Estimation of the chlorophyll content of micropropagated potato plants using RGB based image analysis

A method has been developed for rapid and non invasive determination of chlorophyll content of leaves of micropropagated potato plants using RGB based image analysis. Among the trichomatic colors, R and G negatively correlated with the chlorophyll content, while a positive correlation was observed with B chromate. Compared to mean brightness value, the use of mean brightness ratio considerably improved the relationship of the tricolors with chlorophyll content. The brightness values and ratios of the primary colors are modeled as linear correlation functions for chlorophyll content. A significant correlation was observed between the model predicted chlorophyll content with the chlorophyll content measured by chlorophyll content meter. Spectral properties such as luminosity and saturation were also found to be negatively correlated with the chlorophyll content. The relationship was improved by combining the mean brightness ratio at B band region with luminosity. The potential of the imaging system in micropropagation has been discussed.     

Development of a digital image analysis method for real-time estimation of chlorophyll content in micropropagated potato plants

The present work describes a digital image analysis method based on leaf color analysis to estimate chlorophyll content of leaves of micropropagated potato plantlets. For estimation of chlorophyll content, a simple leaf digital analysis procedure using a simple digital still camera was applied in parallel to a SPAD chlorophyll content meter. RGB features were extracted from the image and correlated with the SPAD values. None of the mean brightness parameters (RGB) were correlated with the actual chlorophyll content following simple correlation studies. However, a correlation between the chromaticity co-ordinates ‘r’, ‘b’ and chlorophyll content was observed, while co-ordinate ‘g’ was not significantly correlated with chlorophyll content. Linear regression and artificial neural networks (ANN) were applied for correlating the mean brightness (RGB) and mean brightness ratio (rgb) features to chlorophyll content of plantlet leaves determined through a SPAD meter. The chlorophyll content as determined by the SPAD meter was significantly correlated (RMSE = 3.97 and 3.59, respectively, for linear and ANN models) to the rgb values of leaf image analysis. Both the models indicate successful prediction of chlorophyll content of leaves of micropropagated plants with high correlation. The developed RGB-based digital image analysis has the advantage over conventional subjective methods for being objective, fast, non-invasive, and inexpensive. The system could be utilized for real-time estimation of chlorophyll content and subsequent analysis of photosynthetic and hyperhydric status of the micropropagated plants for better ex vitro survival.     

Tissue proliferation condition in micropropagated ericaceous plants

Tissue proliferation (TP) is an abnormal tumor-like growth produced at or near the crown of the plant, but may also be found on aerial plant parts of some genotypes. Basal tumors may or may not be accompanied by proliferation of compact shoots with short internodes and a whorled leaf arrangement. Genera exhibiting TP include Rhododendron, Kalmia and Pieris of the Ericaceae family. Development of TP symptoms in a plant is highly correlated to a history of micropropagation and also to genetic background of the genotype. Similarities of TP symptoms to crown gall caused by Agrobacterium tumefaciens led many to initially believe TP to be a form of crown gall, but all evidence suggests that a pathogen is not involved in the TP disorder. Abnormal lignotuber formation is another possible cause for TP for which little supportive evidence could be developed. An epigenetic condition, possibly cytokinin habituation, is the possible cause of TP that is best supported by the majority of research evidence. It is likely that TP is induced in vitro by adventitious shoot formation, resulting from high cytokinin concentrations used to rapidly proliferate shoots. Some nursery production practices may result in increased TP symptoms development post-propagation. TP is well managed now due to greater awareness and adoption of sound micropropagation practices for ericaceous plants by tissue culture labs.     

Biosynthesis of cytokinins by potato cell cultures

Potato cells grown in liquid culture incorporated mevalonic acid lactone-[2-¹⁴C] into free cytokinin (zeatin riboside and zeatin and the cytokinin of RNA (zeatin riboside). The cytokinin liberated by catabolism of RNA can account for no more than 40% of the free cytokinins.     

