GreenTray ® , a TIS Bioreactor for Plant Micropropagation and Abiotic or Biotic Stress Bioassays

: The temporary immersion system bioreactor named GreenTray ® presents advantages over the existing ones. Firstly, there is no need to use forceps to move the shoots or plantlets in or out of the recipient, nor is the use of a scalpel necessary to divide them. Secondly, the basis of the shoot can remain in the GreenTray ® and can sprout again over several cycles of growth. These two characteristics reduce the costs in the process of micropropagation. In addition, shoot or plantlet development is enhanced by the aeration of the recipient, thus enriching the recipient’s atmospheric CO 2 . This article describes and provides images and values of interest in (1) the micropropagation process of some commercial Prunus or Pyrus rootstocks; (2) the plantlet growth under autotrophic conditions; and (3) the inoculation of plants with pathogens that reproduce in vitro the symptoms observed in vivo, using the GreenTray ® bioreactor.


Introduction
Plant propagation through in vitro culture has been adopted by commercial propagation companies because it presents some advantages over conventional propagation with soft or hard wood cuttings.It is a weather-unaffected propagation system, enables the conservation of the initial plant material's sanitary conditions, and exponentially multiplies the selected plant materials every two-to four-week-long cycle.However, in vitro propagation systems based on semisolid agar-containing media require deft manipulation in sterile conditions with appropriate tools and expert hand labor.To prevent contamination but allow plant material aeration, the conventional micropropagation techniques employ glass or plastic containers, flasks or tubes, recycled or disposable, with lids that loosely enclose the recipients, with differential passive aeration.Caps need to be removed in a laminar flow hood cabinet before introducing or extracting the cultured plant portions with the help of forceps, orientating them, dividing them with the help of scalpels using a sterile hard surface, and culturing them in fresh medium with sterile forceps.Therefore, conventional in vitro plant propagation requires a great amount of hard labor input.

Materials and Methods
The patented GreenTray ® [29,30] design is characterized by the ability to access the interior of the plant cultivation container.Its design derives from a previously handmade assemblage [31], and another adaptation of a plastic container [32].When opening the cultivation container of the actual GreenTray ® , the lid and the cultivation tray are always joined, and therefore, the sprouts or plants in cultivation are oriented with the movement of the lid, with just one hand.In the model presented in this work, the GreenTray ® is adapted to a commercial glass flask, but the GreenTray ® design is applicable to any container or bag, of variable size or shape, as long as the culture surface or tray is attached to the container lid, like, for instance, the one presented in [32].

GreenTray ® Parts and Assembly
A commercial cylindrical glass jar with a capacity of 3895 mL, slightly bigger than a US gallon, with a Twist Off 110 lid, is used as the culture container for the plant materials.Its dimensions (15.9 cm in diameter, 25.45 cm in height) are the basis for building each unit of the GreenTray ® bioreactor, with automated modules grouping 7 to 21 bioreactors.Plant materials are contained in this glass flask, while the culture medium is autoclaved and placed in 500 mL ISO GL45 graduated transparent flasks, commonly used in laboratories.
To allow the introduction, cultivation, and extraction of plant materials in the culture container, five pieces were designed (Figure 1), which were manufactured by HP Multi Jet Fusion 3D printing, using PA12 for its manufacture, a material that can withstand autoclave sterilization temperatures, which is necessary for the in vitro cultivation of plants.Each unit was assembled as shown in Figure 2.
Appl.Sci.2024, 14, x FOR PEER REVIEW 3 of 2 autoclave sterilization temperatures, which is necessary for the in vitro cultivation o plants.Each unit was assembled as shown in Figure 2.   Appl.Sci.2024, 14, x FOR PEER REVIEW 3 of 20 autoclave sterilization temperatures, which is necessary for the in vitro cultivation of plants.Each unit was assembled as shown in Figure 2.

Autonomous and Automated Controller with Compressed Air and Lighting
A PLC+HMI TM Touchscreen model, "Samba TM SM35-J-R20" (Unitronics ® , Tel Aviv, Israel), was programmed to control an air diaphragm compressor and vacuum model, "H5P3" (EAD Pumps, Masquefa, Barcelona, Spain), as well as four electro valves and external LED lighting.All components were assembled inside an enclosure for low-voltage switch-gear and control-gear assemblies, the "Spacial CRN" (Schneider Electric Industries SAS, Rueil Malmaison, Paris, France), from which two clear Polyamide Legris (Parker Legris, Terrassa, Spain) compressed air pipes came out, both with 6 and 4 mm external and internal diameters.The first tube provides compressed air at positive pressure (+5 to +6 psi), and when chosen, the same tube exerts suction, or negative pressure (−1 to −1.5 psi).Both air movements arrived at the entrance of the 500 mL ISO GL45.The second tube provides compressed air at positive pressure (+5 to +6 psi) to the entrance of the one-gallon cylindrical jar.Both air entrances were protected from contamination with 25-30 mm diameter 0.22 µm Teflon-PTFE air filters (Filtros Anoia, S.A., Barcelona, Spain).The assemblage is portable and requires only an AC power plug and socket, becoming a module for 7 GreenTray ® units (Figure 3)., x FOR PEER REVIEW 4 of 20

