International Journal of Biological Macromolecules 220 (2022) 135–146

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Agarose hydrogel composite supports microgreen cultivation with

enhanced porosity and continuous water supply under terrestrial and
microgravitational conditions
Zi Teng a, b, Yaguang Luo a, c, *, Daniel J. Pearlstein c, Bin Zhou a, Christina M. Johnson d,
Joseph Mowery e, Qin Wang b, Jorge M. Fonseca a
a
Food Quality Laboratory, U. S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, MD, United States of
America
b
Department of Nutrition and Food Science, University of Maryland, College Park, MD, United States of America
c
Environmental Microbial and Quality Safety Laboratory, U. S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center,
Beltsville, MD, United States of America
d
NASA Postdoctoral Program, Kennedy Space Center, FL, United States of America
e
Electron & Confocal Microscopy Unit, U. S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, MD, United
States of America

A R T I C L E I N F O A B S T R A C T

Keywords: Hydrogels are attractive soilless media for plant cultivation with strong water and nutrient retention. However,
Agarose pristine hydrogels contain mostly ultra-micro pores and lack air-filled porosity for root zone aeration. Herein we
Hydrogel report a porous hydrogel composite comprising an agarose network and porous growing mix particle (GMP)
Microgreens
fillers. The agarose backbone allowed the composite to sustain a 12-d growth cycle for red cabbage microgreens
Soilless culture medium
Pore size distribution
without the need for watering or crew interaction. Moreover, the GMP induced greater total pore volume and
Microgravity increased the prevalence of pores >30 μm by 8-fold. Further investigation suggested that the nutrients from GMP
accounted for a 54 % increase in microgreen yield over pristine hydrogel, while the porous structure introduced
by GMP improved the yield by another 44 %. Increased air-filled porosity accelerated the water transport and
loss of hydrogel but maintained favorable water potential levels for plant extraction. Finally, the hydrogel
composite supported microgreen growth satisfyingly under simulated microgravity despite some morphological
changes. Results of this study reveal a novel growth substrate that is lightweight, convenient, and water-efficient,
while effectively sustaining plant growth for multiple applications including indoor farming and space farming.

1. Introduction
Controlled environment agriculture (CEA) has developed rapidly as a
promising approach to feeding global population with minimal land and
water usage [1]. These operations predominantly use soilless substrates
such as perlite, peat moss, coconut coir, and rock wool [2–4]. Despite the
well-established success achieved by the abovementioned substrates,
there are several issues associated with these materials. For instance, the
exploitation of peat moss has had negative ecological impacts on har­
vesting areas [5]; coconut coir is biodegradable and cost effective but
has high salt concentrations and promotes fungal and bacterial growth
[6,7]. In addition to these concerns, many CEA systems such as nutrient
film technique (NFT) require constant delivery of water to support plant
growth. This adds to operational costs associated with equipment such
as pumps and sensors and increases the risk of unsatisfactory plant
growth or even plant death in case of power failures. Consequently, it is
of great importance to develop a biodegradable, cost-effective, and
reliable substrate to replace or complement existing materials to
improve the efficiency and sustainability of CEA.
Hydrogels are polymeric matrices with strong water holding capac­
ity. Such ability originates from the high porosity (typically >90 %)
which contributes to a strong capillary force to retain water and nutri­
ents [8,9]. Agarose is a linear polysaccharide consisting of D-galactose
and 3,6-L-galactose. Agarose hydrogel has been widely used in tissue

* Corresponding author at: Food Quality Laboratory, U. S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center,
Beltsville, MD, United States of America.
E-mail address: Yaguang.Luo@usda.gov (Y. Luo).

https://doi.org/10.1016/j.ijbiomac.2022.08.046
Received 20 May 2022; Received in revised form 6 August 2022; Accepted 7 August 2022
Available online 10 August 2022
0141-8130/Published by Elsevier B.V.
Z. Teng et al. International Journal of Biological Macromolecules 220 (2022) 135–146

culturing owing to its biocompatibility, stability, and sustained release Table 1

of water and nutrients [10]. However, pristine agarose hydrogel Formulas, pH, TDS, and nitrate/nitrite values of modified agarose hydrogel
comprising mostly ultra-micropores (typically 5 μm or below) and lacks samples.*
air-filled porosity (30 μm or above) at swollen state [11]. This leads to Sample ID Agarose Solvent** GMP pH TDS NO3/
poor gas exchange and suboptimal root growth from the perspectives of (mg/mL) (%) (mg/ NO2
food production. Addressing that concern requires a balanced pore size L) (mg/L)

