**Contents**



## *Editorial* **Modern Seed Technology**

**Alan G. Taylor 1,\*, Masoume Amirkhani <sup>1</sup> and Hank Hill <sup>2</sup>**


Modern Seed Technology (MST) includes a wide range of technologies and practices to upgrade seed quality, enhance seedling and plant growth, and assessing seed quality using imaging technology. Another key topic of MST is Seed Enhancements. First defined as post-harvest methods that improve germination and seedling growth or facilitate the delivery of seeds at the time of sowing [1]. The broader topic of MST includes pre-harvest treatments to hasten seed maturation and post-sowing methods to enhance seed viability and vigor for greenhouse and field production. This special issue of MST has a total of 12 papers with 10 research papers and 2 review articles. Papers were submitted from five countries: Brazil, China, Denmark, Pakistan, and four papers were invited from colleagues in the United States, Multi-State project W-4168. The papers in the special issue of MST were grouped into four categories: Pre- and Post-sowing Seed Enhancements, New Crop Seed Technology, Seed Treatments, and Systemic Uptake, Seed Priming and Seed Imaging. This editorial encompasses perspectives from academia (Taylor and Amirkhani) and industry (Hill) for the future vision of Modern Seed Technology.

The first category has a paper in each sub-heading: Pre- and Post-sowing Seed Enhancements and New Crop Seed Technology.

The first opportunity to manipulate seed quality is while the seeds are still on the mother plant. The use of chemical defoliants can accelerate corn (*Zea mays* L.) seed maturation and drying and thus avoid loss of quality by an early frost. Dean et al. at Iowa State University describe the effect of a selective chemical defoliant on the migration of oil bodies, a sub-cellular event that is a prerequisite for viability and vigor [2]. The major finding was the lack of differences in migration of these oil bodies between treated and nontreated controls. Thus, chemical defoliant did not harm corn seed quality, while still protecting the seed from the damage of an early frost.

The importance of the above article is that most published research concerning Modern Seed Technologies is on post-harvest seed technology because of the emphasis on seed enhancement. Therefore, the opportunity is missed to enhance quality prior to harvesting. The authors feel that future MST research should have a better balance between pre-and post-harvest technology. Moreover, a combination of pre-and post-harvest strategies in the same investigation has the greatest potential to enhance seed performance. Thus, we expand the definition of pre-harvest strategies to include plant-breeding efforts to improve seed quality and vigor as will be cited later.

The second paper by Qin and Leskovar at Texas A&M University focused on improved transplant quality of containerized vegetable crop plants by the addition of humic substances (HS), as a biostimulant, to the plug media [3]. Humic acid has been known for some time to enhance germination and seedling growth. The incorporation of 1% HS (*v*/*v*) into the growing media was demonstrated to have a biostimulant effect and enhanced several plant parameters, and modulated both root and shoot growth. The HS biostimulant effect was particularly effective in mitigating the negative effects of drought and heat stress on growth.

**Citation:** Taylor, A.G.; Amirkhani, M.; Hill, H. Modern Seed Technology. *Agriculture* **2021**, *11*, 630. https://doi.org/10.3390/ agriculture11070630

Received: 28 June 2021 Accepted: 2 July 2021 Published: 6 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The above article focuses on enhancing germination and plant growth under the environmental stress of drought and heat stress because of their negative impact on stand establishment and ultimately yield. A paper from the first two authors of this article also demonstrated the positive effects of a bio-stimulant. They used a seed coating formulation composed of soy flour and vermicompost that served as a biostimulant under optimal growth conditions [4]. These biostimulant- seed treatments and coatings need to be tested under environmental stress to explore their full potential as the above authors demonstrated.

The third paper was from Mi et al., at Cornell and was on hemp (*Cannabis sativa* L.) as a new crop, or at least the reintroduction of a crop first grown in China 6000 years ago. The research was focused on the cultural practices for growing baby leaf hemp including the effect of seed size on germination and fresh and dry seedling weight [5]. Three hemp varieties were studied. The seed size distribution was determined by hand sorting with round hole sieves based on width. The distribution pattern was similar for all three varieties with a normal distribution skewed with a small percentage of small seeds. The small seed sizes had a lower percent germination and slower seedling growth than the larger-sized seeds. Thus, discarding the small percentage of small-sized seeds would upgrade the quality of the lot.

In conclusion, though the importance of seed size has been known for centuries, there is little scientific research published on hemp, and information available online may have questionable validity. Moreover, the hemp seed industry is relatively young compared to the vegetable and field crop industries, so researching the effects of seed size is important to both the seed industry and hemp growers. Continued seed technology research is needed on hemp including the development of treatments to control soil-borne pathogens responsible for damping-off. The goal is to have labeled seed treatments in the conventional and organic production of hemp.

The second category is on Seed Treatments, and Systemic Uptake (of seed treatments).

This category contains half of the papers in this special issue. The first paper by Afzal, Javed, Amirkhani, and Taylor is a joint paper from the University of Agriculture, in Pakistan and Cornell AgriTech and is a review paper on seed coating technologies [6]. For the first time, equipment and processes are described for five major seed coating technologies: dry coating, seed dressing, film coating, encrustments, and seed pelleting. Comparisons are made between each coating type with respect to weight increase after application, relative amounts of loading active ingredients, and time required performing each coating. The trend is to reduce chemical seed treatments and move to active ingredients that are organically approved. The major impetus is that organic seed treatments must be used for certified organic crop production. For organic certification, seed treatment binders and filler coating components must also be approved for organic use. This review paper presented a list of plant protectant groups, seed treatment binders, and fillers, and denotes those materials that may be approved. Seed coatings can be custom designed. Dry seed coating compositions may be required for the application of beneficial fungi that cannot withstand hydration and dehydration without loss of viability. In particular, the Entomopathogenic fungi (EPF), *Metarhizium* and *Beauveria* both require dry-coating technologies in the seedcoating process. Thus, the other four coating techniques: seed dressing, film coating, encrustments, and seed pelleting cannot be used for EPF seed treatment application as water is used in each.

The future of plant protection may well lie in the discussion above. The seed becomes the delivery system for crop protection. The controlled release of microencapsulated pesticides is just one example [7]. Already seed coating enables the additions of fungicides and insecticides to be applied in a far lower dosage on a per acre basis than with in-furrow or foliar applications [8]. Discussion of current progress will allow the seed industry to scale up and implement these new technologies in agriculture.

The second seed-treatment paper in this category is by Averitt et al. and is based on soybean lines with modified seed composition achieved through the use of mutant lines [9]. The larger context is that plant breeding may be used to improve seed quality and stand establishment when standard varieties have inherent low seed-quality potential and are also susceptible to both biotic and abiotic stress. For example, white-seeded snap bean (*Phaseolus vulgaris* L.) varieties are used in the processing vegetable industry but have lower seed quality potential than dark-seeded varieties. Dickson at Cornell summarized research using conventional plant breeding to improve white-seeded bean seed quality over 40 years ago [10]. Plant breeding may also be used to alter the composition of reserve materials in seeds for the purpose of improved taste in vegetable crops, and genetic improvements have greatly enhanced the flavor and shelf-life of fresh market sweetcorn [11].

In many cases where plant breeding alters seed composition for enhanced human and animal consumption, seed quality is compromised. This paper examines the use of soybean genotypes with low phytic acid (LPA) in comparison with normal phytic acid (NPA), and LPA lines have lower germination and low field emergence. The research presented in this paper focused on the use of chemical seed treatment fungicides and seed treatment combinations to compensate for the inherent low seed quality. Collectively, selected seed treatment combinations improved the field emergence of LPA genotypes. Further, seed priming (described later) by itself had a negative impact on stand establishment in LPA genotypes, while first priming followed with a formulation of three seed treatment fungicides improved field emergence.

The next two papers focus on seed coat- permeability and systemic uptake of seed treatments. The experimental approach in both papers used fluorescent tracers to mimic active ingredients to visualize movement within seed and seedlings and thus avoid the use of chemical pesticides. These two papers build on the characterization of the physical/chemical properties responsible for seed-coat permeability of crop seeds. Taylor and Salanenka developed a system to classify seed coat permeability based on the diffusion of ionic and nonionic compounds through the seed coat or seed covering layers [12]. Seed-coat permeability of seeds were grouped as permeable, selective permeability, and nonpermeable. Seeds with permeable seed coats allowed both ionic and nonionic compounds to diffuse through the seed coat, such as soybean and snap beans, while selective seed coat permeability only allowed nonionic compounds to pass including tomato (*Solanum lycopersicum* L.), onion (*Allium cepa* L.), and corn (*Zea mays* L.). Nonpermeable seeds blocked both ionic and nonionic compounds from entering the embryo from the environment and included cucurbits and lettuce (*Lactuca sativa* L.). A simple lab test was proposed to test the seed-coat permeability of any plant species [12].