Autonomous and Automated Controller with Compressed Air and Lighting
A PLC+HMI TM Touchscreen model, "Samba TM SM35-J-R20" (Unitronics ® , Israel), was programmed to control an air diaphragm compressor and vacuum model, "H5P3" (EAD Pumps, Masquefa, Barcelona, Spain), as well as four electro valves and external LED lighting.All components were assembled inside an enclosure for low-voltage switch-gear and control-gear assemblies, the "Spacial CRN" (Schneider Electric Industries SAS, Rueil Malmaison, Paris, France), from which two clear Polyamide Legris (Parker Legris, Terrassa, Spain) compressed air pipes came out, both with 6 and 4 mm external and internal diameters.The first tube provides compressed air at positive pressure (+5 to +6 psi), and when chosen, the same tube exerts suction, or negative pressure (−1 to −1.5 psi).Both air movements arrived at the entrance of the 500 mL ISO GL45.The second tube provides compressed air at positive pressure (+5 to +6 psi) to the entrance of the one-gallon cylindrical jar.Both air entrances were protected from contamination with 25-30 mm diameter 0.22 µm Teflon-PTFE air filters (Filtros Anoia, S.A., Barcelona, Spain).The assemblage is portable and requires only an AC power plug and socket, becoming a module for 7 Green-Tray ® units (Figure 3).

Light Source
The LED lighting used in the GreenTray ® modules is exchangeable between the models of different light spectra produced by Gealed S.L. (Catajorra, Valencia, Spain), namely "Supernova Lima" (Ref.SUP12100LDC), "Alnayr" (Ref.AN-NAYYIR 60S), and "Meissa" (Ref.AL-MAISAN 100S).The last two, with a Cool White 5000K sun-like spectrum, can provide up to 900 µEm-1s-1 PAR 20 cm from the light source.The light intensity and photoperiod is regulated from the previously described automated controller.

Light Source
The LED lighting used in the GreenTray ® modules is exchangeable between the models of different light spectra produced by Gealed S.L. (Catajorra, Valencia, Spain), namely "Supernova Lima" (Ref.SUP12100LDC), "Alnayr" (Ref.AN-NAYYIR 60S), and "Meissa" (Ref.AL-MAISAN 100S).The last two, with a Cool White 5000K sun-like spectrum, can provide up to 900 µEm-1s-1 PAR 20 cm from the light source.The light intensity and photoperiod is regulated from the previously described automated controller.

Immersion and Aeration Frequencies
The automated controller facilitates temporizing the waiting time between explant immersions (WTI), the duration of explant immersion (TI), the waiting time between explant aerations (WTA), and the duration of explant aeration (TA).In addition, the TI can last 90 s during passive movement, driven by gravity, or 30 s during active movement, forced by air impulsion into the culture jar (35 s) or air suction out of the culture medium bottle (30 s).
The WTI, TI, WTA, and TA depend on the plant material's capacity for optimal growth, avoiding waterlog or water loss.Such times are adjusted to the plant materials, and for the present work, they were set up according to what is indicated in Table 2. Depending on the plant material volume occupying the tray, 250 to 200 mL of medium is required to immerse the whole culture tray containing the explants.

Establishment of Shoot Cultures in the GreenTray ®
Apical and internodal segments derived from in vitro shoot tip cultures in agarcontaining medium were used to establish micropropagation cultures in the GreenTray ® , called the 1st generation (G1) herein.Depending on the shoot explant size, each GreenTray ® unit contained between 30 and 40 shoot explants when the explants were between 1 and 2 cm long, and up to 80 or 90 explants when they were 1 cm long or less.

Sequential Number of Shoot Cuts without Subculture
GreenTray ® 's design allows new shoots to be extracted and cut and grown above the culture tray, followed by the introduction of the remaining basal part back into the culture glass jar (Figure 4A,C).Once this basis regrows, the shoots are cut the same way, several times, depending on the plant material's capacity to keep growing.Compared to the conventional in vitro propagation procedures in which shoots are cultured in agarcontaining medium, propagation in the GreenTray ® needs no subculture of the shoot bases, since they remain in the culture tray for several generations, keeping its axillary sprouting capacity, like a hedge from which new shoots are cut.
tween 1 and 2 cm long, and up to 80 or 90 explants when they were 1 cm long or less.

Sequential Number of Shoot Cuts without Subculture
GreenTray ® 's design allows new shoots to be extracted and cut and grown above the culture tray, followed by the introduction of the remaining basal part back into the culture glass jar (Figure 4A,C).Once this basis regrows, the shoots are cut the same way, several times, depending on the plant material's capacity to keep growing.Compared to the conventional in vitro propagation procedures in which shoots are cultured in agar-containing medium, propagation in the GreenTray ® needs no subculture of the shoot bases, since they remain in the culture tray for several generations, keeping its axillary sprouting capacity, like a hedge from which new shoots are cut.

Plantlet Development under Autotrophic Conditions
Rotted plantlets were moved to GreenTray ® , kept vertical with the help of an upper grid, and with their roots laid on the culture tray.The conditions to promote autotrophic growth were as follows: culture liquid: Murashige and Skoog (MS) medium with 0 g/L sucrose; air renewal of the culture jar at 1.5 L/min, forcing aeration in the regime indicated in Table 2 for the 6 weeks of culture; and light intensity: 400 µEm-2s-1 PAR 20 cm from the LED light.

