Callus Fresh Weight Research Articles

Effect of NaCl and Sorbitol on the Production of Some Alkaloids of Fenugreek Cotyledons Derived Callus

Alkaloids often had pharmacological effects. The aim at this study is to increase some alkaloids in fenugreek. Callus induction occurred on cotyledon segments 5 mm long. Murshige and Skoog medium (MS) supplied with 1 mg.lˉ¹ of 2, 4 – D and 0.4 mg.lˉ¹ of kinetin was prepared. The best medium maintained callus was using MS medium supplied with 0.5 mg.lˉ¹ 2, 4-D and 0.5 mg.lˉ¹ kinetin. Callus formation was allowed to take place for a period of thirty days in the dark at 25°C±2. Calli were analyzed using high performance liquid chromatography (HPLC).Methanol callus extract showed high concentrations of some alkaloids compared to the methanol extract of cotyledons. For increasing, the concentration of secondary metabolites, NaCl was added at concentrations 0, 1, 1.5 or 2 g.lˉ¹, sorbitol at concentrations 0, 7, 8 or 9 g.lˉ¹. NaCl at 2 g.lˉ¹ led to significant increase in trigonelline reaching 1354.72 µg per 100 mg fresh weight of callus. NaCl at 1g.lˉ¹ increased choline and carpaine reached 400.82, 483.92 µg.mlˉ¹ per 100 mg respectively. Treatment with 9 g.lˉ¹ sorbitol increased trigonelline and carpaine reached 1666.41, 742.67 µg per 100 mg respectively, while treatment 8 g.lˉ¹ sorbitol significantly increased in choline recorded 588.87 µg per 100 mg.

Trends in accumulation of ephedrine in callus cultures of Ephedra major Host

Ephedra major Host, a medicinal plant, belongs to the family of Ephedraceae. Ephedrine is the main alkaloid in Ephedra, which has different medicinal properties. However, the amount of ephedrine in plant material is low and callus culture can be a way to increase the alkaloid content. The aim of this research was to compare Murashige and Skoog (MS) and Gamborg’s B5 culture media for callus induction and ephedrine production. For this purpose, stem explants were cultured on MS or B5 media containing 0.0, 0.5, 1.0, 2.0, or 3.0 mg L−1 of kinetin (Kin) either alone or in combination with 0.0, 0.5, 1.0, or 2.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and/or naphthalenacetic acid (NAA), in five replicates. MS medium containing 1.0 or 2.0 NAA and 0.5 mg L−1 Kin were the most effective for callus induction. The highest percentage of callus induction (100%) on B5 culture medium was obtained with 2.0 2,4-D and 0.5 mg L−1 Kin treatments. The results showed that there was no significant difference between MS and B5 media for callus induction, and fresh and dry weight production. High-performance liquid chromatography was conducted for the identification and quantification of ephedrine in the obtained callus. The highest level of ephedrine (7.38 mg g−1 DW) was found in callus grown on MS medium containing 0.5 mg L−1 of 2,4-D. The results revealed that ephedrine can accumulate in callus cultures to levels much higher than in E. major wild plants.

In vitro-examination of genetic parameters and estimation of seedling physiological traits under drought and normal conditions in durum wheat

An investigation was carried out to evaluate genetic parameters of in-vitro traits including callus size (CS), fresh weight (FW), dry weight (DW) and relative water content (RWC) under normal and drought conditions for seventeen durum wheat genotypes in a factorial experiment. Analysis of variance revealed significant differences between the genotypes for all traits. The results indicated that the phenotypic coefficient of variation was higher than the genotypic coefficient of variation for all traits. High heritability and genetic advance of traits showed relatively low environmental effects on the traits and thus the chance of effective selection. Genotypic response to drought stress under in-vitro condition in terms of mean comparison demonstrated that the genotypes, 19E-M84859 (G6) and 19E-M141994 (G9) achieved higher levels of CS and RWC under both normal and stress conditions. The genotypes, 19E-TOPDY (G4) and 19E-142069 (G16) had minimum difference between fresh and dry weights in normal and stress conditions. These genotypes had higher genetic potential for response to drought stress. The germplasm evaluation based on physiological traits showed variation for some antioxidant enzyme activities and biomass of genotypes under both normal and stress conditions. The results of present study on genetic diversity among the genotypes may be utilized in wheat breeding programs.

