Hyperhydricity Research Articles

Towards automated detection of hyperhydricity in plant in vitro culture

Hyperhydricity (HH) is one of the most important physiological disorders that negatively affects various plant tissue culture techniques. The objective of this study was to characterize optical features to allow an automated detection of HH. For this purpose, HH was induced in two plant species, apple and Arabidopsis thaliana, and the severity was quantified based on visual scoring and determination of apoplastic liquid volume. The comparison between the HH score and the apoplastic liquid volume revealed a significant correlation, but different response dynamics. Corresponding leaf reflectance spectra were collected and different approaches of spectral analyses were evaluated for their ability to identify HH-specific wavelengths. Statistical analysis of raw spectra showed significantly lower reflection of hyperhydric leaves in the VIS, NIR and SWIR region. Application of the continuum removal hull method to raw spectra identified HH-specific absorption features over time and major absorption peaks at 980 nm, 1150 nm, 1400 nm, 1520 nm, 1780 nm and 1930 nm for the various conducted experiments. Machine learning (ML) model spot checking specified the support vector machine to be most suited for classification of hyperhydric explants, with a test accuracy of 85% outperforming traditional classification via vegetation index with 63% test accuracy and the other ML models tested. Investigations on the predictor importance revealed 1950 nm, 1445 nm in SWIR region and 415 nm in the VIS region to be most important for classification. The validity of the developed spectral classifier was tested on an available hyperspectral image acquisition in the SWIR-region.

Effects of Monochromatic Light on Growth and Quality of Pistacia vera L.

Light-emitting diodes (LEDs) are popular as a light source for in vitro plants because they save energy and allow the morphology of the plant to be altered. The purpose of this study was to show that switching from classical fluorescent light (FL) to LED light can have both beneficial and adverse effects. Pistacia vera plantlets were exposed to FL, monochromatic Blue LED light (B), monochromatic Red LED light (R), and a 1:1 mixture of both B and R (BR). R increased the total weight, shoot length, number of shoots ≥ 1 cm, and proliferation. It also reduced hyperhydricity (HH), but also dramatically increased shoot tip necrosis (STN) and leaf necrosis (LN). B cured plants of HH and STN, but hardly enabled proliferation. It did not solve the problem of LN, but the plants were high in total chlorophyll and carotenoids. BR reduced HH but enabled limited proliferation, high STN, and LN. All three LED treatments reduced HH compared to FL. B induced both high total phenolic and flavonoid content and high DPPH-scavenging activity. These results show that switching from FL to LED can have a significant positive or negative effect on proliferation and quality. This suggests that finding an optimal lighting regimen will take a lot of trial and error.

Nanoparticulas de prata eliminam hiperhidricidade em Lavanda micropropagada

Abstract It is challenging to plant lavender outside of suitable conditions. This economically important plant requires optimal conditions to germinate and produce a high yield. To increase the quality of this plant, tissue culture is utilized. The most problematic aspect of lavender micropropagation is that cytokinin (CK) causes hyperhydricity (HH) during the shooting process. Consequently, this study was conducted to resolve HH in micropropagated lavender plantlets. Different concentrations of silver nanoparticles (AgNPs) were applied in conjunction with 1.0 mg L-1 6-benzylaminopurine (BA). Then, the performance of HH, growth and development, as well as total phenolic (TPC) and total flavonoid (TFC) content, were evaluated. The application of 20 mg L-1 of AgNPs was found to be the optimal method for halting HH. Although shoot proliferation was lower than in BA-supplemented media-grown plants, adding this concentration of AgNPs improved shoot and root quality. An increase in secondary metabolites and antioxidant activity may have caused the remedy of HH.

Critical roles of the activation of ethylene pathway genes mediated by DNA demethylation in Arabidopsis hyperhydricity.

Hyperhydricity (HH) often occurs in plant tissue culture, seriously influencing the commercial micropropagation and genetic improvement. DNA methylation has been studied for its function in plant development and stress responses. However, its potential role in HH is unknown. In this study, we report the first comparative DNA methylome analysis of normal and hyperhydric Arabidopsis thaliana (L.) Heynh. seedlings using whole-genome bisulfite sequencing (BS-seq). We found that the global methylation level decreased in hyperhydric seedlings, and most of the differentially methylated genes were CHH hypomethylated genes. Moreover, the bisulfite sequencing results showed that hyperhydric seedlings displayed CHH demethylation patterns in the promoter of the ACS1 and ETR1 genes, resulting in upregulated expression of both genes and increased ethylene accumulation. Furthermore, hyperhydric seedling displayed reduced stomatal aperture accompanied by decreased water loss and increased phosphorylation of aquaporins accompanied by increased water uptake. While silver nitrate (AgNO3 ) prevented HH by maintained the degree of methylation in the promoter regions of ACS1 and ETR1 and downregulated the transcription of both genes. AgNO3 also reduced the content of ethylene together with the phosphorylation of aquaporins and water uptake. Taken together, this study suggested that DNA demethylation is a key switch that activates ethylene pathway genes to enable ethylene synthesis and signal transduction, which may subsequently influence aquaporin phosphorylation and stomatal aperture, eventually causing HH; thus, DNA demethylation plays a crucial role in HH. These results provide insights into the epigenetic regulation mechanism of HH and confirm the role of ethylene and AgNO3 in hyperhydricity control.

Hypolignification: A Decisive Factor in the Development of Hyperhydricity.

One of the characteristics of hyperhydric plants is the reduction of cell wall lignification (hypolignification), but how this is related to the observed abnormalities of hyperhydricity (HH), is still unclear. Lignin is hydrophobic, and we speculate that a reduction in lignin levels leads to more capillary action of the cell wall and consequently to more water in the apoplast. p-coumaric acid is the hydroxyl derivative of cinnamic acid and a precursor for lignin and flavonoids in higher plant. In the present study, we examined the role of lignin in the development of HH in Arabidopsis thaliana by checking the wild-types (Ler and Col-0) and mutants affected in phenylpropanoid biosynthesis, in the gene coding for cinnamate 4-hydroxylase, C4H (ref3-1 and ref3-3). Exogenously applied p-coumaric acid decreased the symptoms of HH in both wild-type and less-lignin mutants. Moreover, the results revealed that exogenously applied p-coumaric acid inhibited root growth and increased the total lignin content in both wild-type and less-lignin mutants. These effects appeared to diminish the symptoms of HH and suggest an important role for lignin in HH.

Effective reversal of hyperhydricity leading to efficient micropropagation of Dianthus chinensis L.

The online version contains supplementary material available at 10.1007/s13205-021-02645-7.

Role of Biogenic Silver Nanoparticles on Hyperhydricity Reversion in Dianthus chinensis L. an In Vitro Model Culture

Hyperhydricity (HH) is one of the major problems in plant tissue culture. Increased frequency (75%) of HH in the cultures of. Dianthus chinensis L. cultivar telstar scarlet raised from nodal segments on agar (0.85%) gelled, benzyladenine (2.5 µM) supplemented Murashige and Skoog (MS) medium was noticed in the third subculture onwards. To reduce HH and to resume normal growth, the addition of silver nitrate is routinely recommended in the culture medium. However, the impact of biogenic silver nanoparticles (AgNPs) on HH reversion has not been well investigated. In this study, biogenic AgNPs prepared from leaf extract of D. chinensis were used to control HH in D. chinensis cultures. The characterization of bioynthesized AgNPs has been done by UV–Vis spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis, energy-dispersive X-ray spectrum, transmission electron microscopy, and atomic force microscopy. Higher water content or addition of ethylene precursor, ethephon, in culture medium induced plantlet with unhealthy, pale green, glassy shoots (HH). Due to the biological activity of Ag+ ions and water regulating mechanism, AgNPs treatment on hyperhydric explant (4 weeks) resulted in high retroversion coupled with reduced relative water content. Supplementation of 100 µg L−1 AgNPs in MS medium significantly reduced the percentage of HH to 13.3%, in contrast to control (100%). Addition of AgNPs effectively reduced hydrogen peroxide (H2O2) content (50%) characterized by green, healthy shoots with proper stomata in contrast to hyperhydric shoots. The gene expression pattern of 1-aminocyclopropane-1-carboxylase synthase (ACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1) showed reduced expression after the retroversion of microshoots in 100 µg L−1 AgNPs medium compared to hyperhydric shoot. The relative gene expression profile of ACS1 and ACO1 at 100 µg L−1 AgNPs treatment was 9.8 and 1.8-fold over normal shoots (1), respectively. Inter simple sequence repeat (ISSR) analysis on AgNPs directed HH reverted shoots showed genetic stability thus proved safe to adopt this technique to produce true to type plants. Combined manipulation, coordination of water permeability, and anti-ethylene activity of AgNPs were responsible for effective HH reversion. These finding will put forward the future utilization of bioencapsulated AgNPs in plant tissue culture and agriculture practices.

Influence of polyamines on hyperhydricity reversion and its associated mechanism during micropropagation of China pink (Dianthus chinensis L.).

Hyperhydricity (HH) is a physiological disorder that frequently occurs in plant tissue cultures, affecting healthy growth and development of clonal plants. The primary cultures raised in Murashige and Skoog (MS) medium supplemented with 2.5µM N6-benzyladenine (BA) produced normal microshoot (6.3 shoots/ culture) with least HH. However, the third subculture onwards, HH becomes a major problem. The role of ethylene on HH induction through stomatal closure mechanism were proved by the supplementation of ethephon (5µM) in the culture medium containing 2.5µM BA. In the present study, the application of polyamines (putrescine, spermidine, or spermine) to minimize the HH was examined. Supplementation of 5µM spermine in MS medium significantly reduced the percentage of HH to 0.33%, in contrast to control (100%), while a maximum number of healthy reverted shoots (11.0) were observed in 5µM spermidine treatment. The addition of polyamines effectively reduced H2O2 content (50%) characterized by increased chlorophyll content with proper stomatal morphology. The relative gene expression profile of ethylene biosynthesis genes, 1-Aminocyclopropane-1-carboxylase synthase (ACS1) and 1-Aminocyclopropane-1-carboxylic acid oxidase (ACO1) at 5µM spermine added medium was 1.09 and 1.3 over normal (1) or HH cultures (1.93 and 2.53) respectively, andthus directed restoration of normal morphology of shoots. The present finding in brief, forward a novel method to regulate HH in terms of endogenous ethylene by adopting polyamines exposure and the procedure can be applied to many other plants facing similar HH problems.

Trichloroacetate, an inhibitor of wax biosynthesis, prevents the development of hyperhydricity in Arabidopsis seedlings

Arabidopsis seedlings developed severe hyperhydricity (HH) when 0.2% Gelrite was used to solidify the medium instead of 0.7% agar. One mM trichloroacetate (TCA, an inhibitor of wax synthesis) strongly reduced the amount of wax. TCA also strongly increased the permeability of leaves for water as shown by a decrease of the water retention capacity. One mM TCA also fully prevented HH: the visible symptoms of HH were absent and the amounts of water and air in the intercellular spaces were as in normal, nonhyperhydric seedlings. Apparently, the excess of water that caused the symptoms of HH had been removed by the increased cuticular transpiration in the presence of 1 mM TCA.

Hyperhydricity: underlying mechanisms

The apoplast consists of the cell wall continuum and the intercellular spaces. Flooding of the apoplast is the major causative factor of the hyperhydricity (HH) syndrome. Flooding reduces gas exchange by cells. This leads to anaerobiosis and to accumulation within cells of gases like ethylene. The flooding is caused by the ample availability of water in tissue culture and occurs more on liquid medium and Gelritesolidified medium than on agar-solidified medium. Gelrite causes more HH since medium solidified with Gelrite liquefies, probably because of chelating compounds excreted by plant tissues. Root pressure causes translocation of water from the roots to the apoplast. At the same time, the water that has entered the apoplast cannot easily be removed in vitro by transpiration due to the very high relative humidity in the headspace. When the stomata are forced to close by addition of salicylic acid, transpiration is even more impaired. This results in an increase of HH.

Decreasing the Hyperhydricity and Rooting of Scutellariae baicalensis Georgi

For reducing hyperhydric rate and improving rooting percentage of in vitro regenerated shoots from Scutellariae baicalensis Georgi,with the regenerated shoots induced from aseptic seedling stems,we studied the effect of N6-Benzyladenine( 6-BA),sucrose,agar and paclobotrazol( PP333) on hyperhydricity of regenerated shoots in S. baicalensis and the effects of IBA on rooting of regenerated shoots. The hyperhydric rate significantly decreased with lower 6-BA concentration on the medium. The lowest hyperhydric rate and the highest multiplication rate were on the medium containing 0. 2 mg · L~(-1)6-BA. With the sucrose concentration increasing,the hyperhydric rate of S. baicalensis adventitious shoots was reduced,while their multiplication coefficient was also reduced. The best multiplication coefficient in vitro shoots without hyperhydricity were found on the medium containing 25 g·L~(-1)sucrose. On the medium containing 7. 5 g·L~(-1)agar,the hyperhydric rate of S. baicalensis adventitious shoots was the lowest with higher proliferation coefficient. The medium containing PP333 positively affected the hyperhydric occurrence of regenerated shoots. With the increase of PP333 concentration on the medium,the hyperhydric rate was decreased gradually and in vitro shoots were stronger and healthier. The optimal rooting was got from the medium containing 0. 1 mg·L~(-1)IBA,with the rooting rate of100% and seven roots per shoot. The survival rate of rooting plantlets was over 95% after transferred to soil.

In vitro regeneration and hyperhydricity reduction of Lobelia Chinenss Lour

In vitro regeneration and hyperhydricity reduction of Lobelia Chinenss Lour

Influencing factors and structural characterization of hyperhydricity of in vitro regeneration in Brassica oleracea var. italica

Yu, U., Zhao, Y.-Q., Zhao, B., Ren, S. and Guo, Y.-D. 2011. Influencing factors and structural characterization of hyperhydricity of in vitro regeneration in Brassica oleracea var. italica. Can. J. Plant Sci. 91: 159-165. This study examines factors that affect the occurrence of hyperhydric tissue in in vitro cultures of Brassica oleracea variety italica. The anatomy of normal and hyperhydric leaves of plantlets regenerated from the hypocotyls was compared using scanning electron microscopy and transmission electron microscopy. In hyperhydric leaves palisade tissue was absent and the spongy mesophyll displayed large, unorganized intercellular spaces. Hyperhydric leaves had abnormal stomata with deformed guard cells. Significant ultrastructural differences were observed between chloroplasts in normal and hyperhydric leaves. The effects of zeatin, indoleacetic acid, silver nitrate and sucrose on the formation of hyperhydric shoots were studied. Zeatin was the most important factor, followed by sucrose concentration, AgNO3 and indoleacetic acid. The process of hyperhydricity was found to be reversed by increasing the agar concentration and eliminating NH4NO3 from the macro-elements in the MS medium. This is the first report of hyperhydricity in Brassica oleracea, and our study gives a better understanding of the factors that influence hyperhydricity during in vitro regeneration in Brassica crops.

Comparison of Shoot Regeneration on Different Concentrations of Thidiazuron from Shoot Tip Explant of Cowpea on Gelrite and Agar Containing Medium

Cowpea (Vigna unguiculata L. Walp) is an important legume grown all over the world as grain crop, animal fodder, cover crop, gren manure and vegetable. The present study compares effects of agar and gelrite on micropropagation from shoot tip explant of two Turkish cowpea cultivars Akkiz and Karagoz using 0.15, 0.15, 0.35 mg/l Thidiazuron (TDZ), 3 g/l activated charcoal, 2 mg/l yeast extract with and without 1.25 mg/l Polyvinylpyrrolidone (PVP). To overcome problem of endogenic bacterial contaminations, all cultures contained 500 mg/l augmentin and incubated at 24 ± 2o C in 16 h light photoperiod for eight weeks. Therafter, all explants were transferred to MS medium for two weeks for shoot regeneration and elongation under same incubation and photoperiod conditions. The results showed that frequency of shoot regeneration increased with increase in TDZ concentrations in both cultivars on both agar and gelrite gelled medium. Both cultivars showed maximum mean number of shoots per explant in gelrite compared to agar gelled medium. Maximum number of 4.72 and 2.86 shoots per explant were recorded on MS medium containing 0.25 mg/l TDZ in cv. Akkiz and cv. Karagoz respectively. Hyperhydricity was recorded on some regenerated shoots, which was more prominant on agar. Agar gelled medium had greater shoot length compared to gelrite medium in both cultivars. Regenerated shoots were rooted easily on MS medium containing 0.50 mg/l IBA with regeneration of mean number of 4 secondary shoots on cv. Akkiz and 3 on cv. Karagoz. Rooted plantlets were successfully hardened in the growth chamber and subsequently established in the greenhouse; where they flowered and set seeds. The recorded survival rate of the plants was 100%. Plants looked healthy with no visible detecTab. phenotypic variations.