Low-cost media for in vitro conservation of turmeric ( Curcuma longa L. ) and genetic stability assessment using RAPD markers

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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
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  MICROPROPAGATION Low-cost media for  in vitro  conservation of turmeric( Curcuma longa L. ) and genetic stability assessmentusing RAPD markers Rishi K. Tyagi  &  Anuradha Agrawal  &  C. Mahalakshmi  & Zakir Hussain  &  Husnara Tyagi Received: 7 July 2006 /27 November 2006 / Published online: 9 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 carbonsource and gelling agent. Laboratory reagent-grade sucrosewas replaced by locally available commercial sugar (market sugar or sugar cubes) as carbon source and bacteriologicalgrade agar by isabgol (also named isubgol) as gelling agent. No adverse effects on shoot regeneration and conservationon isabgol-gelled low-cost media were observed as com- pared to that on agar-gelled control medium (CM). Some33  –  56% cultures of   C. longa  survived up to 12 mo. onisabgol-gelled medium in comparison to only 16% on CM.Genetic stability of 12-month-old  in vitro -conserved plantswas assessed using 25 random amplified polymorphic DNA(RAPD) primers; no significant variation was observed inRAPD profiles of mother plants and  in vitro -conserved plantlets on CM and low-cost media. Keywords  Curcuma .Isabgol.Isubgol.Low-costtissueculture media.Matricpotential.Osmoticpotential.Psylliumhusk  Introduction  In vitro  techniques have been successfully applied as arelatively safe method for conservation and internationalexchange with limited quarantine of various vegetatively propagated crop germplasm (Ashmore, 1997) including inour laboratory also (Mandal et al., 2000). An often-citeddisadvantage of   in vitro  genebanks is the relatively higher costs involved as compared to other methods (Jarret andFlorkowski, 1990; Koo et al., 2003). The need for low-cost   plant tissue culture systems, applicable for micropropaga-tion 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 inculture media, i.e., carbon sources, gelling agents, inorganicand organic supplements, and growth regulators. Sucrose isusually used as a source of carbon and agar as the gellingagent, and together they constitute the most expensivecomponents of the culture media.The genus  Curcuma  (family Zingiberaceae) comprisesmore than 80 species of rhizomatous perennial herbs andhas a widespread occurrence in the tropics of Asia andextends to Africa and Australia (Purseglove et al., 1981). Curcuma longa  L. (syn.  Curcuma domestica  Val.), com-monly known as turmeric, is an important commercial spicecrop of India, Sri Lanka, Pakistan, Bangladesh, and China,which has traditionally been used in the Ayurvedic andChinese systems of medicine since the ancient times. Alarge number of accessions (2,368) of turmeric and relatedspecies (all vegetatively propagated) are maintained in fieldgenebanks 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 diverseaccessions of   Curcuma  are conserved as  in vitro  cultures inthe  In Vitro  Genebank of National Bureau of Plant GeneticResources following periodic subculturing. As the number of accessions continues to increase in the  In Vitro In Vitro Cell.Dev.Biol.  —  Plant (2007) 43:51  –  58DOI 10.1007/s11627-006-9000-yR. K. Tyagi ( * ) : A. Agrawal :  C. Mahalakshmi : Z. Hussain : H. TyagiTissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, New Delhi 110012, Indiae-mail: rktyagi@nbpgr.ernet.ine-mail: rishiktyagi@email.com  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 sucrosewas replaced by locally available commercial sugar (market sugar or sugar cubes with a standard trade name) as carbonsource and bacteriological-grade agar by isabgol (alsonamed isubgol) as gelling agent. Isabgol or psyllium, thehusk derived from the seeds of the medicinal plant   Plantago ovata  Forsk (family Plantaginaceae), has beensuccessfully 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 mucilaginoushusk in seeds. Mucilage yield amounts to approximately25% (by weight) of the total seed yield and is obtained bymechanical milling/grinding of the outer layer of the seed.The milled seed mucilage is a white fibrous material that ishydrophilic.