Assays for Abiotic and Biotic Stress Agents
Two Prunus rootstocks, namely Garnem ® and Rootpac ® 20, and three apple varieties, namely Carrandona (M0676), Florina (M0216), and Rozona (M0163), were established in vitro, multiplied, and rooted in agar-containing medium following the protocol described in [38].The plantlets were transferred to a GreenTray ® and cultured under autotrophic conditions during the assays to characterize the plant materials' ability to withstand abiotic (high pH, low Ca 2+ concentration) or biotic stress (inoculation with fire blight).

Subculture in New GreenTray ® and Number of Generations
Shoots produced from the GreenTray ® can either be moved to soil for rooting in an acclimation facility, or placed in another GreenTray ® bioreactor, what herein is called a generation (Figure 4B) The number of generations is recorded for each plant material.

Plantlet Development under Autotrophic Conditions
Rotted plantlets were moved to GreenTray ® , kept vertical with the help of an upper grid, and with their roots laid on the culture tray.The conditions to promote autotrophic growth were as follows: culture liquid: Murashige and Skoog (MS) medium with 0 g/L sucrose; air renewal of the culture jar at 1.5 L/min, forcing aeration in the regime indicated in Table 2 for the 6 weeks of culture; and light intensity: 400 µEm-2s-1 PAR 20 cm from the LED light.

Assays for Abiotic and Biotic Stress Agents
Two Prunus rootstocks, namely Garnem ® and Rootpac ® 20, and three apple varieties, namely Carrandona (M0676), Florina (M0216), and Rozona (M0163), were established in vitro, multiplied, and rooted in agar-containing medium following the protocol described in [38].The plantlets were transferred to a GreenTray ® and cultured under autotrophic conditions during the assays to characterize the plant materials' ability to withstand abiotic (high pH, low Ca 2+ concentration) or biotic stress (inoculation with fire blight).

Abiotic Stress Conditions: Rhizosphere pH and Ca 2+ on Plantlet Growth
To determine the differential tolerance of the two Prunus rootstocks to high pHs and Ca 2+ availability, as determined by the shoot fresh weights, 15 plantlets per rootstock were cultured in eight pH x Ca 2+ combinations.The liquid culture (Murashige and Skoog (MS) medium) was adjusted before autoclaving to pH 5.7 (standard) or pH 7.7 (alkaline) and used to test for tolerance to high pHs.In both pH conditions, the standard Ca 2+ concentration in the MS medium (3.0 mM CaCl 2 •2H 2 O) was compared with 1/2 (1.5 mM), 1/3 (1 mM), and 1/4 (0.75 mM) Ca 2+ concentrations in the rhizosphere.Two weeks after culture in the GreenTray ® , the shoot length (cm) of each plantlet was measured.

Abiotic Stress Conditions: In Vitro Atmospheric Humidity on Ca 2+ Uptake
Liquid culture media adjusted to pH 5.7, before autoclaving, and with Ca 2+ at the MS standard concentration (3.0 mM CaCl 2 •2H 2 O) were used to determine the differential ability of the two Prunus rootstocks to uptake and transport Ca 2+ from the rhizosphere to the roots and shoots.Since leaf transpiration is a determinant for Ca 2+ uptake and mobilization to the aerial part, a high aeration regime in the GreenTray ® was set for this assay, as described in [40].
2.6.3.Biotic Stress Conditions: Tolerance to Fire Blight An inoculum of fire blight (Erwinia amylovora) was prepared as described in [37].After three days of adaptation to the GreenTray ® under autotrophic conditions, 15 plantlets of each variety were inoculated with the bacteria with a pair of scissors (Figure 5), with two cuts in opposite sites perpendicular to the mid rip in the upper first expanded leaf.The level of affection of each plantlet was measured ten days post inoculation to determine the differential tolerance of both varieties to the bacteria, following the same levels of affectation described in [37].Briefly, susceptibility was recorded ten days post inoculation by dividing necrosis progression by shoot length.Necrosis progression was measured using the following scale: 0-no visible symptoms; 0.5-necrosis only affected veins of the inoculated leaf; 1-necrosis reached the petiole of the inoculated leaf; and 1+-necrosis reached the stem.
To determine the differential tolerance of the two Prunus rootstocks to high pHs and Ca 2+ availability, as determined by the shoot fresh weights, 15 plantlets per rootstock were cultured in eight pH x Ca 2+ combinations.The liquid culture (Murashige and Skoog (MS) medium) was adjusted before autoclaving to pH 5.7 (standard) or pH 7.7 (alkaline) and used to test for tolerance to high pHs.In both pH conditions, the standard Ca 2+ concentration in the MS medium (3.0 mM CaCl2•2H2O) was compared with 1/2 (1.5 mM), 1/3 (1 mM), and 1/4 (0.75 mM) Ca 2+ concentrations in the rhizosphere.Two weeks after culture in the GreenTray ® , the shoot length (cm) of each plantlet was measured.

Abiotic Stress Conditions:
In Vitro Atmospheric Humidity on Ca 2+ uptake Liquid culture media adjusted to pH 5.7, before autoclaving, and with Ca 2+ at the MS standard concentration (3.0 mM CaCl2•2H2O) were used to determine the differential ability of the two Prunus rootstocks to uptake and transport Ca 2+ from the rhizosphere to the roots and shoots.Since leaf transpiration is a determinant for Ca 2+ uptake and mobilization to the aerial part, a high aeration regime in the GreenTray ® was set for this assay, as described in [40].