The first paper on Systemic Uptake by Mayton et al., at Cornell AgriTech, was on tomato seed coat permeability and drilled down on a compound's lipophilicity measured as the log *Kow* for optimal seed uptake [13]. This research was all possible with the synthesis of a series of 11 fluorescent; n-alkyl piperonyl amides ranging from log *Kow* 0.02 to 5.66 by Stephen Donovan (co-author). The optimal log *Kow* for tomato seed uptake was in the range of 2.9 to 3.8. However, less than 5% of the applied compound was measured in the embryo. Therefore, for control of internal seed-borne pathogens, both the log *Kow* is important for targeting pathogens residing in the embryo and adequate dosage for efficacy.

The next paper by Wang et al., at Cornell AgriTech, investigated the uptake of 32 fluorescent tracers representing 10 chemical families on soybean seed and seedling uptake [14]. Most zanthene and coumarin compounds tested displayed both seed and seedling uptake. Though the log *Kow* of a compound is well established to govern root uptake, the log *Kow* alone could not predict seed uptake. Therefore, the physical/chemical properties for uptake of organic compounds by plant roots are not the same as uptake in seeds during the early stages of germination. Seedling uptake of zanthene compounds, Rhodamine B and Rhodamine 800, a NIR fluorescent tracer were further studied and detected in the true leaves of soybean.

The third category is on Seed Priming as seed enhancements.

There were two papers on Seed Priming. Seed priming is a general term that includes several techniques to hydrate seeds under controlled conditions so physiological processes of germination can occur without the completion of radicle emergence (Phase III, or visible germination) [15]. Common to all seed priming techniques is that radicle emergence is arrested due to restricted water uptake.

In the two papers in this section, seeds were allowed to imbibe in a dilute solution of potassium nitrate [16] or zinc sulfate [17], but germination was arrested prior to drying. In these studies, the concentration of KNO<sup>3</sup> or ZnSO<sup>4</sup> in solution was not sufficient to lower osmotic potential to arrest Phase III germination [16]. Thus the seed priming techniques described in the two papers may be considered as seed steeping [18]. There is not a review paper on seed priming in this special issue, so the reader is referred to previous reviews published from 1977 to 2010 cited in [15].

The first seed priming paper by Ali et al. used a range of potassium nitrate concentrations and 0.75% was optimal for germination, seedling growth, and other physiological attributes [16]. The objective of enhancing tomato seed germination is not new and an early paper reported the use of potassium nitrate and other salt solutions to enhance tomato seed germination almost 60 years earlier [19]. Another objective of seed priming is to improve germination under low temperatures.

The second priming paper by Imran et al., [17] investigated spinach seeds imbibed in dilute ZnSO4 solutions. The optimal concentration was found to be 6 mM resulting in enhanced germination at 8 ◦C. Collectively, both 'nutrient priming' techniques provided enhanced germination and seedling performance. Optimal efficacy required a precise concentration.

The last subject area was Seed Imaging using multispectral imaging (MSI) and nearinfrared spectroscopy (NIRS).

The first paper in this section from Mortensen et al., at Aarhus University, Denmark was an invited review paper on both MSI and NIRS [20]. These technologies are nondestructive and noninvasive tools and have the potential in seed testing for rapid and reproducible results. Applications of MSI in seed testing include varietal identity and purity, detecting seed damage from mechanical abuse and insects, and seed health in detecting fungal infection. Both MSI and NIRS have the potential to detect seed viability on a single-seed basis, and germinating seeds validated predicted seed viability. Combining imaging with seed sorting technology could effectively upgrade seed-lot quality by detecting and removing nonviable seeds.

The second paper in this section by Rego et al. in Brazil focused on seed health using MSI for detecting seed-borne fungi in cowpea (*Vigna unguiculata* L.). MSI was able to detect seeds inoculated with *Fusarium*, *Rhizoctonia*, and *Aspergillus* [21]. A key finding was that if seeds were first imbibed and then frozen at −20 ◦C, pathogen detection was enhanced.

The last paper in this category is from Bello and Bradford at UC Davis. The paper was on investigating and detecting a physiological abnormality in *Brassica oleracea* called "blindness" [22]. MSI was used along with two other modern seed testing techniques: chlorophyll fluorescence and oxygen consumption. All data collection was done on a singleseed basis. In general, more immature seeds were detected by chlorophyll fluorescence; and at specific wavelengths from the MSI were associated with greater occurrence of blindness. The bigger story is that nondestructive and noninvasive imaging technologies have the potential to detect poor-quality seed lots and poor-quality seeds within a seed lot. Seed imaging integrated with seed sorting technology could upgrade seed-lot quality.

In summary, the first and third authors of this article experienced an evolution in seed technology research and development over the past 40 years. Papers in this special issue of Modern Seed Technology are an excellent illustration of current research findings in several categories from many seed research groups throughout the world. Drs. Taylor and Amirkhani are proud to contribute several papers to this special issue of Modern Seed Technology. Future research in this area will be driven by the integration of new technologies from other disciplines with seed technology. We look forward to future developments that move from evolutionary to revolutionary in exploiting seeds as the delivery systems in agriculture.

**Author Contributions:** Conceptualization, A.G.T. and H.H.; investigation, A.G.T. and M.A.; writing—original draft preparation, A.G.T.; writing—review and editing, M.A. and H.H.; visualization, A.G.T., M.A. and H.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This material is based upon work that is supported by the United States Hatch Funds under Multi-state Project W-4168 under accession number 1007938, and Multi-state Project NE-1832 under the accession number 1021019.

**Acknowledgments:** We would like to sincerely thank all authors who submitted papers to the special issue of *Agriculture* entitled "Modern Seed Technology", to the reviewers of these papers for their constructive comments and thoughtful suggestions, and the editorial staff of *Agriculture*.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Migration of Oil Bodies in Embryo Cells during Acquisition of Desiccation Tolerance in Chemically Defoliated Corn (***Zea mays* **L.) Seed Production Fields**

**Ashley N. Dean 1,2, Katharina Wigg 1,3, Everton V. Zambiazzi 1,4,5, Erik J. Christian <sup>1</sup> , Susana A. Goggi 1,4,\* , Aaron Schwarte <sup>6</sup> , Jeremy Johnson <sup>6</sup> and Edgar Cabrera <sup>6</sup>**


**Citation:** Dean, A.N.; Wigg, K.; Zambiazzi, E.V.; Christian, E.J.; Goggi, S.A.; Schwarte, A.; Johnson, J.; Cabrera, E. Migration of Oil Bodies in Embryo Cells during Acquisition of Desiccation Tolerance in Chemically Defoliated Corn (*Zea mays* L.) Seed Production Fields. *Agriculture* **2021**, *11*, 129. https://doi.org/10.3390/ agriculture11020129

Academic Editors: Alan G. Taylor and Les Copeland

Received: 11 January 2021 Accepted: 26 January 2021 Published: 5 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Abstract:** Chemical defoliation of seed corn production fields accelerates seed maturation and desiccation and expedites seed harvest. Early seed harvest is important to minimize the risk of frost damage while in the field. This newly adopted seed production practice also allows seed companies to plan harvest and manage dryer space more efficiently. However, premature defoliation may interfere with the migration of oil bodies within embryo cells during desiccation and affect seed germination and vigor. The objective of this study was to investigate the effect of chemical defoliation on the migration patterns of oil bodies within embryo cells during desiccation. Chemically defoliated and non-defoliated plants from five commercial hybrid seed corn fields were sampled in 2014 and 2015. Whole ears with husks were harvested before and after defoliant application at 600 g H2O kg−<sup>1</sup> fresh weight (fw), and weekly thereafter until seed reached approximately 300–350 g H2O kg−<sup>1</sup> fw. Ten embryos extracted from center-row seeds were fixed to stop metabolic processes, then sliced, processed, and photographed using scanning transmission electron microscopy. The oil bodies within embryo cells followed normal migration patterns according to seed moisture content, regardless of defoliation treatment. Seed germination and vigor were verified and were not significantly affected by defoliation. Chemical defoliation is a viable production practice to accelerate seed corn desiccation and to manage harvest and seed dryer availability more efficiently without negatively affecting seed germination and vigor.

