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Enhancement of callus induction and cucurbitacin content in Citrullus colocynthis L. (Schrad) using plant growth regulators.

ABSTRACT

The objectives of this study were to investigate the effect of growth regulators and different explant type in callus induction and to increase the yield of cucurbitacin in leaf, stem and root explants of Citrullus colocynthis. The specimens were 15 days old and were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin (kin), Benzyl adenine (BA) and [alpha]-naphthalene acetic acid (NAA) The different concentrations of 2, 4-D + kin and BA + NAA as well as different explant organ types increased the callus fresh weight and dry weight. The callus cultures derived from stem explants grown on BA + NAA were proved to be an appropriate protocol for callus induction, while 2,4-D failed to stimulate callus growth in the same manner. Different callus explants and the in vitro raised seedling leaf, stem and root were harvested and subjected to extraction of active principle compounds. The results revealed that stem-derived callus cultured on 2,4-D (2 mg/1) + kin (4 mg/1) produced the highest total cucurbitacins content with values reaching 10.89% compared to the control seedling stem, leaf and root which produced (4.95, 4.97 and 5.12%), respectively. The HPLC analysis of cucurbitacin-E showed distinct changes in the different cultures initiated from various explants.

INTRODUCTION

Many desert plants are major sources of natural products used as pharmaceuticals, agrochemicals, flavors, fragrance ingredients and food additives. Citrullus colocynthis L. (Schrad) is commonly known as Sherry or Handal and is widely distributed in the Egyptian deserts. The plant fruits are used in folk medicine by nomads and people in rural areas as a purgative, anthelmintic, carminative, anti-rheumatic, anti-diabetic and antipyretic (Maatooq et al 1997; Nmila et al., 2000; Hegazy, 2007). Phytochemical analysis of the fruits and leaves of the plant demonstrated the presence of flavonoids, caffeic acid derivatives and terpenoids such as cucurbitacins (Hatam et at., 1989; Seger et al., 2005). The cucurbitacins (highly oxygenated tetracycilic triterpens) were investigated for their cytotoxic, anti-inflammatory, hepatoprotective and cardiovascular effects (Liu et al., 2008). Additionally, several studies indicated that different forms of cucurbitacins inhibit the proliferation of cancer cells and induce cell apoptosis (Jing and Tweardy, 2005; Sun et al, 2005).

In order to protect the medicinal plant resources, cell culture has been used as an alternative source of active constituents of medicinal plants with a view of enhancing secondary metabolites. The potential benefits of stimulating the formation of desirable medicinal compounds from plants, especially by using biotechnological approaches such as plant tissue culture has been recognized (Ramachandra Rao and Ravishankar, 2002). For example, ginsenoside from Panax gingseng, rosmarinic acid from Coleus blumei, shikonin from Lithospermum erythrohizon, diosgenin from Dioscorea, ubiquinone-10 from Nicotiana tabacum, berberin from Coptis japonica, and podophyllotoxin from Juniperus chinensis all accumulated at much higher levels in cultured cells than in intact plants (Misawa et al., 1985; Smith, 2002; Premjet et al., 2002). Several attempts were made in order to obtain a high yield of secondary metabolites by applying exogenous phytohormonon.es or chemical treatments in the growth media (Satdive et al., 2007; Shinde et al., 2009). Establishment of an efficient callus protocol is a prerequisite for harnessing the advantages of cell and tissue culture. Induction of callus biomass and physical disorganization of cultured cells is thought to be a result of the breakdown of intercellular physical and chemical communication (Lindsey and Jones, 1992).

Plant growth regulators such as auxins (2,4-D and NAA) and cytokinins (BA and kin) have different effects on plant growth and are often crucial factors in secondary metabolite accumulation (DiCosmo and Towers, 1984). On the other hand, NAA stimulates water absorption, enhances cell wall plasticity and initiates callus formation (Aki, 2005). The quality and quantity of plant growth regulators initially presented in the media or administered during the course of in vitro culture proved to have a significant effect on the metabolism of secondary metabolites (Zia et al, 2007). Since the production of secondary metabolites in plant cell culture is a function of both cell multiplication and division, growth regulators are bound to play a major role in determining the production potential of a given culture (Staba, 1980). The effect of auxin type on secondary metabolite synthesis was studied in the tissue culture of Ecballium elaterium. Higher yields of cucurbitacin B (1.126%) were obtained from stem node callus cultures in the presence of BA (1 mg/1) and NAA (0.1 mg/1) compared with the yield obtained from raw plant material (0.01%) as stated by Toker et al. (2003).

