Callus Induction Stage Research Articles

In vitro sterilization and callus induction from Cananga odorata forma genuina steenis explants

Cananga odorata (ylang-ylang) is a plant with numerous uses, including ornamental use, traditional medicinal ingredient, as fragrance, and cosmetics. The high market demand for ylang-ylang essential oil needs to be supported by information on the in vitro process of callus formation to supports the secondary metabolites production without having to cut or harvest the trees so that biodiversity is maintained. The aims of this study were to analyze the explants sterilization method using sodium hypochlorite (NaOCl) solution that was most suitable for the ylang-ylang initiation, and to analyze the effect of BAP and NAA treatment on the formation of ylang-ylang callus. The design used is a completely randomized design. The methods used included the preparation, sterilization, and callus induction using a combination of BAP and NAA hormones with twenty four treatment combinations. The results are: the sterilization of explants with sodium hypochlorite (NaOCl) solution at a concentration of 20% (v/v) 10 minutes followed by a concentration of 15% (v/v) 20 minutes was able to produce sterile explants in as much as 67.5% of cases. At the callus induction stage, the highest score of callus formation in leaf explants was in media-23 (BAP 4 ppm plus NAA 5 ppm).

Somatic embryogenesis and optimization of regeneration system from immature embryos in maize inbred lines

Maize production and productivity is on a sharp decline due to abiotic and abiotic stresses, therefore, an efficient regeneration protocol is an important tool that can contribute to maize improvement and gene-function studies to improve food security for the ever-growing population. The objective of this study was to optimize a regeneration system for CML 444 inbred line with CML 442 maize inbred line used as a reference. Callus was generated by incubation of immature embryos in Murashige and Skoog (MS) medium with vitamins supplemented with 0 - 4 g L-1 of 2, 4-D hormones, 900 mg L-1 proline, 250 mg L-1casein hydrolysate and 10 mg L-1 of filter sterilized AgNO3, 30 g L-l of sucrose and 3 g L-1 gelrite. Somatic embryo maturation was achieved by transferring 6-week old callus to MS medium with vitamins prepared as previously in callus induction with 60 g L-1 of sucrose and zero plant growth regulators (PGR). Shoot initiation was conducted in MS medium with vitamins supplemented with BAP, NAA at varied concentrations and a 0 mg L-1 control. Plants at a 3-leaf stage that had not rooted were transferred to MS media with vitamins with IBA at a concentration of 0 - 0.3 mg L–l. The 2, 4-D rates were significantly (p≤0.001) different for callus onset and callus induction. The genotype × rate interaction effects showed that 0 and 2 g L-1 2, 4-D had the lowest and highest mean, respectively in both lines during onset and induction of callus. The lines had significant (p≤0.001) effects on shooting induction, however, their means were not significantly different. Similarly, the means for the hormones were not significantly different for shooting induction. The lines, IBA rate and their interaction were significantly (p≤0.05) different for rooting induction. The means for the lines were significantly different for rooting induction in different IBA rates. Conversely, the mean for the IBA rates was significantly different for rooting induction. This study found that plant growth regulators rates during the callus induction stage play a key role during regeneration. This protocol was a success and could provide a fundamental platform for future transformation in this line.

THE EFFECT OF CULTURE MEDIA TYPE AND PLANT GROWTH REGULATORS ON CALLUS INDUCTION OF OIL PALM (Elaeis guineensis Jacq) PISIFERA TYPE

Oil palm has a high economic value since it is one of the highest vegetable oil-producing plants compared to other oil-producing plants. The provision of good planting material for oil palm plantations is one of the determining factors to increase productivity. The parent Pisifera type of oil palm is important to develop the high-yielding oil palm Tenera varieties. The provision of oil palm material could be done through generative system with seeds and also could be done through vegetative approaches using tissue culture techniques. It aims to get plants that are genetically the same as their parents (true to type). The purpose of this study was to see the effect of different type of tissue culture media and plant growth regulators (PGR’s) on the callus induction stage of Pisifera type oil palm. The results show that the treatment using Murasige and Skoog (MS) culture media is able to give a better effect when compared to the use of Eeuwens and Blake (Y3) basic media. While the use of PGR treatment (H6 = NAA 6 mg/l + 2.4-D 0.5 mg/l) showed a callus growth percentage of 76%, which was better than (H1 = Kin 0.1 mg/l + 2.4-D 100 mg/l) by 42%.

