Medium For Callus Formation Research Articles

Induksi dan Multiplikasi Kalus Eucalyptus sp. pada Berbagai Media Callus Induction Medium (CIM) Secara In Vitro

Callus Induction and Multiplication of Eucalyptus sp. on Various Media Callus Induction Medium (CIM) In Vitro. Callus induction in vitro can be done by planting specific part of plant such as leaf, root, stem, etc in aseptic condition by using medium that contains 2,4-D, a plat regulator for promoting callus formation. One of the objectives of callus production in vitro is to form secondary metabolites, because eucalyptus is known as a producer of essential oils. This research is an initial study for in vitro production of secondary metabolites for further research. The aim of this study was to determine the most suitable callus induction medium for callus formation and callus multiplication for Eucalyptus grown in vitro. This research was designed as a Completely Randomized Design (CRD), five treatments, repeated five times. Woody Plant Medium (WPM) was used as the basic media. Several Callus induction Medium (CIM) were used as treatments, i.e. CIM1 = 1 ppm 2,4-D; CIM2 = 1,5 ppm 2,4-D; CIM3 = 2 ppm 2,4-D; CIM4 = 1 ppm NAA+1 ppm BAP; and CIM5 = 2 ppm NAA+ 2 ppm BAP. Variables observed were the time of curly leaf formation, time of the emerging callus, colour and texture of the callus, callus’s weight, callus surface area, the weight of callus after multiplication. The result showed that CIM4 and CIM5 had the fastest emerging callus (7 days after planting), CIM1 and CIM2 formed green and crumb texture of callus, CIM1 produced the highest of callus’s weight, and CIM2 resulted in the highest of callus’s surface area. In conclusion, the most suitable CIM for callus induction was CIM1, while for callus multiplication was CM2.

Rosmarinic acid inhibits programmed cell death in Solanum tuberosum L. calli under high salinity

Oxidative stress induced by salinity is a prime cause of cell death when Na+ toxicity becomes unbearable. We explored the effect of rosmarinic acid (RA) on the Solanum tuberosum L. cv. Desiree calli against salt-induced programmed cell death (PCD). We showed that PCD events were triggered in calli under 250 mM NaCl by the loss of plasma membrane integrity as measured by the amount of malondialdehyde (MDA) in the cytoplasm, the degree of DNA degradation resulting from the cleavage of nuclear DNA into oligonucleosomal fragments in apoptotic cells, the presence of TUNEL-positive nuclei (90 ± 0.005%) damage in genomic DNA, and activation of caspase 3-like protease. Callus Formation Medium (CFM) supplemented with RA led to the suppression of salt-induced cell death and a dramatic decrease in the MDA level and frequency of TUNEL-positive nuclei under salinity to 4 ± and 7.3 ± % in the presence of 50 and 350 μM RA, respectively. The application of RA also resulted in an increase in GSH content and maintenance of a high GSH/GSSG ratio. Interestingly, these reductions in PCD were accompanied by inhibiting caspase 3-like protease activities due to RA under salinity. Molecular docking predicted high binding energies of RA for binding to subtilisin-like protease (StSCTc-3), which has caspase-3 like activity in Solanum tuberosum, near the active site. This finding supports the notion of a role for RA in PCD protection in plants, which is consistent with earlier reports in animal cells.

Bazı siklamen türlerinde farklı doku kültürü ortamlarının gynogenesis üzerine etkileri