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   invitro  plantlet regeneration and conservation of   C. longa  cvPrathibha. The effect of the low-cost substitutes on the geneticstability of   in vitro -conserved plants was also assessed usingrandom amplified polymorphic DNA (RAPD) markers. Materials and Methods  Initiation of cultures. In vitro  shoot cultures of   C. longa  cvPrathibha were established using the rhizome buds collectedfrom sprouted rhizomes that were harvested from plantsgrowing 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 ontoMurashige and Skoog (1962) medium (MS) supplementedwith 2.5 mg l − 1  N 6  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 4 yr through periodic subculture at 7  –  8 mo. intervals before usingthe explants for the present study.  Plantlet regeneration and conservation.  For all experi-ments, shoots were excised from 4-week-old cultures main-tained through periodic subculture on MS+2.5 mg l − 1 BA.The basal swollen portion of shoot containing the shoot tipencircled by whorls of leaves was cut to 1  –  2 cm above the base (referred to as shoot tip explant hereafter) and culturedon various test media. Table 1 presents media codes anddetails of various combinations of carbon sources andgelling agents, which were tested in the present study. Thetreatment SAD served as the control medium (CM).Sucrose [Qualigens, Mumbai, India, (LR grade)] or inexpensive market crystalline sugar (Mawana CrystalSugar  ™ , India) or sugar cubes (Daurala Sugars ™ , India)were used as carbon sources. Sucrose (LR) is made from canesugar and contains 99.98% sucrose and 0.01% reducingsugars. 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 ismade from fine grains of refined crystalline sugar by shakingit mechanically into cubes, and it contains 99.5% sucrose and0.03% reducing sugars. Itisconsidered tobe ofhigherqualitythan 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 weredrawn for testing their viability and genetic stability; theremaining cultures were allowed to grow beyond 12 mo. ontheir 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 tipexplants from each media were subcultured on their respective parent media (isabgol-gelled) and 4  –  11 shoot tip explants onCM (SAD). No attempt was made to record the detailed data;only the occurrence of shoot and root regeneration wasobserved after 8 wk of subculturing the explants. Table 1.  Details of media tested for   in vitro  plantlet regeneration andconservation of   C. longa Medium code MS+2.5 mg l − 1 BA +Carbon source a  Gelling agent   b SAD (CM) Sucrose Agar MAD Market sugar Agar CAD Sugar cubes Agar SID Sucrose IsabgolMID Market sugar IsabgolCID Sugar cubes Isabgol a  Concentration of carbon sources=3% ( w /  v  )  b Concentration agar=0.7% ( w /  v  ), isabgol=3.5% ( w /  v  )CM=Control medium; MS=Murashige and Skoog ’ s medium [20]52 TYAGI ET AL.   Determination of pH, electrical conductivity, osmotic potential, and relative matric potential of medium.  The pH of the medium was determined before (srcinal) and after autoclavingbyadigitalpHmeter(Chemtron,India,modelno.9140). Immediately after autoclaving, 20 ml of each mediumwas homogenized with 30 ml of distilled water (DW) using amagnetic stirrer. Electrical conductivity (EC) (mS cm − 1 ) of each medium was measured at 25°C by a digital conductiv-ity meter (Control Dynamics, India). Using the EC values,osmotic potential (OP) of the same was calculated accordingto the formula derived by Janardhan et al. Janardhan et al.(1975) and adopted by Bhattacharya et al. (1994): OP  ¼  EC   0 : 36  dilutionfactor  ð Þ 0 : 987Relative matric potential of each medium was measuredfollowing 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 thesurface 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 themedium. Culture conditions.  Unless otherwise stated for a particular treatment, all other chemicals used for preparing the mediawere of LR grade (Qualigens, Mumbai, India) and BA fromSigma, 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 auto-claving. Agar was dissolved in hot (80  –  90°C) DW, whereasisabgol was suspended in DW at room temperature (30  –  32°C) before autoclaving. Approximately 20 ml of the mediumwas dispensed into culture tubes (25×150 mm, Borosil,India). The medium was autoclaved at 121°C and 106 kPa for 20 min. After autoclaving, culture tubes containing isabgol-gelled media were stirred manually to suspend the isabgoluniformly before its gelling. Each culture tube received oneexplant and was closed with polypropylene caps and coveredwith cling film. Cultures were incubated at 25±2°C and alight intensity of 40  μ  mol m − 2 s − 1 , provided by cool whitefluorescent 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 andthe actual amount or volume used to prepare 10 l of finalmedium and were compared. Initially, the costs werecalculated in Indian rupee (INR) which was converted intoUS 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 wasconsidered constant for all the media tested and not includedin the total costs for comparison. 