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 are available (Figure 2). 
	NOTE: Timers should allow temperature and light switch (on/off) control at least every 30 min.</li>
</ol>
2. Plant material
<ol>
	<li>Obtain the plant material from registered nurseries with a virus-free certification (e.g., six mandarin trees cv. &#8216;Clemenules&#8217; and 6 mandarin trees cv. &#8216;Nova&#8217;). 
	NOTE: Mandarin trees can be young (e.g., 1- or 2-year-old varieties grafted onto rootstocks).</li>
	<li>Use appropriate pots (e.g., a plastic pot of 22 cm x 20 cm (diameter x height) and prepare 5 L of standard substrate based on high quality white peat (50%) and coconut fiber (50%).</li>
	<li>Use trees that are around 1.5 m high with a well-developed spherical crown from 1 m to 1.5 m. Plants should be completely healthy, and be pest-, pathogen- or disease-free.</li>
</ol>
3. First irrigation
<ol>
	<li>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 min.</li>
	<li>Keep the plants outside in half shade without irrigation for 3-5 days (Table 1).</li>
</ol>
4. Springtime conditions in the phytotron
<ol>
	<li>Review the site&#8217;s springtime conditions to determine the average day and night temperature, photoperiod and relative humidity (e.g., at the working latitude (39&#176; 28&#8242; 53.95&#8243; N, 0&#176; 20&#8242; 37.71&#8243; W) with only one bloom per year the citrus tree flowering period extends from mid-March to the end of April with some annual variations. Therefore, these dates were checked in several meteorological stations (e.g. w.s. 38&#176; 57&#8217; 51.77&#8243; N, 0&#176; 15&#8217; 02.24&#8243; W 113 m.a.s.l.) for at least 10 years, and the average day and night temperature, photoperiod and relative humidity were determined).</li>
	<li>Program the growth chamber for mandarin trees with the following conditions: (i) temperature of 22 &#176;C/11 &#176;C (day/night); (ii) photoperiod of 13/11 h (light/dark); (iii) relative humidity around 60% and no less than 50% (Figure 3).
	<ol>
		<li>Use two electronic controllers with dual output, one for day and one for night humidity. Use a timer to change from day to night humidity. Set up minimum and maximum humidity for day and night.
		<ol>
			<li>For minimum humidity, press and release (single press) the Set button; SP 1 (set point 1) will appear; press and release the Set button and press the UP key or DOWN key to change the SP1 value (50%).</li>
			<li>For maximum humidity, press and release (single press) the Set button; SP 1 (set point 1) will appear; press the UP key or DOWN key to change to SP 2; SP 2 (set point 2) will appear; press and release the Set button and press the UP key or DOWN key to change the SP2 value (60%).</li>
		</ol>
		</li>
		<li>Use an electronic controller with 2 set points and a differential set point adjustment to set up temperature. Use a timer to change from the day to night temperature.
		<ol>
			<li>Set up the desired day temperature (22 &#176;C). Press and release the Set button; SP 1 (set point 1) will appear; press the Set button; press the UP key or DOWN key to change the SP1 value.</li>
			<li>Set up the regulation band, for example db1 and dF1 parameters. Refrigeration will start when Set point 1 (SP1) plus db1 is reached and will stop at a temperature equal to SP1 plus db1 minus dF1. Press the Set button for 5 s; rE1 will appear; press Set; press the UP key; db1 will appear; press Set and press the UP key or DOWN key to change the db1 value (2 &#176;C); press Set | UP; dF1 will appear; press Set and press the UP or DOWN to change dF1 value (2 &#176;C).</li>
			<li>To set up the desired night temperature (11 &#176;C), access OS1 parameter (Offset Set point 1). Press the Set button for 5 s; press DOWN 3 times; cnF will appear; press Set | DOWN; PA2 will appear; press Set; rE1 will appear; press Set; OS1 will appear; press Set and press UP or DOWN to change the OS1 value (-11 &#176;C); press the fnc button (ESC function (exit)).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Increase the temperature by 1 &#176;C (23/12 &#176;C day/night) after 4 weeks and add a half hour of light (13.5/10.5 light/dark). 
	NOTE: As the phytotron has variation ranges, the nighttime temperature may vary from 11 &#176;C to 14 &#176;C, and the daytime temperature from 19 &#176;C to 22 &#176;C (Figure 3).</li>
	<li>Use two light kits with a reflector, an electric ballast sodium halide and high-pressure sodium (HPS) 600 W lamp to obtain the appropriate light intensity (Figure 4). Light intensity is essential for flowering.</li>
	<li>Modify the distance between the lamp and the crown to obtain the desired light intensity and set up the photoperiod with the timer.</li>
	<li>Check illuminance with a luxmeter. At the top of the crown, 55,000 lux (671 &#181;mol m-2 s-1) should be attained, with 40,000 lux (488 &#181;mol m-2 s-1) at the crown-base.</li>
</ol>
5. Placing trees inside the phytotron
<ol>
	<li>Place trees inside the phytotron and keep them for several weeks without watering them (Figure 5A).</li>
	<li>Distribute trees regularly so that each has the same available space and light (e.g., trees were uniformly distributed inside the growth chamber into three lines and at four positions. The distance between lines was 0.46 cm, while the distance between positions was 0.37 cm) (Figure 1).</li>
	<li>Distribute individuals and varieties randomly among positions (Figure 1).</li>
</ol>
6. Floral induction
<ol>
	<li>Use water stress for floral induction. After the first irrigation, do not irrigate trees until the water stress period is considered to have finished.</li>
	<li>Check the water stress intensity every day by looking at leaf turgidity.</li>
	<li>Consider enough water stress for floral induction when most leaves are flaccid, but have not started to fall (e.g., after 22 days without watering, leaves were flaccid and a few started falling) (Table 1). 
	NOTE: If water stress is excessive (many leaves fall), plant survival can be compromised, whereas if water stress is insufficient (not enough flaccid leaves), poor flowering may take place.</li>
	<li>Irrigate the trees abundantly after the water stress period. For this first irrigation, water by immersion. Cover pots with water halfway for 20 min.</li>
	<li>Measure the water stress intensity for each individual by noting the total number of fallen leaves (Figure 5B,C). The percentage of fallen leaves is an indirect measurement of the water stress suffered by each individual. Estimate the percentage of fallen leaves by comparing the total amount of leaves before and after the water stress period.</li>
</ol>
7. Flower harvesting if necessary for other experiments
<ol>
	<li>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 7 days a week.</li>
	<li>Harvest flowers by hand and keep them at -20 &#176;C in a labeled plastic bag (Figure 5D). The flower production of six mandarin trees can vary from 25 to more than 200 flowers per day.
	<ol>
		<li>Choose the exact flower state when collecting.</li>
		<li>Use flowers for in vitro pollen germination assays or for any other purpose with a pollen viability that equals fresh pollen.</li>
	</ol>
	</li>
</ol>
8. Other management tasks
<ol>
	<li>Water trees approximately once a week after the water-stress period depending on requirements.</li>
	<li>Check the presence of pests and disease every 2-3 days (e.g., only a small population of Icerya purchasi Maskell was observed in this experiment and was manually removed to avoid using chemical treatments (Figure 5E)).</li>
	<li>Check the temperature and humidity settings with a data logger (Figure 3).</li>
</ol>

