This project was financially supported by the National Natural Science Foundation of China (32122075, 32072523).

1. Preparation for aniline blue staining
<ol>
	<li>Prepare the following reagents and tools for the experiment: a pollinator brush, tweezers, a pencil, sulfate paper, a pollination bag, zip lock bags, paper clips, formaldehyde, glacial acetic acid, absolute ethanol, centrifuge tubes, forceps, glue droppers, glass slides, coverslips, scalpels, and polyethylene glycol.</li>
	<li>Prepare in vitro germination medium containing 0.02% MgSO4, 0.01% KNO3, 0.03% Ca(NO3)2, 0.01% H3BO3, 20% PEG-4000, and 15% sucrose. Adjust the pH to 6.0-6.2 with KOH. Use a magnetic stirrer as PEG-4,000 is difficult to dissolve.</li>
	<li>Prepare Carnoy&#39;s fixative solution, which is absolute ethanol and glacial acetic acid mixed at a ratio of 3:1. Prepare FAA fixation solution, which is 40% formaldehyde, 80% ethanol, and glacial acetic acid (1:8:1). Prepare 4 M sodium hydroxide (NaOH) and aniline blue solution, which is 0.1% aniline blue in 0.1 M K3PO4. Use an amber bottle to store the aniline blue solution because it is light-sensitive.</li>
</ol>
2. Pollen collection
<ol>
	<li>Know the flowering period of the experimental trees (Ma jia pummelo in this study) in advance. Collect mature unopened flowers from the beginning of the flowering period to the peak flowering stage, and put them in a zip lock bag. The flowers can be stored at 4 &#176;C in the refrigerator for 24 h.</li>
	<li>Take the flowers to the lab. Use tweezers to collect the anthers, and place them in a Petri dish containing filter paper. Collect anthers from 20 to 30 flowers.</li>
	<li>Place the Petri dish containing the anthers in a 28 &#176;C oven for 24 h until the pollen grains dry. For the detailed flower organization, see Hu et al.9.</li>
	<li>Put the dried pollen into a 1.5 mL centrifuge tube. Save the pollen in an air-tight zip lock bag containing color-changing silica gel (desiccant). Close the bag, and label the bag with the name of the pollen variety and the date of storage. The dried pollen can be stored in a &#8722;20 &#176;C refrigerator for 96 weeks16.</li>
</ol>
3. In vitro pollen germination test
<ol>
	<li>Pour 300 &#181;L of liquid medium into a cell culture dish or the cap of a 2 mL centrifuge tube, and sprinkle the pollen evenly with the help of a pollination brush. Incubate the pollen at 28 &#176;C in a humidified dark environment for 12 h.</li>
	<li>Remove the top 1 mm of the tip of a 1,000 &#181;L pipette tip. Use the pipette to absorb the pollen with a small amount of culture solution and move it to the center of the microscope slide. Cover it with a coverslip. Observe the specimen with an inverted microscope using a 10x objective.</li>
	<li>Perform three independent replicates using approximately the same pollen density for each replicate17. Visually manage the amount of pollen, making sure that whole of the Petri dish is covered with pollen and that each Petri dish has an almost equal amount of pollen.</li>
	<li>The germinated pollen produces a pollen tube with a length approximately twice its diameter. Calculate the germination rate from 20 visual fields, which gives the percentage of germinated pollen in all pollen fields.</li>
</ol>
4. Pollination
<ol>
	<li>Choose a sunny day without wind for pollination. Select 10 fully developed buds about to open, peel back the petals carefully, and take care not to bruise them.</li>
	<li>Use a pollination brush to spread a sufficient amount of viable pollen onto the surface of the stigma, and take care not to damage the pistil. For self-pollination, use the pollen from the same plant/cultivar. For cross-pollination, use the pollen from a plant with a different genotype.</li>
	<li>Cover the pollinated flowers with a sulfate paper bag. Use a paper clip to seal the bag and to prevent pollination by genotypically distinct pollens.</li>
	<li>Write the name of the species and the number and time of pollination on the label. Hang the label on the branches near the pollinated flowers.</li>
</ol>
5. Sampling, fixation, and preservation
<ol>
	<li>Remove the pollination bags approximately 3-4 days after pollination, and collect the pollinated flowers in zip lock bags.</li>