Temporal Control of Morphogenic Factor Expression Determines Efficacy in Enhancing Regeneration.

Background: Regeneration of fertile plants from tissue culture is a critical bottleneck in the application of new plant breeding technologies. Ectopic overexpression of morphogenic factors is a promising workaround for this hurdle. Methods: Conditional overexpression of WUS and ARF5Δ was used to study the effect of timing the overexpression of these morphogenic factors during shoot regeneration from root explants in Arabidopsis. In addition, their effect on auxin-signaling activation was examined by visualization and cytometric quantification of the DR5:GFP auxin-signaling reporter in roots and protoplasts, respectively. Results: The induced expression of both WUS and ARF5Δ led to an activation of auxin signaling in roots. Activation of auxin signaling by WUS and ARF5Δ was further quantified by transient transformation of protoplasts. Ectopic overexpression of both WUS and ARF5Δ enhanced regeneration efficiency, but only during the shoot-induction stage of regeneration and not during the callus-induction stage. Conclusions: The overexpression of WUS and ARF5Δ both lead to activation of auxin signaling. Expression during the shoot-induction stage is critical for the enhancement of shoot regeneration by WUS and ARF5Δ.

The regeneration of Acer rubrum L. \u201cOctober Glory\u201d through embryonic callus

BackgroundTissue culture and rapid propagation technology is an important way to solve the difficulties of plant propagation. This experiment aims to explore the appropriate conditions at each stage of the red maple’s tissue culture process and to obtain plantlets, thus providing a theoretical basis for the establishment of the red maple’s tissue culture system.ResultsThe results showed that the stem segment is the most suitable explant for inducing embryogenic callus. The MS (Murashige&Skoog) + 0.8 mg/L TDZ (Thidiazuron) + 1.0 mg/L 6-BA (6-Benzylaminopurine) + 0.5 mg/L IAA(Indole-3-acetic acid) + 35 g/L sucrose+ 7.5 g/L semi-fixed medium was the best for callus formation. When selecting type VI callus as embryonic callus induction material, MS + 0.6 mg/L TDZ + 0.5 mg/L 6-BA + 2.0 mg/L IAA + 35 g/L sucrose+ 7.5 g/L semi-fixed medium can get embryonic callus. The optimal medium for adventitious bud induction is MS + 1.0 mg/L TDZ + 3.0 mg/L 6-BA+ 0.2 mg/L NAA (1-Naphthaleneacetic acid) + 1.2 mg/L IAA + 35 g/L sucrose+ 7.5 g/L semi-fixed medium. The induction rate of adventitious roots in MS + 0.6 mg/L TDZ + 1.0 mg/L 6-BA+ 3 mg/L NAA + 35 g/L sucrose+ 7.5 g/L semi-fixed medium was the highest, reaching 76%.ConclusionsIn the course of our research, we found that PGRs play an important role in the callus induction stage, and the effect of TDZ is particularly obvious; The callus cells grow and proliferate according to the “S” growth curve, and can be sub-cultured when the highest growth point is reached to maintain the rapid proliferation of the callus cells and to avoid inactivation of callus caused by tight niche.

Global profiling of N6 -methyladenosine methylation in maize callus induction.