Biotic Stress Conditions: Tolerance to Fire Blight
An inoculum of fire blight (Erwinia amylovora) was prepared as described in [37].After three days of adaptation to the GreenTray ® under autotrophic conditions, 15 plantlets of each variety were inoculated with the bacteria with a pair of scissors (Figure 5), with two cuts in opposite sites perpendicular to the mid rip in the upper first expanded leaf.The level of affection of each plantlet was measured ten days post inoculation to determine the differential tolerance of both varieties to the bacteria, following the same levels of affectation described in [37].Briefly, susceptibility was recorded ten days post inoculation by dividing necrosis progression by shoot length.Necrosis progression was measured using the following scale: 0-no visible symptoms; 0.5-necrosis only affected veins of the inoculated leaf; 1-necrosis reached the petiole of the inoculated leaf; and 1+-necrosis reached the stem.

Statistical Analysis
The experiment was set up as a completely randomized design (CRD), and the data were analysed by a one-way ANOVA.Statistical significance was judged at the level p < 0.05.When the analysis was statistically significant, a Tukey-Kramer HSD test was used for the separation of means.Data analysis was performed using JMP ® software (version 16.0.0,SAS Institute Inc., Cary, NC, USA).

Statistical Analysis
The experiment was set up as a completely randomized design (CRD), and the data were analysed by a one-way ANOVA.Statistical significance was judged at the level p < 0.05.When the analysis was statistically significant, a Tukey-Kramer HSD test was used for the separation of means.Data analysis was performed using JMP ® software (version 16.0.0,SAS Institute Inc., Cary, NC, USA).

Shoot Development
Optimal shoot development from internodal shoot explants cultured in the GreenTray ® includes multiplication by axillary branching, shoot elongation, and leaf development with-out vitrification (Figure 6).This was depended on the plant material, as well as on the frequencies of immersion and aeration, the composition of the culture medium, and the light intensity.Following the methodology described herein, a good development was accomplished for the almond × peach (Prunus dulcis × P. persica) hybrid rootstocks Intensia ® , Garnem ® , and GF677 (Figure 6A-C), as well as for the plum hybrid (P.besseyi × P. cerasifera) rootstock Rootpac ® 20 and the plum x almond hybrid (P.cerasifera × P. dulcis) rootstock Rootpac ® R (Figure 6D,E), along with the plum selection (P.cerasifera) rootstock Adara.

Extraction and Division of Shoots without Forceps
With all plant materials, it was easy to extract all shoots and cut the new growth with the use of a pair of scissors, returning the shoot bases back to the culture flask of the Green-Tray ® , as shown in Figure 4.  Other plant materials that showed good shoot elongation and leaf development but require more research on adjusting the culture media and immersion frequencies are the pear (Pyrus communis) rootstock OHF 87 TM (Figure 6F) and the pear rootstock Py170.

Extraction and Division of Shoots without Forceps
With all plant materials, it was easy to extract all shoots and cut the new growth with the use of a pair of scissors, returning the shoot bases back to the culture flask of the GreenTray ® , as shown in Figure 4.

Sequential Shoot Cuts without Subculture and Multiplication Rates
All plant materials used herein had the same multiplication rates in the GreenTray ® than in flasks with agar-containing medium.However, with the utilized culture media and protocols of immersion and aeration, the diverse plant materials tested had different capacities to sprout after being cut.The most productive was Rootpac ® R, which was able to stand seven cuts in a 19-week period of culture, producing 650 shoots from 25 initial explants, which equals 93 shoots for each cut, with an overall multiplication rate of 26, or a multiplication rate of four for each 3-week-long culture period, equivalent to the subculture time with agar-containing medium flasks.
Rootpac ® 20, GF677, and Intensia ® regrew for two consecutive cuts.The Cannabis sativa variety "Ferimon" was also able to regrow for two consecutive cuts, while Garnem ® , Adara, OHF 87 TM , and Py170 could not sprout after a cut.More work is required to develop better culture media and conditions in order favor the growth after being cut.

Hand Labor Reduction and Produced Shoots during Micropropagation
As shown in Figure 7, using the GreenTray ® system, there is a reduction in hand labor, accumulated along the subculture time, for the manipulation required to extract the shoots from the flask, cut them on a sterile surface, and culture them in a new flask.When using the GreenTray ® to establish a new generation (Figure 4B), equivalent to a new subculture in semisolid medium, the time needed is reduced by 65% compared to that required with semisolid medium.
Appl.Sci.2024, 14, x FOR PEER REVIEW 10 of 20 capacities to sprout after being cut.The most productive was Rootpac ® R, which was able to stand seven cuts in a 19-week period of culture, producing 650 shoots from 25 initial explants, which equals 93 shoots for each cut, with an overall multiplication rate of 26, or a multiplication rate of four for each 3-week-long culture period, equivalent to the subculture time with agar-containing medium flasks.Rootpac ® 20, GF677, and Intensia ® regrew for two consecutive cuts.The Cannabis sativa variety "Ferimon" was also able to regrow for two consecutive cuts, while Garnem ® , Adara, OHF 87 TM , and Py170 could not sprout after a cut.More work is required to develop better culture media and conditions in order favor the growth after being cut.