EFFECTS OF DIFFERENT CROTON (Codiaeum variegatum L.) GENOTYPES AND GROWTH REGULATORS ON CALLUS INDUCTION, MICRO PROPAGATION AND ANTIBACTERIAL ACTIVITIES

The Main aim of this study was determine the effect of different croton(Codiaeum variegatum) genotypes (Gold Dust and Petra) and growth hormones on callus induction, micro propagation and antibacterial activities. Murashige and skoog (MS) medium with four compositions of growth hormones for callus induction, murshM1 (2 mg/l 2, 4 Dichlorophenoxy acetic acid (2, 4-D), M2 (3 mg/l 2, 4-D), M3 (2mg/l 1-Naphthalen acetic acid (NAA), M4 (1mg/l 2, 4-D + 1 mg/l NAA) were used. For micro propagation, four different hormone balance were used, i.e., M1 (1 mg/l Benzyl adenine (BA) + 25 mg/l peptone), M2 (1mg/l BA + 50mg/l peptone), M3 (3mg/l BA + 25mg/l peptone), M4 (3mg/l BA + 50mg/l peptone).For rooting induction the combinations of growth regulators were used as follows, Mr1 (1mg/lIndolbuteric acid(IBA), Mr2 (2mg/l IBA), and Mr3 (1mg/l IBA+ 1mg/l NAA). Results showed that the highest genetic response for callus induction frequencies were seen for Petra genotype (91.25%) followed by Gold Dust. Medium M3 showed the highest response for callus induction frequencies (82.4%) followed by M1.Interaction between genotypes and media each alone were highly significant in callus fresh weight character. Regarding the micro propagation, Petra genotype gave highly genetic response followed by Gold Dust, also medium (M1) gave highly response for micro propagation followed by M2. In vitro roots were successfully induced by (1 or 2) mg/l of IBA which gave longest and few roots, while 1mg/l NAA gave shorter and more root number. Plants with roots were moved to the green house for acclimatize in pots contain sand /farmyard manure (8:2VN). Alkaloids extracted from callus for both genotypes were detected for antibacterial activities against Gram positive and negative bacteria. Alkaloids extracted from calli for media M1 and M2 were gave a highest antibacterial activity. Finally the present results gives scientific evidence on the ideal composition of the media to product the alkaloid extracts from C. variegatum (Gold Dust and Petra) as medicinal plants to be used as antibacterial agents against different pathogens.

In Vitro Adventitious Shoot Regeneration through Direct and Indirect Organogenesis from Seedling-derived Hypocotyl Segments of Ficus religiosa L.: An Important Medicinal Plant

