Calli Fresh Weight Research Articles

Increased genetic variability of sugarcane through gamma ray irradiation

To increase the genetic diversity of sugarcane can be done through induced mutation using gamma ray irradiation. This research was carried out to determine the response and radiosensitivity of calli sugarcane variety (Kidang Kencana) to gamma ray irradiation, and knowing the diversities of phenotypic mutant of sugarcane. The research was conducted at BATAN and ICECRD, from August 2012 until March 2013. This research was arranged in Completely Randomized Design with 6 doses of gamma ray irradiation (0, 10, 20, 30, 40 and 50 Gy). Each treatment consisted of 10 replications. Each replication consists of 5 clumps of calli. The observed variables were calli fresh weight, percentage of regenerated calli, number of shoots, shoot height, leaves number, roots number and plantlets number, calli and mutant phenotype. The results showed that the ability of calli to regenerate and shoot growth decreased with increasing doses of gamma ray irradiation. Radiosensitivity (LD20-LD50) of sugarcane calli Kidang Kencana variety to gamma irradiation were in the range of 10 and 30 Gy doses. Gamma irradiation 10 and 20 Gy doses caused the variability mutant phenotype were very high. It means that gamma irradiation can be used to increase the genetic variability of sugarcane.

The induction and development of somatic embryos from the in vitro culture of Catharanthus roseus (L.) G. Don

Catharanthus roseus is containing anticancer alkaloids vincristine and vinblastine as is an important medicinal plant. Several studies have conducted on in-vitro culture of this plant. To optimize the somatic embryogenesis, a factorial based on CRD experiment with 10 replications was conducted. Root, hypocotyl and leaf explants grown in-vitro were transferred and cultured on MS media containing different combinations of 2,4-D, NAA and 2,4-D×BAP. The results revealed that in callogenesis, the interaction effects of root and hypocotyl explants×2,4-D and NAA as well as hypocotyl×(1 mg l-1 NAA+1 mg l-1 BAP) was superior than other treatments (p≤0.01). For calli fresh weight, hypocotyl×NAA and hypocotyl×(1 mg l-1 NAA+1 mg l-1 BAP) was the treatment of choice (p≤0.01). The calli produced were sub-cultured to attain the pre-embryos and somatic embryos. For the number of pre-embryos and somatic embryos; the interaction of hypocotyl×2,4-D was the best and most efficient treatment. Seemingly, the production of somatic embryos is accessible in this plant by the logical management of growth regulator combinations. Furthermore, the production and genetic engineering of the somatic embryos could be a promising trend in the subsequent production of high-valued metabolites from this plant.

Enhancement of Calli Production from Three Cultivars of Jerusalem Artichoke

Jerusalem artichoke (JA) family (Asterceae) is a perennial species known for its tubers rich in inulin, a valuable source of fructose for diabetics.This study aimed to establish an applicable protocol for calli induction and production from different explants of Jerusalem artichoke (Balady, Fuza and Alba) cultivars. For this purpose; leaf, stem and root explants derived from in vitro growing plantlets were cultured on Murashige and Skoog (MS medium) augmented with different combinations of benzyl adenine (BA) and naphthalenacetic acid (NAA).The highest percentage of calli induction (100%) was recorded with all tested media except MS free growth regulators medium and MS fortified with 1mg L-1 BA. On the other hand, the maximum value of calli fresh weight was obtained by culturing stem explants on MS supplemented with 1 mg L-1 BA + 2 mg L-1 NAA for all cultivars. NAA for all cultivars. Moreover and concerning calli growth dynamic for each cultivar, the recorded data clearly showed that gradually increase in calli fresh weights by week reaching the maximum value at eighth week for all used explants cultured on the best medium for calli initiation. The results proved that Balady cultivar recorded the best results for calli induction and production compared with Fuza and Alba cultivars. In the same context, stem explant was the superior explant when it compared with root and leaf explants for all examined cultivars. Moreover and concerning calli growth dynamic for each cultivar, the recorded data clearly showed that gradually increase in calli fresh weights by week reaching the maximum value at eighth week for all used explants cultured on the best medium for calli initiation. The results proved that Balady cultivar recorded the best results for calli induction and production compared with Fuza and Alba cultivars. In the same context, stem explant was the superior explant when it compared with root and leaf explants for all examined cultivars. MS medium fortified with 1 mg L-1 BA + 2 mg L-1 NAA was the most suitable medium compared to the other tested media for inducing calli and to enhance calli production from stem explant for all cultivars.