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The authors have nothing to disclose.

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 ...

forced-flowering-in-mandarin-trees-under-phytotron-conditions

Research

JoVE Journal

Environment

8.9K Views.  Universitat Politècnica de València. 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. 

Video: Forced Flowering in Mandarin Trees under Phytotron Conditions

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

A Venturi Effect Can Help Cure Our Trees

In woody plants, xylem sap moves upwards through the vessels due to a decreasing gradient of water potential from the groundwater to the foliage. According to these factors and their dynamics, small amounts of sap-compatible liquids (i.e. pesticides) can be injected into the xylem system, reaching their target from inside. This endotherapic method, called &#34;trunk injection&#34; or &#34;trunk infusion&#34; (depending on whether the user supplies an external pressure or not), confines the applied chemicals only within the target tree, thereby making it particularly useful in urban situations. The main factors limiting wider use of the traditional drilling methods are related to negative side effects of the holes that must be drilled around the trunk circumference in order to gain access to the xylem vessels beneath the bark.
The University of Padova (Italy) recently developed a manual, drill-free instrument with a small, perforated blade that enters the trunk by separating the woody fibers with minimal friction. Furthermore, the lenticular shaped blade reduces the vessels&#39; cross section, increasing sap velocity and allowing the natural uptake of an external liquid up to the leaves, when transpiration rate is substantial. Ports partially close soon after the removal of the blade due to the natural elasticity and turgidity of the plant tissues, and the cambial activity completes the healing process in few weeks.

Compared to the more traditional hole-based methods, most of which require the tree to be drilled, tools with lenticular blades transform the basics of endotherapy easing the closure of the wound and allowing the natural uptake of the solutions.

In woody plants, xylem sap moves upwards through the vessels due to a decreasing gradient of water potential from the groundwater to the foliage. ...

Sediment Core Sectioning and Extraction of Pore Waters under Anoxic Conditions

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is built and can be transported to a temporary work space close to field sampling site(s) to facilitate rapid analysis. Cores are extruded into a portable glove bag, where they are sectioned and each 1-3 cm thick section (depending on core diameter) is sealed into 50 ml centrifuge tubes. Pore waters are separated with centrifugation outside of the glove bag and then returned to the glove bag for separation from the sediment. These extracted pore water samples can be analyzed immediately. Immediate analyses of redox sensitive species, such as sulfide, iron speciation, and arsenic speciation indicate that oxidation of pore waters is minimal; some samples show approximately 100% of the reduced species, e.g. 100% Fe(II) with no detectable Fe(III). Both sediment and pore water samples can be preserved to maintain chemical species for further analysis upon return to the laboratory.