Callus induction is a dedifferentiation process that accompanies a cell fate transition, and epigenetic regulation plays a crucial role in the process. N6 -methyladenosine (m6A) methylation is an important mechanism in post-transcriptional epigenetic regulation and functions in cell reprogramming. However, the function of m6A methylation during callus induction is still unknown. Here, we performed transcriptome-wide m6A-seq on immature maize embryos after culturing for 2, 4, or 8 days with or without the auxin analogue 2,4-D. A total of 26,794 unique m6A peaks were detected from 17,456 maize genes; and 2,338 specific, 2,4-D-induced m6A peaks (D-specific m6A) were detected only in embryos cultured with 2,4-D. Furthermore, a positive correlation between m6A methylation and mRNA abundance was discovered in the genes with D-specific m6A deposition, especially at the beginning of callus induction. Key genes involved in callus induction, i.e. BABY BOOM and LBD transcription factors, underwent m6A methylation, increasing their transcript levels, thus improving callus induction. These results revealed the importance of m6A methylation during the early stage of callus induction and provided new insights into the molecular mechanism of callus induction at an epitranscriptomic level.

In Vitro Selection of Local Maize (Zea mays) on NaCl Stress and Its Genetic Characterization using RAPD

<p>Maize (<em>Zea mays</em>) is one of graminae plants that widely spread for many purposes wheter in food industry, feed, or bioenergy. Those high utilization required an increment in production, but unfortunately the needs were not meet to the demands since conversion of agricultural area increase lately. Indonesia has 99.093 km of shoreline that recognize as marginal land where the salinity is high as well. This research try to obtain tolerant variant from two local cultivars that planted in Madura Island. Manding and Talango varieties were used as an explant for callus induction stage in MS supplemented with 2.4 D. The result show that 4 ppm of 2.4 D were the best concentration to induce the callus in both varieties. The induced callus were exposed to medium MS that contain NaCl (0, 2500, 5000, and 7500 ppm). In 7500 ppm of NaCl, Manding variety has 100% of surviving callus, while Talango variety only 66.7%. Furthermore, Manding variety shows a better performance in callus weight improvement with 170 mg, while Talango shows no improvement of callus weight. The result of RAPD analysis indicate that the genome characteristic were different between initial callus and surviving callus. Out of ten primers, only five primers shows polymorphism i.e OPA 13, OPB 07, OPC 02, OPK 20, and OPU 19. Manding can be further developed as high tolerance variety in Salinity stress, thus it proposed to be developed furthermore.</p><p>How to Cite</p><p>Saputro, T. B., Finariyah, F., Dianawati, S., Sholihah, N. F., & Ermavitalini, D. (2016). <em>In Vitro</em> Selection of Local Maize (<em>Zea mays</em>) on NaCl Stress and Its Genetic Characterization using RAPD. <em>Biosaintifika: Journal of Biology & Biology Education</em>, 8(3), 344-352. </p>

Genome‐wide analysis of transcription factors involved in maize embryonic callus formation

In this study, a maize inbred line with a strong capacity to induce embryonic callus, 18-599R, was used to analyze the transcription factors expressed during embryonic callus formation. A total of 1180 transcription factors were found to be expressed during three key stages of callus induction. Of these, compared with control, 361, 346 and 328 transcription factors were significantly downregulated during stages I, II and III, respectively. In contrast, 355, 372 and 401 transcription factors (TFs) were upregulated during the respective stages. We constructed a transcription factor-mediated regulatory network and found that plant hormone signal transduction was the pathway most significantly enriched among TFs. This pathway includes 48 TFs regulating cell enlargement, cell differentiation, cell division and cell dedifferentiation via the response to plant hormones. Through real-time polymerase chain reaction (PCR) and degradome sequencing, we identified 23 transcription factors that are regulated by miRNA. Through further analysis, ZmMYB138, a member of the MYB transcription factor family localized in the nucleus, was verified to promote embryonic callus formation in the maize embryo through GA signal transduction.

PEMBENTUKAN KALUS REMAH DARI EKSPLAN DAUN RAMIN (Gonystylus bancanus (Miq) Kurz.)