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 , acomparison of RAPD profiles of mother plants (source of explants used for   in vitro  plantlet regeneration andconservation) and the plantlets conserved  in vitro  for 12 mo. on SAD (CM), MAD, CAD, SID, MID, and CID(low-cost media) was carried out.  DNA extraction.  Total genomic DNA was extracted from1 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 deter-mined following the method of  Brunk et al. (1979) using a fluorometer (Amersham Biosciences, USA) and bisbenzi-mide (Hoechst dye 33258) as the fluorescent dye. TheDNA samples were stored at   − 20°C until further use.Working solutions of genomic DNA (5 ng/  μ  l) were prepared after dilution with sterile DW and stored at 4°Cfor subsequent use in RAPD analyses.  RAPD analysis.  PCR reaction was carried out in a DNAThermal Cycler (GeneAmp 9600 PCR system, Perkin-Elmer Cetus, Norwalk, CT, USA). Each 25  μ  l reaction mixcontained 1× PCR reaction buffer (10 mM Tris  –  HCl,50 mM KCl, and pH 8.3), 3 mM MgCl 2,  0.5 U of TaqDNA polymerase, 200  μ  M each of dATP, dTTP, dCTP anddGTP (all reagents from Bangalore Genei, India), 0.6  μ  Mof primer (Operon Technologies, USA), and approximately50 ng of template DNA.A total of 200 primers (Operon Technologies, USA) werescreenedfor RAPD analysis.Out ofthese,some25primers  —  OPA 03, OPA 10, OPC 01, OPC 03, OPC 04, OPC 05, OPC08, OPC 09, OPC 1, OPC 11, OPC 14, OPC 18, OPC 20,OPD 05, OPD 07, OPD 11, OPD 18, OPK 17, OPK 19, OPL03, OPM 14, OPO 06, OPO 13, OPO 16, and OPW 12, werefound to be polymorphic and used for further analyses. ThePCR conditions were as follows: initial extended step of denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 1 min, primer annealing at 36°C for 1 min, primer elongation at 72°C for 2 min, followed by anextended elongation step at 72°C for 10 min. Reaction products were mixed with 2.5  μ  l of 10× loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, and 40% sucrose, w /  v  ) using a microfuge. The mixture was electrophoresed on1.4% agarose gel at 90 V (Bio  –  Rad subcell GT) followed bystaining 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 LOW-COST MEDIA FOR TURMERIC CONSERVATION 53  lanes, and they were compared with that of mother plantsand plants conserved on CM. Each band was treated as oneRAPD 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 threetimes. The experiments were conducted in completelyrandomized block design. Observations were recorded at  periodic intervals. The data for plant regeneration werecomputed as mean values of the total 36 cultures for thenumber of shoots/culture, shoot length (centimeter), num- ber of roots, and root length (centimeter) up to 8 wk, andmean values were subjected to Tukey ’ s test (Table 2).Shoots were allowed to grow on their respective mediawithout transfer or subculture and studied for conservation.Cultures that contained at least two green shoots wereconsidered as surviving. To determine conservation period,survival of cultures (per cent) was recorded at 1-monthinterval (Fig. 2). ANOVA was carried out for the effect of carbon sources, gelling agents, and their interaction on  invitro  regeneration and conservation. Data for pH, EC, OP,and relative matric potential (Table 3) were recorded for five determinations each, and mean values were subjectedto Duncan ’ s multiple range test (DMRT) using standardsoftware SPSS 10.0 to test the significance. Results The isabgol-gelled (3.5%,  w /  v  ) media solidified faster thanagar-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 aweek and gave rise two or three shoot buds per explant. In3  –  4 wk, 1- to 2-cm long shoots were obtained; these shootswere subcultured on the same medium repeatedly to obtaina sufficient stock of explants for   in vitro  plantlet regener-ation and conservation experiments using low-cost media.  Plantlet regeneration.  Shoot buds were induced from theswollen basal portion of the explants in all the six mediatested within 2 wk. After 8 wk of culture initiation, 83.3 to97% cultures exhibited shoot regeneration ranging from 2.6to 4.6 shoots per culture. In general, longer shoots wererecorded on isabgol-gelled media (3.2  –  4.1 cm) in comparisonto agar-gelled media (2.3  –  3.0 cm) (Table 2). Figure 1 a ,  b represents 10-week-old cultures showing plantlet regenera-tion on all the media tested. Rooting was observed in allshoot-forming cultures on their respective parent media,ranging from 2.1 roots per shoot on SAD medium to 3.4roots per shoot on SID medium (Table 2). Generally, thequality of sugars and type of gelling agent did not influencethe rooting (Fig. 1 a ,  b ). ANOVA shows that the differencesin 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 weresignificant. In contrast, differences for the above parametersdue to various carbon sources used were nonsignificant at   P  ≤ 0.05. Interaction between the carbon source and gellingagent was nonsignificant for all the above parameters except shoot length (  F  =5.8,  df   =5,  P  ≤ 0.05). Conservation.  