Influence of PVP/PEG impregnated CuO NPs on physiological and biochemical characteristics of Trigonella foenum‐graecum L

Incorporation of nanoparticles into a number of manufacturing products raised the concern of environmental release via deliberate or accidental routes. Here, experiments were performed to examine the effect of copper oxide nanoparticles (CuO NPs), and polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG) impregnated CuO NPs on seed germination and growth of Trigonella foenum-graecum L. as well as on callus induction through tissue culture technique. Seed germination frequency, length, and weight parameters did not inhibit at higher extent by application of NPs; however, copper acetate, PVP, and PEG significantly decreased the values of all parameters. In all the cases, negative effects were observed concentration-dependent. PVP and PEG impregnated CuO were found less toxic for calli fresh and dry weight induced from leaf and stem explants. The 2, 2-diphenyl-1-picrylhydrazyl reagent-free radical scavenging activity, total antioxidative potential, and total reducing power potential along with total flavonoid and phenolic contents are found elevated in root when compared with shoot. Furthermore, impregnation of PVP and PEG on CuO NPs increases the oxidative response. The results conclude that impregnation of organic molecules on nanoparticles does not reduce the toxicity though can be exploited for enhanced production of secondary metabolites for medicinal purposes.

Optimal concentration of selective agents for inhibiting in vitro growth of Urochloa brizantha embryogenic calli

The ability to distinguish transgenic cells of untransformed cell mass is a key step for the production of transgenic plants. Thus, the use of selection marker genes for identification of genetically modified plants is necessary. The aim of this study was to determine the optimal concentration of four selective agents (kanamycin, hygromycin, phosphinothricin and mannose) to inhibit in vitro growth of Urochloa brizantha cv. Marandu calli. Embryogenic calli were obtained from mature seeds inoculated in MS medium supplemented with 30 g/L sucrose, 3 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 300 mg/L hydrolysate casein and their growth rate was monitored for 74 days by measuring calli fresh weight. It was demonstrated that U. brizantha calli are more sensitive to low concentrations of hygromycin than kanamycin (25 and 50 mg/L, respectively). For the herbicide phosphinothricin, 5 mg/L was enough to prevent the calli growth, but allowed escape. Mannose should be used as the only carbon source on the plant tissue culture medium. All selective agents tested here, in the appropriate concentration, could be used in experiments aiming to produce transgenic signal grass. However, mannose selection might reduce environmental concerns about gene flow and development of herbicide resistance in escaped Urochloa populations. Key words: Signal grass, transformation, marker genes, selection.

Osmotic stress stimulates shoot organogenesis in callus of rice (Oryza sativa L.) via auxin signaling and carbohydrate metabolism regulation

This study aimed to clarify the possible mechanism of endogenous phytohormone signaling and carbohydrate metabolism during shoot organogenesis induced by osmotic stress in rice (Oryza sativa L. cv. Tainung 71) callus. Non-regenerable calli derived from Tainung 71 immature embryos were inoculated on Murashige and Skoog medium containing 10 μM 2,4-D. They turned to highly regenerable calli (HRC) (regeneration frequency more than 75 %) with lower calli fresh weight and water content when 0.6 M sorbitol was supplemented into the medium. The regeneration frequency was prominently decreased to 25 % while an auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), was added into the sorbitol-treated medium. It suggested that endogenous auxin signal may be involved in the induction of HRC under osmotic stress treatment. As well, HRC showed high levels of glucose, sucrose, and starch and high expression of cell wall-bound invertase 1, sucrose transporter 1 (OsSUT1), OsSUT2, PIN-formed 1, and late embryogenesis abundant 1 (OsLEA1) genes. Their expressions are all dramatic inhibited except OsLEA1 under TIBA treatment. It suggests a key role of auxin may be linked to the effect of shoot regeneration under osmotic stress treatment. Therefore, we present a putative hypothesis for regenerable calli induction by osmotic stress treatment in rice. Osmotic stress may regulate endogenous levels of auxin interacting with abscisic acid, then affect carbohydrate metabolism to trigger callus initiation and further shoot regeneration in rice.

Influence of abscisic acid and sucrose on somatic embryogenesis in Cactus Copiapoa tenuissima Ritt. forma mostruosa.

Having produced the embryos of cactus Copiapoa tenuissima Ritt. forma monstruosa at the globular stage and callus, we investigated the effect of abscisic acid (ABA) in the following concentrations: 0, 0.1, 1, 10, and 100 μM on successive stages of direct (DSE) and indirect somatic embryogenesis (ISE). In the indirect somatic embryogenesis process we also investigated a combined effect of ABA (0, 0.1, 1 μM) and sucrose (1, 3, 5%). The results showed that a low concentration of ABA (0-1 μM) stimulates the elongation of embryos at the globular stage and the number of correct embryos in direct somatic embryogenesis, while a high ABA concentration (10–100 μM) results in growth inhibition and turgor pressure loss of somatic embryos. The indirect somatic embryogenesis study in this cactus suggests that lower ABA concentrations enhance the increase in calli fresh weight, while a high concentration of 10 μM ABA or more changes calli color and decreases its proliferation rate. However, in the case of indirect somatic embryogenesis, ABA had no effect on the number of somatic embryos and their maturation. Nevertheless, we found a positive effect of sucrose concentration for both the number of somatic embryos and the increase in calli fresh weight.

Effects of pulsed magnetic field treatment of soybean seeds on calli growth, cell damage, and biochemical changes under salt stress

The effects of magnetic field (MF) treatments of soybean seeds on calli growth, cell damage, and biochemical changes under salt stress were investigated under controlled conditions. Soybean seeds were exposed to a 1.0 Hz sinusoidal uniform pulsed magnetic field (PMF) of 1.5 µT for 5 h/day for 20 days. Non-treated seeds were considered as controls. For callus regeneration, the embryonic axis explants were taken from seeds and inoculated in a saline medium with a concentration of 10 mM NaCl for calli growth analysis and biochemical changes. The combined treatment of MF and salt stress was found to significantly increase calli fresh weight, total soluble sugar, total protein, and total phenol contents, but it decreased the ascorbic acid, lipid peroxidation, and catalase activity of calli from magnetically exposed seeds compared to the control calli. PMF treatment significantly improved calli tolerance to salt stress in terms of an increase in flavonoid, flavone, flavonole, alkaloid, saponin, total polyphenol, genistein, and daidzein contents under salt stress. The results suggest that PMF treatment of soybean seeds has the potential to counteract the adverse effects of salt stress on calli growth by improving primary and secondary metabolites under salt stress conditions.

In Vitro Selection of Foeniculum vulgare for Salt Tolerance

In vitro selection of Foeniculum vulgare for salt tolerance was undertaken by the use of somaclonal variation. In this idea, explants of root, hypocotyl and cotyledon of sterilized seedling were transferred to callus and regeneration media with concentrations of 0, 50, 100 and 150 mM of NaCl. After 4 weeks, calli induction, regeneration frequency and calli fresh and dry weights, in both control and stress conditions, were measured. The results showed that salinity caused a significant decrease in the callus induction and shoot regeneration of fennel. However, in the presence of 100 and 150 mM NaCl, the highest frequency of callus induction in hypocotyl and cotyledon explants was recorded on the media supplemented with 1 mg l-1 IAA (Indol-3-Acetic Acid) plus 1 mg l-1 2,4-D (2, 4- dichlorophenoxyacetic acid) and 2 mg l-1 kinetin. Among different growth regulator treatments, the combination of 2 mg l-1 NAA (Naphtaleneacetic acid) and 0.5 mg l-1 kinetin was found to be the most effective for shoot regeneration under stress condition. The highest dose of NaCl (150 mM) inhibited callus induction and shoot regeneration compared to control with 41% and 96% respectively. The calli fresh and dry weights of all explants were decreased with the increas of NaCl concentration. The highest and the lowest of dry and fresh weight of calli were observed in 0 and 150 mM respectively.

The influence of flask sealing on in vitro morphogenesis of eggplant (Solanum melongena L.)

The influence of flask sealing on eggplant morphogenic responses and morpho-anatomical characteristics was evaluated. Eggplant seeds from the cultivar Embu were disinfected and inoculated in MS medium supplemented with B5 vitamins, 0.55 mM myo-inositol, 2% (w/v) sucrose, and 0.65% (w/v) agar. NAA (53.7 μM) and IAA (0.57 μM) were added to the medium to elicit morphogenic responses from cotyledon and hypocotyl explants via somatic embryogenesis and organogenesis, respectively. The plates were sealed with Micropore® 3M, Parafilm®, or polyvinyl chloride (PVC) film. The effect of glass vessel capping on morphogenesis was also evaluated for shoot apexes inoculated on medium containing half-strength MS where the capping consisted of polypropylene lids with or without two vents (0.45-μm MilliSeal® air vent) and PVC film. Leaf histological analysis and leaf bleaching from each treatment were performed. No significant differences were observed in the number of embryos and root primordia in media containing either 53.7 μM NAA or 0.57 μM IAA. However, embryogenic calli fresh weight was higher for PVC and Parafilm®. Morphogenesis from the shoot apex was influenced, except the plant height. Plants maintained in glass flasks capped with vented lids showed more vigorous growth and differentiated anatomical structures compared to plants under other treatments. This treatment resulted in more expanded leaves, wider stems, and higher dry and fresh weights. In all treatments, the number of stomata was higher in the abaxial surfaces of leaves. Our results indicate that the flasks with vents provided air exchange beneficial for plant morphogenesis.

