<i>In Vitro</i> Propagation of <i>Agave guiengola</i> Gentry Using Semisolid Medium and Temporary Immersion Bioreactors

Agave guiengolaGentry is an endemic plant from a very small locality in Oaxaca, Mexico. Its conservation status is fragile and can rapidly worsen. Because of its scarcity, this agave has been used solely for ornamental purposes, but it could have other uses if more plants were available. In vitro propagation by enhanced axillary sprouting from stem segments was attained using Murashige and Skoog Basal Medium (MS) as well as basal medium supplemented with cytokinins 6-Benzylaminopurine (BA) or 6-(γ,γ-Dimethylallylamino)purine (2iP). The best treatment for shoot induction in semisolid medium consisted in MS supplemented with 2 mg l^–1 BA, obtaining a mean of 3.7 shoots per explant. Other interesting responses were observed, such as nodular callus induction using combinations of BA and 2,4-Dichlorophenoxyacetic acid (2,4-D); root induction without Plant Growth Regulators (PGR); and generation of shoot clusters. These clusters constituted an excellent explant for micropropagation in temporary immersion bioreactors, obtaining a propagation rate of 43 shoots per explant with 1 min immersion and 6 h immersion frequencies. All new plants rooted and survived the transfer to soil. This study developed an in vitro propagation scheme to produce individuals that can be used either for reforestation, economical purposes, or to carry out studies in this species to assess its full potential, avoiding exploitation from wild plants.     

<i>In Vitro</i>-Propagation of <i>Agave tequilana</i> Weber cv. azul in a Temporary Immersion System

In Mexico, there is a need to produce large quantities of plantlets for the establishment and replanting of blue (cv. azul) agave production areas. Most of these plots are within the origin denomination area (DOT, Spanish acronym) of the distilled product of this plant, known as tequila. The objective of this study was to develop an in vitro-propagation protocol for Agave tequilana Weber cv. azul using segmented stems in both: solid and liquid media. A disinfection and in vitro technique were developed to obtain shoots, through plantlets collected in commercial plots, which attained 100% surface-disinfection and budding rate. At the multiplication stage, the effects of 6-Benzylaminopurine (BA) (0.0, 4.4 and 13.2 μM) and kinetin (0.0, 9.4, 18.8 and 37.6 μM) were evaluated on lateral-shoot production of segmented sagittal stems. These were cultivated on Murashige & Skoog (MS) medium, with the addition of 3.0% sucrose and 8 g L⁻¹ agar. It was observed that BA and kinetin increased the number of shoots per explant, obtaining up to 18 and 26, respectively. Furthermore, it was found that just the sagittal segmentation of explants increased axillary budding. On the other hand, segmented-stem bases were grown in MS liquid medium with 3.0% sucrose, inside a RITA® system, programmed by a 5 min immersion step with a frequency of every 4 h. The effect of Indole−3-Acetic acid (IAA) (0.57, 2.9, 5.7 μM) was evaluated, while maintaining a concentration of BA (13.2 μM). It was observed that the greatest concentration of IAA led to the formation of more than 20 buds per explant. These results offer a new methodology to increase the efficiency of A. tequilana Weber cv. azul-in vitro multiplication by sagittal segmentation of stems and the addition of BA and/or IAA.     

Conservation Strategy for African Medicinal Species: <i>In Vitro</i> Biotechnological Approach

The use of medicinal plants for different therapeutic values is well documented in African continent. African diverse biodiversity hotspots provide a wide range of endemic species, which ensures a potential medicinal value. The feasible conservation approach and sustainable harvesting for the medicinal species remains a huge challenge. However, conservation approach through different biotechnological tools such as micropropagation, somatic embryogenesis, synthetic seed production, hairy root culture, molecular markers based study and cryopreservation of endemic African medicinal species is much crucial. In this review, an attempt has been made to provide different in vitro biotechnological approaches for the conservation of African medicinal species. The present review will be helpful in further technology development and deciding the priorities at decision-making levels for in vitro conservation and sustainable use of African medicinal species.     