Cyclamen breeding is difficult due to selfing depression different ploidy levels, self-incompatibility and abortive embryo formation. Therefore, in vitro haploidisation is commonly used in cyclamen breeding. In this study, we investigated effects of different tissue culture media and plant hormones as a first of in vitro haploidisation and propagation. We used ovule explants from Cyclamen persicum and Cyclamen pseudibericum on ½MS and B5 culture mediums supplemented with different concentration of 2,4-dichlorophenoxyaceticacid (2,4-D) and 6-(γ,γ –dimethylallylamino) purine (2iP). According to results best callus formation rate was 70% obtained from B5 medium supplemented with 1.5 mg L-1 2,4-D and 0.5 mg L-1 2iP in Cyclamen persicum. It was 48% on B5 medium supplemented with 1 mg L-1 2,4-D and 0.3 mg L-1 2iP in Cyclamen pseudibericum. In Cyclamen persicum, highest embryo induction rate was 55% obtained from callus regenerated on ½MS medium supplemented with 1.5 mg L-1 2,4-D and 0.8 mg L-1 2iP. However, embryo induction was not obtained on any medium for Cyclamen pseudibericum. Overall, ½MS culture medium supplemented with high auxin and low cytokinin concentration is better for embryo induction although B5 medium was better than ½MS medium for callus formation and 42 plantlets obtained only from callus regenerated on ½MS medium supplemented with 2.0 mg L-1 2,4-D and 0.5 mg L-1 2iP.

In vitro micropropagation of Anthurium andraeanum through thin cell layer culture

Anthurium is a kind of major cut flower species and economically important genera in the family Araceae. A regeneration system of Anthurium andreanum from callus culture is studied along with improving propagation process. Optimal medium for callus formation from the longitudinal thin cell layer leaf culture was ½ MS (Murashige, Skoog, 1962) supplemented with 30 g/l sucrose, 1 g/l casein hydrolysate (CH), 8 g/l agar, 1.5 mg/1 BA and 0.2 mg/1 2,4-D (the induction callus rate of 77.33%). Calli multiplied with 21.45-fold fresh mass increase when they were subcultured once 60 days on the ½ MS supplemented with 1 g/l CH, 30 g/l sucrose, 1,5 mg/l BA and 8 g/l agar. The highest ratio of shoot induced from callus culture was also on this medium (18.54 shoots per explants). The medium ½ MS supplemented with 20 g/l sucrose, 1 g/l AC, 8 g/l agar and without plant growth regulators is the most suitable one for root formation of these multiple-shoots. The highest survival rate is observed with substrate mixture of fern moss and rice husk ash in the ratio of 1:1 (gave the best regeneration rate of 100% in the ex vitro culture) and no morphological variations were observed in these plantlets after about thirty days,acclimatized in the greenhouse. Histological observation showed that these calli contained embryogenic cells with their fast growth.It could be an alternative tool for propagation and preservation of Anthurium andreanum, a valuable cultivar.

Devolopment of Some Canola Genotypes to Salinity Tolerance Using Tissue Culture Technique

T HIS WORK was carried out during 2011- 2015 in the laboratories of Cell Research Department (CRD), Field Crops Research Institute (FCRI), Agricultural Research Center (ARC), Giza, as well as, in the greenhouse of Agronomy Department, Faculty of Agriculture, Ain Shams University, Egypt, to study the variation among canola varieties in response to salt tolerance through tissue culture propagation. This work was aimed to establish a regeneration protocol for canola genotypes, production of salt-tolerance plants through selection of salt-tolerance callus via somaclonal variation and subsequent regeneration of plantlets from such callus. Four genotypes of canola were used for establishment a regeneration protocol, Bingo and Torpe were uesed as highly salt tolerant genotypes while, Conny and Siberian as susceptible genotypes. The results indicated that MS medium supplemented with 2 mg/l 2,4-D was the successive medium for callus formation and shoot regeneration was obtained from MS medium supplemented with 5 mg/l BA + 0.05 NAA, while, 1mg/l IBA was the better growth regulator for rooting.For in vitro selection of salt tolerant calli, Torpe (genotype overcomes the others in calli survival percentages under 8000, 12000 and 16000 ppm NaCl concentrations. The results of shoot frequency from tolerant calli cleared that Torpe genotype recorded the highest value of shoot percentage followed by Bingo genotype, whereas, Conny followed by Siberian genotypes were recorded the lowest values. At 16000 ppm NaCl concentration, only tolerant calli of Torpe genotype was able to initiat shoot under this concentration. For acclamatization after regeneration from the tolerant calli, only five salt tolerant plantlets developed from Torpe genotype and eight salt tolerant plantlets developed from Siberian genotype that succeeded to complete their life cycle and reached maturity stage and produced seeds. These regenerated genotypes were referred as T1, T2, T3, T4 and T5, the five regenerated plants developed from Torpe genotype that tolerate NaCl concentration up to 16000 ppm, as well as, S1, S2, S3, S4, S5, S6, S7 and S8, the eight regenerated plants developed from Siberian genotype that tolerate NaCl concentration up to 12000 ppm.

