Wound Healing In Potato Tubers Research Articles

Altered Metabolism in Knockdown Lines of Two HXXXD/BAHD Acyltransferases During Wound Healing in Potato Tubers.

Suberin biosynthesis involves the coordinated regulation of both phenolic and aliphatic metabolisms. HXXXD/BAHD acyltransferases occupy a unique place in suberization, as they function to crosslink phenolic and aliphatic monomers during suberin assembly. To date, only one suberin-associated HXXXD/BAHD acyltransferase, StFHT, has been described in potatoes, whereas, in Arabidopsis, at least two are implicated in suberin biosynthesis. RNAseq data from wound-induced potato tubers undergoing suberization indicate that transcripts for 28 HXXXD/BAHD acyltransferase genes accumulate in response to wounding. In the present study, we generated RNAi knockdown lines for StFHT and another highly wound-induced HXXXD/BAHD acyltransferase, designated StHCT, and characterized their wound-induced suberin phenotype. StFHT-RNAi and StHCT-RNAi knockdown lines share the same aliphatic suberin phenotype of reduced esterified ferulic acid and ferulates, which is similar to the previously described StFHT-RNAi knockdown suberin phenotype. However, the phenolic suberin phenotype differed between the two knockdown genotypes, with StHCT-RNAi knockdown lines having proportionately more p-hydroxyphenyl-derived moieties than either StFHT-RNAi knockdown or empty vector control lines. Analysis of soluble polar metabolites revealed that StHCT catalyzes a step upstream from StFHT. Overall, our data support the involvement of more than one HXXXD/BAHD acyltransferase in potato suberin biosynthesis.

The combination treatment of low voltage electrostatic field and Ultraviolet-C could accelerate the process of wound healing of potato tubers

Low voltage electrostatic field (LVEF) and ultraviolet-C (UV–C) are non-thermal, safe, and environmentally friendly preservation technologies. This study explored the efficacy of LVEF, UV-C (0.1 kJ m−2), and their combination (LVEF + UV-C) in regulating the wound-healing response of injured potato tubers over a 21-day healing period at 20 °C. Results demonstrated that LVEF + UV-C treatment showed superior efficacy in accelerating wound healing of tubers compared to single-treatment and control groups, mitigating weight loss and disease index, promoting suberin and lignin accumulation, and enhancing firmness, browning index, and H2O2 content. Biochemical changes observed during tuber healing exhibited a positive correlation with increased enzyme activities (PAL, 4CL, CAD, POD, and PPO) and metabolite levels (phenolic compounds, flavonoids, lignin) associated with phenylpropane metabolism. Our study findings suggest that the application of LVEF + UV-C stimulates phenylpropanoid metabolism, accelerates suberin and lignin accumulation at wounds, thus facilitates wound healing in potato tubers.

Modulatory role of nitric oxide in wound healing of potato tubers

IntroductionWounding of potato (Solanum tuberosum L.) tubers affects the postharvest storage qualities and marketability. To mitigate wound-related losses, it is necessary to accelerate wound healing (WH) responses of potato tubers. While role of nitric oxide (NO) in healing of wounded biological tissues is well known, its impact on WH responses of potato tubers has not been examined thoroughly. The aim of this study was to elucidate the potential modulatory role of NO in WH response of potato tubers by determining the activity of key enzymes associated with NO biosynthesis.MethodsUsing a model wounding system, tuber tissue discs were extracted from mini tubers of cv. Russet Burbank to examine their WH responses. Tuber discs were treated with NO related chemicals, such as NO scavenger, nitric oxide synthase (NOS) inhibitor, and NO donors. The effect of these treatments on in vitro production of NO, activity of nitrate reductase, NOS, and phenylalanine ammonia-lyase were determined. The accumulation of suberin polyphenolics (SPP) in wounded parenchyma cells, an indicator of early suberization process, was analyzed histologically.ResultsTreatment of tuber tissues with the higher dose of an NO scavenger (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) at the time of wounding completely ceased SPP accumulation between 0 h to 3 d after wounding. Increased production of NO and enhanced activity of NOS were also observed immediately after wounding of the tuber tissues. The results suggest that NO is a key biological modulator in WH responses of potato tubers and could be optimized as a potential exogenous treatment to counter wound-related losses.

