Potato Calli Research Articles

Optimization of core phases of biolistic transformation of potato

We have established the optimal exposure time for internodes and callus plant material of Aksor potato variety on the OSS osmotic medium before and after biolistics and the type of plant explant that is most suitable for that. The optimal objects were explants from internodes of test plants. The best time for exposure of plant material to the osmotic medium before bombardment is 24 hours, and after biolistics – 48 hours. Callus tissue developed on the MS medium with zeatin two weeks later in internodes that were cultivated before bombarding on the OSS for 24 hours and we observed an increase in the callus mass by the end of fourth week. Not a single callus was obtained with rhizogenesis on the R4 medium from internodes and potato calli that were on the osmotic medium within 24 and 72 hours after biolistics. The regenerative capacity of internodes after cultivation on an osmotic medium for 48 hours after bombarding was higher than that of calli. We obtained 12 regenerant plants out of 200 internode explants and 4 regenerant plants out of 150 calli that were used in the experiment.

Selection and regeneration of purple sweet potato calli against drought stress simulated by polyethylene glycol

Background: Water shortage due to natural and/or technical drought stress, widespread throughout Sumatra, Java, Sulawesi and Kalimantan islands, significantly reduces crop production. The development of varieties tolerant to drought stress is important since it is more effective rather than improving irrigation infrastructure to increase the sweet potato productivity. Methods: Selection and regeneration experiments assessing purple sweet potato callus tolerance of drought stress, simulated by polyethylene glycol (PEG), were conducted to generate new variant plants tolerant of drought stress. Sterile explants (leaf and petiole) generated from previous in vitro culture were inoculated to the Murishage and Skoog (MS) medium containing plant growth regulator combination as treatments to induce calli. The calli were then transferred to half-MS medium containing 0, 5, 10, 15 and 20% PEG as selection agent for drought tolerance. The surviving calli were regenerated in the MS medium containing 0, 0.5, 1 or 1.5 mg l-1 6-benzylaminopurine (BAP). The callus formation, growth and survivability during in vitro culture were measured. Results: Calli were successfully formed in almost all media containing 2,4-Dichlorophenoxyacetic acid (2,4-D ) with the concentration of 1, 2, 3 and 4 mg l-1 and BAP (concentration: 0.5 and 1 mg l-1), but the medium of MS + 2 mg l-1 2,4-D + 0.5 mg l-1 BAP resulted in the highest number of induced calli per treatment (mean=11.36), with the percentage of responsive explants standing at around 96%. The higher the concentration of PEG, the lower the number of surviving calli. At 20% PEG, only 54.42% calli survived. There were five plants successfully regenerated from the survived calli at 20% PEG, using MS medium containing 1.5 mg l-1 BAP. Conclusions: The experiment has successfully produced putative drought-tolerant plants by callus screening using PEG as drought-tolerance-selecting agent in purple sweet potato.

Selection and regeneration of purple sweet potato calli against drought stress simulated by polyethylene glycol

Background: Water shortage due to natural and/or technical drought stress, widespread throughout Sumatra, Java, Sulawesi and Kalimantan islands, significantly reduces crop production. The development of varieties tolerant to drought stress is important since it is more effective rather than improving irrigation infrastructure to increase the sweet potato productivity. Methods: Selection and regeneration experiments assessing purple sweet potato callus tolerance of drought stress, simulated by polyethylene glycol (PEG), were conducted to generate new variant plants tolerant of drought stress. Sterile explants (leaf and petiole) generated from previous in vitro culture were inoculated to the Murishage and Skoog (MS) medium containing plant growth regulator combination as treatments to induce calli. The calli were then transferred to half-MS medium containing 0, 5, 10, 15 and 20% PEG as selection agent for drought tolerance. The surviving calli were regenerated in the MS medium containing 0, 0.5, 1 or 1.5 mg l-1 6-benzylaminopurine (BAP). The callus formation, growth and survivability during in vitro culture were measured. Results: Calli were successfully formed in almost all media containing 2,4-Dichlorophenoxyacetic acid (2,4-D ) with the concentration of 1, 2, 3 and 4 mg l-1 and BAP (concentration: 0.5 and 1 mg l-1), but the medium of MS + 2 mg l-1 2,4-D + 0.5 mg l-1 BAP resulted in the highest number of induced calli per treatment (mean=11.36), with the percentage of responsive explants standing at around 96%. The higher the concentration of PEG, the lower the number of surviving calli. At 20% PEG, only 54.42% calli survived. There were five plants successfully regenerated from the survived calli at 20% PEG, using MS medium containing 1.5 mg l-1 BAP. Conclusions: The experiment has successfully produced putative drought-tolerant plants by callus screening using PEG as drought-tolerance-selecting agent in purple sweet potato.