REGENERATION OF CALLUS AND SHOOTS FROM THE OVULES AND OVARIES OF CHRYSANTHEMUM IN VITRO

Unfertilized chrysanthemum ovaries and ovules were investigated in terms of callus and shoots regeneration in vitro on media with a different plant growth regulators content. Ligulate florets of cultivar ‘Capitola’ were collected from five peripheral whorls of mature inflorescences and sterilized. The ovules were isolated from sterile florets under spectroscopic microscope, while the ovaries were precisely cut off from the florets. The explants were inoculated onto six MS based induction media, solidified with 0.8% agar, supplemented with different concentrations of BAP and 2,4-D (1+1;1+2;1+3; 2+1; 2+2; 2+3 mg/L of BAP + 2,4-D, respectively). For 5 weeks cultures were kept under 24°C, light was supplied by fluorescent tubes emitting daylight, photon flux density was 12 µmol/(m2s), 16/8 h of day/night photoperiod. Afterwards, one half of the explants were transferred onto regeneration medium supplemented with 2 mg/L kinetin, 1 mg/L IAA and 4 mg/L glycine. Starting from the fifth week of culture up to the fifteenth week, both, transferred and non-transferred explants were maintained at 32 µmol/(m2s) photon flux density. 90% of ovaries and 38% ovules formed callus. The best medium for callus formation was supplemented with 1 mg/L 2,4-D and 2 mg/L BAP. The shoots regenerated only from ovaries, 10% of ovaries produced shoots; a total number of 103 shoots were formed. The transfer of ovaries onto the regeneration medium containing kinetin and IAA promoted shoots formation; most shoots regenerated from the ovaries subcultured from the medium containing 1 mg/L 2,4-D and 1 mg/L BAP. Mean numbers of shoots per inoculated explant from non-transferred and transferred ovaries were 0.10 and 0.25, respectively.

Establishment of a Simple Plant Regeneration System Using Callus from Apomictic and Sexual Seeds of Guinea Grass (Panicum maximum)

Aims: For analysis of apomixis genes, as the first step, an efficient and simple plant regeneration system has been established using callus from apomictic and sexual seeds of guinea grass (Panicum maximum). Study Design: The best basic medium for callus formation from matured seeds of guinea grass was selected, and then, the best combinations of growth regulators on different media were selected for plant regeneration by indirect organogenesis. Methodology: Guinea grass accessions of sexual and apomict of seeds were used for culture. Seeds sterilized were cultured in Murashige and Skoog [10] medium (MS) and in that proposed by Chu et al. [12] (N6D) for callus formation. The best medium and, the effects of L-proline and growth Original Research Article Chen et al.; BBJ, 7(4): 183-190, 2015; Article no.BBJ.2015.059 184 regulators’ type and concentrations on callus formation and plant regeneration in 3 accessions were examined, respectively. After the plantlets rooting in MS hormone-free medium, the complete plants were planted in pots for hardening. Results: N6D medium has given better rates for callus formation, that is, 97.1% in sexual N68/968-o-5, and 91.7% and 84.6% in apomict N68/96-8-o-11 and ‘Natsukaze’, respectively. MS medium with 0.2 mg/l of Kinetin has given the best rate or plant regeneration among the used 4 kinds of the media. For each material, the best results were obtained on MS with 0.2 mg/l of Kinetin for N68/968-o-5, and MS with 2.0 mg/l of L-proline and 0.2 mg/l of Kinetin for ‘Natsukaze’. After hardening of the regenerated plants in soil, 100% of surviving rates were obtained, and showed normal growth comparing with the plants-derived from seeds. Conclusion: We have established, as the first case, an efficient and simple plant regeneration system by using callus from apomictic and sexual seeds of guinea grass for analysis of apomixis genes, consisting of analysis of the best media, L-proline usages and phytohormone combinations for callus formation, plant regeneration and hardening in different stages, respectively.