Quality of micropropagated plants—Vitrification

Summary Vitrification-Hyperhydrous shoot development, effects the survival and quality of several micropropagated plants ex-vitro. The leaves which are the immediate organ to be affected, exhibit abnormal morphology and physiology. Leaf malfunction is apparently a stress response to very rich media and high relative humidity. The understanding of the underlying mechanism of vitrification and its control in vitro can contribute to a more efficient micropropagation. Vitrification was found to be associated with elevated ethylene production which was related to hypolignification and poor cell wall development. Liquid and low agar media induced callose formation along with reduced and disoriented cellulose biosynthesis, manifested also in non-functioning guard cells. Malfunctioning stomata, in addition to defective cuticle contributed to increased transpiration and desiccation of in vitro formed leaves. The activity of various enzymes, associated with cell wall synthesis, was low and total proteins in normal leaves was higher than in vitreous ones. Various measures were found to reduce vitrification; lowered matrix and water potential in the medium, reduction in RH, low NH ₄ ⁺ , changes in Ca⁺⁺ levels and the removal of ethylene. These measures improved leaf morphogenesis, survival and the quality of several micropropagated plant species. Presented in the Session-in-Depth Transition of Plants From Culture to Establishment In Vivo,“ at the 41st Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990.     

Degradation of cytokinins by cytokinin oxidases in plants

The degradation metabolism of cytokinins is an important process that controls the levels of cytokinin active forms and their distribution in plant tissues. It appears to be due, in large part, to the activity of a specific enzyme, cytokinin oxidase. This review attempts to collate the limited information available about this enzyme and introduce new facts, obtained in our laboratory, concerning the mechanism of degradation of cytokinins bearing unsaturated isoprene side chains. However, complete clarification of the effects of cytokinin oxidase on cytokinin regulation and its molecular and biochemical properties will be dependent upon the purification of the protein with cytokinin oxidase activity to homogeneity and progress in the development of requisite molecular probes.     

Role of cytokinins in stress resistance of plants

The facts of both positive and negative influences of cytokinins on stress resistance of plants are known today. Without pretending to a final choice between these points of view, we have made an attempt to analyze the details of the experiments that gave rise to conclusions about the nature of the effect of cytokinins on the resistance to stress-causing influences with a focus on their intensity and duration. The review deals with the data concerning the influence of different adverse factors on the content of endogenous cytokinins and transduction of cytokinin signals, examines the influence on plant resistance of treatment with exogenous hormone, and the effects of genetic modifications causing changes in cytokinin content and signaling. Resistance is considered not only as a mean of plant survival under severe stress but also as an instrument of maintaining growth rate in plants exposed to moderate stress. Literature data and our own results make it possible to conclude that cytokinins play an important role in formation of plant resistance to adverse influences; however, the effect of these hormones depends on stress intensity. Under moderate stress, cytokinins ensure maintenance of plant growth, whereas a drop in cytokinins hampers growth under a strong influence of adverse factors, which is a prerequisite for mobilization of limited resources characteristic of severe stress and ensures preservation of plant viability.     

Changes in cytokinins during initiation and development of potato tubers

Cytokinin-like activity was assayed in stolons and tubers of Solanum tuberosum L. ssp. andigena (Juz. et Buk.) Hawkes cv. 165 grown in pots under controlled environment conditions. The plants were allowed to tuberise without the application of environmental or other external stimuli. The soluble sugar and starch contents of stolon tips and tubers were measured. Starch accumulation was a precise indicator of tuber initiation. Cytokinin-like activity began to increase in tubers with a diameter greater than 7.5 mm and, as assessed on a per tuber basis, was greatest in the largest size-category analysed. However, expressed as a function of fresh and dry weight, activity was greatest in tubers of 15–20 mm in diameter. Increases in cytokinin-like activity occurred subsequent to tuber formation, indicating that the tuberisation stimulus is unlikely to be cytokinin-like in nature.     