All the tested media supported growthof almost 100% shoot-forming cultures up to 6 mo. After 8 mo., the survival pattern of the cultures maintained onvarious media tested is shown in Fig. 2 a ,  b . With theincrease in age of the cultures beyond 8 mo., survival ratesgenerally declined. In 12-month-old cultures, higher sur-vival (50.0  –  56.4%) was observed on media gelled withisabgol (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 MIDmedium (Fig. 2). ANOVA shows that the differences insurvival of cultures (  F  =115.3,  df   =1,  P  =0.01) due to thetwo gelling agents were highly significant. However, nosignificant differences were observed due to either sourceof carbon or interactions of carbon source and gellingagent. At the end of 14 mo., approximately 22% cultures onMID and 12  –  13% cultures each on SID and CID survived,whereas no cultures or <5% cultures survived on all theagar-gelled media. Viability of in vitro-conserved plantlets.  Upon subculturingthe shoot tip explants excised from 12-month-old  in vitro -conserved plantlets on low-cost media, shoot regeneration(two to four shoots per culture) was obtained in almost all Table 2.  Plantlet regeneration in  C. Longa  after 8 wk of culture oncontrol (SAD) and various low-cost mediaMediumcode *  No. of shoots/ cultureShoot length (cm) No. of roots/shoot Root length(cm)SAD 4.6±0.7 a  2.3±0.1 c 2.1±0.4 a  1.1±0.1 c MAD 4.5±0.4 ab 2.5±0.1  bc 2.7±0.3 a  1.2±0.1 c CAD 4.5±0.6 ab 3.0±0.3  bc 2.6±0.3 a  1.4±0.1  bc SID 2.6±0.5  b 4.1±0.4 a  3.4±0.1 a  1.6±0.2 ab MID 4.1±0.3 ab 3.3±0.2 ab 2.7±0.3 a  1.9±0.2 a  CID 3.2±0.6 ab 3.2±0.1  b 2.9±0.5 a  1.9±0.2 a * See Table 1 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).54 TYAGI ET AL.  the cultures on parent media as well as on CM (SAD). This phenomenon was observed after both the first subcultureand the second subculture. Irrespective of the media, profuse rooting also occurred within 8 wk (Fig. 1 d  ).  pH, electrical conductivity, osmotic potential, and relativematric potential   The changes in pH, EC, OP, and relativematric potential of post-autoclaved media and their effectson  in vitro  plantlet regeneration and conservation were Table 3.  Values of pH, electrical conductivity (EC), osmotic potential (OP), and relative matric potential of different media (at 25°C) used for   invitro  plant regeneration and conservation of   C. longa Medium code pH EC (mS cm − 1 ) OP (MPa) Relative matric potentialOriginal* Post-autoclaveSAD 4.35 5.75 3.802±0.051 a  − 0.347±0.005 a  1.272±0.051 a  MAD 4.37 5.74 3.920±0.028 a  − 0.357±0.003 a  1.247±0.018 a  CAD 4.30 5.72 3.768±0.046 a  − 0.344±0.004 a  1.218±0.016 a  SID 5.02 5.17 2.530±0.030  b − 0.231±0.003  b 0.780±0.006  b MID 5.04 5.11 2.758±0.129  b − 0.251±0.012  bc 0.782±0.005  b CID 5.10 5.12 2.690±0.028  b − 0.245±0.003  bc 0.798±0.009  b *Original pH of all media was adjusted to 5.8 before autoclaving using 0.1 N NaOH or HCl.Values are mean±SE of five replicates of each medium.Values superscripted with the same letter in each column are not significantly different on the basis of DMRT analysis (  P  ≤ 0.05). Figure 1.  ( a  –  b ) Ten-week-old cultures of   C. longa  cv Prathibha on:( a ) SAD, MAD, CAD agar-gelled media; ( b ) MID, CID, SID isabgol-gelled media, all of which showing normal plantlet regeneration; ( c )12-month-old  in vitro -conserved cultures on control (SAD) andvarious low-cost media; ( d  ) an 8-week-old plantlet regenerated after subculturing of a shoot tip explant from 12-month-old  in vitro -conserved plantlets on isabgol-gelled medium; ( e ) RAPD profilesgenerated from mother plants (M) and 12-month-old  in vitro -conserved plants on SAD (CM) and MID. Amplification pattern inlanes 1  –  3 obtained by primer OPC 05, lanes 4  –  6 by primer OPC 08,and lanes 7  –  9 by primer OPC 10; (  f    ) as in ( e ) except the profiles of  plants on SID. Amplification pattern obtained by using primer OPW12. Each bar on a, b, c, and d=2.5 cm. See Table 1 for media codes.LOW-COST MEDIA FOR TURMERIC CONSERVATION 55
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