<i>In Vitro</i> and <i>in Silico</i> Insights on the Biological Activities,Phenolic Compounds Composition of <i>Hypericum perforatum</i> L. Hairy Root Cultures

Three Hypericum perforatum hairy root lines (HR B, HR F and HR H) along with non-transformed roots were analyzed for phenolic compounds composition and in vitro enzyme inhibitory properties. In silico molecular modeling was performed to predict the interactions of the most representative phenolic compounds in HR clones with enzymes related to depression, neurodegeneration and diabetes. Chromatographic analyses revealed that HR clones represent an efficient source of quinic acid and hydroxybenzoic acids, epicatechin and procyanidin derivatives, quercetin and kaempferol glycosides, as well numerous xanthones. In vitro antidepressant activity of HR extracts through monoamine oxidase A (MAO-A) inhibition was attributed to the production of oxygenated and prenylated xanthones. The neuroprotective potential of HR extracts was related to the accumulation of quercetin 6-C-glucoside, epicatechin, procyanidins and γ-mangostin isomers as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Vanillic acid and prenylated xanthones in HR clones as promising inhibitors of tyrosinase additionally contributed to the neuroprotective activity. Five preeminent xanthones in HR (γ-mangostin, mangiferin, garcinone C, garcinone E and 1,3,7-trihydroxy-6-metoxy-8-prenyl xanthone) along with the flavonol quercetin 6-C-glucoside effectively inhibited α-amylase and α-glucosidase indicating the antidiabetic properties of HR extracts. Transgenic roots of H. perforatum can be exploited for the preparation of novel phytoproducts with multi-biological activities.     

Rapid Propagation of <i>Rhynchostylis retusa in Vitro</i>

An efficient regeneration system of Rhynchostylis retusa was established to provide technical reference for the application of tissue culture tube seedlings in production. The mixtures of callus and protocorm from aseptic germination were used as explants. The optimal media of each stage was selected for callus proliferation, protocorm occurrence and growth, rejuvenation and rooting via a single, complete combination and orthogonal experiment. The results showed that the optimal medium for callus proliferation, protocorms occurrence and growth was 1/2 Murashige and Skoog (MS) medium adding 50 g·L⁻¹ banana puree, 0.1 mg·L⁻¹ α-naphthaleneacetic acid (NAA), 1.5 mg·L⁻¹ 6-benzylaminopurine (6-BA) and 1.0 mg·L⁻¹ kinetin (KT) with 17.33 proliferation coefficient of callus and 19.63 occurrence coefficient of buds after 90 days. Then the buds occurred from protocorm were cultured on 1/2 MS medium including 100 g·L⁻¹ banana puree, 1.0 mg·L⁻¹ NAA, 2.0 mg·L⁻¹ 6-BA and 0.05 mg·L⁻¹ KT, in which the proliferation coefficient of callus was 10.32 and occurrence coefficient of buds reached 17.87. In the further subculture, the same medium was simultaneously used for callus proliferation, protocorm occurrence and bud growth. The plantlets developed roots in 1/2 MS medium containing 70 mL·L⁻¹ coconut water and 1.5 mg·L⁻¹ NAA with 100% rooting rates after 90 days. The survival rate was more than 90% after domestication and transplantation. This regeneration protocol will provide technique foundation for protecting wild resource and developing artificial cultivation.     

<i>In Vitro</i> Propagation,Isolation and Expression Studies of <i>Suaeda edulis</i> Genes Involved in the Osmoprotectants Biosynthesis

Halophytes are an excellent choice for the study of genes conferring salt tolerance to salt-sensitive plants and, they are suitable for reclamation and remediation of saline soil. We develop an in vitro plant propagation protocol and studies of genes involved with GB and Pro biosynthesis in Suaeda edulis. Axillary buds were used as explants and cultured in different treatments on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators. The highest number of multiple shoots was on MS medium containing 1 mg/L Benzyladenine (BA) and / or 2 g/L activated carbon with 5.5 ± 06 shoots per explant. The identification and expression analysis of genes involved in glycine betaine (GB) biosynthesis were S-adenosylmethionine synthetase (SAMS), choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), and for proline (Pro) was pyrroline 5-carboxylate synthetase (P5CS). These sequences shared 90–95% of identity with others plant homologous in public databases. The amino acids sequence analysis showed that all these peptides contain some of the conserved motifs of those kinds of enzymes. The qRT-PCR analysis revealed a higher expression of SeBADH, SeCMO, and, SeP5CS genes in the roots and leaves from plants collected in the field in contrast with from in vitro plants. However, the expression level of SeSAMS was higher only in the leaves of plants collected in the field when compared to those cultivated in vitro.     