Essential oils from micropropagated plants of Lavandula viridis

The essential oils of Lavandula viridis were analysed by GC and GC-MS. Comparisons were made between three types of plant material from the same clone: field-grown plant, in vitro shoot cultures and micropropagated plants of the same clone. The most common components usually found in lavender oils were present in the oil samples analysed and more than 45 constituents were identified, representing more than 80% of the essential oil. The essential oils analysed consisted mainly of monoterpenes (75.4-76.3%), where oxygenated and hydrocarbons identified ranged from 41.8 to 57.3% and 18.1 to 34.2%, respectively. The major components found were 1.8-cineole (18.2-25.1%), camphor (9.1-15.7%), alpha-pinene (8.8-14.1%), borneol (4.1-4.8%), beta-pinene (1.2-5.6%), delta 3-carene (1.0-6.5%) and alpha-terpineol (0.8-4.2%). The monoterpene fraction of the in vitro shoot cultures showed different relative amounts of hydrocarbons and oxygenated components in relation to the parent plant and to micropropagated plants. In the sesquiterpene hydrocarbon fraction of the oil samples analysed (6.1-8.2%), 7-epi-alpha-selinene (1.6-4.8%) was the most important component and the oxygenated sesquiterpenes were found in small amounts (1.1-1.7%). The essential oils from field-grown plants of L. viridis, when compared with those obtained from in vitro shoot cultures or micropropagated plants of the same clone, demonstrated that the same major components were found without significant compositional variations.     

Endogenous cytokinins in the ribosomal RNA of higher plants

Endogenous cytokinin-active ribonucleosides were isolated from the rRNA and tRNA of pea epicotyls (Pisum sativum L., var Alaska) and of wheat germ (Triticum aestivum). The RNA preparations were analyzed for cytokinins by enzymic hydrolysis, ethyl acetate extraction, and Sephadex LH-20 fractionation in several solvents. Tentative identification of the cytokinins was based on cochromatography with synthetic cytokinin standards in several systems and on activity in the tobacco bioassay. Both the rRNA and tRNA from 10 day old pea epicotyls contained ribosylzeatin, isopentenyladenosine, and 2-methylthioribosylzeatin. The latter compound was the most active fraction in the pea rRNA, but was the least active fraction in the tRNA, where isopentenyladenosine activity was predominant. The 2-methylthioribosylzeatin from pea rRNA was identified by gas chromatography-mass spectrometry. Wheat germ rRNA contained cis and trans ribosylzeatin and 2-methylthioribosylzeatin. The tRNA contained isopentenyladenosine in addition. The specific cytokinin activity (activity per A₂₆₀ unit) of the tRNA was over forty times that of the rRNA. Significant contamination of the rRNA preparations by cytokinin-containing tRNA is considered unlikely on the basis of quantitative differences in the cytokinin content of the rRNA and tRNA preparations, electrophoretic analysis of rRNA purity and cytokinin analysis of fractionated oligonucleotide digests.     

A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants

A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora , including subsp. carotovora and subsp. atroseptica , and all pathovars/biovars of Erwinia chrysanthemi , on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2·0 × 10²–3·4 × 10³ cfu ml⁻¹ (equivalent to 1–17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2·3 × 10²–1·9 × 10⁴ cfu per microplant sample (equivalent to 5 cfu – 3·8 × 10² cfu per PCR). To improve the sensitivity of the method in planta , to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was <10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material.     

Physiological and biochemical responses of micropropagated tea plants

Summary Physiological and biochemical responses of micropropagated tea plants grown under field conditions were investigated in comparison to vegetatively propagated (VP) plants. No significant variation was observed between tissue culture raised (TC) and VP plants in terms of photosynthetic carbon assimilation rate. However, clones showed significant variation among themselves. Carbon assimilation studies carried out with a radiotracer technique revealed that ‘Assam’ cultivar UPASI-27 assimilated a higher amount of labeled carbon dioxide followed by UPASI-3. However, UPASI-27 was marginally better than UPASI-3 in terms of mobilization of assimilates to the growing sinks. Both, UPASI-3 and UPASI-27 reassimilated higher quantities of photosynthates followed by BSB-1 and UPASI-26. Though there was a marginal variation in photosynthetic pigments of TC and VP plants, it was not statistically significant. Similarly, no significant variations were observed in certain substrates (polyphenols, catechins and amino acids) and enzymes (polyphenol oxidase, peroxidase and phenylalanine ammonia-lyase) except protease involved in the formation of quality constituents of made tea. However, clonal variation was evident with respect to photosynthetic pigments, substrates/enzymes. Under soil moisture stress, no significant variation was observed between VP and TC plants in terms of proline accumulation.     

