Use of Response Surface Methodology (RSM) to Optimize Culture Media for in vitro Callogenesis in Ipomoea obscura (L.) Ker-Gawl, a Little Known Medicinal Plant | Chapter 4 | Current Strategies in Biotechnology and Bioresource Technology Vol. 1

Aims: The present study was undertaken to explore the potentiality of in vitro micropropagation (by callus induction) of Ipomoea obscura(L.) Ker-Gawl (Convolvulaceae), a perennial herb. Method: Axenically grown cotyledonary leaves regenerated into profuse calli, in various combinations of naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 2,4dichlorophenoxyacetic acid (2,4 D) with benzylaminopurine (BAP). Microroots emerged from these calli when subjected to various combinations of NAA and BAP. Maximum number of root meristems and microroots were formed in 1.25 mg/L NAA with the combination of 0.5 mg/L BAP (1:4 v/v). Leaf, nodal and internodal segments as explants were cultured on MS media containing 2,4 D at 0.5, 1.25, 2.50, 5.0 and 10.0 mg/L, for callus induction. Only leaf tips initiated callogenesis with highest response at 10 mg/l 2,4 D and lowest with 0.5 mg/l 2.4 D. This is the first report of in vitro response of I. obscura.  Results: All the results were analyzed on the context of response surface methodology (RSM) and simultaneously by contour plot. Two variables lay out in X and Y axes and a third variable Z is plotted for contour level as concentric values. An efficient and effective micropopagation protocol was developed from the leaf of Ipomoea obscura (L.) Ker-gawl, an important medicinal plant. Transplantation of regenerated plants under in vivo condition has been done successfully. RSM has been used for modeling, analyzing and optimizing in vitro responses of Ipomoea obscura. The specific target response surface plot and contour plot are focused to determine optimum shoot and root length against optimum hormone concentration, number of shoot and root length   in best concentration of MS medium and finally mean number of root and shoot in optimum NAA concentration. Depending upon variances of dry weight of callus, rooting response and root length, response surface plots (RSM) and contour plots were drawn  where four types of hormone combinations  (IAA- BAP, IBABAP, NAA-BAP, 2,4D-BAP) were used in different combinations.

Author(s) Details
Dr. Tanmoy Sinha
Cytogenetics and Molecular Biology Unit, Department of Botany, The University of Burdwan, Burdwan-713104, West Bengal, India

A. Bandyopadhyay
Cytogenetics and Molecular Biology Unit, Department of Botany, The University of Burdwan, Burdwan-713104, West Bengal, India.


In vitro Seed Germination Behaviour of Simmondsia chinensis | Chapter 15 | Modern Research in Botany Vol. 1

Simmondsia chinensis (Link) Schneider (Simmondsiaceae) is a evergreen shrub of desert and marginal land of India. It produces a unique liquid-wax which has commercial potential in pharmaceutical industry. It is difficult to propagate vegetatively as it is a seasonal procedure and grows slowly. Therefore, there is a need to improve the propagation method through seed germination and seedling behaviour. The present investigation was carried out to assess viability and in vitro germination tests of fresh and old seeds. It was found that light (16/8-h day/night photoperiod) conditions resulted in early and higher seed germination percentage as compared to total dark period under in vitro culture conditions. Improvement in germination percentage of one-year-old seeds was observed using Gibberellic acid (GA3), Benzylaminopurine (BAP) and Thidiazuron (TDZ) under given photoperiod conditions. Interestingly, TDZ 1.0 mg/L-1 were produced highest germination rate of seeds (92%), as well as better seedling growth, followed by BAP with 70% germination rate at 0.5 mg•L-1 concentration. The method of propagation through in vitro seed germination could be effectively employed for large scale production of plant material.

Author(s) Details

Dr. Raman Bala
Department of Environmental Science, Maharshi Dayanand University, Rohtak, India.

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In vitro Impact Assessment of Aqueous Extract of Sida cordifolia Linn. Upon Rat Spermatozoa Parameters | Chapter 07 | Emerging Research in Medical Sciences Vol. 1

Sida cordifolia Linn. finds frequent mention in Ayurvedic System of Medicine for many therapeutic properties including beneficial effect upon male reproductive processes. Aim of present in vitro study was to evaluate impact of aqueous extract of Sida cordifolia roots on rat spermatozoa in terms of count, motility & morphology and through HOS study following standard assessment methods to assess any likely reproductive toxicity, spermicidal action, lethality or abnormalities in animal experimentation. Sexually mature (18-22 weeks old) male Wister rats weighing 180 to 260 gm were used. Sperms were collected from cauda epididymis of rat testes and sperm suspension was prepared by mixing them with 2 ml of 5% Sucrose at 37°C, dividing into 4 samples – control (A) and 3 drug treated samples (B, C & D) containing 10, 20 or 30 mg/ml of aqueous extract of Sida cordifolia. The various sperm parameters were observed under Olympus CX41 microscope. Sperm concentration, Progressive and Non-Progressive motility were calculated using the Neubauer Chamber. The viability and morphology of sperm suspensions were assessed using Eosin Y and Nigrosin staining method using 400 X magnification under Phase contrast microscope. Hypo-osmotic swelling (HOS) test was used to evaluate the functional integrity of plasma membrane. Treatment with aqueous extract of Sida cordifolia resulted in no significant adverse impact upon sperm count up to 30 mg/ml dose, suggesting no noticeable spermicidal effect. Progressive motility increased marginally due to intervention of Sida cordifolia in a concentration-dependent manner from 40.00±1.07 in Sample A to 43.50± 2.05 in Sample D. Similarly, percentage of normal sperms and their viability exhibited steady enhancements with increasing concentrations of research drug. The HOS test indicated no adverse structural changes in plasma membrane integrity of sperms up to 30mg/ml level. Thus, in vitro assessment of sperm motility, morphology and viability due to treatment with Sida cordifolia aqueous extract indicated no spermicidal or toxic effect and indicated small but significant enhancement in various parameters which was concentration-dependent.

Author(s) Details

Dr. Mradu Gupta
Institute of Post Graduate Ayurvedic Education and Research, 294/3/1, A. P. C. Road, Kolkata, 700009, India.

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