	<li>Immediately remove the petals, receptacles, and ovaries from the flowers, and immerse the stigmas fused to styles in a centrifuge tube containing a freshly prepared fixative solution. Incubate the stigmas and styles in the fixative solution overnight at 4 &#176;C.</li>
	<li>The next day, discard the fixative solution, and wash the stigmas and styles two to three times in 95% ethanol.</li>
	<li>Transfer the styles to a 70% ethanol solution. Ensure that the sample is completely immersed in the solution. The styles at this stage can be stored at 4 &#176;C for 1-2 months.</li>
</ol>
6. Aniline blue staining
<ol>
	<li>Wash the style samples stored in 70% ethanol with distilled water three to four times. Immerse in 4 M NaOH solution, seal, and incubate in a 65 &#176;C water bath for 60 min. During this step, the color of the style changes from yellow-white to orange-red.</li>
	<li>Soak the styles in distilled water for 30 min. Discard the distilled water, and wash the styles with distilled water three to four times or until the color of the style becomes yellow.</li>
	<li>Place the sample in a 10 mL tube, add the aniline blue until the sample has been dipped, and dye for 12 h in the dark.</li>
	<li>Observe the pollen tube growth with a fluorescence microscope.</li>
</ol>
7. Fluorescence microscopy
<ol>
	<li>Before observing the specimens, place the slide on a flat table, and add two to three drops of polyethylene glycol to the surface of the slide.</li>
	<li>Wash the style to be observed with distilled water. Use a scalpel to divide it into two halves along the longitudinal axis. Place one half of the style on the glass slide, and cover with a coverslip.</li>
	<li>Place the slide on the stage of the microscope above the aperture, and visualize using a 10x objective. Use the DAPI filter (excitation: 325-375 nm; emission: 435-485 nm). Observe five styles for each type of pollination. Observe the pollen tube growth.</li>
</ol>
8. PCR-based S genotype identification
<ol>
	<li>Extract the genomic DNA from the stigma sample using the CTAB method18.
	<ol>
		<li>Put the collected leaves into a 2 mL centrifuge tube, and snap-freeze in liquid nitrogen. Prepare HCl:EDTA:NaCl:H2O buffer at a ratio of 1:1:3:5 and a chloroform isoamyl alcohol mixture at a ratio of 24:1</li>
		<li>Add 10 mL of the prepared buffer, 0.2 g of CTAB, and 200 &#181;L of mercaptoethanol to a 50 mL centrifuge tube, and put them in a water bath at 65 &#176;C for 5 min until the solution becomes clear and transparent.</li>
		<li>Put the blades into a mortar, add the frozen samples, add liquid nitrogen, and grind. Put the ground sample into a 2 mL centrifuge tube, add 600 &#181;L of CTAB mixture, put it in a water bath at 65 &#176;C for 60 min, and mix it upside down every 30 min.</li>
		<li>Add 700 &#181;L of the chloroform isoamyl alcohol mixture (24:1), and mix upside down for 10 min. Centrifuge at 24 &#176;C at 12,000 x g for 10 min, pipette the supernatant, and transfer to a 1.5 mL centrifuge tube.</li>
		<li>Add 60 &#181;L of 5 M NaCl solution and 1 mL of absolute ethanol, and mix upside down. Freeze at &#8722;20 &#176;C for 30 min, and centrifuge at 24 &#176;C and 9,000 x g for 5 min.</li>
		<li>Discard the supernatant, add 1 mL of 70% ethanol solution, and leave at room temperature for 1-2 h. Centrifuge at 24 &#176;C, 9,000 x g for 5 min, discard the supernatant, aspirate the excess ethanol solution, and air-dry for 5 min.</li>
		<li>Add 100 &#181;L of sterile water to dissolve, measure the DNA concentration with a spectrophotometer, and freeze in a -4 &#176;C freezer for long-term storage.</li>
		<li>Configure the RT-PCR reaction system. Prepare the following reaction mix for 10 &#181;L containing 5 &#181;L of 2x PCRMix, 0.25 &#181;L each of forward and reverse primer, 1 &#181;L of DNA (100 ng/ &#181;L), and 3.5 &#181;L of H2O.</li>
	</ol>
	</li>
	<li>Set up the PCR program as per Table 1. The PCR program for all isoforms was 95 &#176;C for 5 min 32x (95 &#176;C for 30 s, 55 &#176;C for 30 s, and 72 &#176;C for 1 min) and 72 &#176;C for 5 min. Separate the products on 1.5% TAE-agarose gels and photograph9. Verify the specified S genotype using genomic DNA.</li>
</ol>

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The authors declare that they have nothing to disclose.

