Indian Journal of Experimental Biology

http://www.niscair.res.in; http://nopr.niscair.res.in

 

Total visitors: 3756 since 7-10-2014

 

VOLUME 52

NUMBER 10

OCTOBER 2014

CODEN: IJEB (A6) 52 (10) 921-1016 (2014)

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

CONTENTS

Papers

 

 

 

An ideal oocyte activation protocol and embryo culture conditions for somatic cell nuclear transfer using sheep oocytes

925

 

 

Hiren Patel, Shruti Chougule, Parul Chohan, Naval Shah & Deepa Bhartiya

 

 

 

Development of dog mammary tumor xenograft in immunosuppressed Swiss albino mice

935

 

 

R S Rajmani, Prafull Kumar Singh, Sanjay Kumar, G Ravi Kumar, Aditya P Sahoo,
Lakshman Santra, Shikha Saxena, Lakshya Veer Singh, Uttara Chaturvedi, Lovleen Saxena,
G S Desai, Shishir Kumar Gupta, Amit Kumar, N S Jadon & Ashok K Tiwari

 

 

 

Evaluation of target mRNA cleavage by Aurorakinase B specific siRNA in prostate and
hepatic cancer cells and its therapeutic potential in mouse models of liver cancer

943

 

 

Shailaja A Chile, Kriti B Ray, Sameer Shaikh, Vikram Rajagopal, Harinarayana S Rao,

Venkata Ramana & A S Manoj Kumar

 

 

 

Seabuckthron (Hippophae rhamnoides L) leaf extract ameliorates the gamma radiation mediated DNA damage and hepatic alterations

952

 

 

Amitava Khan, Krishnendu Manna, Chinchubose, Dipesh Kr Das, Mahuya Sinha,
Swaraj Bandhu Kesh, Ujjal Das, Rakhi Sharma Dey, Asoke Banerji & Sanjit Dey

 

 

 

Hypolipidemic and hypoglycemic effects of Centella asiatica extract in vitro and in vivo

965

 

 

Nattapon Supkamonseni, Aree Thinkratok, Duangdeun Meksuriyen & Rungrudee Srisawat

 

 

 

Disruption of glucose tolerance caused by glucocorticoid excess in rats is partially prevented, but not attenuated, by arjunolic acid

972

 

 

Luiz M Gon軋lves-Neto, Francielle BD Ferreira, Luiz Souza, Cristiane dos Santos,
Antonio C Boschero, Valdir A Facundo, Adair R S Santos, Everson A Nunes & Alex Rafacho

 

 

Rapid flow cytometry based cytotoxicity assay for evaluation of NK cell function

983

 

 

Snehal Mhatre, Manisha Madkaikar, Kanjaksha Ghosh, Mukesh Desai,
Vaishali Pujari & Maya Gupta

 

 

 

Safety assessment of hydroethanolic rambutan rind extract: Acute and sub-chronic
toxicity studies

989

 

 

Aree Thinkratok, Parin Suwannaprapha & Rungrudee Srisawat

 

 

Population genetic structure of malaria vector Anopheles stephensi using mitochondrial Cytochrome oxidase II gene in Indian populations

996

 

 

Arvind Sharma, Arunaditya Deshmukh, Richa Sharma, Ashwani Kumar, Sayantan Mukherjee, G C Chandra, S K Gakhar

 

 

 

In vitro culture of immature embryos of Cinnamomum tamala Nees.裕he role of
different factors

1003

 

 

Madhabi S Deb, N S Jamir & Chitta Ranjan Deb

 

 

 

Cross-species amplification of microsatellite markers in Mycteria leucocephala Pennant 1769: Molted feathers as successful DNA source

1011

 

 

Bharat Bhushan Sharma, Mohd. Mustafa, Tusha Sharma, Basu Dev Banerjee &
Abdul Jamil Urfi

 

 

 

Announcement

 

 

 

SERB School in Neuroscience, 8th edition: Brain Circuits

924

覧覧覧覧覧

Announcement

SERB School in Neuroscience, 8th edition: Brain Circuits

8-21 December, IISER, Pune

Sponsored by the Department of Science and Technology, Govt. of India, the Science and Engineering Research Board (SERB) School in Neuroscience, 8th edition will be conducted at the Indian Institute of Science Education and Research (IISER) Pune during 8-21 December 2014. The focus of this edition is Brain Circuits: development, organization, plasticity and computation in neuronal circuits.