Ficus religiosa is an important industrial, medicinal, and ornamental plant, so in vitro regeneration is of high paramount in this valuable germplasm. Two efficient protocols were developed for indirect and direct shoot organogenesis through hypocotyl explants. In the first experiment, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and indole butyric acid (IBA) (0.5, 1.0, and 1.5 mg·L−1) in combination with 6-benzyl amino purine (BAP) (ratio 10:1, respectively) were used for callus formation. Two types of callus were obtained from different concentrations of plant growth regulators (PGRs). Also, 2,4-D produced yellow-brownish and friable callus (Type I), whereas the green and compact callus (Type II) was achieved in IBA. The highest callus fresh weight (2.43 g) was observed in Murashige and Skoog (MS) medium containing 0.5 mg·L−1 2,4-D plus 0.05 mg·L−1 BAP. In the later experiments, various concentrations of thidiazuron (TDZ), 6-furfuryl amino purine (KN), and BAP (0.25, 0.5, 1.0, and 1.5 mg·L−1) in combination with IBA (ratio 10:1, respectively) were applied for shoot regeneration (direct and indirect organogenesis). In shoot regeneration from callus, the highest regeneration frequency (86.66%) and shoot number per callus (4.13) were achieved in MS medium supplemented with 1.5 mg·L−1 BAP plus 0.15 mg·L−1 IBA from type I calli. However, no regeneration was observed in type II calli. In direct shoot regeneration, the highest regeneration frequency (96.66%) and shoot number (6.26) were obtained in the medium mentioned previously. In root induction experiment, different concentrations of naphthalene acetic acid (NAA) and IBA alone or in combination were applied, and MS medium containing 2.0 mg·L−1 IBA along with 0.1 mg·L−1 NAA was the best hormonal balance for root induction. The rooted plantlets’ survival rate was more than 95% in the acclimatization stage. These results demonstrated that the direct regeneration method provides more shoot regeneration frequency and take a less time for shoot organogenesis than the indirect regeneration method. Based on our knowledge, this study is the first report of direct and indirect shoot organogenesis of F. religiosa via hypocotyl from in vitro–grown seedling.

The Effect of Explant Types and Kinetin Concentration on In Vitro Callus Induction in Vetiveria zizanioides (L.) Nash

The aims of this research were to evaluate the effect of explant types and several kinetin concentrations on in vitro induction and growth of callus vetiver ( Vetiveria zizanioides (L.) Nash). Crown and tiller of vetiver were cultured on Murashige and Skoog’s (MS) media supplemented with combination of 2,4-D 0.75 ppm and several kinetin concentrations (0, 0.3, 0.5, 0.75, and 1) ppm. The induction and growth of callus were influenced by type of explant and concentration of kinetin. Formation and growth of callus on tiller explant were faster than crown explant. Callus on tiller explant were formed one week after culture, while callus from crown explant were formed at four weeks after culture. Callus growth on tiller explant also was better than crown explant. Eight weeks after culture, callus fresh weight from tiller explant was 0.35 ± 0.09 g, while callus fresh weight from crown explant was only 0.16 ± 0.08 g. The addition of kinetin in the medium combined with 2,4-D was able to increase callus growth and the optimum concentration of kinetin used was 0.5 ppm. The addition of kinetin more than 0.5 ppm in the medium decreased the callus fresh weight. Keywords: callus, crown, in vitro culture, tiller, Vetiveria zizanioides .

Peningkatan Induksi Kalus Embriogenik dan Konversi Embrio Somatik Kopi Robusta Klon BP 308

<em>Robusta coffee </em>(Coffea canephora)<em> is a cross-pollinated plant, therefore vegetative propagation is necessary to ensure identical traits with parents, such as tissue culture techniques through somatic embryo. The study aimed to find the effect of plant growth regulator 2.4-D and thidiazuron in inducing embryogenic callus, by adding incision area on leaf explant, and to evaluate addition of GA<sub>3</sub> in increasing somatic embryo conversion. The study was conducted from December 2014 to June 2016 in the Tissue Culture Laboratory, IAARD, Bogor. The research consisted of 2 stages. Stage 1 used a complete randomized design of 2 factors; the first factor was a combination of plant growth regulator 2.4-D (1.0 and 2.0 mg/l) and thidiazuron (1.0; 3.0; and 5.0 mg/l), the second factor was leaf incision (slashed and unslashed). Stage 2 used a complete randomized design, with GA<sub>3 </sub>treatment at different concentrations (0.0; 0.5; and 1.0 mg/l). Observed variables were percentage of callus formation, fresh weight of callus, number of torpedoes, number of somatic embryos at cotyledon stage, and number of germinated embryo. The results showed growth regulatory treatments influenced the percentage of embryogenic callus formation and fresh weight of callus. Extra incision on leaf showed no effect in embryogenic callus induction. Embryogenic callus inducted using 2.4-D 1.0 mg/l + thidiazuron 5.0 mg/l medium which then regenerated in ½ MS medium added with kinetin 2 mg/l exhibited the highest number of germination. Adding GA<sub>3</sub> 0.1 mg/l in regeneration medium is recommended to increase somatic embryos of Robusta coffee BP 308 clone.</em>