distribution, with a mixture of micro- and macropores offering optimal A125 12.5 Water 0 6.80 49 ± <LOD**
water delivery and root zone aeration. ± 3d
0.02c
Beyond the Earth, the production of fresh vegetables during deep
A125FE 12.5 Fine GMP 0 6.76 774 42.5 ±
space exploration has been recognized as a critical element for the extract ± ± 16a 2.1a
physical and psychological health of astronauts on missions [12]. 0.03c
Hydrogels are a promising candidate as a plant growth medium as they A125E 12.5 Crude GMP 0 7.12 720 44.3 ±
may address two challenges associated with existing growth systems: (1) extract ± ± 20a 1.4a
0.05a
inconsistent water transfer leading to either root hypoxia or salt burn A125P25 12.5 Water 2.5 6.85 158 13.1 ±
[13], and (2) the mass of equipment and substrates (perlite, peat moss, ± ± 9c 1.2b
and arcillite) [14] compared to the limited volume and lift capacity of 0.05c
space launches. Agarose hydrogel has been employed in various A125P100 12.5 Water 10 6.81 674 46.2 ±
± 12ab 1.5a
research systems, including the Plant Growth Unit (PGU) and Biological ±
0.04c
Research in Canisters (BRIC) series including BRIC-60, BRIC-100, BRIC- A75P25 7.5 Water 2.5 6.95 166 12.5 ±
100VC, BRIC-PDFU, and BRIC-LED [15,16]. However, severe root zone ± ± 14c 0.8b
hypoxia has occurred due to the lack of convection under microgravity, 0.03b
leading to delayed plant development and increased ethanol fermenta­ A75P100 7.5 Water 10 6.75 656 45.5 ±
± 30b 0.9a
tion [16,17]. We hypothesized that the root zone hypoxia is exacerbated
±
0.05c
by the inadequate air-filled porosity in the hydrogel-based medium
*
[15,16], which may be addressed by tuning the pore size distribution in Data are presented as mean ± standard error (n = 3). Different letters with
the gel matrix. each column denote statistically significant difference (P < 0.05).
**
Below limit of detection.
In this study, we aimed to develop and evaluate an agarose-based
plant culturing medium with enhanced air-filled porosity. We added a
small amount of growing mix particles (GMP) as natural fillers to alter 2.2.1. Agarose control
not only the porosity but also the nutrient composition of the hydrogel, Agarose was suspended in water at 7.5 or 12.5 mg/mL. The sus­
both of which could facilitate plant tissue development. To separate the pension was heated to and held at 95 ◦ C for 30 min, and the resultant
effects of the nutrient and porosity, we compared three types of solution was incubated in water bath at 45 ◦ C for 60 min. Subsequently,
hydrogels: pristine agarose (control), agarose with GMP extract (altered 160 mL of the solution was transferred to a paper cup (7.6 cm diameter
nutrient composition), and agarose with GMP (altered nutrient × 9 cm height, same hereinafter), which was held on ice bath to allow
composition and porosity). The hydrogel samples were examined for gelation.
their chemical composition, morphology, porosity profile, surface area,
texture property, and water potential, all of which are critical parame­ 2.2.2. Agarose with GMP extract
ters for plant tissue development. Red cabbage (Brassica oleracea var. An aqueous extract of the GMP was prepared by immersing 100 g
capitata f. rubra) microgreens were chosen for proof-of-concept due to GMP in 1 L water in a Whirl-Pak ® sample bag with 0.33 mm pore size
their ease of measurement (short growth cycle and intense color) and (Cole-Parmer, Vernon Hills, IL, USA). The suspension was held at room
practical values (bold flavor and high nutrient level) [18]. These qual­ temperature for 24 h, after which the filtrate (defined as crude extract)
ities also make red cabbage microgreens a candidate crop for spaceflight was collected. A portion of the extract was further filtered through a 1.5-
applications [19]. The growth of microgreens on those media was μm syringe filter (Pall Corporation, New York, NY, USA) to produce a
evaluated by yield, stem length, leaf area, and stem and leaf color. clear yellow solution defined as fine extract. Hydrogel was prepared
Finally, the suitability of the hydrogel composite under simulated following the same procedures as agarose control (12.5 mg/mL
microgravitational condition was assessed. agarose), except that water was replaced with crude (A125E) or fine
extract (A125FE).
2. Materials and methods
2.2.3. Agarose with GMP
2.1. Materials Agarose solution was prepared, heated, and incubated as with the
control. The solution (160 mL) was transferred to a paper cup containing
Agarose (biomolecular biology grade, EEO < 0.13, gel point 36 ± 4 g (2.5% in solution) or 16 g (10% in solution) GMP. The mixture was
1.5 ◦ C, gel strength ≥1200 g/cm2 at 1 %) was purchased from Sigma stirred manually on an ice bath for approximately 7 min until gelation
Aldrich (St. Louis, MO, USA). Sungro Metro-Mix ® 360 growing mix was complete.
particles (GMP) were purchased from Griffin's Greenhouse Supply Co.
(Morgantown, PA, USA). The main components of the GMP are 2.3. Characterization of growth substrates
vermiculite, peat moss, tree bark, and dolomite lime. Red cabbage seeds
suitable for microgreen culturing were provided by Johnny's Selected The hydrogel samples were characterized at different stages of
Seeds (Waterville, ME, USA). Deionized water was used throughout the preparation. The GMP were analyzed without pre-treatment. Detailed
study. testing procedures are provided below.

2.2. Preparation of agarose hydrogels
Three types of hydrogels were prepared with agarose as described
below. The formulation and denotation for all samples are summarized
in Table 1.
2.3.1. pH, TDS, and nitrate content
All measurements were performed with gelling solutions or mixtures
incubated at 50 ◦ C for 30 min. The pH was analyzed with a digital pH
meter (Oakton Instruments, Vernon Hills, IL, USA). The TDS was
measured using a TDS meter (135A, Orion, Thermo Fisher Scientific,