Chitooligosaccharide accelerated wound healing in potato tubers by promoting the deposition of suberin polyphenols and lignin at wounds

Chitooligosaccharide (COS) is a low molecular weight product of chitosan degradation. Although COS induces plant resistance by activating phenylpropanoid metabolism, there are few reports on whether COS accelerates wound healing in potato tubers by promoting the deposition of phenolic acids and lignin monomers at wounds. The results showed that COS activated phenylalanine ammonialyase and cinnamate 4-hydroxylase and promoted the synthesis of cinnamic, caffeic, p-coumaric, ferulic acids, total phenolics and flavonoids. COS activated 4-coumaric acid coenzyme A ligase and cinnamyl alcohol dehydrogenase and promoted the synthesis of sinapyl, coniferyl and cinnamyl alcohols. COS also increased H2O2 levels and peroxidase activity and accelerated the deposition of suberin polyphenols and lignin on wounds. In addition, COS reduced weight loss and inhibited lesion expansion in tubers inoculated with Fusarium sulfureum. Taken together, COS accelerated wound healing in potato tubers by inducing phenylpropanoid metabolism and accelerating the deposition of suberin polyphenols and lignin at wounds.

A Genome-Wide View of the Transcriptome Dynamics of Fresh-Cut Potato Tubers.

Fresh fruits and vegetable products are easily perishable during postharvest handling due to enzymatic browning reactions. This phenomenon has contributed to a significant loss of food. To reveal the physiological changes in fresh-cut potato tubers at the molecular level, a transcriptome analysis of potato tubers after cutting was carried out. A total of 10,872, 10,449, and 11,880 differentially expressed genes (DEGs) were identified at 4 h, 12 h and 24 h after cutting, respectively. More than 87.5% of these DEGs were classified into the categories of biological process (BP) and molecular function (MF) based on Gene Ontology (GO) analysis. There was a difference in the response to cutting at different stages after the cutting of potato tubers. The genes related to the phenol and fatty biosynthesis pathways, which are responsible for enzymatic browning and wound healing in potato tubers, were significantly enriched at 0-24 h after cutting. Most genes related to the enzymatic browning of potato tubers were up-regulated in response to cut-wounding. Plant hormone biosynthesis, signal molecular biosynthesis and transduction-related genes, such as gibberelin (GA), cytokinin (CK), ethylene (ET), auxin (IAA), jasmonic acid (JA), salicylic (SA), and Respiratory burst oxidase (Rboh) significantly changed at the early stage after cutting. In addition, the transcription factors involved in the wound response were the most abundant at the early stage after cutting. The transcription factor with the greatest response to injury was MYB, followed by AP2-EREBP, C3H and WRKY. This study revealed the physiological changes at the molecular level of fresh-cut potato tubers after cutting. This information is needed for developing a better approach to enhancing the postharvest shelf life of fresh processed potato and the breeding of potato plants that are resistant to enzymatic browning.

UV-C irradiation accelerated the deposition of suberin and lignin at wounds by activating phenylpropanoid metabolism in potato tubers

UV-C could directly inactivate pathogens, and reduce postharvest diseases of fruits and vegetables by inducing resistance. To date, no studies have been carried out to verify whether UV-C may accelerate the wound healing of potato tubers. The present results demonstrated that UV-C irradiation up-regulated StPAL, St4CL, and StCAD expression and activated phenylalanine ammonia-lyase (PAL), 4-coumarate: coenzyme A ligase (4CL), and cinnamyl alcohol dehydrogenase (CAD). Meanwhile, UV-C irradiation elevated the levels of four phenolic acids (p-coumaric acid, caffeic acid, sinapic acid, and ferulic acid) and three lignin monomers (coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol), and accelerated the suberin polyphenolic (SPP) and lignin accumulation at wounds, thereby decreasing weight loss and disease index. The weight loss and disease index in the UV-C irradiated tubers were 36.48% and 56.25% lower than that of the control on the 14 d of healing, respectively. Taken together, UV-C irradiation promoted the process of wound healing in potato tubers by stimulating phenylpropanoid metabolism as well as accelerating the SPP and lignin accumulation at wounds. Therefore, UV-C irradiation could be employed as a simple, safe, and low-cost handling technology for accelerating wound healing of potato tubers.

