Indian Journal of Biotechnology Vol 5 October 2006, pp 535-542

In vitro corm induction and genetic stability of regenerated plants in taro [Colocasia esculenta (L.) Schott]

Z Hussain and R K Tyagi*

Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, New Delhi 110 012, India

Received 9 February 2005; revised 6 September 2005; accepted 25 November 2005

In vitro corm formation was achieved on Murashige and Skoog’s (MS) medium, containing 8-10% sucrose, 22 µM N6-benzyl aminopurine (BAP), 0.6 µM α-naphthaleneacetic acid (NAA) and 0.8% agar, in taro (IC 420791). The corm forming cultures could be conserved up to 15 months at 25+2oC, whereas shoot-forming cultures could last for only 6 months on MS medium, containing 3% sucrose + 2.2 µM BAP + 0.6 µM NAA + 0.8% agar. Plantlets with in vitro-formed corms showed 100% survival in the field, and developed normal uniform corm-producing plants. Uniformity of tissue culture regenerated plants with corm (Ro) was determined on the basis of 12 qualitative and 10 quantitative morphological traits related to leaf, petiole, corm and root. Additionally, two PCR-based markers—RAPD and ISSR were also applied to determine the genetic stability of Ro plants. A total of 13 RAPD primers (of 35 tested initially) and 6 ISSR primers gave 111 distinct bands in RAPD and 43 in ISSR, and exhibited uniform RAPD and ISSR banding patterns for Ro plants tested. Our results suggested that present protocol used for in vitro corm formation may cost-effectively be applied for conservation of taro germplasm along with maintaining the genetic stability and functionality of plants. Further possibility may also be explored to use this protocol for production of disease-free planting materials. This will facilitate the national and international exchange of taro germplasms.

Keywords: conservation, genetic integrity, inter-simple sequence repeats, random amplified polymorphic DNA, taro, tissue culture

IPC Code: Int. Cl.8 A01H4/00, 5/04

Introduction Taro (Colocasia esculenta (L.) Schott) is a root

crop with edible corms and leaves, belonging to the family Araceae. It is one of the most ancient cultivated crops, which is originated in the Indo-Malayan region1,2. India, being one of the centers of origin of taro, is endowed with immense genetic diversity3. Considerable variability has been recorded in the crop at morphological, agronomical and molecular level4.

Of the 4,000 accessions of taro3,5, about 1,500 accessions are collected from different parts of India and being maintained in the field genebanks at various centres of Indian Council of Agricultural Research (ICAR), including Central Tuber Crop Research Institute (CTCRI), Thiruvananthapuram and National Bureau of Plant Genetic Resources (NBPGR), New Delhi. Maintenance of germplasm in the field genebank demands not only greater resources, but also the germplasm is always at risk

due to pest and disease infestation and other natural calamities. A significant amount of germplasm of taro has been lost due to natural vagaries as well as anthropogenic activities5.

Application of plant tissue culture has been found very useful for rapid multiplication, safe exchange and conservation of germplasm of many vegetatively propagated crops6,7. Micropropagation systems in taro have been developed that can be used for a wide range of cultivars7,8. A few reports are also available on in vitro conservation of taro at low temperatures (at 9 & 4oC)9,10. Further, induction of in vitro storage organs has been found useful for conservation of germplasm of many vegetatively propagated crops, for example, potato11, sweet potato12, yams13,14 and tannia15.

Since occurrence of somaclonal variations in in vitro cultures are available in the literature16,17, it is important to ascertain the genetic fidelity of in vitro conserved germplasms. Apart from morphological assessment, the genetic stability of the plants regenerated with in vitro corms should also be assessed by using two simple polymerase chain

______________ *Author for correspondence: Tel: 91-11-25848955; Fax: 91-11- 25851495 E-mail: rktyagi@nbpgr.delhi.nic.in

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reaction (PCR)-based molecular marker techniques, i.e. random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR).

In the present study, we report a protocol for in vitro induction and conservation of corms of taro at 25 + 2oC (ambient culture room temperature) and genetic stability of the plant regenerated with in vitro-formed corms after in vitro conservation.