136
Z. Teng et al.
Waltham, MA, USA). The nitrate level was analyzed by simplified TKN
method (Hach method 10,242) [20].
2.3.2. Elemental composition
The elemental compositions of GMP and freeze-dried hydrogels were
measured by inductively coupled plasma (ICP) analysis. Samples (500
mg) were digested per EPA 200.2 method [21] and analyzed with an
ICPE-9000 ICP Atomic Emission Spectrometer (Shimadzu Scientific In­
struments, Columbia, MD, USA) by previously described procedures
[22]. The measured levels of elements were converted to fresh weight-
based values using the dry matter contents of samples measured
gravimetrically.
2.3.3. Cryo-SEM
The morphology of GMP and fresh hydrogel was observed with a
Hitachi SU-7000 SEM (Hitachi Ltd., Tokyo, Japan) equipped with
Quorum PP3010 Cryo Prep system (Quorum Technologies, Lewes, UK).
Samples were frozen in liquid nitrogen and freeze-fractured by previ­
ously described procedures [23] and then coated with a 10 nm layer of
conductive platinum using a magnetron sputter head. The resultant
specimen was observed in the SEM on a − 170 ◦ C cryostage, using an
accelerating voltage of 15 kV. Representative images were reported.
2.3.4. Mercury intrusion porosimetry (MIP)
The porosity of GMP and freeze-dried hydrogel was measured with a
Micromeritics Autopore V 9600 mercury intrusion porosimeter (Micro­
meritics Instrument Corporation, Norcross, GA, USA). Pore size distri­
bution and total porosity were determined following previously
described procedures [24] assuming cylindrical pore shape. The per­
centages of areas attributed to macropores (>75 μm), mesopores (30–75
μm), micropores (5–30 μm), ultramicropores (0.1–5 μm), and crypto­
pores (<0.1 μm) were calculated from the pore size distribution.
2.3.5. Nitrogen adsorption and BET analysis
Nitrogen adsorption and BET (Brunauer-Emmett-Teller) analysis
were performed on GMP and freeze-dried hydrogels with a Micrometric
ASAP 2020 adsorption analyzer (Micrometrics Instrument Corp, Nor­
cross, GA, USA). Samples (100 mg) were degassed at 180 ◦ C for 6 h and
measured for their nitrogen adsorption isotherm from which BET sur­
face area, total pore volume were derived.
2.3.6. Texture analysis
The stress-strain curves of freshly prepared hydrogels were measured
using a TA-XT21 texture analyzer (Texture Technologies Corp., Hamil­
ton, MA, USA). Cylindrical samples (34 mm diameter x 25 mm height)
were analyzed by the compression test under the following conditions:
load cell of 2 kg, probe diameter of 10 mm, test speed of 0.5 mm/s, and
maximal strain of 30 %.
2.4. Germination test
Hydrogel samples were equilibrated at 25 ◦ C for 16 h, after which
twenty red cabbage seeds were sown evenly and pressed gently against
the gel surface. Seeded substrates were incubated at 25 ◦ C and 70–80 %
relative humidity (RH) for 4 d, during which the germination rate (GR)
was recorded as the percentage of germinated seeds relative to the total
number (20). A seed was deemed germinated as the radicle (1–2 mm)
emerged [25]. For comparison purpose, GMP saturated with DI water
(2.2 g water per gram GMP) was tested following the same procedure.
2.5. Microgreen growth tests under terrestrial conditions
2.5.1. Seeding and growth
Prior to seeding, hydrogel samples were incubated at 25 ◦ C for 16 h,
and the weight of each sample was recorded. Thereafter, 1.5 g of red
cabbage seeds were sown evenly on each gel sample and gently pressed
137
International Journal of Biological Macromolecules 220 (2022) 135–146
to enhance contact. The seeded samples were covered with aluminum
foil and incubated in darkness at 25 ◦ C for 60 h. Subsequently, the foil
was removed, and the samples were placed in an enclosed growth space
illuminated by eight EnviroGro T5 bulbs (454 W total) from Hydrofarm
(Fairless Hills, PA, USA). As measured with a Sekonic C-7000 light meter
(Sekonic Corporation, North White Plains, NY, USA), the bulbs provided
a color temperature of 6129 K and a spectrum consisting of 35 % blue
(400–500 nm), 42 % green (500–600 nm), 21 % red (600–700 nm), and
2 % far red (700–800 nm). The growth condition was as follows: tem­
perature of 25–29 ◦ C, RH of 75–85 %, photosynthetic photon flux den­
sity (PPFD) of 230 ± 20 μmol/s/m2, and photoperiod of 12 h. The water
potential of hydrogel samples was monitored throughout a 12-d growth
cycle with a TEROS 31 tensiometer connected to a ZL6 data logger
(Meter Group Inc., Pullman, WA, USA). No watering was performed
during the growth cycle.
2.5.2. Harvest and characterization
The microgreens were harvested manually on Days 7 and 12 at
approximately 1 cm above the hydrogel surface. The total fresh weight
was measured, and the remaining hydrogel was cleaned for plant tissue
and weighed to determine water loss. Ten harvested plants were placed
on a matte white paper sheet situated in a portable photography tent
with controlled lighting [26]. Photos were taken and analyzed for stem
length and color (CEILab 1976) with ImagePro 10 software (Media
Cybernetics, Rockville, MD, USA) following our previously established
protocols [26]. Thereafter, the cotyledons were excised carefully from
the plants, flattened with forceps, photographed, and analyzed by
ImagePro 10 for leaf area and color.
2.6. Preliminary test under simulated microgravity
A random positioning machine (RPM, version 2.0, Airbus, Leiden,
Netherlands) was used to simulate microgravitational conditions. The
RPM consists of two independently rotating frames, which are
controlled by the manufacturer-developed algorithms to offset the
directional effect of gravity. Hydrogel samples (16 mL) including A125,
A125FE, and A125P100 were prepared in 20-mL polypropylene cups
and seeded with 0.2 g red cabbage seeds. The samples were fixed onto
the sample stage located on the primary rotating frame, after which the
RPM was set to run program “p0b” (simulated zero gravity) for 12 d.
Samples were covered by aluminum foil in the first 60 h of clinorotation
to block light. The growth conditions were as follows: temperature of
23–25 ◦ C, RH of 65–80 %, PPFD of 40 ± 3 μmol/s/m2, and photoperiod
of 12 h. The ground control was performed by the same procedure
except that the samples were not placed on the RPM. The PPFD applied
on the ground control was 29 ± 2 μmol/s/m2, which was chosen by
averaging the PPFD measured at the rotating stage over 3 h.
2.7. Statistics
All treatments and analyses were performed in triplicate, except for
MIP and BET which were conducted only once. Data were presented as
the mean ± standard error. Statistical significance was determined by
one-way ANOVA followed by Tukey post-hoc test at an experiment-wise
significance level (α) of 0.05, using SigmaPlot 13.0 (Systat Software,
Palo Alto, CA, USA).
3. Results
3.1. Physicochemical properties of gelling solution
Table 1 delineates the formulas, pH, TDS, and nitrate levels of
hydrogel samples. The control (A125) showed a pH of 6.80 and TDS of
49 ± 3 mg/L. Replacement of water with fine GMP extract (A125FE) did
not lead to a significant (p < 0.05, same hereinafter) drop in pH, while
replacement of water with crude extract (A125E) led to a slight increase
Z. Teng et al. International Journal of Biological Macromolecules 220 (2022) 135–146

in pH to 7.12. The GMP-containing hydrogels showed similar pH to the
control, although sample A75P25 exhibited slightly higher pH of 6.95.
In addition, samples prepared with the extract (A125E and A125FE)
showed much higher TDS and nitrate levels than A125 but comparable
values to A125P100 and A75P100. The TDS and nitrate levels of
hydrogel composites containing 2.5 % GMP were approximately one
fourth those of composites containing 10 % particles. Lastly, at each
level of GMP, there was no significant difference between formulas
containing 7.5 or 12.5 mg/mL agarose.
of different types of pores of hydrogels and GMP. GMP-free samples,
including A125, A125FE, and A125E, showed porosity above 90 %.
However, over 98 % of the pore area was occupied by cryptopores,
namely, the pores that are smaller than 0.1 μm. On the other hand,
sample A125P100 showed slightly lower porosity at 86 %, but the
percentage of cryptopores fell to 11 %, and the proportions of macro-,
meso-, micro-, and ultramicropres all increased significantly. Lastly, the
GMP while exhibiting the lowest porosity (66 %) showed the greatest
ratios of macro-, meso-, and micropores.

3.2. Elemental composition 3.5. Nitrogen adsorption and BET surface area

Table 2 summarizes the contents of selected elements in the hydrogel As shown in Fig. 3, all tested hydrogels and GMP exhibited a Type II
and GMP. The control (A125) contained trace amounts of P and S, while isotherm. The adsorption at low relative pressure (P/P0) was attributed
the GMP exhibited K, Ca, and Mg levels over 10,000 mg/kg FW, as well to the filling of micropores with nitrogen, which followed similar dy­
as P and S contents above 1000 mg/kg FW. The hydrogel prepared with namics among all samples. As the pressure increased, the isotherms
10 % GMP showed P, K, Ca, Mg, and S contents that were between 10 % started to diverge, with GMP and A125P100 absorbing the largest
and 12 % those of the GMP. In contrast, the samples prepared with GMP quantity of nitrogen. This phenomenon suggested different rates of
extract (A125FE and A125E) had much lower contents in those ele­ monolayer and multilayer formation, as well as capillary condensation,
ments, which suggested limited water solubility of GMP constituents. among the tested samples.
Table 4 summarizes the BET surface area, pore volume, and average
pore size of different samples measured by nitrogen adsorption. It should
3.3. Morphology
be noted that this measurement provides information on pores in the
nanometer range, which complements the MIP analysis that mainly
Fig. 1 shows the cryo-SEM images of hydrogel samples and GMP. The
covers the micron range of pore size distribution. As shown in the table,
cryo-fracture process allowed cross-sectional observation of hydrogel
all hydrogel samples and GMP measured similar BET surface area which
samples while maximally preserving their structures at hydrated state.
was derived from the linear portion of the adsorption isotherm. In
As can be seen, the control (A125) exhibited a smooth cross section
addition, sample A125FE and A125E showed 11 % and 44 % greater
(Fig. 1A and B). Similar structure was observed on A125FE (Fig. 1C and
pore volume respectively, as well as 132 % and 139 % greater average
D). The hydrogel prepared with crude GMP extract (A125E) showed
pore diameter respectively, compared to A125. Sample A125P100 had
slightly more roughness (Fig. 1E and F) probably due to the incorpora­
217 % higher total pore volume and 282 % greater average pore
tion of fine particulates during preparation. Lastly, the hydrogel pre­
diameter compared to A125, and a similar trend was observed with
pared with GMP (A125P100) exhibited more complex structure with
GMP.
increased roughness and pore size ranging from several microns
(Fig. 1H) to approximately 100 μm (Fig. 1G). This additional porosity
3.6. Texture analysis
was probably attributable to the constituents of GMP (Fig. 1I).
Fig. 4 displays the compression stress-strain curves of hydrogel
3.4. Porosity by MIP samples. The control (A125) showed the greatest stress at break (69.1
kPa) followed by A125FE (61.1 kPa) and A125E (35.3 kPa). Incorpo­
Fig. 2 shows the pore size distribution of hydrogels and GMP ration of GMP generally induced a decrease in stress at break, with
measured by MIP. GMP-free samples (A125, A125FE, and A125E) A125P25 and A125P100 showing maximal stress of 24.9 kPa and 30.6
possessed pores predominantly in the range of 0.01 to 0.1 μm. In kPa, respectively. Lastly, reducing the agarose level to 7.5 mg/mL
contrast, sample A125P100 demonstrated a wider pore size distribution further lowered the maximal stress of hydrogel to 10.7 kPa.
together with an increase in average pore size. This change was in line
with the broad pore size distribution of GMP which ranged between 0.1 3.7. Germination test
and 100 μm.
Table 3 summarizes the porosity, average pore size, and percentages As shown in Fig. 5, the GR on all substrates changed in similar
temporal trends, i.e., a low GR (5 to 20 %) on Day 1, followed by a rapid
Table 2 increase on Day 2 (up to 90 %) and subsequent plateau on Days 3 and 4.
Elemental composition of different agarose hydrogel samples and growing mix The greatest difference in GR was observed on Day 1, with hydrogels
particles (GMP).* with GMP extract (A125FE and A125E) showing higher GR (20 % and
Sample ID P K Ca Mg S 28 % respectively) than A125P100 (10 %) and GMP (5 %) that yielded
the lowest GR. On Day 4, A75P25 showed the highest GR of 95 %,
mg/kg mg/kg FW mg/kg FW mg/kg FW mg/kg
FW FW whereas A125 and A125E both yielded the lowest GR of 83 %.
A125 0.16 ± <LOD** <LOD** <LOD** 2.8 ±
0.01e 0.1e 3.8. Microgreen growth
GMP 1074 ± 19,123 ± 14,570 ± 37,520 ± 1782 ±
95a 305a 60a 527a 78a Fig. 6 displays the growth of red cabbage microgreens on seven
A125FE 4.46 ± 130 ± 2d 157 ± 2d 66 ± 1d 122 ± 1d hydrogel substrates. The control (A125) produced the shortest stems and
0.01d
A125E 6.60 ± 162 ± 1c 220 ± 1c 98 ± 2c 160 ± 2c
limited root development. The hydrogel prepared with fine (A125FE)
0.07c and crude (A125E) GMP extract showed significantly longer stems and
A125P100 113 ± 3b 2282 ± 95b 1725 ± 4498 ± 84b 177 ± 2b greater amount of root through and around the matrix. The hydrogel
57b supplemented with 10 % GMP (A125P100 and A75P100) yielded the
*
Data are presented as mean ± standard error (n = 3). Different letters with greatest stem length and best root development, and those samples also
each column denote statistically significant difference (P < 0.05). showed the highest degree of shrinkage due to water loss. Formulas with
**
Below limit of detection. 2.5 % GMP (A125P25 and A75P25) yielded results superior to A125 and

138
Z. Teng et al.
Fig. 1. Cryo-SEM images for different hydrogel samples: A125 (A, B), A125FE (C, D), A125E (E, F), A125P100 (G, H), and GMP (I).
A125FE, similar to A125E, and inferior to those with 10 % GMP.
Fig. 7 compares the fresh weight, stem length, leaf area, and stem
and leaf colors yielded by different hydrogel samples. As shown in
Fig. 7A, the fresh weight increased continuously and ranked similarly
among treatments between Days 7 and 12. The control (A125) yielded
the lowest fresh weight (4.22 g) on Day 12, followed by A125FE (6.50 g),
A125P25 (7.54 g), A125E (7.62 g), and A125P100 (9.35 g). As indicated
by those results, the GMP and its fine extract improved the yield of red
cabbage microgreens by 122 % and 54 % over the control, respectively.
Finally, decreasing the agarose level from 12.5 to 7.5 mg/mL improved
the yield by a further 22 % and 24 % in the presence of 2.5 % and 10 %
GMP, respectively.
The trends of stem length and leaf area (Fig. 7B) was overall
consistent with that of the fresh weight: GMP-containing hydrogels
yielded the longest stems and largest leaves, followed by A125E,
A125FE, and lastly A125. However, decreasing agarose level from 12.5
to 7.5 mg/mL in the presence of GMP did not lead to a significant change
in stem and leaf dimensions despite yielding higher fresh weight. It is
also noteworthy that the treatment A125P100 did not result in the
139
International Journal of Biological Macromolecules 220 (2022) 135–146
highest fresh weight or stem length but produced the largest leaf area.
Fig. 7C, D, and E depict the effect of hydrogel formula on the color of
stems and leaves of red cabbage microgreens. Compared to the control,
sample A125FE did not induce a significant change in L*, a*, or b* of
stems or leaves despite its effects on microgreen yield and size. Samples
A125E led to a slight decrease in a* of leaves but did not show significant
effect on other color parameters. On the contrary, GMP-containing
hydrogels caused an increase in L* values by approximately 25 % and
34 % for stems and leaves, respectively (Fig. 7C). Moreover, GMP-
containing formulas produced lower a* (Fig. 7D) and higher b* values
(Fig. 7E) for both stems and leaves, although there was no significant
difference in color parameters within this group of samples. Overall,
these results suggest that hydrogel composites yielded lighter, yellower,
and greener color in both stems and leaves of red cabbage microgreens.
3.9. Water equilibrium of hydrogel
Fig. 8A shows the mass of hydrogel on Days 7 and 12. The initial
mass of all samples was 159 ± 3 g. After seven days of microgreen
Z. Teng et al. International Journal of Biological Macromolecules 220 (2022) 135–146

Fig. 2. Pore size distribution of hydrogel samples.

