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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<li>Reece, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fruit+set+in+the+sweet+orange+in+relation+to+flowering+habit%5BTitle%5D)+AND+%22Proceedings+of+the+American+Society+for+Horticultural+Science%22%5BJournal%5D)">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>
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<li>Jones, W., Embleton, T., Garber, M., Cree, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Creasing+of+orange+fruit%5BTitle%5D)+AND+%22Hilgardia%22%5BJournal%5D)">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/pubmed?term=((The+morphology+of+epicuticular+wax+and+albedo+cells+of+orange+fruit+in+relation+to+albedo+breakdown%5BTitle%5D)+AND+%22Journal+of+Horticultural+Science%22%5BJournal%5D)">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>
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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><tbody><tr><td>Data-logger</td><td>Testo&amp;nbsp;</td><td>Testo 177-H1</td><td>Testo 177-H1, humidity/temperature logger, 4 channels, with internal sensors and additional external temp</td></tr><tr><td>Data-logger sotfwae</td><td>Testo</td><td>Software Comsoft Basic Testo 5</td><td>Basic software for the programming and reading of the data loggers Testo</td></tr><tr><td>Electronic controller differential</td><td>Eliwell&amp;nbsp;</td><td>IC 915 (LX)&amp;nbsp; (cod. 9IS23071)</td><td>Electronic controller with 2 set points and differential set point adjustment&amp;nbsp;</td></tr><tr><td>Electronic controller dual&amp;nbsp;</td><td>Eliwell&amp;nbsp;</td><td>IC 915 NTC-PTC</td><td>Electronic controllers with dual output</td></tr><tr><td>Growth chamber - phytotron</td><td>Rochina</td><td></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.&amp;nbsp;</td></tr><tr><td>Light kit</td><td>Cosmos Grow/Bloom Light</td><td></td><td>Light kit with reflector, electric ballast sodium/halide and high-pressure sodium (HPS) 600W lamp&amp;nbsp;</td></tr><tr><td>Luxmeter</td><td>Delta OHM</td><td>HD 9221</td><td>HD 9221 Luxmeter to measure the light intensity</td></tr><tr><td>Plant material</td><td>Beniplant S.L (AVASA)</td><td></td><td>Mandarin trees from registered nurseries with a virus-free certification&amp;nbsp;</td></tr><tr><td>Substrate</td><td>Plant Vibel</td><td></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

9.0K Views.  Universitat Politècnica de València. Experimenten waarbij citrusbloeiingen betrokken zijn, zijn meestal beperkt tot de lente. Met deze methoden, zullen onderzoekers in staat zijn om te controleren en te ontwerpen wanneer de bloemen te verkrijgen om mee te werken. Het is een duidelijke en eenvoudige techniek die het mogelijk maakt om bloemen te verkrijgen van jonge Mandarijnen op elk moment van het jaar.Onderzoekers kunnen data en bloemintensiteit controleren. Deze techniek kan niet alleen worden gebruikt voor Mandarijnbom...

Video: Forced Flowering in Mandarin Trees under Phytotron Conditions

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

Robotic Sensing and Stimuli Provision for Guided Plant Growth

Robot systems are actively researched for manipulation of natural plants, typically restricted to agricultural automation activities such as harvest, irrigation, and mechanical weed control. Extending this research, we introduce here a novel methodology to manipulate the directional growth of plants via their natural mechanisms for signaling and hormone distribution. An effective methodology of robotic stimuli provision can open up possibilities for new experimentation with later developmental phases in plants, or for new biotechnology applications such as shaping plants for green walls. Interaction with plants presents several robotic challenges, including short-range sensing of small and variable plant organs, and the controlled actuation of plant responses that are impacted by the environment in addition to the provided stimuli. In order to steer plant growth, we develop a group of immobile robots with sensors to detect the proximity of growing tips, and with diodes to provide light stimuli that actuate phototropism. The robots are tested with the climbing common bean, Phaseolus vulgaris, in experiments having durations up to five weeks in a controlled environment. With robots sequentially emitting blue light-peak emission at wavelength 465 nm-plant growth is successfully steered through successive binary decisions along mechanical supports to reach target positions. Growth patterns are tested in a setup up to 180 cm in height, with plant stems grown up to roughly 250 cm in cumulative length over a period of approximately seven weeks. The robots coordinate themselves and operate fully autonomously. They detect approaching plant tips by infrared proximity sensors and communicate via radio to switch between blue light stimuli and dormant status, as required. Overall, the obtained results support the effectiveness of combining robot and plant experiment methodologies, for the study of potentially complex interactions between natural and engineered autonomous systems.

Distributed robot nodes provide sequences of blue light stimuli to steer the growth trajectories of climbing plants. By activating natural phototropism, the robots guide the plants through binary left-right decisions, growing them into predefined patterns that by contrast are not possible when the robots are dormant.

Robot systems are actively researched for manipulation of natural plants, typically restricted to agricultural automation activities such as harvest, ...

Engineering

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. ...