Growth kinetics and diosgenin estimation from callus cultures of Costus speciosus (Koen. ex. Retz.)

The present investigation reports the growth kinetics and diosgenin accumulation in callus cultures of Costus speciosus. Effect of explants, media and plant growth regulators was evaluated with respect to callus induction and growth. Out of the two explants viz pseudostem and seed, pseudostem showed maximum callus induction frequency of 90% on MS medium. The fresh weight of callus was maximum (9-folds) on 28th day on 1.0 mg/L picloram containing medium. The callus obtained was white compact hard (WCH). For growth kinetics study pseudostem derived callus was transferred on different media supplemented with 1.0 mg/L picloram. All phases of growth were seen in callus inoculated on all the three media except the absence of stationary phase on MS and SH media. MS medium proved to be the best for maximum biomass accumulation (9-fold) on 28th day of culture and callus in post-exponential phase showed maximum diosgenin accumulation (33 ppm).

A Novel Medium for Enhancing Callus Growth of Hazel (Corylus avellana L.)

Paclitaxel is a powerful antimitotic agent with excellent activity against a range of cancers. Hazel has been described as a paclitaxel-producing species among angiosperms. Fast-growing callus is a prerequisite for the success of callus production and then paclitaxel production. Therefore, optimizing the medium culture for enhancing callus growth is a crucial step for paclitaxel production. In this research, Murashige and Skoog (1962) (MS) medium was optimized for improving callus growth of hazel (Corylus avellana L.). The M10 medium (MS medium with pH 6.0 and supplemented with 1000 mg l−1 spirulina powder, 1000 mg l−1 casein hydrolysate and 3 g l−1 gelrite) significantly improved hazel callus growth. This modified MS medium increased callus fresh weight (55.8%) as compared to the control. M10 medium increased fatty acids yield of callus (66.7%) as compared to the control. Liquid M10 medium maintained growth over a longer period of time and also increased slightly, the paclitaxel production as compared to the control. This novel medium is promising for facilitating the mass production of hazel callus as a source of valuable metabolites including paclitaxel, linoleic and oleic acids.

Callus Induction and Plantlet Regeneration Systems in Indica Rice (Oryza sativa L.) Cultivar Sangyod

This study was conducted to determine the optimum concentrations of 2, 4-D, L-proline and casein hydrolysate (CH) for efficient callus induction and plantlet regeneration from culturing mature embryos of Sangyod, an economically important indica rice variety in Thailand. The highest frequency of callus induction (73.08 ± 2.65 %) was obtained from MS medium supplemented with 2 mg/L 2,4-D, 750 mg/L CH and 200 mg/L L-proline. The combination of 1 mg/L BA, 0.5 mg/L Kn and 0.5 mg/L NAA containing solidified MS medium gave the maximum mean fresh weight of callus (938.9 ± 44 mg), green spot formation (64.17 ± 7.08 %), shoot induction frequency (66.25 ± 6.80 %) and mean number of shoots/explant (6.12 ± 0.36 shoots). The greatest mean number of shoots/explant (14.93 ± 0.97 shoots) and root formation percentage (82.71 ± 3.03 %) was observed in liquified MS medium supplemented with 0.5 mg/L NAA and 1 mg/L BA.