Encapsulation of Immature Somatic Embryos of <i>Coffea arabica</i> L. for <i>in Vitro</i> Preservation

The present study aimed to develop a protocol for somatic embryogenesis and encapsulation of coffee embryos (Coffea arabica L.), for the conservation of genotypes with characteristics of commercial interest. Somatic embryos were induced from leaf explants in Murashige and Skoog medium (MS) supplemented with 1 mg · L⁻¹ of 2,4-dichlorophenoxiacetic acid (2,4-D) combined with 2 mg · L⁻¹ of benzyladenine (BA). Somatic embryos (SE) at the globular stage were encapsulated in a sodium alginate matrix; two treatments were tested: MS + 5 mg · L⁻¹ BA + 1 mg · L⁻¹ NAA + 3% (w/v) alginate, and MS + 7 mg · L⁻¹ BA + 5.7 mg · L⁻¹ indoleacetic acid (IAA) + 3% (w/v) alginate. Alginate was complexed with 100 mM calcium chloride (CaCl₂). Viability of the encapsulated SE was determined by staining with 0.01% fluorescein diacetate (FDA) after 0, 15, 30, and 45 days of storage at 4°C. Embryo viability was 100% in both treatments.     

<i>In vitro</i> Antibacterial Activity of <i>Moringa oleifera</i> Ethanolic Extract against Tomato Phytopathogenic Bacteria

The tomato (Solanum lycopersicum L.) is one of the world’s most important vegetable crops. Still, phytopathogenic bacteria affect the yield and quality of tomato cultivation, like Agrobacterium tumefeciens (At), Clavibacter michiganensis subsp. michiganensis (Cmm), Pseudomonas syringae pv. tomato (Pst), Ralstonia solanacearum (Rs), and Xanthomonas axonopodis (Xa). Synthetic chemical products are used mostly on disease plant control, but overuse generates resistance to bacterial control. This study aimed to evaluate the in vitro antibacterial activity of the ethanolic extract of Moringa oleifera Lam. leaves against At, Cmm, Pst, Rs, and Xa, as well as information about this plant species’ chemical composition. Antibacterial activity against pathogens observed by microplate technique, phytochemical screening, and FTIR analysis revealed different bio-active compounds on ethanolic extracts with antibacterial activity. The growth inhibition rate ranged between 0.08% and 99.94%. The inhibitory concentration, IC50, required to inhibit 50% of At, Cmm, Pst, Rs, and Xa bacterial growth, was 276.67, 350.48, 277.85, 351.49, and 283.22 mg/L, respectively. Inhibition of phytopathogen bacteria’s growth increased as the concentrations of the extract also increased. Moringa oleifera extract can be recommended as a potent bio-bactericide.     

Antifungal Potential of <i>Beauveria bassiana</i> on <i>Solanum lycopersicum</i> L. Infected with <i>Fusarium oxysporum</i> f. sp. <i>lycopersici</i>

The objective of this work was to evaluate the effect of Beauveria bassiana (Bb 1205) on controlling Fusarium oxysporum f. sp. lycopersici (Fol 17108) in tomato plants in greenhouse conditions. Inoculation of Bb 1205 was the most promising among the agronomic variables and expression of the activity of the enzymes β-1,3-glucanases and chitinases. Inoculation of Bb 1205 occurred at a concentration of 1 × 10⁸ conidia·mL⁻¹, which was administered onto the leaves, directly into the soil and via injection. Infection with Fol 17108 occurred with 1 × 10⁶ spores·mL⁻¹, which were added directly to the soil. Spectrophotometry was used for measuring agronomic parameters, namely activity of chitinases and β-1,3-glucanases in foliage and roots. When Bb 1205 was added to the soil, the chlorophyll index and aerial part length showed significant differences. In addition, it was determined that root length, fresh weight of foliage, flower, and fruit count increased 82 days after inoculation (dai). Chitinase activity induced by Bb 1205 in leaves and roots of tomato plants infected with Fol 17108 was observed when injected into the stem at 32 dai (41.8 and 11.6-fold, respectively). Inoculation on the foliage showed a 10-fold increase of β-1,3-glucanases in the roots after 82 dpi. As for leaves, a 3.8-fold increase was found when the stem was inoculated. In the different in vivo applications, Bb 1205 activated its defenses by expressing the chitinase enzymes and β-1,3-glucanase, thus reducing the damage caused by Fol 17108, demonstrating increase plant growth thereafter.     

