Subculture Of Callus Research Articles

Insights into total phenolic, flavonoid, and antioxidant activity of callus subculture frequency in rare endemic Ajuga xylorrhiza Kit Tan

This study assessed the callus morphology, growth index, callus diameter, total antioxidant activity, and total phenolic and flavonoid contents (TPC and TFC) of callus extracts from Ajuga xylorrhiza, a severely endangered plant found in small populations in Türkiye. Calluses obtained from a total of 18 applications formed in culture media supplemented with Kin and 2,4-d at six ratios (A, B, C, D, E, and F) and their subcultures (A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1, and F2) were used for examination. Extracts A and D had a higher TFC than the other groups (27.26 and 18.08 µg QEs/mg extract, respectively). Cultures D, A, and B had high TPCs of 66.29, 58.85, and 51.41 GAEs/mg extract, respectively, while F, F2, and E1 had the lowest. Total antioxidant activity varied by the method (DPPH, ABTS, and CUPRAC). Extracts A, D, and B (IC50 = 4.52, 9.51, and 16.18 µg/mL, respectively) demonstrated higher radical scavenging activity with the ABTS technique than the positive controls, BHT and BHA. The findings showed that an increase in plant growth regulators negatively impacted TPC, TFC, and total antioxidant activity. A. xylorrhiza extracts displayed higher antioxidant activity than their respective subcultures, indicating that the plant has a significant phytochemical content (total phenol and flavonoid contents). Moreover, the data suggest a possible relationship between TPC and TFC in extracts A, B, and D and radical scavenging activity (total antioxidant), which is worth investigating in further research.

Kemiri Sunan Buds Induction (Reutealis trisperma (Blanco) Airy Shaw) For In Vitro in Ms Media with Different Concentrations of the Substance Growing Manager

Need oil fuel in Indonesia has increased. Oil production reached only 410,000 barrels per day while the needs of up to 1 million barrels per day. One plant is capable of producing biofuels is Pecan sunan. Vegetative reproduction has been hampered by the limited number of obtained so that necessary research in vitro. This research aims to know the concentration of the substance growing balance sitokinin (BA, TDZ and kinetin) and Auxin (NAA) in Pecan sunan buds induces on the MS medium. Experimental design used was Complete Random Design simple. The treatment is the composition of the basic MS medium with medium that is combined with the astringent balance grow NAA (0.5 mg/l), BA (1-3 mg/l), TDZ (0.2 mg/l) and kinetin (2 mg/l). The result of the observation form buds, but callus changes color and size kalus. Before and after color callus subculture of potentially forming buds is the A9 (MS + 0.5 mg/l NAA + 3 mg/l BA + 2 mg/l kinetin). While kalus response tends to be good on the A5 is 0 MS media (MS + 0.5 mg/l NAA + 2 mg/l BA + 0.2 mg/l TDZ) and G media is A2 (MS + 0.5 mg/l NAA + 2 mg/l BA).

Callus induction, subculture, and browning inhibition of the anticancer plant Taxus media

In order to obtain more effective cell culture parameters of Taxus anticancer plants, we optimized the callus induction and subculture conditions of the explants (stem segments with buds) of the anticancer medicinal plant Taxus media by using the plant tissue culture technology and orthogonal test. Furthermore, we studied the method to inhibit browning in the culture. The results indicated that the optimal conditions for inducing callus was culture in the medium composed of B5+0.25 mg/L 2, 4-D+1.5 mg/L NAA+0.5 mg/L KT in the dark, which showed short induction time and a high induction rate (86.7%). The formula of the optimal medium for subculture was B5+0.5 mg/L 2, 4-D+2.0 mg/L NAA+1.5 mg/L KT.The proliferation multiple of callus cultured by subculture on the 10th day of callus growth was the highest. Activated carbon inhibited the browning in callus subculture, with the optimal inhibitory concentration of 0.8 g/L. The results of this study lay a foundation for the production of taxol by suspension culture of T. media cells.

