Anuradha Agrawal Abstract Up to 73% decrease in cost of media for plant regeneration and in vitro conservation was achieved in Curcuma longa cv Prathibha by using inexpensive carbon source and gelling agent. Laboratory reagentgrade sucrose was replaced by locally available commercial sugar (market sugar or sugar cubes) as carbon source and bacteriological grade agar by isabgol (also named isubgol) as gelling agent. No adverse effects on shoot regeneration and conservation on isabgolgelled lowcost media were observed as com pared to that on agargelled control medium (CM). Some 33–56% cultures of C. longa survived up to 12 mo. on isabgolgelled medium in comparison to only 16% on CM. Genetic stability of 12monthold in vitroconserved plants was assessed using 25 random amplified polymorphic DNA (RAPD) primers; no significant variation was observed in RAPD profiles of mother plants and in vitroconserved plantlets on CM and lowcost media
See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/227105130 Low-cost media for in vitro conservation of turmeric (Curcuma longa L.) and genetic stability assessment using RAPD markers Article in In Vitro Cellular & Developmental Biology - Plant · March 2007 DOI: 10.1007/s11627-006-9000-y CITATIONS READS 88 750 authors, including: Rishi Tyagi Anuradha Agrawal Asia-Pacific Association of Agricultural Research Institutions (APAARI) National Bureau of Plant Genetic Resources 99 PUBLICATIONS 648 CITATIONS 25 PUBLICATIONS 383 CITATIONS SEE PROFILE Zakir Hussain Indian Agricultural Research Institute 25 PUBLICATIONS 224 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Molecular breeding in tomato for resiliency for biotic and abiotic stresses View project NICRA, CRIDA, HYDERABAD View project All content following this page was uploaded by Rishi Tyagi on 11 December 2014 The user has requested enhancement of the downloaded file SEE PROFILE In Vitro Cell.Dev.Biol.—Plant (2007) 43:51–58 DOI 10.1007/s11627-006-9000-y MICROPROPAGATION Low-cost media for in vitro conservation of turmeric (Curcuma longa L.) and genetic stability assessment using RAPD markers Rishi K Tyagi & Anuradha Agrawal & C Mahalakshmi & Zakir Hussain & Husnara Tyagi Received: July 2006 / 27 November 2006 / Published online: February 2007 / Editor: A Vieitez # The Society for In Vitro Biology 2007 Abstract Up to 73% decrease in cost of media for plant regeneration and in vitro conservation was achieved in Curcuma longa cv Prathibha by using inexpensive carbon source and gelling agent Laboratory reagent-grade sucrose was replaced by locally available commercial sugar (market sugar or sugar cubes) as carbon source and bacteriological grade agar by isabgol (also named isubgol) as gelling agent No adverse effects on shoot regeneration and conservation on isabgol-gelled low-cost media were observed as compared to that on agar-gelled control medium (CM) Some 33–56% cultures of C longa survived up to 12 mo on isabgol-gelled medium in comparison to only 16% on CM Genetic stability of 12-month-old in vitro-conserved plants was assessed using 25 random amplified polymorphic DNA (RAPD) primers; no significant variation was observed in RAPD profiles of mother plants and in vitro-conserved plantlets on CM and low-cost media Keywords Curcuma Isabgol Isubgol Low-cost tissue culture media Matric potential Osmotic potential Psyllium husk Introduction In vitro techniques have been successfully applied as a relatively safe method for conservation and international R K Tyagi (*) : A Agrawal : C Mahalakshmi : Z Hussain : H Tyagi Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, New Delhi 110012, India e-mail: rktyagi@nbpgr.ernet.in e-mail: rishiktyagi@email.com exchange with limited quarantine of various vegetatively propagated crop germplasm (Ashmore, 1997) including in our laboratory also (Mandal et al., 2000) An often-cited disadvantage of in vitro genebanks is the relatively higher costs involved as compared to other methods (Jarret and Florkowski, 1990; Koo et al., 2003) The need for low-cost plant tissue culture systems, applicable for micropropagation and in vitro conservation of plant genetic resources, has been emphasized to allow the