Differentiation of Embryonic and Non-Embryonic Callus of Convolvulus Pluricaulis Choisy using

International Journal of Botany and Research (IJBR) ISSN (P): 2277–4815; ISSN (E): 2319–4456 Vol. 10, Issue 1, Jun 2020, 25–32 © TJPRC Pvt. Ltd.

DIFFERENTIATION OF EMBRYONIC AND NON-EMBRYONIC CALLUS OF CONVOLVULUS PLURICAULIS CHOISY USING BIOCHEMICAL STUDIES SANTOSHKUMAR SINGH1 & ZANKHANA RATHOD2 1 2

Assistant Professor, Department of Botany, D. N. Science College, Dabhoi, Vadodara Gujarat, India

Assistant Professor, Department of Botany, M. G. Science Institute, Navrangpura, Ahmedabad, Gujarat, India

ABSTRACT Convolvulus pluricaulis Choisy (syn Convolvulus microphyllus L.) of family Convolvulaceae commonly known as ‘Shankhpushpi’ is an indigenous plant commonly mentioned in ancient system of Indian medicine as a rasayan which is mainly advocated to use as brain tonic. Plant tissue culture techniques were employed for conservation and multiplication of Convolvulus pluricaulis. During the studies, biochemical profile of non-embryonic and embryonic callus was studied at weekly intervals and it revealed remarkable changes right at the end of second week. In the two weeks, old culture the activity of Peroxidase, Polyphenol Oxidase (PPO), Catalase and enzyme protein was higher in non-embryonic callus and activity of IAA Oxidase and Protease was higher in embryonic callus. Total Amylase activity of wall bound fractions was higher in non-embryonic and embryonic callus. Variations in enzymatic activities could well

KEYWORDS: Convolvulus Pluricaulis, Embryonic and Non- Embryonic Callus & Enzymes as Biochemical Markers

Received: Dec 15, 2019; Accepted: Jan 05, 2020; Published: Feb 20, 2020; Paper Id.: IJBRJUN20204

INTRODUCTION

Original Article

serve as a biochemical marker to determine the embryonic and non-embryonic callus at very early stage of culture.

According to Ayurveda Convolvulus pluricaulis (L.) Choicy (Shankhpushpi) of family Convolvulaceae is an important and well known traditional source of medhya rasayan drug (that counter act stress, improve intelligence and memory) used for rejuvenation of brain and mental health and also to promote intellect and memory1,2 It is also used as a psycho-stimulant and tranquilizer. It is reported to reduce mental tension3,4 An alkaloid Sankhpushpine is the major component of phytoactive compounds along with the several other alkaloids reported from this plant like convolamine, phyllabine, convolidine, confoline, convoline, subhirsine, convosine, and convolvidine with scopoline and β- sitosterol.5, 6 Plant tissue culture technology offers an alternative to fulfill the demand of raw material with continues supply and also in conservation of the endangered medicinal plants7, 8 Callus culture is an effective tool to obtain the pharmaceutically important compounds in-vitro condition which enables the plants to be preserved in their natural habitat. Somatic embryogenesis has proved to be useful for micropropagation, production of mutants, artificial seed and material for use in the plant genetic engineering. Several biochemical markers have been shown to discriminate between embryonic and non-embryonic tissues like distinct proteins9 isozyme forms and ethylene are among them10 Physiological conditions of embryonic and non-embryonic callus are different so the enzymes and their activity rates also vary in the system. The variation in activity of enzymes could serve as an effective tool as ‘Biochemical Marker’ for early differentiation of embryonic and non-embryonic callus at very early stage of culture. It also aids our basic understanding of the biochemical changes underlying the formation of somatic and

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Santoshkumar Singh & Zankhana Rathod

zygotic embryos11 Hence the present studies on somatic embryogenesis, the analysis of cytoplasmic, wall bound enzymes between embryonic and non-embryonic callus were undertaken on Convolvulus pluricaulis.

MATERIALS AND METHODS Plants of Convolvulus pluricaulis were collected from the Gujarat University botanical garden. They were thoroughly washed under running tap water for 10 mins. followed by double distilled water. Leaves were surface sterilized with 0.1% (w/v) systemic fungicide - bavistin followed by 0.1% (w/v) HgCl2, 5% (v/v) sodium hypochlorite (NaOCl) and 5% (v/v) Tween-20 solution respectively. Finally, these were rinsed 4-5 times with sterile double distilled water and used for culture on Murashige and Skoog12 medium supplemented with 2,4-D and kinetin in combination of 1:1 to 5:5 mg/l. According to the need of the different experiments, the formulations of MS were suitably modified. 4% (w/v) sucrose and 0.8% (w/v) agar-agar were used. The cultures were maintained in culture room at 25±1° C and exposure of 12 hrs to fluorescent light.

The explant swelled up within a week of inoculation and exhibited callus initiation from the cut margins of leaf. After 3035 days of inoculation the compact slows growing the callus produced white hard notched structure, the somatic embryos, on its surface. This was distinguished as embryonic callus. The enzymatic activities taking place in embryonic and nonembryonic callus estimations were carried out for Peroxidase;13 IAA-Oxidase;14 Invertase;15 Protease;16,17 α and β Amylase;18 Polyphenol oxidase (PPO);19 Catalase;20 Enzyme Protein.21

RESULTS AND DISCUSSIONS During the present work, Murashige and Skoog’s medium supplemented with 2,4-D and kinetin in combination of 1:1, 2:2, 3:3, 4:4 and 5:5 mg/l medium was used for callus production. Lower concentrations of PGRs formed non-embryonic callus while higher concentrations 4:4 and 5:5 mg/l of concentration formed embryonic callus. Non-embryonic callus was green, friable while globular embryonic callus was compact, green with pink pigmentation. However, the callus differentiation was seen after 30-35 days of inoculation.

Table 1: Effect of 2, 4-D and Kinetin on the Callus Induction in Leaf Culture 2,4-D Kinetin Callus Media Remarks (mg/l) (mg/l) Induction 1 1 ++++ Non embryonic, green, friable Non embryonic, whitish green, 2 2 +++ friable Non embryonic whitish yellow, MS 3 3 +++ mucilaginous Basal Embryonic whitish green 4 4 ++ compact Embryonic, compact, globular, 5 5 ++ green, with pink pigmentation

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Differentiation of Embryonic and Non-Embryonic Callus of Convolvulus Pluricaulis Choisy using Biochemical Studies

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Figure 1: Biochemical Analysis of Embryonic and Non- Embryonic Convolvulus pluricaulis.

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Santoshkumar Singh & Zankhana Rathod

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Figure 9

Analysis of enzymatic patterns from the non-embryonic callus and embryonic callus revealed that total amylase content of cytoplasmic fraction of embryonic callus was very close to the wall bound fraction of the non-embryonic callus and was at highest. Invertase activity was highest in the wall bound fraction of the embryonic callus. Polyphenol activity was distinctly higher in cytoplasmic and wall bound fraction of the non-embryonic callus. No large variations was observed in the protease content of the two materials, however, the wall bound fractions showed the higher activity.

Enzyme protein content was higher in both the fractions of the non-embryonic callus. Peroxidase activity was notably higher in the non-embryonic callus. IAA oxidase activity was higher in the embryonic callus were as the Catalase activity was higher in the cytoplasmic and the wall bound fractions of the non-embryonic callus.

1 mg/l 2,4-D + 1 mg/l kinetin

CONCLUSIONS Many researchers have tried to understand the biochemical profile of embryonic and non-embryonic callus to understand the development of stage-specific biochemical markers that differentiates between embryonic and nonembryonic conditions of a callus. Storage proteins were the first compounds used as markers in comparing the developmental programs of two types of embryogenesis.22,

23

Higher peroxidase activity were reported from the

embryonic callus of Bougainvillea spectabillis L.24 The presence of homologous proteins in the mature somatic embryos along with their triglyceride content indicates embryo quality.25 The presence of peroxidase and esterase, isozymes patterns reflects the embryogenic potential of Medicago sativa and Dactylis glomerata suspension cultures.26 Peroxidase was shown the difference between the developmental stages of Picea abies somatic embryogenesis.27 Thus, the present study indicates that the process of somatic embryogenesis is characterized by biochemical changes in the enzymes which is induced by plant growth regulators and in turn can be successfully utilized to differentiate embryonic and nonembryonic callus.

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Differentiation of Embryonic and Non-Embryonic Callus of Convolvulus Pluricaulis Choisy using Biochemical Studies

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AUTHORS PROFILE

Dr. Santoshkumar Singh, currently working as Assistant Professor in the subject of Botany at D. N. Science College, Dabhoi, Vadodara, Gujarat. He has Ph. D. awarded for the thesis entitled “Physiological and Phytochemical Studies of Bacopamonnieri Wetist, Centellaasiatica (L.) Urban and Convolvuluspluricaulis Choicy” in the subject of Botany by Gujarat University, Ahmedabad – 380 009, Gujarat, India. He has contributed in 11 publications which includes two chapters in Book. Research articles are published in reputed journals like NASI – Nat. AcadSciLett and J of Indian Botanical Society.

Research Projects Completed •

“Phytochemical studies of Cassia fistula” UGC sponsored Minor Research Project

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Differentiation of Embryonic and Non-Embryonic Callus of Convolvulus Pluricaulis Choisy using Biochemical Studies

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“Study of Floral Diversity in Jambughoda Wildlife Sanctuary” funded by Gujarat Forest Department, Gandhinagar

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“Constitution of BMCs and Preparation of PBRs in few selected Village Panchayats of Dabhoi Taluka of Vadodara District, Gujarat State” funded by Gujarat State Biodiversity board.

Achievements •

December-2006, first prize in Best Poster Presentation at All India Seminar on Advances in Biotechnological and Microbiological Researches in India During last Decade (1996-2006), UGC Sponsored, Bikaner, Rajasthan.

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March 2007, First Prize in Poster Presentation at Gujarat Science Congress, Patan, North Gujarat University

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Nov. 2007, First Prize of Young Scientist Award at Annual meeting of Indian Botanical Society, Jiwaji University, Gwalior.

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February 2019, Third prize for oral presentation at National Seminar on Innovative approaches in Plant Tissue Culture, Virani Science College, Rajkot.

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October 2019, Third Prize for presentation AICTE sponsored International Conference on Bimolecular and Biotechnological Aspects of medicinal plants, L M. College of Pharmacy, Ahmedabad

Dr. Zankhana Rathod, currently working as Assistant Professor in the subject of Botany, M. G. Science College, Ahmedabad, Gujarat. She has Ph. D. awarded for the thesis entitled “Studies on medicinal plant having anti-diabetic activities” in the subject of Botany by Gujarat University, Ahmedabad – 380 009, Gujarat, INDIA. She has contributed in 20 publications which includes two chapters in Book. Research articles are published in reputed journals like Indian Journal of Plant Physiology (IJPP); NASI – Nat. AcadSciLett; J of Indian Botanical Society. She is a Life member: Indian Science Congress Association, Indian Botanical Society,

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