This review presents a comprehensive and systematic study of the field of plant biostimulants and considers the fundamental and innovative principles underlying this technology. The elucidation of the biological basis of biostimulant function is a prerequisite for the development of science-based biostimulant industry and sound regulations governing these compounds. The task of defining the biological basis of biostimulants as a class of compounds, however, is made more complex by the diverse sources of biostimulants present in the market, which include bacteria, fungi, seaweeds, higher plants, animals and humate-containing raw materials, and the wide diversity of industrial processes utilized in their preparation. To distinguish biostimulants from the existing legislative product categories we propose the following definition of a biostimulant as ‘a formulated product of biological origin that improves plant productivity as a consequence of the novel or emergent properties of the complex of constituents, and not as a sole consequence of the presence of known essential plant nutrients, plant growth regulators, or plant protective compounds’. The definition provided here is important as it emphasizes the principle that biological function can be positively modulated through application of molecules, or mixtures of molecules, for which an explicit mode of action has not been defined. Given the difficulty in determining a ‘mode of action’ for a biostimulant, and recognizing the need for the market in biostimulants to attain legitimacy, we suggest that the focus of biostimulant research and validation should be upon proof of efficacy and safety and the determination of a broad mechanism of action, without a requirement for the determination of a specific mode of action. While there is a clear commercial imperative to rationalize biostimulants as a discrete class of products, there is also a compelling biological case for the science-based development of, and experimentation with biostimulants in the expectation that this may lead to the identification of novel biological molecules and phenomenon, pathways and processes, that would not have been discovered if the category of biostimulants did not exist, or was not considered legitimate.

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Biostimulants in Plant Science: A Global Perspective.

Fertilizer use in modern agriculture is highly inefficient; much of the applied fertilizer is released into the environment, causing environmental degradation. One way in which fertilizer use can be reduced without damaging plant nutrition is to enhance crop uptake of nutrients through the use of biostimulants. A broad definition of plant biostimulants, including substances sometimes categorized as biofertilizers or biopesticides, is used throughout this review: “Plant biostimulants are substances or materials, with the exception of nutrients and pesticides, which, when applied to plants, seeds, or growing substrates in specific formulations, have the capacity to modify physiological processes in plants in a way that provides potential benefits to growth, development, or stress response.” This definition includes a variety of substances, four of which will be reviewed in this article: seaweed extract, humic substances, amino acids, and plant-growth-promoting bacteria. We will concentrate on the positive effects of biostimulant application on plant nutrient uptake, and the underlying mechanisms, which include positive changes in soil structure or nutrient solubility, root morphology, plant physiology, and symbiotic relationships, will be discussed. Recommendations for future research directions include finding the most promising substances, isolating the active ingredients and clearly demonstrating the mechanisms by which they affect nutrient uptake. The beneficial effects and mechanisms must be consistently demonstrated in greenhouse and field experiments.

The Use of Biostimulants for Enhancing Nutrient Uptake

Although marine seaweeds have been used as biostimulants since the beginning of modern agriculture, studies have only recently focused on the ability of seaweed extracts and their polysaccharides to enhance growth of plants. In this work, two bioassays were carried out to study the growth-promoting activity of seaweed extracts and polysaccharide-enriched extracts from Ulva lactuca and Padina gymnospora, obtained in neutral and alkaline conditions. Initially, the effect of seaweed extracts and polysaccharide-enriched extracts on seed germination and growth-promoting activity on tomato (Solanum lycopersicum cv. Rio Grande) plants under in vitro conditions was studied. Half-strength Murashige-Skoog (MS) medium with or without sucrose was supplemented with different concentrations of seaweed extracts (2, 4, and 10 mg mL−1) or polysaccharide-enriched extracts (0.2, 0.4, and 1.0 mg mL−1). The parameters evaluated were germination percentage, radicle and shoot length, and dry weight. In a second experiment, polysaccharide-enriched extracts at 1.0 mg mL−1 and indole-3-butyric acid as the control were studied for root inducer activity in mung bean (Vigna radiata). The majority of seaweed extracts had an inhibitory effect on seed germination. However, a significant effect (P ≤ 0.05) on tomato seedling growth (except for dry weight) was shown with seaweed extracts at 2 mg mL−1 included in half-strength MS medium with sucrose (30 g L−1). Moreover, 10 mg mL−1 neutral and alkaline seaweed extracts had an inhibitory effect on the parameters evaluated. In contrast, polysaccharide-enriched extracts obtained from U. lactuca and P. gymnospora promoted germination and stimulated growth of tomato plants compared to the controls. Additionally, treatment of mung bean hypocotyl cuttings with polysaccharide-enriched extracts of U. lactuca and P. gymnospora induced rooting more rapidly and in greater number compared to the controls. These results provide evidence that polysaccharide-enriched extracts act as an effective growth-promoting treatment.

