Name: forced flowering in mandarin trees under phytotron conditions
Uploaded: 2019-03-06T15:00:00
Description: Watch this Scientific Journal Video about Forced Flowering in Mandarin Trees under Phytotron Conditions at JoVE.com

The authors thank Jos&#233; Javier Zaragoz&#225; Dolz for providing technical assistance and helping in the management tasks. This research was partially supported by the Asociaci&#243;n Club de Variedades Vegetales Protegidas as part of a project undertaken with the Universitat Polit&#232;cnica de Val&#232;ncia (UPV 20170673).

1. Growth chamber characteristics and regulation requirements
<ol>
	<li>Use a growth chamber measuring 1.85 m x 1.85 m x 2.5 m (L x W x H) with a total volume of 8.56 m3 (Figure 1). A bigger or smaller growth chamber can be resorted to if necessary. 
	NOTE: Almost any room, or even a greenhouse, can be adapted to be used as a growth chamber.</li>
	<li>Check if regulations such as temperature (day/night), photoperiod (day/night), light intensity and minimum relative humidity...

<ol>
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Effects of temperature and photoperiod on the flower formation.</a> Tohoku Journal of Agricultural Research. 13 (3), 191-203 (1962).</li><li>Shillo, R., Halevy, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+photoperiod+and+temperature+in+flowering-control+of+Gypsophila+paniculata+L.">Interaction of photoperiod and temperature in flowering-control of Gypsophila paniculata L.</a> Scientia Horticulturae. 16 (4), 385-393 (1982).</li><li>Nunn, A. 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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+temperature+and+photoperiod+on+flower+induction+and+inflorescence+development+in+sweet+orange+(Citrus+sinensis+L.+Osbeck).">Influence of temperature and photoperiod on flower induction and inflorescence development in sweet orange (Citrus sinensis L. Osbeck).</a> Journal of Horticultural Science. 44 (4), 311-320 (1969).</li><li>Moss, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effects+on+flower+initiation+in+sweet+orange+(Citrus+sinensis).">Temperature effects on flower initiation in sweet orange (Citrus sinensis).</a> Australian Journal of Agricultural Research. 27 (3), 399-407 (1976).</li><li>Reece, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+set+in+the+sweet+orange+in+relation+to+flowering+habit.">Fruit set in the sweet orange in relation to flowering habit.</a> Proceedings of the American Society for Horticultural Science. 46, 81-86 (1945).</li><li>Khan, S. A., Perveen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+pollen+germination+of+five+citrus+species.">In vitro pollen germination of five citrus species.</a> Pak. J. Bot. 46 (3), 951-956 (2014).</li><li>Planes, L., Catal&#225;n, J., Jaques, J. A., Urbaneja, A., Tena, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pezothrips+kellyanus+(Thysanoptera:+Thripidae)+nymphs+on+orange+fruit:+importance+of+the+second+generation+for+its+management.">Pezothrips kellyanus (Thysanoptera: Thripidae) nymphs on orange fruit: importance of the second generation for its management.</a> Florida Entomologist. , 848-855 (2015).</li><li>Carimi, F., Caleca, V., Mineo, G., De Pasquale, F., Crescimanno, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+of+Prays+citri+on+callus+derived+from+lemon+stigma+and+style+culture.">Rearing of Prays citri on callus derived from lemon stigma and style culture.</a> Entomologia Experimentalis et Applicata. 95 (3), 251-257 (2000).</li><li>Jones, W., Embleton, T., Garber, M., Cree, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creasing+of+orange+fruit.">Creasing of orange fruit.</a> Hilgardia. 38 (6), 231-244 (1967).</li><li>Storey, R., Treeby, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphology+of+epicuticular+wax+and+albedo+cells+of+orange+fruit+in+relation+to+albedo+breakdown.">The morphology of epicuticular wax and albedo cells of orange fruit in relation to albedo breakdown.</a> Journal of Horticultural Science. 69 (2), 329-338 (1994).</li><li>Rewald, B., Raveh, E., Gendler, T., Ephrath, J. 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It was possible to force the flowering of young citrus trees (only 2 years old) quickly and at any time with profuse flower production (around 216 flowers per tree). In previous studies14,15, flower initiation was induced by low temperatures and the process lasted around 120 days. The combination of a short water stress period with spring conditions in the phytotron allowed this time to be significantly reduced, with mandarin trees (cv. Nova) flourishing after 68...

Phytotron has been widely used to assess the effect of numerous parameters on the development of many herbaceous and bulb plants. Species such as rice1, lily2, strawberry3 and many others4 have been evaluated under phytotron conditions. Chamber experiments on forest trees have also been carried out to evaluate ozone sensitivity on juvenile beech5,6, and to assess the influence of temperatures on frost hardening in seedlings of Scots pine and Norway spruce7. Less information is available about how to obtain fast profuse flowering in young fruit trees via growth chambers. 
The flowering of citrus trees, and its relationship with many endogenous and exogenous factors, have long since been broadly studied. Temperatures8, water availability9, carbohydrates10, auxin and gibberellin contents11,12, abscisic acid13, and many other factors that affect citrus reproductive systems have been studied. Temperature and photoperiod effects on flower initiation have been studied in sweet orange (Citrus &#215; sinensis (L.) Osbeck)14,15. In these experiments, long inductive conditions (5 weeks at 15/8 &#176;C) were used and the temperature during shoot development influenced inflorescence type14. During citrus flowering, the term "inflorescence" has been applied to all types of flower-bearing growth that arise from axillary buds, as used by Reece16.
Having a clear precise methodology to force flowering over a short time period and at other times other than spring can provide many advantages for researchers. Save tropical areas, the flowering of fruit trees occurs only once a year, which limits the number of experiments that can be done. 
Flowers obtained by forced methods can be used for a wide variety of experiments to: obtain viable pollen for in vitro growth and germination experiments in any month17; run experiments with pests that affect early fruit development stages, even before petal fall, such as Pezothrips kellyanus Bagnall18, or Prays citri Milli&#232;re19; study the effect of temperatures, chemical treatments, natural predators or just insects rearing; assess the influence of numerous factors on the physiological alterations that disturb early fruit development stages, such as "creasing" in sweet orange20,21; help plant breeders to shorten times to obtain male and female gametes to perform forced-crosses.
This paper aims 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. To achieve this main objective, details on artificial lighting (lumens), photoperiod, temperatures, plant size and age, induction strategy, days for induction, days for sprouting, days for flowering, and the total amount of flowers per variety are provided. Water stress induction intensity was also recorded and related with inflorescence type, dates and amounts of flowers.

<table border="0" cellpadding="0" cellspacing="0" style="width:1309px;" width="1309">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Data-logger</td>
			<td style="width:188px;">Testo&#160;</td>
			<td style="width:227px;">Testo 177-H1</td>
			<td style="width:619px;">Testo 177-H1, humidity/temperature logger, 4 channels, with internal sensors and additional external temp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Data-logger sotfwae</td>
			<td style="width:188px;">Testo</td>
			<td style="width:227px;">Software Comsoft Basic Testo 5</td>
			<td>Basic software for the programming and reading of the data loggers Testo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Electronic controller differential</td>
			<td style="width:188px;">Eliwell&#160;</td>
			<td style="width:227px;">IC 915 (LX)&#160; (cod. 9IS23071)</td>
			<td>Electronic controller with 2 set points and differential set point adjustment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electronic controller dual&#160;</td>
			<td style="width:188px;">Eliwell&#160;</td>
			<td style="width:227px;">IC 915 NTC-PTC</td>
			<td>Electronic controllers with dual output</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Growth chamber - phytotron</td>
			<td style="width:188px;">Rochina</td>
			<td style="width:227px;"></td>
			<td>Chamber measuring 1.85 x 1.85 x 2.5 m (L x W x H) with a total volume of 8.56 m3. With temperature (day/night), photoperiod (day/night), light intensity and minimum relative humidity control.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Light kit</td>
			<td style="width:188px;">Cosmos Grow/Bloom Light</td>
			<td style="width:227px;"></td>
			<td>Light kit with reflector, electric ballast sodium/halide and high-pressure sodium (HPS) 600W lamp&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Luxmeter</td>
			<td style="width:188px;">Delta OHM</td>
			<td style="width:227px;">HD 9221</td>
			<td>HD 9221 Luxmeter to measure the light intensity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Plant material</td>
			<td style="width:188px;">Beniplant S.L (AVASA)</td>
			<td style="width:227px;"></td>
			<td>Mandarin trees from registered nurseries with a virus-free certification&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Substrate</td>
			<td style="width:188px;">Plant Vibel</td>
			<td style="width:227px;"></td>
			<td>Standard substrate based on quality 50% white peat and 50% coconut fiber</td>
		</tr>
	</tbody>
</table>

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.

The experiment was carried out in the plant growth chamber located at the Valencia Polytechnic University&#39;s Gand&#237;a Campus (municipality of Gand&#237;a) in the province of Valencia, Spain (39&#176; 28&#8242; 53.95&#8243; N, 0&#176; 20&#8242; 37.71&#8243; W), in autumn and winter (2017 Oct. 26 - 2018 Feb. 5) (Table 1). Six mandarin trees cv. &#39;Clemenules&#39; (a bud mutation of Citrus clementina hort. ex Tanaka) and six mandarin trees cv. &#39;Nova&#39;...