Hand Labor Reduction and Produced Shoots during Micropropagation
As shown in Figure 7, using the GreenTray ® system, there is a reduction in hand labor, accumulated along the subculture time, for the manipulation required to extract the shoots from the flask, cut them on a sterile surface, and culture them in a new flask.When using the GreenTray ® to establish a new generation (Figure 4B), equivalent to a new subculture in semisolid medium, the time needed is reduced by 65% compared to that required with semisolid medium.
When additional shoot cuts were made from one GreenTray ® , while only one cut was possible from each flask with agar-containing medium, the time saved in the procedure was reduced and the number of shoots produced with the same number of subcultures increased in the GreenTray ® .As shown in Figure 7, with a second shoot cut of the Roopac ® R, the number of shoots produced in the seventh subculture with the GreenTray ® approaches 2500 × 10 3 , 20% more than with the conventional semisolid-containing flasks.When additional shoot cuts were made from one GreenTray ® , while only one cut was possible from each flask with agar-containing medium, the time saved in the procedure was reduced and the number of shoots produced with the same number of subcultures increased in the GreenTray ® .As shown in Figure 7, with a second shoot cut of the Roopac ® R, the number of shoots produced in the seventh subculture with the GreenTray ® approaches 2500 × 10 3 , 20% more than with the conventional semisolid-containing flasks.
3.1.5.Number of Generations in the GreenTray ® , without Establishing from Agar Cultures Up to six generations of the rootstock Rootpac ® R were initiated, from one GreenTray ® to another, without having to start from cultures maintained in agar-containing medium.The procedure was carried out as indicated in Figure 4B, and there were no contaminations in either the GreenTray ® source of shoots or the newly established GreenTray ® .
With other plant materials, the number of generations was lower.For Rootpac ® 20, the maximum number of generations was four, while for Intensia ® , Garnem ® , GF667, Adara, OHF87 TM , and Py170, it was possible to establish only one generation.

Autotrophic and Long-Term Cultures
In the conditions stated to promote autotrophic growth, excellent root and shoot development were obtained in the GreenTray ® with rooted shoots of Rootpac ® 20 and Garnem ® , used for abiotic stress assays [40].
The three apple varieties, Carrandona, Florina, and Rozona, doubled their size after two weeks of culture and before inoculation with the biotic stress agent (Figure 8).

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With other plant materials, the number of generations was lower.For Rootpac ® 20, the maximum number of generations was four, while for Intensia ® , Garnem ® , GF667, Adara, OHF87 TM , and Py170, it was possible to establish only one generation.

Autotrophic and Long-Term Cultures
In the conditions stated to promote autotrophic growth, excellent root and shoot development were obtained in the GreenTray ® with rooted shoots of Rootpac ® 20 and Garnem ® , used for abiotic stress assays [40].
The three apple varieties, Carrandona, Florina, and Rozona, doubled their size after two weeks of culture and before inoculation with the biotic stress agent (Figure 8).Kalanchoe daigremontiana propagules collected from the leaves of a mature plant, established in the GreenTray ® , developed and produced new propagules in their leaves, which were spontaneously dropped on the culture tray and became new mature plants, in a cycle that was repeated four times over a year-long period without needing any manipulation inside the culture flask (Figure 9).We can observe the propagules at the edges of the mature leaves and the small plants growing on the surface of the culture tray, in which the white-yellowish film observed is not a contamination but as a result of the natural degradation of the previous generation of plants.Kalanchoe daigremontiana propagules collected from the leaves of a mature plant, established in the GreenTray ® , developed and produced new propagules in their leaves, which were spontaneously dropped on the culture tray and became new mature plants, in a cycle that was repeated four times over a year-long period without needing any manipulation inside the culture flask (Figure 9).We can observe the propagules at the edges of the mature leaves and the small plants growing on the surface of the culture tray, in which the white-yellowish film observed is not a contamination but as a result of the natural degradation of the previous generation of plants.

Rhizosphere pH and Ca 2+ on Plantlet Growth
Both Prunus rootstocks behaved differently to the high pH and lower Ca 2+ availability in the liquid culture medium.Garnem ® (Figure 10A), an almond x peach hybrid, showed a significantly (p < 0.001) better shoot growth at basic pH 7.5 than at the conventional culture pH 5.7, without interacting with the Ca 2+ concentration (p = 0.100) and without the effect of the lower Ca 2+ availability (p = 0.977).Instead, the plum hybrid Rootpac ® 20 (Figure 10B) was negatively affected in terms of its shoot growth when the plantlets were depleted of Ca 2+ (p = 0.001) to 0.75 mM, 25% of the usual 3 mM present in the medium, and also when the pH was alkaline (7.5) compared to the conventional pH 5.7 (p = 0.001).

Rhizosphere pH and Ca 2+ on Plantlet Growth
Both Prunus rootstocks behaved differently to the high pH and lower Ca 2+ availability in the liquid culture medium.Garnem ® (Figure 10A), an almond × peach hybrid, showed a significantly (p < 0.001) better shoot growth at basic pH 7.5 than at the conventional culture pH 5.7, without interacting with the Ca 2+ concentration (p = 0.100) and without the effect of the lower Ca 2+ availability (p = 0.977).Instead, the plum hybrid Rootpac ® 20 (Figure 10B) was negatively affected in terms of its shoot growth when the plantlets were depleted of Ca 2+ (p = 0.001) to 0.75 mM, 25% of the usual 3 mM present in the medium, and also when the pH was alkaline (7.5) compared to the conventional pH 5.7 (p = 0.001).