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

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

Establishment of in Direct Propagation of Mandarin (Citrus reticulata L.) Using Tissue Culture

THE PRESENT investigation deals with development of an efficient micropropagation protocol for mandarin (Citrus reticulata L.) via in direct propagation using cotyledons and juice vesicles as explant. The establishment of in vitro callus induction from leaf, stem segments and cotyledon system were excised from seedlings has been done. Best results for induction of callus were observed from cotyledons and stem segments 100% on MS medium supplemented 1 or 2mg/l 2,4-D. MS medium supplemented with 300mg/l glutamine or 1.5mg/l casein hydrolysate proved to be the most efficient additive in promoting callus formation from juice vesical with 4.22, 4.23g/callus fresh weight, 100% response and was the most favorable medium. Maximum callus fresh weight (7.96 g) and 100% response were obtained from juice vesicles when cultured on MS medium supplemented with 2mg/l 2,4-D and 10% coconut water. The highest callus regeneration and weight of callus observed from juice vesicles by adding 2mg/l 2,4-D, 300 or 400 mg/l malt extract. Callus raised from juice vesicles showed maximum shoots (96.67%) with 0.5mg/l Kin, 400mg/l malt extract and 2mg/l NAA. Regenerated shoots raised from juice vesicles showed better shoot multiplication and highest length of shoots on MS medium with 2mg/l BA and 0.4mg/l NAA. Regenerated shoots were rooted on MS medium supplemented with different concentrations of NAA and best response (98%) was observed with NAA (2 mg/l) and gave maximum, roots number, roots length and leaves number. Rooted plantlets were successfully acclimatized with survival rate reaching almost 87%. These plants grew normally without showing any morphological variation. Developed protocol can be useful for application of somatic embryogenesis from juice vesicles to improve mandarin.

THE USE OF EMBRYO CULTURES FOR STUDYING SALT STRESS TOLERANCE OF FOUR DIFFERENT BREAD WHEAT VARIETIES AND THEIR PROMISING MUTANTS

The present study was carried out to determine the genetic response of four different bread wheat varieties and their mutants for callus induction using mature embryo culture and salt stress tolerance and to assess the genetic diversity between them using ISSR markers. The results of the ability of callus induction revealed that differences among genotypes, callus induction media (CI) and their interactions were highly significant for callus induction frequencies (CIF). Also in relation to the callus fresh weight (CFW), the differences between genotypes and their interactions with media were highly significant, whereas insignificant differences were found between media after 14 at 28 days of culture. The best medium for CIF was CIM2 containing 2 mg/l of 2,4-D for all genotypes except Gemiza 9-1 and Sakha 93-3 had the best CIF of CIM3 containing 3 mg/l of 2,4-D. Seds 12-7 had the highest CIF, whereas Sakha 93-3 had the lowest CIF of CIM2. Gemiza 9-1 had the highest CFW, whereas Sakha 93-3 had the lowest CFW after 28 days of culture. The fresh callus produced after month was transferred to salt stress media at 10000ppm NaCl level. Giza 168, Gemiza 9 and Sakha 93 showed increase in CFW, whereas other genotypes strongly decreased. Sakha 93 and Gemiza 9 gave the lowest value of callus necrosis (CN) and the highest value of callus in vitro tolerance (CINTOL) at the 10000ppm NaCl level after 28 days on salt stress media. The salt stressed callus was tested for shoot induction ability; callus of Gemiza 9 and Sakha 93 showed the best shoot induction frequencies at 10000 ppm level. Days to shoot induction were decreased with the increase of salt levels compared to normal conditions. Transfer of shooty callus on plant regeneration media to be grown and development failed. The callus turned brown and some of the resulting growths were weak and white. The use of ISSR technique illustrated the genetic similarities between the wheat genotypes, which ranged from 0.638 to 0.979 with an average of 0.809, suggesting a high genetic diversity between the wheat genotypes. Cluster analysis performed to generate dendrogram showed that wheat genotypes had been clustered into two main clusters. From the previous results, we recommended that Gemiza9 and Sakha93 are good materials for salt stress tolerance of bread wheat breeding programs.