A protocol for sectioning sediment cores and extracting pore waters under anoxic conditions in order to permit analysis of redox sensitive species in both solids and fluids is presented.

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is ...

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The system comprises a high-pressure rheometer which is connected to a circulation loop. The rheometer has a rotational flow-through measurement cell with two alternative geometries: coaxial cylinder and double gap. The circulation loop contains a mixer, to bring the crude oil sample into equilibrium with CO2, and a gear pump that transports the mixture from the mixer to the rheometer and recycles it back to the mixer. The CO2 and crude oil are brought to equilibrium by stirring and circulation and the rheology of the saturated mixture is measured by the rheometer. The system is used to measure the rheological properties of Zuata crude oil (and its toluene dilution) in equilibrium with CO2 at elevated pressures up to 220 bar and a temperature of 50 &#176;C. The results show that CO2 addition changes the oil rheology significantly, initially reducing the viscosity as the CO2 pressure is increased and then increasing the viscosity above a threshold pressure. The non-Newtonian response of the crude is also seen to change with the addition of CO2.

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A method for measuring the rheology of crude oil in equilibrium with carbon dioxide at reservoir conditions is presented.

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

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 herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

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 area. Biomass capacitance plays an integral role in the avoidance of hydraulic impairment to transpiration. However, high temporal resolution measurements of dynamic changes in the hydraulic capacitance of large trees are rare. Here, we present procedures for the calibration and use of capacitance sensors, typically used to monitor soil water content, to measure the volumetric water content in trees in the field. Frequency domain reflectometry-style observations are sensitive to the density of the media being studied. Therefore, it is necessary to perform species-specific calibrations to convert from the sensor-reported values of dielectric permittivity to volumetric water content. Calibration is performed on a harvested branch or stem cut into segments that are dried or re-hydrated to produce a full range of water contents used to generate a best-fit regression with sensor observations. Sensors are inserted into calibration segments or installed in trees after pre-drilling holes to a tolerance fit using a fabricated template to ensure proper drill alignment. Special care is taken to ensure that sensor tines make good contact with the surrounding media, while allowing them to be inserted without excessive force. Volumetric water content dynamics observed via the presented methodology align with sap flow measurements recorded using thermal dissipation techniques and environmental forcing data. Biomass water content data can be used to observe the onset of water stress, drought response and recovery, and has the potential to be applied to the calibration and evaluation of new plant-level hydrodynamics models, as well as to the partitioning of remotely sensed moisture products into above- and belowground components.

The hydraulic capacitance of biomass is a key component of the vegetation water budget, which serves as a buffer against short and long-term drought stresses. Here, we present a protocol for the calibration and use of soil moisture capacitance sensors to monitor water content in the stems of large 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 ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Cereal Crop Ear Counting in Field Conditions Using Zenithal RGB Images

Ear density, or the number of ears per square meter (ears/m2), is a central focus in many cereal crop breeding programs, such as wheat and barley, representing an important agronomic yield component for estimating grain yield. Therefore, a quick, efficient, and standardized technique for assessing ear density would aid in improving agricultural management, providing improvements in preharvest yield predictions, or could even be used as a tool for crop breeding when it has been defined as a trait of importance. Not only are the current techniques for manual ear density assessments laborious and time-consuming, but they are also without any official standardized protocol, whether by linear meter, area quadrant, or an extrapolation based on plant ear density and plant counts postharvest. An automatic ear counting algorithm is presented in detail for estimating ear density with only sunlight illumination in field conditions based on zenithal (nadir) natural color (red, green, and blue [RGB]) digital images, allowing for high-throughput standardized measurements. Different field trials of durum wheat and barley distributed geographically across Spain during the 2014/2015 and 2015/2016 crop seasons in irrigated and rainfed trials were used to provide representative results. The three-phase protocol includes crop growth stage and field condition planning, image capture guidelines, and a computer algorithm of three steps: (i) a Laplacian frequency filter to remove low- and high-frequency artifacts, (ii) a median filter to reduce high noise, and (iii) segmentation and counting using local maxima peaks for the final count. Minor adjustments to the algorithm code must be made corresponding to the camera resolution, focal length, and distance between the camera and the crop canopy. The results demonstrate a high success rate (higher than 90%) and R2 values (of 0.62-0.75) between the algorithm counts and the manual image-based ear counts for both durum wheat and barley.

We present a protocol for counting durum wheat and barley ears, using natural color (RGB) digital photographs taken in natural sunlight under field conditions. With minimal adjustments for camera parameters and some environmental condition limitations, the technique provides precise and consistent results across a range of growth stages.