Overexpression of StCDPK23 promotes wound healing of potato tubers by regulating StRbohs

Calcium-dependent protein kinase (CDPK) is a Ca2+ sensor that can phosphorylate and regulate respiratory burst oxidase homolog (Rboh), inducing the production of O2−. However, little is known about how StCDPK23 affects ROS production in the deposition of suberin at potato tuber wounds by regulating StRbohs. In this study, we found that StCDPK23 was induced significantly by the wound in potato tubers, which contains a typical CDPK structure, and was highly homologous to AtCDPK13 in Arabidopsis. Subcellular localization of results showed that StCDPK23 was located in the nucleus and plasma membrane of N. benthamiana epidermis cells. StCDPK23-overexpressing plants and tubers were obtained via Agrobacterium transformation. The expression of StCDPK23 was significantly upregulated in the overexpressing tubers during healing and increased 2.3-fold at 5 d. The expression levels of StRbohs (A-E) were also upregulated in the overexpressing tubers. Among them, StrbohA showed significant expression in the early stage of healing, which was 16.3-fold higher than that of the wild-type tubers at 8 h of healing. Moreover, the overexpressing tubers produced more O2− and H2O2, which are 1.1-fold and 3.5-fold higher than that of the wild-type at 8 h, respectively. More SPP deposition was observed at the wounds of the overexpressing tubers. The thickness of SPP cell layers was 53.2% higher than that of the wild-type after 3 d of the wound. It is suggested that StCDPK23 may participate in the wound healing of potato tubers by regulating Strbohs, which mainly contributes to H2O2 production during healing.

Transcriptome sequencing and differential expression analysis of natural and BTH-treated wound healing in potato tubers (Solanum tuberosum L.)

BackgroundWound healing is a representative phenomenon of potato tubers subjected to mechanical injuries. Our previous results found that benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) promoted the wound healing of potato tubers. However, the molecular mechanism related to inducible wound healing remains unknown.ResultsTranscriptomic evaluation of healing tissues from potato tubers at three stages, namely, 0 d (nonhealing), 5 d (wounded tubers healed for 5 d) and 5 d (BTH-treated tubers healed for 5 d) using RNA-Seq and differentially expressed genes (DEGs) analysis showed that more than 515 million high-quality reads were generated and a total of 7665 DEGs were enriched, and 16 of these DEGs were selected by qRT-PCR analysis to further confirm the RNA sequencing data. Gene ontology (GO) enrichment analysis indicated that the most highly DEGs were involved in metabolic and cellular processes, and KEGG enrichment analysis indicated that a large number of DEGs were associated with plant hormones, starch and sugar metabolism, fatty acid metabolism, phenylpropanoid biosynthesis and terpenoid skeleton biosynthesis. Furthermore, a few candidate transcription factors, including MYB, NAC and WRKY, and genes related to Ca2+-mediated signal transduction were also found to be differentially expressed during wound healing. Most of these enriched DEGs were upregulated after BTH treatment.ConclusionThis comparative expression profile provided useful resources for studies of the molecular mechanism via these promising candidates involved in natural or elicitor-induced wound healing in potato tubers.

Brassinosteroid Accelerates Wound Healing of Potato Tubers by Activation of Reactive Oxygen Metabolism and Phenylpropanoid Metabolism

Wound healing could effectively reduce the decay rate of potato tubers after harvest, but it took a long time to form typical and complete healing structures. Brassinosteroid (BR), as a sterol hormone, is important for enhancing plant resistance to abiotic and biotic stresses. However, it has not been reported that if BR affects wound healing of potato tubers. In the present study, we observed that BR played a positive role in the accumulation of lignin and suberin polyphenolic (SPP) at the wounds, and effectively reduced the weight loss and disease index of potato tubers (cv. Atlantic) during healing. At the end of healing, the weight loss and disease index of BR group was 30.8% and 23.1% lower than the control, respectively. Furthermore, BR activated the expression of StPAL, St4CL, StCAD genes and related enzyme activities in phenylpropanoid metabolism, and promoted the synthesis of lignin precursors and phenolic acids at the wound site, mainly by inducing the synthesis of caffeic acid, sinapic acid and cinnamyl alcohol. Meanwhile, the expression of StNOX was induced and the production of O2− and H2O2 was promoted, which mediated oxidative crosslinking of above phenolic acids and lignin precursors to form SPP and lignin. In addition, the expression level of StPOD was partially increased. In contrast, the inhibitor brassinazole inhibited phenylpropanoid metabolism and reactive oxygen metabolism, and demonstrated the function of BR hormone in healing in reverse. Taken together, the activation of reactive oxygen metabolism and phenylpropanoid metabolism by BR could accelerate the wound healing of potato tubers.

