Forced Flowering in Mandarin Trees under Phytotron Conditions

Summary

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.

Abstract

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 °C, night 11 h, 12 °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.

Introduction

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 beech^5,6, 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 contents^11,12, 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 × sinensis (L.) Osbeck)^14,15. In these experiments, long inductive conditions (5 weeks at 15/8 °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 Millière¹⁹; 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 orange^20,21; 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.

Protocol

1. Growth chamber characteristics and regulation requirements

1. 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.
2. 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.

2. Plant material

1. Obtain the plant material from registered nurseries with a virus-free certification (e.g., six mandarin trees cv. ‘Clemenules’ and 6 mandarin trees cv. ‘Nova’).
   NOTE: Mandarin trees can be young (e.g., 1- or 2-year-old varieties grafted onto rootstocks).
2. 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%).
3. 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.

3. First irrigation

1. 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.
2. Keep the plants outside in half shade without irrigation for 3-5 days (Table 1).

4. Springtime conditions in the phytotron

1. Review the site’s springtime conditions to determine the average day and night temperature, photoperiod and relative humidity (e.g., at the working latitude (39° 28′ 53.95″ N, 0° 20′ 37.71″ 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° 57’ 51.77″ N, 0° 15’ 02.24″ W 113 m.a.s.l.) for at least 10 years, and the average day and night temperature, photoperiod and relative humidity were determined).
2. Program the growth chamber for mandarin trees with the following conditions: (i) temperature of 22 °C/11 °C (day/night); (ii) photoperiod of 13/11 h (light/dark); (iii) relative humidity around 60% and no less than 50% (Figure 3).
   1. 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.
      1. 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%).
      2. 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%).
   2. 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.
      1. Set up the desired day temperature (22 °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.
      2. 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 °C); press Set | UP; dF1 will appear; press Set and press the UP or DOWN to change dF1 value (2 °C).
      3. To set up the desired night temperature (11 °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 °C); press the fnc button (ESC function (exit)).
3. Increase the temperature by 1 °C (23/12 °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 °C to 14 °C, and the daytime temperature from 19 °C to 22 °C (Figure 3).
4. 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.
5. Modify the distance between the lamp and the crown to obtain the desired light intensity and set up the photoperiod with the timer.
6. Check illuminance with a luxmeter. At the top of the crown, 55,000 lux (671 µmol m^-2 s^-1) should be attained, with 40,000 lux (488 µmol m^-2 s^-1) at the crown-base.

5. Placing trees inside the phytotron

1. Place trees inside the phytotron and keep them for several weeks without watering them (Figure 5A).
2. 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).
3. Distribute individuals and varieties randomly among positions (Figure 1).

6. Floral induction

1. Use water stress for floral induction. After the first irrigation, do not irrigate trees until the water stress period is considered to have finished.
2. Check the water stress intensity every day by looking at leaf turgidity.
3. 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.
4. Irrigate the trees abundantly after the water stress period. For this first irrigation, water by immersion. Cover pots with water halfway for 20 min.
5. 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.

7. Flower harvesting if necessary for other experiments

1. 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.
2. Harvest flowers by hand and keep them at -20 °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.
   1. Choose the exact flower state when collecting.
   2. Use flowers for in vitro pollen germination assays or for any other purpose with a pollen viability that equals fresh pollen.

8. Other management tasks

1. Water trees approximately once a week after the water-stress period depending on requirements.
2. 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)).
3. Check the temperature and humidity settings with a data logger (Figure 3).

Results

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

Discussion

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 studies^14,15, 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...

Materials

Name	Company	Catalog Number	Comments
Data-logger	Testo	Testo 177-H1	Testo 177-H1, humidity/temperature logger, 4 channels, with internal sensors and additional external temp
Data-logger sotfwae	Testo	Software Comsoft Basic Testo 5	Basic software for the programming and reading of the data loggers Testo
Electronic controller differential	Eliwell	IC 915 (LX)  (cod. 9IS23071)	Electronic controller with 2 set points and differential set point adjustment
Electronic controller dual	Eliwell	IC 915 NTC-PTC	Electronic controllers with dual output
Growth chamber - phytotron	Rochina		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.
Light kit	Cosmos Grow/Bloom Light		Light kit with reflector, electric ballast sodium/halide and high-pressure sodium (HPS) 600W lamp
Luxmeter	Delta OHM	HD 9221	HD 9221 Luxmeter to measure the light intensity
Plant material	Beniplant S.L (AVASA)		Mandarin trees from registered nurseries with a virus-free certification
Substrate	Plant Vibel		Standard substrate based on quality 50% white peat and 50% coconut fiber