat.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Authors declare no conflict of interest\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eI.K performed the experiment, analyzed data and wrote the first draft and M.S conceptualized the study, supervised the experiments and improved the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShewry PR. 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Plant Physio. 2015; 10(2):55-67.\u003c/li\u003e\n\u003cli\u003eDevrnja N, Milojević J, Tubić L, Zdravković-Korać S, Cingel A, Ćalić D. Pollen morphology, viability, and germination of \u003cem\u003eTanacetum vulgare\u003c/em\u003e L. HortScience. 2012; 47(3):440-442.\u003c/li\u003e\n\u003cli\u003eMulcahy GB, Mulcahy DL.The effect of supplemented media on the growth \u003cem\u003ein vitro\u003c/em\u003e of bi-and trinucleate pollen. Plant Science. 1988;55(3): 213-216.\u003c/li\u003e\n\u003cli\u003eSong J,Tachibana S. Loss of viability of tomato pollen during long-term dry storage is associated with reduced capacity for translating polyamine biosynthetic enzyme genes after rehydration. Journal of Experimental Botany. 2007;58(15-16): 4235-4244.\u003c/li\u003e\n\u003cli\u003eD\u0026apos;SouzA L. Studies on the suitability of wheat as pollen donor for cross pollination, compared with rye, Triticale and Secalotricum. Zeitschrift fur Pflanzenzuchtung. 1970; 63: 246-69.\u003c/li\u003e\n\u003cli\u003eParzies HK, Schnaithmann F, Geiger HH. Pollen viability of Hordeum spp genotypes with different flowering characteristics. Euphytica.2005; 145: 229-235.\u003c/li\u003e\n\u003cli\u003eTuinstra MR ,Wedel J. Estimation of pollen viability in grain sorghum.Crop science. 2000;40(4): 968-970.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pollen viability, wheat, germination, spike, anther, preservation","lastPublishedDoi":"10.21203/rs.3.rs-2659708/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2659708/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\n\u003cp\u003eWheat pollen grains have very short longevity and are non-viable after ~30 minute at room temperature and ~60 minutes at 4\u003csup\u003e°\u003c/sup\u003eC. Pollen grain viability can be preserved maximum to ~24 hrs with existing preservation methods.\u003c/p\u003e\n\u003cp\u003eResults\u003c/p\u003e\n\u003cp\u003eHerein, we developed two simple methods-anther preservation and spike preservation- to preserve the pollen grain viability in wheat. \u0026nbsp;The methods were validated using viability and \u003cem\u003ein vitro \u003c/em\u003egermination of pollen grains of 50 diverse spring wheat genotypes. Anthers and spikes were collected for anther preservation and spike preservation methods, respectively, and stored at room temperature (22°C) and fridge (4°C) for 0 and 1-week. Pollen viability were assessed using Alexander staining techniques at two storage temperatures 22°C and 4°C. \u003cem\u003eIn vitro\u003c/em\u003e germination was determined using liquid germination medium at 4°C. After 1 week, the pollen viability and \u003cem\u003ein vitro \u003c/em\u003egermination as determined with Spike preservation method were higher as compared with those of anther preservation method at 4°C. In addition, \u003cem\u003ein vitro \u003c/em\u003epollen germination and pollen viability significantly reduced as storage duration increased. After 1-week, the pollen grains preserved with anther preservation method at 4°C failed to germinate.\u003c/p\u003e\n\u003cp\u003eConclusion\u003c/p\u003e\n\u003cp\u003eThe Spike preservation method is effective for preserving the pollen grain viability and \u003cem\u003ein vitro \u003c/em\u003egermination in a large panel of wheat genotypes. This new method is instrumental to further our understanding on pollen grain viability and germination.\u003c/p\u003e","manuscriptTitle":"Spike preservation: A simple method to preserve pollen viability and in vitro germination in wheat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-13 23:07:07","doi":"10.21203/rs.3.rs-2659708/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"16f83206-a9a6-4a55-889b-90b1047a547b","owner":[],"postedDate":"March 13th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-03-13T23:07:10+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-13 23:07:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2659708","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2659708","identity":"rs-2659708","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}