Activity of seaweed extracts and polysaccharide-enriched extracts from Ulva lactuca and Padina gymnospora as growth promoters of tomato and mung bean plants

Cold is a major constraint for tomato growth and productivity; as it is a cold sensitive crop. DREB1A plays a key role in generating cold tolerance in tomato by regulating the response of multiple genes under chilling stress. In this study, cold tolerant gene (DREB1A) driven by Lip9 promoter, was transformed in three tomato genotypes through Agrobacterium tumefaciens, employing tissue culture independent transformation strategy. Overnight imbibed seeds of tomato were surface sterilized, 3-day old shoot apical meristem of the developing seedling were pierced and incubated for twenty minutes with A. tumefaciens strain EHA-105 having OD600 nm of 1.0. The treated seeds were co-cultivated with Agrobacterium for 2 days. The regenerated shoots were subjected to 35 mg/l hygromycin as a selection for a period of 2–3 weeks. The presence of DREB1A and hpt genes in the hygromycin resistant (T0) transgenic plants was evaluated by PCR analysis. The transgene activity was detected in T1 plants by Reverse Transcriptase Polymerase Chain Reaction and Southern blotting that showed the stable integration of the transgene to the next generation. Physiological analysis of T2 transgenic plants depicted that increased expression of DREB1A induced only during chilling stress. After various chilling stresses, stomatal conductance, transpiration rate and relative water contents of transgenic lines were significantly higher than those of NT plants. While leaf osmotic potential of transgenic plants was lower as compared to NT plants. The established procedure is novel and can produce stable transgenic tomato plants in efficient manner by saving potential resources in terms of cost and time.

Piercing and incubation method of in planta transformation producing stable transgenic plants by overexpressing DREB1A gene in tomato ( Solanum lycopersicum Mill.)

The beneficial effects of seaweeds (macroalgae) and their extracts on plant systems has been used for many centuries to improve soil properties and to enhance the productivity and quality of agricultural crops and ornamental plants and turf grass However, it has only been recently that advances in scientific research have uncovered the potential applications for commercially produced macroalgal extracts as plant biostimulants Extensive literature on seaweed-derived extracts as biostimulants to improve plant tolerance to abiotic and/or biotic stress, plant growth promotion and their effects on root/microbe interactions, have demonstrated the varied roles in plant health, quality/biofortification and soil improvement These effects have been supported by physiological, biochemical and molecular mechanisms, as evident through investigations using model plants This review discusses selected aspects of the role of seaweeds and their extracts on plant growth; it also provides an overview of mechanisms of activity and potential bioactive components The review is presented to stimulate further studies on the use of macroalgal-derived extracts for applications in agriculture and horticulture

Seaweeds (Macroalgae) and Their Extracts as Contributors of Plant Productivity and Quality: The Current Status of Our Understanding