**Keywords:** corn; seed acquisition of desiccation tolerance; oil-bodies migration; physiological maturity; seed quality

## **1. Introduction**

Seed corn (*Zea mays* L.) is harvested close to physiological maturity and dried artificially in specialized seed dryers before storage. Physiological maturity is the developmental stage at which seeds reach maximum dry weight [1,2]. At this developmental stage, seed moisture content ranges from 300 to 380 g H2O kg−<sup>1</sup> fresh weight (fw) depending on the genetic background of the plant and environmental conditions during seed development and maturation [3]. Seed corn is harvested early to avoid possible seed freezing injury caused by an early frost event [4]. The seed industry in the US Upper Midwest experiences significant monetary losses from early frost events every five to six years [5].

Many seed companies have adopted a new seed production practice of chemical defoliation to accelerate seed corn harvest. The defoliant is applied to the plants when seed corn is close to 600 g H2O kg−<sup>1</sup> fw or approximately 14 days before normal seed corn harvest. The seed moisture content of chemically defoliated plants decreases more rapidly than in untreated plants because of earlier senescence (personal observation). Chemical defoliation expedites harvest by two to five days, thus widening the harvest window of optimal seed moisture in different hybrid fields. This practice also facilitates harvest schedules and management of seed dryer space.

Although defoliants have been used in cotton to accelerate plant senescence and facilitate mechanical harvest in the US since 1945 [6–8], little is known about the use of defoliants in seed corn production. Drexel Defol® 5, a chemical defoliant salt solution used in the US, has not been readily adopted or widespread used. Moreover, the effect of this defoliation treatment on seed quality (seed germination and vigor) has not been fully investigated.

Orthodox seeds, such as corn, undergo a desiccation phase towards the end of seed development. These seeds survive desiccation through physiological changes called acquisition of desiccation tolerance [9]. Seed dehydration is an adaptive mechanism that allows seeds to survive unfavorable weather conditions common in temperate zones. These physiological changes are essential to the normal development of high seed quality. Seed quality in this work is defined as seed germination and vigor.

One important physiological change during the acquisition of desiccation tolerance is the migration and alignment of oil bodies along the cell membrane in corn embryo cells. These oil bodies are accumulated in the cytoplasm of the embryo cell during seed development and, as seeds dehydrate, they migrate to the cell membrane to protect cells from dehydration [10,11]. This migration of oil bodies and alignment alongside of the cell membrane is essential to seed quality.

The objective of this study was to document the migration of oil bodies in embryo cells from chemically defoliated and untreated plants.

#### **2. Materials and Methods**

#### *2.1. Seed Production and Defoliation Treatment*

A commercial hybrid seed field was sampled in 2015 near Nevada, Iowa. The field was planted in blocks with a 4:2 female-to-male ratio and managed by the seed company Corteva (Johnston, IA, USA) according to their established hybrid seed production practices.

The chemical defoliant Drexel Defol® 5 (42.3 ai NaClO3) (Drexel Chemical Company, Memphis, TN, USA) was applied to the corn plants when seed moisture content was approximately 600 g H2O kg−<sup>1</sup> fw with a Hagie high-clearance sprayer (Hagie Manufacturing Co., Clarion, IA, USA) equipped with a 27.4 m boom and 68 L water tank. A strip that was two to three female blocks wide and 800 m long was not sprayed as a control. Two replications of twenty ears were hand-harvested from the treated and control areas, once prior to the application of the defoliant and at least once a week after application. Sampling continued until the field was mechanically harvested by the seed company when seed moisture content reached approximately 350–370 g H2O kg−<sup>1</sup> fw. Therefore, harvest dates are 31 August 2015; 4 September 2015; 11 September 2015; 18 September 2015; and 22 September 2015. Field replications were maintained separately throughout the experiment.

#### *2.2. Seed Moisture Determination and Seed Drying*

At each sampling date, the sampled ears were brought immediately into the Iowa State University Seed Science Center for processing. All ears were husked by hand within 1 h after sampling the field. To consistently evaluate seeds at the same developmental stage within the ear [12], seed moisture content was determined on forty seeds removed from the center portion of five ears. Seed were divided into two 5 cm diameter aluminum trays and placed inside of an 80 ◦C oven and weighed daily until seed reached constant weight.

Moisture content was calculated on a fresh weight basis by using the following formula: (fresh weight − dry weight) fresh weight−<sup>1</sup> .

#### *2.3. Ultrastructure Determinations*

Ten embryos extracted from seed in the central portion of the ear were prepared for microscopy following the protocol described in Perdomo and Burris [10] with the following modifications. The extracted embryos were dissected in two through the point of attachment perpendicular to the embryo axis to allow the fixative solution to penetrate rapidly throughout the embryo axis. Embryo halves were immediately placed in freshly prepared fixative solution (3% glutaraldehyde (*w*/*v*) and 2% paraformaldehyde (*w*/*v*) in 0.1 M cacodylate buffer, pH 7.2). Embryos in fixative solution were stored in a refrigerator at 4 ◦C for 12–24 months before they were processed for microscopy. Once fixed, all metabolic processes within the embryo ceases.

For microscopy, samples were dissected and fixed with 3% glutaraldehyde (*w*/*v*) and 2% paraformaldehyde (*w*/*v*) in 0.1 M cacodylate buffer, pH 7.2 for 48 h at 4 ◦C. Fixed samples were rinsed three times in 0.1 M cacodylate buffer and then post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at room temperature. The samples were rinsed in deionized distilled water and enbloc stained with 2% aqueous uranyl acetate for 1 h, dehydrated in a graded ethanol series, cleared with ultra-pure acetone, infiltrated and embedded using Spurr's recipe epoxy resin (Electron Microscopy Sciences, Ft. Washington, PA, USA). Resin blocks were polymerized for 48 h at 65 ◦C. Thick and ultrathin sections were made using a Leica UC6 ultramicrotome (North Central Instruments, Minneapolis, MN, USA). Thick sections were stained with 1% toluidine blue stain and imaged with an Olympus BX-40 light microscope (Olympus Scientific Solutions Technologies, Waltham, MA, USA). Ultrathin sections were collected onto copper grids and images were captured using a JEOL 2100 scanning and transmission electron microscope (Japan Electron Optic Laboratories, Peabody, MA, USA). Images were captured using an UltraScan 1000 camera (Gatan, Inc., Pleasanton, CA, USA).

#### *2.4. Seed Quality Determination*

Standard germination tests were conducted on seed from the last harvest according to the Association of Official Seed Analysts (AOSA) rules for testing seeds [13]. One hundred seeds per each treatment and field replication were planted on crepe cellulose paper media (Kimberly Clark Corp., Neenah, WI, USA) moistened with 800 mL of tap water on fiberglass trays (45 cm × 66 cm × 2.54 cm). Seeds were lightly pressed into the media to create good seed–media contact. After planting, the trays were placed inside germination carts, and the carts were placed inside a walk-in germination chamber at constant 25 ◦C with alternating 8 h of light and 16 h of darkness d−<sup>1</sup> . Final seedling evaluation was performed at 7 days after planting.

Seed vigor was evaluated using the tray-method cold test [14]. One hundred seeds from each treatment and field replication were planted on top of crepe cellulose paper media watered with 1100 mL of water pre-chilled for 24 h at 10 ◦C on fiberglass trays (45 cm × 66 cm × 2.54 cm). After planting, trays were covered with approximately 1 cm of dry 80% sand: 20% soil mixture. The trays were placed inside enclosed germination carts, and the carts were placed inside a dark walk-in chamber at constant 10 ◦C for 7 days and then moved to a constant 25 ◦C walk-in germination chamber with alternating 8 h of light and 16 h of darkness d−<sup>1</sup> . Normal seedlings [13] were evaluated and recorded at 7 days after placing in the constant 25 ◦C walk-in germination chamber.

#### *2.5. Statistical Analysis for Seed Quality*

The two field replications were maintained throughout the experiment, and data were analyzed as a completely randomized design (CRD). The main effects were harvest time and defoliation treatment. All main effects were fixed, and replications were random.

Data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Carey, NC, USA) [15]. The analysis of variance was estimated using the restricted maximum likelihood method after testing the data for normality and homozygous error variances. Mean comparisons were made using Fisher's protected least significant difference (LSD) test (*p* < 0.05).

#### **3. Results**

Light micrographs show the different radicle tissues (Figure 1). Transmission electron microscopy (TEM) micrographs were recorded from the epidermis and cortex cells of the radicle (Figure 1).