The Interaction Between StCDPK14 and StRbohB Contributes to Benzo-(1, 2, 3)-Thiadiazole-7-Carbothioic Acid S-Methyl Ester-Induced Wound Healing of Potato Tubers by Regulating Reactive Oxygen Species Generation

Reactive oxygen species (ROS) production is essential for both physiological processes and environmental stress in diverse plants. Previous studies have found that benzo-(1, 2, 3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH)-inducible ROS were associated with wound healing of potato tubers. Calcium-dependent protein kinases (CDPKs), the important calcium receptors, are known to play a crucial part in plant development and adaptation to abiotic stresses. However, whether CDPK-mediated ROS generation induced by BTH is involved in wound healing is elusive. In this study, we measured Solanum tuberosum CDPKs (StCDPKs) expression using real-time PCR, and it was found that the transcriptional levels of StCDPKs from BTH-treated tissues were significantly induced, among which StCDPK14 presented the most increased level. Subcellular localization results showed that StCDPK14 is located in the nucleus and membrane. The transgenic potato plants and tubers were developed using interference-expression of StCDPK14 by Agrobacterium tumefaciens–mediated transformation. The St respiratory burst oxidase homologs (StRbohs) expression showed a remarkable decrease in StCDPK14 transgenic tubers, notably, H2O2 content and suberin deposition were also significantly declined. To confirm the relationship between StCDPK14 and StRbohB, yeast-two-hybrid and bimolecular fluorescence complementation were used to examine the interaction, and it was shown that StCDPK14 interacted with the specific Ca2 + -binding motif (helix-loop-helix, called EF-hand) of StRbohB N-terminus. The above results unraveled that StCDPK14 functions in ROS generation via interacting with StRbohB during wound healing of potato tubers.

Candida Oleophila Proliferated and Accelerated Accumulation of Suberin Poly Phenolic and Lignin at Wound Sites of Potato Tubers.

Candida oleophila is a type of biocontrol yeast offering effective postharvest disease control. To the best of our knowledge, the effect of C. oleophila upon the healing of tubers is yet to be studied. The present study addresses the existing knowledge gap by investigating the effect of C. oleophila on wound healing in potato tubers. The results show that C. oleophila colonized and proliferated at the wound sites during the early and intermediate stages of healing. In addition, C. oleophila reduced weight loss of wounded tubers, decreased disease index of inoculated tubers with Fusarium sulphureum, and accelerated accumulation of suberin poly phenolic (SPP) and lignin at wound sites. C. oleophila activated phenylpropanoid metabolism and increased the content of SPP monomers, total phenol, flavonoids, and lignin. Furthermore, the yeast increased H2O2 content as well as peroxidase activity.

Hot water dipping stimulated wound healing of potato tubers

This study examines the effects of hot water dipping on wound healing of harvested potato tubers and to explore partial mechanisms. The potato ‘cv. Longshu No. 7′ was used as materials, which were artificially wounded and then dipped into 45 °C hot water for 10 min. The wounded tubers were stored at ambient temperature (20−25 °C, RH 70–80%) in dark for wound healing. The weight loss of wounded tubers and the disease index of wounded tubers inoculated with Fusarium sulphureum were determined. The accumulation of suberin poly phenolic, suberin poly aliphatic and lignin at wound sites were observed. The activity of phenylalanine ammonia lyase, peroxidase activity, the content of H2O2, total phenols, flavonoids and lignin were also measured. We found that hot water dipping effectively reduced the weight loss and the disease index of wounded potato tubers. The weight loss of treated tubers was 45.1% lower than the control on the 14 d of healing, and the disease index of treated tubers was 18.2% lower than the control on the 21 d of healing. Hot water dipping significantly stimulated the accumulation of suberin poly phenolic, suberin poly aliphatic and lignin at wound sites. The thickness of suberin poly phenolic, suberin poly aliphatic and lignin cell layers of treated tubers were 28.9%, 23.2% and 18.9% higher than the control on the 21 d of healing, respectively. Moreover, hot water dipping significantly enhanced the activity of phenylalanine ammonia lyase and peroxidase. Meanwhile, the H2O2 content, total phenols, flavonoids and lignin were also significantly promoted at wound sites by hot water dipping treatment during healing. These results suggested that hot water dipping promotes wound healing of potato tubers by activating phenylpropanoid metabolism, increasing H2O2 content and peroxidase activity, promoting the suberin and lignin accumulation at wound sites and decreasing the weight loss and the disease index of tubers during healing.