Atmospheric Humidity on Ca 2+ Uptake
As described in [40], GreenTray ® established a TA of 1 min and a WTA of 15 min, resulting in lowering the relative humidity in the culture flask to 60% during aeration and which recovered afterwards to 80% during the WTA.Instead, the non-aerated GreenTray ® had a constant 100% relative humidity.

Atmospheric Humidity on Ca 2+ Uptake
As described in [40], GreenTray ® established a TA of 1 min and a WTA of 15 min, resulting in lowering the relative humidity in the culture flask to 60% during aeration and which recovered afterwards to 80% during the WTA.Instead, the non-aerated GreenTray ® had a constant 100% relative humidity.

Tolerance to Fire Blight
For the apple plantlets inoculated as shown in Figure 5, after three days of culture in the GreenTray ® , the first symptoms of infection in the leaf blade were visible through the culture flask glass.At 7 and 10 d of culture, necrosis reached the leaf mid rib (Figure 11A) and shoot (Figure 11B) of the balanced apple variety Florina.
Ten days post inoculation, the different levels of susceptibility to Erwinia amylovora of the three apple varieties tested, Carrandona, Florina and Rozona, were evident (Figure 12), with increasing levels of susceptibility to fire blight, respectively.Shoot and root development was clearly better for Carrandona and Florina (Figure 12A,B) than for Rozona (Figure 12C), which showed evident growth reduction, wilting, and shoot and root necrosis.
For the apple plantlets inoculated as shown in Figure 5, after three days of culture in the GreenTray ® , the first symptoms of infection in the leaf blade were visible through the culture flask glass.At 7 and 10d of culture, necrosis reached the leaf mid rib (Figure 11A) and shoot (Figure 11B) of the balanced apple variety Florina.Ten days post inoculation, the different levels of susceptibility to Erwinia amylovora of the three apple varieties tested, Carrandona, Florina and Rozona, were evident (Figure 12), with increasing levels of susceptibility to fire blight, respectively.Shoot and root development was clearly better for Carrandona and Florina (Figure 12A,B) than for Rozona (Figure 12C), which showed evident growth reduction, wilting, and shoot and root necrosis.The affectation levels obtained in the GreenTray ® (Figure 13A) showed a significant (p < 0.001) difference among the three apple varieties.A significantly (p = 0.050) higher tolerance to fire blight was observed for Carrandona and Florina than Rozona.With a level of R 2 = 0.68 (Figure 13B), the level of susceptibility to fire blight observed in the GreenTray ® was significantly (p < 0.0001) and linearly correlated with the level of tolerance observed in potted plants grown in soil inside a climatic growth chamber of a BSL3 facility [37].The affectation levels obtained in the GreenTray ® (Figure 13A) showed a significant (p < 0.001) difference among the three apple varieties.A significantly (p = 0.050) higher tolerance to fire blight was observed for Carrandona and Florina than Rozona.With a level of R 2 = 0.68 (Figure 13B), the level of susceptibility to fire blight observed in the GreenTray ® was significantly (p < 0.0001) and linearly correlated with the level of tolerance observed in potted plants grown in soil inside a climatic growth chamber of a BSL3 facility [37].

Discussion
The extraction of shoots with forceps from in vitro culture containers, as well as from TIS bioreactors, followed by arranging them on sterile surfaces in order to divide them with the help of surgical scalpels and their subsequent introduction into new culture containers, a process known as subculturing in the conventional in vitro culture process, is time-consuming and represents a significant portion the hand labor involved in most in vitro plant culture processes.The components of the actual GreenTray ® TIS bioreactor described herein, which is a further evolution of the ones presented in the past [31,32], make it significantly improved and versatile compared to other commercial TIS bioreactors, such as RITA ® , Matis ® , Plantform TM , SETIS TM [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17], or the TIS developed by several in vitro plant culture laboratories oriented to solve particular propagation objectives for diverse plant species [18][19][20][21][22][23][24][25][26][27][28].
GreenTray provides the opportunity to automate the in vitro clonal plant propagation process because of its new methods: the disposition of the plant material, extraction from the culture vessel, easy manipulation for reorientation, shoot cutting, and reintroduction for the further growth of the shoot basis.These facts represent a significant reduction in the time required for conventional subcultures, implying a reduction in hand labor and an increase in the number of shoots produced per container, which reduce the cost per plant produced and therefore facilitates the application of micropropagation to more plant species.
Although excellent shoot development and sequential shoots cuts were obtained for Rootpac ® R, Rootpac ® 20, GF677, Intensia ® , and Ferimon, further research is needed to improve the culture media, the frequencies of immersion, and aeration for Garnem ® , Adara, OHF 87 TM , and Py170.Indeed, this observation is applicable to all plant species for which a great amount of experience has been accumulated during previous decades of work with semisolid culture media, compared to TIS culture systems.
As reported in previous works [41 -46], autotrophic in vitro culture is facilitated by aeration in the culture vessel and increasing the PAR radiation without stressing the plant materials.GreenTray ® 's design is particularly focused on improving aeration through two