An efficient and reproducible two-step in vitro propagation system for tomato (Lycopersicon esculentum) was developed by using the combinations of seaweed biostimulant (Gracilaria edulis and Sargassum wightii) extracts and plant growth regulators (PGRs). Double cotyledonary nodal (DCN) explants of Co-3 cultivar were initially cultured on Murashige and Skoog (MS) and Gamborg’s medium (B5) containing thidiazuron (TDZ) and 6-benzylaminopurine (BA); the best responding cytokinin was tested in combinations with different auxins (NAA, IAA and IBA), and seaweed extracts (G. edulis and S. wightii) of about basal MS medium +10–70% was used for shoot proliferation. The best organogenic culture response was obtained on MS medium fortified with 1.5 mg L−1 TDZ and 1.5 mg L−1 IBA. Up to 24 shoots per explants were formed at an optimal duration of exposure to 35 days. Mini shoots of about 3–4 cm were transferred to medium supplemented with MS + iP, MS + zeatin, MS + G. edulis and MS + S. wightii at different concentrations. High frequency of shoot elongation was observed in the medium supplemented with 30% G. edulis (15.2 cm), and profuse rooting was observed in the medium supplemented with 50% S. wightii of about 16.1 cm. Shoot elongation and rooting were observed in the medium supplemented with seaweed extracts. The plantlets were transferred to the plant growth chamber (70% of relative humidity and 9 light cycles) and maintained in it for a week, and then they were transferred to a greenhouse condition. The plant growth chamber to green house transferred plantlets showed an increase in the survival rate from 70 to 85%. Thus a two-step regeneration protocol was developed in this study with a combination of seaweed extracts and PGRs, which provides a basis for the production of transgenics with high frequency and survivability of tomato plants.

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Effect of seaweed extracts and plant growth regulators on high-frequency in vitro mass propagation of Lycopersicon esculentum L (tomato) through double cotyledonary nodal explant

In the present study a simple and efficient somatic embryogenesis system was developed from leaf explants of Lycopersicon esculentum L. The protocol has been developed by using plant growth regulators and seaweed extracts a natural biostimulant. The leaf sections were initially cultured on to leaf embryogenic callus induction medium fortified with various concentration and combinations of 2,4-dichlorophenoxy acetic acid (0.2–1.0 mg L−1), picloram (0.2–1.0 mg L−1), and kinetin (0.1–0.5 mg L−1). The best responding concentration in induction of friable embryogenic callus was tested for the proliferation. The friable cultures were detached from the mother culture and inoculated in three different media supplemented with plant growth regulators, plus 0–25 % Caulerpa scalpelliformis or 0–25 % Gracilaria corticata extracts for embryo development. A twofold increase in maturation and germination of somatic embryos was observed in the media containing seaweed extracts (MSMG2 and MSMG3) than the control (MSMG1). The plantlets transferred from plant growth chamber to greenhouse conditions exhibited higher survival rate (90 %) than directly shifted plantlets.

https://link.springer.com/content/pdf/10.1007%2Fs10811-013-0151-z.pdf

Optimization of somatic embryogenesis protocol in Lycopersicon esculentum L. using plant growth regulators and seaweed extracts

Effect of cyanobacterial extracellular products on high-frequency in vitro induction and elongation of Gossypium hirsutum L organs through shoot apex explants

Synthesis of zinc oxide nanoparticles mediated by Dictyota dichotoma endophytic fungi and its photocatalytic degradation of fast green dye and antibacterial applications

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD600 = 0.2–1.0). The germinated seeds were cocultivated in the MS medium fortified with (0–200 mM) acetosyringone and minimal concentrations of (0–20 mg L−1) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD600 = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg L−1 thidiazuron, 1.5 mg L−1 indole-3-butyric acid, 30 mg L−1 kanamycin, and 0–1.5 mg L−1 adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.

P. Gurusaravanan

Papers

Effect of seaweed extracts and plant growth regulators on high-frequency in vitro mass propagation of Lycopersicon esculentum L (tomato) through double cotyledonary nodal explant

Optimization of somatic embryogenesis protocol in Lycopersicon esculentum L. using plant growth regulators and seaweed extracts

Effect of cyanobacterial extracellular products on high-frequency in vitro induction and elongation of Gossypium hirsutum L organs through shoot apex explants

Synthesis of zinc oxide nanoparticles mediated by Dictyota dichotoma endophytic fungi and its photocatalytic degradation of fast green dye and antibacterial applications

Optimization of Factors Influencing Microinjection Method for Agrobacterium tumefaciens-Mediated Transformation of Tomato