The present study was carried out to develop an efficient protocol for callus induction, total cucurbitacins and cucurbitacin-E accumulation under the effect of different growth regulators and explant types of Citrullus colocynthis (Handel).

MATERIALS AND METHODS

Standards and Reagents

Cucurbitacin-E, purity (HPLC)>98% was obtained from Carl Roth GmbH+Co. KG. (Karlsruhe, Germany); MS media, 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin (kin), Benzyl adenine (BA) and [alpha]-naphthalene acetic acid (NAA) were obtained from Sigma Chemical Co. (St. Louis, Mo. USA); and phosphomolybdic acid was obtained from BDH, Dorset, UK. The HPLC-grade organic solvents methanol and acetonitrile were obtained from Merck. All other chemicals were of analytical-grade purity and available from the Centre of Excellence for Advanced Sciences, National Research Centre, Dokki, Egypt.

Plant Materials

Mature seeds of Handal were collected from naturally growing plant populations in Wadi Soule, Sinai, Egypt. The collected seed materials were botanically authenticated (collection No. 655) by the Herbarium of the Botany Dept., Faculty of Science, Cairo University. The collected seed material was kept in dark bottles and preserved under normal lab conditions until used.

Culture Media

MS-media (Murashige and Skoog, 1962) was used as the base medium. The medium was solidified with 0.7% agar and pH adjusted to 5.8 before autoclaving. The prepared media were poured into sterilized 200 ml glass jars. Each jar contained 50 ml solidified medium; the media were then autoclaved at 121 [degrees] C under pressure 1.2 Kg/[cm.sup.2] for 20 min.

Seed Germination and Explants

Seeds of C. colocynthis were manually decoated and then transferred to the laminar air flow cabinet. The seeds were kept in 70% ethanol for 30 sec followed by rinsing three times in sterile distilled water. The seeds were then immersed in a continuously stirred 50% (v/v) commercial Clorox solution (5.25% NaOCl) for 30 min and finally washed several times with sterile distilled water to remove any traces of Clorox. Sterilized seeds were cultured (10 seeds/jar) into 200 ml glass jars containing 50 ml of solidified basal MS-medium supplemented with 3% sucrose and 0.7% agar without growth regulators. The jars containing the seeds were incubated for seed germination in the dark at 28 [+ or-] 2 [degrees] C for five days. Jars with germinated seeds were then transferred to the growth chamber at 25[+ or -] 2 [degrees] and 16/8 h light/dark regime at light intensity 2000 Lux produced from cool white fluorescent lamps. After 15 days, the in vitro raised seedlings were used as the plant material source for callus induction.

Callus Induction

Three explants (leaf, stem and root) were excised from in vitro raised seedlings and cultured in 200 ml jars containing 50 ml MS-medium. To assess the effect of auxin in combination with cytokinin on callus induction, eight different medium formulations were used. All the formulations consisted of MS -medium supplemented with different concentrations of 2, 4-D and Kin as well as BA and NAA (Table 1). The cultures were incubated for callus induction in the growth chamber for three weeks. After 21 days, callus tissues characteristics were determined. After obtaining the fresh weights, the samples were then air dried at 40 [degrees] C till they reached a constant dry weight.
Table 1. Murashige and Skoog media supplemented with different
concentration of growth regulators for callus induction of
Citrullus colocynthis.