Materials and Methods Plant Material and Micropropagation

The corms of a taro accession IC 420791, obtained from CTCRI, Thiruvananathapuram, were planted in earthen pots (30 cm diam), filled with soil and farmyard manure (FYM) (3:1), and allowed to develop into plants in the net house at NBPGR, New Delhi. The explants consisting of a shoot tip, embedded deep into the corm tissue encircled with leaf primordial, and basal part of the petioles (size: 5-10 mm diam, 20-30 mm length) were used to establish the tissue cultures in rainy season (July-August) and subsequently in winter season (November-December). The explants were immersed in 0.05% Tween 20 for 10 min with frequent agitation, sterilized with 0.1% HgCl2 for 10 min and rinsed 4 times with sterilized distilled water. These explants were then cultured on Murashige and Skoog’s (MS) medium18 containing 3% sucrose, 2.2 µM N6-benzyl aminopurine (BAP), 0.6 µM α-naphthaleneacetic acid (NAA) and gelled with 0.8% bacteriological grade agar-agar (M1). The cultures were maintained by periodic subculture at 45 d intervals on M1 and sufficient number of explants required for further experiments were regenerated. In vitro Corm Induction, Conservation and Hardening of Plantlets

For in vitro induction of corms, the explants were excised from 4-week-old in vitro raised plantlets on M1 medium. Basal part of shoot (5 mm) containing shoot tip (hereafter referred to as shoot tip) after removal of leaves and roots were used as explant. Such shoot tip explants were cultured on the following media: M1 = MS (3% sucrose) + 2.2 µM BAP + 0.6 µM NAA + 0.8% agar, M2 = MS (8% sucrose) + 22 µM BAP + 0.6 µM NAA + 0.8% agar, and M3 = MS (10% sucrose) + 2.2 µM BAP + 0.6 µM NAA + 0.8% agar. The shoot tip cultures were allowed to develop the shoots, roots and corms without subculturing on aforementioned media. M1 medium served as control. Each culture vessel received one explant. The observations for number of shoots/culture, shoot length, number of

corms/culture were recorded for a given culture at 3 and 6 months. The period between initiation of culture for in vitro corm induction and transplanting the corms in the potted soil for hardening was considered as conservation period for a given culture.

For all the media tested, the pH was adjusted to 5.8 with 1 M HCl or 1 M NaOH prior to autoclaving. The culture vessels (25 × 150 mm; Borosil, Mumbai, India) containing about 20 mL medium, enclosed with polypropylene caps, were used and autoclaved at 121oC and 1.1 kg cm-2 for 17 min. Further, all the cultures were incubated in a culture room at 25+2oC with 10 h photoperiod (40 µmol m-2 s-1) provided by cool light fluorescent lamps (40 W, Philips, Mumbai, India).

Plantlets with induced corms and without corms were transplanted in pots, containing mixture of soil and SoilriteTM (1:1), and covered with transparent porous polythene bags (10" × 7") for maintaining proper humidity and aeration. After 4-5 d, polythene bags were removed and the planlets were transplanted directly to the field. A total of 30 plantlets (with corms) and 15 (without corms) were transferred and survival was recorded after 1 month of transplantation in the field.

All the treatments were replicated thrice and data were presented as mean values of 20 cultures for each treatment. Analysis of variance (ANOVA) and Duncan's multiple range test (DMRT) were used to determine whether the differences were significant. Genetic Stability Assessment

Morphological Markers One-month-old plants (8) transplanted in the field

were used to study the genetic stability. The observations on 12 qualitative morphological traits—(i) predominant position of leaf lamina, (ii) leaf blade margin, (iii) leaf blade colour, (iv) leaf vein pattern, (v) petiole colour, (vi) corm infestation, (vii) corm branching, (viii) corm weight (g), (ix) corm cortex colour, (x) corm skin surface, (xi) corm bud colour, (xii) root colour, and 10 quantitative morphological traits—(i) plant height (cm), (ii) fresh weight of plant (g), (iii) leaf length (cm), (iv) leaf width (cm), (v) corm length (cm), (vi) corm weight (g), (vii) no. of cormels/plant, (viii) no. of eyes/corm, (ix) no. of roots/plant, and (x) root length (cm), were recorded for the individual regenerant to assess the uniformity among them19. In addition, genetic stability of the regenerated plantlets with in vitro corms was also assessed using RAPD and ISSR markers.