The course is aimed at PhD students, post-doctoral fellows and junior faculty. For details of School and application, please visit: www.iiserpune.ac.in/~serbneuro2014

覧覧覧覧

Errata

Suppression of Eis and expression of Wag31 and GroES in Mycobacterium tuberculosis cytosol under anaerobic culture conditions, by Vineet K Maurya, Kavita Singh & Sudhir Sinhaク Indian J Exp Biol, Vol. 52, August 2014, pp. 773-780.

The figure 1 appearing on p. 775 may be replaced with the following figure:

 

覧覧覧覧

43 kDa and 66 kDa, two blood stage antigens induce immune response in Plasmodium berghei malaria, by Chhaya Pirta & H S Banyalク Indian J Exp Biol, Vol. 52, August 2014, pp. 781-786.

In figure 2 (p. 783) the legends may be read as:

■ Prechallenge □ Postchallenge

覧覧覧覧覧覧覧

 

Author Index

Amit Kumar

935

Antonio C Boschero

972

Ashwani Kumar

996

 

 

Banerjee Basu Dev

1011

Banerji Asoke

952

Bhartiya Deepa

925

 

 

Chandra G C

996

Chaturvedi Uttara

935

Chile Shailaja A

943

Chinchubose

952

Chohan Parul

925

Chougule Shruti

925

 

 

Das Dipesh Kr

952

Das Ujjal

952

Deb Chitta Ranjan

1003

Deb Madhabi S

1003

Desai G S

935

Desai Mukesh

983

Deshmukh Arunaditya

996

Dey Rakhi Sharma

952

Dey Sanjit

952

 

 

Facundo Valdir A

972

Ferreira Francielle BD

972

 

 

Gakhar S K

996

Ghosh Kanjaksha

983

Gon軋lves-Neto Luiz M

972

Gupta Maya

983

Gupta Shishir Kumar

935

 

 

Jadon N S

935

Jamir N S

1003

 

 

Kesh Swaraj Bandhu

952

Khan Amitava

952

 

 

Madkaikar Manisha

983

Manna Krishnendu

952

Manoj Kumar A S

943

Meksuriyen Duangdeun

965

Mhatre Snehal

983

Mukherjee Sayantan

996

Mustafa Mohd.

1011

 

 

Nunes Everson A

972

 

 

Patel Hiren

925

Pujari Vaishali

983

 

 

Rafacho Alex

972

Rajagopal Vikram

943

Rajmani R S

935

Ramana Venkata

943

Rao Harinarayana S

943

Ravi Kumar G

935

Ray Kriti B

943

 

 

Sahoo Aditya P

935

Sanjay Kumar

935

Santos Adair R S

972

Santos Cristiane dos

972

Santra Lakshman

935

Saxena Lovleen

935

Saxena Shikha

935

Shah Naval

925

Shaikh Sameer

943

Sharma Arvind

996

Sharma Bharat Bhushan

1011

Sharma Richa

996

Sharma Tusha

1011

Singh Lakshya Veer

935

Singh Prafull Kumar

935

Sinha Mahuya

952

Souza Luiz

972

Srisawat Rungrudee

965,989

Supkamonseni Nattapon

965

Suwannaprapha Parin

989

 

 

Thinkratok Aree

965,989

Tiwari Ashok K

935

 

 

Urfi Abdul Jamil

1011

 

 

Keyword Index

Acarbose

965

Acute toxicity

989

Anopheles stephensi

996

Antioxidant activity

989

Arjunolic acid

972

Aurorakinase B

943

 

 

Centella asiatica

965

Cinnamomum tamala

1003

Cytochrome oxidase

996

 

 

DNA damage

952

Dog mammary tumour

935

 

 

Embryo culture

925,1003

 