Preserving Nature’s Treasure: A Journey into the In Vitro Conservation and Micropropagation of the Endangered Medicinal Marvel—Podophyllum hexandrum Royle

Podophyllum hexandrum Royle, also known as Podophyllum emodi Wall, holds significant ecological, ornamental, and medicinal values. However, it has become endangered due to overexploitation, prolonged seed dormancy, slow natural regeneration, and climate change. This study developed an efficient in vitro protocol for callogenesis and micropropagation of P. hexandrum to conserve germplasm in in vitro conditions. Callus formation from various plant parts, including the leaf, stem, rhizome, radicle, and cotyledon, was induced using Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The combination of benzyladenine at 1 mg L−1 and 4-dichlorophenoxy acetic acid at 3 mg L−1 was optimal for biomass production, yielding 215.88 ± 0.31 mg, with growth per gram at 8.32 ± 0.32 and a growth rate of 13.62 ± 0.25 mg/day on MS medium. For shoot proliferation, benzyladenine (3.5 mg L−1) and naphthalene acetic acid (0.5 mg L−1) combined with activated charcoal showed the highest shoot induction percentage per explant. For shoot regeneration from calluses, 6-benzylaminopurine (0.5 mg L−1) and thidiazuron (2 mg L−1) were most effective, producing superior shoot length, number of regenerations, and regeneration percentage. Root induction was successful with α-naphthalene acetic acid supplementation (0.5 to 1.5 mg L−1) in MS medium, resulting in the highest number per explant (4.08 ± 0.08), length (5.45 ± 0.15 cm), and rooting rate (87.00 ± 1.66%) of roots in plantlets. Subculturing for callus culture was performed every 28 days for up to four subcultures to prevent nutrient depletion and toxic metabolite accumulation, ensuring tissue health and viability. Continuous subculturing of callus on MS medium maintained healthy P. hexandrum germplasm in vitro. Overall, this micropropagation protocol provides a rapid system for conserving P. hexandrum germplasm.

Long-lasting bioactive compounds production from Malus x domestica Borkh var. Mela Rosa Marchigiana pulp callus culture with antioxidant activity: In vitro, ex vivo and in vivo validation

Long-lasting bioactive compounds production from Malus x domestica Borkh var. Mela Rosa Marchigiana pulp callus culture with antioxidant activity: In vitro, ex vivo and in vivo validation

Development and Optimization of a Rapid In Vitro Micropropagation System for the Perennial Vegetable Night Lily, Hemerocallis citrina Baroni

The perennial herbaceous night lily, Hemerocallis citrina Baroni, is an important vegetable crop with an increasing production and consumption in China. The long lifecycle and slow growth of the night lily are becoming bottlenecks for the large-scale production of elite lines and various genetic and breeding studies. There is a lack of a protocol for rapid and efficient micropropagation for this crop. Here, we reported the systematic investigation and optimization of in vitro plant regeneration through tissue-culture-based organogenesis in the night lily variety ‘Datong Huanghua’. We evaluated various factors affecting the efficiency of callus induction and subculture, shoot regeneration, rooting and plantlet establishment, including explant type and age, inoculation methods, basal culture media and the type and concentration of plant growth regulator (phytohormones) in various growth media. We developed an optimized protocol, as follows. The highest efficiency of callus induction was observed on Murashige and Skoog (MS) medium supplied with 22.7 µM TDZ (thidiazuron) using the young scape (flower stem or stalk) as the explant, which was cut longitudinally in half to produce a segment approximately 0.5 cm in length. Callus subculture and proliferation were more efficient on MS medium containing 9.0 µM 2,4-D (2,4-dichlorophenoxyacetic acid) under light culture conditions. Shoot regeneration showed the highest efficiency on MS medium supplemented with 8.9 µM 6-BA (6-benzylaminopurine) + 5.4 µM NAA (α-naphthaleneacetic acid), while the best rooting medium was MS medium containing 2.7 µM NAA. After transplanting, the transplanted regenerated seedlings showed the highest survival rate (96%) on a substrate mixture with a 2:1:1 ratio of peat/perlite/vermiculite. A protocol and flowchart for the rapid in vitro micropropagation of night lily plants is proposed that will facilitate various genetic, genomic and breeding studies on this crop.