Relationships among iron deficit-induced potato callus growth inhibition, Fe distribution, chlorosis, and oxidative stress amplified by reduced antioxidative enzyme activities

Callus cultures were used to investigate and delineate responses of potato to iron (Fe) deficiency conditions over different culture durations. The morphological responses included chlorotic symptoms, reduced fresh weight and area of callus growth on Fe-deficient medium compared to calli grown under Fe sufficient conditions. Biochemically, potato calli under Fe deficit exhibited decreases in chlorophyll and carotenoid contents, reduction in activities of antioxidant enzymes (peroxidase, catalase and ascorbate peroxidase), as well as an increase in ferric chelate reductase (FCR) activity, lipid peroxidation, phenolic production and hydrogen peroxide (H2O2) level. Perls staining revealed sparse Fe distribution in Fe-deficient callus cells whereas Fe was widely distributed and intensely stained among numerous actively dividing cells in Fe-sufficient calli. These responses of calli to Fe deficiency were more pronounced with prolonged exposure to such stress leading to severe chlorosis and/or death of cells in chlorosis-susceptible calli but potential chlorosis-tolerant callus cells maintained their greenness and viability. Over a prolonged period in culture, significantly positive correlations were found among callus fresh weight, chlorophyll and carotenoid contents, antioxidant enzyme activities and lipid peroxidation as Fe supplies to the medium was increased. FCR activity was strongly correlated in a negative manner with Fe deficiency, chlorophyll content and peroxidase activity. The responses of calli to Fe supply can serve as reliable indicators for detecting chlorosis tolerance and/or nutrient deficiency stress.

Overexpression of the sweet potato IbOr gene results in the increased accumulation of carotenoid and confers tolerance to environmental stresses in transgenic potato

In a previous study, we have evidenced that the overexpression of the IbOr gene isolated from sweet potato conferred a tolerance activity against salinity and methyl viologen (MV) treatment in transgenic sweet potato calli along with an enhanced carotenoid content. In this study, to further examine the function of the IbOr gene in heterologous organism, we transformed the IbOr gene into potato under the direction of SWPA2 promoter, a strong inducible promoter upon treatment with various environmental stresses. Consistently with our previous study of sweet potato calli, the level of total carotenoid was elevated up to 2.7-fold (38.1μgg−1DW) compared to the non-transgenic control, Atlantic cultivar. However, the composition of carotenoid was not influenced by the overexpression of the IbOr gene since only pre-existing carotenoids in the non-transgenic control including violaxanthin, lutien and β-carotene were elevated at a similar level of total carotenoids. In general, the transcript levels for most of carotenogenesis-related genes were elevated in transgenic tuber, whereas they remained at similar levels in transgenic leaf tissues compared to those of non-transgenic controls. The increased levels of carotenoid content in the leaf or tuber tissue of transgenic lines were correlated with the enhanced tolerance activity against salt- or MV-mediated oxidative stresses and DPPH radical-scavenging activity. Our preliminary results suggest that further investigation is required for the development of a crop tolerant to salinity and other environmental stresses through the overexpression of the IbOr gene.

ASSESSMENT OF GENETIC VARIATION IN POTATO CALLUS TISSUE UNDER SALINITY USING RAPD MARKERS

ISHS XXVIII International Horticultural Congress on Science and Horticulture for People (IHC2010): International Symposium on New Developments in Plant Genetics and Breeding ASSESSMENT OF GENETIC VARIATION IN POTATO CALLUS TISSUE UNDER SALINITY USING RAPD MARKERS

Proline, sodium and potassium concentration changes in gamma rays and NaCl

In this study, the effects of gamma rays on changes of proline, sodium and potassium in potato calli (Solanum tuberosoum L.) cultivar White Desiree were investigated. After irradiation of calli by 60 and 80 Gy of gamma radiation, irradiated and non irradiated calli were transferred to a callus production medium containing 0, 30, 60, 90, 120 and 150 mM NaCl. After 26 days post treatment amounts of proline, Na+ and K+ were measured. The results showed that generally in each of gamma ray treatment, amounts of proline at 120 and 150 mM NaCl were significantly more than 0, 30, 60 and 90 mM salt. A significant difference observed only at 150 mM NaCl between irradiated and non irradiated calli. This result could be related to the proline role in osmotic adjustment at high concentration of salt. The amounts of sodium increased by increasing salt the concentration but the amount of potassium decreased. These results showed that the sodium influx inhibited the potassium uptake, and may be because of strong inhibitory effect of sodium on the potassium uptake system or efflux potassium from the cells. The Na+/K+ ratio decreased at 150 mM by 60 and 80 Gy of gamma radiation.