No. of media  Media code  Growth regulator concentration (mg/l)

1             MD1         MS + 2.0 2,4-D + 1.0 kin *
2             MD2         MS + 1.0 2,4-D + 1.0 kin
3             MD3         MS + 6.0 2,4-D + 2.0 kin
4             MD4         MS + 2.0 2,4-D + 4.0 kin

5             MB1         MS + 0.0 BA + 5.0 NAA
6             MB2         MS + 0.01 BA + 1.0 NAA
7             MB3         MS + 0.1 BA + 5.0 NAA
8             MB4         MS + 1.0 BA + 0.1 NAA

* Kinetin (kin), Benzyl Adenine(BA), [alpha]-Naphthalenc Acetic
Acid (NAA)


Determination of Total Cucurbitacins

Different callus explants and the in vitro raised seedling stem, leaf and root were harvested and subjected to extraction of cucurbitacins. Exactly 100 mg of each air-dried callus culture and in vitro seedlings stem, leaf and root organ (used as control) were ground to fine powder, followed by extraction with absolute ethanol. Each sample was extracted with 5 ml absolute ethanol for 2 hr; then the extract was filtered using Whatman No. filter paper. The filtrates were dried under vacuum at 40[degrees]C using a rotary evaporator (Buchi, Switzerland) for 30 mints. The extraction was repeated twice, and the resulting residue was re-dissolved with ethanol. An equal volume of each extract and 2% solution of phosphomolybdic acid in absolute ethanol were mixed at room temperature, and the absorbance was measured by a spectrophotometer (UNICAM UV 300) at 492 nm after 5 min (Attard and Scicluna-Spiteri, 2001). Standard stock solution of cucurbitacin-E was prepared in the range between 0.5 to 5.00 mg/ml. The results were expressed as percentage (w/w) based on dry weight.

Determination of Cucurbitacin-E

Cucurbitacin-E was determined in the extract of both derived calli and in vitro seedling organs by High Performance Liquid Chromatography (HPLC) according to Toker et al. (2003). Dried tissues were ground to fine powder in a mortar; 50 mg of this powder was extracted with 5 ml chloroform for 2 hr, and then the extract was filtered using Whatman No. 4 filter paper. The chloroform extract was evaporated at 45 [degrees] C till dryness. HPLC was performed using an Agilent 1100 series (Waldbronn, Germany) equipped withquaternary pump (G 13 11 A), degasser (G 1329A) and auto sampler (G 1329A). Dry samples were re-dissolved in HPLC-grade methanol and filtered through a Millipore filter membrane (0.45 [micro] m pore size) directly into auto sampler vials. Optimal separation of cucurbitacin-E was obtained with isocratic mobile phase: acetonitrile / water (70:30). Flow rate was adjusted to 1 ml/min and the column was equilibrated for 10 min with the mobile phase before each run. Detection was achieved by variable wave length detector (G 1314A) at wave length 229 nm. Cucurbitacin-E was identified through its retention time by comparison with the cucurbitacin-E reference standard, and quantified on the basis of the integrated peak area. The results were expressed as percentage (w/w) based on dry weight.

Statistical Analysis

The experiment was conducted as Completely Randomized Design (CRD) with five replications. The results were reported as mean [+ or -] standard deviation (SD). The significance of differences among different combinations of growth regulators and explant types was analyzed by one-way ANOVA using SPSS 10.0 Windows package. The least significant difference (LSD) at P= 0.05 level was used for mean separations for the variables with significant F values, at 5 % level of probability.

RESULTS AND DISCUSSION

Induction of Callus Cultures and Biomass Accumulation

A reliable protocol for the in vitro callus induction of Handal was optimized. For callus induction, eight different media formulation were used (Table 1). Callus tissues began to appear on wounded explants within one week of culturing. Callus induction efficiency expressed as fresh and dry weight differed significantly (P[less than or equal to] 0.05) among the different concentrations of growth regulators and explant types. Efficiency of the stem explants was significantly higher than that of leaf and root explants for all media. The highest yield of callus biomass (581 mg fw/inoculum and 31 mg dw/inoculum) was obtained from stem explants raised on MD4 media (Table 2). This callus was solid in texture and pale green in color. The maximum fresh biomass in the leaf explants reached 355 mg fw/inoculum and 29 mg dw/inoculum in MB3 media (Table 3); this callus was solid in texture and white in color. The root explants produced the highest fresh biomass (173 mg fw/inoculum and 11 mg dw/inoculum) when grown on MB2 media (Table 3).
Table 2. Callus fresh and dry weight (mg/inoculum) of Citrullus
colocynthis raised on Murashige and Skoog media supplemented by
different combinations of 2,4-D and Kinetin.