The effect of benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) treatment on regulation of reactive oxygen species metabolism involved in wound healing of potato tubers during postharvest

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) can improve wound healing of potato tubers; however, how the chemical regulates reactive oxygen species (ROS) generation and scavenging during wound healing is not completely understood. BTH at 100 mg·L−1 regulated changes in ROS generation and scavenging in healing tissues of potato tubers. A higher H2O2 content was presented in healing tissues of potato tubers, while cell membrane permeability and malondialdehyde content declined due to BTH treatment. Additionally, the activities and transcript level of enzymes related with ROS generation, including NADPH oxidase, peroxidase and polyamine oxidase, as well as enzymes involved in ROS scavenging, such as superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase, were significantly enhanced by BTH treatment. It is suggested that ROS metabolism might play a crucial role in wound healing of potato tubers mediated by BTH during postharvest.

Biological differences that distinguish the 2 major stages of wound healing in potato tubers

ABSTRACTThe two stages of potato tuber wound healing, closing layer formation (CLF) and wound periderm formation (WPF), have critical biological differences. The first stage, CLF, involves early induction of DNA synthesis and nuclear division in the absence of cell division. The transition phase from CLF to the second stage, WPF, is marked by a transient decrease in expression of suberin-specific genes. The second stage involves cell division. Although biologically active cytokinins (CKs) are not present in quantifiable amounts during this stage, the presence of precursor and catabolic products suggest the presence of trace amounts of active CKs that, in conjunction with increased auxin (indole acetic acid), provide necessary signals for meristematic activity. Augmenting these putative trace amounts with exogenous biologically active CK inhibits WPF; this suggests that the CK requirements for meristematic activity are finely controlled and sensitive to extremely low concentrations. Evidence is discussed for separate biological processes and signals that distinguish the 2 stages of wound healing.

Metal nanoparticles induce germination and wound healing in potato tubers

Metal nanoparticles induce germination and wound healing in potato tubers

Wound‐induced superoxide production and PAL activity decline with potato tuber age and wound healing ability

Wound healing of potato tubers involves the concerted action of several enzymes that facilitate polymerization of phenolics into suberin at the wound site. A decline in the efficiency of healing and resistance to pathogens with advancing tuber age was associated with reduced ability of older tubers to produce superoxide radicals (FRs) in response to wounding. Autophotographs of luminol‐treated longitudinal sections of tissue from 6‐, 18‐ and 30‐month‐old tubers revealed a substantial decline in superoxide production at the wound surface with advancing age. Older tubers were less able to respond to wounding by increasing phenylalanine ammonia lyase (PAL) activity. This enzyme produces t‐cinnamic acid, which constitutes a component of the phenolic domain of suberin, and is normally induced by wounding and/or ethylene. Interestingly, the ability of wounded tissue to oxidize exogenous 1‐aminocyclopropane‐1‐carboxylic acid (ACC) to C2H4 also decreased with advancing tuber age. The oxidation of ACC was inhibited by the FR scavenger, n‐propyl gallate (PG), and inhibition was greatest in tissue from younger tubers, reflecting their greater ability to produce superoxide radicals upon wounding. Regardless of tuber age, 1‐aminocyclobutane‐1‐carboxylic acid, an ACC oxidase inhibitor, did not inhibit C2H4 generation from exogenous ACC. Hence, C2H4 production from ACC by wounded tuber tissue is largely non‐enzymatic and FR‐driven, and thus serves as an indicator of the ability of wounded tissue to produce superoxide. Age‐induced reduction in PAL activity and FR production at the wound surface probably limited the oxidative polymerization of phenolics into suberin during wound periderm formation. The age‐induced loss in ability of wounded tissue to heal and resist pathogens is thus consistent with reduced synthesis and polymerization of phenolic adducts into suberin, a consequence of reduced FR and PAL activity at the wound surface.