Establishment of callus and cell suspension culture of Scrophularia striata Boiss.: an in vitro approach for acteoside production.

In the present study, a protocol was optimized for establishment of callus and cell suspension culture of Scrophularia striata Boiss. as a strategy to obtain an in vitro acteoside producing cell line for the first time. The effects of growth regulators were analyzed to optimize the biomass growth and acteoside production. The stem explant of S. striata was optimum for callus induction. Modified Murashige and Skoog medium supplemented with 0.5mg/l naphthalene acetic acid+2.0mg/l benzyl adenine was the most favorable medium for callus formation with the highest induction rate (100%), the best callus growth and the highest acteoside content (1.6μg/g fresh weight). Incompact and rapid growing suspension cells were established in the liquid medium supplemented with 0.5mg/l naphthalene acetic acid+2.0mg/l benzyl adenine. The optimum time of subculture was found to 17-20days. Acteoside content in the cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The maximum content of acteoside (about 14.25μg/g cell fresh weight) was observed on the 17th day of the cultivation cycle. This study provided an efficient way to further regulation of phenylethanoid glycoside biosynthesis and production of valuable acteoside, a phenylethanoid glycoside, on scale-up in S. striata cell suspension culture.

Mutagenesis in olive (Olea europaea L.) calli caused by sodium azide and detection of mutants using ISSR and RAPD markers

SummaryIn vitro callus formation was established in the olive (Olea europaea L.) cultivar ‘Kronaki’ and in vitro mutagenesis of calli was carried out using five different concentrations (1.0 – 5.0 mM) of sodium azide (NaN3). Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to detect genetic polymorphism among the olive mutants produced. Six RAPD and seven ISSR primers were used to amplify DNA segments from the genomes of untreated and NaN3-treated calli. The optimum medium for callus formation was 0.5× Murashige and Skoog (MS) medium, supplemented with 2 mg l–1 αnaphthaleneacetic acid and 0.5 mg l–1 2-isopentenyl-adenine. A total 60 RAPD amplicons and 60 ISSR fragments were detected. The sizes of the PCR amplicons varied from 200 – 3,000 bp in length. Fifteen of the 60 RAPD (40%) and ten of the 60 ISSR (16.6%) fragments were polymorphic. The results showed that 4.0 mM and 5.0 mM NaN3 were the most effective doses for mutagenesis. Based on RAPD, ISSR, and combined RAPD and ISSR marker analysis, it was shown that all NaN3 treatments caused detectable levels of mutation. The use of NaN3 for in vitro mutagenesis in olive calli resulted in genetic dissimilarity values of 0.14 – 0.21, and represents a useful way to induce mutants in olive which could be exploited in the future for trait improvement and in olive breeding programmes.

Introduction to the cell culture and production of brachycome iberidifolia and chrysanthemum carinatum

Brachycome iberidifolia and chrysanthemum carinatum as cell cultures. The optimal medium for callus formation, regeneration and rooting of regenerated plants are found.

Improvement of caper ( Capparis spinosa L.) propagation using in vitro culture and gamma irradiation

Some of the factors influencing the propagation of caper ( Capparis spinosa L.) plants in vitro and germination of the seed were studied. The number of adventitious shoots emerging from caper stems cultured in vitro increased from 2.2 shoots per explant when the growth medium contained 2 mg/L of gibberellic acid (GA3) to 5.5 when the growth medium contained 2 mg/L zeatin riboside (ZR) and 1 mg/L naphthalene acetic acid (NAA). The best medium for callus formation from leaf and stem parts contained the growth regulators 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L NAA and the best medium for plant regeneration contained 1 mg/L kinetin and 0.1 mg/L indole-3-acetic acid (IAA). The effect of gamma irradiation on the growth of caper shoots in vitro was also studied. A 10 Gy dose of gamma irradiation stimulated growth of shoots up to 200% and increased shoot rooting percentage from 75 to 100%. Methods of scratching the seed coat with iron particles and treating the whole seeds with concentrated H 2SO 4, ultrasonic waves and gamma rays were employed for breaking the seed dormancy. Treating the seed with H 2SO 4 for 20 min along with scratching was very effective in stimulating germination (46% as compared with 0% for the control). Irradiating caper seeds with 100 Gray (Gy) dose of gamma rays led to 50% seed germination in vitro and 70% on peatmoss one month after culturing.