In fruit crops, both parthenocarpy and SI are important traits because they pave the way for seedless fruits - a trait that is highly appreciated by consumers. Self-incompatibility promotes the rejection of self-pollen and, thus, prevents inbreeding20. Among citrus, pummelo is a self-incompatible variety7. Almost 40% of all angiosperm species exhibit SI21. This trait prevents fruit setting, lowers the yield, and brings huge economic losses to growers. To solve this problem, farmers include pollinizer trees throughout their orchards. However, the selection of suitable pollinizer trees is a challenging task that requires time-consuming laboratory experiments. To solve these issues, we have developed a rapid method for identifying SI genotypes and determining the pollen compatibilities and incompatibilities of different citrus varieties to facilitate the accurate selection of pollinizer trees. Moreover, the pollen viability and germination rates can also be determined using the in vitro method described in this protocol.
There are some reports on the determination of SI genotypes and self-(in)compatibility and inter-(in)compatibility using a combination of different methods in Japanese plum and apricot22,23. The development of S-specific primers relies on the identification of the S genotype. In citrus, the transcriptome analysis of stigma and pollen from 64 pummelo accessions has identified nine S-RNases specifically expressed in the styles and one variant of the S-RNase. Another 12 pairs of S-specific primers were developed later in citrus by Liang et al.7 and Wei et al.19. However, relative to pear and apple, fewer S genotypes have been identified in citrus4. The identification of PCR-based S genotypes is a critical step, as it provides a basis for compatible/incompatible combinations. There are also some limitations to this protocol. The S genotypes of some citrus varieties cannot be identified using this method. This finding indicates that further expansion of the S genotype library in citrus is required. Additionally, the S-specific primers cannot distinguish the S genotypes of cultivars with highly similar S sequences and, thus, nonspecifically amplify similar S sequences.
Altogether, due to its cost-effectiveness and ease of use, this method is an effective tool for determining pollen compatibility and incompatibility for different citrus varieties. This protocol can be used for the selection of suitable pollinizer trees and in breeding research programs. It can be applied to several species from the Rutaceae family (e.g., Citrus trifoliata and Fortunella japonica) for the selection of pollinizer trees.

Self-incompatibility (SI) is a genetically controlled mechanism present in approximately 40% of angiosperm species. In this process, the pistil rejects pollen from a plant with the same SI genotype and, thus, prevents self-fertilization1,2. Ma jia pummelo is a local variety in Jinagsu province, China, with the excellent qualities of large, pink fruit, a rich juice content, a sweet and sour taste, and a thick peel3. Although SI promotes outcrossing, it negatively impacts the yield and quality of fruits4 and necessitates suitable pollinizer trees with distinct SI genotypes for reliable fruit-setting rates and high yields. At present, there are two main types of SI, sporophytic self-incompatibility (SSI), represented by Brassicaceae, and gametophytic self-incompatibility (GSI), represented by Rosaceae, Papaveraceae, Rutaceae, and Solanaceae5,6,7,8.
Citrus is one of the most important fruit crops in the world. The S-RNase-based GSI system is found in many citrus accessions and negatively influences the fruit-setting rate9. In this system, SI is controlled by the S locus, a single polymorphic locus with two complex alleles that carry pistil S determinants and pollen S determinants7. The female determinant is the S ribonuclease (S-RNase), and the male determinant is the S locus F-box (SLF)7. The cells of the pistil secrete S-RNase proteins. The non-self S-RNases are recognized by the SLF proteins, which leads to the ubiquitination and degradation of the non-self S-RNases by the 26S proteasome pathway. In contrast, the self S-RNases are able to accumulate and inhibit pollen tube (PT) growth because they evade the SLF proteins and, therefore, are prevented from ubiquitinzation10,11,12,13.
Here, we report an in vivo technique that is useful for identifying S-genotypes and degrees of pollen compatibility and incompatibility. The protocol involves extracting total DNA from leaves and predicting the S genotype using S-specific primers. Moreover, aniline blue staining and fluorescence microscopy followed by hand pollination provide evidence for the degree of compatibility and incompatibility. The semi in vivo pollination procedure, which involves the manual pollination of flowers in the laboratory14,15,&#160;has also been adapted to assess the degrees of self-compatibility and incompatibility. However, we have also used field pollination followed by the bagging of flowers to avoid contamination from undesired pollen to allow the pollen tubes to develop in natural conditions. This protocol is simple and straightforward and provides the information necessary for the successful selection of suitable pollinizer trees.