Table 3
Porosity, average pore size, and percentages of area occupied by different categories of pores of hydrogel samples.
Label Porosity% Area-averaged Pore size (μm) Macropores% Mesopores% Micropores% Ultramicro-pores% Crypto-pores%

A125 96.42 0.27 0.13 0.15 0.41 0.25 99.06

A125FE 96.22 0.18 0.13 0.03 0.08 0.19 99.56
A125E 92.09 0.22 0.16 0.04 0.13 0.83 98.84
A125P100 85.65 2.70 0.86 1.63 5.95 80.11 11.45
GMP 66.20 4.15 9.43 3.91 24.87 54.45 7.34

Table 4
Surface area, pore volume, and pore size of different agarose hydrogel samples
and growing mix particles (GMP) at nanoscale measured by BET surface area
analysis.
Sample ID BET surface BJH adsorption total BJH adsorption average
area pore volume pore diameter
(m2/g) (cm3/g) (nm)

A125 2.922 0.00133 2.702

A125FE 2.843 0.00147 6.282
A125E 2.889 0.00192 6.454
A125P100 2.701 0.00422 10.32
GMP 2.846 0.00520 10.87

growth, all hydrogel samples lost approximately 30 g water, of which

between 1.95 and 9.15 g was attributable to the weight gain of micro­
greens (Fig. 7A). On Day 12, samples A125 lost the smallest amount of
water (44 g), followed by A125FE (46 g) and A125E (52 g). Hydrogel
composites with 2.5 % GMP lost 73 g (A125P25) or 86 g (A75P25) of
water, while the ones with 10 % GMP lost 103 (A125P25) or 100 g
Fig. 3. Nitrogen adsorption isotherms of hydrogels and GMP.
(A75P100) of water on average.

140
Z. Teng et al.
Fig. 4. Compression stress-strain curves of different hydrogel samples
Fig. 5. Germination rate of red cabbage seeds on hydrogels and saturated GMP
(GMP/H2O).
Fig. 8B compares the temporal change in water potential of various
hydrogels. All samples showed similar water potential values close to 0
kPa at the germination stage (Days 0–2). During the growth period (Days
2–12), samples A125 and A125FE maintained stable water potential at
approximately − 2 kPa, while A125E exhibited a slight decline in water
potential to − 3.78 kPa. In comparison, a sharp decline in water potential
to − 31.5, − 41.9, − 35.5, and − 40.9 kPa was observed for samples
A125P25, A125P100, A75P25, and A75P100, respectively.
3.10. Performance under simulated microgravity
Fig. 9 compares the growth of red cabbage microgreens on three
hydrogel samples under normal gravity and simulated microgravity.
Regardless of the gravity level, the hydrogel substrates maintained their
structural integrity relatively well and supported satisfactory micro­
green growth. Moreover, sample A125P100 yielded the longest stems at
both gravity levels, followed by A125FE and A125. The difference in
plant growth under the two tested conditions lied in two aspects. First,
the stems grew in a more random pattern under simulated microgravity.
This effect was observed on all samples but was most pronounced on
substrate A125: while most stems were elongated vertically against the
hydrogel surface under normal gravity, some stems grew sideways or
spirally upwards under simulated microgravity, and a few stems even
141
International Journal of Biological Macromolecules 220 (2022) 135–146
grew towards and penetrated the hydrogel surface. The second differ­
ence lies in root orientation, in that microgravity yielded less vertical
and more randomly oriented root development.
4. Discussion
4.1. Formulation of hydrogel material
As mentioned in Section 1, the key intent for this study was to
develop a hydrogel formula that balances aeration (macro-/mesopores)
and water storage and delivery (smaller pores) [27]. Various natural and
synthetic polymers were screened in our preliminary study, including
agarose, gellan gum, sodium alginate, locust bean gum, polyacrylamide,
sodium polyacrylate, and polyvinyl alcohol. Agarose was chosen as a
model material in this study since it yielded the highest germination rate
and stem length, which is in line with a previous study [28]. However,
polymers such as gellan gum and polyvinyl alcohol can also benefit from
GMP inclusion according to our preliminary study.
The GMP used in our study consist mainly of vermiculte (metal ox­
ides), peat moss (organic acids, phenolic compounds, aldehydes, and
alcohols) [29], and tree bark (polysaccharides, acids, and other organic
compounds) [30]. This material is characterized by its abundance of Ca,
Mg, K, N, P, and S (Table 2) and a variety of pores (Tables 3 and 4). Since
the microgreen growth can be affected by both the nutrient and porosity
from GMP, a series of hydrogels were tested to separate the above two
factors (Table 1).
The hydrogel composite is more convenient to prepare and handle
than mechanically extruded agar beads [31], and it avoids some issues
associated with porogen leaching-based techniques [32], such as the
extensive chemical/water usage leading to high cost and potential
pipeline erosion. There have been several reports on supplementing
tissue culture media with porous materials, especially activated charcoal
(AC) [33–35]. However, the function of AC in those formulas is to
reinforce the gel structure [36] or absorb detrimental compounds
secreted at later stages of plant development [35,37]. In addition, the
level of AC in existing reports ranges typically from 0.5 to 1 % which, per
our preliminary study, is insufficient to improve the porosity and boost
the microgreen growth on the hydrogel.
4.2. Effects of GMP and its extract on agarose hydrogel structure
As shown in Fig. 1, replacing water with fine GMP extract led to
minimum change in the macroscopic and microscopic morphology. This
was expected since agarose does not possess easily ionizable functional
groups. Thus, the gelation process of agarose is not altered by most ions
except for sulfate [34], whose concentration was relatively low in the
GMP extract (Table 2). This explains why A125FE and A125 exhibited
similar morphology (Fig. 1), pore size distribution (Fig. 2), and
compression stress (Fig. 4) despite having different TDS (Table 1) and
elemental compositions (Table 2). Unlike the fine extract, the crude
extract (A125E) introduced fine particles (smaller than 300 μm) that
partially disrupted the hydrogel network (Fig. 1), increased the total
volume of pores in the nanometer range (Fig. 3 and Table 4), and low­
ered the compression stress by 49 % (Fig. 4). However, such structural
disruption was insufficient to alter the pore size distribution beyond 1
μm per MIP analysis (Table 3). In comparison, the addition of GMP
introduced a significant change in chemical composition (Table 2), total
pore volume (Fig. 3 and Table 4), and a shift of MIP pore size distribu­
tion towards greater diameters (Fig. 2 and Table 3), all of which had led
to a dramatic decrease in compression stress (up to 64 %, Fig. 4).
4.3. Effects of GMP and its extract on microgreen growth
As shown in Fig. 5, GMP and its extract led to decreased and
increased GR respectively on the first day, but the difference among
substrates diminished on Day 2. The fact that all other formulas showed
Z. Teng et al.
Fig. 6. Digital photos of red cabbage microgreens grown on different agarose-based substrates. Photos were on Day 12. Sample denotation is provided in Table 1.
higher GR than A125 on Day 4 suggested the beneficial role of GMP and
its extract in seed germination. Regarding seedling growth, A125FE
produced significantly faster growth (Fig. 7B) and 54 % higher yield
than A125 (Fig. 7A), which is attributable to the nutrients in GMP
extract (Table 2). Furthermore, A125P100 showed an improvement in
yield over A125FE by 44 % (Fig. 7A). It is noteworthy that A125P100
and A125FE showed quite similar TDS (Table 1) despite significantly
different elemental compositions (Table 2). This fact suggests that the
two formulas contained comparable levels of water-soluble nutrients,
which led to the assumption that the superior performance of A125P100
was mainly ascribed to its structural properties. These may include (1)
the introduction of larger pores (Tables 3 and 4) leading to improved gas
[6], nutrient, and water exchange [38,39], and (2) decreased
compression stress (Fig. 4) facilitating root penetration. Finally,
lowering the agarose concentration to 7.5 mg/mL further improved the
total yield (Fig. 7A), which was possibly due to the a looser polymeric
network [40] that further increased porosity and lowered substrate
stiffness (Fig. 4).
4.4. Effects of GMP and its extract on water equilibrium
The investigation on water equilibrium shed light on the interplay
between plants and hydrogel and the effect of GMP thereon. The fact
that all hydrogels exhibited water potentials near zero in the first six
days (144 h, Fig. 8B) suggested the ease of water extraction by seeds and
roots from those substrates [41,42]. In the following days, GMP-
incorporated hydrogels lost significantly more water than GMP-free
counterparts, which can be explained by two reasons. First, the larger
pore volume and size of GMP-incorporated hydrogels facilitated evap­
oration from the substrate. Second, the increased porosity and decreased
compression stress promoted root development (Fig. 6), which did not
only lead to greater water uptake (Fig. 7A) and transpiration by the
plants, but also caused greater disruption of hydrogel network thus
accelerating the evaporation. As a result, GMP-incorporated hydrogels
exhibited much more negative water potential on Day 12 than GMP-free
142
International Journal of Biological Macromolecules 220 (2022) 135–146
samples (Fig. 8). Nonetheless, since such negative water potential can be
overcome easily by roots [42], the GMP was not expected to limit the
water accessibility to plants.
4.5. Merits, drawbacks, and potential applications of hydrogel composites
There are several major advantages of the agarose hydrogel com­
posite over traditional substrates (e.g., peat or rock wool). First, the
system maintains a delicate balance between water retention and
aeration, supporting plant growth for prolonged periods under normal
and microgravitational conditions without human intervention or so­
phisticated instrumentation. Second, the hydrogel features very low dry
weight and thus minimal amount of residue after use. Such a property is
critical for space farming, in which weight efficiency of the substrate is
paramount due to the limited payload capacity at launch. The third
advantage is that the ability of hydrogel matrices in encapsulating and
delivering functional compound, such as fertilizers, antimicrobials, or
other bioactive agents [43,44]. Lastly, agarose-based hydrogels being
biocompatible and biodegradable are more suitable for producing
organic edible plants than commercial water absorbent materials (e.g.,
polyacrylamide or polyacrylate). The only inorganic ingredient in the
current formula (vermiculite) may be eliminated or replaced with
organic materials (e.g., biochar) in the future.
Despite the aforementioned advantages, there are three potential
issues that need to be addressed. First, the cost of polymers used in this
study is significantly higher than that of conventional substrates. This
issue can be addressed by finding reusable and cost-effective alterna­
tives. Second, the hydrogel formula is rather sensitive to RH: lower RH
leads to rapid evaporation of stored water and thus insufficient water
supply to the plants, while higher RH increases the chance of mold
growth. This issue may be addressed by (1) optimizing the hydrogel
porosity and growing conditions on a cultivar-specific basis, (2) devel­
opment of rehydratable formulas capable of being recharged after
extended time of use, and (3) incorporation of antimicrobial materials
(e.g., silver nanoparticles) into the hydrogel matrix. The third issue was
Z. Teng et al. International Journal of Biological Macromolecules 220 (2022) 135–146

Fig. 7. Growth of red cabbage microgreens on different hydrogel substrates. Fig. 4A shows the fresh weight of harvested microgreens on Days 7 and 14. Fig. 4B, C, D,
and E present the stem lengths and leaf areas, L*, a*, and b* values of microgreens on Day 14. Sample name explanations are provided in Table 1.

that the addition of GMP only led to limited increase in the proportion of
meso- and macropores (Table 3), which was probably due to the infil­
tration and subsequent solidification of gelling solution blocking some
pores on GMP. The air-filled porosity of the hydrogel composite may be
further improved by modulating the gelation process, e.g., manipulating
the viscosity of gelling solution and gelation under reduced pressure.
These approaches will be explored in future studies.
5. Conclusion
A novel growth substrate was developed by incorporating GMP in
agarose hydrogel. The GMP altered the chemistry of the hydrogel
significantly, together with a remarkable change in pore size,
morphology, and compression stress. The hydrogel supplied water to red
cabbage microgreen plants during a 12-d growth cycle without any need
for watering or attendance. Furthermore, the nutrients and porous

143
Z. Teng et al. International Journal of Biological Macromolecules 220 (2022) 135–146

Fig. 8. Water loss (A) and water potential (B) of hydrogel samples during the growth cycle. The initial weight of all hydrogel samples shown in Fig. 8A was 160 g.
The inset diagram in Fig. 8B was intended to visualize the water potential change at earlier time. Sample name explanations are provided in Table 1.

Fig. 9. Growth of red cabbage microgreens on different hydrogel samples on Day 12 under normal gravity (top) and simulated microgravity (bottom).

structure from the GMP increased microgreen yield by 54 % and 44 %,
respectively, with a combined effect of 121 % improvement. Since all
hydrogels maintained relatively high water potential, the GMP had most
likely improved microgreen growth by enhancing root aeration rather
than water extraction. Finally, the novel substrate demonstrated effi­
cient, passive water delivery under simulated microgravitational con­
ditions. The developed substrate may find its use in various applications,
especially space farming where minimal human intervention and low
dry weight are critical for the mission.
CRediT authorship contribution statement
Zi Teng was responsible for the conceptualization, methodology,
investigation, formal analysis, validation, and manuscript writing and

144
Z. Teng et al.
editing. Yaguang Luo was responsible for the supervision, conceptuali­
zation, funding acquisition, resources, project administration, and
manuscript editing. Daniel J. Pearlstein was involved in the methodol­
ogy, investigation, validation, and manuscript editing. Bin Zhou
contributed to the methodology, investigation, and manuscript editing.
Christina M. Johnson contributed to methodology and manuscript
editing. Joseph Mowery contributed to the methodology, investigation,
and formal analysis. Qin Wang was responsible for the supervision and
manuscript editing. Jorge Fonseca was responsible for the supervision,
resources, project administration, and manuscript editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We acknowledge the support of the Maryland NanoCenter and its
FabLab on nitrogen adsorption analysis. We appreciate the aid from Ms.
Eunhee Park, Ms. Ellen R. Turner, and Mr. James D.D. Fitzwater for their
support in texture analysis. We thank Ms. Marya Orf Anderson for her
assistance in ICP analysis. Mention of trade names or commercial
products in this publication is solely for the purpose of providing specific
information and does not imply recommendation or endorsement by the
USDA. USDA is an equal opportunity provider and employer.
References
[1] K. Benke, B. Tomkins, Future food-production systems: vertical farming and
controlled-environment agriculture, sustainability: science, Pract. Policy 13 (1)
(2017) 13–26.
[2] Y.M. Awad, S.-E. Lee, M.B.M. Ahmed, N.T. Vu, M. Farooq, I.S. Kim, H.S. Kim,
M. Vithanage, A.R.A. Usman, M. Al-Wabel, Biochar, a potential hydroponic growth
substrate, enhances the nutritional status and growth of leafy vegetables, J. Clean.
Prod. 156 (2017) 581–588.
[3] D. Dannehl, J. Suhl, C. Ulrichs, U. Schmidt, Evaluation of substitutes for rock wool
as growing substrate for hydroponic tomato production, J. Appl. Bot. Food Qual.
88 (1) (2015).
[4] J. Xiong, Y. Tian, J. Wang, W. Liu, Q. Chen, Comparison of coconut coir, rockwool,
and peat cultivations for tomato production: nutrient balance, plant growth and
fruit quality, Front. Plant Sci. 8 (2017) 1327.
[5] C. Ouellette, S. Courtenay, A. St-Hilaire, A. Boghen, Impact of peat moss released
by a commercial harvesting operation into an estuarine environment on the sand
shrimp Crangon septemspinosa, J. Appl. Ichthyol. 22 (1) (2006) 15–24.
[6] F. Di Gioia, P. De Bellis, C. Mininni, P. Santamaria, F. Serio, Physicochemical,
agronomical and microbiological evaluation of alternative growing media for the
production of rapini (Brassica rapa L.) microgreens, J. Sci. Food Agric. 97 (4)
(2017) 1212–1219.
[7] M. Prasad, Physical, chemical and biological properties of coir dust, in:
International Symposium Growing Media and Plant Nutrition in Horticulture 450,
1996, pp. 21–30.
[8] K. Yan, F. Xu, S. Li, Y. Li, Y. Chen, D. Wang, Ice-templating of chitosan/agarose
porous composite hydrogel with adjustable water-sensitive shape memory property
and multi-staged degradation performance, Colloids Surf. B: Biointerfaces 190
(2020), 110907.
[9] M. Du, Z. Xiao, Y. Luo, Advances and emerging trends in cultivation substrates for
growing sprouts and microgreens towards safe and sustainable agriculture, Current
Opinion in Food Science (2022) 100863.
[10] L. Cao, N. Li, Activated-carbon-filled agarose hydrogel as a natural medium for
seed germination and seedling growth, Int. J. Biol. Macromol. 177 (2021)
383–391.
[11] J.S. Varghese, N. Chellappa, N.N. Fathima, Gelatin–carrageenan hydrogels: role of
pore size distribution on drug delivery process, Colloids Surf. B: Biointerfaces 113
(2014) 346–351.
[12] F.A. Oluwafemi, R. Abdelbaki, J.C.-Y. Lai, J.G. Mora-Almanza, E.M. Afolayan,
A review of astronaut mental health in manned missions: potential interventions
for cognitive and mental health challenges, Life Sci. Space Res. 28 (2021) 26–31.
[13] G.D. Massa, N.F. Dufour, J.A. Carver, M.E. Hummerick, R.M. Wheeler, R.
C. Morrow, T.M. Smith, VEG-01: veggie hardware validation testing on the
international Space Station, Open Agric. 2 (1) (2017) 33–41.
[14] G.D. Massa, G. Newsham, M.E. Hummerick, J.L. Caro, G.W. Stutte, R.C. Morrow, R.
M. Wheeler, Preliminary species and media selection for the veggie space
hardware, Gravitational Space Res. 1 (1) (2013).
145
International Journal of Biological Macromolecules 220 (2022) 135–146
[15] D. Porterfield, G. Neichitailo, A. Mashinski, M. Musgrave, Spaceflight hardware for
conducting plant growth experiments in space: the early years 1960–2000, Adv.
Space Res. 31 (1) (2003) 183–193.
[16] C.M. Johnson, A. Subramanian, S. Pattathil, M.J. Correll, J.Z. Kiss, Comparative
transcriptomics indicate changes in cell wall organization and stress response in
