Name: establishing pollination requirements in japanese plum by phenological monitoring, hand pollinations, fluorescence microscopy and molecular genotyping
Uploaded: 2020-11-09T15:30:00
Description: Watch this Scientific Journal Video about Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping at JoVE

This research was funded by Instituto Nacional de Investigaci&#243;n y Tecnolog&#237;a Agraria y Alimentaria (RFP2015-00015-00 and RTA2017-00003-00); Gobierno de Arag&#243;n&#8212;European Social Fund, European Union (Grupo Consolidado A12-17R), and Junta de Extremadura &#8212;Fondo Europeo de Desarrollo Regional (FEDER), Plan Regional de Investigaci&#243;n (IB16181), Grupo de Investigaci&#243;n (AGA001, GR18196). B.I. Guerrero was supported by a fellowship of Consejo Nacional de Ciencia y Tecnolog&#237;a of M&#233;xico (CONACYT, 471839).

1. Hand-pollination in the field
<ol>
	<li>Pollen extraction
	<ol>
		<li>To obtain pollen, collect flower buds at stage D24, according to stage 57 on the BBCH scale25,26. 
		NOTE: More flower buds are necessary in Japanese plum than in other Prunus species because their anthers produce less pollen.</li>
		<li>Remove the anthers using a plastic mesh (2 mm x 2 mm pore size) and place them on paper at room tem...

The methodology described herein for pollination requirements of Japanese plum cultivars requires determining the self-(in)compatibility of each cultivar by controlled pollinations in the field or the laboratory, and the subsequent observation of pollen tube growth with fluorescence microscopy. The incompatibility relationships are established by the characterization of the S-alleles by molecular genotyping. Finally, the selection of pollinizers is performed by the monitoring phenology to detect those cultivars ...

Japanese plum (Prunus salicina Lindl.) is native to China1. In the 19th century, this crop was introduced from Japan to the United States, where it was intercrossed with other North American diploid plums2. In the 20th century, some of these hybrids were spread to temperate regions around the world. Nowadays, the term &#8220;Japanese plum&#8221; refers to a wide range of interspecific hybrids derived from crosses between the original P. salicina with up to 15 other diploid Prunus spp.3,4,5.
Japanese plum, like other species of the Rosaceae family, exhibits Gametophytic Self-Incompatibility (GSI), which is controlled by a single and highly polymorphic S-locus containing multiple alleles6. The S-locus contains two genes that encode a ribonuclease (S-RNase) expressed in the pistil, and an F-box protein (SFB) expressed in the pollen grain7. In the self-Incompatibility reaction, when the S-allele expressed in the pollen grain (haploid) is the same as one of the two expressed in the pistil (diploid), the growth of the pollen tube across the style is arrested due to the degradation of the pollen tube RNA by the action of the S-RNase8. Since this process prevents fertilization of the female gametophyte in the ovule, GSI promotes the outcrossing between cultivars.
Although some Japanese plum cultivars are self-compatible, most cultivars currently grown are self-incompatible, and need pollen from inter-compatible donors to fertilize their flowers3. In stone fruit species of genus Prunus such as almond9, apricot10,11,12 and sweet cherry13, pollination requirements of cultivars can be established by different approaches. Self-(in)compatibility can be determined by self-pollination of flowers in the field and subsequent monitoring of fruit set, or by semi-in vivo self-pollinations at controlled conditions in a laboratory and the observation of pollen tubes under the microscope14,15,16,17,18. Incompatibility relationships among cultivars can be determined by cross pollinations in the field or the laboratory using pollen of the potential pollinizer cultivar, and by the identification of S-alleles of each cultivar by PCR analysis14,15,16,19,20,21,22. In species such as sweet cherry or almond, self-(in)compatibility can be also assessed by the identification of particular S alleles associated to self-compatibility, as S4&#8217; in sweet cherry13 or Sf in almond23.
Several plum breeding programs from the main producing countries are releasing a number of new cultivars2,14, many of them 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 self-pollinations in both the field and the laboratory, followed by observations of pollen tubes under the fluorescence microscopy. Selection of pollinizer cultivars combines the identification of S-genotypes by PCR analysis with the monitoring of flowering time in the field.

<table>
	<tbody>
		<tr>
			<td>Acetic Acid Glacial</td>
			<td>Panreac</td>
			<td>131008.1611</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>iNtRON Biotechnology</td>
			<td>25999</td>
			<td></td>
		</tr>
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			<td>Aniline blue</td>
			<td>Difco</td>
			<td>8504-88</td>
			<td></td>
		</tr>
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			<td>Boric Acid (H3BO4)</td>
			<td>Panreac</td>
			<td>131015.1210</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Nitrate 4-hydrate (Ca(NO3)2&#183;4H2O)</td>
			<td>Panreac</td>
			<td>131231.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverglass</td>
			<td>Deltalab</td>
			<td>D102460</td>
			<td>24 mm x 60 mm</td>
		</tr>
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			<td>Digital Camera</td>
			<td>Imaging Developmet Systems</td>
			<td>UI-1490SE</td>
			<td></td>
		</tr>
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			<td>Digital Camera Software Suite</td>
			<td>Imaging Developmet Systems</td>
			<td>4.93.0.</td>
			<td></td>
		</tr>
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			<td>DNA Oligos</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
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			<td>dNTP Mix, 10 mM each</td>
			<td>ThermoSischer Scientific</td>
			<td>R0193</td>
			<td></td>
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			<td>DreamTaq Green DNA polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td>EP0713</td>
			<td></td>
		</tr>
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			<td>Ethanol 96&#176;</td>
			<td>VWR-Chemicals</td>
			<td>83804.360</td>
			<td></td>
		</tr>
		<tr>
			<td>1Kb DNA Ladder (U.S. Patent No. 4.403.036) (500pb-12Kb)</td>
			<td>Invitrogen</td>
			<td>15615-016</td>
			<td>Size: 250&#181;g; Conc: 1.0 &#181;g/&#181;l</td>
		</tr>
		<tr>
			<td>Gel Documentation System</td>
			<td>Bio-Rad</td>
			<td>1708195</td>
			<td></td>
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			<td>Hand Counter</td>
			<td>Tamaco</td>
			<td>TM-4</td>
			<td></td>
		</tr>
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			<td>Image Lab Software</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>Image Analyse System for Gel Documentation System</td>
		</tr>
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			<td>MetaPhor Agarose</td>
			<td>Lonza</td>
			<td>50180</td>
			<td></td>
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			<td>Microcentrifuge 5415 R</td>
			<td>Eppendorf</td>
			<td>Z605212</td>
			<td></td>
		</tr>
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			<td>Microscope with UV epiflurescence</td>
			<td>Leica</td>
			<td>DM2500</td>
			<td>Exciter filter BP340-390, Barrier filter LP425</td>
		</tr>
		<tr>
			<td>Microslides</td>
			<td>Deltalab</td>
			<td>D100004</td>
			<td>26 mm x 76 mm</td>
		</tr>
		<tr>
			<td>Mini Electrophoresis System</td>
			<td>Fisherbrand</td>
			<td>14955170</td>
			<td></td>
		</tr>
		<tr>
			<td>Minicentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>15334204</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Deltalab</td>
			<td>200201</td>
			<td>55 mm x 14 mm</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Tribasic (K3PO4&#183;1.5H2O)</td>
			<td>Panreac</td>
			<td>141513</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer forward &#39;Pru C2&#39;</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer forward Pru T2&#39;</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer reverse &#39;PCER&#39;</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
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			<td>RedSafe Nucleic Acid Staining Solution</td>
			<td>iNtRON Biotechnology</td>
			<td>21141</td>
			<td></td>
		</tr>
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			<td>Saccharose</td>
			<td>Panreac</td>
			<td>131621.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulphite anhydrous (Na2SO3)</td>
			<td>Panreac</td>
			<td>131717.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Speedtools plant DNA extraction Kit</td>
			<td>Biotools</td>
			<td>21272</td>
			<td></td>
		</tr>
		<tr>
			<td>TBE Buffer (10X)</td>
			<td>Panreac</td>
			<td>A0972,5000PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler T100</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer comfort</td>
			<td>Eppendorf</td>
			<td>T1317</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Autoclave Presoclave II</td>
			<td>JP Selecta</td>
			<td>4001725</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisherbrand</td>
			<td>11746744</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Each Japanese plum flower bud contains an inflorescence with 1&#8211;3 flowers. As in other stone fruit species, each flower is made up of four whorls: carpel, stamens, petals, and sepals, which are fused forming a cup at the base of the flower. Flower structures are smaller than other stone fruits, with a short and fragile pistil surrounded by the stamens that contain a small amount of pollen grains. At full bloom, the flowers of each inflorescence appear separated on short stalks, showing the white petals forming a bal...

Watch this Scientific Journal Video about Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping at JoVE

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

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. ...

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