In vitro propagation, genetic stability, and secondary metabolite analysis of wild lavender (Lavandula coronopifolia Poir.)

Lavenders (Lavandula species) are important aromatic ornamental medicinal plants with wide ranging applications in perfume and pharmaceutical industries. We developed an in vitro propagation protocol for Lavandula coronopifolia. Murashige and Skoog (MS) medium supplemented with 0.5 mg·L-1 N 6 -benzyladenine was the best medium for the proliferation of microshoots, while the highest rooting frequency was obtained using MS medium supplemented with 1.0 mg·L-1 indole-3-butyric acid. Inter-simple sequence repeat analysis revealed that the in vitro-propagated microshoots were highly genetically stable, even after subculture. The highest callus fresh weight (667.9 mg) was obtained by propagating on medium supplemented with a combination of 1.0 mg·L-1 naphthaleneacetic acid and 0.5 mg·L-1 butyric acid. Using the Folin-Ciocalteu method, methanolic extracts of wild L. coronopifolia revealed total phenolic content of 4.9 mg expressed in gallic acid equivalents (GAE) (mg GAE·g-1 dry matter). Radical scavenging activity was estimated at 85% using the free radical 2,2-diphyenyl-picrylhydrazyl assay. Using the brine shrimp assay for cytotoxicity, the methanolic extract was found to be nontoxic. Finally, liquid chromatography tandem mass spectrometry with standard reference compounds was used to quantify the key phenolic compounds in both in vitro and in vivo-grown L. coronopifolia. Six major phenolic compounds were identified: caffeic acid, rosmarinic acid, rutin, quercetin, luteolin, and hesperidin. Levels of these phenolic compounds were highest in wild plant extracts.

Effect of Bacterial Cell Wall Components on Callus Growth of Phoenix dactylifera L. cv. Barhee Propagated Through In vitro

This study was conducted on shoot tip explants of date palm cv. Barhee to investigate effect of bacterial cell wall components on initial callus growth. Three concentrations of bacterial media ( 0.5 and 1.0 and 2.5) v/v were used for growing callus by tissue culture technique . MS media was served as a control treatment.Results showed that all bacterial media treatments increased callus fresh weight and nitrogen concentration of media significantly as compared to that of MS control media. Bacterial media treatment of 2.5 (v/v) gave the highest values of callus fresh weight and nitrogen concentration (518 mg and 3.3089%) respectively, whereas MS control media had the lowest values (193 mg and 0.01%) respectively. There was a significant increase in callus fresh weight at 60 days incubation over that of 30 days incubation. The combination of bacterial media treatment at 2.5 (v/v) and incubation period of 60 days recorded the highest significant increase in callus fresh weight (631 mg), whereas the combination of MS control media treatment and incubation period of 30 days had the lowest value (166 mg) in this respect.

ROLE OF PLANT GROWTH REGULATORS IN CALLUS INITIATION AND PLANTLETS REPRODUCING OF TWO ALFALFA CULTIVARS

An experiment was conducted at the central lab of Graduate studies - College of Agriculture, University of Baghdad during 2015-2016. The objective was to study the effect of growth regulator on inducing callus from embryos of two alfalfa cultivars (PAC-78001 and local) variety by tissue culture technique using factorial experiments according to CRD. Seeds sterilized by NaOCl at 4.5% for 15 minutes, then, cultured on MS media free of growth regulators to embryo induced. The embryos were cultured on MS media at different concentrations of 2,4-D (0, 1, 2, 3 and 4) mg Li-1. Induced callus from embryos was cultured on MS media supplementing with Kin. at 0, 0.25, 0.50, 0.75 and 1 mg Li-1 to determine the best concentration. The results showed that best concentration of 2,4-D and Kin were 2 and 0.5 mg L-1 respectively which gave highest callus fresh weight and callus dry weight (204.65 and 21.50) mg Li-1, respectively. BA at 2 mg Li-1 gave the highest plantlets reproducing from callus amounted 5.5 vegetative growth tube-1.