**Figure 1.** Light microscopy image showing the different tissues of the radicle tip: (**A**) pericycle; (**B**) cortex; (**C**) epidermis; (**D**) root cap. Magnification = 10×.

− − Prior to defoliation, the oil bodies in epidermis and cortex cells were located randomly throughout the cytoplasm of the cells (Figure 2). The moisture content of the seed was approximately 600 g H2O kg−<sup>1</sup> fw. Four days after defoliant application, seed moisture content decreased to 517 and 509 g H2O kg−<sup>1</sup> fw in the untreated and treated samples, respectively. The oil bodies in epidermis cells showed the initiation of migration and alignment alongside the cell membrane for both treatments, defoliated and non-defoliated plants (Figure 2). However, the oil bodies in cells from the cortex did not show oil bodies migration for the same seed moisture content.

At 11 days after defoliant application, seed moisture content decreased to 434 and 400 g H2O kg−<sup>1</sup> fw in seed samples from the untreated and treated plants, respectively. The migration and alignment of oil bodies along the cell membrane was evident in both tissues, epidermis, and cortex cells. These oil bodies remained aligned along the cell membrane, as observed 18 days after defoliant application (Figure 2). The seed moisture content at this stage was 375 and 368 g H2O kg−<sup>1</sup> fw in the untreated and treated seed samples, respectively (Figure 3). The seed field was harvested immediately after these samples were collected.

− − − − **Figure 2.** Transmission electron microscopy images of radicle epidermis and cortex cells. Oil body migration is recorded as seed desiccate. All images are taken at 1000×, except for cells in the cortex of untreated plants, which were photographed at 1500×. (Day) Days from defoliant application on the treated plants: day (−1) seeds were harvested and artificially dried with forced ambient air before defoliant application; days (+3), (+11), (+18) indicate seeds were harvested and artificially dried with forced ambient air at 3, 11, and 18 days after defoliant application, respectively. Seed moisture content was expressed on a fresh weight basis as gr H2O kg seed−<sup>1</sup> for all treatments. The seed moisture content of untreated plants was 605 on date (−1); 517 on date (+3); 434 on date (+11); and 375 on date (+18). The seed moisture content for seed of plants treated with a defoliant were 581 on date (−1); 509 on date (+3); 430 on date (+11); and 368 on date (+18).

−

−

≤ **Figure 3.** Mean moisture content in percentage at each harvest date for corn hybrid seeds harvested at different harvest dates in 2015. 31-Aug (Pre) refers to harvest before defoliant application; all other harvest dates are post-defoliant application. Aug and Sep indicate the months of August and September. The blue line represents seed moisture values for seed harvested from plants treated with a defoliant; the yellow line represents seed moisture values for seed harvested from the untreated control plants. Means are not significantly different (*p* ≤ 0.05).

≤ The germination (Figure 4) and cold test (Figure 5) values of seed harvested from defoliated and non-defoliated areas were not significantly different (*p* ≤ 0.05).

≤ **Figure 4.** Mean standard germination test values in percentage for corn hybrid seeds harvested at different harvest dates in 2015. 31-Aug (Pre) refers to harvest before defoliant application; all other harvest dates are post-defoliant application. Aug and Sep indicate the months of August and September. Blue columns are the values for seed harvested from plants treated with a defoliant; yellow columns are the values for seed harvested from the untreated control plants. Bars indicate standard error of the mean (*SEM*). Means are not significantly different (*p* ≤ 0.05).

≤

≤

**Figure 5.** Mean cold test values in percentage for corn hybrid seeds harvested at different harvest dates in 2015. 31-Aug (Pre) refers to harvest before defoliant application; all other harvest dates are post-defoliant application. Aug and Sep indicate the months of August and September. Blue columns are the values for seed harvested from plants treated with a defoliant; yellow columns are the values for seed harvested from the untreated control plants. Bars indicate standard error of the mean (*SEM*). Means are not significantly different (*p* ≤ 0.05).

≤

#### **4. Discussion**

The US seed corn market is very competitive [16]. Farmers expect rapid and uniform field emergence of their crop under a wide range of environmental conditions. Cold and wet conditions at planting are common in the upper Midwest of the USA [17]. The use of seeds with high physiological potential is essential to achieve rapid and uniform emergence under these stressful environmental conditions [18]. Seed physiological potential is the maximum at physiological maturity [1,3]. Physiological maturity is defined as the developmental stage at which the seed reaches maximum dry weight [1]. Seed physiological potential for this article comprises an active seed metabolic system capable of producing a healthy seedling under a range of environmental conditions in the field (seed germination and vigor). In corn, this developmental stage coincides with black layer formation or the formation of callus tissue that marks the end of seed development and severs the connection between the seed and female parent [3].

The environmental conditions during seed development play a crucial role in seed physiological potential. Abiotic stresses such as plant defoliation during the critical stages of flowering, seed development, and seed maturation can reduce seed yield and seed physiological potential. Freezing temperatures in early fall may cause irreversible damage to cells and reduces seed physiological potential when seed moisture content is greater than 350 g H2O kg−<sup>1</sup> fw [4]. These freezing events cause intercellular and intracellular ice formation within the seed embryo, which results in irreversible damage to cells and reduces seed physiological potential [19]. Consequently, seed corn is harvested on or before physiological maturity and dried artificially. At this developmental stage, seed is also at high moisture content, approximately 300 to 400 g H2O kg−<sup>1</sup> fw [20]. Seed corn is harvested on the cob and artificially dried until seed reaches a safe moisture content for storage, approximately 120 g H2O kg−<sup>1</sup> fw [16]. Seed dryer space may become a limiting factor at the peak of seed corn harvest. In these instances, an early fall frost event can threaten the physiological potential of seed in the field.

Plant defoliation accelerates senescence and seed maturation. Defoliation early in seed development can trigger seed abortion, which lowers seed yields and seed physiological potential. The defoliation stress restricts photosynthesis and reduces the production of sugars necessary for the developing seeds. In sorghum, plants subjected to severe defoliation stress early during seed formation produced larger proportions of low specific gravity

seeds with extensive hollow areas in the endosperm [21]. In corn, severe defoliation stress approximately 3 weeks after pollination accelerated seed maturation and reduced seed weight [3]. As seed approaches physiological maturity, however, defoliation accelerates seed maturation, with no negative effects on seed physiological potential.

Seed dehydration capacity is unique to orthodox seeds. These seeds are named "orthodox" because they have the capability to dehydrate to very low moisture content of 40 to 50 g H2O kg−<sup>1</sup> fw, while remaining alive. These seeds undergo a series of metabolic changes known as acquisition of desiccation tolerance. The seeds accumulate protective compounds and inactive forms of germination-promoting compounds as they lose water [22]. Also, lipid bodies from the cytoplasm of embryo cells migrate to align along the plasma membranes of the cells [10]. Cells in the root meristem exhibit a distinct migration of the lipid bodies towards the cell walls in response to desiccation. This lipid alignment is essential to seed survival and optimal seed physiological potential [11]. Seeds where lipid alignment is incomplete exhibit an increase in seed leakage during imbibition. The authors theorized that the alignment of lipid bodies along the plasma membrane leads to a more organized dehydration during seed drying [11].

#### **5. Conclusions**

In our study, plant defoliation late in seed development did not change patterns of lipid-body migration and alignment along the cell membrane. The application of a defoliant resulted in slow plant senescence and seed dehydration. The treated plant senesced a few days earlier, but the difference in moisture content between seeds from the untreated and treated plants remained within 10 to 20 g H2O kg−<sup>1</sup> fw. However, the faster seed dehydration time was enough to allow one or two days harvest-date difference between treated and untreated plants. Our study also demonstrated that chemical defoliation did not reduce seed quality, which was defined as germination and vigor in this article. The use of a defoliant allows seed companies to harvest seed earlier, thus reducing the chance of seed deterioration in the field. Farmers also benefit from this technology, as high-quality seed of multiple genetic backgrounds are available for planting.

Even though this defoliation method is not available for use in EU countries, alternative defoliation methods should be investigated to broaden seed harvest timelines and reduce the need for building additional seed dryers when dryer space is limited. These expensive buildings are an additional cost for the seed companies, which may lead to increased production costs and higher seed price.

**Author Contributions:** Conceptualization: E.J.C., S.A.G., A.S., J.J., and E.C.; Formal analysis: E.V.Z.; Funding acquisition: S.A.G., A.S., J.J., and E.C.; Investigation: A.N.D., K.W., E.J.C., and S.A.G.; Methodology: E.V.Z. and S.A.G.; Project administration: E.J.C., E.V.Z., and S.A.G.; Writing, review and editing: S.A.G., A.N.D., K.W., and E.V.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** The APC and Students were funded by Hatch Projects IOW03814, IOW04114, and IOW05594; hybrid corn seed was harvested from Corteva Agriscience Seed Production Fields.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data available upon request from corresponding author.