In Vitro Callogenesis of Murici-Pitanga (Byrsonima gardneriana a. Juss.) Malpighiaceae

Objective: The present work aimed to evaluate the callogenesis of murici-pitanga in different explants and growth regulators. since this species has great medicinal, environmental and economic importance, however the cultivation process and extractivism make its propagation difficult. Therefore, in vitro cultivation provides conditions that can optimize its propagation. Among the techniques used in in vitro micropropagation, callogenesis allows the regeneration of tissues, organs or plants, becoming a cultivation alternative for the species under study. Method: Explants from seedlings cultivated in vitro were inoculated in MS 1/2 medium with different types, concentrations and combinations of growth regulators, namely: zygotic embryos under 6-benzylaminopurine x α-naphthaleneacetic acid (BAP x ANA) (0.0 ; 11.1, 22.2 and 44.4 µM); leaf and stem segments under NAA (0.0; 13.42; 26.85 and 40.27 µM) and root and leaf segments under 2,4-dichlorophenoxyacetic acid (2,4-D) (0.0; 2. 26; 4.52; 9.05 and 18.10 µM), with subcultures in cell suspensions (0.0; 4.52; 9.05; 18.10 and 3620 µM) and callus growth curve (4. 52 µM). Resultsandconclusion: The percentage of induction and mass of fresh callus matter were evaluated. The callogenesis of murici-pitanga occurs more efficiently in the root segment in a medium supplemented with 4.52 µM of 2,4-D. Where, the calluses presented friable characteristics, in addition to better averages of induction percentage and fresh matter mass. For cell suspension, a concentration of 4.52 µM of 2,4-D is indicated, and in the growth curve, the best period for callus subculture may occur on the 56th day of cultivation, established based on the callus growth pattern sigmoidal with three distinct phases. Therefore, the murici-pitanga can be regenerated through calluses originating from root segments. Originality/value: Studies that address the in vitro propagation of murici-pitanga are scarce, and the present work reports significant results, with new explant options and different types of growth regulators to be used. In addition to advances in studies of callus subcultures with great potential for regeneration. These results become important, since the species needs techniques that facilitate its propagation, and could support future morphogenetic studies, obtaining secondary metabolites, in addition to establishing a regeneration protocol via somatic embryogenesis.

Alteration in the callogenesis, tropane alkaloid formation, and gene expression in Hyoscyamus niger under clinorotation.

This study aimed to investigate the effects of clinorotation induced by 2-D clinostat on the growth, tropane alkaloid production, gene expression, antioxidant capacity, and cellular defense responses in the callus tissue of Hyoscyamus niger. Callus induction was conducted by putting hypocotyl explants in the MS culture medium supplemented with 1 mgL-1 2,4-D and 1 mgL-1 BAP growth regulators. The sub-cultured calli were placed on a clinostat for 0, 3, 7, and 10 days (2.24 × 10-5 g on the edge of the callus ring). Clinorotation significantly increased callus fresh weight, dry weight, protein, carbohydrate, and proline contents compared to the control, and their maximum contents were obtained after 7 and 10 days. H2O2 level enhanced under clinorotation with a 76.3% rise after 10 days compared to control and positively affected the atropine (77.1%) and scopolamine (69.2%) productions. Hyoscyamine 6-beta hydroxylase and putrescine N-methyltransferase gene expression involved in the tropane alkaloid biosynthesis were upregulated markedly with 14.2 and 17.1-folds increase after 10 days of clinorotation, respectively. The expressions of jasmonic acid, mitogen-activated protein kinase, and ethylene-responsive element-binding transcription factor were upregulated, and the activity of peroxidase and catalase showed a 72.7 and 80% rise after 10 days. These findings suggest that microgravity can enhance callogenesis by stimulating the ROS level, which can impact the antioxidant enzymes, tropane alkaloid formation, and gene expression.

Effect of Gamma Irradiation on Callus of Handeuleum (Graptophillum pictum L. Griff) Kalimantan and Papua Accession

Handeuleum (Graptophyllum pictum L.Griff) is a medicinal plant that is widely used as a traditional medicine for its healing properties, hence it should be developed as one of the leading Indonesian medicinal plants. The combination of tissue culture and gamma irradiation are two common methods to improve vegetatively-propagated cultivars. This experiment aims to determine the effect of gamma irradiation dose-rate on the callus culture of the two handeuleum accessions. Callus generated from proliferation were irradiated with gamma rays (0 Gy, 15 Gy, 25 Gy, and 35 Gy) and then grown on regeneration medium containing BAP (4.44 μM and 8.88 μM) and TDZ (4.44 μM and 8.88 μM). The result of the experiment shows that RD50 was at19.25 Gy. The interaction among the Papua accession, gamma irradiation dose-rate of 15 Gy, and regeneration media 4.44 μM BAP produced the highest weight of callus subculture 1 and 2. Irradiation dose-rate of 25 Gy on Kalimantan accession resulted in the highest relative fresh weight growth of irradiated callus. The treatment with a 25 Gy dose-rate produced brownish-white callus and produced the highest value of variability in the relative growth rate of irradiated callus. 
 Keywords: accession, irradiation dose-rate, purple leaves, variability

An Efficient Agrobacterium-Mediated Transformation Method for Hybrid Poplar 84K (Populus alba × P. glandulosa) Using Calli as Explants.