Morphogenesis of potato plants in vitro. II. Endogenous levels,distribution, and metabolism of IAA and cytokinins

The levels of endogenous IAA and cytokinins (zeatin, zeatin riboside, isopentenyladenine, and isopentenyladenosine) were determined in potato plants cultured in vitro under red light (R) and blue light (B) on medium with or without hormones. On medium without hormones in B, plants contained much higher cytokinin levels, particularly in leaves and roots, and also slightly elevated IAA levels. Kinetin in the medium in B changed the distribution of cytokinins and significantly increased IAA level in roots. In R, the presence of kinetin led to an increased cytokinin level in the whole plant, while the IAA level was slightly lower. IAA in the medium in B decreased cytokinin level in all plant parts, while the IAA level did not change significantly. In R, the presence of IAA in the medium led to a moderate increase of CK level and to a significant increase in IAA level, especially in roots. Uptake of 1-¹⁴C-IAA and of ³H-zeatin was generally higher in B than in R. Higher percentage of IAA taken up in B was converted to conjugates in the roots. Metabolism of ³H-zeatin was similar in R and B with only slight differences in metabolite amounts.Thus, in all experimental situations in which tuber formation was stimulated, IAA level in roots and stolons rose significantly, stressing the importance of an IAA gradient for tuber formation.     

High level of endogenous cytokinins in transgenic potato plantlets limits photosynthesis

Introduction of the gene for cytokinin synthesis into potato genome lead to a manifold increase in the level of cytokinins (zeatin, zeatin riboside, isbpentenyl-adenine, isopentenyladenosine) in plantlets grownin vitro.The increasing cytokinin level was associated with increasing tendency to teratoma formation, to decreasing leaf net photosynthetic rate and to increasing dark and light respiration rates and CO₂ compensation concentration. During plantlet (or teratoma) ontogeny, net photosynthetic rate increased simultaneously with the decrease in cytokinin level. High level of endogenous cytokinins was associated also with lower photochemical activities of both photosystems in isolated chloroplasts.     

Micropropagation and field evaluation of micropropagated plants of turmeric

A protocol was developed for in vitro propagation of turmeric cv `elite' using young vegetative buds from sprouting rhizomes. The shoot buds produced multiple shoots when cultured on MS solid medium supplemented with benzyladenine and 1-naphthalene acetic acid. The effect of various cytokinins on shoot multiplication was studied by culturing the shoot tips on MS liquid medium supplemented with benzyladenine, benzyladenine riboside, kinetin, kinetin riboside, zeatin, 6-,-dimethylallylaminopurine, adenine, adenine sulfate or metatopolin each at 10 M in combination with 1-naphthalene acetic acid (1 M). Significant differences were observed between the treatments. Liquid medium was more favourable than agar medium for shoot multiplication. Among the various concentrations of agar tested, 0.4% and 0.6% were the best and produced the highest number of shoots per explant. Among the different carbohydrates tested, sucrose, fructose, glucose, sugar cubes, maltose, levulose and market sugar were found to be equally effective for shoot multiplication and xylose, rhamnose, lactose and soluble starch were inhibitory. Ninety five percent of the micropropagated plants survived in sterilized soil in paper cups and all of them survived in the field. Among 48 plants, two plants showed variegated leaves on the tillers. The micropropagated plants showed a significant increase in shoot length, number of tillers, number and length of leaves, number of fingers and total fresh rhizome weight per plant when compared with conventionally propagated plants. RAPD analysis of 11 regenerated plants using sixteen 10-mer primers did not show any polymorphism.     