Differential responses of the antioxidant defence system and ultrastructure in a salt-adapted potato cell line

Changes in lipid peroxidation and ion content and the possible involvement of the antioxidant system in salt tolerance at the cellular level was studied in a potato (Solanum tuberosum L.) callus line grown on 150 mM NaCl (salt-adapted) and in a non-adapted line exposed to 150 mM NaCl (salt-stressed). Salinity reduced the growth rate and increased lipid peroxidation in salt-stressed line, which remained unaltered in the adapted line. Na⁺ and Cl⁻ content increased due to salinity in both lines, but the adapted line displayed greater K⁺/Na⁺ ratio than the stressed one. Total superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2) activities decreased in both salt-exposed lines; catalase (CAT, EC 1.11.1.6) activity did not change in the adapted line, but decreased in the stressed cell line. Salinity caused the suppression of one GR isoform, while the isozyme patterns of SOD, APX, and CAT were not affected. Ascorbate and reduced glutathione increased in both salt-exposed calli lines. α-Tocopherol increased as a result of salt exposure, with higher levels found in adapted calli. Electron microscopy showed that neither the structural integrity of the cells nor membrane structure were affected by salinity, but plastids from adapted cells had higher starch content. The results suggest that the enzymic and non-enzymic components of the antioxidant system are differentially modulated by salt. Different concentrations of antioxidant metabolites are more relevant to the adaptive response to salinity in potato calli than the differences in activity of the antioxidant enzymes.

Changes in carbohydrate content of potato calli during osmotic stress induced by mannitol

Endogenous carbohydrate (fructose, glucose and sucrose) fractions were measured in calli of potato genotypes with different field tolerance to drought. Under in vitro stress conditions induced by 0.8 M mannitol, sucrose level of calli increased extremely in medium-tolerant (by 424.5%) and sensitive (by 302.7%) genotypes whilst the rate of increase was 12-18-times lower in the drought tolerant variety. Results suggest the applicability of sucrose as biochemical marker for distinguish drought tolerant genotypes from great population in callus culture.

Occurrence of plant-uncoupling mitochondrial protein (PUMP) in diverse organs and tissues of several plants.

The presence of plant-uncoupling mitochondrial protein (PUMP), previously described by Vercesi et al. (1995), was screened in mitochondria of various organs or tissues of several plant species. This was done functionally, by monitoring purine nucleotide-sensitive linoleic acid-induced uncoupling, or by Western blots. The following findings were established: (1) PUMP was found in most of the higher plants tested; (2) since ATP inhibition of linoleic acid-induced membrane potential decrease varied, PUMP content might differ in different plant tissues, as observed with mitochondria from maize roots, maize seeds, spinach leaves, wheat shoots, carrot roots, cauliflower, broccoli, maize shoots, turnip root, and potato calli. Western blots also indicated PUMP presence in oat shoots, carnation petals, onion bulbs, red beet root, green cabbage, and Sedum leaves. (3) PUMP was not detected in mushrooms. We conclude that PUMP is likely present in the mitochondria of organs and tissues of all higher plants.

Changes in the Cellular Ultrastructure of Potato Calli Induced by a Culture Filtrate of Phytophthora Infestans

ABSTRACTThe effect of Phytophthora infestons culture filtrate (CF) upon the ultrastructure of cellular organelles in potato tuber and stem derived calli has been studied. During the callus growth at light a transformation of proplastides in chloroplasts has been observed: first, continuous thylakoid membranes are formed around the starch inclusion, secondly, the assembly in grana of thylakoid took place. In CF treated calli the following changes were noted: (a) transformation of the cellular content into amorphous mass indicating the necrosis of the tissues; (b) distrophy of the nucleus; (c) disintegration of the plastid and peroxisome envelopes and of the cytoplasmic membrane, which resulted in vesicles and (d) swelling of the mitochondria followed by fragmentations of cristae and matrix lysis. CF resistant calli having normal growth presented more developed photosynthetic apparatus in stem derived calli.

Starch-accumulating Sweet Potato Callus Tissue Devoid ofβ-Amylase but with Two Starch Phosphorylase Isozymes

By controlling the concentrations of kinetin, auxin, and sucrose in the Murashige–Skoog medium, starch contents in callus culture induced from sweet potato tissues could be manipulated. Activity staining and Western analysis on PAGE plates and activity assays made on starch phosphorylase in the presence and absence of mercuric ions showed that β-amylase is absent in callus cultures regardless of whether their starch content is high or low. This would imply that β-amylase induction in sweet potato calli is not linked to the metabolic control through which the expression of storage function is associated, as proposed by Nakamura et al. [Plant Physiol., 96, 902 (1991)] for sweet potato leaf-petiole cuttings. Analyses of starch phosphorylase in crude extracts suggested the presence of a new starch phosphorylase in tuberous root and callus tissue. This phosphorylase is immunologically different from the tuberous root and leaf enzymes that we studied previously.