<i>Insilico</i> Anticancer Peptide Prediction from <i>Curcuma longa</i>

Cancer is the second biggest cause of death globally, and the use of therapeutic peptides to specifically target and destroy cancer cells has gotten much interest. Cancer peptides or vaccinations are utilized to treat cancer nowadays, apart from chemotherapy, which has significant discomfort, side effects and costly. It is time demanding to identify and predict potential anticancer peptides using computational biology approaches. Thus, 3-D molecular modeling is being used to find possible ACP candidates. In this research, Curcuma longa has predicted peptide sequences were docked on breast cancer receptors and used a molecular docking technique to assess the anticipated peptides’ binding affinities to MHC molecules. A similar approach was utilized to simulate the interactions of the chosen peptide with the TCR. Additionally, the Pep10 LIRQHVASNIGIAKSKIREPIV was examined, and our findings indicated interaction with MHC classes I and II. However, the maximum binding energy was obtained with TCR at 695.61, giving strength through eight hydrogen bonds. Similarly, the Pep20, GAIIGNRKIKLQPHIIIRID, the projected, has the most significant overall binding energy with MHC classes I and II but a lower global E total value with TCR, namely −600.97 kj/Mol, and also four hydrogen bonds. This research could lead to the development of novel anticancer drugs based on the anticancer activity of the Curcuma longa medicinal plant.     

Micropropagation of Daylily (<i>Hemerocallis fulva</i>) from Crown-Tip Explants and Assessment of Somaclonal Variation of <i>in Vitro</i>-Propagated Plants Using SCoT Markers

Determination of the somaclonal variation of in vitro-propagated plants is crucial to determine the appropriate micropropagation protocol and growth regulators for commercial scale multiplication. In this research, nine multiplication media (MM) augmented with different concentrations of 6-benzyl adenine (BA), Kinetin (Kin), and Thidiazuron (TDZ), Three rooting media (RM) supplemented with three levels of α-naphthalene acetic acid (NAA) and three types of soil mixtures (v/v); Coco peat/Vermiculite/Sand (CVS), Peat moss/Perlite/Sand (PPS) and Peat moss/Perlite (PP) were used in the micropropagation protocol of daylily plants. MM2 showed the maximum shoot length and the number of leaves, while MM9 showed the maximum number of shoots. The RM1 showed the maximum root length and the number of roots. During acclimatization, CVS, PPS, and PP soil mixture showed similar performance except the CVS mixture showed lower performance regarding plant height and diameter. The genetic fidelity of micropropagated plants was evaluated using Start Codon Targeted (SCoT) Markers. Six SCoT primers amplified 51 scorable bands with an approximate range from 146 bp to 1598 bp size. Thirty one out of 51 loci were presented in the mother plants. 40 loci were polymorphic, 11 were monomorphic and 7 were unique. The amplification patterns of the micropropagated plants demonstrated genetic integrity to the mother plant ranging from 84.32 to 47.06 and somaclonal variations ranging from 52.94 with 5 mg/l BA pathway to 15.68 with 1mg/l TDZ pathway, thus demonstrating that the homogeneity and the variation of the micropropagated plants affected by the type and the quantity of the plant growth regulator used during multiplication subcultures. This research can be successfully used for other ornamental and medicinal plants’ bulk multiplication, germplasm conservation, and future genetic improvement.     