Ramin (Gonystylus bancanus (Miq.) Kurz) is the most favourite and the most over exploited woody species. This species has been listed in CITES APPENDIX II from 2004. The purpose of this experiment is to obtain the best treatment for friable and embryogenic callus formation that can develop to somatic embryo. Basal Murashige and Skoog (MS) media was used as growth medium. The experiment was conducted in three stages : callus induction and propagation stage, friable callus induction stage and embryogenic callus induction stage. The treatment of 3.0 – 5.0 mg/l 2,4-D was used for callus induction. The best of callus was propagated in 5.0 mg/l 2,4-D + 1.0 – 2.0 mg/l thidiazuron. The best of callus was subcultured for callus friable induction used 6.0 mg/l 2,4-D + 1.0 – 2.0 mg/l thidiazuron + 1.0 – 2.0 mg/l biotin. The best friable callus was subcultured for embryogenic callus induction used 7.0 – 8.0 mg/l 2,4-D + 1.0 – 2.0 mg/l biotin. The observation was made on texture, percentages, performance and color of friable callus. The results showed that callus can be induced on 5.0 mg/l 2,4-D. The treatment of 6.0 mg/l 2,4-D + thidiazuron produced friable callus yellowish on color. 2,4-D + biotin treatment produced very friable callus with yellowish on color and with no embryogenic callus development.

Influence of isolation technique of half-anthers and of initiation culture medium on callus induction and regeneration in Anthurium andreanum

Optimization of the isolation technique and initiation culture medium are two critical aspects that can determine the success of anthurium half-anther culture. Both aspects in half-anther culture of Anthurium andreanum Linden ex Andre cv. ‘Tropical’ were studied and successfully improved. Untreated half-anthers, when cultured abaxial side down on medium, was the most suitable means of inducing callus. Callus formation was further improved by culturing half-anthers adaxial side down on Winarto-Teixeira (WT) medium (Winarto et al. in Plant Growth Regul 65:513–529, 2011b) supplemented with 0.01 mg/l α-naphthaleneacetic acid (NAA), 1.0 mg/l 6-benzyladenine (BA) and 0.5 mg/l thidiazuron (TDZ). Gelrite enhanced callus formation (compared to agar) when the concentration was reduced from 2.0 to 1.5 g/l on WT medium. Application of 0.5 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) in WT medium increased callus formation. Most callus formed when half-anthers were cultured adaxial-side down on WT medium supplemented with 0.5 mg/l 2,4-D in combination with 0.01 mg/l NAA, 1.0 mg/l BA and 0.5 mg/l TDZ using 1.5 g/l Gelrite. This ideal medium induced the growth of half-anthers, with as much as 38 % of half-anthers producing callus, or, on average, 3.4 half-anthers/treatment. Callus derived from this optimized protocol regenerated easily and could be multiplied on New Winarto-Teixeira medium (NWT) (Winarto et al. in Plant Growth Regul 65:513–529, 2011b) containing 0.25 mg/l 2,4-D, 0.02 mg/l NAA, 0.75 mg/l BA, 1.5 mg/l TDZ and 2.0 g/l Gelrite. Shoots rooted well on hormone-free NWT medium with 2.0 g/l Gelrite. The plantlets could be easily acclimatized in a substrate of raw rice husk, burned-rice husk and organic manure (1:1:1, v/v/v) with a high survival (100 %) ex vitro in a greenhouse. The results of this study would benefit half-anther culture of other Anthurium cultivars, particularly at the initial stage of callus induction.

Genome scale transcriptome analysis of shoot organogenesis in Populus.