Shoot tip and cotyledon explants of Eucalyptus saligna Sm. cultivated on different kanamycin levels

An efficient selec tive agent helps to obtain transgenic plant s; the most used selective agent for genetic transformation of Eucalypt is kanamycin. The aim of this research was to determine ideal dose of kanamycin in shoot tip and cotyledon explants of E. saligna for use as selective agent in genetic transformation. Two experiments were carried out, first in order to test effects of the kanamycin in cotyledons and second, in order to test effects of the kanamycin in shoot tip. In the cotyledons experiment were tested 0, 12.5, 25, 37.5 and 50 mg.L-1 kanamycin on a callus formation medium and for shoot tip were tested 50, 75, 100, 125 and 150 mg.L-1 kanamycin on a multiplication medium. Cotyledons can be selected more quickly than shoot tip, moreover needs lower kanamycin levels than shoot tip, what results more speed and economy during genetic transformation process. It is probable that levels lower than 12.5 mg.L-1 kanamycin must be ideal for the cotyledons selection and 50 mg.L-1kanamycin for shoot tip.

Efficient transformation and regeneration of fig (Ficus carica L.) via somatic embryogenesis

Fig is one of the most important fruit trees in Egypt. It used to constitute the major source of income for the inhabitants of the western north coast of Egypt. Since 1993 fig cultivations were threatened by a number of factors including virus, insect and mite infections. An efficient system for regeneration and transformation of the common fig Ficus carica L. cultivar Sultani (fresh consumption) was required to conserve fig cultivation in the area. The effect of different combinations of BA and NAA/2,4-D and kinetin on callus formation from leaf segments were studied. Results showed that the best medium for callus formation was MS supplemented with 2.0 mg/l 2,4-D and 0.2 mg/l kinetin. The best plantlet differentiation was obtained at concentrations of 30 mg/l 2iP and 7 mg/l TDZ with 0.25 mg/l NAA (with a regeneration efficiency of 83 and 79%, respectively). On the other hand, the obtained callus failed to induce organogenesis on media containing a combination of BA and kinetin. The highest shoot formation percentage (89%) was obtained when using 2 mg/l TDZ and 4 mg/l 2iP. The highest percentage of shoots forming roots (95%) was obtained when using MS medium supplemented with 1.0 mg/l IBA. Explants were transformed using Agrobacterium and microprojectile bombardment using the plasmid pISV2678 which harbors the gus-intron and bar genes. Results showed that the highest transformation efficiency using the Agrobacterium (17.5%) was obtained when explants were co-cultivated with the bacteria for 30 min. The highest transformation efficiency recorded using the microprojectile bombardment (12%) was obtained with 2.0 μg DNA per shot at 1,100 psi and a distance of 6 cm repeated twice. The transgenic nature of regenerated plants was confirmed by PCR analysis, histochemical GUS assay and leaf painting assay.

Callus induction and plant regeneration of two Cuban rice cultivars using different seed explants and amino acid supplements

Most of Cuban rice cultivars are classified into indica subspecies, and they are inclined to poor in vitro response. In this paper we studied the role of endosperm and amino acids on callus formation of two Cuban rice cultivars: J-104 and IACuba-28. Callus cultures were initiated from three treatments for mature seed: intact seed, embryo with scutellum but without endosperm, and endosperm alone. It demonstrated the direct incidence of endosperm on in vitro seed contamination. But the higher percentage of embryogenic calli was obtained from intact seeds, despite of 12.94 % of seed contamination. Callus formation from endosperm alone did not occur. The role of endosperm to successful callus formation from scutellum was discussed. Effect of amino acids on rice callus growth from intact seeds was examined by supplying callus formation medium with glutamine and proline, separately or in combination, in both cultivars. Callus formation of J-104 was improved considerably with 500 mg/l of proline and glutamine in the culture medium, but in IACuba-28 were not observed significant changes. The percentage of embryogenic callus and the increase of fresh weight of calli were correlated with genotype and amino acid supplement in culture medium.

Establishment of Callus and Cell Suspension Cultures of Corydalis saxicola Bunting, a Rare Medicinal Plant

An efficient procedure has been developed for callus induction and cell suspension cultures of C. saxicola for the first time. Explant selection was carried out among leaf, stem and root to select a suitable type of explants capable of higher callus formation. Leaf explants thus selected showed maximum response to callus induction (67.1%). Modified B5 medium supplemented with 0.5 mg l(-1) 2,4-D plus 2 mg l(-1) BA was the most favorable medium for callus formation with the highest induction rate (94.8%) and greatest fresh weight of callus (1.7 g per explant). Cell suspension cultures were established by transferring 2-8 g fresh callus to 80 ml liquid B5 medium. An inoculum size of 8 g produced the greatest biomass accumulation, dehydrocavidine and berberine productions, which was 13.1 g l(-1), 8.0 mg l(-1) and 4.1 mg l(-1), respectively. In response to various sucrose concentrations from 10 g l(-1) to 80 g l(-1), cultures with 60 g sucrose l(-1) not only produced the highest dry biomass (18.5 g l(-1)) but also the highest formation of dehydrocavidine (11.6 mg l(-1)) and berberine (7.6 mg l(-1)). These prepared cell suspension cultures provided a useful material for further regulation of alkaloid biosynthesis and for enhanced production of valuable alkaloids on a large scale.

Regeneration potential of CIMMYT durum wheat and triticale varieties from immature embryos

AbstractTwenty‐five durum wheat elite advanced lines and released varieties, and five triticale varieties were evaluated for their ability to produce embryogenic callus using three different media. For callus initiation and maintenance there were basal Murashige and Skoog (MS) medium containing double strains of macroelements and 2.5 mg/l 2,4D (DW1), basal MS medium containing 2.0 mg/l 2,4D (DW2), or basal MS medium supplemented with 1.0 mg/l 2,4 D and coconut milk (DW3). Plant regeneration was achieved on basal MS medium with indoleacetic acid and 6‐benzylaminopurine, and plants rooted on MS with 1‐naphthale‐neacetic acid. DW3 medium proved better than the other media tested for embryogenic callus initiation and maintenance. Regeneration rates varied widely with both genotype and initiation medium, with values ranging from no regeneration to 100% regeneration; the plantlets produced per embryo ranged from five to 20. Fourteen of the durum wheat genotypes showed 63–100% regeneration from DW3 callus formation medium, four lines from DW1 medium, and two lines from DW2. Four of the triticale varieties had regeneration of 48–100% from DW3 medium. After six subcultures, over a 6‐month period, genotypes lost their ability to regenerate plants. Only 10 lines retained some plant regeneration potential but regeneration was at reduced levels. Successful regeneration of durum wheat and triticale varieties will be used as an integral part of the transformation process.

Establishment of Culture Medium for Protoplasts and Plant Regeneration in Japanese Bunching Onion (Allium fistulosum L.).

For the purpose of establishing protoplast culture systems in Japanese bunching onion (Allium fistulosum L.), based on BDS inorganic salts (Dunstan and Short, 1977), nutrient media suitable for protoplast culture were investigated. The optimum cell division was achieved with 5 mM potassium nitrate and a combination of 2μM 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.2 or 1 μM 6-benzylaminopurine (BAP). The suitable osmolality for protoplast culture was around 0.60 osmol·kg-1, whereas a combination of 0.2 M sucrose and 0.2 M glucose accelerated cell division. The protoplasts developed into colonies by gradual reduction of sugar concentration. After 45 days of culture, numerous micro-calli formed which were subsequently transferred to a callus formation medium. Plantlets were regenerated by transferring protoplast-derived calli of ca. 2 mm in diameter to a modified Murashige-Skoog medium. Of the 75 calli inoculated, 24 (32%) gave rise to green shoots on a regeneration medium 2 to 3 months after inoculation. These shoots rooted when cultured on a hormone-free medium. These plantlets were successfully acclimatized and grew normally in a greenhouse.