large-scale application of such technology in developing countries (IAEA, 2004) Recurring costs of micropropagation and in vitro conservation include those for chemicals that are used in culture media, i.e., carbon sources, gelling agents, inorganic and organic supplements, and growth regulators Sucrose is usually used as a source of carbon and agar as the gelling agent, and together they constitute the most expensive components of the culture media The genus Curcuma (family Zingiberaceae) comprises more than 80 species of rhizomatous perennial herbs and has a widespread occurrence in the tropics of Asia and extends to Africa and Australia (Purseglove et al., 1981) Curcuma longa L (syn Curcuma domestica Val.), commonly known as turmeric, is an important commercial spice crop of India, Sri Lanka, Pakistan, Bangladesh, and China, which has traditionally been used in the Ayurvedic and Chinese systems of medicine since the ancient times A large number of accessions (2,368) of turmeric and related species (all vegetatively propagated) are maintained in field genebanks in India; the conservation of these is of high priority for their use in future crop improvement programs (Ravindran et al., 2005) Currently, 137 genetically diverse accessions of Curcuma are conserved as in vitro cultures in the In Vitro Genebank of National Bureau of Plant Genetic Resources following periodic subculturing As the number of accessions continues to increase in the In Vitro 52 TYAGI ET AL Genebank, the cost of conservation on long-term basis becomes a major concern In our study, to reduce the cost of plant regeneration and in vitro conservation, laboratory reagent (LR)-grade sucrose was replaced by locally available commercial sugar (market sugar or sugar cubes with a standard trade name) as carbon source and bacteriological-grade agar by isabgol (also named isubgol) as gelling agent Isabgol or psyllium, the husk derived from the seeds of the medicinal plant Plantago ovata Forsk (family Plantaginaceae), has been successfully used as gelling agent in tissue culture media (Babbar and Jain, 1998; Jain and Babbar, 2005) The efficacy of isabgol is due to the presence of mucilaginous husk in seeds Mucilage yield amounts to approximately 25% (by weight) of the total seed yield and is obtained by mechanical milling/grinding of the outer layer of the seed The milled seed mucilage is a white fibrous material that is hydrophilic In the present study, we aimed to determine a low-cost media using readily available and inexpensive alternatives of carbon sources and gelling agents in culture medium for in vitro plantlet regeneration and conservation of C longa cv Prathibha The effect of the low-cost substitutes on the genetic stability of in vitro-conserved plants was also assessed using random amplified polymorphic DNA (RAPD) markers Materials and Methods Initiation of cultures In vitro shoot cultures of C longa cv Prathibha were established using the rhizome buds collected from sprouted rhizomes that were harvested from plants growing in a net house of the National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India The buds (2–3 cm) were excised and thoroughly washed under running tap water, surface-sterilized using 0.1% (w/v) mercuric chloride and two or three drops of Tween-20 for 10–15 min, and subsequently thoroughly washed seven or eight times with sterilized distilled water to remove traces of mercuric chloride The sterilized buds were cultured onto Murashige and Skoog (1962) medium (MS) supplemented with 2.5 mg l−1 N6 benzyladenine (BA) to obtain the in vitro shoots, which were further used to study in vitro plant regeneration and conservation (Balachandran et al., 1990, Tyagi et al., 2004) In fact, such shoot cultures were maintained on the above medium for approximately yr through periodic subculture at 7–8 mo intervals before using the explants for the present study Plantlet regeneration and conservation For all experiments, shoots were excised from 4-week-old cultures maintained through periodic subculture on MS+2.5 mg l−1 BA The basal swollen portion of shoot containing the shoot tip Table Details of media tested for in vitro plantlet regeneration and conservation of C longa Medium code SAD (CM) MAD CAD SID MID CID MS+2.5 mg l−1 BA + Carbon sourcea Gelling agentb Sucrose Market sugar Sugar cubes Sucrose Market sugar Sugar cubes Agar Agar Agar Isabgol Isabgol Isabgol a Concentration of carbon sources=3% (w/v) Concentration agar=0.7% (w/v), isabgol=3.5% (w/v) CM=Control medium; MS=Murashige and Skoog’s medium [20] b encircled by whorls of leaves was cut to 1–2 cm above the base (referred to as shoot tip explant hereafter) and cultured on various test media Table presents media codes and details of various combinations of carbon sources and gelling agents, which were tested in the present study The treatment SAD served as the control medium (CM) Sucrose [Qualigens, Mumbai, India, (LR grade)] or inexpensive market crystalline sugar (Mawana Crystal Sugar™, India) or sugar cubes (Daurala Sugars™, India) were used as carbon sources Sucrose (LR) is made from cane sugar and contains 99.98% sucrose and 0.01% reducing sugars Market crystalline sugar (referred to as market sugar hereafter) is made from cane syrup treated with sulfur dioxide (sulfitation) or carbon dioxide (carbonation) and contains 96– 97% sucrose and 0.75–1% reducing sugars A sugar cube is made from fine grains of refined crystalline sugar by shaking it mechanically into cubes, and it contains 99.5% sucrose and 0.03% reducing sugars It is considered to be of higher quality than crystalline sugar and is hard enough not to break but dissolves instantly in water (WOI, 1981) The media used for the present study were either gelled with 0.7% (w/v) agar (bacteriological grade; Qualigens, Mumbai, India) or 3.5% (w/v) isabgol (Sat-Isabgol™ Sidhpur, Gujarat, India) After 12 mo of conservation, some of the cultures were drawn for testing their viability and genetic stability; the remaining cultures were allowed to grow beyond 12 mo on their respective media without subculture Viability of in vitro-conserved plantlets After 12–13 mo of conservation, plantlets conserved on CM (SAD) and low-cost media (SID, MID, and CID) were subcultured to test their viability through shoot regeneration Some 4–12 shoot tip explants from each media were subcultured on their respective parent media (isabgol-gelled) and 4–11 shoot tip explants on CM (SAD) No attempt was made to record the detailed data; only the occurrence of shoot and root regeneration was observed after wk of subculturing the explants LOW-COST MEDIA FOR TURMERIC CONSERVATION 53 Determination of pH, electrical conductivity, osmotic potential, and relative matric potential of medium The pH of the medium was determined before (original) and after autoclaving by a digital pH meter (Chemtron, India, model no 9140) Immediately after autoclaving, 20 ml of each medium was homogenized with 30 ml of distilled water (DW) using a magnetic stirrer Electrical conductivity (EC) (mS cm−1) of each medium was measured at 25°C by a digital conductivity meter (Control Dynamics, India) Using the EC values, osmotic potential (OP) of the same was calculated according to the formula derived by Janardhan et al Janardhan et al (1975) and adopted by Bhattacharya et al (1994): considered constant for all the media tested and not included in the total costs for comparison EC 0:36 dilution factorị OP ẳ 0:987 DNA extraction Total genomic DNA was extracted from g of leaves of three to five plantlets for each treatment using a modified Saghai-Maroof et al (1984) method After RNAse treatment, the DNA concentrations were determined following the method of Brunk et al (1979) using a fluorometer (Amersham Biosciences, USA) and bisbenzimide (Hoechst dye 33258) as the fluorescent dye The DNA samples were stored at −20°C until further use Working solutions of genomic DNA (5 ng/μl) were prepared after dilution with sterile DW and stored at 4°C for subsequent use in RAPD analyses Relative matric potential of each medium was measured following the method using filter-paper discs (Whatmann No 3) as described by Owens and Wozniak (1991) A precisely moistened filter-paper disc was placed on the surface of the medium, allowed to equilibrate, removed, and weighed The relative gain or loss of water from the paper discs was a measure of the matric potential of the medium Culture conditions Unless otherwise stated for a particular treatment, all other chemicals used for preparing the media were of LR grade (Qualigens, Mumbai, India) and BA from Sigma, St Louis, MO, USA The pH of all media used for raising the cultures (Table 1) was adjusted to 5.8 with 0.1 N NaOH or HCl before adding the gelling agent and autoclaving Agar was dissolved in hot (80–90°C) DW, whereas isabgol was suspended in DW at room temperature (30–32° C) before autoclaving Approximately 20 ml of the medium was dispensed into culture tubes (25×150 mm, Borosil, India) The medium was autoclaved at 121°C and 106 kPa for 20 After autoclaving, culture tubes containing isabgolgelled media were stirred manually to suspend the isabgol uniformly before its gelling Each culture tube received one explant and was closed with polypropylene caps and covered with cling film Cultures were incubated at 25±2°C and a light intensity of 40 μmol m−2 s−1, provided by cool white fluorescent lamps (Philips, India) at a 16-h photoperiod Comparison of costs of media Costs of carbon sources (sucrose, market sugar, sugar cubes) and gelling agents (agar, isabgol) were calculated on the basis of prevailing costs and the actual amount or volume used to prepare 10 l of final medium and were compared Initially, the costs were calculated in Indian rupee (INR) which was converted into US dollar (US$) by dividing with a factor of 45 (US$ 1=INR 45) Because equal amounts of other chemicals [MS salts, inorganic and organic supplements, and BA (2.5 mg l−1)] were used in each treatment, the cost of these chemicals was Genetic stability assessment To ascertain whether low-cost carbon sources and isabgol used in media have any effect on the genetic stability of plantlets conserved in vitro, a comparison of RAPD profiles of mother plants (source of explants used for in vitro plantlet regeneration and conservation) and the plantlets conserved in vitro for 12 mo on SAD (CM), MAD, CAD, SID, MID, and CID (low-cost media) was carried out RAPD analysis PCR reaction was carried out in a DNA Thermal Cycler (GeneAmp 9600 PCR system, PerkinElmer Cetus, Norwalk, CT, USA) Each 25 μl reaction mix contained 1× PCR reaction buffer (10 mM Tris–HCl, 50 mM KCl, and pH 8.3), mM MgCl2, 0.5 U of Taq DNA polymerase, 200 μM each of dATP, dTTP, dCTP and dGTP (all reagents from Bangalore Genei, India), 0.6 μM of primer (Operon Technologies, USA), and approximately 50 ng of template DNA A total of 200 primers (Operon Technologies, USA) were screened for RAPD analysis Out of these, some 25 primers— OPA 03, OPA 10, OPC 01, OPC 03, OPC 04, OPC 05, OPC 08, OPC 09, OPC 1, OPC 11, OPC 14, OPC 18, OPC 20, OPD 05, OPD 07, OPD 11, OPD 18, OPK 17, OPK 19, OPL 03, OPM 14, OPO 06, OPO 13, OPO 16, and OPW 12, were found to be polymorphic and used for further analyses The PCR conditions were as follows: initial extended step of denaturation at 94°C for followed by 35 cycles of denaturation at 94°C for min, primer annealing at 36°C for min, primer elongation at 72°C for min, followed by an extended elongation step at 72°C for 10 Reaction products were mixed with 2.5 μl of 10× loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, and 40% sucrose, w/v) using a microfuge The mixture was electrophoresed on 1.4% agarose gel at 90 V (Bio–Rad subcell GT) followed by staining with ethidium bromide and photographed under ultraviolet light using a Digital Imaging System (UltraCam) The amplification products of the in vitro-conserved plantlets derived from MID medium were scored across the 54 TYAGI ET AL lanes, and they were compared with that of mother plants and plants conserved on CM Each band was treated as one RAPD marker and scored as present or absent from the photographs Amplifications were repeated twice Data recording and statistical analyses Each treatment comprised a set of 12 cultures and was replicated three times The experiments were conducted in completely randomized block design Observations were recorded at periodic intervals The data for plant regeneration were computed as mean values of the total 36 cultures for the number of shoots/culture, shoot length (centimeter), number of roots, and root length (centimeter) up to wk, and mean values were subjected to Tukey’s test (Table 2) Shoots were allowed to grow on their respective media without transfer or subculture and studied for conservation Cultures that contained at least two green shoots were considered as surviving To determine conservation period, survival of cultures (per cent) was recorded at 1-month interval (Fig 2) ANOVA was carried out for the effect of carbon sources, gelling agents, and their interaction on in vitro regeneration and conservation Data for pH, EC, OP, and relative matric potential (Table 3) were recorded for five determinations each, and mean values were subjected to Duncan’s multiple range test (DMRT) using standard software SPSS 10.0 to test the significance Results The isabgol-gelled (3.5%, w/v) media solidified faster than agar-gelled (0.7%, w/v) media However, both types of media were firm enough to hold the explant vertically Initiation of cultures Rhizome buds, cultured on MS (3% sucrose) + 2.5 mg l−1 BA, started sprouting within a Table Plantlet regeneration in C Longa after wk of culture on control (SAD) and various low-cost media Medium code* SAD MAD CAD SID MID CID * No of shoots/ culture 4.6±0.7a 4.5±0.4ab 4.5±0.6ab 2.6±0.5b 4.1±0.3ab 3.2±0.6ab Shoot length (cm) No of roots/shoot 2.3±0.1c 2.5±0.1bc 3.0±0.3bc 4.1±0.4a 3.3±0.2ab 3.2±0.1b 2.1±0.4a 2.7±0.3a 2.6±0.3a 3.4±0.1a 2.7±0.3a 2.9±0.5a Root length (cm) 1.1±0.1c 1.2±0.1c 1.4±0.1bc 1.6±0.2ab 1.9±0.2a 1.9±0.2a See Table for medium codes Values are mean±SE of 36 replicate cultures Values superscripted with the same letter in each column are not significantly different on the basis of Tukey’s test analysis (P≤0.05) week and gave rise two or three shoot buds per explant In 3–4 wk, 1- to 2-cm long shoots were obtained; these shoots were subcultured on the same medium repeatedly to obtain a sufficient stock of explants for in vitro plantlet regeneration and conservation experiments using low-cost media Plantlet regeneration Shoot buds were induced from the swollen basal portion of the explants in all the six media tested within wk After wk of culture initiation, 83.3 to 97% cultures exhibited shoot regeneration ranging from 2.6 to 4.6 shoots per culture In general, longer shoots were recorded on isabgol-gelled media (3.2–4.1 cm) in comparison to agar-gelled media (2.3–3.0 cm) (Table 2) Figure 1a, b represents 10-week-old cultures showing plantlet regeneration on all the media tested Rooting was observed in all shoot-forming cultures on their respective parent media, ranging from 2.1 roots per shoot on SAD medium to 3.4 roots per shoot on SID medium (Table 2) Generally, the quality of sugars and type of gelling agent did not influence the rooting (Fig 1a, b) ANOVA shows that the differences in the number of shoots (F=8.8, df=1, P≤0.05), shoot length (F=24.5, df=1, P≤0.01), root length (F=29.5, df=1, P ≤ 0.01) due to isabgol used as gelling agent were significant In contrast, differences for the above parameters due to various carbon sources used were nonsignificant at P≤0.05 Interaction between the carbon source and gelling agent was nonsignificant for all the above parameters except shoot length (F=5.8, df=5, P≤0.05) Conservation All the tested media supported growth of almost 100% shoot-forming cultures up to mo After mo., the survival pattern of the cultures maintained on various media tested is shown in Fig 2a, b With the increase in age of the cultures beyond mo., survival rates generally declined In 12-month-old cultures, higher survival (50.0–56.4%) was observed on media gelled with isabgol (SID, MID, and CID) as compared to 11.2–16.6 % survival on agar-gelled media (SAD, MAD, and CAD), with the highest value (56.4%) being recorded on MID medium (Fig 2) ANOVA shows that the differences in survival of cultures (F=115.3, df=1, P=0.01) due to the two gelling agents were highly significant However, no significant differences were observed due to either source of carbon or interactions of carbon source and gelling agent At the end of 14 mo., approximately 22% cultures on MID and 12–13% cultures each on SID and CID survived, whereas no cultures or