                   Fresh and dry weight
                 ([+ or -]SD) mg/inoculum

                   Explant types

Media code              Leaves

                   F.W             D.W

   MD1         194 [+ or -] 6   17[+ or -]0.2
   MD2         134 [+ or -] 2   13 [+ or -] 1
   MD3          91 [+ or -] 8   14 [+ or -] 1
   MD4         107 [+ or -] 10  16 [+ or -] 2
LSD at 0.05         6.824           2.833

                   Fresh and dry weight
                 ([+ or -]SD) mg/inoculum

                      Explant types

Media code               Stems

                      F.W            D.W

   MD1          362 [+ or -] 9  24 [+ or -] 0.1
   MD2          323 [+ or -] 2  27 [+ or -] 0.1
   MD3          299 [+ or -] 5  23 [+ or -] 0.2
   MD4          581 [+ or -] 9  31 [+ or -] 1
LSD at 0.05          6.799          1.136

                   Fresh and dry weight
                 ([+ or -]SD) mg/inoculum

                   Explant types

Media code               Roots

                  F.W             D.W

   MD1          41 [+ or -] 9   0.7[+ or -] 0.1
   MD2          78 [+ or -] 7   0.8 [+ or -] 0.3
   MD3          56 [+ or -] 8   0.8[+ or -] 0.2
   MD4         168[+ or -] 16   1.5 [+ or -] 0.7
LSD at 0.05         8.156            0.525

Table 3 Callus fresh and dry weight (mg/inoculum) of Citrullus
colocynthis raised on medium supplemented by different combinations
of Benzyl Adenine and alpha-Naphthalene Acetic Acid.

                 Fresh and dry weight
               ([+ or -]SD) mg/inoculum

                      Explant types

Media code                 Leaves

                   F.W               D.W

   MB1       251[+ or -] 19     16[+ or -] 1
   MB2        74 [+ or -] 9    12 [+ or -] 2
   MB3       355 [+ or -] 9   29 [+ or -] 0.9
   MB4       151 [+ or -] 10    17[+ or -] 1
LSD at 0.05       8.728            1.037

                 Fresh and dry weight
               ([+ or -]SD) mg/inoculum

                    Explant types

Media code                Stems

                    F.W              D.W

   MB1        157 [+ or -] 6   21 [+ or -] 4
   MB2        179 [+ or -] 6   25 [+ or -] 1
   MB3        257 [+ or -] 5   27 [+ or -] 1
   MB4        424 [+ or -] 4  35 [+ or -] 0.6
LSD at 0.05      12.248           3.153

                 Fresh and dry weight
               ([+ or -]SD) mg/inoculum

                    Explant types

Media code                  Roots

                   F.W              D.W

   MB1        77 [+ or -] 6   5 [+ or -] 0.5
   MB2       173 [+ or -] 10  11 [+ or -] 0.8
   MB3        82 [+ or -] 5   4 [+ or -] 0.9
   MB4        69 [+ or -] 9    4[+ or -] 0.7
LSD at 0.05       14.636         0.341


This callus was white in color and less solid in texture. The callus characteristics on MS-medium supplemented by different combinations of growth regulators were summarized (Tables 4&5).
Table 4. Callus characteristics of Citrullus colocynthis raised on
Murashige and Skoog media supplemented by different combinations of
2,4-D and Kinetin.

                  Callus characteristics

                    Explant types

                   Leaves               Stems

Media code   Texture     Colour    Texture    Colour

   MD1      solid     light brown  solid    pale green
   MD2      solid     light brown  solid    pale yellow
   MD3      solid     light brown  solid    pale green
   MD4      solid     light brown  solid    pale green

           Callus characteristics

               Explant types

Media code       Roots

           Texture   Colour

   MD1      solid    brown
   MD2      solid    brown
   MD3      solid    brown
   MD4      solid    brown

Table 5. Callus characteristics of Citrullus colocynthis raised on
Murashige and Skoog media supplemented by different combinations of
Benzyl Adenine and [alpha]-Naphthalene Acetic Acid.