Plant Regeneration from Mesophyll Protoplasts of Spinacia oleracea L.

Mesophyll protoplasts of Spinacia oleracea L. (cv. JIROMARU) were isolated from the in vitro-grown young seedlings by treatment of the enzyme solution containing 0.1% Pectolyase Y-23, 0.3% Cellulase ONOZUKA RS, CPW salts and 0.5M mannitol (pH 5.8). The most efficient cell division and colony formation occurred in a modified 1/2MS medium containing 5mg/l BA and 1mg/l 2, 4-D. Shoots were induced in the following four treatments: a combination of the callus formation medium containing 1mg/l BA and 10mg/l NAA with the 2 kinds of shoot formation media containing 1mg/l kinetin or 5mg/l zeatin; a combination of the callus formation medium containing 5mg/l BA and 5mg/l NAA with the shoot formation medium containing 5mg/l BA; a combination of the callus formation medium containing 5mg/l BA and 10mg/l NAA with the shoot formation medium containing 1mg/l kinetin. Regenerated shoots developed roots in the MS liquid medium containing 1mg/l IAA or IBA.

Isozyme Patterns in Tobacco Plant Parts and Their Derived Calli1

This study was initiated to determine if different tissues of the same plant maintain persistent tissue specific patterns of gene expression during in vitro culture or if in vitro cultures are characterized by identical patterns of gene expression no matter what their tissue of origin. Two‐millimeter sections were excised from the root, lower leaf, upper leaf, pith, sepal, ovary, and anthers of a mature ‘Wisconsin 38’ tobacco (Nicotiana tobacum L.) plant and were grown on a common medium for callus formation. Transfer to fresh medium was made when the calli reached about 8 mm in diameter. One to five such transfers were made depending upon the growth rate of different explants. Extracts from the original explants and each callus transfer were assayed for several isozyme systems using horizontal starch‐gel electrophoresis. Malate dehydrogenase and peroxidase isozymes were found to have good xesointion and to be suitable for the study. The patterns of the isozymes displayed tissue specific differences at the explant level; however, substantial differences between the isozyme patterns of the calii and that of the original explant were displayed. Initially there were differences in isozyme patterns among the calli from different origins and within the different transfers of the same explant callus. However, after the second or third callus transfer, isozyme patterns became more uniform for calli of the same explant.

Regulation of organogenesis in small explants of superficial tissue of Nicotiana tabacum L.

Small explants composed of 3-6 layers of epidermal and subepidermal cells excised from floral branches of Nicotiana tabacum are capable of de novo organogenesis. Continuous mitoses without organogenesis, or mitoses followed by the formation of floral buds, vegetative buds or roots can be obtained by varying the auxin-cytokinin-sucrose ratio in the medium. The physiological stage of the floral branches, and light and darkness had also an influence on the de novo organogenesis. Best floral-bud formation was obtained when explants were taken from plants in which the terminal bud was in the green-fruit stage, and were cultured on a medium with indole-3-acetic acid and kinetin both at 10(-6) M and with 3% sucrose, in the light. For differentiation of vegetative buds, the optimal auxin/cytokinin ratio was one tenth of that for floral-bud formation, while the sucrose concentration was the same. Optimal conditions for root formation were: explants from plants in which the terminal bud carried a mature fruit; 10(-7) M kinetin, 10(-5) M indole-3-butyric acid, and 1% sucrose in the medium; and culture in darkness. The optimal medium for callus formation contained 5×10(-6)M 2,4-dichlorophenoxyacetic acid, 10(-7) M kinetin and 3% sucrose.