Discussion
The extraction of shoots with forceps from in vitro culture containers, as well as from TIS bioreactors, followed by arranging them on sterile surfaces in order to divide them with the help of surgical scalpels and their subsequent introduction into new culture containers, a process known as subculturing in the conventional in vitro culture process, is time-consuming and represents a significant portion the hand labor involved in most in vitro plant culture processes.The components of the actual GreenTray ® TIS bioreactor described herein, which is a further evolution of the ones presented in the past [31,32], make it significantly improved and versatile compared to other commercial TIS bioreactors, such as RITA ® , Matis ® , Plantform TM , SETIS TM [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17], or the TIS developed by several in vitro plant culture laboratories oriented to solve particular propagation objectives for diverse plant species [18][19][20][21][22][23][24][25][26][27][28].
GreenTray provides the opportunity to automate the in vitro clonal plant propagation process because of its new methods: the disposition of the plant material, extraction from the culture vessel, easy manipulation for reorientation, shoot cutting, and reintroduction for the further growth of the shoot basis.These facts represent a significant reduction in the time required for conventional subcultures, implying a reduction in hand labor and an increase in the number of shoots produced per container, which reduce the cost per plant produced and therefore facilitates the application of micropropagation to more plant species.
Although excellent shoot development and sequential shoots cuts were obtained for Rootpac ® R, Rootpac ® 20, GF677, Intensia ® , and Ferimon, further research is needed to improve the culture media, the frequencies of immersion, and aeration for Garnem ® , Adara, OHF 87 TM , and Py170.Indeed, this observation is applicable to all plant species for which a great amount of experience has been accumulated during previous decades of work with semisolid culture media, compared to TIS culture systems.
As reported in previous works [41 -46], autotrophic in vitro culture is facilitated by aeration in the culture vessel and increasing the PAR radiation without stressing the plant materials.GreenTray ® 's design is particularly focused on improving aeration through two mechanisms, suction from the glass bottle containing the medium or the propulsion of air into the glass culture jar.PAR radiation was significantly increased with LED lights, with a variable and high range of intensities, paired with low heating effects.Aeration of the culture flask induces transpiration in the plantlet's leaves; therefore, water exits through the open stomas and CO 2 diffuses in, which, in return, favors photosynthesis [41].
Rooted plantlets of the Prunus rootstocks Rootpac ® 20 and Garnem ® , or the apple varieties Carrandona, Florina, and Rozona, grew in the GreenTray ® without sucrose in the culture medium during the biotic and abiotic stress agent assays that lasted several weeks.As an example of a long-term in vitro autotrophic culture, the Kalanchoe daigremontiana species demonstrated that the autotrophic culture conditions in the GreenTray ® are feasible for at least one year.GreenTray ® 's design allows access to shoots or plantlets by moving the container's lid and attached tray sideways.With this simple movement, all plants are pulled out of the culture recipient and displayed in front of the observer, so they can easily be treated, inoculated, phenotyped, sampled, or pictured.By pulling the holding racks, plantlet roots can be observed, sampled, or pictured the same way as the aerial part of the plants.Since the container holding the culture medium is independent from the cultured plantlets, it can be aliquoted, supplemented, inoculated, and observed at any time during culture [39,42].These characteristics make the GreenTray ® TIS superior to other commercial TIS bioreactors and containers to perform studies on abiotic [43][44][45][46] or biotic [47][48][49][50][51][52][53][54][55] stress agents.
The experimental conditions to assay the effects of pH and Ca 2+ availability in the rhizosphere were easily established and repeated with the GreenTray ® design.The differential capacities of the Prunus rootstocks Garnem ® and Rootpac ® 20 to grow in alkaline conditions and Ca 2+ depletion, reported herein, were in accordance with what is known from agronomic trials in calcareous soils [56,57].Therefore, GreenTray ® could be useful for many nutritional studies, including the characterization and selection of plant materials [36].
Forcing a reduction in the atmospheric humidity inside the GreenTray ® 's culture vessel, although periodic and recovered rapidly through the evaporation of wet surfaces and transpiration through the plantlet leaf stomas, implies a lower water potential, which induces higher transpiration of the plantlet's leaves and a higher water uptake at the roots, reinforcing Ca 2+ transport to the shoots [41].In these conditions, the rootstock Rootpac ® 20 presented a significantly higher shoot Ca 2+ uptake than Garnem ® [40].All mechanisms of ventilation inside the bioreactor, for which the GreenTray ® is especially suited for, increase the water potential in the atmospheric culture recipient, and this favors water and nutrient root uptake and translocation to the leaves.
In vitro assays for tolerance to Erwinia amylovora have always been approached in heterotrophic conditions [47][48][49][50][51][52][53][54].Although the results presented herein are for a reduced number of apple varieties, this is the first time that the test has been performed in the in vitro autotrophic conditions of the GreenTray ® , and the same plant material was tested against the same Erwinia amylovora strain in a confined growth chamber with grafted potted plants.Since a very good correlation was found in terms of susceptibility to fire blight between the in vitro and in vivo growth, it is recommendable to use the GreenTray ® bioreactor to select seedlings and obtain apple or pear varieties with higher tolerance to fire blight through cross-breeding.Further improvements will be needed in inoculating all plants with the same amount and concentration of the bacterial strain or obtaining a more uniform plant development before their inoculation.
Consequently, the GreenTray ® , as it is developed, is a good tool to propagate plants in vitro at a lower cost and higher multiplication rates.Its design favors the in vitro growth of plantlets in autotrophic conditions, like hydroponic cultures, but in a highly controlled environment and with reduced space requirements.Bioassays performed in the GreenTray ® to characterize the responses of plants to biotic and abiotic stress agents are more reliable

Figure 2 .
Figure 2. Assembled GreenTray ® , sterilized and ready for the cultivation of plants.