<table><tbody><tr><td>absolute ethanol</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10009218</td><td></td></tr><tr><td>Aniline blue</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td></td><td></td></tr><tr><td>Boric acid, H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10004818</td><td></td></tr><tr><td>Brown bottle</td><td>Labgic Technology Co., Ltd</td><td></td><td></td></tr><tr><td>Calcium nitrate tetrahydrate, Ca(NO3 )2</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>80029062</td><td></td></tr><tr><td>Centrifugal tube</td><td>Labgic Technology Co., Ltd</td><td></td><td></td></tr><tr><td>centrifuge tubes</td><td>Labgic Technology Co., Ltd</td><td></td><td></td></tr><tr><td>CTAB</td><td>GEN-VIEW SCIENTIFIC INC</td><td>57-09-0(CAS)</td><td></td></tr><tr><td>Dropping</td><td>Jiangsu Songchang Medical Equipment Co., Ltd</td><td></td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid, EDTA</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10009617</td><td></td></tr><tr><td>Forceps</td><td>LUXIANZI Biotechnology Co., Ltd</td><td></td><td></td></tr><tr><td>formaldehyde</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10010018</td><td></td></tr><tr><td>Fully automatic sample fast grinder</td><td>Shanghai Jingxin Industrial Development Co., Ltd</td><td>Tissuelyser-96</td><td></td></tr><tr><td>glacial acetic acid</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10000218</td><td></td></tr><tr><td>Grinding Tube</td><td>Shanghai Jingxin Industrial Development Co., Ltd</td><td></td><td></td></tr><tr><td>Isoamyl alcohol</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10003218</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>80109218</td><td></td></tr><tr><td>label</td><td>M&amp;amp;G Chenguang Stationery Co., Ltd.</td><td></td><td></td></tr><tr><td>Leica DMi8</td><td>Shanghai Leica Co.,Ltd</td><td>21903797</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate, MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10013018</td><td></td></tr><tr><td>MICROSCOPE Cover glass</td><td>Zhejiang Shitai Industrial Co., Ltd</td><td></td><td></td></tr><tr><td>NaCl</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10019318</td><td></td></tr><tr><td>paper clips</td><td>M&amp;amp;G Chenguang Stationery Co., Ltd.</td><td></td><td></td></tr><tr><td>pencil</td><td>M&amp;amp;G Chenguang Stationery Co., Ltd.</td><td></td><td></td></tr><tr><td>pollinator brush</td><td>Shanghai Yimei Plastics Co., Ltd</td><td></td><td></td></tr><tr><td>Polyethylene glycol, PEG 6000</td><td>Beijing Dingguo Changsheng Biotechnology Co., Ltd</td><td>DH229-1</td><td></td></tr><tr><td>Polyethylene glycol, PEG-4000</td><td>Guangzhou saiguo biotech Co., Ltd</td><td>1521GR500</td><td></td></tr><tr><td>Potassium hydroxide, KOH</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10017008</td><td></td></tr><tr><td>Potassium nitrate, KNO3</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10017218</td><td></td></tr><tr><td>Scalpel</td><td>Jiangsu Songchang Medical Equipment Co., Ltd</td><td></td><td></td></tr><tr><td>Slide</td><td>Zhejiang Shitai Industrial Co., Ltd</td><td></td><td></td></tr><tr><td>Sodium hydroxide, NAOH</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10019718</td><td></td></tr><tr><td>Sucrose</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10021418</td><td></td></tr><tr><td>sulfate paper</td><td>Taizhou Jinnong Mesh Factory</td><td></td><td></td></tr><tr><td>Thermostat water bath</td><td>Shanghai Jinghong Experimental Equipment Co., Ltd</td><td>L-909193</td><td></td></tr><tr><td>Trichloromethane</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10006818</td><td></td></tr><tr><td>Tripotassium phosphate tribasic trihydrate, K&lt;sub&gt;3&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Shanghai Lingfeng Chemical Reagent Co.,Ltd</td><td>20032318</td><td></td></tr><tr><td>Tris-HCl</td><td>GEN-VIEW SCIENTIFIC INC</td><td>1185-53-1</td><td></td></tr><tr><td>zip lock bags</td><td>M&amp;amp;G Chenguang Stationery Co., Ltd.</td><td></td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>GEN-VIEW SCIENTIFIC INC</td><td>60-24-2(CAS)</td><td></td></tr></tbody></table>

determination of self-(in)compatibility and inter-(in)compatibility relationships in citrus using manual pollination, microscopy, and s-genotype analyses