Lignin biosynthesis during wound healing of potato tubers in response to gamma irradiation

Abstract The effect of gamma irradiation on lignin biosynthesis during wound healing in potato tubers was studied by [U- 14 C] phenylalanine incorporation into lignin and monitoring the activities of key enzymes involved in the lignification process. There was a 40% reduction in lignin biosynthesis during wound healing in response to gamma irradiation. The level of the first enzyme involved in lignin biosynthesis, phenylalanine ammonia lyase (PAL), was five-fold higher in irradiated potatoes than in control tubers during wound healing. However the level of another key enzyme, cinnamyl alcohol dehydrogenase (CAD) was found to be 30% less in irradiated potatoes. There was no significant change in peroxidase activity and its isozyme pattern during wound healing. Differential regulation of different enzymes of lignin biosynthesis in response to gamma irradiation may be responsible for the decreased lignin biosynthesis.

Biochemical Characterization of the Suberization-Associated Anionic Peroxidase of Potato

The anionic peroxidase associated with the suberization response in potato (Solanum tuberosum L.) tubers during wound healing has been purified and partially characterized at the biochemical level. It is a 45-kD, class III (plant secretory) peroxidase that is localized to suberizing tissues and shows a preference for feruloyl (o-methoxyphenol)-substituted substrates (order of substrate preference: feruloyl > caffeoyl > p-coumaryl approximately syringyl) such as those that accumulate in tubers during wound healing. There was little influence on oxidation by side chain derivatization, although hydroxycinnamates were preferred over the corresponding hydroxycinnamyl alcohols. The substrate specificity pattern is consistent with the natural substrate incorporation into potato wound suberin. In contrast, the cationic peroxidase(s) induced in response to wound healing in potato tubers is present in both suberizing and nonsuberizing tissues and does not discriminate between hydroxycinnamates and hydroxycinnamyl alcohols. A synthetic polymer prepared using E-[8-(13)C]ferulic acid, H(2)O(2), and the purified anionic enzyme contained a significant amount of cross-linking through C-8, albeit with retention of unsaturation.

S-Carvone Inhibits Phenylalanine Ammonia Lyase (PAL) Activity and Suberization During Wound Healing of Potato Tubers

Summary The periderm formation of wound tissue of potato tuber was inhibited temporarily after exposure to S-carvone. In wound tissue of control tubers a suberized cell layer was visible after 4 days whereas in S-carvone treated tissue this was observed only after 14 days in most of the tubers. At day 21 all the S-carvone treated tubers had formed suberin but only 10 % of the tubers had developed a (begin of a) cambium layer. The appearance of suberin was related to the activity of phenylalanine ammonia lyase (PAL); in control and S-carvone treated tissue, maximum PAL activity preceded the appareance of the first suberized cell layers by 2 and 5 days, respectively. The specific activity reached the same level in both control and S-carvone treated wound tissue. A short term treatment (2, 4 and 7 days) of tuber wound tissue with S-carvone delayed suberin formation. All the treated tubers showed suberization about 4 days after removal of S-carvone and this was closely related to the increase in PAL-activity. After the S-carvone treatment, the glutathione metabolism increased: after an initial decrease, as observed in the control, the total glutathione content increased fourfold after 9 days and remained high during the treatment. However, the length of the wound healing period before the start of a S-carvone treatment was of great influence on the increase of the glutathione metabolism and on the induction of glutathione reductase.

Vaughn and Lulai: Suberin stains comparison of fluorescent stains for the detection of suberin in potato periderm

Several fluorescent staining procedures to detect suberin in normal periderm of potato were assessed. Among the stains tested, treatment with the alkaloid fluorochrome berberine followed by counterstaining with crystal violet appears to be the most sensitive combination. Crystal violet increased contrast by quenching lignin fluorescence which is also present in the periderm cell walls. This technique may be useful for studying the role of suberin in disease resistance and wound healing of potato tubers and other tissues.