Figure 2 .
Figure 2. Assembled GreenTray ® , sterilized and ready for the cultivation of plants.Figure 2. Assembled GreenTray ® , sterilized and ready for the cultivation of plants.

Figure 2 .
Figure 2. Assembled GreenTray ® , sterilized and ready for the cultivation of plants.Figure 2. Assembled GreenTray ® , sterilized and ready for the cultivation of plants.
2.5.5.Subculture in New GreenTray ® and Number of Generations Shoots produced from the GreenTray ® can either be moved to soil for rooting in an acclimation facility, or placed in another GreenTray ® bioreactor, what herein is called a generation (Figure4B) The number of generations is recorded for each plant material.

Figure 4 .
Figure 4. Propagation of Rootpac ® 20 in the GreenTray ® : (A) culture tray extraction from the glass flask; (B) orientation of the culture tray to facilitate shoot cut, for the next GreenTray ® generation; (C) introduction of the culture tray holding the shoot basis into the culture glass jar.

Figure 4 .
Figure 4. Propagation of Rootpac ® 20 in the GreenTray ® : (A) culture tray extraction from the glass flask; (B) orientation of the culture tray to facilitate shoot cut, for the next GreenTray ® generation; (C) introduction of the culture tray holding the shoot basis into the culture glass jar.

Figure 5 .
Figure 5. Inoculation of apple leaves with fire blight, grown in the GreenTray ® .

Figure 5 .
Figure 5. Inoculation of apple leaves with fire blight, grown in the GreenTray ® .

Figure 7 .Figure 7 .
Figure 7. Time required for manipulation (lines) and number of shoots produced (bars) of Rootpac ® R grown in flasks with agar-containing medium (segmented) or in GreenTray ® (solid) during the first 7 subcultures.

Figure 8 .
Figure 8. Plantlets of the apple variety Carrandona, grown under autotrophic conditions for three weeks in the GreenTray ® .

Figure 8 .
Figure 8. Plantlets of the apple variety Carrandona, grown under autotrophic conditions for three weeks in the GreenTray ® .

Figure 9 .
Figure 9. Plantlets of Kalanchoe daigremontiana grown in the GreenTray ® over a year-long period under autotrophic conditions.

Figure 9 .
Figure 9. Plantlets of Kalanchoe daigremontiana grown in the GreenTray ® over a year-long period under autotrophic conditions.

Figure 10 .
Figure 10.Shoot length of (A) Garnem ® and (B) Rootpac ® 20 plantlets cultured in the GreenTray ® at pH 5.7 (light gray) or pH 7.5 (dark gray) as affected by lowering the Ca 2+ concentration from 3 to 1.5, 1, and 0.75 mM, in the culture medium.Mean values come from the measures of 15 replicates per genotype, pH and Ca 2+ concentration.Bars represent the standard error.For each rootstock, Ca 2+ concentrations with different letter (a,b), and pH with asterisk (*) are significantly different (Tukey-Kramer HSD, p < 0.05).

Figure 10 .
Figure 10.Shoot length of (A) Garnem ® and (B) Rootpac ® 20 plantlets cultured in the GreenTray ® at pH 5.7 (light gray) or pH 7.5 (dark gray) as affected by lowering the Ca 2+ concentration from 3 to 1.5, 1, and 0.75 mM, in the culture medium.Mean values come from the measures of 15 replicates per genotype, pH and Ca 2+ concentration.Bars represent the standard error.For each rootstock, Ca 2+ concentrations with different letter (a,b), and pH with asterisk (*) are significantly different (Tukey-Kramer HSD, p < 0.05).

Figure 11 .
Figure 11.Symptoms of fire blight on (A) leaves, seven days post inoculation, and on (B) shoot and leaves, ten days post infection of the apple variety Florina grown in the GreenTray ® .

Figure 11 . 20 Figure 12 .
Figure 11.Symptoms of fire blight on (A) leaves, seven days post inoculation, and on (B) shoot and leaves, ten days post infection of the apple variety Florina grown in the GreenTray ® .Appl.Sci.2024, 14, x FOR PEER REVIEW 15 of 20

20 Figure 13 .
Figure 13.Levels of affectation by Erwinia amylovora on three apple varieties (A) tested for tolerance in the GreenTray ® , and (B) their correlation with the known effect levels observed with potted plants.Mean values come from the measures of 15 replicates per genotype.Bars represent the standard error.Means with different letters (a, b) are significantly different (Tukey-Kramer HSD, p < 0.05).

Figure 13 .
Figure 13.Levels of affectation by Erwinia amylovora on three apple varieties (A) tested for tolerance in the GreenTray ® , and (B) their correlation with the known effect levels observed with potted plants.Mean values come from the measures of 15 replicates per genotype.Bars represent the standard error.Means with different letters (a, b) are significantly different (Tukey-Kramer HSD, p < 0.05).

Table 1 .
Plant material used herein for propagation, and biotic or biotic stress assays.