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and fertilization. However, identifying suitable varieties to serve as pollinizers is a time-consuming process. To solve this problem, we have developed a rapid method for identifying pollination-compatible citrus cultivars that utilizes agarose gel electrophoresis and aniline blue staining. Pollen compatibility is determined based on the identification of S genotypes by extracting total DNA and performing PCR-based genotyping assays with specific primers. Additionally, styles are collected 3-4 days after manual pollination, and aniline blue staining is performed. Finally, the growth status of the pollen tubes is observed with a fluorescence microscope. Pollen compatibility and incompatibility can be established by observing whether the pollen tube growth is normal or suppressed, respectively. Due to its simplicity and cost-effectiveness, this method is an effective tool for determining the pollen compatibility and incompatibility of different citrus varieties to establish incompatibility groups and incompatibility relationships between different cultivars. This method provides information essential for the successful selection of suitable pollinizer trees and, thus, facilitates the establishment of new orchards and the selection of appropriate parents for breeding programs.

For the experiments done here, mature flowers were selected, the anthers&#160;were collected, dried in an oven, and the pollen was germinated at 28&#176;C for 12 h. The pollen viability and germination rates were quantified as shown in Figure 1.
Citrus was manually pollinated, and the pollen compatibility and incompatibility were assessed using aniline blue staining and fluorescence microscopy. The compatible pollen could germinate on the surface of the stigma and produce a normal pollen tube that could grow and ultimately lead to fertilization in the ovary. In contrast, incompatible pollen tubes grew through approximately two-thirds of the style and then stopped growing (Figure 2).
To identify the S genotype, total DNA was extracted from the plant. Specific primers were designed based on the sequence of the S locus that were useful for amplifying part of the S locus in the PCR reactions. The amplification products were analyzed using agarose gel electrophoresis. The amplified bands were detected (between 500-1,000 bp). The corresponding S genotype was identified (Figure 3). By this method, we have identified the S genotypes of 63 pummelo germplasm resources7. Our group has identified 21 S-haplotypes in different citrus species using this method19 (Table 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65056/65056fig01.jpg" /> 
Figure 1:&#160;Different rates of pollen germination. Germination and growth of the pollen. (A) The viable pollen has a higher germination rate, and a normal pollen tube can be grown. (B) The non-viable or less viable pollen has a much lower germination rate, and few pollen tubes can grow. Scale bars = 100 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65056/65056fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65056/65056fig02.jpg" /> 
Figure 2:&#160;Fluorescence microscopy images of pollen tubes in pistils after pollination. (A) Self-compatible pistil with numerous growing pollen tubes. (B) Self-incompatible pistil with pollen tube growth arrested within the style. Abbreviations: Pt = pollen tube; vb = vascular bundle. Scale bars = 1 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65056/65056fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65056/65056fig03.jpg" /> 
Figure 3:&#160;Specific amplification of the S-RNase gene from Ma jia pummelo. After PCR amplification and gel electrophoresis, it was found that the two amplified bands S10 and S16 were the brightest. These data indicate that the genotype of Ma jia pummelo was S10 and S16. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65056/65056fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: The reaction system used for the PCR-based S genotype identification. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65056/Table 1 PCR reaction system.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: List of the primers for 21 S genotypes in citrus identified by our group. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65056/Table 2 The specific primers for S-genotypes identification.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Determination of Self-Incompatibility and Inter-Incompatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses at JoVE.

Determination of Self-Incompatibility and Inter-Incompatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

This protocol provides a rapid method for determining pollen compatibility and incompatibility in citrus cultivars.

Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and ...

determination-self-compatibility-inter-compatibility-relationships

Universidad San Sebastian

Research

JoVE Journal

Biology

2.2K Views.  Huazhong Agricultural University. This protocol provides a rapid method for determining pollen compatibility and incompatibility in citrus cultivars.