Watch this Scientific Journal Video about Forced Flowering in Mandarin Trees under Phytotron Conditions at JoVE.com

Forced Flowering in Mandarin Trees under Phytotron Conditions

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 ...

Experiments that involve citrus flowerings are usually limited to springtime. With these methodologies, researchers will be able to control and design when to obtain the flowers to work with. It is a clear and easy technique that allows to obtain flowers from young Mandarins at any time of the year.Researchers can control dates and flower intensity. This technique can be used not only for Mandarin trees but also for other citrus trees as lemons or grapefruits and other fruit trees. To prepare, use a growth chamber measuring length of 1.85 meters, width of 1.85 meters, and height of 2.5 meters with a total volume of 8.56 cubic meters.Check if regulations such as temperature for day and night, photo period, light intensity, and minimum relative humidity are available. Obtain the plant material from registered nurseries with a virus-free certification. For example, six Mandarin trees cultivars Clemenules and six Mandarin trees cultivars Nova.Mandarin trees can be young, for example one or two-year-old varieties grafted onto root stocks. Use appropriate pots such as a plastic pot with diameter of 22 centimeters and height of 20 centimeters and prepare five liters of standard substrate based on 50%of high-quality white peat and 50%of coconut fiber. Irrigate the plants for the first time as soon as they arrive from the nursery to standardize moisture content.Water by immersion. Cover the pots with water halfway for 20 minutes. Keep the plants outside in half shade without irrigation for three to five days.Program the growth chamber for Mandarin trees based on the site's springtime conditions. Use two electronic controllers with dual output, one for day and one for night humidity. Use a timer to change from day to night.To set up minium humidity, press and release the set button. When SP1 appears, press and release the set button and press the up key or down key to change the SP1 value to 50%For maximum humidity, press and release the set button and then press the up key or down key to change to SP2. Press and release the set button and press the up key or down key to change the SP2 value to 60%To set up the desired day temperature to 22 degrees Celsius, press and release the set button.When SP1 appears, press the set button and then press the up key or down key to change the SP1 value. To set up the desired night temperature, offset set point one to access OS1 parameter. Press the set button for five seconds then press the down key three times.When CnF appears, press the set button and then press the down button. When PA2 appears, press set two times with rE1 and OS1 appearing in sequence. Press set again and press up or down key to change the OS1 value to minus 11 degrees Celsius and then press the FNC button to exit.After four weeks, increase the temperature by one degree Celsius and add a half hour of light. As the phytotron has variation ranges, the nighttime temperature may vary from 11 to 14 degrees Celsius and the daytime temperature from 19 to 22 degrees Celsius. Use two light kits with a reflector, an electric ballast sodium halide and high-pressure sodium 600 watt lamp to obtain the appropriate light intensity.Use a lux meter to check illuminance. It is desired to be attained 55, 000 lux at the top of the crown and 40, 000 lux at the crown base. Before floral induction, place trees inside the phytotron and keep them for several weeks without watering them.Distribute trees regularly so that each has the same available space and light. Distribute individuals and varieties randomly among positions. To use water stress for floral induction, after the first irrigation, do not irrigate trees until the water stress period is considered to have finished.Look at leaf turgidity to check the water stress intensity everyday. When most leaves are flaccid but not have started to fall, consider enough water stress for floral induction. This usually happens after 22 days without watering.Irrigate the trees abundantly after the water stress period. For this first irrigation, cover pots with water halfway to immerse for 20 minutes. Compare the total amount of leaves before and after the water stress period.Calculate the percentage of fallen leaves which is an indirect measurement of the water stress suffered by each individual. At the beginning and the end of flowering periods, collect flowers once a day. On the days of maximum flower production, collect flowers twice a day and seven days a week.Perform other management tasks such as to water trees approximately once a week after the water stress period and check the temperature and humidity settings with a data logger. In this experiment, three levels of water stress intensity were established for the same number of days without irrigation, low intensity which has five to 10%leaf fall of the Clemenules individuals, medium to high intensity which has 50 to 60%leaf fall of the Nova individuals, very high intensity which has 80 to 90%leaf fall of the Nova individuals. The vast majority of flowers were complete and viable.Some small leafy flowers with very short petals were observed at the beginning of the flowering period probably due to the partial induction of some buds. The higher the leaf fall percentage the more water stress and therefore the greater floral induction intensity. Individuals with high induction displayed mainly leafless buds with one flower or several.The individuals with intermediate induction showed mainly buds with a balanced number of leaves and flowers but also leafless buds with one flower or several and very few buds with many leaves and a few flowers. Individuals with low induction showed mainly buds with much more leaves than flowers. Water stress level can be estimated with leaf turgidity and will be correlated with floral intensity.Dates, amount of flowers, and inflorescence type will be determined. This technique can be useful for any other fruit trees to obtain flowers at any time.

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Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus ...