Indian Journal of Experimental Biology

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VOLUME 50

NUMBER 12

DECEMBER 2012

CODEN: IJEB (A6) 50 (12) 833-942 (2012)

ISSN: 0019-5189 (Print); 0975-1009 (Online)

 

CONTENTS

 

Papers

 

 

 

Optimal concentration of hyaluronan and plant protein in different culture systems for in vitro maturation of bovine oocytes

839

Jolanta Opiela, Ewa Latasiewicz & Zdzisław Smorąg

 

 

 

Effects of sodium dodecyl sulphate on enhancement of lipoxygenase activity of hemoglobin

847

F C Ezebuo, S O O Eze & F C Chilaka

 

 

 

Curcumin sensitizes lung adenocarcinoma cells to apoptosis via intracellular redox status mediated pathway

853

Gaurav Kaushik, Toshi Kaushik, Subodh Kumar Yadav, Sanjeev Kumar Sharma, Pavitra Ranawat, Krishan Lal Khanduja & Chander Mohan Pathak

 

 

 

Neuropharmcological potential of methanolic extract and a triterpene isolated from Madhuca longifolia L leaves in mice

862

Triveni S Inganakal, Md. Liyakhat Ahmed & Paramjyothi Swamy

 

 

 

Effect of long acting insulin supplementation on diabetic nephropathy in Wistar rats

867

Shiju Thomas Michael, Rajesh Nachiappa Ganesh & Pragasam Viswanathan

 

 

 

Protective effects of different extracts of Eucommia ulmoides Oliv. against thioacetamide-induced hepatotoxicity in mice

875

Jie Zheng, Qiuping Yan, Kun Zhang, Yang Zheng & Suqing Zhao

 

 

 

Endothelium-dependent and independent vasorelaxant effects of aqueous extract of Tridax procumbens Lin leaf in rat aortic rings

883

Hussein M Salahdeen, Gbolahan O Idowu, & Babatunde A Murtala

 

 

 

Microwave radiation induced oxidative Stress, cognitive impairment and inflammation in brain of Fischer rats

889

Kanu Megha, Pravin Suryakantrao Deshmukha, Basu Dev Banerjeea, Ashok Kumar Tripathi & Mahesh Pandurang Abegaonkar

 

 

 

Protective effect of Spirulina and tamarind fruit pulp diet supplement in fish (Gambusia affinis Baird & Girard) exposed to sublethal concentration of fluoride, aluminum and aluminum fluoride

897

K P Sharma, N Upreti, Shweta Sharma & S Sharma

 

Induction of somatic embryogenesis in endangered butterfly ginger Hedychium coronarium J. Koenig

904

Manju Verma & Y K Bansal

 

 

 

Direct and indirect method of plant regeneration from root explants of Caesalpinia bonduc (L.) Roxb.輸 threatened medicinal plant of Western Ghats

910

S R Santosh Kumar, V Krishna , Venkatesh, K Pradeepa, K Girish Kumar & A U Gnanesh

 

 

 

Annual Index

 

Contents

918

 

 

Keyword Index

931

 

 

Author Index

934

 

 

List of Experts

937

 

覧覧覧覧覧

NISCAIR Policy on Plagiarism

 

The system of formal communication in science through publication in primary journals is based on originality and quality of information being the only criteria for publication. However, there have been tendencies to misuse the system and vitiate the process of science communication for personal benefits. One of the ills afflicting science communication is plagiarism. Attempts at plagiarism may range from verbatim, copying of extensive material of other authors, misappropriating results/data of others with minor changes in language/presentation without giving credit to original source, to publish essentially the same information more than once.

As the premier publisher in India of primary scientific journals in various disciplines of science and technology, NISCAIR strongly reiterates its policy of discouraging plagiarism of all kinds. All efforts are made detect and frustrate attempts at plagiarism through editorial screening and rigorous peer review in respect of communications received for publication in NISCAIR publications. Cooperation of the scientific community is sought in our efforts to frustrate all attempts at plagiarism.

It is mandatory on the part of the corresponding author to furnish the following certificate at the time of submission of the manuscript for publication:

 

[This is to certify that the reported work in the article entitle, (give full title with all the authors name) submitted for publication in the journal, the. is an original one and has not been submitted for publication elsewhere. I/we further certify that proper citation to the previously reported work have been given and no data/table/figures have been quoted verbatim from other publications without giving due acknowledgement and without the permission of the author(s). The consent of all the authors of this article has been obtained for submitting the article to the journal, 套.

Signatures and names of all the authors]

 

In case any attempt to plagiarize is brought to our attention accompanied with convincing evidence, following steps would be taken:

(a)        After consulting the respective Editorial Board Members, authors guilty of plagiarism will be debarred from publishing their papers in NISCAIR journals.

(b)        Heads of the departments/institutes of the offending authors will be intimated of such incidences of plagiarism.

(c)        Such incidents of plagiarism will be publicized through the concerned NISCAIR journals in consultation with the respective Editorial Board Members.

 

 

 

 

Author Index

Abegaonkar Mahesh Pandurang

889

Ahmed Md. Liyakhat

862

Ashok Kumar Tripathi

889

Banerjee Basu Dev

889

Bansal Y K

904

Chilaka F C

847

Deshmukh Pravin Suryakantrao

889

Eze S O O

847

Ezebuo F C

847

Ganesh Rajesh Nachiappa

867

Girish Kumar K

910

Gnanesh A U

910

Idowu Gbolahan O

883

Inganakal Triveni S

862

Kanu Megha

889

Kaushik Gaurav

853

Kaushik Toshi

853

Khanduja Krishan Lal

853

Krishna V

910

Latasiewicz Ewa

839

Michael Shiju Thomas

867

Murtala Babatunde A

883

Opiela Jolanta

839

Pathak Chander Mohan

853

Pavitra Ranawat

853

Pradeepa K

910

Salahdeen Hussein M

883

Santosh Kumar S R

910

Sharma K P

897

Sharma S

897

Sharma Sanjeev Kumar

853

Sharma Shweta

897

Smorąg Zdzisław

839

Swamy Paramjyothi

862

Upreti N

897

Venkatesh

910

Verma Manju

904

Viswanathan Pragasam

867

Yadav Subodh Kumar

853

Yan Qiuping

875

Zhang Kun

875

Zhao Suqing

875

Zheng Jie

875

Zheng Yang

875

 

 

Keyword Index

A549 cells

853

Acetylcholine

883

Adenocarcinoma

953

Aluminum

897

Aluminum fluoride

897

Antioxidant activity

875

Aortic ring

883

Apoptosis

853

Bovine oocytes

839

Caesalpinia bonduc

910

Callus culture

904

Chlorpromazine

862

Chronic toxicity

897

Cognitive function

889

Curcumin

853

Cytokines

889

Diabetic nephropathy

867

Diazepam

862

DNA fragmentation

839

Eucommia ulmoides

875

Fluoride

897

Glargine

867

Hedychium coronarium

904

Hemoglobin

847

Hyaluronan

839

Hypertension

883

Inflammation

889

IVM

839

Linoleic acid

847

Lipoxygenase

847

Long acting insulin

867

Madhuca longifolia

862

Madhucic acid

862

MAPK signaling

853

Medicinal plant

904

Met-hemoglobin

847

Microwave radiation

889

Neuropharmacological effect

862

Oxidative stress

889

Oxodienes

847

Phenobarbitone

862

Plant regeneration

910

Protective effect

875

Rat model

867

Reactive oxygen species

853

Redox status

853

Root explants

910

Smooth muscle

883

Sodium dodecyl sulphate

847

Somatic embryogenesis

904

Spirulina

897

Streptozotocin

867

Tamarind

897

Thioacetamide-induced hepatotoxicity

875

Tridax procumbens

883

TUNEL

839

 

Correspondent author is marked by *

 

 

Indian Journal of Experimental Biology

Vol. 50, December 2012, pp. 839-846

 

 

 

Optimal concentration of hyaluronan and plant protein in different
culture systems
for in vitro maturation of bovine oocytes

Jolanta Opiela*, Ewa Latasiewicz & Zdzisław Smorąg

Department of Biotechnology of Animal Reproduction; National Research Institute of Animal Production;
Krakowska 1 st., Balice/Krakw, Poland

Received 16 April 2012; revised 17 August 2012

With a view to search for optimal concentration of hyaluronan (HA) and plant protein (PP) in different culture systems for in vitro maturation of bovine oocytes, cumulus-oocyte complexes (COCs) were matured in vitro in 2 culture systems (first co-cultured with granulose cells and estrus calf serum (ECS) in 2 mL volume, second without co-culture where ECS was replaced by exogenous hormones and BSA or PP in 100 オL dose under mineral oil). Seven types of media were used; 3 in first system and 4 in second system. To evaluate HA and PP effect on oocytes after in vitro culture an estimation of meiosis stage and a level of DNA fragmentation was performed by TUNEL staining. The highest meiotic maturation (84%) was observed in oocytes cultured in medium enriched with ECS in co-culture with granulose cells (1st system). The lowest meiotic maturation was noted in medium with addition of BSA (43%). The addition of HA in the medium enriched with BSA significantly increased the rate of matured oocytes (67%) and also didn稚 affect the chromatin quality of individual oocytes. The addition of HA to the culture medium supplemented with a PP decreased the rate of matured oocytes to 54% but no statistical differences were noted. The results of the present study showed that HA supplementation didn稚 have a detrimental impact on oocyte chromatin integrity and improved bovine oocytes meiotic maturation in medium supplemented only with BSA without co-culture of granulose cells.

 

 

Indian Journal of Experimental Biology

Vol. 50, December 2012, pp. 847-852

 

 

Effects of sodium dodecyl sulphate on enhancement of lipoxygenase
activity of hemoglobin

F C Ezebuo, S O O Eze* & F C Chilaka

Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria

Received 12 April 2012; revised 9 September 2012

Lipoxygenases comprise a family of non-heme iron-containing enzymes that catalyze the stereospecific dioxygenation of polyunsaturated fatty acids with 1, 4-cis-cis-pentadiene structure. Hemoglobin, a heme iron-containing protein has been reported to have lipoxygenase activity but the assay conditions that could enhance the activity remain obscure. Therefore, establishment of optimum assay conditions for lipoxygenase activity of hemoglobin could allow modeling of hemoglobin as lipoxygenase. Hemoglobin was extracted from blood of an identified individual of genotype AA. The hemoglobin was dialyzed at 4 oC for 24 h against 50 mM Tris-HCl buffers (pH 8.5 and 7.2) and effects of sodium dodecyl sulphate (SDS) and linoleic studied at pH 5.0 and 7.2 with UV坊IS Titration Spectrophotometry. The results show that 3.3, 8.6 and 88.1% concentrations of met-hemoglobin were found in presence of 0.0 mM SDS at pH 5.0 and 7.2, 1.043 mM SDS at pH 7.2 and 0.404 mM SDS at pH 5.0 respectively. Also, the difference spectra of hemoglobin in presence of linoleic acid showed positive peak at 285 nm which suggest the presence of oxodienes紡 reaction product of hydroperoxidase activity of lipoxygenase. Formation of met-hemoglobin/met-myoglobin is highly correlated with lipid oxidation. Since highest concentration of met-hemoglobin (88.1%) was observed in presence of 0.404 mM SDS at pH 5.0, lipoxygenase activity of hemoglobin was enhanced in presence of SDS under these conditions.

 

 

Indian Journal of Experimental Biology

Vol. 50, December 2012, pp. 853-861

 

 

Curcumin sensitizes lung adenocarcinoma cells to apoptosis via
intracellular redox status mediated pathway

Gaurav Kaushik, Toshi Kaushik, Subodh Kumar Yadav, Sanjeev Kumar Sharma, Pavitra Ranawat,
Krishan Lal
Khanduja & Chander Mohan Pathak*

Department of Biophysics, Post Graduate Institute of Medical Education and Research, Chandigarh, 160 012, India

Received 6 June 2012; revised 5 September 2012

The present study demonstrates that curcumin acts as pro-oxidant and sensitizes human lung adenocarcinoma epithelial cells (A549) to apoptosis via intracellular redox status mediated pathway. Results indicated that curcumin induced cell toxicity (light microscopy and MTT assay) and apoptosis (AnnexinV-FITC/PI labeling and caspase-3 activity) in these cells. These events seem to be mediated through generation of reactive oxygen species (ROS) and superoxide radicals (SOR) and enhanced levels of lipid peroxidation. These changes were accompanied by increase in oxidized glutathione (GSSG), reduced glutathione (GSH) and γ-glutamylcysteine synthetase (γ-GCS) activity, but decrease in GSH/GSSG ratio. The induction of apoptosis and decrease in GSH/GSSG ratio was also accompanied by sustained phosphorylation and activation of p38 mitogen activated protein kinase (MAPK). On the other hand, addition of N-acetyl cysteine (NAC), an antioxidant, blocked the curcumin-induced ROS production and rescued malignant cells from curcumin-induced apoptosis through caspase-3 deactivation. However, L-buthionine sulfoximine (BSO), a GSH synthesis blocking agent, further enhanced curcumin-induced ROS production and apoptosis in A549 cells. Decreased GSH/GSSG ratio seems to be a crucial factor for the activation of MAPK signaling cascade by curcumin. The study therefore, provides an insight into the molecular mechanism involved in sensitization of lung adenocarcinoma cells to apoptosis by curcumin.

 

 

Indian Journal of Experimental Biology

Vol. 50, December 2012, pp. 862-866

 

 

Neuropharmcological potential of methanolic extract and a triterpene isolated from Madhuca longifolia L leaves in mice

Triveni S Inganakal1, Md. Liyakhat Ahmed2 & Paramjyothi Swamy1*

1Department of Biochemistry, Gulbarga University, Gulbarga 585106, India

2Department of Pharmacology, Luqman College of Pharmacy, Gulbarga 585106, India

Received 6 June 2012; revised 10 September 2012

The methanolic extract of M. longifolia (MLME) and a compound a triterpene, derivative of madhucic acid (dMA) isolated from the leaves of M. longifolia, were investigated for their possible neuropharmacological activities in mice using phenobarbitone induced sleeping time, spontaneous motor activity, marble burying test and Eddy痴 hot plate method. LD50 for MLME and dMA were 100 and 10 mg/kg of body weight, respectively. Both MLME and dMA (10 mg/kg and 2 mg/kg oral route respectively) exhibited significant increase in phenobarbitone induced sleeping time, greater reduction in spontaneous motor activity and marble burying activity, confirming their sedative nature. Both MLME and dMA also exhibited considerable antinociceptive activity in experimental animals. The results suggest that both MLME and dMA have CNS depressant activity in mice.

 

 

Indian Journal of Experimental Biology

Vol. 50, December 2012, pp. 867-874

 

 

Effect of long acting insulin supplementation on diabetic nephropathy
in Wistar rats

 

Shiju Thomas Michaela, Rajesh Nachiappa Ganeshb & Pragasam Viswanathana*

aRenal Research Lab, Center for Bio Medical Research, School of Bio Sciences and Technology,
VIT University, Vellore 632 014, India

bDepartment of Pathology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER),
Dhanvantri Nagar, Puducherry 605 006, India

Received 21 May 2012; revised 22 August 2012

This study was designed to check whether insulin supplementation is crucial for inducing diabetic nephropathy (DNP) in Wistar rats. Diabetes was induced by a single intraperitoneal injection of streptozotocin. The urinary biochemical parameters such as albumin, creatinine and urea nitrogen were monitored every two weeks. The histological changes in the kidney were observed at the end of both fifth and seventh month. Immunohistochemical analyses of VEGF, ERK-1 and NF-κB expression were performed to demonstrate mesangial expansion and glomerulosclerosis, which are the defining histological features of nephropathy. A significant change in the urinary biochemistry was observed in diabetic animals at the end of four months, but the aforementioned quantitative changes were delayed in diabetic animals treated with insulin. At the end of seven months, the diabetic animals showed prominent histological changes such as glomerular basement membrane thickening, nodular glomerulosclerosis and mesangial expansion. However, these changes were not observed in diabetic animals treated with insulin even at the end of the study. From the results, it can be concluded that there is no need of insulin supplementation for inducing DNP, when the animals are induced with an optimal dose of 45 mg/kg body weight of streptozotocin.

 

 

Indian Journal of Experimental Biology

Vol. 50, December 2012, pp. 875-882

 

 

Protective effects of different extracts of Eucommia ulmoides Oliv. against thioacetamide-induced hepatotoxicity in mice

Jie Zheng, Qiuping Yan, Kun Zhang*, Yang Zheng & Suqing Zhao*

Department of Pharmaceutical Engineering, School of Chemical Engineering and Light Industry,
Guangdong University of Technology, Guangzhou, 510006, P.R. China

Received 12 April 2012; revised 13 September 2012

A comparison of analysis in evaluating the hepatoprotective action of fractional ethanolic (F0), ethyl acetic (F1),
n-
butanol (F2) and aqueous (F3) extracts of E. ulmoides Oliv. (EUO) against thioacetamide (TAA) induced hepatic damage was studied in mice. The extract (453 mg/kg-F0, 104 mg/kg-F1, 95 mg/kg-F2 and 237 mg/kg-F3 body weight, po, once daily for 15 days) restored serum marker enzymes levels to normal in TAA treated mice. The biochemical biomarkers viz., total protein, albumin and total bilirubin were also restored forward normal level expression pattern of liver protein profile of mice by using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis showed 144 spots in TAA administered group which were significantly reduced in EUO extracts treated group. Among the four extracts ethyl acetate (F1) and n-butanol (F2) extracts showed more significant liver protection. TAA induced injury can be correlated with its high phenolic content in these extracts which may have hepatoprotective effects in regulating liver proteins by scavenging free radicals.

 

 

Indian Journal of Experimental Biology

Vol. 50, December 2012, pp. 883-888

 

 

Endothelium-dependent and independent vasorelaxant effects of aqueous
extract of Tridax procumbens Lin. leaf in rat aortic rings

Hussein M Salahdeen1*, Gbolahan O Idowu2 & Babatunde A Murtala1

1Department of Physiology, College of Medicine, Lagos State University, Ikeja, Lagos, Nigeria

2Department of Physiology, Olabisi Onabanjo University, Ikene, Ogun State, Nigeria

Received 1 March 2012; revised 12 September 2012

Tridax procumbens leaf extract induced aortic relaxation in a concentration-dependent manner, for both phenylephrine (PE) and KCl- induced contractions in isolated rat aortic rings. The relaxation effect of the extract on PE-induced contraction was 57% greater than that on KCl- induced contraction. The extract caused dose-dependent relaxations in precontracted isolated rat aorta with phenylephrine; the relaxation was attenuated by the removal of endothelium. However, the relaxation responses to sodium nitroprusside were not significantly abolished by the removal of endothelium. The vasorelaxatory effect of the extract was completely abolished in presence of L-NAME. The results indicate that the vasorelaxant effect of T. procumbens extract is probably mediated by both endothelium-dependent and-independent mechanisms.

 

 

Indian Journal of Experimental Biology

Vol. 50, December 2012, pp. 889-896

 

 

Microwave radiation induced oxidative stress, cognitive impairment and inflammation in brain of Fischer rats

Kanu Meghaa, Pravin Suryakantrao Deshmukha, Basu Dev Banerjeea*, Ashok Kumar Tripathia &
Mahesh Pandurang Abegaonkarb

a Environmental Biochemistry and Molecular Biology Laboratory, Department of Biochemistry,
University College of Medical Sciences & G.T.B. Hospital (University of Delhi), Dilshad Garden, Delhi 110 095, India

bCentre for Applied Research in Electronics (CARE), Indian Institute of Technology, Hauz Khas, New Delhi 110 016, India

Received 4 June 2012; revised 21 September 2012

Public concerns over possible adverse effects of microwave radiation emitted by mobile phones on health are increasing. To evaluate the intensity of oxidative stress, cognitive impairment and inflammation in brain of Fischer rats exposed to microwave radiation, male Fischer-344 rats were exposed to 900 MHz microwave radiation (SAR = 5.953ラ10-4 W/kg) and 1800 MHz microwave radiation (SAR = 5.835ラ10-4 W/kg) for 30 days (2 h/day). Significant impairment in cognitive function and induction of oxidative stress in brain tissues of microwave exposed rats were observed in comparison with sham exposed groups. Further, significant increase in level of cytokines (IL-6 and TNF-α) was also observed following microwave exposure. Results of the present study indicated that increased oxidative stress due to microwave exposure may contribute to cognitive impairment and inflammation in brain.

 

 

Indian Journal of Experimental Biology

Vol. 50, December 2012, pp. 897-903

 

 

Protective effect of Spirulina and tamarind fruit pulp diet supplement in fish (Gambusia affinis Baird & Girard) exposed to sublethal concentration of fluoride, aluminum and aluminum fluoride

K P Sharma1,* N Upreti1, Shweta Sharma2 & S Sharma2

1Department of Botany, 2Department of Zoology, University of Rajasthan, Jaipur 302 004, India

Received 7 March 2012; revised 3 September 2012

Protective role of diet supplements (Spirulina, tamarind fruit pulp and their combination) on a freshwater fish G. affinis exposed at sublethal concentration of fluoride (F-) (10 ppm), Al+3 (3 ppm) and aluminum fluoride (AlF3) (35.4 ppm) in the microcosms (15 L sized) for 30-60 days in winter (90 days in summer) has been reported. Toxic effects of chemicals were manifested as higher fish mortality (4-50%) and acid (≈-30%) and alkaline phosphatase (25-50%) contents, but reduction in RBC counts (5-55%) and protein content (≈-29%) compared with controls. Alterations in values of these parameters were found maximum in aluminum exposed fish suggesting it as the most toxic among the tested chemicals. Diet supplements reduced toxicity of tested chemicals, especially when Spirulina and tamarind were given together.

 

 

Indian Journal of Experimental Biology

Vol. 50, December 2012, pp. 904-909

 

 

Induction of somatic embryogenesis in endangered butterfly ginger
Hedychium coronarium J. Koenig

Manju Verma & Y K Bansal*

Plant Tissue Culture Laboratory, Department of P.G. Studies and Research in Biological Science,
R. D University, Jabalpur 482 001, India

Received 7 May 2012; revised 16 August 2012

An efficient protocol has been developed for regeneration of complete plants through somatic embryogenesis in
H. coronarium. Creamish white, pale yellow and brown calli were obtained on MS medium supplemented with different concentrations of auxins [2, 4-Dichlorophenoxy acetic acid (2, 4-D), Indole-3 acetic acid (IAA) and 1-Naphthylacetic acid (NAA)] after 4 weeks. Creamy white calli developed on
0.5 mg L-1 2, 4-D turned embryogenic when subcultured on basal medium and produced small globular somatic embryos after 6 weeks. Further growth of somatic embryos required their transfer to medium containing 6-benzylaminopurine (BAP) or kinetin (KN). BAP was more effective than KN in promoting shoot proliferation. Maximum shoot length was obtained with 0.5 mg L-1 BAP whereas maximum shoot number was obtained with 1.0 mg L-1 BAP. The plantlets thus formed were successfully hardened, and transferred to sand-soil and farm yard manure (1:1:1) with 95% survival.

 

 

Indian Journal of Experimental Biology

Vol. 50, December 2012, pp. 910-917

 

 

Direct and indirect method of plant regeneration from
root explants of Caesalpinia bonduc (L.) Roxb.
A threatened medicinal plant of Western Ghats

S R Santosh Kumar, V Krishna*, Venkatesh, K Pradeepa, K Girish Kumar & A U Gnanesh

P.G. Department of Studies and Research in Biotechnology and Bioinformatics, Kuvempu University,
Shankaraghatta 577 451, India

Received 21 May 2011; revised 7 September 2012

An in vitro regeneration protocol has been standardized via direct and indirect methods from excised root explants of
C. bonduc,
a threatened woody legume used for the treatment of contagious diseases, inflammation, leprosy, antiperiodic, febrifuge, anthelmenthic, urinary disorders, leucorrhoea, piles and to heal wounds. MS medium supplemented with 17.75 オmol BAP and 2.46 オmol IBA, induced a mean of 3.40 ア 1.07 shoots directly from the surface of excised root explant. Subsequently, the shoots rooted readily on MS half strength medium with out growth regulators. In indirect organogenesis, callogenic frequency was optimized (96.66%) at the concentration of 9.04 オmol 2, 4-D and 0.88 オmol BAP. An average, 15.30 ア 5.25 shoots were differentiated from the root callus at the concentration of 17.57 オmol BAP and 2.85 オmol IAA. Shoots regenerated through callus were rooted well on MS half strength medium with growth regulators at 2.95 オmol IBA. Rooted plantlets were transferred to the pots containing sterilized soil and were successfully hardened at greenhouse condition for three weeks then exposed to the natural environment. Survival rate was more (95%) in plantlets derived through direct organogenesis than (60%) the plantlets regenerated through root calli.