Video: Determination of Self-Incompatibility and Inter-Incompatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 &deg;C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

Environment

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify the pollination effectiveness of each pollinator for visited flowers. Pollinator observations have been one of the most common methods to identify pollinators, and bagging and cage experiments have been conducted to show the effectiveness of specific pollinators. In a previous study of Lycoris sanguinea var. sanguinea, its effective pollinators, the visitation frequencies of each floral visitor, and its reproductive strategies were not identified. This study reports the observation that small bees visited flowers that were partially opened (breaking buds). To the best of our knowledge, this phenomenon has not been reported previously. Further, this study investigates the hypothesis that small bees can pollinate at that flowering stage. This study demonstrates the basic methods of field experiments in pollination ecology using L. sanguinea var. sanguinea. Pollinator observations and digital video showed the visitation frequencies of each floral visitor. Bagging and cage experiments revealed that these flowers could be pollinated fully and that breaking-bud pollination could be important for the pollination of this plant species. The advantages and disadvantages of each method are discussed, and recent developments, including laboratory experiments, are described.

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

To reveal the pollinator effectiveness of a given plant species, multiple methods of field experiments have been developed. This study demonstrates the basic methods of field experiments for pollination ecology using the case study of Lycoris sanguinea var. sanguinea and the novel pollination mechanism, breaking-bud pollination.

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify ...

Forced Flowering in Mandarin Trees under Phytotron Conditions

Phytotron has been widely used to assess the effect of numerous parameters on the development of many species. However, less information is available on how to achieve fast profuse flowering in young fruit trees with this plant growth chamber. This study aimed to outline the design and performance of a fast clear methodology to force flowering in young mandarin trees (cv. Nova and cv. Clemenules) and to analyze the influence of induction intensity on inflorescence type. The combination of a short water stress period with simulated spring conditions (day 13 h, 22 &#176;C, night 11 h, 12 &#176;C) in the phytotron allowed flowers to be obtained only after 68-72 days from the time the experiment began. Low-temperature requirements were adequately replaced with water stress. Floral response was proportional to water stress (measured as the number of fallen leaves): the greater the induction, the larger the quantity of flowers. Floral induction intensity also influenced inflorescence type and dates for flowering. Details on artificial lighting (lumens), photoperiod, temperatures, plant size and age, induction strategy and days for each stage are provided. Obtaining flowers from fruit trees at any time, and also several times a year, can have many advantages for researchers. With the methodology proposed herein, three, or even four, flowering periods can be forced each year, and researchers should be able to decide when, and they will know, the duration of the entire process. The methodology can be useful for: flower production and in vitro pollen germination assays; experiments with pests that affect early fruit development stages; studies on fruit physiological alterations. All this can help plant breeders to shorten times to obtain male and female gametes to perform forced-crosses.

Here, we present a protocol to force flowering in mandarin trees under phytotron conditions. Water stress, high illuminance and a simulated spring photoperiod allowed viable flowers to be obtained in a short period of time. This methodology allows researchers to have several flowering periods in 1 year.

Phytotron has been widely used to assess the effect of numerous parameters on the development of many species. However, less information is available on ...

Comparative Karyotype Analysis of Lycoris aurea Herb Populations Using Fluorochrome Banding and 45S and 5S rDNA-FISH

To understand karyotype variation in eight populations, detailed karyotypes were meticulously established using chromosomal measurements, fluorescence bands, and rDNA FISH signals. The number of 45S rDNA sites varies from one to five pairs per population, with the most common number per karyotype being four pairs. The 45S rDNA locus is predominantly located in the short arms and terminal regions of chromosomes, while the 5S rDNA locus is found mainly in the short arm and the terminal or proximal regions. Populations HBWF, HNXN, HBBD, and HNZX showed a similar distribution of 45S rDNA sites, as did GXTL, HBFC, and SCLS, indicating a close relationship between populations with similar 45S rDNA site distributions. The karyotypes of all studied populations are symmetrical, comprising stable and metastable centromeres or exclusively stable centromeres. Scatter plots of MCA and CVCL effectively distinguish their karyotypic structures. The analysis includes six quantitative parameters (x, 2n, TCL, MCA, CVCL, CVCI). Additionally, the results indicate that PCoA based on these six parameters is a robust method for determining biological karyotype relationships among the eight populations. The chromosome number in Lycoris populations is x = 6-8. Based on the current study and literature, genomic differentiation of these populations is discussed in terms of genome size, heterochromatin, 45S and 5S rDNA sites, and karyotype asymmetry.

This protocol investigates the heterochromatin distribution in eight populations of Lycoris aurea (L&#8242;Her) Herb, using combined PI and DAPI chromosome staining, fluorescence in situ hybridization (FISH) with 18S-5.8-26S rRNA gene regions (45S rDNA regions), and 5S ribosomal DNA probes.