**Acknowledgments:** The authors thank Harry Horner and Tracey P. Stewart at the Roy J. Carver High Resolution Microscopy Facility, Iowa State University, Ames IA, for providing equipment and advice for the microscopy work reported.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


## *Article* **Humic Substances Improve Vegetable Seedling Quality and Post-Transplant Yield Performance under Stress Conditions**

## **Kuan Qin and Daniel I. Leskovar \***

Texas A&M AgriLife Research and Extension Center, Texas A&M University, Uvalde, TX 78801, USA; qinkuan@tamu.edu

**\*** Correspondence: d-leskovar@tamu.edu

Received: 4 June 2020; Accepted: 29 June 2020; Published: 1 July 2020

**Abstract:** Vegetable growers require vigorous transplants in order to reduce the period of transplant shock during early stand establishment. Organic media containing solid humic substances (HS) are amendments that have not been comprehensively explored for applications in containerized vegetable transplant production systems. In this study, HS (1% *v*/*v*) were applied to a peat-based growth medium to evaluate pre- and post-transplant growth modulation of four economically important vegetable species. Those were: pepper, tomato, watermelon, and lettuce. Seeding for all species was performed in two periods in order to evaluate their post-transplant yield performance under drought (water deficit vs. well-watered) and heat (hot vs. cool season) stresses. Compared with control, HS-treated plants had: (1) increased leaf and root biomass after transplanting due to faster growth rates; (2) lower root/shoot ratio before transplanting, but higher after 10 days of field establishment; and (3) increased root length and surface area. The negative effects of heat and drought stresses on crop yield were more prominent in control plants, while HS-treated transplants were able to mitigate yield decreases. The results clearly demonstrated the benefits of using solid HS as a management input to improve transplant quality in these crop species.

**Keywords:** containerized transplants; humic acids; relative growth rate (RGR); specific root length (SRL); heat and drought stresses; heatmaps

#### **1. Introduction**

In vegetable production, the use of containerized transplants is a standard practice to establish crops in open fields and protected environments. The advantages of transplants over direct seeding have been recently reviewed by Leskovar [1]: transplanting can optimize the timing and scheduling for field cultivation, shorten the cropping period, increase growth cycles, provide uniform, rapid growth and phenological synchrony (flowering, fruit set), and enhance yield and earliness. However, transplants will inevitably suffer from the mechanical damage of root tips and hairs due to the removal of seedlings from the tray, disturbing the root/shoot balance and causing transplant shock and transiently shoot growth stunting [2,3]. Poorly grown transplants will negatively affect plant performance (or tolerance) in post-field establishment environments which is often accompanied by different abiotic stresses. Therefore, a high-quality transplant should have an ability to bear transient or long-lasting field environmental changes, better survival and uniform stand establishment, and higher resource use efficiency, which will eventually achieve high and profitable yield [4]. Transplants are typically grown in multicell trays. Due to the limited volume of cells and short growing cycle (4 to 6 weeks), transplant quality is often determined by root developmental traits and root-to-shoot balance in the confined cells; high transplant quality is typically associated with vigorous root growth

such as higher root length, surface area, and dry weight accumulation [4,5]. For example, lettuce seedlings grown with a proper level of N fertilization (60 mg/L) in the growing media produced better quality transplants with higher root dry weight, and subsequent yield performance as compared with seedlings grown with excessive or low N inputs [6]. It has been recognized that large root systems (represented by biomass) could benefit transplant growth with higher growth rate and improved water and nutrient capture in the soil [2].

Several management factors are known to affect transplant quality (root and shoot developmental traits), such as nitrogen fertilization rate [7], irrigation systems [8], container cell size [9], and light quality [10]. In addition, organic sources such as plant (sesame and alfalfa meal, wood fiber, coconut coir) and animal (fish meal and animal manure)-based compost, and vermicompost, are media amendments that can be potentially used in transplant production due to their potential roles for biostimulation, biofertilization, and plant pathogen suppression [11]. Organic sources can affect germination and emergence rates, and physical and chemical structures of the growth media and rhizosphere shortly after transplanting in the field, which ultimately could be translated into improved plant growth and biomass and early yield. For example, Jack et al. [12] used plant- and animal-based vermicompost (earthworm-driven) and thermogenic compost (self-heating), and found that a small level of additional sesame compost (1–2.5% *v*/*v*) in peat-based commercial media significantly increased tomato transplant shoot biomass. However, the use of organic substrates has to be thoroughly tested and validated since certain amended levels could negatively affect seedling growth due to their bound or unbound high salt content [11].

Humic substances (HS), resulting from the decomposition of plant and animal residues, have been widely reported to be used as organic amendments for their biostimulation (auxin-like) effects on enhancing plant root development, nutrient acquisition, and shoot growth [13]. In vegetable transplant production, HS have been used as liquid extractants (humic acids, HA) and applied as foliar sprays. Hartwigsen and Evans [14] used 2.5 and 5 g/kg HA in cucumber and squash seedlings, which resulted in significantly higher root fresh weight and lateral root length; Turkmen et al. [15] used 1 g/kg HA in tomato seedlings, which resulted in improved seedling growth and nutrient contents; Osman and Rady [16] used 0.5 g/L HA as an additive to growing media and found the dry weights, relative water contents, and NPK uptake of tomato and eggplant transplants were all increased. However, no research has been found using solid HS in seedling production. Compared with liquid HS, which can be dissolved easily and normally have quick and profound effects on plant growth [13], solid forms of HS containing humin have less intense effects, but they could increase media water holding capacity due to increased cellulose contents, and nutrient retention due to their cation exchange capacity (CEC) with much longer existence in soil solutions [17–19], which could make them suitable as supplementary amendments for growing media. Therefore, the potential use of solid HS products with the composition of both HA and humin could improve growing media properties and vegetable seedling quality traits, and the beneficial effects on transplants could last longer, even after field establishment.

In this study, we evaluated how and to what extent solid HS added to a peat-based growing media affected root and shoot developmental traits pre- and post-transplanting, as well as subsequent yield of four vegetable species: tomato, pepper, watermelon, and lettuce. We hypothesized that media amended with HS would improve root development and root-to-shoot growth modulation of containerized seedlings during the nursery period (pre-transplanting), as well as long-standing growth during field establishment (post-transplanting), which in turn will increase yield performance.

#### **2. Materials and Methods**

#### *2.1. Plant Materials, Growing Media, and Amendment Treatments*

We selected four commercial vegetable species representing high-value vegetable crops, each with two distinctive cultivar types (Figure S1): *Capsicum annuum* with cv. Hunter as bell pepper and cv. Jalafuego as jalapeño pepper; *Solanum lycopersicum* with cv. HM1823 as round tomato and cv. Sakura

as cherry tomato; *Citrullus lanatus* with cv. Estrella as diploid (seeded) watermelon and cv. Fascination as triploid (seedless) watermelon; *Lactuca sativa* with cv. Sparx as romaine lettuce and cv. Buttercrunch as butterhead lettuce. Jalafuego, Sakura, Sparx, and Buttercrunch seeds were obtained from Johnny's Selected Seeds (Winslow, ME, USA); Hunter, Estrella, and Fascination from Syngenta (Minneapolis, MN, USA); and HM1823 from Clifton Seed Company (Faison, NC, USA).

Speedling (Ruskin, FL, USA) polystyrene 200-cell trays with inverted pyramid cells (Model TR200A, 2.5 × 2.5 cm<sup>2</sup> × 7.6 cm deep with 32 cm<sup>3</sup> volume per cell) were used for transplant growth in pepper, tomato, and lettuce. Watermelon seeds were sowed into 128-cell trays (Model TR128A, 3.1 × 3.1 cm<sup>2</sup> × 6.4 cm deep with 43 cm<sup>3</sup> volume per cell). Lambert Germination, Plugs and Seedlings (LM-GPS) growing media (90% sphagnum peat moss, 10% perlite and vermiculite; Lambert, Québec, Canada) were used as control (C). Lignite-derived solid humic substances (Novihum Technologies, Salinas, CA, USA), with a composition of 32% humic acid, 3% fulvic acid, and 24% humin, were mixed with the control growing media as an amendment treatment (HS) at the rate of 1% by volume (*v*/*v*) basis. The basic physical and chemical properties of the commercial media and humic substances were measured and are shown in Tables 1 and 2.



