Abstract
Tylophora indica Burm F. Merrill. is widely used against various diseases owing to the presence an array of medicinally important secondary metabolites. Its stem is bitter, stomachic, stimulates bile secretion, enriches the blood and cures diseases like diabetes, fever, flatulence, hypertension, jaundice, leucorrhoea, urinary disease and upper respiratory tract infection. It is neglected for tissue culture work because of deciduous nature of climbing shrub, facing problems for micropropagation. Hence, in vitro regeneration of complete plantlets was done through indirect organogenesis in Tylophora indica. Calli were produced from in vivo leaves of T. indica on MS medium supplemented with 6-Benzylaminopurine (BAP: 2.0 mg l-1) and Indole-3-butyric acid (IBA: 0.5 mg l-1). The multiple shoots (12.00 ± 1.50) emerged and elongated on MS medium fortified with Thidiazuron (TDZ: 0.1 mg l-1). They were rooted on half strength MS medium having IBA (0.5 mg l-1) (7.75 ± 0.25) after 20 days of sub-culturing followed by hardening and acclimatization. During indirect regeneration of plants, chances of somaclonal variations may arise. These variations should be identified to produce true to type plants. Plantlets raised through tissue culture were used to validate the clonal fidelity through Inter simple sequence repeat markers (ISSR). Clonal fidelity is a major consideration in commercial micropropagation using in vitro tissue culture methods. During the study, total 71 clear and distinct bands were produced using 6 primers. The banding pattern of each primer was uniform and comparable to mother plant and showed about 93% homology using un-weighted pair group method with arithmetic averaging (UPGMA). ISSR analysis confirmed the genetic stability of in vitro raised plants.
Highlights
To ascertain genetic variations in number as well as sizes of repeat units, we produced in vitro plants of T. indica through callus culture and detected genomic variation through reliable, reproducible, low cost and successful molecular markers technology i.e. Inter simple sequence repeat markers (ISSR) which are useful in areas of genetic diversity, phylogenetic studies, gene tagging, genome mapping and evolutionary biology in a wide range of crop species
The present study was conducted with an aim of ascertaining the genetic uniformity of the tissue culture raised plantlets with respect to mother plant- T. indica using ISSRs markers
In vitro regenerated shoots were rooted on half strength MS medium supplemented with Indole-3-butyric acid (IBA) (0.05-0.5 mg l−1) and indole-3acetic acid (IAA: 0.05-0.5 mg l−1)
Summary
To ascertain genetic variations in number as well as sizes of repeat units, we produced in vitro plants of T. indica through callus culture and detected genomic variation through reliable, reproducible, low cost and successful molecular markers technology i.e. ISSR which are useful in areas of genetic diversity, phylogenetic studies, gene tagging, genome mapping and evolutionary biology in a wide range of crop species. The present study was conducted with an aim of ascertaining the genetic uniformity of the tissue culture raised plantlets with respect to mother plant- T. indica using ISSRs markers. Plant material The experiments were conducted to initiate and produce stock callus through leaf explants for further morphogenetic differentiation.
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