 

Flow cytometry

983

 

 

Gamma radiation

952

Gene flow

996

Genetic differentiation

996

Glucocorticoid

972

Glucose tolerance

972

 

 

Haplotype diversity

996

Hepatic alteration

952

Hepatocellular carcinoma

943

Hypoglycemia

965

Hypolipidemia

965

 

 

Immunosuppression

935

Insulin sensitivity

972

 

 

Lipid emulsion-fed rat

965

 

 

Metabolism

972

Micropropagation

1003

Morphogenesis

1003

 

 

Natural Killer cell cytotoxicity

983

 

 

Nephelium lappaceum

989

Non-invasive sampling

1011

 

 

Obesity

965

Oocytes

925

Orlistat

965

 

 

Painted Stork

1011

Parthenogenesis

925

Population genetics

1011

 

 

Rambutan

989

Reactive oxygen species

952

Recalcitrant seed

1003

RNAi

943

Rutin

965

 

 

Seabuckthron

952

Sheep

925

Simple Sequence Reapeats

1011

siRNA

943

Somatic cell nuclear transfer

925

Spice yielding plant

1003

Sub-chronic toxicity

989

 

 

Whole blood

983

Wood Stork

1011

 

 

Xenograft

935

 

 

Correspondent author is marked by *

Papers

 

Indian Journal of Experimental Biology

Vol. 52, October 2014, pp. 925-934

 

 

An ideal oocyte activation protocol and embryo culture conditions for
somatic cell nuclear transfer using sheep oocytes

Hiren Patel, Shruti Chougule, Parul Chohan, Naval Shah & Deepa Bhartiya*

Stem Cell Biology Department, National Institute for Research in Reproductive Health,
Jehangir Merwanji Street, Mumbai, 400 012, India

Received 24 June 2014; revised 11 August 2014

Pluripotent stem cells are possibly the best candidates for regenerative medicine, and somatic cell nuclear transfer (SCNT) is one of the viable options to make patient-specific embryonic stem cells. Till date efficacy of SCNT embryos is very low and requires further improvement like ideal oocyte activation and in vitro culture system. The aim of the present study was to evaluate ideal oocyte activation using different stimulation protocols and to study the effect of cumulus co-culture conditions on embryo development. Results demonstrate that between electric stimulation and chemical stimulation using calcium ionomycin and ionophore, best oocyte activation was obtained using calcium ionomycin (5 μM for 5 min) which resulted in 83% cleavage followed by 7% of early blastocyst which further increased to 15% when a cumulus bed was also introduced during embryo culture. Sequential modified Charles Rosenkrans 2 (mCR2) medium was used for embryo culture in which glucose levels were increased from 1 mM to 5 mM from Day 3 onwards. SCNT using cumulus cells as donor somatic cell, calcium ionomycin to activate the reconstructed oocyte and embryo culture on a cumulus bed in sequential mCR2 medium, resulted in the development of 6% embryos to early blastocyst stage. Such technological advances will make SCNT a viable option to make patient-specific pluripotent stem cell lines in near future.

 

 

Indian Journal of Experimental Biology

Vol. 52, October 2014, pp. 935-942

 

 

Development of dog mammary tumor xenograft in immunosuppressed
Swiss albino mice

R S Rajmania, Prafull Kumar Singha, Sanjay Kumarb, G Ravi Kumara, Aditya P Sahooa, Lakshman Santraa,
Shikha Saxenaa, Lakshya Veer Singha, Uttara Chaturvedia, Lovleen Saxenaa, G S Desaia, Shishir Kumar Guptaa,
Amit Kumarc, N S Jadonb & Ashok K Tiwaria*

aMolecular Biology Laboratory, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar,
Bareilly 243 122, India

bDivision of Surgery, Govind Ballabh Pant University of Agriculture & Technology, Pantnagar, India

cDivision of Animal Genetics and Breeding, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India

Received 1 October 2013

Development and study of dog mammary tumour xenograft in immunosuppressed Swiss Albino Mice adds a new dimension in cancer research as dog tumors have many similarities with human tumors regarding progression, histopathology, molecular mechanism, immune response and therapy. Failure of the immune system to recognize and eliminate cancer cells leads to cancer progression and the fight between immune cells and cancer cells has a great role in understanding the mechanism of cancer progression and elimination. Rejection and acceptance of tumour xenograft depends on efficiency of CD4+, CD8+ and NK cell populations. In the present investigation, dog mammary tumor xenograft in cyclosporine-A and γ-irradiated, immunosuppressed Swiss Albino mice was developed and the immune cell status of graft accepted and rejected mice was assessed. It was observed that all the major immune cells (CD4+, CD8+ and NK cells) play an equal role in tumour rejection.

 

 

Indian Journal of Experimental Biology

Vol. 52, October 2014, pp. 943-951

 

 

Evaluation of target mRNA cleavage by Aurorakinase B specific siRNA in prostate and hepatic cancer cells and its therapeutic potential in
mouse models of liver cancer

Shailaja A Chile1, Kriti B Ray1, Sameer Shaikh2, Vikram Rajagopal1,3, Harinarayana S Rao2,
Venkata Ramana1 & A S Manoj Kumar1*

1Therapeutics Proteins Group; 2Laboratory of Animal Research Services, Reliance Life Sciences Pvt. Ltd.,
Dhirubhai Ambani Life Sciences Center, Thane-Belapur Road, Rabale, Navi Mumbai 400 701, India

Received 10 March 2014; revised 12 June 2014

The anti proliferative potential of siRNA26, targeted to Aurora kinase B, in prostate cancer cells is known from a previous study from our laboratory. Here we first show that siRNA26 cleaves at the same position of the target mRNA in the prostate cancer and hepatocellular carcinoma cell lines, PC3 and HepG2 respectively. Aurorakinase B specific siRNA, but not a control siRNA, inhibited PC3 and HepG2 cell proliferation and cell migration. These effects correlated to RNA silencing of Aurorakinase B in both the cell lines. Intra-tumoral administration of HiPerfect complexed siRNA26 inhibited the growth of HepG2 xenografts in SCID mice. In an orthotopic setting, intravenous administration of HiPerfect encapsulated siRNA26 appeared to reduce the severity of multifocal lesions.

 

 

Indian Journal of Experimental Biology

Vol. 52, October 2014, pp. 952-964

 

 

Seabuckthron (Hippophae rhamnoides L.) leaf extract ameliorates the gamma radiation mediated DNA damage and hepatic alterations

Amitava Khan1, Krishnendu Manna1, Chinchubose2, Dipesh Kr Das1, Mahuya Sinha1, Swaraj Bandhu Kesh1,
Ujjal Das1, Rakhi Sharma Dey3, Asoke Banerji2 & Sanjit Dey1*

1Department of Physiology, UCSTA, University of Calcutta, 92 A P C Road, Kolkata 700 009, India

2Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Amritapuri, Kollam 690 525, India

3Department of Food and Nutrition, Barrackpore Rastraguru Surendranath College, Kolkata 700 120, India

Received 9 August 2013; revised 11 August 2014

In vitro assessment showed that H. rhamnoides (HrLE) extract possessed free radical scavenging activities and can protect gamma (γ) radiation induced supercoiled DNA damage. For in vivo study, Swiss albino mice were administered with HrLE (30 mg/kg body weight) for 15 consecutive days before exposing them to a single dose of 5 Gy of γ radiation. HrLE significantly prevented the radiation induced genomic DNA damage indicated as a significant reduction in the comet parameters. The lipid peroxidation, liver function enzymes, expression of phosphorylated NFκB (p65) and IκBα increased whereas the endogenous antioxidants diminished upon radiation exposure compared to control. Pretreatment of HrLE extract ameliorated these changes. Based on the present results it can be concluded that H. rhamnoides possess a potential preventive element in planned and accidental nuclear exposures.

 

 

Indian Journal of Experimental Biology

Vol. 52, October 2014, pp. 965-971

 

 

Hypolipidemic and hypoglycemic effects of Centella asiatica (L.) extract
in vitro and in vivo

Nattapon Supkamonseni1, Aree Thinkratok1, Duangdeun Meksuriyen2 & Rungrudee Srisawat1*

1Institute of Science, Suranaree University of Technology, 111 University Avenue, Suranaree District, Amphur Muang,
Nakhon Ratchasima 30000, Thailand

2Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phyathai Road, Pathumwan, Bangkok 10330, Thailand

Received 15 October 2013; revised 18 July 2014

In vitro study revealed that pancreatic lipase inhibitory activity of C. asiatica extract was significantly higher than rutin but lower than orlistat, an anti-obesity drug. α-Amylase inhibitory activities of C. asiatica extract and rutin were significantly lower than acarbose, an anti-diabetic drug. Inhibition of α-glucosidase activity by C. asiatica extract, rutin, and acarbose was not different. The in vivo study substantiated the in vitro results. C. asiatica extract
(1000 and 2000 mg/4 mL/kg), rutin (1000 mg/4 mL/kg), and orlistat (45 mg/4 mL/kg)
significantly decreased plasma glucose, triglyceride and total cholesterol levels in lipid emulsion-induced hyperlipidemic rats at 3 h. However, plasma aspartate aminotransferase and alanine aminotransferase levels did not show significant change. The present work further supports that the C. asiatica extract and its bioactive rutin may help managing hypolipidemic and hypoglycemic effects.

 

 

Indian Journal of Experimental Biology

Vol. 52, October 2014, pp. 972-982

 

 

Disruption of glucose tolerance caused by glucocorticoid excess in rats is
partially prevented, but not attenuated, by arjunolic acid

Luiz M Gon軋lves-Neto1, Francielle BD Ferreira1, Luiz Souza2, Cristiane dos Santos1, Antonio C Boschero3,
Valdir A Facundo4, Adair R S Santos1, Everson A Nunes1 & Alex Rafacho1*

1Department of Physiological Sciences and 2Biochemistry, Centre of Biological Sciences, 88040-900,
Federal University of Santa Catarina剖FSC, Florianpolis, Brazil

3Department of Structural and Functional Biology, Institute of Biology, 13083-862,
State University of Campinas剖NICAMP, Campinas, Brazil

4Department of Chemistry, 78900-500, Federal University of Rondnia剖NIR, Porto Velho, Brazil

Received 2 August 2013; revised 4 August 2014

Arjunolic acid (AA) obtained from plants of the Combretaceae family has shown anti-diabetic effects. Here, we analyzed whether the diabetogenic effects of dexamethasone (DEX) treatment on glucose homeostasis may be prevented or attenuated by the concomitant administration of AA. Adult Wistar rats were assigned to the following groups: vehicle-treated (Ctl), DEX-treated (1 mg/kg body weight intraperitoneally for 5 days) (Dex), AA-treated (30 mg/kg body weight by oral gavage twice per day) (Aa), AA treatment previous to and concomitant to DEX treatment (AaDex), and AA treatment after initiation of DEX treatment (DexAa). AA administration significantly ameliorated (AaDex) (P>0.05), but did not attenuate (DexAa), the glucose intolerance induced by DEX treatment. AA did not prevent or attenuate the elevation in hepatic glycogen and triacylglycerol content caused by DEX treatment. All DEX-treated rats exhibited hepatic steatosis that seemed to be more pronounced when associated with AA treatment given for a prolonged period (AaDex). Markers of liver function and oxidative stress were not significantly altered among the groups. Therefore, AA administered for a prolonged period partially prevents the glucose intolerance induced by DEX treatment, but it fails to produce this beneficial effect when given after initiation of GC treatment. Since AA may promote further hepatic steatosis when co-administered with GCs, care is required when considering this phytochemical as a hypoglycemiant and/or insulin-sensitizing agent.

 

 

Indian Journal of Experimental Biology

Vol. 52, October 2014, pp. 983-988

 

 

Rapid flow cytometry based cytotoxicity assay for evaluation of NK cell function

Snehal Mhatre, Manisha Madkaikar, Kanjaksha Ghosh, Mukesh Desai*, Vaishali Pujari & Maya Gupta

Department of Pediatric Immunology and Leukocyte Biology, National Institute of Immunohaematology (ICMR),
13th floor, Multistoreyed Building, KEM Campus, Parel, Mumbai 400 012, India

Received 30 September 2013; revised 7 August 2014

Assessment of natural killer cells (NK-cell) cytotoxicity is used not only in research settings but is also important in diagnosis of various diseases. NK-cell cytotoxicity assays are based on measurement of target cells killed by cytotoxic cells analyzed either by chromium (51Cr) release assay or flow cytometry. Both these methods use peripheral blood mononuclear cells (PBMC) or pure NK-cell population and hence require large volume of blood sample which is difficult to obtain in pediatric patients and patients with cytopenia. Hence, a flow cytometric assay was designed to determine NK cell activity using whole blood, eliminating the need for isolation of PBMCs or pure NK cells. This assay is based on a dual fluorescent staining of target cells (K562 cell line). The DIOC18 dye labeled K562 cells are incubated with whole blood and then counterstained with 7-AAD enabling the measurement of dead target cell and then percent cytotoxicity is calculated. This study compared the NK cell cytotoxicity using PBMC and whole blood in clinically relevant samples. There was no significant difference between two assays in the measurement of lytic activity or in reproducibility in the repeated samplings of healthy individuals. The whole blood assay required less volume of blood and also less processing time as compared to PBMC assay. It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis expected to have low NK-cell activity. This assay is rapid, sensitive and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK-cell function.

 

 

Indian Journal of Experimental Biology

Vol. 52, October 2014, pp. 989-995

 

 

Safety assessment of hydroethanolic rambutan rind extract: Acute and sub-chronic toxicity studies

Aree Thinkratok1, Parin Suwannaprapha2 & Rungrudee Srisawat1*

1Institute of Science, Suranaree University of Technology, 111 University Avenue, Suranaree District, Amphur Muang,
Nakhon Ratchasima Province 30000, Thailand

2Faculty of Veterinary Science, Mahidol University, 999 Phutthamonthon Sai 4 Road, Salaya, Phutthamonthon,
Nakhonpathom Province 73170, Thailand

Received 15 October 2013; revised 18 July 2014

This study evaluated the safety of rambutan rind extract (RRE) in male Wistar rats. While acute toxicity was evaluated by feeding the rats with single doses of RRE (1000, 2000, 3000, 4000, and 5000 mg/kg) and its sub-chronic toxicity was observed in rats orally administered with RRE (500, 1000, and 2000 mg/kg) daily for 30 days. In acute toxicity study, the LD50 was found to be greater than 5000 mg/kg of RRE. In sub-chronic toxicity study, no mortality and sign of toxicity was found up to 1000 mg/kg/day of RRE. At 2000 mg/kg/day dose, the mortality rate was 12.5%. Significant decreases in body weight gain and food consumption were found in both acute and sub-chronic toxicity studies. In acute toxicity study, all the studied doses of RRE did not alter serum levels of triglyceride (TG), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In sub-chronic toxicity study, all studied doses of RRE significantly decreased plasma levels of TG and blood urea nitrogen, but did not alter plasma levels of AST and ALT. TC levels did not show any significant change in both the studies. The obtained results provide basic information for in vivo experimental studies of the pharmacological potentiality of RRE.

 

 

Indian Journal of Experimental Biology

Vol. 52, October 2014, pp. 996-1002

 

 

Population genetic structure of malaria vector Anopheles stephensi using mitochondrial Cytochrome oxidase II gene in Indian populations

Arvind Sharma, Arunaditya Deshmukh1, Richa Sharma, Ashwani Kumar,
Sayantan Mukherjee2, G C Chandra2, S K Gakhar*

Centre for Biotechnology, Maharshi Dayanand University, Rohtak, 123 4001

Received 1 November 2013; revised 17 June 2014

The genetic differentiation in A. stephensi based on haplotype diversity using Restriction Fragment Length Polymorphism and by sequencing of CO II gene across different localities in India has been analyzed. The presence of only one DraI restriction site in CO II gene conferred to haplotype B indicating that the gene is very much conserved and the gene flow is not affected even by a major geographical distance barrier. The sequencing and analysisof various population parameters revealed seven haplotypes in all populations. The West Bengal population was found to be more genetically diverse than others. The geographic distance between populations was found to be contributing to the genetic differentiation. The sign of demographic expansion were found in three of the five populations. The local geographic barriers were found to be ineffective in prevention of gene flow.

 

 

Indian Journal of Experimental Biology

Vol. 52, October 2014, pp. 1003-1010

 

 

In vitro culture of immature embryos of Cinnamomum tamala Nees.
The role of different factors

Madhabi S Deb, N S Jamir & Chitta Ranjan Deb*

Department of Botany, Nagaland University, Lumami 798 627, India

Received 3 June 2013; revised 8 August 2014

Seed characteristics and in vitro culture of C. tamala embryos were studied. Embryos desiccated below 50%
(fresh weight) exhibited poor morphogenetic response in vitro and confirmed the recalcitrant nature of seeds. The immature embryos of various developmental ages (4-16 week after flowering, WAF) were cultured on different strengths of MS medium. Morphogenesis responses were recorded after 10 days of culture. The best culture responses were achieved from the immature embryos of 12 WAF on MS medium with sucrose (3%, w/v), polyvinyl pyrollidone (100 mg L-1) and benzyl adenine (12 オM). Under optimum condition ~60% explants responded; and ~7.3 shoots buds developed per explants after 35 days of culture initiation. The shoot buds could be converted into micro-shoots on MS medium with sucrose (3%) and kinetin (3 オM). About 5.3 micro-shoots/shoot buds sprouted per sub-culture. The micro-shoots were rooted by maintaining them on MS medium with α-naphthalene acetic acid (3 オM) where within 6-8 wk of culture ~8-10 roots developed. The rooted plantlets were acclimatized in vitro before they were transferred to community potting mix and maintained in the poly-shade ca 75% shading. The transplants registered ~70% survival after two months of transfer.

 

 

Indian Journal of Experimental Biology

Vol. 52, October 2014, pp. 1011-1116

 

 

Cross-species amplification of microsatellite markers in Mycteria leucocephala Pennant 1769: Molted feathers as successful DNA source

Bharat Bhushan Sharma1,2, Mohd. Mustafa2, Tusha Sharma2, Basu Dev Banerjee2 & Abdul Jamil Urfi1*

1Department of Environmental Studies, University of Delhi, New Delhi 110 007, India

2Environmental Biochemistry & Molecular Biology Laboratory, Department of Biochemistry,
University College of Medical Sciences & Guru Teg Bahadur Hospital,
University of Delhi, Dilshad Garden, Delhi 110 095, India

Received 26 July 2013; revised 6 August 2014

DNA from molted feathers is being increasingly used for genetic studies on birds. However, the DNA obtained from such non-invasive sources is often not of enough quantity and quality for isolation of new microsatellite markers. The present study examined the potential of shed feathers of near threatened Painted Stork as a source of its DNA for cross-species amplification of microsatellites. Thirty-one shed feathers of varying conditions (good and deteriorated) and sizes (large, intermediate and small) collected in a north Indian population were used to isolate DNA by a standard isopropanol method and 11 microsatellite markers already developed in the Wood Stork were screened for amplification. Nine plucked feathers from two dead Painted Storks were also used to compare the DNA yield and amplification success. The DNA yield of feathers varied significantly in relation to the calamus size and condition. Among molted feathers, good and large samples provided more DNA than deteriorated and small ones, respectively. Large plucked feathers yielded more DNA than large molted feathers. DNA was almost degraded in all the samples and ratio of absorbance at 260/280 nm varied from 1.0 to 1.8, indicating impurity in many samples. Independent of DNA yields, all microsatellites were cross-amplified in all kinds of feathers, with >80% success in different feather categories. It is concluded that the shed feathers can be successfully used to isolate DNA in the Painted Stork and for cross-species amplification of microsatellites.