Treatment Of Plantlets Research Articles

Crosstalk between H2O2 and Ca2+ signaling is involved in root endophyte-enhanced tanshinone biosynthesis of Salvia miltiorrhiza

Tanshinones are bioactive ingredients derived from the herbal plant Salvia miltiorrhiza and are used for treating diseases of the heart and brain, thus ensuring quality of S. miltiorrhiza is paramount. Applying the endophytic fungus Trichoderma atroviride D16 can significantly increase the content of tanshinones in S. miltiorrhiza, but the potential mechanism remains unknown. In the present study, the colonization of D16 effectively enhanced the levels of Ca2+ and H2O2 in the roots of S. miltiorrhiza, which is positively correlated with increased tanshinones accumulation. Further experiments found that the treatment of plantlets with Ca2+ channel blocker (LaCl3) or H2O2 scavenger (DMTU) blocked D16-promoted tanshinones production. LaCl3 suppressed not only the D16-induced tanshinones accumulation but also the induced Ca2+ and H2O2 generation; nevertheless, DMTU did not significantly inhibit the induced Ca2+ biosynthesis, implying that Ca2+ acted upstream in H2O2 production. These results were confirmed by observations that S. miltiorrhiza treated with D16, CaCl2, and D16+LaCl3 exhibit H2O2 accumulation and influx in the roots. Moreover, H2O2 as a downstream signal of Ca2+ is involved in D16 enhanced tanshinones synthesis by inducing the expression of genes related to the biosynthesis of tanshinones, such as DXR, HMGR, GGPPS, CPS, KSL and CYP76AH1 genes. Transcriptomic analysis further supported that D16 activated the transcriptional responses related to Ca2+ and H2O2 production and tanshinones synthesis in S. miltiorrhiza seedlings. This is the first report that Ca2+ and H2O2 play important roles in regulating fungal-plant interactions thus improving the quality in the D16-S. miltiorrhiza system.

Calcium-Dependent Changes in Cellular Redox Homeostasis and Heat Resistance of Wheat Plantlets under Influence of Hemin (Carbon Monoxide Donor)

Carbon monoxide (CO) is considered to be an important molecule-gasotransmitter involved in the regulation of the functional activity of plants, including in the processes of adaptation to stress factors. However, the associations of CO with other participants of signaling in the plant cells remain poorly studied. Using an inhibitory method, the role of different calcium pools in realization of the influence of hemin (carbon monoxide donor) on generation and neutralization of reactive oxygen species (ROS) in the root cells of wheat (Triticum aestivum L.) plantlets and their resistance to damaging heating (45°C, 10 min) was studied. The treatment of plantlets with 5 μM hemin caused a transient increase in the activity of extracellular peroxidase in roots and an increase in the generation of ROS with the maximum in 1.5–2 h after the beginning of treatment. The chelator of extracellular calcium EGTA and the inhibitor of inositol-1,4,5-phosphate formation neomycin, which reduces calcium influx into the cytosol from intracellular compartments, almost completely eliminated an increase in the activity of extracellular peroxidase caused by exogenous CO. At the same time, EGTA (completely) and neomycin (partially) leveled an increase in the content of hydrogen peroxide in the roots of plantlets occurring under the influence of CO donor. The treatment of plantlets with hemin also induced an increase in the activity of superoxide dismutase, catalase, and intracellular peroxidase in roots. Both calcium antagonists eliminated these effects. In the presence of EGTA and neomycin, there was also no positive effect of the treatment of hemin on the state of biomembranes and survival of plantlets after the damaging heating. It was concluded that both extracellular calcium and that deposited in intracellular compartments is involved in CO donor-induced increase in the formation of ROS, induction of the antioxidant system, and development of heat resistance of wheat plantlets.

State of enzymatic antioxidative system and heat resistance of wheat plantlets treated by combination of 24-epibrassinolide and NO donor

The influence of 24-epibrassinolide (24-EBL) and nitric oxide donor sodium nitroprusside (SNP) on the state of antioxidative system and heat resistance of wheat (Triticum aestivum L.) plantlets has been investigated. The treatment with 24-EBL (20–200 nM) or with SNP (0.1–2.0 mM) increased the survival of plantlets after 10-min damaging heating at 46 °C in the water thermostat. The cotreatment with 20 nM of 24-EBL and 0.2 mM of SNP caused more essential protective influence in comparison with the treatment by each compound separately. At the same time, joint influence of high concentrations (200 nM of 24-EBL and 2 mM of SNP) reduced the heat resistance of plantlets. Under the influence of 24-EBL and SNP in concentrations that have protective effect, as well as their combinations, the activity of superoxide dismutase (SOD) in roots increased. After damaging heating, the highest value of SOD activity remained in the variant with cotreatment of plantlets with 20 nM of 24-EBL and 0.2 mM of SNP. The activity of catalase and guaiacol peroxidase (GPX) in plantlets roots increased under the influence of various concentrations of SNP and its combinations with 24-EBL. At the same time, 24-EBL alone did not exert the impact on the catalase activity and caused the decrease in GPX activity. The activity of these enzymes decreased in all variants of experiment after heating. The heat stress caused the increase in the content of lipid peroxidation product malonic dialdehyde. The treatment of plantlets with 24-EBL and also its combination with SNP in low concentrations removed this effect, but in high concentrations, on the contrary, increased it. The conclusion was made that intensifying of protective influence of 24-EBL and SNP on wheat plantlets at their combined application in low concentrations is substantially caused by stabilization of pro-/antioxidative balance.

Participation of Hydrogen Sulfide in Induction of Antioxidant System in Roots of Wheat Plantlets and Their Heat Resistance by Salicylic Acid

The possible role of hydrogen sulfide as a signal mediator in the induction of heat resistance of wheat (Triticum aestivum L.) plantlets by salicylic acid (SA) has been studied. The treatment of plantlets with SA (1 and 10 μM) or a hydrogen sulfide donor (NaHS, 0.1 and 0.25 mM) increased their resistance to damaging heating (10 min at 45°C). Under the influence of SA, a transient increase in the hydrogen sulfide content occurred in roots with the maximum effect in 2–3 h after the start of treatment. The treatment of roots with SA increased the activity of superoxide dismutase (SOD), catalase, and guaiacol peroxidase in them. Under the influence of the hydrogen sulfide donor NaHS, the activity of SOD and catalase increased significantly. Also, the treatment of roots with SA and NaHS reduced the effect of the accumulation of the product of lipid peroxidation, malondialdehyde, which is caused by heating. The inhibitors of hydrogen sulfide synthesis, hydroxylamine and potassium pyruvate, partially eliminated the effects of the increase in the activity of antioxidant enzymes and the development of heat resistance caused by SA. At the same time, the combined treatment of intact plantlets with 10 μM SA and 0.1 mM NaHS contributed to the additional increase in the activity of antioxidant enzymes and increased plantlet survival after heating. It is concluded that hydrogen sulfide participates in the implementation of the protective influence of SA on wheat plantlets under heat stress.

Productive potential of cassava plants (Manihot esculenta Crantz) propagated by leaf buds.

New techniques of rapid multiplication of cassava (Manihot esculenta Crantz) have been developed, requiring technical support for large-scale use. This work main to evaluate the agronomic performance of plantlets obtained by leaf buds technique against stem cuttings in the field conditions. The work was conducted using the randomized block design in a factorial scheme with 3 varieties (BRS Kiriris, 98150-06, 9624-09) × 4 origins of the plantlets (conventional - stem cuttings of 20 cm length, leaf buds of the upper, middle and inferior stem part) × 2 agrochemicals (control and treated). There was a remarkable decrease in some agronomic traits that ranged from 23% (number of branches) to 62% (shoot weight) when using leaf buds plantlets. The treatment of plantlets with agrochemicals promoted significant increases in all traits, ranging from 26% (number of roots per plant) to 46% (shoot weight). The plantlets originating from leaf buds of the upper and middle parts were able to generate stem-like plants similar to stem-derived ones. Despite its lower agronomic performance under field conditions, multiplication by leaf buds may generate five times the number of propagules in comparison with the conventional multiplication, and therefore it could be a viable alternative for rapid cassava multiplication.

SIGNAL MEDIATORS AT INDUCTION OF HEAT RESISTANCE OF WHEAT PLANTLETS BY SHORT-TERM HEATING.

The effects of functional interplay of calcium ions, reactive oxygen species (ROS) and nitric oxide (NO) in the cells of wheat plantlets roots (Triticum aestivum L.) at the induction of their heat resistance by a short-term influence of hyperthermia (heating at the temperature of 42 degrees C during 1 minute) have been investigated. The transitional increase of NO and H2O2 content, invoked by heating, was suppressed by the treatment of plantlets with the antagonists of calcium EGTA (chelator of exocellular calcium), lanthanum chloride (blocker of calcium channels of various types) and neomycin (inhibitor of phosphatidylinositol-dependent phospholipase C). The rise of hydrogen peroxide content, caused by hardening, was partially suppressed by the action of inhibitors of nitrate reductase (sodium wolframate) and NO-synthase (N(G)-nitro-L-arginine methyl ester--L-NAME), and the increasing of nitric oxide content was suppressed by the treatment of plants with the antioxidant ionol and with the scavenger of hydrogen peroxide (dimethylthiourea). These compounds and antagonists of calcium also partially removed the effect of the rise of plantlets' heat resistance, invoked by hardening heating. The conclusion on calcium's role in the activation of enzymatic systems, generating reactive oxygen species and nitric oxide, and on the functional interplay of these signal mediators at the induction of heat resistance of plantlets by hardening heating is made.

Enhanced salt-induced antioxidative responses involve a contribution of polyamine biosynthesis in grapevine plants

The possible involvement of polyamines in the salt stress adaptation was investigated in grapevine (Vitis vinifera L.) plantlets focusing on photosynthesis and oxidative metabolism. Salt stress resulted in the deterioration of plant growth and photosynthesis, and treatment of plantlets with methylglyoxal-bis(guanylhydrazone) (MGBG), a S-adenosylmethionine decarboxylase (SAMDC) inhibitor, enhanced the salt stress effect. A decrease in PSII quantum yield (Fv/Fm), effective PSII quantum yield (Y(II)) and coefficient of photochemical quenching (qP) as well as increases in non-photochemical quenching (NPQ) and its coefficient (qN) was observed by these treatments. Salt and/or MGBG treatments also triggered an increase in lipid peroxidation and reactive oxygen species (ROS) accumulation as well as an increase of superoxide dismutase (SOD) and peroxidase (POX) activities, but not ascorbate peroxidase (APX) activity. Salt stress also resulted in an accumulation of oxidized ascorbate (DHA) and a decrease in reduced glutathione. MGBG alone or in combination with salt stress increased monodehydroascorbate reductase (MDHAR), SOD and POX activities and surprisingly no accumulation of DHA was noticed following treatment with MGBG. These salt-induced responses correlated with the maintaining of high level of free and conjugated spermidine and spermine, whereas a reduction of agmatine and putrescine levels was observed, which seemed to be amplified by the MGBG treatment. These results suggest that maintaining polyamine biosynthesis through the enhanced SAMDC activity in grapevine leaf tissues under salt stress conditions could contribute to the enhanced ROS scavenging activity and a protection of photosynthetic apparatus from oxidative damages.

Glycyrrhizin production by in vitro cultured Glycyrrhiza glabra elicited by methyl Jasmonate and salicylic acid

Licorice (Glycyrrhiza glabra L. var. glabra, Fabaceae) is considered as a model plant synthesizing triterpenoid secondary compounds. It is known that glycyrrhizin is accumulated in thickened intact licorice roots. The effects of methyl jasmonate (MeJa) and salicylic acid (SA) on plant growth and production of glycyrrhizin in the roots of in vitro cultured 65-day-old plants were studied. Increasing amounts of glycyrrhizin in the roots treated with MeJa inhibited root growth, while SA increased the amount of glycyrrhizin without negative effects on growth. Treatment of plantlets with 0.1–2 mM MeJa and 0.1 and 1 mM SA enhanced the production of glycyrrhizin by 3.8 and 4.1 times, respectively, as compared to the controls. Results support the hypothesis that production of glycyrrhizin is related to a defense response system of the licorice.

Regulation of growth of Lilium plantlets in liquid medium by application of paclobutrazol or ancymidol, for its amenability in a bioreactor system: growth parameters

Effect of growth retardants (paclobutrazol or ancymidol) was studied in Lilium plantlets growing in liquid culture. A significant increase in leaf chlorophyll, epicuticular wax, plant dry weight and bulb starch contents were found in plantlets treated with growth retardants. A similar increase in the number of leaves, roots and bulbs was also noted. However, total leaf area and the fresh weight increased only marginally. These features resulted in robust plantlets that showed significantly improved ex vitro survival. Based on these features, a comprehensive index (CI) was calculated as a measure of quality of the plantlets, and it correlated well with their ex vitro survival. Treatment of plantlets with 3.4 microM paclobutrazol was found to be the best and its carry over effects were also minimal.

Shoot water status and ABA responses of transgenic hybrid larch Larix kaempferi x L. decidua to ectomycorrhizal fungi and osmotic stress

It has been postulated that osmotic effects on plant tissue are mediated by abscisic acid (ABA). Hybrid larch (Larix kaempferi (Lambert) Carr. x L. decidua Mill.) plantlets, transformed with the ABA-inducible wheat Em promoter associated with the Gus reporter gene, were axenically inoculated with two ectomycorrhizal fungi: Cenococcum geophilum Fr., considered tolerant to water stress, and Laccaria bicolor (Marie) Orton, considered less tolerant to drought. The mycorrhizal and non-mycorrhizal transgenic plantlets were subjected to osmotic stress by adding polyethylene glycol (PEG) to the culture medium. In the presence of PEG, L. bicolor and C. geophilum reduced shoot water potential and turgor potential, but increased host osmotic potential. Treatment of plantlets with PEG induced a significant increase in endogenous ABA concentrations. Laccaria bicolor and C. geophilum behaved similarly and significantly decreased the ABA response of plantlets to PEG treatment. Moreover, inoculation with either fungus regulated the ABA response of the plantlets even when the fungus was separated from the host by a cellophane sheet that prevented mycorrhiza formation. Although the wheat Em promoter was inducible in larch plantlets, it was not regulated by endogenous ABA. Induction of the wheat Em promoter in larch plantlets depended on organ type, with maximum induction in the root apex. Induction of the Em promoter was significantly decreased by mycorrhizal inoculation.

Morphology of potato (Solanum tuberosum L. cv. Sante) stem node cultures in relation to the level of endogenous cytokinins

Stem node culture of the potato (Solanum tuberosum) cv. Sante was used to examine the phenotypical alterations due to different levels of endogenous cytokinins. The altered phenotype, which dramatically deviates from the control phenotype, was induced after treatment of plantlets with 1 μm jasmonic acid. Plantlets grown on the medium supplemented with jasmonic acid were taller, with well developed root systems, expanded leaves, thickened stems, and they showed hyperhydric symptoms. Their cytokinin content was about half that of the control plantlets. Morphologic characteristics corresponding to transgenic plants that overproduce cytokinins, including release of axillary buds and inhibited rooting, correlated with the high cytokinin levels in control plants.