<sup>1</sup> TP: total porosity; <sup>2</sup> AS: air space; <sup>3</sup> CWHC: container water holding capacity; <sup>4</sup> BD: bulk density.


**Table 2.** Basic chemical properties of the commercial media (CM), humic substances (HS), and field soil (FS).

<sup>1</sup> EC: electrical conductivity; <sup>2</sup> OC: organic carbon; <sup>3</sup> N/A: not available.

#### *2.2. Growth Environments and Stress Treatments*

After sowing seeds, all trays received irrigation to about 60% water holding capacity and were incubated in a growth chamber (PGR15, Conviron, Winnipeg, Canada) in darkness at 25 ◦C for 48 h. All trays were then transferred to a greenhouse with an overhead motorized spraying boom system (total length 7.1 m with two long arms at sides and operating orbit at center; each arm has 3.2 m length with 13 sprinkler units) for delivering uniform irrigation and fertilization. Environmental conditions (temperature and humidity) inside the greenhouse were controlled by a Wadsworth control system (Arvada, CO, USA) and hourly monitored by a weather station WatchDog (Spectrum Technologies Inc., Aurora, IL, USA) (Figure 1). After six weeks of growth, seedlings were transplanted in a field with raised beds at the Texas A&M AgriLife Research and Extension Centers in Uvalde, Texas (29.21◦ N, 99.79◦ W) with a clay soil type (41% clay, 31% sand, 28% silt) (Table 2). The field was prepared using ridge tillage. Planting configuration–number of rows per bed, distance between plants and beds for peppers were double-row, 0.3 m and 1.8 m; for tomatoes were single-row, 0.46 m and 1.8 m; for watermelons were single-row, 0.6 m and 2.4 m; for lettuces were double-row, 0.25 m and 0.9 m, respectively. Drip irrigation with emitter rate at 0.87 L per hour and emitter spacing at 30 cm (Netafim, Fresno, CA, USA) was installed at 10–15 cm below the soil surface in the center bed and was used for all vegetables tested in this experiment. White plastic mulch was used for pepper and tomato, black for watermelon, and bare soil for lettuce.

**Figure 1.** Temperature, daily light integral, and growing cycles (cool and hot seasons indicated by arrows) of greenhouse (**A**,**B**) and field (**C**,**D**) from 1 February 2019 to 30 August 2019.

During the field growing period, all transplants were subjected to two environmental treatment factors: heat and drought stresses. Heat stress was naturally imposed by growing seedlings during a hot season as compared with no stress with seedlings grown during a cool season. The average field maximum, mean, and minimum temperatures for the cool season were 30.4 ◦C, 24.1 ◦C, and 18.7 ◦C and for the hot season were 35.2 ◦C, 28.6 ◦C, and 22.9 ◦C, respectively (Figure 1). Drought stress was imposed by applying deficit irrigation using an evapotranspiration (ET)-based irrigation scheduling (deficit 50% ET vs. full irrigation at 100% ET). The ET crop water requirement was calculated based on the specific crop coefficients (Kc), flow rate of the drip tape, mulch covering, and precipitation [20]. The differential irrigation treatments started 10 days after transplanting, while fertilization was kept the same among treatments and other standard management practices (weeding, pest and disease control, pruning, trellis, etc.) were followed during the growing period. Within each cultivar/crop/growing season (cool vs. hot) after transplanting, the field layout was a split-plot design with four blocks–irrigation level (50% ET vs. 100% ET) as the whole-plot factor and amendment treated transplants (control vs. HS) as the split-plot factor.

#### *2.3. Seedling and Transplant Quality Evaluation and Yield Performance*

− − − Seedling emergence was counted for all crops within 1 to 2 weeks after seeding. During each growing cycle, 4 plants per cultivar/crop from each treatment (C and HS) were randomly sampled from the growing trays at 4 weeks after seeding (WAS), 5 WAS, 6 WAS, and 10 days after transplanting (DAT) for seedling (plants defined as before transplanting) and transplant (after transplanting) evaluation. Plants were removed from the trays and separated by leaf, stem, and root components. The whole roots were carefully washed, scanned using an EPSON V700 scanner (Epson, Long Beach, CA, USA), and then root length (RL), root surface area (RSA), and root average diameter (RAD) were obtained by using WinRHIZO software (Regent Instruments, Québec, Canada). After taking pictures of all leaves with a 1 cm<sup>2</sup> square scale, ImageJ [21] was used for measuring leaf area (LA). Leaf, stem, and root dry weight (LDW, SDW, RDW) were measured after oven drying at 75 ◦C for 2 days. Leaf area ratio (LAR, ratio of leaf area to plant total dry weight), root/shoot ratio (R:S, ratio of root to shoot dry weight), specific root length (SRL, ratio of root length to root dry mass) were then calculated. Relative growth rate (RGR, calculated based on leaf, stem, root, and total plant) and net assimilation rate (NAR, the increases in plant dry mass per unit leaf area and time) were also calculated based on the following equations. For convenience, all abbreviations are listed in Table S1.

$$\text{RGR} = ((\ln(\text{DW}\_{\text{time1}}) - \ln(\text{DW}\_{\text{time2}})) ((\text{time1} - \text{time2}) \tag{1}$$

$$\text{VAR} = ((\text{DW}\_{\text{time1}} - \text{DW}\_{\text{time2}}) \times ((\ln(\text{LA}\_{\text{time1}}) - \ln(\text{LA}\_{\text{time2}}))) \newline ((\text{LA}\_{\text{time1}} - \text{LA}\_{\text{time2}}) \times (\text{time1} - \text{time2})) \newline \tag{2}$$

All plants were kept growing in the field under the two treatment factors (cool vs. hot season; well-watered vs. deficit irrigation) until final harvest. Pepper, tomato, and watermelon were harvested at different times during the growing season, while lettuce was once-over harvested when the majority of heads reached maturity. The total yield was calculated and the average fruit weight (AFW) for pepper, tomato, and watermelon and average head weight for lettuce were calculated based on the total number of fruits (or heads) harvested.

#### *2.4. Statistical Analysis*

Seedling and transplant evaluation parameters were analyzed considering media-amendment (Control vs. HS) as the main factor with 8 replications from both growing seasons; while yield performance was analyzed following the split-plot design. R [22] was used for performing ANOVA and means were separated by the least significant difference (LSD) test at 4 levels: *P* ≤ 0.1, 0.05, 0.01, 0.001.

#### **3. Results**

Based on the two cycles of growth, there were no significant differences of seedling emergence percentage between control and humic substances (HS)-treated growing media (Table S2). Pre- and post-transplanting time-course growth data for each crop species and cultivars are presented in separate graphs.

#### *3.1. Pepper*

Compared with untreated control plants (Figure 2), bell pepper (cv. Hunter) grown in HS-added substrate had significantly higher LDW before transplanting and RDW after transplanting (*P* < 0.001). Although there were no significant differences in SDW, HS-treated seedlings had a faster stem RGR than control before transplanting (*P* < 0.05). Lower root-to-shoot ratio (R:S) was observed in HS-treated plants before transplanting compared with control, but the difference disappeared after transplanting, which may be caused by the increases in root growth (RDW). There were no significant differences in NAR, SRL, and RAD. Regarding yield responses, HS-treated transplants had higher yield compared with control under water stress (50% ET) in both cool (*P* < 0.1) and hot seasons (*P* < 0.05), but no differences were found in well-watered treatment (100% ET). HS amendments decreased bell pepper AFW under well-watered treatment in hot season (*P* < 0.1). In bell pepper, the highest RGR increase between 5 and 6 weeks of growth was mostly due to stem rather than root or leaf growth.

Similar RGR trends were observed in HS-treated jalapeño pepper (cv. Jalafuego), which in addition showed a significantly faster RGR in roots after transplanting (*P* < 0.05). Lower R:S were also observed in HS-treated plants before transplanting, but R:S significantly increased after transplanting as compared with control (*P* < 0.1), which could be explained by the significant enhancement of root growth traits (RDW, RL, RSA, *P* < 0.05). There were no significant differences in NAR, SRL, yield, and average fruit weight (AFW) due to the HS application. In field production, both bell and jalapeño peppers had lower yield and AFW in hot temperature as compared with the cool season (*P* < 0.001), and in water stress compared with no stress (*P* < 0.01) (Figure 2 and Table 3).

ff≤ **Figure 2.** Pepper seedling and transplant quality traits as affected by media amendments, yield traits as affected by amendments and irrigation during the two growing seasons. †, \*, \*\*, \*\*\* show significant difference comparing HS to control (C) at*P*≤0.1, 0.05, 0.01, and 0.001, respectively.