Impact of Zeatin and Thidiazuron on Phenols and Flavonoids Accumulation in Callus Cultures of Gardenia (Gardenia jasminoides).

Gardenia (Gardenia jasminoides) has many pharmacological actions such as anti-inflammatory, antioxidant and fibrolytic activities and cytotoxic effects, etc. This study was conducted to recognize the effect of zeatin and thidiazuron (TDZ) on callus proliferation, total phenolic content, total flavonoids and DPPH scavenging activity of gardenia callus cultures. Calli were cultured on Murashige and Skoog (MS) medium supplement with different concentrations (2, 4 or 6 mg L-1) of zeatin or TDZ individually as well as combination of 2 mg L-1 zeatin+4 mg L-1 TDZ. Cultures contained 4 mg L-1 TDZ gave the highest callus fresh weight followed by those contained 2 mg L-1 zeatin then that cultured on 4 mg L-1 zeatin. Data reported as Mean±Standard Deviation (SD). Data were subjected to one-way analysis of variance (p< 0.05). Results were processed by Excel (2010) and SPSS Version 17.0. It was found that callus growing on medium supplemented with 4 mg L-1 zeatin gave the maximum value (14.93%) of yield extract. Callus cultured on 4 mg L-1 zeatin recorded the maximum total phenol (268.33 mg GAE/100 g FW of callus) and total flavonoids (2703.33 μg QE/100 g FW of callus) accumulation. The antioxidant activity of each extract was determined through DPPH radical scavenging activity. Callus cultured on 4 mg L-1 TDZ showed the highest antioxidant activity then those cultured on 4 mg L-1 zeatin. The HPLC analysis for phenolic acids showed that chlorogenic acid, rosmarinic acid and cinnamic reached their highest contents with callus cultured on 4 mg L-1 TDZ (123.24, 322.14 and 278.22 μg g-1, respectively). Regarding flavonoids and using HPLC analysis, rutin, apigenin-7-glucoside and kaempferol were detected. Callus cultured with 4 mg L-1 TDZ gave the highest rutin and kaempferol contents (287.76 and 10.38 μg g-1, respectively). However, apigenin-7-glucoside was detected with high content (129.86 μg g-1) in callus culture with 4 mg L-1 Zeatin. The HPLC analysis recommended that TDZ is more effective in accumulation of individual phenolic and flavonoid than Zeatin. The present study provided a useful system for further study on in vitro culture of G. jasminoides as alternative and new source for important secondary products.

Improvement of Plant Regeneration Frequency from Carbon Sources in Aromatic Rice (Oryza sativa L.)

The objective of this research is to investigate carbon sources for optimum callus induction and plant regeneration frequencies. The effect of carbon sources on callus induction and plant regeneration from mature seeds of aromatic rice (Oryza sativa L.) cultivar Khao Dawk Mali 105 (KDML105) was studied in this experiment. For callus induction, the different types of carbon sources along with 30 g L−1 of sucrose or maltose were evaluated. NSI and NMI medium were used for callus induction in three replicates (sucrose and maltose as carbon sources, respectively). The calli derived from NMI medium showed the higher percentage of callus induction, fresh weight, dry weights, callus size, green spots and regeneration frequency than calli derived from NSI medium. For plant regeneration, the 3-week-old calli were transferred to the regeneration medium which was different in carbon sources (NSR, NMR, NSMR0.5, NMMR0.5, NSMR1, NMMR1, NSMR1.5, NMMR1.5, NSMR2, NMMR2, NSMR2.5 and NMMR2.5 medium) and tested to investigate the effect of carbon sources in three replicates. The percentage of regeneration (60.71%) and the ratio of the number of seedlings to the number of regenerated calli (3.02) obtained from NMR medium showed the highest values. The percentage of callus induction and morphological performance (weight and size) were correlated with plant regeneration frequency. The results showed that using maltose as a carbon source gave a better result than using sucrose as a carbon source for callus induction and plant regeneration in aromatic rice cultivar KDML105. The regenerated plants were successfully rooted and well grown in greenhouse conditions.