Infectivity and effectiveness of Glomus intraradices on micropropagated plants

Colonization by Glomus intraradices takes place very early within the root system of micropropagated plantlets of strawberry (var. avanta, elsanta), raspberry (var. himboqueen, Zeva I), and hortensia (var. leuchtfeuer). The arbuscular mycorrhizal fungus (AMF) did not colonize roots of the different hosts to the same extent, and considerable differences were observed between the varieties. The results reported here confirm that endomycorrhizal root colonization is affected by the host-fungus combination. The effects ranged from mutualistic (hortensia), through neutral (strawberry var. avanta, raspberry var. Zeva I) to negative (raspberry var. himboqueen and strawberry var. elsanta). Non-mycorrhized (control) plants of strawberry produced more runners than mycorrhized plants under controlled growth conditions (phytotron). Transfer of the potted plants to the field resulted in drastic alterations in overall growth and development within 4 weeks. Mycorrhized plants became healthy, and mycorrhized strawberry plants produced many stout runners. The number of the runners and their biomass were almost the same (var. avanta) and treated plants produced even more runners than the controls (var. elsanta). The authors have demonstrated the need to determine the specific effects of each species of AMF on individual prospective host plants prior to their utilization in the micropagation of plantlets.     

In vitro nodulation of micropropagated plants ofLeucaena leucocephala byRhizobium

Micropropagated plants ofLeucaena leucocephala nodulated with Rhizobium during thein vitro hardening stage, grew well on N-free-substrate. This is the first report ofin vitro nodulation of micropropagated plants by Rhizobium.     

Field performance and biochemical evaluation of micropropagated mulberry plants

Microclones of different mulberry genotypes were successfully transferred to the field. The same genotypes were raised through conventional methods (cuttings). A comparative study using morphological and biochemical tests of field established micropropagated and cutting derived plants of mulberry genotypes was conducted. Micropropagated mulberry plants showed significant morphogenic vigour when compared to plants raised through cuttings. Biochemical tests of leaves revealed that, there was no significant nutritional difference between micropropagated plants and those originated from cuttings. This revised version was published online in June 2006 with corrections to the Cover Date.     

Proteinase activity in potato plants

Several vegetative tissues of potato plants were screened for proteinase activity. Both endopeptidase and exopeptidase activities were investigated using gelatin and L-amino acid-4-nitroanilides (benzoyl-L-arginine-4-nitroanilide/BAPA, glutaryl-L-phenyl-alanine-4-nitroanilide/GLUPHEPA, alanine-4-nitro-anilide/APA, leucine-4-nitroanilide/LPA, and benzoyl-L-tyrosine-4-nitroanilide/BTPA) as substrates. Leaves and rootes were found to contain the highest levels of endopeptidase activity; lesser activities were detected in flower petals, sprouts, and tubers. Three different types of proteinases, L-BAPAase (serine proteinase), APAase (thiol proteinase), and BTPAase (sensitive to reducing agents), were characterized in various physical and chemical properties. Their temperature optima were determined to be 25° (L-BAPAase) and 40° (BTPAase, APAase) respectively; their pH optimum was between 8.6 and 9.0, their isoelectric points were between pH 4.25 and 6.0, and their molecular weight was estimated 70,000 (L-BAPAase, APAase) and between 150,000–250,000 (BTPAase). The trypsin-like activity against L-BAPA was inhibited by diisopropylfluorophosphate and by tosyllysine-chloromethyl ketone, but not by trypsin inhibitors from potato and legume.Abbreviations APA alanine-4-nitroanilide - BAPA benzoyl-L-arginine-4-nitroanilide - BTPA benzoyl-L-tyrosine-4-nitroanilide - DFP diisopropylfluorophosphate - DMF dimethyl formamide - EDTA ethylenedinitrilotetraacetic acid - GLUPHEPA glutaryl-L-phenylalanine-4-nitroanilide - LPA leucine-4-nitroanilide - PHMB p-hydroxy-mercuribenzoate - PI-I potato chymotrypsin inhibitor I - PPI potato proteinase leaf - PPr potato proteinase root - PPt potato proteinase tuber - PVP polyvinylpyrrolidone - TLCK tosyl-L-lysinechloromethyl ketone - TPCK tosyl-L-phenylalanyl chloromethane