BackgroundOur aim is to improve knowledge of gene regulatory circuits important to dedifferentiation, redifferentiation, and adventitious meristem organization during in vitro regeneration of plants. Regeneration of transgenic cells remains a major obstacle to research and commercial deployment of most taxa of transgenic plants, and woody species are particularly recalcitrant. The model woody species Populus, due to its genome sequence and amenability to in vitro manipulation, is an excellent species for study in this area. The genes recognized may help to guide the development of new tools for improving the efficiency of plant regeneration and transformation.ResultsWe analyzed gene expression during poplar in vitro dedifferentiation and shoot regeneration using an Affymetrix array representing over 56,000 poplar transcripts. We focused on callus induction and shoot formation, thus we sampled RNAs from tissues: prior to callus induction, 3 days and 15 days after callus induction, and 3 days and 8 days after the start of shoot induction. We used a female hybrid white poplar clone (INRA 717-1 B4, Populus tremula × P. alba) that is used widely as a model transgenic genotype. Approximately 15% of the monitored genes were significantly up-or down-regulated when controlling the false discovery rate (FDR) at 0.01; over 3,000 genes had a 5-fold or greater change in expression. We found a large initial change in expression after the beginning of hormone treatment (at the earliest stage of callus induction), and then a much smaller number of additional differentially expressed genes at subsequent regeneration stages. A total of 588 transcription factors that were distributed in 45 gene families were differentially regulated. Genes that showed strong differential expression included components of auxin and cytokinin signaling, selected cell division genes, and genes related to plastid development and photosynthesis. When compared with data on in vitro callogenesis in Arabidopsis, 25% (1,260) of up-regulated and 22% (748) of down-regulated genes were in common with the genes regulated in poplar during callus induction.ConclusionThe major regulatory events during plant cell organogenesis occur at early stages of dedifferentiation. The regulatory circuits reflect the combinational effects of transcriptional control and hormone signaling, and associated changes in light environment imposed during dedifferentiation.

Light control of shoot regeneration in callus cultures derived from barley (Hordeum vulgare L.) immature embryos

Callus growth and shoot regeneration were examined in cultures of immature barley embryos incubated under various combinations of a week under a 16-h photoperiod and a week under continuous darkness. Incubation in darkness for four weeks, during which calli were formed, enhanced shoot regeneration in ‘K-3’ and ‘Kanto Nijo-5’, but inhibited it in ‘Lenins’. ‘K-3’ and ‘Kanto Nijo-5’, incubated in darkness during the first two weeks of callus induction, followed by two weeks of a 16-h photoperiod (D2L2), showed a higher percentage of shoot regeneration than those incubated for two weeks with a 16-h photoperiod followed by two weeks of darkness (L2D2). Nevertheless, when the light conditions, D2L2 and L2D2, were combined into the same periods of a 16-h photoperiod and continuous darkness, except for the order, light conditions affected shoot regeneration differently. The early stage of callus induction seems to be sensitive to light. The expression ratios of the auxin-responsive gene (AUX/IAA) to the cytokinin-responsive gene (WPK4) were increased in continuous darkness in ‘K-3’ and ‘Kanto Nijo-5’. The effects of light qualities (white, red, far-red, and blue) on callus growth and shoot regeneration were also examined. Blue light inhibited shoot regeneration, as did white light, in ‘K-3’ and ‘Kanto Nijo-5’, and far-red light in ‘Kanto Nijo-5’. Light probably controls shoot regeneration from calli by modifying cytokinin levels and/or response; blue light signals act in photo-inhibition of shoot regeneration in immature barley embryo culture.

Agrobacterium-mediated transformation of Solanum phureja

A population of transgenic plants was produced by the transformation of internodal explants of Solanum phureja, DB337/37 (the cultivar Mayan Gold) using an Agrobacterium tumefaciens LBA4404-based vector containing a phytoene synthase gene (crtB). The regeneration strategy utilised a two-step protocol, with a 12-day callus induction stage mediated by 1.07 microM alpha-napthaleneacetic acid (NAA), 7.10 microM zeatin riboside and 0.06 microM gibberellic acid (GA3), followed by a prolonged (up to 90 day) shoot induction stage on medium containing 0.11 microM NAA, 7.10 microM zeatin riboside and 0.06 microM GA3 supplemented with kanamycin at 50 mg l(-1) as the selection agent. Southern analysis of the transgenic population revealed that the transgene copy number varied between one and five in the lines tested. Northern blot analysis showed significant expression of the introduced crtB gene in some lines during tuber development. Cytological analysis of the material showed a high incidence of chromosome doubling in the transgenic population with over 80% of all lines tested having doubled their chromosome complement during the transformation process.