                   Callus characteristic

                      Explant types

Media code          Leaves                     Stems

             Texture     Colour      Texture          Colour

   MB1        solid    light brown    solid        pale yellow
   MB2        solid    light brown    solid        pale yellow
   MB3        solid    white          solid     Fluorescent green
   M34        solid    white        less solid        white

            Callus characteristic

                 Explant types

Media code           Roots

                Texture   Colour

   MB1        less solid  white
   MB2        less solid  white
   MB3        less solid  white
   M34        less solid  white


The effect of auxins and cytokinins and their combinations on callus fresh and dry weight indicated that kin concentration outweighs the 2,4-D in the medium. This combination produced significant effects on the callus induction in both stem and root explants. The higher concentration of 2, 4-D over kin in the medium produced significant callus biomass in the leaf explants. These results are in agreement with the findings of Halaweish and Tallamy, (1998). They confirmed that MS-medium supplemented by 2 mg/1 2,4-D in combination with 1 mg/1 kin produced the highest biomass of callus tissues when induced from the rootless seedlings explants Cucurbita andreana. The optimum medium for callus induction of Eremochloa ophiuroides (Munro) was MS media supplemented with 2,4-D at 1.0 mg/1 (Yuan et al., 2009).

In the present study, increasing the NAA concentration in the medium amplified the callus induction in the leaf and root explants, whereas the higher ratio of BA in the medium had a positive effect on the callus induction in the stem explants. The media supplemented with BA alone or in combination with NAA were better for callus induction from seedling explants of Ecballium elaterium (Attard and Scicluna-Spiteri, 2001). Moreover, the highest callus growth of Solanum nigrum in terms of fresh and dry weight (1818.20[+ or -]63.30 and 1317.18[+ or -]42.83 mg, respectively) was observed in MS medium fortified with 3 mg/l IAA and 0.5 mg/1 BAP, respectively (Yogananth et al., 2009).

Total Cucurbitacins Formation

Tissues obtained from stem, leaf and root organs of in vitro raised C. colocynthis seedlings and their derived callus cultures were analyzed for their total cucurbitacins content. The total cucurbitacin content in the ethanolic extracts differed significantly (P<0.05) depending on the explant type and the different concentration of growth regulators. Data presented in Figures land 2 shows that the highest content of total cucurbitacins (10.89%) was produced in the stem-derived calli cultured on MD4 media. This content was higher than that in the control seedling stem, leaf and root. The in vitro production of total cucurbitacins was increased from 0.02 [+ or -]0.003 mg/g, fresh weight with 2 mg/L 2, 4-D and 1 mg/1 kin to 0.04[+ or -]0.004 mg/g, fresh weight with 6 mg/1 2, 4-D and 2 mg/I kin in callus cultures of Cucurbita andreana as reported previously (Halaweish and Tallamy, 1998).

[FIGURE 1 OMITTED]

The effect of various concentrations of BA and NAA treatments on total cucurbitacins production presented in Figure 2 shows that the total cucurbitacins content attained the highest value (8.68%) in the root-derived calli raised on MB2 media. The higher concentration of NAA over BA in the medium (MB2) caused significant total cucurbitacins production in the callus cultures of leaf and root explants (5.19 and 8.68%) compared to the yields obtained from in vitro seedling leaf and root (used as control) 4.97 and 5.12%, respectively. The high concentration of BA in the medium (MB4) had a greater effect on the production of total cucurbitacins in the stem explants (6.93%) than in the in vitro seedling stem used as control (4.95 %).

[FIGURE 2 OMITTED]

These results are in agreement with those reported by Attard and Scicluna-Spiteri, (2001). They demonstrated that media containing 5 mg/1 NAA optimized the total cucurbitacin content (3.064%) in the undifferentiated callus cultures of Ecballium elaterium. Additionally, increasing the level of NAA on the MS media supplemented with 2 mg/1 NAA and 0.5 mg/1 BA produced the highest accumulation of Phyllanthusol A (20.23 mg/g, dry weight) in callus cultures of Phyllanthus acidus Skeels (Duangporn and Siripong, 2009). It is evident from the data of Figures I&2 that 2, 4-D in combination with kin have a greater effect on the in vitro production of total cucurbitacins than BA in combination with NAA. Secondary metabolites are enhanced by over-expression of the enzymes that often link the secondary metabolic pathways to the primary metabolic pathways viz, cucurbitadienol synthase is the first pathway-specific enzyme in cucurbitacin biosynthesis (Balliano et al., 1983). The enhancement of total cucurbitacins in C. colocynthis callus cultures may be attributed to the positive effect of the plant growth regulators on cucurbitadienol synthase activity.