seedlings during spaceflight, Am. J. Bot. 104 (8) (2017) 1219–1231.
[17] A.-L. Paul, A.K. Zupanska, D.T. Ostrow, Y. Zhang, Y. Sun, J.-L. Li, S. Shanker, W.
G. Farmerie, C.E. Amalfitano, R.J. Ferl, Spaceflight transcriptomes: unique
responses to a novel environment, Astrobiology 12 (1) (2012) 40–56.
[18] M.C. Kyriacou, S.De Pascale, A. Kyratzis, Y. Rouphael, Microgreens as a component
of space life support systems: a cornucopia of functional food, Front. Plant Science
(2017) 1587.
[19] L. Poulet, K. Engeling, T. Hatch, S. Stahl-Rommel, Y. Velez Justiniano, S. Castro-
Wallace, J. Bunchek, O. Monje, M. Hummerick, C. Khodadad, Large-scale crop
production for the moon and Mars: current gaps and future perspectives, Front.
Astron. Space Sci. 8 (2022), 733944, https://doi.org/10.3389/fspas.
[20] Hach.Company, Nitrogen, Simplified TKN (s-TKNTM), Method 10242,
DOC316.53.01258, Edition 4. https://www.hach.com/asset-get.download-en.jsa?
id=7639982525, 2017.
[21] USEPA, Method 200.2, Revision 2.8: Sample Preparation Procedure for
Spectrochemical Determination of Total Recoverable Elements, Accessed at,
https://www.epa.gov/sites/default/files/2015-08/documents/method_200-2_re
v_2-8_1994.pdf, 1994.
[22] O. Adesanwo, D. Ige, L. Thibault, D. Flaten, W. Akinremi, Comparison of
colorimetric and ICP methods of phosphorus determination in soil extracts,
Commun. Soil Sci. Plant Anal. 44 (21) (2013) 3061–3075.
[23] R. Aston, K. Sewell, T. Klein, G. Lawrie, L. Grøndahl, Evaluation of the impact of
freezing preparation techniques on the characterisation of alginate hydrogels by
cryo-SEM, Eur. Polym. J. 82 (2016) 1–15.
[24] C.C. Chu, S.h. Kim, Pore structure analysis of swollen dextran-methacrylate
hydrogels by SEM and mercury intrusion porosimetry, J. Biomed. Mater. Res. 53
(3) (2000) 258–266.
[25] H. Zhang, M. Yang, Q. Luan, H. Tang, F. Huang, X. Xiang, C. Yang, Y. Bao, Cellulose
anionic hydrogels based on cellulose nanofibers as natural stimulants for seed
germination and seedling growth, J. Agric. Food Chem. 65 (19) (2017) 3785–3791.
[26] Z. Teng, Y. Luo, E.R. Bornhorst, B. Zhou, I. Simko, F. Trouth, Identification of
romaine lettuce (Lactuca sativa var. longifolia) cultivars with reduced browning
discoloration for fresh-cut processing, Postharvest Biol. Technol. 156 (2019),
110931.
[27] S.S.G.T. Committee, S.S.S.o. America, Glossary of Soil Science Terms 2008, ASA-
CSSA-SSSA, 2008.
[28] L.B. Johansson, Increased induction of embryogenesis and regeneration in anther
cultures ofSolanum tuberosum L, Potato Res. 31 (1) (1988) 145–149.
[29] S.C. Moldoveanu, 11 - Analytical pyrolysis of several organic geopolymers, in: S.
C. Moldoveanu (Ed.), Analytical Pyrolysis of Natural Organic Polymers, Second
Edition, Elsevier, 2021, pp. 403–425.
[30] A.M.A. Hamad, S. Ates, Ç. Olgun, M. Gur, Chemical composition and antioxidant
properties of some industrial tree bark extracts, Bioresources 14 (3) (2019)
5657–5671.
[31] E.G. Barrett-Lennard, M. Dracup, A porous agar medium for improving the growth
of plants under sterile conditions, Plant Soil 108 (2) (1988) 294–298.
[32] J.H. Park, B.G. Chung, W.G. Lee, J. Kim, M.D. Brigham, J. Shim, S. Lee, C.
M. Hwang, N.G. Durmus, U. Demirci, Microporous cell-laden hydrogels for
engineered tissue constructs, Biotechnol. Bioeng. 106 (1) (2010) 138–148.
[33] T.D. Thomas, The role of activated charcoal in plant tissue culture, Biotechnol.
Adv. 26 (6) (2008) 618–631.
[34] T. Singh, R. Meena, A. Kumar, Effect of sodium sulfate on the gelling behavior of
agarose and water structure inside the gel networks, J. Phys. Chem. B 113 (8)
(2009) 2519–2525.
[35] Y. Mohamed-Yasseen, Influence of agar and activated charcoal on uptake of
gibberellin and plant morphogenesis in vitro, In Vitro Cell Dev. Biol. Plant 37 (2)
(2001) 204–205.
[36] A. Saha, S. Sekharan, U. Manna, Superabsorbent hydrogel (SAH) as a soil
amendment for drought management: a review, Soil Tillage Res. 204 (2020),
104736.
[37] J.C. Sorgato, J.S. Soares, C.R. Damiani, L.M. Ribeiro, Effects of light, agar,
activated charcoal, and culture medium on the germination and early development
of'Dendrobium'seedlings, Aust. J. Crop. Sci. 14 (4) (2020) 557–564.
[38] N. Gruda, W. Schnitzler, Suitability of wood fiber substrate for production of
vegetable transplants: I. Physical properties of wood fiber substrates, Sci. Hortic.
100 (1-4) (2004) 309–322.
[39] L. Toková, D. Igaz, J. Horák, E. Aydin, Effect of biochar application and re-
application on soil bulk density, porosity, saturated hydraulic conductivity, water
content and soil water availability in a silty loam haplic luvisol, Agronomy 10 (7)
(2020) 1005.
[40] C.T. Buckley, S.D. Thorpe, F.J. O’Brien, A.J. Robinson, D.J. Kelly, The effect of
concentration, thermal history and cell seeding density on the initial mechanical
properties of agarose hydrogels, J. Mech. Behav. Biomed. Mater. 2 (5) (2009)
512–521.
[41] C.E. Evans, J.R. Etherington, The effect of soil water potential on seed germination
of some British plants, New Phytol. 115 (3) (1990) 539–548.
Z. Teng et al. International Journal of Biological Macromolecules 220 (2022) 135–146

[42] M. Aston, D.W. Lawlor, The relationship between transpiration, root water uptake,
and leaf water potential, J. Exp. Bot. 30 (1) (1979) 169–181.
[43] D.W. Davidson, M.S. Verma, F.X. Gu, Controlled root targeted delivery of fertilizer
using an ionically crosslinked carboxymethyl cellulose hydrogel matrix,
Springerplus 2 (1) (2013) 1–9.
[44] R. Rajakumar, J. Sankar, Hydrogel: novel soil conditioner and safer delivery
vehicle for fertilizers and agrochemicals–a review, Int. J. Appl. Pure Sci. Agric. 2
(9) (2016) 163–172.

146