To understand karyotype variation in eight populations, detailed karyotypes were meticulously established using chromosomal measurements, fluorescence ...

Genetics

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. In apricot, the determination of self- and inter-(in)compatibility relationships is increasingly important, since the release of an important number of new cultivars has resulted in the increase of cultivars with unknown pollination requirements. Here, we describe a methodology that combines the determination of self-(in)compatibility by hand-pollinations and microscopy with the identification of the S-genotype by PCR analysis. For self-(in)compatibility determination, flowers at balloon stage from each cultivar were collected in the field, hand-pollinated in the laboratory, fixed, and stained with aniline blue for the observation of pollen tube behavior under the fluorescence microscopy. For the establishment of incompatibility relationships between cultivars, DNA from each cultivar was extracted from young leaves and S-alleles were identified by PCR. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars, which provides a valuable information to choose suitable pollinizers in the design of new orchards and to select appropriate parents in breeding programs.

Determination of Self- and Inter-incompatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

We present a methodology to establish the pollination requirements of apricot (Prunus armeniaca L.) cultivars combining the determination of self-(in)compatibility by fluorescence microscopy with the identification of the S-genotype by PCR analysis.

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. ...

Developmental Biology

Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping

The Japanese plum cultivars commonly grown are interspecific hybrids derived from crosses between the original Prunus salicina with other Prunus species. Most hybrids exhibit gametophytic self-incompatibility, which is controlled by a single and highly polymorphic S-locus that contains multiple alleles. Most cultivated hybrids are self-incompatible and need pollen from a compatible donor to fertilize their flowers. Establishing pollination requirements in Japanese plum is becoming increasingly important due to the high number of new cultivars with unknown pollination requirements. In this work, a methodology for the determination of pollination requirements in Japanese plum-type hybrids is described. Self-(in)compatibility is determined by hand-pollinations in both the field and in the laboratory, followed by monitoring pollen tube elongation with fluorescence microscopy, and also monitoring fruit maturation in the field. Selection of pollinizer cultivars is assessed by combining the identification of S-genotypes by PCR analysis with the monitoring of flowering time in the field. Knowing the pollination requirements of cultivars facilitates the selection of cultivars for the design of new orchards and allows the early detection of productivity problems related with pollination deficiency in established orchards.

A methodology for the determination of pollination requirements in Japanese plum-type hybrids is described, which combines field- and laboratory-pollinations and observations of pollen tubes under the fluorescence microscopy with the identification of S-genotypes by PCR and the monitoring of flowering for the selection of pollinizers.

The Japanese plum cultivars commonly grown are interspecific hybrids derived from crosses between the original Prunus salicina with other Prunus species. ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae

Saccharomyces cerevisiae is a widely used model organism in genetics, evolution, and molecular biology. In recent years, it has also become a popular model organism to study problems related to speciation. The life cycle of yeast involves both asexual and sexual reproductive phases. The ease of performing evolution experiments and the short generation time of the organism allow for the study of the evolution of reproductive barriers. The efficiency with which the two mating types (a and &#945;) mate to form the a/&#945; diploid is referred to as the mating efficiency. Any decrease in the mating efficiency between haploids indicates a pre-zygotic barrier. Thus, to quantify the extent of reproductive isolation between two haploids, a robust method to quantify the mating efficiency is required. To this end, a simple and highly reproducible protocol is presented here. The protocol involves four main steps, which include patching the haploids on a YPD plate, mixing the haploids in equal numbers, diluting and plating for single colonies, and finally, calculating the efficiency based on the number of colonies on a drop-out plate. Auxotrophic markers are employed to clearly make the distinction between haploids and diploids.

Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae

In this work, a robust method for the quantification of mating efficiency in the yeast Saccharomyces cerevisiae is described. This method is particularly useful for the quantification of pre-zygotic barriers in speciation studies.

Saccharomyces cerevisiae is a widely used model organism in genetics, evolution, and molecular biology. In recent years, it has also become a popular ...

An Improved Method to Measure Pollen Hydration Profiles in Arabidopsis thaliana

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

Author Spotlight: A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...

Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

Summary

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Chapters in this video

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Forced Flowering in Mandarin Trees under Phytotron Conditions

Comparative Karyotype Analysis of Lycoris aurea Herb Populations Using Fluorochrome Banding and 45S and 5S rDNA-FISH

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana