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Effect of GH p.L127V Polymorphism and Feeding Systems on Milk Production Traits and Fatty Acid Composition in Modicana Cows

Simple Summary Growth hormone (GH) participates in the regulation of lactation and lipid metabolism. Milk traits and fatty acid composition were investigated in Modicana cows, in relation to the genetic polymorphism at GH locus p.L127V and its interaction with the feeding system. It has been compared 8 hours of grazing without concentrate (EX), versus 2 h of grazing with concentrate (SI). The frequencies of LL, LV and VV genotypes were, respectively, 0.64, 0.34, 0.02. The GH polymorphism and its interaction with feeding system did not influence milk yield and gross composition. LL cows showed higher levels of total unsaturated and monounsaturated fatty acids, thus suggesting a potential role of the GH polymorphism on improving the healthy quality of milk. A higher level of 18:1 in the LL cows was evident only in the extensive system. The lower energy input in the extensive system, compared to the semi-intensive system, could justify this result. Abstract Growth hormone participates in the regulation of lactation and lipid metabolism. A trial study was conducted to evaluate the effects of genetic polymorphism at GH p.L127V and its interaction with feeding system (extensive, EX; semi-intensive, SI) on milk traits and fatty acids composition in Modicana cows. In the semi-intensive farm (SI) diet consisted of hay, concentrate and 2 h of grazing. In the extensive farm (EX) feeding consisted in 8 h of grazing and hay. The frequencies of LL, LV and VV genotypes were, respectively: 0.64, 0.34, 0.02. GH polymorphism and its interaction with feeding system did not influence milk yield and composition. Cows carrying LL genotype produced milk with lower 6:0 and 8:0 and higher 16:1 c9 and 18:1 c9, total UFA and total MUFA. Feeding significantly affected fatty acids: in EX cows lower SFA and higher PUFA and UFA were found, compared to SI cows. The lower, more favorable atherogenic index of milk from EX system was coherent with the improved healthy characteristics of milk from animals fed almost exclusively on pasture. A significant interaction genotype x feeding system was evident for 18:1, higher in the LL cows only in the EX system, but not in the SI system.

Introduction
Growth hormone (GH) is a member of the somatotropin/prolactin family of hormones secreted in acidophilic cells of the anterior pituitary gland of mammals. It plays an important role in growth, lactation, milk production, reproduction processes, protein, lipid and carbohydrate metabolism [1][2][3][4].
As a result of these physiological effects, GH is considered a candidate gene for marker-assisted selection in livestock [5][6][7]. The bovine growth hormone (GH) gene (Gen Bank Acc. Num. M57764.1) is located in BTA19; it includes five exons that encode a polypeptide of 217 amino acids [8,9]. Among the different polymorphisms characterized at the GH locus, the missense single nucleotide polymorphism (SNP) C > G in exon 5, that changes leucine to valine in position 127, is of particular interest [10]. Many studies have focused on this polymorphic site, named GH p.L127V, in order to evaluate the allele frequency distribution in dairy, meat and dual-purpose cattle breeds. Schlee et al. [11] reported that the leucine homozygous genotype (p.127LL) was associated with a higher GH concentration in the bloodstream. The research has investigated the effects of the polymorphism GH p.L127V on production traits, such as milk production, growth or carcass traits in cattle. The influence of GH p.L127V polymorphism on milk production traits is not conclusive, and seems to be rather breed-specific [12,13]. Furthermore, the p.127V variant is present at low frequency in many breeds; therefore, the association analysis is often limited by the lack of homozygous p.127VV animals, which disables a direct comparison of both homozygous variants.
Growth hormone is involved in lipid metabolism [14]. Due to this effect, a direct or indirect role of GH on the acidic composition of milk could be hypothesized.
The role of pasture feeding on milk traits and fatty acid composition is well known. Milk fat from ruminants fed with pasture is generally characterized by higher levels of polyunsaturated fatty acids, isomers of conjugated linoleic acid (CLA) and omega-3 fatty acids, compared to milk from stall-fed ruminants [15].
In Italy, despite of the wide diffusion of cosmopolite specialized breeds, several local cattle breeds are still farmed in marginal areas [16]. Most of them are traditionally reared in extensive and semi-intensive systems. In Sicily, the Modicana breed is reared for milk production, that is mainly addressed to the production of "Ragusano", a protected designation of origin (PDO) cheese, ageing from 4 to 12 months. During the grazing season, especially in the area of origin, the Hyblaean plateau, feeding system of Modicana cows is essentially based on pasture with no, or limited supplementation of concentrate; however, semi-intensive farming is also present where the time spent on pasture is lower and level of concentrate supplementation is higher than in the extensive system. Despite it is important role in the local economy, the Modicana cattle breed has been scarcely investigated for its genetic polymorphism, except for milk protein genes [17], the MC1R gene [18] and, more recently, for some genes involved in lipid metabolism [19].
The aim of this research was to study the effects of the genetic polymorphism GH p.L127V and its interaction with the feeding system on milk traits and fatty acids composition in Modicana cows. In particular, it has been studied if a different level of pasture in the diet could interfere on the effect of GH polymorphism on milk yield and its nutritional quality and acidic profile.

Material and Methods
The experiment lasted 7 months, from October to April in two Modicana dairy cow farms located in the province of Ragusa (Southern of Italy) and characterized by two different feeding systems: a semi-intensive system, SI (36 • 56 47 N and 14 • 41 50 E; 639 m above sea level) and an extensive system, EX (36 • 52 53 N and 14 • 33 51 E; 308 m above sea level), In the semi-intensive farm (SI) the diet consisted of stall feeding (hay and concentrate) and 2 h of daily grazing on natural pasture. In the extensive farm (EX) feeding consisted primarily of natural pasture (8 h per day) and hay.

Genetic Characterization
The DNA of 188 cows (specifically 118 from SI and 70 from EX) was obtained from milk samples by using the Milk DNA Preservation and Isolation Kit (Norgen Biotek), following the manufacturer's instructions. The concentration of extracted DNA was measured by using the NanoDrop 1000 Spectrophotometer and brought to 30-40 ng/µL.
Single Nucleotide Polymorphism C > G at the V exon of the GH locus (Acc. Num. M57764.1) has been analyzed by PCR-RFLP according to Komisarek et al. [20]. The polymorphism, which causes an amino acid substitution in the protein sequence, is identified as p.L127V. Amplifications were performed in a 30 µL reaction volume using a GenAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) thermal cycler. Amplified fragments 404 bp long were digested for 2 h at 37 • C with 5 units of AluI (New England BioLabs Inc., Ipswich, MA, USA). Restriction patterns were visualized and recorded after electrophoretic separation in 3% GelRed Nucleic Acid Gel Stain (Biotium, Inc., Fremont, CA, USA) agarose gel (Bio-Rad Laboratories, Inc., Segrate, Italy) using the ChemiDoc TM System with Image Lab TM Software (Bio-Rad Laboratories, Inc., Segrate, Italy).
The polymorphic region of GH exon V was sequenced in six samples (2 for each genotype p.127LL, p.127LV, p.127VV) in order to check potential new SNPs. The fragments of 404 bp were sequenced on both strands on an ABI PRISM 3130 Genetic Analyser, equipped with Sequencing Analysis software (Applied Biosystems, Foster City, CA, USA) and aligned with the reference sequence (GenBank Acc. Num. M57764.1) by using the software CLUSTALX 1.8 [21].

Animals and Experimental Design
According to the 188 observed genotypes at the GH locus, two experimental groups were formed. A total of 66 cows, 36 from SI and 30 from EX, at their third or fourth lactation, homogeneous for days of lactation (88.2 ± 22.1 and 95.7 ± 31.2 days for SI and EX, respectively) and milk yield (11.0 ± 3.0 and 12.2 ± 6.3 kg/d for SI and EX, respectively) were selected. Within each farm, the GH genotypes were distributed as follows: -SI: LL, 21 cows; LV, 13 cows; VV, 2 cows; -EX: LL, 19 cows; LV, 10 cows, VV, 1 cow.
In the two farms, the cows were fed as follows: SI cows were fed with 5 kg of vetch and oats hay, 5 kg of a commercial concentrate and grazed 2 h per day on natural pasture; EX cows were fed with 5 kg of vetch and oats hay and grazed 8 h per day on natural pasture.

Data Collection and Chemical Analysis
Every month individual milk productions were recorded and the relative milk samples, consisting of proportional volumes from the morning and evening milk, were collected. Fat, protein and lactose were analyzed using an automated Fourier transform infrared absorption spectrophotometric analyzer (Combi-foss 6000, Foss Electric, Hillerød, Denmark). Milk fat was extracted and converted to fatty acid methyl esters (FAME) [22]. Nonadecanoic acid was used as an internal standard (Sigma-Aldrich, St. Louis, MO, USA). FAME were analysed on a Trace Thermo Finnigan GC equipped with a flame ionization detector and a 100 m × 0·25 mm i.d. fused-silica capillary column (SP-2560, Supelco, Inc., Bellefonte PA, USA) according to Valenti et al., [19]. Fatty acids were expressed as g/100 g of total fatty acids.

Statistical Analysis
Genotypic and allelic frequencies were calculated, and the Chi-squared test was used to test whether the population deviated from Hardy-Weinberg equilibrium. Individual data for milk yield and composition (fat, protein, lactose, fatty acids) were analyzed using the GLM procedure for repeated measures of Statistical Package for Social Science (SPSS for Windows, SPSS Inc., Chicago IL, USA). The analysis included main effects of GH genotype, feeding system, period and the interaction genotype × feeding system. Pre-experimental data of daily milk production were used as a covariate for milk yield and gross composition. When the covariance was not significant (p > 0.05) it was removed from the statistical model. When interaction was significant, differences between means were assessed using the Tukey's adjustment for multiple comparisons. Significance was declared when p < 0.05.

Results
In the whole sample of 188 Modicana cows, three GH genotypes were found; the distribution of individual genotypes, with genotype and allele frequency in the two farms are reported in Table 1. The genotype p.127LL was the most frequent in Modicana cows. The genotypes resulted well distributed in the two farms (SI and EX) and no significant deviation from the Hardy-Weinberg equilibrium was observed (Table 1). No new SNPs were found by sequencing the analyzed exonic region of the GH gene. Table 2 reports the main effects of GH genotype and feeding system on milk yield and gross composition. Since, in our case, the VV Modicana cows were only three, this genotype was excluded from the statistical analysis. In this sample of Modicana cows milk yield and gross composition were not influenced by GH p.L127V polymorphism. The feeding system affected milk yield and protein content, that were higher in the SI reared cows, and lactose, that was higher in the EX reared cows. No significant interaction was found between GH genotype and feeding system.

GH p.L127V Polymorphism in Modicana Cows
In Modicana breed GH gene, a potential candidate gene for production traits, has been investigated for the first time. The p.127LL genotype was the most frequent in our sample, whereas p.127V allele was found at low frequency (0.20). This evidence confirmed the previous studies that reported GH p.127V variant as the minor allele in a wide range of Bos genus breeds.
In the present study, the Modicana GH p.L127V polymorphism was similar to that described earlier in German Black and White [11], Ayrshire [12], Jersey [27], and in two rustic Turkish breeds [30]. Instead, considerably higher frequencies of the V allele (≥0.28) were observed in Charolais [25] Piemontese [31], Limousine [32]. According to Shariflou et al. [33] GH p.127L allele was the most frequent in almost all the reported breeds but with averagely lower values in meat breeds. This has led the authors to hypothesize that the superiority in milk production of cows carrying L allele has induced a major selection towards the subjects carrying the L allele in milk breeds, but not in meat breeds. As confirmed byČítek et al. [34], the spread of both alleles in the German Holstein population was affected indirectly by selection for milk production in the past, resulting in a higher occurrence of L allele.

GH Polymorphism Effects on Milk Yield, Composition and Fatty Acids Content
Lucy et al. [12] reported that GH release in German Black-White cows carrying L genotype was higher than in cows carrying V genotype. Coherently, Sorensen et al. [13] found that LL Danish Jersey calves released 57% more GH than LV calves, which, in turn, released 53% more GH than VV calves. However, this effect seems to be breed-specific since they did not find similar differences in GH release between these genotypes in Danish Red and Holstein breeds.
Taking into account the positive effect of circulating GH on milk yield [35], a production increase would be expected in LL cows. Dario et al. [36] in Jersey and Akyuz et al. [28] in Holstein cows found higher milk production in LL genotype. Coherently, Lucy et al. [12] and Heidari et al. [37] found a genetic advantage for milk production in LL Holstein cows. Dybus [38] found higher milk yield in LL cows only during the first lactation. Shariflou et al. [33] found that LL and LV Australian Holstein-Friesian cows produced more milk than VV cows. Contrasting with the above results, Kovacs et al. [39] found that heterozygous cows (LV) had significantly higher 305 days of lactation milk yield, compared to LL cows. In Modicana cows the GH p.L127V polymorphism did not determine significant variations in milk yield. These results are in line with other authors [40,41].
Conflicting results are also reported on the effects of GH polymorphism on milk gross composition: in our study, no significant effect was evident. Dario et al. [36] found higher milk fat and protein content in LV genotype. Dybus et al. [38] and Kovacs et al. [39] report higher milk fat and protein in LL cows. Yardibi et al., [42] reported that LL genotype had significantly higher milk fat level compared to the other genotypes.
To our knowledge, no studies report the effects of the GH Alul polymorphism on milk fatty acid profile. The GH p.L127V polymorphism showed no effects on the fatty acid composition of meat in Korean cattle [43]. To better understand the effects of a greater release of somatotropin on the acid composition of milk fat, it is possible to refer to studies on the role of exogenous use of bovine somatotropin (bST) on this aspect. Taking into account the fact that the LL genotype would appear to be associated with a higher somatotropic hormone release [13], one could hypothesize an analogy of response in the milk fatty acid composition between subjects carrying the LL genotype and subjects treated with exogenous bST.
Linch et al. [44] did not find any difference in cows fatty acid composition as a consequence of treatment with bovine somatotropin. As reported by Lough et al. [45], the treatment of lactating dairy cows with somatotropin did not show any influence on the mechanisms of fatty acid synthesis or mammary lipid metabolism. Marty and Block [46] found a low effect of bST treatment on the milk fatty acid profile, whereas Apps et al. [47] found that cows given bST during late lactation, when in positive energy balance, produced milk with lower levels of C4 and long chain fatty acids and higher levels of medium-chain fatty acids, compared to untreated cows.
Eppard et al. [48] administering subcutaneous injections of 0 IU/d, 5 IU/d, 10 IU/d, 25 IU/d, 50 IU/d, and 100 IU/d of growth hormone report that the secretion of short and medium chain fatty acids in milk was increased but the increase has stabilized between 50 and 100 IU/d, whereas the secretion of long-chain fatty acids progressively increased until the highest level of injection.
In our conditions, Modicana cows carrying the LL genotype produced milk with lower levels of 6:0 and 8:0, but no differences were found for medium and saturated long chain fatty acids. On the contrary, 16:1 c9 and 18:1 c9, total UFA, and total MUFA were higher in LL cows. These results seem to be in line with the possible negative energy balance induced by a hypothetical increase of GH release in the LL cows. In fact, in general, it has been widely demonstrated that a higher level of GH (for example during a bST treatment) induces a negative energy balance status, due, among other causes, to a lower insulin sensitivity. This metabolic status induces an increase of body fat reserves mobilization and, as a consequence, an increase of monounsaturated fatty acids, especially 16:1 c9 and 18:1 c9 [49,50]. Indeed, as reported by Palmquist et al. [51], long-chain FA deriving from plasma are incorporated into milk fat, so they inhibit the de novo synthesis of short-chain FA by the mammary gland. As a consequence, one would expect a reduction of the short chain fatty acids, as occurred in our experimental conditions for 6:0 and 8:0. A similar trend of short chain fatty acids as effect of the energy balance is reported by Nogalski et al. [49].

Feeding Effects on Milk Yield and Composition and Fatty Acid Profile
The significantly higher levels of milk yield and protein percentage found in cows fed with hay, concentrate and few hours of pasture are the probable consequence of the higher energy and protein input, compared to cows fed mainly with pasture. Surprisingly no differences were found between feeding systems in the fat percentage. In fact, one would have expected a reduction of milk fat content in the animals that received feeds, such as concentrates, poorer in structural carbohydrates. The significant differences between the two feeding systems for most fatty acids are associated with the different proportion of pasture in the two diets. In fact, it is well known that the role of pasture feeding on milk fat composition: the higher levels of 14:1, 16:0 and total SFA and the lower levels of 18:0, 18:1 t11, 18:2 c9 t11, 18:3, branched chain fatty acids (15:0 iso, 17:0 iso and 17:0 anteiso) total UFA and total PUFA in cows fed in the semi intensive system are in line with results reported for stall fed cows, compared to pasture fed cows, by Villeneuve et al. [52]. The higher UFA/SFA ratio and the lower, more favorable atherogenic index of milk from the EX system is consistent with the improved healthy characteristics of milk from animals fed almost exclusively on pasture, compared to confined animals, as previously reported [53]. The significantly higher level of 18:1 in the LL cows was evident only in the extensive system, thus demonstrating a significant genotype x feeding interaction. The lower level of energy input in the extensive system, compared to the semi-intensive system, could somehow justify this result. In fact, Bitman et al. [54] found that C18:1 cis increased after a short term administration of bST when the cows were in negative energy balance; however, the differences with the untreated cows gradually decreased as energy balance became less negative.

Conclusions
In the present study, novel information about the GH p.L127V polymorphism and its interaction with the feeding system was investigated, for the first time in the Modicana breed. In particular, how different levels of pasture in the diet could interfere with the GH polymorphism on milk traits and fatty acid composition was studied. The tendency of LL cows towards higher levels of total unsaturated and monounsaturated fatty acids seems to suggest a possible role of the GH polymorphism on improving the healthy quality of milk. This research, conducted on a local breed reared in traditional systems, represents a first approach to the study of the role of the GH p.L127V polymorphism on the acidic composition of milk, and could represent a starting point for its extension to cosmopolitan breeds


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

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Aspects in Tobamovirus Management in Intensive Agriculture

In the recent years, disease spread of old and newly evolved tobamoviruses has occurred worldwide, affecting production of various vegetable and ornamental crops. The tobamoviruses are highly stable plant viruses that could cause severe disease symptoms. The well-known tobamovirus Cucumber green mottle mosaic virus (CGMMV) has recently caused severe damages in the cucumber, melon, and watermelon cucurbitaceous crops, worldwide. Similarly, a recent widespread of the newly identified tobamoviruses, Tomato mottle mosaic virus (ToMMV) and Tomato brown rugose fruit virus (ToBRFV), has reduced the solanaceous crop production. The primary route of tobamoviral infection is through mechanical means. These viruses adhere to agricultural facilities, contaminate the soil, infect seeds, and spread via beneficial pollinators and irrigation water. Mechanical plant injury suffices to initiate viral infection. Practicing hygiene by plant growers and in nurseries is currently the main strategy for mitigation of tobamoviral infection. Promoting the production of solanaceous vegetable crops genetically resistant to ToMMV and ToBRFV infection is a promising approach. However, CGMMV-resistant sources of cucurbitaceous vegetable crops are scarce. Conferring resistance to rootstocks and cross-protection strategies are newly implemented approaches that could alleviate tobamovirus disease spread in both solanaceous and cucurbitaceous crops.

Introduction
In the recent years, there has been a growing concern regarding disease damages and losses occurring in vegetable crop production. Plant viruses constitute the major causal factor for the diseases. Tomato plants, belonging to the Solanaceae family, and cucumber, melon, and watermelon plants, belonging to the Cucurbitaceae family, have shown the most severe disease symptoms. These symptoms are primarily attributed to infections by viruses belonging to the Tobamovirus genus, in the Virgaviridae family. The prevalent route of tobamovirus infection is via mechanical plant manipulations [1]. The tobamoviruses are highly stable and kept infectious in soil containing buried virus-contaminated plants [2], on various agricultural facility tool surfaces, in seeds [3], and upon adhering to beneficial pollinator body parts [4,5]. Two tobamovirus species that had a conspicuous effect on vegetable crop production in various countries and caused severe disease symptoms in host plants are the Tomato brown

Tobamovirus worldwide spread
Viruses belonging to the Tobamovirus genus are positive-sense single-stranded RNA viruses that infect a wide range of plant species. Tobacco mosaic virus (TMV), first described by Mayer in 1886 [11], is the prototype of this genus, in the Virgaviridae family. Tobamoviruses infect vegetable crops mostly solanaceous and cucurbitaceous plants, ornamental plants, weeds, and medicinal plants. In the recent years, the spread of tobamoviruses that infect two major cultivated vegetable crops, the solanaceous and cucurbitaceous plants, has increased. The Tomato mottle mosaic virus (ToMMV) that infected tomato plants (Solanum lycopersicum) had spread in America and Spain [12,13]. In the Middle East, ToBRFV had broken the highly durable resistance-conferring allele Tm-2 2 [6] that was introgressed into Lycopersicon esculentum from L. peruvianum [14]. Phylogenetic tree analysis showed that ToBRFV and ToMMV were clustered in separate clades [6]. ToBRFV infection of tomato plants has recently occurred in Mexico [15], Germany [16], and the USA [17]. A worldwide infection of the cucurbitaceous plants has occurred due to the spread of the tobamovirus CGMMV, first reported by Ainsworth in 1935 [8, 18]. Excluding few reports on CGMMV-resistant plants, commercial cultivars resistant to CGMMV are scarce [19,20].

Genome organization
The genome organization of the tobamoviruses ToBRFV and CGMMV resembles in general that of TMV [21,22]. The virus single-stranded RNA genome encodes four known proteins: short (126 or 129 kDa) and long (183 or 186 kDa) replicase-associated proteins. The long component is the outcome of a translational read-through of a termination codon of the short component. In addition, a movement protein (MP) of ~30 kDa and a coat protein (CP) of ~17 kDa are translated from sub-genomic RNA. A putative fifth 54 kDa protein resides between the two replicase-associated proteins [23]. Recently, in Solanaceae-infecting tobamoviruses, a sixth protein of 4-5 kDa has been identified, which is encoded by a region in the genome overlapping the open reading frames (ORFs) of the MP and the CP [24][25][26].

Particle pathogenicity and systemic disease spread
ToBRFV infection of solanaceous plants induced pathogenic systemic symptoms of narrowing leaves and yellow and brown spotted fruits. CGMMV infection of cucurbitaceous plants resulted in systemic mottle mosaic leaves and fruits as well as yellowing fruit flesh combined with necrotic peduncles (Figure 1). Increased severity of the symptoms could occur due to a variety of mixed infections. For example,  (Figure 1d), and the cucurbitaceous cucumber plants infected by both CGMMV and the Pythium species P. spinosum showed plant wilting and collapse [27] (Figure 1j).
The virulence factors that caused the severe symptoms occurring upon tobamovirus infection have not been established yet excluding the virulence factor of TMV upon infection of Nicotiana benthamiana that was identified as the orf6-expressed protein, which occurs in Solanaceae-infecting tobamoviruses [24]. Similarly, the mechanism of Tm-2 2 resistance breaking by ToBRFV has not been discovered yet. The viral MP is the avirulence factor recognized by the plant resistance-conferring protein Tm-2 2 [28]. Mutational analysis of the MP revealed that a change of two amino acids could overcome the resistance conferred by the Tm-2 2 allele [29]. However, the MP modifications that have occurred during the evolvement of ToBRFV are still unknown, although in bioinformatics approach several potential mutations were identified in the MP of ToBRFV. In the Cucurbitaceae-infecting CGMMV, a single amino acid substitution at the replicase site resulted in symptom attenuation [30], conferring a role for the replicase in viral virulence mechanism. The tobamovirus CP molecules constitute the capsid of the virion, which is ~300 nm long and 18 nm wide. For viral RNA encapsidation, ca. 2000 CP subunits form a right-handed helix in which each subunit binds three nucleotides. There are electrostatic interactions between charged amino acid residues that contribute to CP subunit interactions and particle stability, which is strengthened by hydrophobic contacts in the capsid [31] and carboxylate interactions between subunits [32]. Tobamovirus particles can survive 90°C heating and years of storage [31,33].
Tobamovirus encapsidation is necessary for long-distance movement of the virus in the plant but is dispensable for cell-to-cell movement [34,35]. Since viruses are localized in the symplast, it is necessary to maneuver cell-to-cell movement. Viral MP binds RNA and increases the plasmodesmata size exclusion limit [36]. The virus could then be transmitted via the widened cytoplasmic continuity that was formed between cells [37]. In addition to the MP effect on viral cell-to-cell movement, a role for the short replicase-associated protein in cell-to-cell viral movement was also observed, although the mechanism of the replicase effect is still unclear [38].
Tobamovirus CP is required for long-distance viral dissemination [34,39]. It is required for viral movement across the boundary between vascular parenchyma and companion cells [40]. It is not quite clear, however, whether the CP is necessary for interactions with host factors [39]. In the phloem, virus particles follow photoassimilate transportation [41]. However, mechanisms of entry and egress from the phloem differ [42,43]. For example, egress from the phloem involves the activity of the host plant pectin methylesterase [44].
Viral genome replication that occurs in the epidermis and mesophyll cells induces plant resistance programs such as the RNA silencing process [45,46]. Plant RNA silencing is triggered by viral double-stranded RNA precursors, which are processed by RNase III Dicer-like protein to small interfering RNA duplexes (siRNA), 21-24 nucleotide long [47,48]. The siRNAs are stabilized by HUA ENHANCER1 (HEN1), which catalyzes methylation at the 3′ end, generating 2′-O-methylated siRNAs [49]. The methylation prevents uridylation and degradation of the siRNA duplexes [49,50]. Single-stranded siRNAs direct ARGONAUTE (AGO) protein residing in RNA-induced silencing complex (RISC) to silence posttranscriptionally complementary RNA by endonucleolytic activity [51]. Importantly, small RNA duplexes that are formed by the plant silencing mechanism function also as silencing signals that spread via the plasmodesmata between cells and systemically through the phloem [52,53]. Establishing silencing process systemically can lead to degradation of newly infecting viruses prior to viral replication [54]. Viruses counteract the plant silencing process by the expression of silencing suppressors [55,56]. The tobamovirus short protein associated with the replicase, such as the 126 kDa protein of TMV [57] or ToMV [58] interferes with the methylation of the siRNA duplexes catalyzed by HEN1 and thereby induces degradation of the siRNAs [54]. Viral RNA silencing suppressors are therefore positive factors in viral long-distance movement [39]. However, the contribution of the replicase-associated protein to viral entry into the phloem is not clear yet but could not be attributed to suppression of RNA silencing [33].

Seeds
Tobamoviruses are seed-borne viruses, although the average of reported seed to seedling transmission ratios was only 4.1% [3]. Low viral transmission ratios in grow-out experiments are commonly indicative of uninfected embryos. Seed coat contamination could occur due to physical attachment between the seeds and the fruit flesh. However, there were also reports on ToMV infecting the endosperm of tomato plant seeds [1]. The tobamovirus passage through the maternal seed coat to the endosperm, which originates from both maternal and paternal sources, is enigmatic in the face of the uninfected embryos [3]. The consequence of endosperm infection is problematic, in particular, for considerations of the appropriate seed disinfection procedures. CGMMV-infected cucurbitaceous seeds are the most challenging for disinfection practices. Seeds from symptomatic CGMMV-infected cucurbit plants showed tobamoviral infection of both the seed coat and the perispermendosperm (PE) envelope underlying the seed coat [59,60], which is characteristic to cucurbits [61,62]. Importantly, the PE envelope is comprised of endospermic cells on top of which noncellular lipid and callose layers were formed [61,62]. A similar question is raised therefore regarding CGMMV occurrence in the PE envelope in the face of the uninfected embryos [59,60]. Tobamoviral dispersal emerging from infected seeds could occur via physical manipulations of the seeds upon sowing, seed to seedling transmission, and seed coat contamination of the soil.

Soil
Tobamovirus soil contamination primarily occurs due to buried plant debris originated from tobamovirus-infected crops [63]. Using a serological method for ToMV detection, a high primary infection ratio, of up to 80%, apparently occurred in the tomato plants grown in the contaminated soil [63]. Under field conditions as well, ToMV was detected in soil containing tomato debris of crops originated from a previous year planting [2]. ToMV was also recovered from forest soil in which the mineral fraction had more virus than the organic fraction [64]. Clay in the soil adsorbs a high fraction of tobamovirus particles [65], which could be visualized by scanning electron microscopy. Similarly, CGMMV inoculum buried in the ground for overwintering contaminated the soil [66]. Soil contamination was apparent by CGMMV detection in the soil supernatant, by inoculating uninfected cucurbit plants with the soil supernatant and by planting uninfected cucurbit plants in the contaminated soil [66]. Various soil types mediate CGMMV dispersal in various efficiencies, which could be attributed to root damage in the case of rock containing soil.

Beneficial pollinators
Bumblebees (Bombus terrestris) are essential beneficial pollinators of tomato crop cultivation. Bumblebee hives that were placed in ToBRFV-infected tomato-growing areas were ToBRFV contaminated [5]. The virus was detected in the hive components: the comb, the enveloping cotton, and the nectar. ToBRFV in the hives was infectious as studied by inoculating the laboratory test plant Nicotiana tabacum cv. Samsun with virus purified from the hive comb. ToBRFV adhered to the bumblebee body parts, primarily the abdomen, suggesting that ToBRFV could be transmitted by buzz pollination. Importantly, bumblebee hives from ToBRFV-infected tomato greenhouses placed in a new greenhouse of uninfected tomato plants constituted carriers of a primary infectious inoculum. ToBRFV infection ratios of the newly infected tomato plants were 12-60%. The ToBRFV infection ratios were positively correlated with bumblebee activity [5].

Secondary disease spread
Tobamovirus disease spread is abiotic. Mechanical manipulations during crop cultivation and the commonly associated plant injury constitute a major route for Plant Diseases-Current Threats and Management Trends tobamovirus disease spread. Low concentrations of tobamovirus contamination could establish the disease spread in a growing area due to mechanical manipulations [1]. For effective tobamovirus disease transmission, leaf or root injury seems imperative. Although the most common way of tobamovirus disease spread is via physical attachment, root-to-root viral transmission ratios in tobamoviruscontaminated soil are low [67]. Similarly, seeds sown in tobamovirus-contaminated soil showed low infection ratios when compared to seedling planting, which could involve plant injury [68]. However, high concentrations of the contaminating tobamoviruses and repeated exposure to the infectious source reduce the impact of injury as a necessary determinant in tobamovirus disease spread [63,65].

Irrigation water
Humidity preserves tobamovirus particle viability in soil. Infectious tobamovirus particles of TMV and ToMV were found in environmental waters such as ponds and streams. The occurrence of the tobamoviruses was visualized by electron microscopy, and the infectivity of the tobamovirus particles was examined in a biological assay on laboratory test plants [69]. A quantitative and sensitive approach to detect the tobamoviruses in environmental waters was also applied using sensitive reverse-transcription real-time PCR analysis [70]. Apparently, there were environmental water samples that tested positive for ToMV without the usual sample concentration step. Dispersal of the Cucurbitaceae-infecting tobamovirus CGMMV via irrigation water was tested, for example, in laboratory facilities in the Volcani Center, Israel. In the middle of a planting tray of cucumber (Cucumis sativus) plants, one plant was sap-inoculated with the virus and was then separated from adjacent plants by an open plastic vessel to prevent any mechanical transmission of the virus via any other way than that of the irrigation dripping system that was applied. The results inspected a month later showed that CGMMV infection ratios ranged between 36 and 91%, while the control plants were CGMMV free (Dombrovsky and Darzi, unpublished data). CGMMV transmission efficiency via dripping and flooding irrigation systems was examined in a glasshouse experiment. The distances of CGMMV infection of watermelon (Citrullus lanatus Thunb.) plants by dripping and flooding irrigation systems were 1.9 and 2.3 m, respectively [71]. CGMMV was also detected in a river close to a farm of CGMMV-infected muskmelon (Cucumis melo) and watermelon plants [72].

Plant manipulations
Contaminated hands, pruning shears, knives, trellising ropes, and grafting procedures are the most common means for effective tobamovirus transmission. Attempts to quantitate the contribution of mechanical contact to tobamovirus disease spread revealed that a high number of repeated contacts between TMVinfected tobacco leaves and uninfected plants were positively correlated with increased tobamovirus transmission efficiency [63]. Interestingly, there was no correlation between the TMV quantity in the source leaves and the efficiency of viral transmission. Several characteristics of the source of infection could also affect transmission efficiency, such as leaf age and the viral source, whether it was the outcome of systemic spread or it was the primary inoculated material. However, the effects of these parameters on TMV disease spread were not conclusive [73]. Quantitating the disease transmission ratios of the Cucurbitaceae-infecting CGMMV in cucumber plants was conducted, for example, by touching the plants with CGMMV-contaminated hands. CGMMV contamination analyzed serologically 3 weeks post the infection procedure spread down the row, sequentially, and the infection ratio was 86% [68]. The contribution of agro-technical work of pruning and trellising to CGMMV disease spread was monitored in an experiment conducted in commercial cucumber greenhouses [68]. In the greenhouses, 5-11 scattered CGMMV-infected plants, which constituted 0.4-0.5% of the plants, were identified, and a survey was conducted on the effects of the intensive agro-technical activities on CGMMV disease spread. The percent increase in infected plants due to agro-technical practice for 40 days, in the various greenhouses, was in the range of 11-32% [68].

Beneficial pollinators
Beneficial pollinators do not only constitute a primary source of tobamovirus disease spread, as was observed in ToBRFV spread analysis [5], but could also promote secondary viral spread between tobamovirus-infected and uninfected plants. Hives containing bumblebees (Bombus terrestris L.) placed in a greenhouse of TMV-infected tomato plants (Lycopersicon esculentum L. cv. Momotaro) spread the TMV infection to adjacent uninfected tomato plants planted in the greenhouse. TMV viral particles attached to the bumblebee body parts were visualized by electron microscopy and tested positive for TMV in a serological assay. TMV viral particles isolated from the hive components were infectious, as analyzed in a biological assay using Nicotiana glutinosa seedlings for inoculation [74]. Regarding the Cucurbitaceae-infecting tobamovirus CGMMV, the honeybee Apis mellifera promoted disease spread between infected melon seedlings, which constituted a primary viral source, and adjacent uninfected plants [4]. Efficient secondary viral transmission between the plants occurred when the uninfected plants were placed on the path of the honeybee foraging track, between the beehive and the CGMMVinfected plants.

Weeds
Volunteer plants such as weeds could have an important role as tobamovirus reservoirs that may constitute a source of infection. Apparently, weed species could constitute asymptomatic reservoirs of the Cucurbitaceae-infecting tobamovirus CGMMV [75,76]. Among the weed species susceptible to CGMMV infection that did not show any conspicuous symptom development and their susceptibility to CGMMV infection which was confirmed by laboratory mechanical inoculations were Molucella laevis, Amaranthus graecizans, and the medicinal plant Withania somnifera [76]. Overwintering of tobamoviruses in the weed hosts could promote the tobamovirus spread between cultivated crops of consecutive growing seasons.

Management strategies 6.1 Sensitive tobamovirus detection methods
In order to prevent the occurrence and establishment of tobamovirus primary infectious source introduced by virus-infected seeds, the appropriate detection methods should be applied. Viral RNA extraction (using Viral RNA Extraction Kit; Bioneer) from ToBRFV-infected tomato (S. lycopersicum) seeds (Luria and Dombrovsky, unpublished data) and CGMMV-infected cucumber (C. sativus Derben) and melon (C. melo Raanan) seeds were successfully executed [60]. Nextgeneration sequencing (NGS) platform has been successfully applied for detection of the tobamoviruses ToBRFV [6] and CGMMV [77]. The NGS detection method is highly sensitive when compared to the most commonly used serological assays (enzyme-linked immunosorbent assay, Western blot) and the genome sequence analysis performed after PCR amplification. ToBRFV-infected tomato seeds mixed with uninfected seeds in a ratio of 1:600 were successfully detected by the NGS method (Luria and Dombrovsky, unpublished data). Viral RNA extractions, which are easy to perform and need a small amount of starting material, were successfully applied in the NGS analysis [78]. The use of the new technology based on a single-molecule sequencing such as the Oxford Nanopore sequencing platform was successfully applied for detection of plant viruses and bacteria and the detection of the tobamovirus ToBRFV in infected tomato seeds. The sensitivity ratio for detection of ToBRFV-infected tomato seeds by applying the Oxford Nanopore sequencing platform was 1:200 virus-infected seeds mixed with uninfected seeds [10]. Application of sensitive methods for tobamovirus detection in seeds is most critical and needs to be developed for CGMMV-infected cucurbitaceous seeds, in which the viral particles accumulate in the PE envelope underlying the seed coat [59,60].

Alleviating soil-associated tobamovirus infectivity
Soil fumigation with pesticides such as methyl bromide, which had an effect on a wide range of plant pathogens, was successfully used in various crop production facilities. However, since the elimination of its use due to its high toxicity and the detrimental effect on the ozone, several other chemicals such as chloropicrin (trichloronitromethane) had been used [79]. Unlike methyl bromide, many newly used chemicals had no effect on weeds or plant debris, and their efficiency in inactivation of tobamoviruses was questioned. A good alternative to those chemicals are products based on strobilurin fungicide, which originally occurred in the mushroom Strobilurus tenacellus [80]. Synthetic compounds such as pyraclostrobin (F 500) that protected the tobacco plant N. tabacum (cv. Xanthi nc) against TMV infection could be used in soil preplant treatment. Other chemicals that are based on natural products are alkaloids such as phenanthroquinolizidine that can be extracted from several plant families such as the Vitaceae family [81]. Several formulas of the alkaloid exhibited anti-TMV activity [81]. Similarly, quassinoids isolated from Brucea javanica showed anti-TMV activity [82]. Recently, the strong antiviral effect of synthesized bioactive tricyclic spirolactones, which are based on natural polycyclic compounds, has been demonstrated on TMV infection of tobacco plants [83]. Interestingly, the antibiotic Ningnanmycin showed anti-TMV activity both by induction of plant resistance against virus and by inhibition of the virus virulence [84].
Soil steaming treatment using low temperatures (50-60°C) for short time periods (several minutes) had been useful for inactivation of most plant pathogens while preserving soil microflora and minerals [85]. However, these steaming conditions could not be applied for disinfection of tobamovirus-contaminated soil [60]. Interestingly, a combination of mild steaming conditions with chemicals such as potassium hydroxide that are exothermal when reacted with water was effectively applied to disinfection of TMV-infected soil. TMV infectivity ratios were reduced to 3.0%. The increase in the persisted heat in the soil and the higher soil pH could have affected TMV stability [86]. Importantly, regarding CGMMV-contaminated soil, application of intermediate medium composed of CGMMV-free compost, which was prepared from cattle feces, into planting pits prior to planting melon (C. melo) seedlings, significantly reduced the initiation of primary infectious foci in the growing area. When combining removal of newly identified infected plants at early growth stage, before trellising, with the implementation of intermediate medium, CGMMV infection ratio at 60 days post planting was 0.3% [60]. Recently, growers have implemented growth bags that separate between plants, to grow various vegetable crops in order to reduce tobamovirus disease spread via soil (Figure 2).
Grafting vegetable crops on Tobamovirus-resistant rootstocks could also separate the cultivated crops from the contaminated soil. In particular, Solanum gilo accessions were found resistant to the tobamoviruses TMV, ToMV, and the Pepper mild mottle virus (PMMoV). Unfortunately, CGMMV-resistant rootstocks scarcely occur and are difficult to find. Using CGMMV-tolerant rootstocks for grafting cucumber plants in field experiment in Northern Israel resulted in viral infectivity ratios of 0.4-0.8% (2 rows, 250 plants in each row). Concomitant growth of ungrafted cucumber plants had CGMMV infectivity ratios in the range of 16-44% (5 rows, 320 plants in each row) (Dombrovsky and Koren, unpublished data). Importantly, it is preferable to use rootstocks that do not cause any reduction in crop yield. Rotations in crop cultivation could reduce buildup of primary infectious tobamovirus inoculum from contaminated soil [87]. For example, it is possible to plant tomato plants after pepper plant plantings since PMMoV does not infect tomatoes. However, because ToMV infects pepper plants, planting tomato crops should not be followed by pepper plant plantings [87]. Importantly, alternating rice and watermelon cultivation reduced CGMMV infection ratios of the watermelons by tenfold (amounting to 7.3%) when compared to consecutive watermelon cultivation [88].

Hygienic and careful planting procedures
The use of sodium hypochlorite solution on planting facilities and even on seeds was recommended for disinfection of tobamoviruses. However, seeds treated with the hypochlorite solution could show low germination ratios. Trisodium phosphate treatment (TSP) of tobamovirus-infected seeds was also implemented. Importantly, these commonly used disinfection procedures could not be applied for disinfection of tobamovirus-infected cucurbitaceous seeds in which the virus penetrated the seed coat and accumulated in the PE envelope underlying the seed coat [59,60]. TSP (10%) treatment, combined with heating to 72°C for 72 hours, did not disinfect CGMMV-infected melon (C. melo cv. Raanan) seeds. The CGMMV detected in the treated seeds using serological assays and PCR analysis was infectious in a biological assay [60]. However, sodium hypochlorite solution and the new stabilized chlorine product, which has the active ingredient C 3 Cl 3 N 3 NaO 3 , could be useful for general tobamovirus disinfection of planting facilities [8] such as shears, knives, trellising ropes, planting trays, and irrigation pipes. Careful planting procedures should be implemented, avoiding the infliction of any injury to the plants. Dividing tasks between workers during planting could also reduce secondary tobamovirus infection spread. Weeding the weeds that might be reservoirs of tobamoviruses [76] and supplementing new bumblebee hives for tomato plant cultivation [5] could be important in preventing primary inoculum of tobamovirus infection. Identification and removal of tobamovirus-infected plants in crop cultivation facilities at early stages, before trellising, could reduce secondary tobamovirus disease spread.

Near-future management strategies
In the face of the genetic tobamovirus resistance occurring in tomato plants for many years, such as that of the durable Tm-2 2 resistance allele [14], a search for ToBRFV-resistant tomato plants could be successful. However, CGMMV-resistant genetic sources for introgression into commercial cucurbitaceous vegetable crop cultivation are scarce [19,20]. Similarly, sources for CGMMV-resistant rootstocks are limited. Engineering transgenic watermelon rootstocks by transformation of the rootstock Citrullus lanatus (Twinser) cv. Gongdae with CGMMV CP gene conferred viral resistance to the plants [89], similar to the phenomenon observed in TMV CP-mediated resistance against TMV [90]. Another approach that could confer viral resistance to susceptible host plants while avoiding the production of transgenic crops is the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology [91]. This mutagenesis system is targeted by guide RNAs to a desired site in the plant cell genome, where Cas9 endonuclease causes double-stranded DNA breaks. The system exploits host cellular repair mechanisms to confer heritable high fidelity change in the genome. Host endogenous genes, such as the Arabidopsis thaliana TOM1 and TOM3 genes, and their homologues in tomato and melon plants, are necessary for tobamovirus replication [92,93]. The host proteins translated from these genes comprise a complex with the tobamovirus replication protein [94]. These host proteins could be targeted by the CRISPR/Cas9 system. Importantly, RNA silencing of these host genes conferred tobamovirus resistance in Nicotiana tabacum [95] and in tomatoes [96]. Interestingly, RNA silencing, which is systemic [52,53,97], could be transmitted from rootstocks to scions. Hence, engineering rootstocks alone for tobamovirus resistance by RNA silencing of these host genes could confer resistance to the nontransgenic grafted tomato or melon plants. When using the biocontrol approach, plant defense mechanisms that specifically target the infecting tobamovirus, such as the RNA silencing, could be initiated by infecting the susceptible plants with a stable attenuated virus clone or a mutagenized variant of the virus. For example, several attenuated strains were successfully applied to protect tomato plants against ToMV infection, pepper plants against PMMoV infection [98], and muskmelon and cucumber [99] plants against CGMMV infection. However, mutagenized clones might not always be stable, and symptoms might develop in the susceptible plants. Therefore, in order to implement the cross-protection approach, for example, against ToBRFV that infects tomato plants harboring the Tm-2 2 resistance allele, it might be beneficial to infect the tomato plants with the stable ToMV that does not show symptoms in the resistant tomato plants. For that purpose, ToMV needs to infect systemically the Tm-2 2 resistant tomato plants.

Conclusions
In the recent years, tobamovirus disease spread has been one of the core causes for severe damages observed in various vegetable and ornamental crop productions. Concomitantly, there has been an increase in suggested strategies for tobamovirus disease management. The basic approach of implementing hygienic behavior


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Effect of sonic bloom frequency on the growth of red amaranth (Alternanthera amoena Voss)

Red amaranth (Alternanthera amoena Voss) is able to be used not only as food plants but also as herbal plants. Due to a high nutrient content of red amaranth, the demand of this plant is continue to increase. Even though this plant has high demand, its productivity is relatively low. The Sonic-bloom was experimented to be applied on the red amaranth cultivation to improve the productivity. The purpose of this research is to determine the effect of various frequencies and duration of sonic-bloom exposure on the growth of the red amaranth plant (Alternanthera amoena Voss). The frequencies were set at two levels i.e. 3000-5000Hz and 7000-9000 Hz with the exposure duration were 15, 30, 45, 60 and 75 minutes respectively. The results showed that the frequency and duration of exposure have a significant influence on the cultivation parameters of red amaranth plants. Frequency treatment of 3000-5000 Hz and 7000-9000 Hz produced plants with 25.93 cm and 18.83 cm of height, 13 strands of leaves, 42 cm and 30.6 cm of plants length, 2841.01 μm2 and 3688.51 μm2 of stomata area, and 14.84 gr and 7.58 gr of wet plant weight respectively. These results were significantly different compared to the control (without sonic bloom treatment) that have 8.53 cm of height, 7-13 strands of leaves, 14.87 cm of plants length, 972.98 μm2 of stomata area, and 0.55 gr of wet weight. The best treatment in this study is the frequency of 3000-5000 Hz with exposure duration of 75 minutes, concluding that Sonic Bloom technology is able to improve the productivity of red amaranth plants.

Introduction
Sonic bloom is an organic technology that can stimulate plant growth through the application of music frequencies and providing of plant nutrition. This treatment is able to stimulate plant cells to absorb nutrition more effective [1]. Sonic Bloom can be applied to all types of plants from annual crops such as rice, crops, flowers and perennial plants such as coffee, cocoa, rubber, palm oil, teak, and so on. Each plant has specific frequency for up-taking of nutrition [2].
Red Spinach (Amaranthus spp.) is promoted widely as vegetables and as source of nutrition for residents in developing countries. The energy content of red spinach is 55 kcal/100 g better than that of green spinach which only have 15 kcal/100 g. Anthocyanin is a purple red pigment that makes the spinach leaves have red color. This compound is able to prevent oxidation of free radicals that cause various diseases [3]. The Sonic-bloom was experimented in this research to be applied on the red International Conference on Innovation and Technology IOP Conf. Series: Earth and Environmental Science 524 (2020) 012020 IOP Publishing doi:10.1088/1755-1315/524/1/012020 2 amaranth cultivation to improve the productivity. The purpose of this research is to determine the effect of various frequencies and duration of sonic-bloom exposure on the growth of the red amaranth plant (Alternanthera amoena Voss).

Materials and Method
Research was carried out by assembling a set of sonic bloom and preparing the red amaranth seedbed in the planting medium in the laboratory of the laboratory of Mechatronic and Agricultural Machinery, Department of Agricultural Engineering, Universitas Brawijaya from June to July 2019.
Rockwool was prepared as growing media for seeding the red amaranth. Rockwool was cut in small cubes, arranged in a seedling tray and kept in wet condition. The seeds were germinated on rockwool for 10 days and then transplanted to the polybag of 15 cm diameter with planting media. The planting media were a mixture of soil, organic fertilizer, and rice husk with a ratio of 1: 1: 1. Liquid fertilizer was sprayed with appropriate dose and weeding was carried out manually.
The number of leaves and plant height were measured every three days, while the area of stomata opening was observed on days 15 and 30. Measurement of the stomata area was conducted with a microscope that was interfaced with computer. Software of DX-Olympus was used to measure length and wide of stomata. Plants are harvested at the age of 30 days and then measured the length of the plant i.e. the length from the root tip to the longest leaf. Each plant is weighed to measure the wet mass of plants.
Relative humidity, ambient temperature and light intensity were observed. The Environmental parameters are measured in the morning and evening. The level of environmental noise both in the control treatment and in the experimental treatment was measured routinely as well.

Results and Discussion
The range of Relative Humidity (RH), temperature and light intensity during experiments are in the range of 40-50%, 28-32 o C and 23000-27000 lux respectively. The level of environmental noise in the control treatment was 68-76 dB and in the experimental treatment was 55-57 dB.

Plant height
Plant height was measured from ground level to the highest end of the plant. The highest plants were 27.05 cm by the treatment of sonic bloom with a frequency of 3000-5000 Hz with exposure time of 60 minutes. It was extremely height compare to the control that only produce 8.95 cm plant height and 17.25 cm produced by treatment of 7000-9000 Hz frequency and exposure time of 75 minutes. This result was in line with the research conducted by Utami et al. [4] that the treatment of classical and hard rock music has a significant effect on the growth of chili. Figure 1 shows the effect of music frequency on the height of the red amaranth plants.   Figure 1 shows that the treatment of 3000-5000 Hz improves the growth of red amaranth compared to the controls (without treatment). But, it was not as high as that of from the frequency of 7000-9000 Hz. These results are consistent with the results of the research conducted by Bahtiar et al. [5] that the treatment of 4 kHz has effect on growth of rice seed. Sonic bloom technology and organic fertilizer can be applied simultaneously to improve onions productivity [6]. It means that the treatment of specific frequency effects on the growth of rice seed, but also effects on others plants grow include the red amaranth.

Numbers of leaves
The amaranth with treatment of 7000-9000 Hz frequency and 60 minutes exposure has the most leaves, which are 16 leaves as in Figure 2. It is more leaves compared with the other exposure time at the same frequency and also the treatment of 3000-5000 Hz for all exposure time. The control treatment produced only 7leaves, while the treatment with frequency produced leaves in the range of 8 to 16 strands or 11 strands average. This data shows that the sonic bloom frequency effects on the production of leaves number. This result is appropriate with A'mallina and Astono [7], stating that grasshopper makes a sound that can increase the productivity of peanut plants. It is possible because the frequency of grasshopper sound is in the range of 7000 Hz.

Length of plant
Similar to the effect on other variable, the sonic bloom frequency has significant effects on the length of plants. Frequency of 3000-5000 Hz and exposure of 60 minutes stimulate the growth of the red amaranth. The plant length of 46.9 cm can be produced at the frequency treatment and the exposure time. This treatment produces three times compared to the control treatment that only produce plants length of 16 cm.
The red amaranth treated with frequency show average size longer than plants without treatment. These results are consistent with the results of the study conducted by Prasetyo et al. [8]. They applied sonic frequency to improve the productivity of green spinach. The sonic bloom frequency also had positive effect on the growth of red amaranth. Figure 3 shows the effect of sound frequency on the length of red amaranth. Both of low frequency and high frequency produce red amaranth longer than that red amaranth without frequency treatment. Frequency has positive effect on growth parameter, because it has a relation with plant metabolism. It can stimulate gas exchange through openings of stomata.

Area of stomata
The opening areas of stomata are showed in Figure 4. The largest opening area of stomata occurs in the frequency of 7000-9000 Hz and exposure of 75 minutes. It was 3688.51 μm 2 . The smallest opening area occurs in the frequency of 3000-5000 Hz and exposure of 30 minutes, but it was still larger than opening area of control treatment i.e. 972.98 μm 2 . Application of sonic bloom frequency was able to increase opening area between 1.7-2.7 times to the area of the control treatment.   This result was matching with the research carried out by Aditya et al. [9], that music frequencies of 6000-9600 Hz open the stomata of mustard larger than the normal. The application of frequency has effect on the opening area of stomata.
Identical research has been carried out by Widiastuti [10]. Application of frequency of 3000 Hz on corn plant opens the stomata larger than normal condition and application of other frequency. The consequence of the larger stomata area results the smoothness of carbon dioxide mobility for photosynthesis. The exposure of the insect sound manipulated at peak frequencies of 3500 Hz could affect the area of stomata opening [11]. It has effect on the plant height, number of stems, and mass of yields on rice plants. It is logic that the sound treatments in this research increase the growth of red amaranth plants through the opening of stomata. Figure 5 shows the dimension of stomata captured with microscope that is interfaced with computer. The shape of the stomata is ellipse. The dimension is showed by the size of long axis and short axis.

Wet mass
The red amaranth plant was weighed with analytical scales after harvesting. The control treatment produced average weight of 0.4 g for each plant. This result is much smaller compared to the results of the sonic bloom treatment. The treatment of 3000-5000 Hz and exposure of 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 75 minutes produce 9.96g, 3.62 g, 4.92 g, 12.78 g and 17.93 g respectively. It is interesting that high frequency treatment results wet mass smaller than that of low frequencies treatment as shown in Figure 6. Frequency treatment of 7000-9000 Hz with exposure time of 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 75 minutes respectively produce 3.67 g, 3.17 g, 3.22 g, 2.19 g, and 5.79 g wet mass. This wet mass is bigger than the wet mass without frequency treatment, but lower than that medium frequency. Although frequency treatment affects wet mass growth, there is an optimum frequency that must be applied to get maximum results. It means that the frequency is not directly proportional with the mass growth of the red amaranth.  Figure 6. Effect of music frequency on wet mass of red amaranth The research conducted by [8] shows also that the wet mass of green mustard plants with frequency treatment have bigger than that without frequency treatment. The optimum frequency is on the range of 3000 Hz. Experiments show that the optimal frequency range for red amaranth plants is also similar to the optimum frequency range for mustard greens.
The application of sound frequency in general improves the wet mass of plants. Anas and Kadarisman [11] conducted this research on rice plants and shows that the wet mass of rice with sound frequency was better than that without treatment. They use a kind of insect for generating sound frequency. A frequency of 6000-9000 Hz was applied to improve production of mustards plant [9].

Conclusions
Sonic bloom with specific frequency can be applied to improve the growth of red amaranth. All of plant growth parameter i.e. plant height, number of leaves, plant length, stomata area, and wet mass increase significantly after sonic bloom applied. The optimum frequency of sonic bloom was 3000-5000 Hz for the best growth of red amaranth. The optimum exposure time of sonic bloom was 75 minutes.


extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
Enhanced Production of Bryonolic Acid in Trichosanthes cucumerina L. (Thai Cultivar) Cell Cultures by Elicitors and Their Biological Activities

Bryonolic acid is a triterpenoid compound found in cucurbitaceous roots. Due to its biological activities, this compound gets more attention to improve production. Herein, we carried out efficient ways with high bryonolic acid productions from Trichosanthes cucumerina L., a Thai medicinal plant utilizing plant cell cultures. The results showed that calli (24.65 ± 1.97 mg/g dry weight) and cell suspensions (15.69 ± 0.78 mg/g dry weight) exhibited the highest bryonolic acid productions compared with natural roots (approximately 2 mg/g dry weight). In the presence of three elicitors (methyl jasmonate, yeast extract, and chitosan), cell suspensions treated with 1 mg/mL of chitosan for eight days led to higher bryonolic acid contents (23.56 ± 1.68 mg/g dry weight). Interestingly, cell culture and root extracts with high bryonolic acid contents resulted in significantly higher percent cell viabilities than those observed under control (1% v/v DMSO) treatment in Saos-2 and MCF-7 cells. The present study indicated that T. cucumerina L. cell cultures are alternative and efficient to produce the biologically important secondary metabolite.

Introduction
Medicinal plants have been used as major natural sources in drug development over recent decades. Owing to their activities, there are many reports that compounds isolated from plants have been used as modern drugs against many human diseases. As far as everybody knows, the chemical synthesis of valuable natural compounds is difficult because of the structural complexities, especially triterpenoids. For solving this problem with the aim of producing a large amount of natural compound, plant biotechnological knowledge, including plant cell cultures, have been developed. Bryonolic acid is an active triterpenoid compound with various interesting potentials. This compound exerts anti-allergic activity by inhibiting homologous passive cutaneous anaphylaxis and delayed hypersensitivity; this activity is more potent than that of glycyrrhetinic acid with less toxicity [1]. Besides, bryonolic acid reduces nitric oxide by suppressing inducible nitric oxide synthase expression, indicating anti-inflammatory activity [2]. Interestingly, several studies have reported the ability of bryonolic acid in rat adrenal pheochromocytoma (PC12) cells against N-methyl-D-aspartate (NMDA)-induced neurotoxicity, indicating it as a candidate neuroprotective agent for cerebral ischemic treatment [3].
Although the sources of bryonolic acid mainly available on cucurbitaceous roots, it was reported only 1.34% w/w of dry root from Cucurbita pepo [4].
Trichosanthes is the largest genus in the Cucurbitaceae family, with more than 100 species in Indochina [5]. This genus has been widely studied because it has a long history of traditional use to treat a variety of diseases. Trichosanthes cucumerina L. (TC) is one of the widespread species with various varieties and medicinal potentials [6]. The TC variety cultivated in Thailand is commonly called "Buap Khom" which means bitter gourd because all parts of this variety are incredibly bitter and therefore are not consumed as a vegetable like other cucurbitaceous plants such as cucumbers, watermelons, squashes, and pumpkins. This variety showed unique characteristics, especially its small elliptical fruit [7], which is different from variety cultivated in India, China, and Japan [5,8,9]. TC has traditionally been used as a medicinal plant because of its strong laxative, dandruff treatment, and sinusitis relief properties. Moreover, it was previously reported that its anti-diabetic [10,11], cardioprotective [12], gastroprotective [13], hepatoprotective [14], and cytotoxic [15] activities mainly result from two active triterpenoid compounds, cucurbitacin B and bryonolic acid.
Plant cell cultures are potentially valuable sources for the production of secondary metabolites. These cells, including callus and cell suspension, have been developed during the past decades. They are taken advantage of fast-growing and higher-productive properties for the commercial and industrial productions of plant valuable compounds [16]. Moreover, the application of elicitors is also advantageous for improving desire compounds. Elicitors can induce the production of phytoalexins and stimulating plant defenses to protect the cells leading to the promotion of secondary metabolites; therefore, biotic elicitors have been used in plant cell cultures [17]. Owing to the difficulty of obtaining the triterpenoid productions, there are many evident reports studied on the enhancement of those compounds with elicitor treatments. For example, exogenous methyl jasmonate, a plant signaling molecule, increased soyasaponin production up to six-fold with 100 µM in the licorice cell suspension. Moreover, the expressions of enzymes involved in triterpenoid biosynthesis were also upregulated [18]. The presence of yeast extract, a microorganism-derived elicitor, potentially induced gymnemic acid overproduction in Gymnema sylvestre cells reached to six folds [19]. Furthermore, Hu, et al. [20] reported that chitosan treatment was able to induce Panax ginseng cultured cells in the accumulation of triterpenoid saponins through upregulating of squalene synthase and squalene epoxidase expressions. Obviously, the key enzymes in the triterpenoid biosynthetic pathway were induced by those types of elicitors; thus, they should be used to optimize the bryonolic acid production.
Because cucurbitacin B and bryonolic acid exhibit numerous activities, many researchers have attempted to improve these compound quantities. Alternative biotechnological strategies, including plant cell cultures, have been studied. Interestingly, TC is one of the major sources for both active triterpenoid compounds. Furthermore, the use of elicitors for improving cucurbitacin B and bryonolic acid production has not been previously reported. In this study, we reported large quantities of bryonolic acid using TC callus and cell suspension. The use of different elicitors (methyl jasmonate (MJ), yeast extract (YE), and chitosan (CH)) at various concentrations were also tested. The cytotoxic activities of these extracts were studied in breast cancer (MCF-7) and osteosarcoma (Saos-2) cell lines. Furthermore, we investigated the wound healing properties of the extracts in fibroblast (Balb/c 3T3) cell lines.

Effect of Explants on Callus and Cell Suspension Induction
Explants from the leaves, cotyledons, hypocotyls, epicotyls, and roots of plants grown in vitro were cultured to induce callus formation by culturing them on Murashige and Skoog (MS) plant medium containing 0.5 mg/L indole-3-butyric acid (IBA) and 1 mg/L benzylaminopurine (BAP). From the observation, it can be seen that calli from hypocotyls and epicotyls were white and fragile, meaning that they were appropriate for initiating cell suspensions ( Figure 1). Calli from epicotyls were used to establish cell suspensions through culturing in MS liquid medium supplemented with 1 mg/L IBA and 1 mg/L BAP in a shaker at 120 rpm in the dark. The fresh and dry weights of cells were observed for growth rate analyses. The 5-week cell suspension showed the highest fresh and dry weights. The exponential phase of the growth curve was observed from the fourth week to the fifth week, when remarkably increased growth was recorded ( Figure 2). Therefore, the four-week cells were used in other experiments, including the study of elicitor effects for enhancing bryonolic acid content.

Effect of Elicitors on Bryonolic Acid Production
The effects of elicitors were observed using different concentrations of MJ (50-200 µM), YE (0.1-2% w/v), and CH (1-100 mg/mL) detected via HPLC analysis. Cell suspensions elicited with 200 µM of MJ for six days or 0.1% w/v of YE for eight days were able to produce more bryonolic acid (10.33 ± 1.27 and 18.63 ± 5.10 mg/g dry weight, respectively) than the corresponding control groups (5.63 ± 1.44 and 9.11 ± 3.58 mg/g dry weight, respectively). Comparatively, elicitation with CH at 1 mg/mL provided the best conditions for increasing bryonolic acid levels after treatment for eight days (approximately 23 mg/g dry weight) to levels almost two times higher than in the control groups ( Figure 3, Table S6).  (1,50, and 100 mg/mL). Elicitors were added to four-week cell suspensions, which were observed after treatment for 2, 4, 6, and 8 days compared with controls (70% ethanol, sterile water, or 1 N acetic acid, respectively). Data are presented as the mean from three independent cell lines, and error bars show the standard deviations. The asterisk indicates significance (* p-value < 0.05, ** p-value < 0.01 compared with control group by t-test from Microsoft Excel 2016).
It is known that types of elicitors, concentration, and duration of elicitor exposure are important factors affecting the production of secondary metabolites [21]. Plants require different elicited time in response to the different elicitors in any concentrations. From our result, TC cell cultures showed strong responses to MJ elicitation only two days, whereas YE and CH required the delayed responses in the increases of bryonolic acid productions. Sivanandhan, et al. [22] reported that using MJ treatment in Withania somnifera hairy roots only 2-8 h potentially increased withanolide A approximately 50-fold. MJ is known as an endogenous elicitor produced after pathogen attack [23]; thus, it probably requires less duration of time than exogenous elicitors produced by pathogens or microorganisms [24].
The use of elicitors in cell suspension culture also offered large quantities of bryonolic acid. This application is appropriate for upscaling the production of interest compounds from small flask to bioreactor. For example, cell suspension cultures of Withania somnifera, a medicinal plant known as Ashwagandha or winter cherry, were upscaled in a 5 L bioreactor to obtain greater production of withanolides compared with small flask cultures by approximately 1.5-fold [25].

Metabolic Extraction
In HPLC analysis compared with two standards, cucurbitacin B and bryonolic acid, were detected at 5.8 and 14.2 min, respectively. The extracts from natural roots (TC1), leaves (TC2), stem (TC3), and loofahs (TC4), including cultured cell suspension (TC5), and calli (TC6) were analyzed. Cucurbitacin B was only in TC4 with 4.2 mg/g dry weight. Bryonolic acid was detected in TC1, TC5, and TC6. In our experiments, bryonolic acid contents were measured at approximately 15 mg/g dry weight in TC5 and almost 25 mg/g dry weight by in TC6, whereas the roots were only able to produce this compound at approximately 2 mg/g dry weight (Table S5). Although alternative methods have previously been developed for bryonolic acid isolation, the maximum content was only 13.4 mg/g dry weight by root cultures throughout C. pepo with specific conditions [4]. Through the plant cell cultures and hairy roots, bryonolic acid was produced only 10-13 mg/g dry weight [26,27]. Therefore, TC is a valuable novel source in bryonolic acid purification.

Biological Activities
In the presence of the six TC extracts (TC1-TC6), including the bryonolic acid standard, none of the extracts at a concentration of 50 or 100 µg/mL showed toxicity in either the Saos-2 or MCF-7 cell lines. Conversely, the TC1, TC5, and TC6 extracts resulted in a significantly higher percentage of cell viability in MCF-7 cells by 1.4-1.7 times than in the control treatment group (1% v/v DMSO) ( Figure 4A). Moreover, the TC5 and TC6 extracts increased the percentage of cell viability in Saos-2 cells by 1.1-1.3 times compared with 1% v/v DMSO treatment ( Figure 4B). Similarly, bryonolic acid at 100 µg/mL resulted in 1.6 times higher cell viability of both cell lines compared with 1% v/v DMSO. Previously, two antifungal agents, fludioxinil and fenhexamid, which have structurally similar to 17β-estradiol, were reported with the capacity of the induction in MCF-cell proliferations through estrogen receptor by 1.2 to 1.5 times and 2.5 times, respectively, compared to control (0.1% v/v DMSO) treatment [28,29]. Although bryonolic acid has been shown to exert a neuroprotective effect against NMDA in rat adrenal pheochromocytoma (PC12) cell lines [3], it also showed cytotoxic activity against various other cell lines, such as lung cancer cells (A549) and breast cancer cells (MDA-MB435, SKBR3, MCF-7, and T47D) [15]. From these reported findings, it would conclude that bryonolic acid exhibits cell-type specific properties; however, additional experiments are needed to confirm this hypothesis. Because of the presence of bryonolic acid peaks in TC1, TC5, and TC6 ( Figure S3), we assumed that bryonolic acid may have a synergistic effect with other compounds in the extracts increasing MCF-7 cell viability. These observations suggested that TC1, TC5, TC6, and bryonolic acid may induce the proliferations of the treated cell lines. It is well known that cell proliferation is one of the main characteristics of the wound-healing process [30,31]. Based on this assumption, we further examined the wound-healing properties of these three extracts with the hypothesis of cytoprotection.
A wound-healing assay was performed using two-dimensional confluent monolayer Balb/c 3T3 cells. The length or wound width, which represents the migration rate of cells, was evaluated after 14 h of exposure to the extracts. Cells that were treated with all extracts and bryonolic acid showed slightly narrower wound widths than the cells treated with 1% v/v DMSO, except for those treated with TC1 at the concentration of 100 µg/mL. Among all groups tested, the extract from cell suspensions (TC5) resulted in the narrowest wound with no significant difference ( Figure 5). Although our extracts led to the increased cell viability, they could not clearly represent the wound-healing effect. As mentioned before, wound-healing effect can be performed through cell proliferation. Many reports suggested that this activity was also able to process by other mechanisms. It was presented that leaf extract of Chromolaena odorata exerted wound-healing property by the increase of heme oxygenase-1 (HO-1) [32]. Moreover, cell migration was also a key role in this activity [33]. Accordingly, further validated study on cell proliferation would potentially help in the cytoprotective hypothesis.

Plant Materials
The natural plant materials were kindly obtained in 2015 (Nakhon Pathom province, Thailand) from Prof. Dr. Weena Jiratchariyakul, Faculty of Pharmacy, Mahidol University, Thailand (BKF no. 70279). The dried roots, leaves, stems, and loofahs were powdered and extracted for HPLC analysis. The seeds were sterilized with 70% v/v ethanol for 1 min and 0.5% v/v sodium hypochlorite for 5 min. Then, they were washed with sterile water three times. The sterile seeds were soaked in sterile water for two days before culturing on MS hormone-free medium. The pH of the medium was adjusted to 5.7 ± 0.1 before autoclaving at 121 • C for 15 min. The cultures were maintained at 25 ± 2 • C in the dark.

Callus Induction
After seed germination, the seedlings were cut and cultured on MS medium supplemented with 0.5 mg/L IBA and 1 mg/L BAP for inducing callus [34]. The explants from leaves, cotyledons, hypocotyls, epicotyls, and roots (n = 25) were observed under conditions of temperature maintained at 25 ± 2 • C and a 16 h photoperiod (light intensity at 2000 lux).

Cell Suspension Initiation
The calli were used for preparing cell suspension cultures. Approximately 0.5 g of calli were cultured in 125 mL Erlenmeyer flasks containing 30 mL of MS liquid medium supplemented with 1 mg/L of IBA and 1 mg/L of BAP in a shaker at 120 rpm in the dark to establish cell suspensions [34]. The cell suspension cultures were routinely subcultured every 30 days. The cell suspensions were harvested from the first week until the sixth week for a growth rate study. The fresh and dry weights (g in 30 mL) of these cell suspensions were recorded.

Effect of Elicitors on Bryonolic Acid Accumulation
Different concentrations of MJ (50, 100, and 200 µM), CH (1, 50, and 100 mg/L), and YE (0.1, 0.5, and 2% w/v) were added to 4-week cell suspensions cultured in 30 mL of MS liquid medium containing 1 mg/mL IBA and 1 mg/mL BAP. The treated groups of cell suspensions were observed after elicitation for 2, 4, 6, and 8 days compared with the control groups for MJ, CH, and YE administered 70% v/v ethanol, 0.1 N acetic acid, or water, respectively.

Active Compound Analysis Using High-Performance Liquid Chromatography (HPLC)
Approximately 100 mg of each dried sample was extracted with 1 mL of methanol (MeOH) containing 200 µg/mL of progesterone as an internal standard. Each extract was filtered through a 0.45 µm syringe filter before analysis by HPLC (LC-20A, Shimadzu) with a BDS HYPERSIL C18 column (Dim. 250 × 4.6 mm, particle size 5 µm) with an injection volume of 10 µL. The mobile phases were MeOH and water: 0-2.5 min (70% v/v MeOH), 2.51-10 min (100% v/v MeOH) and 10.01-19.00 min (70% v/v MeOH), at a flow rate of 1 mL/min. The extracts were detected at 210 nm compared with cucurbitacin B and bryonolic acid standards for quantification based on standard calibration curves. These two standards were reconfirmed via high-resolution ESI-MS (Figures S1 and S2). The HPLC conditions were validated. Linear regression equation, correlation coefficient (r 2 ), and the limit of quantitation (LOQ) were determined through five concentrations of cucurbitacin B and bryonolic acid (500, 250, 125, 100, and 50 µg/mL). The precision was confirmed by the relative standard deviation (%RSD). The accuracy was performed based on %recovery with three-level concentrations of both compounds (400, 125, and 75 µg/mL) (Tables S1-S3).
Stock solutions (10 mg/mL) of MeOH extracts from the roots (TC1), leaves (TC2), stems (TC3), loofahs (TC4), cell suspensions (TC5), and calli (TC6) were prepared by dissolving the dried methanolic extracts in DMSO. Bryonolic acid was also tested in this experiment for comparison. Two concentrations of TC extracts (50 and 100 µg/mL) were prepared via the following protocol. A 100 µg/mL extract was prepared from the stock solution by a 100-fold dilution in DMEM. Subsequently, this solution was diluted two-fold in DMEM containing 1% v/v DMSO to yield 50 µg/mL of extract. The final concentration of DMSO in all samples was set at a constant 1% v/v.
The toxicity of the extract was tested in the Saos-2 and MCF-7 cell lines. Cells (100 µL) were seeded in 96-well plates at cell densities of 2000 and 4000 cells per well for Saos-2 and MCF-7 cells, respectively. After incubation under a 5% CO 2 humidified atmosphere at 37 • C for 24 h, the medium was discarded, and 100 µL of the sample was added to each well. After 48 h of incubation, 10 µL of PrestoBlue ® reagent was added, followed by incubation for another 50 min. Absorbance values were recorded at 570 and 600 nm as the measurement and reference wavelengths, respectively, in a Tecan Infinite ® 200 microplate reader (NanoQuant, Männedorf, Switzerland). Cells incubated with DMEM containing 1% v/v DMSO and cisplatin at 10 µg/mL were used as negative and positive controls. The measurement was performed in triplicate on three different days.

Wound-Healing Test
Mouse fibroblast cell lines (Balb/c 3T3 close A31, ATCC ® CCL-163TM from American Type Culture Collection (ATCC), MD, USA) were maintained in DMEM. The media were supplemented with 10% bovine calf serum (Donor bovine serum with iron Gibco ® , Life Technologies Corporation, CA, USA) and 1% penicillin/streptomycin. Cells were incubated at 37 • C under 5% CO 2 . The medium was replenished every three days. Subculturing was performed before the cells were completely confluent.
To evaluate the cell migration of a confluent monolayer, an in vitro wound-healing assay was conducted by scratching a two-dimensional confluent culture [35]. Balb/c 3T3 cells were seeded at a concentration of 0.3 × 106 cells per well in 12-well plates one day prior to the experiment. Then, scratches were made using 200 µL micropipette tips (three scratches per well), followed by gently washing off the cell debris with PBS. Cells were replenished with fresh medium containing the extracts from TC1, TC5, and TC6 at concentrations of 50 and 100 µg/mL. Since the TC extracts were dissolved in DMSO, 1% v/v DMSO served as a control. After 14 h of incubation, the cells were washed with PBS and then fixed with 4% paraformaldehyde. Images of the scratched area were captured using a CKX53 inverted microscope (Olympus ® , Shinjuku, Tokyo) at a magnification of 10×. For each concentration of TC extract, nine scratches were made in three 12-well plates, and five images per scratch were obtained. ImageJ (ImageJ 1.50i, National Institutes of Health, USA) was used to measure the wound width (µm).

Statistical Analysis
The experimental results are reported as the mean ± standard deviation. The cytotoxicity data were statistically analyzed with GraphPad Prism ® version 7.00 for Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com). Statistical significance was determined using one-way ANOVA and Dunnett's multiple comparisons test (p-value < 0.05).

Conclusions
In this study, plant cell cultures and the effects of elicitors on bryonolic acid were investigated. The results of the study showed notably amounts of bryonolic acid compared with those of the TC plant parts from nature by way of TC callus and cell suspension cultures. The presence of chitosan in cell suspension culture remarkably increases the production of bryonolic acid compared with the control group. Besides, the cytotoxic activities of the extracts from calli, cell suspensions and roots with high bryonolic acid contents were associated with a significantly greater percentage of cell viability than 1% v/v DMSO treatment. Bryonolic acid has an interesting pleiotropic profile of biological activities, including immunomodulatory, anti-inflammatory, cytoprotective, and cytotoxicity effects, as mentioned above. Thus, bryonolic acid is a promising candidate for further investigation to explore mechanisms underlying biological activities and for the development of new drugs in the future. This finding provides a novel plant source and an alternative method through plant cell culture techniques to improve the production of bryonolic acid for further needs.