**Table 3.** ANOVA of total yield, average fruit weight (AFW) as influenced by amendments (A) and irrigation (IR) treatments during the two growing seasons (S).

†, \*, \*\*, \*\*\* show significant difference at *P* ≤ 0.1, 0.05, 0.01, and 0.001, respectively; NS, not significant at *P* ≤ 0.1.

#### *3.2. Tomato*

Compared with untreated control plants (Figure 3), HS-treated round tomato (cv. HM1823) had significantly higher LDW, SDW, and RDW before and after transplanting (*P* < 0.05). RGR was also higher, especially in stem; however, NAR was lower during early growth (4–5 WAS, *P* < 0.05), but these differences were reversed 10 DAT. Similarly, R:S was lower before transplanting but higher after transplanting (*P* < 0.1). In terms of root traits, RL and RSA were significantly higher, especially after transplanting (*P* < 0.001), RAD was also higher (*P* < 0.1), but SRL was lower. HS-treated transplants had higher yield compared with control under no stress conditions (100% ET and cool season) (*P* < 0.1).

In cherry tomato (cv. Sakura), HS had early beneficial effects on leaf and root growth even at 4WAS, with additional faster root RGR after transplanting and higher RL, RSA, RAD during seedling growth and transplant periods than control (*P* < 0.01). Yield was significantly higher for HS than the control under well-watered conditions (*P* < 0.001). For both round and cherry tomatoes, deficit irrigation treatment (50% ET) had significant negative effects on yield during the cool season but not during the hot season (*P* < 0.001). Under heat stress, plants exhibited a dramatic decreased in tomato yield and AFW, especially on cherry tomato (*P* < 0.001) (Figure 3 and Table 3).

#### *3.3. Watermelon*

Compared with untreated control plants (Figure 4), HS-treated diploid seeded watermelon (cv. Estrella) had lower leaf and root RGR between 4 and 5 WAS, but higher root biomass (*P* < 0.1) and RGR (*P* < 0.05) were observed 10 DAT. R:S was lower before transplanting, but these differences disappeared after transplanting. Similar trends were observed for NAR. HS-treated transplants had higher SRL but lower RAD at 6 WAS, and higher RL and RSA (*P* < 0.05) at 10 DAT. Although not significant, HS-treated plants had a numerical yield increase of diploid watermelon in the cool season regardless of irrigation treatments.

ff≤**Figure 3.** Tomato seedling and transplant quality traits as affected by media amendments, yield traits as affected by amendments and irrigation during the two growing seasons.†, \*, \*\*, \*\*\* show significant difference comparing HS to control (C) at*P*≤0.1, 0.05, 0.01, and 0.001, respectively.

ff≤**Figure 4.** Watermelon seedling and transplant quality traits as affected by media amendments, yield traits as affected by amendments and irrigation during the two growing seasons.†, \*, \*\*, \*\*\* show significant difference comparing HS to control (C) at*P*≤0.1, 0.05, 0.01, and 0.001, respectively.

Triploid seedless watermelon (cv. Fascination) had different root and shoot growth responses as compared with Estrella. HS-treated transplants had faster leaf and stem RGR than control (*P* < 0.1) during the field establishment period (up to 10 DAT), biomass accumulation was accordingly increased although not significant. Before transplanting, RL and RSA were not affected by HS application, but they significantly increased at 10 DAT (*P* < 0.05). These root responses were consistent with those found in the diploid watermelon. SRL was higher for HS plants compared with control at 6 WAS, but similar after transplanting. During the cool season, HS-treated plants had a numerically increased yield under both irrigation rates. HS also increased yield of triploid watermelon in the hot season, particularly for the well-watered treatment (*P* < 0.05). Comparing both stresses, heat stress (high temperature) had more negative dominant effects on yield and AFW of both diploid and triploid watermelons (*P* < 0.001) as compared with water stress (Figure 4 and Table 3).

#### *3.4. Lettuce*

Compared with untreated control plants (Figure 5), HS-treated romaine lettuce (cv. Sparx) had significantly higher LDW (*P* < 0.001), faster leaf RGR (*P* < 0.05), but lower RDW before transplanting; however, RDW and root RGR were significantly higher after transplanting (*P* < 0.05). R:S was significantly lower during seedling development and after transplanting. For root traits, RL and RSA were not affected by HS, but SRL was higher before but lower after transplanting, and the reverse responses were measured for RAD. HS-treated romaine lettuce had a significant increase in yield and average head weight (AHW) in the hot season regardless of irrigation treatments (*P* < 0.05).

Butterhead lettuce (cv. Buttercrunch) had similar results as Sparx, with additional significantly lower NAR (*P* < 0.05) and no differences in final yield (though numerically lower during the cool season) comparing HS- with control-treated plants. Heat stress (hot season) had significant negative effects on yield of both romaine and butterhead lettuce types (*P* < 0.001), while the impacts from irrigation treatments were relatively low (Figure 5 and Table 3).

≤ **Figure 5.** Lettuce seedling and transplant quality traits as affected by media amendments, yield traits as affected by amendments and irrigation during the two growing seasons. †, \*, \*\*, \*\*\* show significant difference comparing HS to control (C) at *P*≤0.1, 0.05, 0.01, and 0.001, respectively.

#### **4. Discussion**

Adding solid organic amendments such as compost and vermicompost (derived from organic waste) in growing media have shown benefits in transplant growth [12,23]. However, it is recognized that these amendments that contain high soluble salts could adversely affect germination by lowering the osmotic potential of the water in the media [24]. Since seed germination and seedling emergence are rapid and powerful ways to test potential substrate phytotoxicity [25], they should be fully examined before evaluating seedling or transplant quality. In our study, there were no significant differences in germination percentage and seedling emergence between control and humic substances (HS)-treated growing media, indicating that 1% (*v*/*v*) HS was safe and not phytotoxic on seeds tested (Table S2). The overall effects of HS amendments on leaf and root traits, RGR, NAR, yield, and average fruit weight are summarized in Table S3. We found that due to the HS application, leaf, stem, and root biomass accumulation were significantly improved, which could have resulted from higher carbon input from leaves and nutrient absorption from the root.

The HS used in this study were obtained by using the ammonoxidation procedure (lignite reacting with oxygen in aqueous ammonia) and resulted in a product with lower hydrophobicity (mainly caused by reduced aromatic compounds) and higher bioactivity than naturally slow-generated HS from lignite [26]. In addition, solid HS contain humin, which has less hydrophilic carboxyl and hydroxyl groups but higher hydrophobic alkyl groups and ash contents [18]. Raw materials also decide HS properties: lignite-derived HS are composed of highly oxidized sulfur-containing molecules and aromatic and aliphatic groups, which can give the products a higher hydrophobic protection than other raw materials (e.g., peat, compost, sludge, leonardite). This makes them more stable in terms of their existence (lifespan) in the soil solutions, having slowly beneficial effects [27,28]. This HS product contained higher N, K, Mg, and Na contents than commercial media, however, by adding HS with 1% *v*/*v*, the nutrient differences compared with control (solely commercial media) were minimized. The similar early growth performance (4 or 5 WAS) also indicated that there were no initial nutrient differences between control and HS-treated trays. During the seedling growth period, the fertilization amount applied for both control and HS trays were exactly the same and sufficient for seedling growth, thus the beneficial effects from HS were probably not related with nutrients. We found the increased seedling biomass in HS-treated trays mainly occurred at a later seedling growth stage (6 WAS) and during early field establishment, with prominent effects on root development. This could indicate the positive results from HS were mainly due to their biostimulation (auxin-like) effects on enhancing plant root development and nutrient acquisition [13], which occurred slowly due to the solid HS product. Since transplant quality was the main focus, below we explain in detail the effects of HS on the specific transplant growth parameters.

As a growth speed index, relative growth rate (RGR) can be affected by internal (species, seed mass, growth cycle) and external physical and environmental factors (pot volume, light, nutrients, and temperature) [29]. Based on the variability, RGR could be used as an indicator for separating functional strategies of plant growth: faster RGR indicates more competition for obtaining growing resources, slower RGR indicates more stress tolerance [30]. Variation of RGR could be predicted by NAR (representing the balance of photosynthetic and respiration rates) or LAR (representing the deployed efficiency of photosynthetic resources) [31–33]. In our study, a significantly positive correlation between RGR and NAR was found only in fruit-based vegetables (pepper, tomato, watermelon), while a significantly positive correlation between RGR and LAR was detected only in the leaf-based vegetable (lettuce) (Figure 6). This could indicate that the growth rate of fruit-based vegetables was determined by both photosynthesis and respiration, while leaf-based vegetables were mainly affected by their photosynthetic resources. In addition, HS-treated transplants had an overall higher RGR (especially root) than control transplants regardless of crop species, which showed a stronger recovery and adaptability (less transplant shock) during the field establishment period, and also indicated a higher nutrient uptake since nutrient absorption correlated with growth rate [34].