In vitro chromosome doubling of Miscanthus sinensis

AbstractThe aim was to develop an efficient chromosome doubling method for Miscanthus sinensis to enable the production of triploids and so avoid seed dispersal to the environment. Antimitotic treatments with colchicine or oryzalin were tested in M. sinensis cl. MS‐88‐110 on: (1) in vitro shoots and plants established in soil; (2) during propagation of embryogenic callus; and (c) during the initial stages of callus induction. All systems produced chromosome‐doubled plants. A higher percentage of tetraploids was found after antimitotic treatment at the explant or callus level compared with treatment of in vitro shoots or plants established in soil. In general, oryzalin was more toxic to plant material than colchicine. A higher frequency of chimeras was found among plants with altered ploidy level when the target was formed shoot buds compared with adventitious shoot formation from callus. Antimitotic treatment of embryogenic callus from shoot apices also resulted in a high degree of albinism.

Overcoming phenolic accumulation during callus induction and in vitro organogenesis in water chestnut (Trapa japonica Flerov)

Procedures for callus induction and subsequent organogenesis in the aquatic plant, water chestnut (Trapa japonica Flerov), were established. Phenolics exuded from explants at the callus-induction stage adversely affect callus growth. For cotyledonary node-derived callus cultured in Murashige and Skoog (MS) medium (full, half or quarter strength) containing 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with benzyladenine (BA), the accumulation of phenolics was reduced and callus induction increased by the addition of 10.8 μM phloroglucinol (PG) to the medium. Ascorbic acid was also effective in reducing phenolic accumulation, but less effective for callus induction than PG. Half-strength MS medium supplemented with 2.7 μM 2,4-D, 108.0 μM casein hydrolyzate, and 10.8 μM PG supported maximum callus induction. Plant organogenesis was increased by addition of vitamins (0.27 μM biotin and 2.7 μM folic acid) to half-strength MS medium supplemented with 0.27 μM BA. Many shoots developed from the regenerated nodal shoot explants in liquid half-strength MS salts medium supplemented with 1.08 μM BA and 0.27 μM naphthaleneacetic acid. Individual shoots were excised and cultured in liquid half-strength MS medium supplemented with 5.4 μM IBA and rooted plantlets (108) were transferred and acclimatized in plastic pots. After 3 wk, the plantlets were transplanted in a water chestnut field and the survival rate was 100%.

Genetic control of in vitro response in wheat (Triticum aestivum L.)

The present state of genetic control of in vitro responses of wheat and of the prospeets of its improvement and practical use are presented. The genetic factors affecting different stages of callus induction and of organogenesis in immature embryos are discussed. The dominant genotypie role in the in vitro processes and the cytoplasmic influence on the latter are shown. Genotype × environment and nucleus × cytoplasm interactions were observed. Some unsolved problems needing further investigations of cereals at the tissue culture level are pointed out.