Content of Cucurbitacin-E

HPLC analysis was used to detect cucurbitacin-E concentration in the callus cultures and in vitro raised seedling leaf, stem and root organs. Generally, the accumulation of cucurbitacins-E was higher under 2, 4-D + kin treatments than BA--NAA treatments (Figure 3). The high concentration of 2,4-D over kin in the media (MD3) showed significant superiority for cucurbitacin-E production (0.16%) in the root explains. This level is higher than those estimated from the in vitro raised seedling stem, leaf and root, which were estimated at 0.023, 0.083, and 0.013%, respectively. The high concentration of kin in the media (MD4) significantly increased cucurbitacin-E concentration (0.026%) in the stem explants whereas the equal concentrations of 2,4-D and kin in the media (MD2) showed significant superiority for cucurbitacin-E production (0.065%) in the leaf explants.

[FIGURE 3 OMITTED]

Data presented in Figure 4 demonstrate the effects of different concentrations of BA and NAA on cucurbitacin-E production. Among single growth regulators, increasing the concentration of NAA in the media (MB3) caused significant increase in cucurbitacin-E in the leaf and root explants with values 0.0337 and 0.0886% respectively, over all the other studied growth regulator treatments. The higher concentration of BA in the medium (MB4) exerted a positive effect on the accumulation of cucurbitacin-E 0.077% in the stem explants.

[FIGURE 4 OMITTED]

These results are in agreement with those of Attard and Scicluna-Spiteri, (2001), who demonstrated that MS media containing 5 mg/l NAA produced cucurbitacin-E content of 2.970% in undifferentiated callus cultures of Ecballium elaterium. Additionally, Nikolaeva et al, (2009) reported that the presence of NAA in the nutrient medium elevated the biosynthetic ability of callus cultures of tea plant (Camellia sinensis L.) and especially stimulated the accumulation of total soluble phenolics. Higher yields of cucurbitacin B (1.126%) were obtained from stem nodes callus cultures of Ecballium elaterium in the presence of 1 mg/l BA and 0.1 mg/l NAA compared to the yield obtained from raw plant material 0.01 % (Toker et al., 2003). In conclusion, the effectiveness of Citrullus colocynthis callus culture induction, total cucurbitacins and cucurbitacin-E depends on the type and concentration of growth regulator as well as the explant source organ. In a majority of cases, the BA in combination with NAA was the best treatment for callus induction. The stem explants demonstrated better callus induction than leaf and root. Also C. colocynthis proved to synthesize total cucurbitacins and cucurbitacin-E in undifferentiated callus. Using of 2, 4-D in combination with kin was found to be the best treatment for cucurbitacins production. The accumulations of total cucurbitacins and cucurbitacin-E in callus tissues Enhancement of Callus Induction in Citrullus colocynthis were higher than those obtained from the in vitro raised seedlings. This study suggested that in vitro secondary metabolites production by C. colocynthis callus cultures could be considered an appropriate alternative method to whole plant extraction.

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Ahmad K. Hegazy * (a), Amal A. Mohamed (b), Sami I. Ali (b), Mahmoud M. Saker (c)

(a) Botany Department, Faculty of Science, Cairo University, Giza, Egypt.

(b) Plant Biochemistry Department, National Research Centre, Dokki, Cairo, Egypt.

(c) Plant Biotechnology Department, National Research Center, Dokki, Cairo, Egypt.

Correspondence: Ahmad K. Hegazy (akhegazy2202@hotmail.com)
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Author:Hegazy, Ahmad K.; Mohamed, Amal A.; Ali, Sami I.; Saker, Mahmoud M.
Publication:Journal of the Alabama Academy of Science
Article Type:Report
Geographic Code:7EGYP
Date:Jan 1, 2010
Words:4694
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