**Figure 6.** Linear regression plotted for net assimilation rate (NAR) and leaf area ratio (LAR) against relative growth rate (RGR) of (**A**,**B**) fruit-based vegetables (pepper, tomato, watermelon) and (**C**,**D**) leaf-based vegetable (lettuce).

Root-to-shoot ratio (R:S) is an important indicator for the allocation of plant organs against limited growing resources. In general, suitable environments rich in nutrients improve shoot (leaf and stem) growth, while poor environments with insufficient nutrients improve root relative to shoot growth. In seedling production, it is well accepted that R:S is found lower with higher substrate nutrient supply, particularly nitrogen [35]. In our study, lower R:S found in HS-treated seedlings before transplanting indicated a rich nutrient environment possibly due to the nutrient retention ability from HS. Although the boundaries of the optimum R:S are difficult to define, transplants with higher R:S are often considered to have better growth capacity and quicker establishment after transplanting [36]. HS-treated plants (except lettuce) had higher R:S than control plants after transplanting, which could explain the improvement in field establishment and yield performance.

Specific root length (SRL) is a trait that identifies the economic return (represented by root length, RL) from the cost (represented by root dry weight, RDW). The increase of SRL is often associated with nutrient limitation or dry environments [37]. However, an increase in nutrients could also lead to a higher SRL, especially when supplied in a localized nutrient patch, but the proliferation of fine root length was not accompanied by more allocation to root biomass [38]; meanwhile, this situation is species-specific [39]. SRL is strongly dependent on fine roots; with decreased RAD, SRL increased [40]. In our study, compared HS- with control-treated seedlings, RAD was lower before but higher after transplanting; in contrast, SRL changed from higher to lower (except for tomato cultivars). In seedling production before transplanting, nutrients provided in the trays are localized, thus the higher SRL was probably due to a better productive environment with HS, but after transplanting in the field, soil nutrient supply was not as localized as in trays, with lower SRL from HS-treated plants regardless of crops, indicating a less initial stress than control during the transplant shock period. In addition, a significantly increased RDW demonstrated that HS improved plant capacity for rapid root regeneration and growth for larger structural roots during field establishment.

Temperature and irrigation play important roles in vegetable production as they modulate vegetative and reproductive development. In general, flowers are the most temperature-sensitive organs, with high temperature (heat stress) decreasing pollen viability and fruit set, disturbing root functional water and nutrient uptakes, as well as causing abnormal development of shoot tip [41]. Drought stress will impair cell division and leaf area expansion, decrease leaf photosynthetic rate, and delay the conversion of vegetative to reproductive stage [42]. In our study, both heat and water stress decreased crop yield and average fruit weight (size), with heat stress having more significant effects than drought stress. Although within each crop, cultivars representing unique types had different responses, we found that stronger transplant quality due to HS application could ameliorate the adverse effects caused by the abiotic stresses, which led to a higher yield compared with control. These included: bell pepper under drought and heat stresses; round and cherry tomatoes under optimized environment (no stress); triploid watermelon under heat stress without irrigation limitation; romaine lettuce in heat stress regardless of irrigation rates.

In order to better understand the general HS effects on all crop cultivars tested and build linkages between measured seedling or transplant quality traits and subsequent yield, heatmaps (Figure 7) were created based on standardized data sets obtained before and after transplanting. Treatments were clustered based on their measured variables, and variables were clustered based on their correlations (closer meant higher positive correlations). We found that either before or after transplanting, HS treatments were clearly distinguished from control in all crops, mainly due to the higher shoot (SDW), root dry weight (RDW), root length (RL), root surface area (RSA), and yield. Yield was highly correlated with shoot growth (SDW) before transplanting, and root growth traits (RL, RDW, RSA) after transplanting, which indicated that during the seedling stage, sufficient nutrients should be kept in the growth media to improve the plant above-ground growth, while after transplanting, management practices aimed at improving root development should be considered. Besides the application of solid HS in this study, the use of other biostimulant substrates (phenols, salicylic acid, humic and fulvic acid, seaweed extracts, protein hydrolases) and microbial inoculants (plant-growth-promoting rhizobacteria and mycorrhizal fungi) have shown to boost root performance [43,44], which can be used for enhancing transplant field establishment and subsequent crop production. Overall, solid HS with shoot and root growth-promoting effects can satisfy the requirements of transplant growth and subsequent yield in both pre- and post-transplanting environments, which makes them suitable and reliable amendments for use in transplant media.

**Figure 7.** Heatmaps and clustering of the amendment treatments (C and HS) based on the (top) before-transplanting traits and (bottom) after-transplanting traits with the consideration of yield components. Each row represents a crop cultivar with or without HS treatment, and each column represents a measured variable, including shoot dry weight (SDW), root dry weight (RDW), root shoot ratio (RSR), root length (RL), specific root length (SRL), root average diameter (RAD), root surface area (RSA) and average fruit weight (AFW). The expression variable values of the heatmaps follow the red (high)–yellow (low) color scale. All data are standardized and measured variables are clustered based on their correlations.

#### **5. Conclusions**

In this study, humic substances (HS) added as a media amendment for growing containerized vegetable transplants were evaluated for their seedling root and shoot growth modulation effects before and after field transplanting. Compared with control, HS: (1) improved plant shoot biomass accumulation of pepper, tomato, and lettuce mostly due to faster shoot growth rates, while these effects were not prominent in watermelon; (2) enhanced pepper and watermelon root developmental traits (RDW, RL, RSA) after transplanting due to faster root growth rates, and tomato root development both before and after transplanting, while these effects were not shown in lettuce; (3) decreased net assimilation rate of tomato, watermelon, and lettuce before transplanting but improved after transplanting, while this effect was not significant for pepper; (4) improved leaf area ratio in all four crops; (5) improved specific root length of tomato, watermelon, and lettuce before transplanting but decreased it after transplanting; (6) lowered root-to-shoot ratio of all the crops before transplanting but reversed it after transplanting, except for lettuce. Based on the field performance, we found suitable R:S ranges for high-quality transplants to be as follows: 0.25–0.35 for pepper, 0.15–0.2 for tomato, 0.1 for watermelon, and 0.15–0.2 for lettuce. This study demonstrated that HS differentially modulated root and shoot growth based on crop species: root performances were outstanding in fruit-based crops (pepper, tomato, watermelon), while leaf performances were significantly improved in the leaf-based crop (lettuce). Overall, imposed heat and drought stresses had significantly negative effects on crop yield and average fruit weight, but HS-treated plants showed more improved stress tolerance than control plants by mitigating the yield loss. This study showed the potential application of solid humic substances as biostimulants for enhancing transplant quality and crop performance in four economically important vegetable species (tomato, pepper, watermelon, and lettuce).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0472/10/7/254/s1, Figure S1: Vegetable crops and cultivars used in this study, Table S1: Abbreviations and their full names used in this study, Table S2: ANOVA and means comparison of germination percentage as affected by amendments (A), Table S3: Summary of HS effects on transplant quality traits (pre- and post-transplanting) and yield components (cool vs. hot seasons, low vs. high irrigation rates) compared to control (higher or lower at significant level *P* ≤ 0.1).

**Author Contributions:** Conceptualization, K.Q. and D.I.L.; methodology, K.Q. and D.I.L.; software, K.Q.; validation, K.Q. and D.I.L.; formal analysis, K.Q. and D.I.L.; investigation, K.Q. and D.I.L.; resources, K.Q. and D.I.L.; data curation, K.Q.; writing—original draft preparation, K.Q.; writing—review and editing, D.I.L.; visualization, K.Q.; supervision, D.I.L.; project administration, D.I.L.; funding acquisition, D.I.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Novihum Technologies GmbH.

**Acknowledgments:** The material is based upon work that is supported by the National Institute of Food and Agriculture, United States Department of Agriculture, Multi-state Project W-4168. We thank Joshua T. Harvey for the proofreading and constructive comments on the paper, and Manuel Figueroa Pagan and Michael Tidwell for their assistance in field experiments.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


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