<i>In vitro</i> Germination and Micropropagation of <i>Aconitum vilmorinianum</i>: An Important Medicinal Plant in China

Aconitum vilmorinianum, a well-known traditional Chinese herb, is recently being threatened by overexploitation and environment disturbance. This study was conducted to provide propagation methods through in vitro germination and explant cultivation. Germination was stimulated up to 66.00% on Murashige and Skoog (MS) medium containing 2.0 mg L⁻¹ 6-benzylaminopurine (BAP), 0.1 mg L⁻¹ 1-napthaleneacetic acid (NAA), and 30 g L⁻¹ sucrose. Three bacteria (Pantoea agglomerans, Erwinia persicina, and Pseudomonas tolaasii) would be responsible for consistent contamination during germination. The latter two were effectively eradicated after disinfected. The influence of explant types and hormone combinations on direct and indirect organogenesis was evaluated in the present work. The frequency of shoot induction from axillary bud explants was 100% on the MS fortified with 2.0 mg L⁻¹ BAP and 0.3 mg L⁻¹ NAA. Shoots multiplication was optimized on MS medium supplemented with 0.1 mg L⁻¹ thidiazuron (TDZ) and 0.1 mg L⁻¹ NAA. High callus induction percentage (96.67%) was obtained from stem segments on MS medium with 2.0 mg L⁻¹ 2,4-D, then successfully regenerated into shoots on MS medium in the presence of 0.1 mg L⁻¹ TDZ and 0.2 mg L⁻¹ NAA. The present work could be useful for the utilization and conservation of this valuable species.     

Cloning and Characterization of <i>EuGID1</i> in <i>Eucommia ulmoides</i> Oliver

Gibberellic acid controlled the key developmental processes of the life cycle of landing plants, and regulated the growth and development of plants. In this study, a novel gibberellin receptor gene EuGID1 was obtained from Eucommia ulmoides Oliver. The cDNA of EuGID1 was 1556 bp, and the open reading frame was 1029 bp, which encoded 343 amino acids. EuGID1 had the homology sequence with the hormone-sensitive lipase family. Amino acid sequence alignment confirmed EuGID1 protein had the highest homology with the GID1 protein of Manihot esculenta. EuGID1 was located in the nucleus and cell membrane and had expression in four plant organs. Overexpression of EuGID1 in transgenic Arabidopsis plants promoted plant elongation and increased siliques yield.     

<i>Arthrobacter agilis</i> UMCV2 accelerates growth of <i>Pinus devoniana</i>

Pinus devoniana is one of the most widely distributed species of Pinus in Mexico, and has a relevant economic and ecological importance. In this work, the effect of inoculating juvenile P. devoniana plants with the rhizobacteria Arthrobacter agilis UMCV2, and its dimethylhexadecylamine compound was studied under greenhouse conditions. Our results showed that A. agilis UMCV2 promoted growth of P. devoniana as a result of increases on height and stem diameter, fresh weight and chlorophyll concentrations. Under our experimental conditions, the bacterial dimethylhexadecylamine compound produced an increase in the concentration of chlorophyll. These data show the feasibility of using A. agilis UMCV to significantly enhance the growth rate of P. devoniana at a greenhouse scale.     

Effects of <i>Piriformospora indica</i> on the Respiration of <i>Taxus chinensis</i> var. <i>mairei</i> under Water Stress

Seedlings of Taxus chinensis var. mairei were used as experimental materials to study the adaptation of Piriformospora indica to this plant under water stress. The materials were divided into two groups, namely, with or without inoculation with P. indica. Each group was subjected to four different levels of water stress. Vitality and physiological and biochemical indexes of the roots of T. chinensis var. mairei were regularly measured. Under water stress, T. chinensis var. mairei had significantly decreased root vitality; root vitality was higher in inoculated roots than in uninoculated roots. Under intense water stress, the inoculated roots had a higher soluble sugar content than the uninoculated roots. Under water stress, T. chinensis var. mairei experienced decreased activity of aerobic respiratory metabolic enzymes. The activity of anaerobic respiratory metabolic enzymes and alcohol dehydrogenase initially increased and then decreased, whereas that of lactate dehydrogenase increased. The inoculated roots had a higher activity of respiratory metabolic enzymes than the uninoculated roots. As water stress was further intensified, the roots had significantly decreased activity of aerobic respiratory metabolic enzymes and significantly increased activity of anaerobic respiratory metabolic enzymes. The activity of respiratory metabolic enzymes decreased faster in the uninoculated roots than in the inoculated roots. This study demonstrated that Piriformospora indica plays a positive role in enhancing the antihypoxic ability of T. chinensis var. mairei, thereby alleviating plant damage due to water stress.     