Developmental Biology

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Biology

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of non-photochemical quenching (NPQ) has proven to be an effective strategy to increase photosynthetic performance. However, the potential to breed for improved NPQ and a complete understanding of the genetic basis of NPQ relaxation is lacking due to limitations of oversampling and data collection from field-grown crop plants. Building on previous reports, we present a high-throughput assay for analysis of NPQ relaxation rates in Glycine max (soybean) using pulse amplitude modulated (PAM) chlorophyll fluorometry. Leaf disks are sampled from field-grown soybeans before transportation to a laboratory where NPQ relaxation is measured in a closed PAM-fluorometer. NPQ relaxation parameters are calculated by fitting a bi-exponential function to the measured NPQ values following a transition from high to low light. Using this method, it is possible to test hundreds of genotypes within a day. The procedure has the potential to screen mutant and diversity panels for variation in NPQ relaxation, and can therefore be applied to both fundamental and applied research questions.

The protocol introduces a high-throughput method for measuring the relaxation of non-photochemical quenching by pulse amplitude modulated chlorophyll fluorometry. The method is applied to field-grown Glycine max and can be adapted to other species to screen for genetic diversity or breeding populations.

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of ...

Deep Fluorescence Observation in Rice Shoots via Clearing Technology

The recently developed clearing technology that eliminates refractive index mismatches and diminishes auto-fluorescent material has made it possible to observe plant tissues in three dimensions (3D) while preserving their internal structures. In rice (Oryza sativa L.), a monocot model plant and a globally important crop, clearing technology has been reported in organs that are relatively easy to observe, such as the roots and leaves. Applications of clearing technology in shoot apical meristem (SAM) and stems have also been reported, but only to a limited degree because of the poor penetration of the clearing solution (CS) in these tissues. The limited efficiency of the clearing solutions in these tissues has been attributed to auto-fluorescence, thickening, and hardening of the tissues in the stem as the vascular bundles and epidermis develop and layering of the SAM with water-repellent leaves. The present protocol reports the optimization of a clearing approach for continuous and 3D observation of gene expression from the SAM/young panicle to the base of the shoots during development. Fixed tissue samples expressing a fluorescent protein reporter were trimmed into sections using a vibrating micro-slicer. When an appropriate thickness was achieved, the CS was applied. By specifically targeting the central tissue, the penetration rate and uniformity of the CS increased, and the time required to make the tissue transparent decreased. Additionally, clearing of the trimmed sections enabled the observation of the internal structure of the whole shoot from a macro perspective. This method has potential applications in deep imaging of tissues of other plant species that are difficult to clear.

Deep Fluorescence Observation in Rice Shoots via Clearing Technology

The present protocol describes a clearing technique for rice shoots, which are difficult to prepare for internal structural observations owing to the hard, thick, or layered nature of the tissues. This method facilitates continuous and deep fluorescence observations, even in adult rice plants.

The recently developed clearing technology that eliminates refractive index mismatches and diminishes auto-fluorescent material has made it possible to ...

Molecule Delivery in Plants Using a Direct Infusion Device

Direct Infusion Device for Molecule Delivery in Plants

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed.
To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant&#39;s trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed.
Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for&#160;testing and facilitate research and screening purposes.

Author Spotlight: Direct Infusion Device for Molecule Delivery in Plants  

This manuscript describes a novel direct plant infusion device for screening the effectiveness of molecules against the bacteria (Candidatus Liberibacter asiaticus) or its insect vector (Diaphorina citri, Kuwayama) that, in combination, are associated with Huanglongbing citrus disease.

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but ...

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.

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.

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

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Developing Gene Transformation and Editing Methods in Bamboo

In Planta Gene Expression and Gene Editing in Moso Bamboo Leaves

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and regeneration processes. This method involves Agrobacterium-mediated gene expression via wounding and vacuum for bamboo seedlings. It successfully demonstrated the expression of exogenous genes, such as the RUBY reporter and Cas9 gene, in bamboo leaves. The highest transformation efficiency for the accumulation of betalain in RUBY seedlings was achieved using the GV3101 strain, with a percentage of 85.2% after infection. Although the foreign DNA did not integrate into the bamboo genome, the method was efficient in expressing the exogenous genes. Furthermore, a gene editing system has also been developed with a native reporter using this method, from which an in situ mutant generated by the edited bamboo violaxanthin de-epoxidase gene (PeVDE) in bamboo leaves, with a mutation rate of 17.33%. The mutation of PeVDE resulted in decreased non-photochemical quenching (NPQ) values under high light, which can be accurately detected by a fluorometer. This makes the edited PeVDE a potential native reporter for both exogenous and endogenous genes in bamboo. With the reporter of PeVDE, a cinnamoyl-CoA reductase gene was successfully edited with a mutation rate of 8.3%. This operation avoids the process of tissue culture or callus induction, which is quick and efficient for expressing exogenous genes and endogenous gene editing in bamboo. This method can improve the efficiency of gene function verification and will help reveal the molecular mechanisms of key metabolic pathways in bamboo.

Author Spotlight: Advancing Gene Editing in Bamboo Leaves for Sustainable Plastic Alternatives 

In this study, a novel in planta gene expression and gene editing method mediated by Agrobacterium was developed in bamboo. This method greatly improved the efficiency of gene function validation in bamboo, which has significant implications for accelerating the process of bamboo breeding.

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and ...

Forced Flowering in Mandarin Trees under Phytotron Conditions

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