Investigation the Antibacterial Activity of Portulaca oleracea L. Tissue Cultures in vitro

The objective of this research was to studying the antibacterial activity of Portulaca oleracea L. in vitro cultures. P. oleracea leaves were collected from the local markets. Callus tissues were induced from the plant leaves (expalnt) through inoculating the explants on MS medium supplemented with different concentrations of 2, 4-D (0.0, 2.0 or 3.0 mg/l) and Kintin (0.0 or 0.5 mg/l). All cultures were incubated at 25 ± 2°C in dark. Water and ethanol extracts of P. oleracea were prepared. Bacterial samples were collected, they consist of gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Pseudomonas aeruginosa). Antibacterail activity of P. oleracea extracts were investigated using diffusion method. Results shown that mean % callus induction, callus fresh weight and callus dry weight inceasred significantly in 2.0 mg/l 2, 4-D or 3.0 mg/l 2, 4-D with 0.5 mg/l kin in comparsion to the control (0.0), and the highest mean obtained at 3.0 mg/l 2, 4-D with 0.5 mg/l kin recording 80% and 94 mg and 52 mg for % callus induction, callus fresh weight and callus dry weight respectively. Also there was no significat differences ocurred in the mean inhibition zone diameter (mm) of the bacterial isolates treated with water or ethanol extracts of callus tissues recording 10 and 9.5 mm respectively compard with control (14.5 mm) and mean inhibition zone (mm) significantly decreased in other treatment types in compasion to the control. The results obtained in this research work clearly indicated a promising potential of P. oleracea extracts as antibacterial agent and inspite of the small inhibition zone diameter obtaind using plant extract but it has proved highly efficiency as an anti bacterial compared with the higher concentration of the standard antibiotic used in this experiment.

Effect of AgNO3 and BAP on Root as a Novel Explant in Date Palm (Phoenix dactylifera cv. Medjool) Somatic Embryogenesis

Somatic embryogenesis techniques are used for cloning a wide range of varieties of date palms around the world. The aim of the present study was to develop an efficient method with the lowest cost and the greatest potential to obtain in vitro plantlets of date palm cv. Medjool. Also, produce embryogenic callus and somatic embryos without using 2,4-dichlorophenoxyacetic acid (2,4-D). In this study, produced plantlets through somatic embryogenesis were used in vitro roots as explant cultured on Murashige and Skoog (MS) media containing three level of Silver Nitrate (AgNO3) (0, 3 and 6 mg L-1) plus two level of 6-benzylaminopurine (BAP) (0 and 2 mg L-1) plus 0.1 mg L-1 1-naphthylacetic acid (NAA) for callus induction. After 12 weeks of culture, callus induction and after 16 weeks, production of embryogenic callus and embryos were occurred from root explants. According to the results, medium containing 2 mg L-1 BAP and 3 mg L-1 silver nitrate+0.1 mg L-1 NAA showed the highest amount of embryogenic callus fresh weight (1.38 g). This treatment also cause the highest number and length of embryos by production of 90.04 embryogenic callus with length of 11.18 mm. On the other hand, shoots were appeared from germinated embryos and white roots began to appear within 8 weeks. Medium contains 3 mg L-1 BAP and 0.1 mg L-1 NAA with average of 12.27 cm shoot length and 15.48 cm root length was the best. Control treatment had the lowest average shoot (3.71 cm) and root (5.03 cm) length. This study showed that certain concentration of silver nitrate and BAP has stimulating effect on growth of produced embryonic callus from root segments of Medjool cultivar of date palm.