<i>Trichoderma</i> spp. fostering growth on <i>Capsicum chinense</i> Jacq. seedlings and antagonistic against <i>Meloidogyne incognita</i>

Fourteen native strains of Trichoderma spp. from wildand agricultural pathosystems in the state of Yucatan, Mexico, with growth-promoting ability of Capsicum chinense Jacq. seedlings were evaluated and antagonistic effect of their filtrate against second-stage juveniles (J2) of Meloidogyne incognita. The strains Th05-02 and Th27-08 showed the best significant effects on plant hight variable increments 55.57 and 47.62%, theTh07-04 with 29.48% more root length, theTh02-01 and Th07-04 isolates increased from 48.71 to 84.61% in volume radical and 53.40% of total dry biomass. Statistical analysis (p≤0.001) of Th43 and Th43-13-14 filtrates caused 100% mortality at 24 and 48h. In the test of reversibility to 24 h after replacing the filtrates Th43-13, Th43-14, TH09-06 and TH20-07 by sterile distilled water, the J2 did not recover their viability, so they were considered as the best potential strains of Trichoderma spp. with antagonistic capacity in J2 of M.incognita.     

Antibacterial Activity and Mechanisms of Ethanol Extracts from <i>Scutellaria baicalensis</i> Georgi and <i>Magnolia officinalis</i> against <i>Phytophthora nicotianae</i>

Phytophthora nicotianae causes substantial economic losses in most countries where tobacco is produced. At present, the control of P. nicotianae mainly depends on chemical methods, with considerable environmental and health issues. We investigated the effects of ethanol extracts from Scutellaria baicalensis Georgi (SBG) and Magnolia officinalis (MO). On mycelial growth, sporangium formation, and zoospore release of P. nicotianae. Both extracts inhibited the growth of P. nicotianae, with mycelial growth inhibition rates of 88.92% and 93.92%, respectively, at 40 mg/mL, and EC50 values of 5.39 and 5.74 mg/mL, respectively. The underlying mechanisms were the inhibition of sporangium formation, the reduction of zoospore number, and the destruction of the mycelium structure. At an SBG extract concentration of 16.17 mg/mL, the inhibition rates for sporangia and zoospores were 98.66% and 99.39%, respectively. At an MO extract concentration of 2.87 mg/mL, the production of sporangia and zoospores was completely inhibited. The hyphae treated with the two plant extracts showed different degrees of deformation and damage. Hyphae treated with SBG extract showed adhesion and local swelling, whereas treatment with MO extract resulted in broken hyphae. Mixture of the extracts resulted in a good synergistic effect.     

<i>In vitro</i> Mutagenesis for the Improvement of Agave Genus

Biotechnological techniques provide a viable alternative to help improve and increase the production of plant species of agricultural and economic importance, which have been affected over the years by climate change, increasing their susceptibility to pests and/or diseases, generating losses in production as well as a decrease in their regenerative and genetic diversity. The application of biotechnological techniques such as in vitro mutagenesis offers a viable option for the generation of crops that are resistant to the different factors caused by abiotic and biotic stress. In vitro mutagenesis has been used in an efficient way to generate genetic changes in different plant species. However, these methods have not been studied thoroughly in crops of agro-industrial interest, such as agave, which represents an economic resource of national importance and are considered as endemic species of Mexico. Therefore, this literary review aimed to focus on the studies that have been used for the genetic improvement of this species via mutagenesis techniques in plants in the agave genus. Therefore, the objective was to set a precedent for future genetic studies that aim to obtain more productive regenerants for various industries, such as food and pharmaceutical. It is also of great interest to compile information from basic research that helps understand and elucidate a model of possible defense mechanisms that are activated in the Agave genus.