Effects of cytokinin-auxin combinations on cell arrangement in the basal stems and tropane alkaloid production in cultured non-rooted shoots of Duboisia myoporoides

In non-rooted shoots regenerated from calluses induced with nine different cyokinin–auxin combinations, basal stem elongation and the cell arrangement in the basal stem sections depended on the cytokinin–auxin combinations used at the callus-induction stage. Elongated basal stems like seedling and large cells in the secondary xylem were found in the non-rooted shoots regenerated from calluses induced with benzyladenine (BA) 10 µM + naphthaleneacetic acid (NAA) 1 µM and BA 10 µM + indole-3-butyric acid (IBA) 0.1 µM. Tropane alkaloids were detected in these shoots. The peak hyoscyamine and scopolamine contents were 38.5 × 10 –4 and 6.5 × 10 –4 % dry weight, respectively. Various patterns of vascular regions without large cells in the secondary xylem were found in the basal stem sections of the non-rooted shoots regenerated from calluses induced with the other cytokinin–auxin combinations. No tropane alkaloids were detected in these shoots.

Effects of cytokinin/auxin combinations on organogenesis, shoot regeneration and tropane alkaloid production in Duboisia myoporoides

Leaf explants of Duboisia myoporoides R. Br. were exposed to 65 different cytokinin/auxin combinations and the morphogenetic responses compared. Two different types of calluses were induced depending on the cytokinin/auxin combinations used for callus induction. Non-rooted shoots were regenerated from calluses induced with 7 different cytokinin/auxin combinations, cultured in the regeneration medium and also from the calluses induced with 2 different cytokinin/auxin combinations, cultured in the callus induction medium. Shoot-bud regenerating ability of the calluses, number of leaves formed/shoot, lengths of the leaves and shoots and tropane alkaloid biosynthetic ability of the non-rooted shoots depended on the cytokinin/auxin combinations used at the callus induction stage. Hyoscyamine and scopolamine were detected in the 11-week-old callus induced with BA 10 μM + NAA 1 μM. These alkaloids were also detected in the 6 and 9-week-old non-rooted shoots regenerated from callus induced with BA 10 μM + NAA 1 μM, cultured in regeneration medium and from the callus induced with BA 10 μM + IBA 0.1 μM, cultured in callus induction medium. Shoots were regenerated in liquid medium supplemented with BA 10 μM + IBA 0.1 μM. No tropane alkaloids were detected in the 4-week-old shoot cultured in liquid medium.

Fluorescent lipid peroxidation products and antioxidant enzymes in tissue cultures of Vitis vinifera L.

Fluorescent lipid peroxidation products were determined in nodal and internodal stem explants from micropropagated Vitis vinifera L. cv. Sultanina. Fluorescent compounds extracted in the acqueous phase increased in both explants during micropropagation. Solvent extractable products initially decreased in internodal explants, but, later increased during micropropagation. Superoxide dismutase (EC 1.15.1.1) activity did not change significantly during micropropagation. Catalase (EC 1.11.1.6) activity was only detectable in nodal stem sections from micropropagated plants, but, was present in nodal and internodal explants undergoing callogenesis. Fluorescent product content changed considerably in both explants during early callus initiation. Superoxide dismutase activities were similar in nodes and internodes undergoing callogenesis. A reduction in explant fluorescent oxidation products during the latter stages of callus induction was concomitant with increased antioxidant activity.

The histological development of the regenerative tissue derived from cultured immature embryos of wheat ( Triticum aestivum L.)

Histological studies were conducted on the embryogenic tissue of wheat ( Triticum aestivum L.), using primary, subcultured and differentiating callus derived from the scutellum and epiblast of immature embryos. Structures with multiple strata were observed in the late stages of callus induction and on subcultured callus; these were characterized by a middle stratum of ground meristematic tissue with a shoot-forming stratum above and a stratum of large cells below. The shoot-forming protuberances appearing on the differentiating callus were morphologically similar to those observed on subcultured and primary callus and also to the expanding shoot meristem of the cultured embryos. This similarity suggests the continuity of the pre-existing shoot forming tissue during culture in vitro. Typical embryoids have been previously reported on both scutellum and epiblast callus hence this callus could be described as embryogenic. However, organogenesis also occured on the callus as some of the protuberances had a vascular connection to the basal callus. Some of the regenerative outgrowths on the callus were broadly based and were not of single cell origin.