A highly efficient Agrobacterium-mediated transformation method is needed for the molecular study of model tree species such as hybrid poplar 84K (Populus alba × P. glandulosa cv. ‘84K’). In this study, we report a callus-based transformation method that exhibits high efficiency and reproducibility. The optimized callus induction medium (CIM1) induced the development of calli from leaves with high efficiency, and multiple shoots were induced from calli growing on the optimized shoot induction medium (SIM1). Factors affecting the transformation frequency of calli were optimized as follows: Agrobacterium concentration sets at an OD600 of 0.6, Agrobacterium infective suspension with an acetosyringone (AS) concentration of 100 µM, infection time of 15 min, cocultivation duration of 2 days and precultivation duration of 6 days. Using this method, transgenic plants are obtained within approximately 2 months with a transformation frequency greater than 50%. Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and β-galactosidase (GUS) histochemical staining analyses confirmed the successful generation of stable transformants. Additionally, the calli from leaves were subcultured and used to obtain new explants; the high transformation efficiency was still maintained in subcultured calli after 6 cycles. This method provides a reference for developing effective transformation protocols for other poplar species.

MicroRNA Zma-miR528 Versatile Regulation on Target mRNAs during Maize Somatic Embryogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.

Induction of cell division and antioxidative enzyme activity of Matricaria chamomilla L. cell line under clino-rotation

This study represents an optimized protocol for cell line culture of Matricaria chamomilla and the impact of clino-rotation on cell division, cell growth, and antioxidant enzyme activities for the first time. The cell suspension was transferred in the solid MS medium supplied with 2, 4-D, and KIN. Then the calli produced from a cell line were selected for callus subculture and clino-rotation treatment for 7 days by a 2D-clinostat. A significant rise of fresh and dry weights, cell division, total soluble sugar, reducing sugar, and starch contents were detected under clino-rotation. Protein content approximately unchanged in microgravity-treated calli. Antioxidant enzymes activities, such as peroxidase, catalase (CAT), and superoxide dismutase were elevated in calli exposed to microgravity. CAT activity showed a more than three-fold increase than that of control. According to native polyacrylamide gel electrophoresis, all the antioxidant enzymes isoforms were stronger in clino-rotated calli than that of the untreated control. Microgravity also stimulated H2O2 production and markedly adjusted lipid peroxidation in calli exposed to clino-rotation. These findings suggest that clino-rotation with stimulation of carbohydrate accumulation and antioxidant enzymes mitigates oxidative stress and improves growth and cell division. The isolation of M. chamomilla cell line with high growth was conducted to study the impact of clino-rotation on some cellular and antioxidative enzyme responses. Clino-rotation stimulated the cell division and growth by induction of antioxidant enzyme activities.

In vitro regeneration system of Halogeton glomeratus: an important halophyte

Halogeton glomeratus (H. glomeratus) is one of the most important halophytes in Asia, and the research on the genes and salt-tolerant mechanisms of this species is limited because of the lack of an optimal and efficient in vitro regeneration system. Here, we developed an efficient plant regeneration protocol using H. glomeratus leaves as explants, which has not been previously reported. High-quality calli were successfully obtained from the H. glomeratus leaves at a frequency of 100% supplemented with 2.00 mg/L 2,4-dichlorophenoxyacetic acid, 0.50 g/L PVP, 5.00 g/L agar, and 30.00 g/L sucrose; the optimum callus subculture medium is 1.00 mg/L 2,4-dichlorophenoxyacetic acid, 0.50 g/L PVP, 5.00 g/L agar, and 30.00 g/L sucrose. Shoots were regenerated on shoot induction medium at a frequency of 100% added with 0.50 mg/L 6-benzylaminopurine, 2.00 mg/L kinetin, 0.20 mg/L naphthaleneacetic acid, 0.50 g/L PVP, 5.00 g/L agar, and 30.00 g/L sucrose; then, shoots grew up on shoot regeneration medium. Finally, roots were regenerated from the shoots on Murashige and Skoog medium with high efficiency. This study firstly offers a rapid and efficient system for plantlet regeneration from leaves of H. glomeratus. Whether our protocol applies well to other tissue regeneration of H. glomeratus needs further confirmation. This work will facilitate basic research and salt-tolerant mechanisms of this important halophyte species.

In vitro plant regeneration and Agrobacterium-mediated genetic transformation of a carnivorous plant, Nepenthes mirabilis

In nutrient-poor habitats, carnivorous plants have developed novel feeding strategies based on the capture and digestion of prey and the assimilation of prey-derived nutrients by specialized traps. The Nepenthes genus, comprising nearly 160 species, presents a remarkable pitcher-shaped trap, leading to great interest among biologists, but the species of this genus are listed as threatened. In this work, we developed a protocol for reproducing Nepenthes mirabilis through shoot regeneration from calli. The cultivation of stem segments of N. mirabilis on MS medium containing thidiazuron induced organogenic calli after 10 weeks. Subcultured calli exposed to 6-benzylaminopurine showed shoot regeneration in 3 weeks with considerable yields (143 shoots/g of calli). Excised shoots transferred to medium with indole-3-butyric acid allowed rooting in 4 weeks, and rooted plantlets had a 100% survival rate. Based on this method, we also developed an Agrobacterium-mediated genetic transformation protocol using calli as explants and ipt as a positive method of selection. Twelve weeks post infection, regenerated shoots were observed at the surface of calli. Their transgenic status was confirmed by PCR and RT-PCR. In conclusion, this study provides an efficient method for regenerating Nepenthes and the first protocol for its stable genetic transformation, a new tool for studying carnivory.

Effects of Different Hormone Concentrations on Callus Induction of Astragalus mongholicus Bunge

A method of callus induction and subculture of Astragalus mongholicus was established. Different methods were used to sterilize seeds to induce sterile seedlings. The different parts of plants and Astragalus aseptic seedlings were used for the explants, different concentrations of auxin NAA and Cytokinin BAP were used in MS medium to induce callus, and then the callus with better growth condition was selected for secondary culture. Results showed that the browning phenomenon in the secondary culture was inhibited and the best disinfectant was mercuric chloride. The germination rate of seeds treated with 75% ethanol for 40 s, 1.5% NaCl for 20 minutes, and 0.1% HgCl2 for 3 minutes can reach up to 66.2%. The hypocotyls of sterile seedlings as explants were the best way to induce callus. The induction rate was close to 100% when 0.5 mg/L BAP and 0.15 mg/L of NAA and 0.01 mg/L of nano-TiO2 were added to MS medium. Besides, Nano-TiO2 can effectively prevent bacterial contamination of MS medium. Cotyledons, induced by cotyledon as explants, showed the most severe browning. Therefore, the best hormone combination of Astragalus subculture was 1.0 mg/L BAP, 1.5 mg/L NAA and 0.01 mg/L nano-TiO2 .

Increasing anthraquinone compounds on callus leaf Morinda citrifolia (L.) by elicitation method using chitosan shell of shrimps (Penaeus monodon)

Elicitation is one method to increase the content of secondary metabolite compounds in plant tissues. Anthraquinone is one of the secondary metabolite compounds that have potential of being a medicinal agent and produced by plants. The purpose of this research is to increase anthraquinone content in Morinda citrifolia (L.) leaf callus using elicitation method with chitosan from shrimp shell extract (Penaeus monodon) as elicitors. M.citrifolia (L.) callus was obtained from young leaf explants grown on Murashige and Skoog (MS) medium with 2.4-D addition of 1.75 mg / L and Kinetin 1.5 mg / L aseptically. The second subculture of callus is elicited by elicitor of chitosan each of 0.5 ml. The concentration of elicitor used was 0, 0.5,1,1.5,2 and 2.5 mg / ml. Harvesting of elicitation result done on day 0, 2, 4 and day 8 after elicitation. The results showed that callus containing anthraquinone could be induced in MS medium with addition of 2,4-D 1.75 and Kinetin 1.5 mg / L. and addition of chitosan at some concentrations may increase anthraquinone. The highest anthraquinone content was produced at concentration 2 mg / ml and 4 days harvest time

Enhanced Production of Berberine Through Callus Culture of Tinospora cordifolia (Willd.) Miers ex Hook F. and Thoms.

Callus culture of elite plant of Tinospora cordifolia selected for high berberine contents was established from nodal explants on agar (0.7%) gelled Murashige and Skoog (MS) medium with different types and concentrations of auxins and 3% sucrose. The best result in terms of callus fresh weight (2531 mg), dry weight (303 mg) and berberine (756 μg g−1dw) content was obtained on a medium with 5 μM α naphthalene acetic acid (NAA). Callus was repeatedly subcultured for five times. Stability of berberine production in the cultures was maintained prior to further experiments. Precursors of berberine viz. tyrosine and phenylalanine were incorporated in the culture medium for further studies. A significant increase (3.19-fold) in berberine production (1894 μg g−1 dw) was achieved by the addition of 40 μM tyrosine in the MS medium as revealed by high-performance liquid chromatography (HPLC) analysis. The berberine production in culture has been stable over continuous subculture of callus. This method for the production of berberine can be used for harvesting of berberine. Such callus cultures can also be used to initiate suspension cultures of T. cordifolia for studies on berberine.

Regeneration of White Lupin, Lupinus albus, Plants form Hypocotyl Stems

This study succeeded in regenerating the white lupin, Lupinus albus, plants from differentiated the hypocotyl stems callus of its sterilized seedlings. The results indicated to superior the solid Murashige and Skoog (MS) medium provided with 1.0 mg L-1 NAA in callus initiation with 90% after 20 days of culture from the other of selected media. The rate of the value of the callus fresh weight was 3.3 gm after 40 days of growth, which gives indication on the viability and activity of callus. The continued of callus subculture on the same its initiated medium led to spontaneous production of 22 shoots. These regenerated shoots rooted when cultivated in MSO with its full strength at ratio 50% after 26 days, producing the whole lupin plants in the culture media. .

Dynamics of the Methylome and Transcriptome during the Regeneration of Rice

Oryza sativa indica (cv. IR64) and Oryza sativa japonica (cv. TNG67) vary in their regeneration efficiency. Such variation may occur in response to cultural environments that induce somaclonal variation. Somaclonal variations may arise from epigenetic factors, such as DNA methylation. We hypothesized that somaclonal variation may be associated with the differential regeneration efficiency between IR64 and TNG67 through changes in DNA methylation. We generated the stage-associated methylome and transcriptome profiles of the embryo, induced calli, sub-cultured calli, and regenerated calli (including both successful and failed regeneration) of IR64 and TNG67. We found that stage-associated changes are evident by the increase in the cytosine methylation of all contexts upon induction and decline upon regeneration. These changes in the methylome are largely random, but a few regions are consistently targeted at the later stages of culture. The expression profiles showed a dominant tissue-specific difference between the embryo and the calli. A prominent cultivar-associated divide in the global methylation pattern was observed, and a subset of cultivar-associated differentially methylated regions also showed stage-associated changes, implying a close association between differential methylation and the regeneration programs of these two rice cultivars. Based on these findings, we speculate that the differential epigenetic regulation of stress response and developmental pathways may be coupled with genetic differences, ultimately leading to differential regeneration efficiency. The present study elucidates the impact of tissue culture on callus formation and delineates the impact of stage and cultivar to determine the dynamics of the methylome and transcriptome in culture.

Production and characterization of lectin from Bauhinia holophylla (Fabaceae:Cercideae) calli

Bauhinia holophylla is a woody plant found in the Brazilian Cerrado used in folk medicine to treat diabetes. Lectins are glycoproteins with several biotechnological applications and play important roles in plant defenses and in medicine. Lectins have been produced in vitro using plant tissue culture techniques. This study shows the production and characterization of lectin from B. holophylla by callus culture. Calli were obtained from leaf segments placed on woody plant and Murashige and Skoog media supplemented with 2,4-dichlorophenoxyacetic acid (0, 4.52, 9.05, 18.10 µM) or 6-benzylaminopurine (0, 4.44, 8.88, 17.75 µM), in the presence or absence of light. The highest concentrations of lectin expressed by hemagglutination activity were observed in green and compact callus induced in woody plant medium supplemented with 6-benzylaminopurine in the presence of light, which showed specificity by all types of erythrocytes tested. The highest concentrations of lectin (128 HU mg−1) and fresh weight of callus were observed in the stationary phase (41st–60th day). Lectin was not detected in subcultured calli. d-Galactose promoted the highest inhibitory effect on the lectin activity in human erythrocytes ABO system, especially B-type. Lectin has been shown to be a stable protein, maintaining its hemagglutination activity after incubation at a wide range of temperatures, pH values and ethylenediaminetetraacetic acid concentrations.