Effect of physical, physiological and genetic factors on callus induction, differentiation and regeneration of winter triticale (× Triticosecale Wittm.)

Callus was initiated from immature embryos of winter hexaploid triticale cultivars Bogo, Moniko, Moreno, Newcale, Salvo and Vero. Salvo was used in its original version (cytoplasm Triticum aestivum) and as alloplasmic (fertile) inbred line (B11) with Triticum timopheevi cytoplasm (SalvoTt). The MS induction medium was supplemented with 4 mg/cm3 of dicamba. Regeneration was performed on hormone free MS medium. In this study, the influence of genotype, embryo size and cold treatment during the induction phase on the culture efficiency were determined. Calli fresh weight, their differentiation pattern and plant regeneration rate were evaluated. On average from 3 to 14 plants per plated embryo were obtained. Genotype, embryo size and their interaction significantly influenced the culture parameters while the treatment of 5 °C (10 days) applied after 4 weeks of induction made no effect. Salvo and SalvoTt responded at the highest rate to the culture conditions, however T. timopheevi cytoplasm influenced negatively the regeneration rate. Embiyo at late globular or early scutellar stage produced calli with the highest regeneration potential.

Growth and physiological responses of chestnut calli to crude extracts of virulent and hypovirulent strains of Cryphonectria parasitica

The effect of crude extracts (CEs) of virulent (V‐E4) and hypovirulent (H‐E13) Cryphonectria parasitica strains on growth and physiological activities of chestnut calli was investigated on cell cultures obtained from either a susceptible Castanea sativa or a resistant Castanea mollissima chestnut species. The V‐E4 CEs significantly reduced calli fresh weight in both species when used at 50 and 500 μg g–1 and, in general, showed an inhibitory effect on either O2 uptake and H+‐ATPase activity. Only the highest (500 μg g–1) concentration of H‐E13 CEs had a detrimental effect on callus growth, whereas the lowest one (5 μg g–1) induced a slight, but significant early increase in callus fresh weight of both genotypes. The O2 uptake and H+‐ATPase activity were enhanced by the presence of H‐E13 CEs. The decrease of callus growth and physiological activities, caused by V‐E4 CEs, was attributed to toxic compounds produced by the C. parasitica virulent strain, whereas the stimulatory effects of H‐E13 CEs on the overall callus metabolism seemed to be due either to the lack or very reduced amount of phytotoxic compounds and to growth‐regulating substances produced only by the hypovirulent strain of the fungus.

Callus initiation and regeneration in a minor tuber crop 'Rizga' (<i>Plectranthus esculentus</i> N. BR.)

Calli were initiated from tuber cuttings, internodes and leaf disc explants of P.esculentus. From tuber cutting, calli were formed when explants were cultured on MS basal salts supplemented with differing regimes of either 2, 4-D and NAA separately or in combination with 0.5mgl-1 BAP. Initiation of callus was best when cultures were incubated on 2, 4-D containing media. 2, 4-D at a concentration of 1mgl-1 and culture condition of total darkness/25oC gave maximum calli fresh weight of 541.5 ± 43.9mg. In the case of internodes, maximum callus (82.3 ± 18.4mg) was obtained when explants were incubated on MS medium supplemented with 50gl-1 sucrose and 1mg-1 2, 4-D. Leaf disc explants gave maximum calli fresh weights of 2,462.8 ± 279.7 and 1,688.8 ± 350.9 mg at sucrose concentration of 50 and 60gl-1, respectively. Histological assessment of calli initiated from explants cultured on MS basal salts supplemented with 50gl-1 sucrose and 1mgl-1 2, 4-D and CP -a cytokinin, suggested caulogenesis and somatic embryogenesis might have occurred on CP and 2, 4-D containing media, respectively. Key words: Callus, regeneration, tuber, Plectrantus esculentus [Global Jnl Agric Sci Vol.1(1) 2002: 55-62]