HUSSAIN & TYAGI: CORM INDUCTION AND GENETIC STABILITY IN IN VITRO PLANTS OF TARO

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RAPD and ISSR Markers Total genomic DNA was extracted individually

from 1 g of leaves of 5 individual regenerated plants and mother plants, which were used to initiate the cultures, using modified Saghai-Maroof et al20 method. After RNAse treatment, the DNA concentrations were determined following the method of Brunk et al21, using a fluorometer (Amersham Biosciences, USA) and bisbenzimide (Hoechst dye 33258) as the fluorescent dye. The DNA samples were stored at −20oC till further use. Working solutions of genomic DNA (5 ng/µL) were prepared after dilution with sterile distilled water and stored at 4oC for subsequent use in RAPD and ISSR analyses.

PCR reaction was carried out in a DNA Thermal Cycler (GeneAmp 9600 PCR system, Perkin-Elmer Cetus, Norwalk, CT). Each 25 µL reaction mix contained 1X PCR reaction buffer (10 mM Tris-HCl, pH 8.3 and 50 mM KCl), 3 mM MgCl2, 0.5 U of Taq DNA polymerase; 200 µM each of dATP, dTTP, dCTP and dGTP (all reagents from Genei, Bangalore); 0.6 µM of primer (Operon Technologies, USA or M/s Genetix, India) and approximately 30 ng of template DNA used for RAPD and ISSR analyses.

A total of 35 primers were used of RAPD. Of the 35 primers, 13 (OPA 10, OPC 01, OPC 11, OPC 12, OPC 14, OPC 19, OPC 20, OPD 02, OPD 10, OPW 15, OPW 16, OPW 17, R 10) were found to be polymorphic and were used for further RAPD analysis. The PCR conditions were as follows: initial extended step of denaturation at 94oC for 5 min followed by 35 cycles of denaturation at 94oC for 1 min, primer annealing at 36oC for 1 min, primer elongation at 72oC for 2 min, followed by an extended elongation step at 72oC for 10 min.

For ISSR analysis, a total of 6 primers were tested22. Only one primer was used in each PCR, and the amplification conditions were as follows: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 60 sec, primer annealing at 36-58°C (depending upon the primer used) for 30 sec, primer elongation at 72°C for 5 min and extended elongation at 72°C for 10 min. For ISSR primers, annealing temperature (Ta) was empirically optimized for number and clarity of bands with melting temperature (Tm) of the oligonuceotide as the reference point. To calculate Tm following thumb rule was applied23: Tm = 81.5 + 16.6 log [Na+ ]) + 0.41 (% G + C) - (675/n)

Where, n = number of bases in oligo and Na+ = molar salt concentration.

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 microfuge24. The amplification products were electrophoresed on 1.4% agarose gels at 90 volts (BIO RAD subcell GT), followed by staining with ethidium bromide and photographed on ultraviolet light using Ultracam digital imaging system. Results and Discussion

Shoot tips embedded in corm tissue grew within 2 weeks on M1 medium and 100% of cultures converted into shoots. Generally, one shoot (4-5 cm) was obtained from one explant. About 95-100% healthy (contamination-free) cultures could be established when the explants were surface-sterilized once with 0.1% HgCl2 and cultured during winter season in comparison to 40-50% in rainy season. The growing shoots were subcultured on fresh M1 medium. As a result, 3-5 shoots/explant were regenerated after 4 weeks of subculture. Such shoots were further subcultured at periodic interval of every 4 weeks to obtain sufficient number of shoots required as explants for corm induction experiments. In vitro Corm Induction and Conservation

In both 3- and 6-month-old cultures of taro, corms (3.8 & 3.6 corms/culture) were formed on M2 and M3 media, whereas their formation was not observed on M1 medium (Table 1; Figs 1A & B). Further, no significant difference was observed in the number of corms formed on M2 and M3 media. Age of the culture (3- to 6-month-old) also had no effect on the number of corms formed on both media. These results clearly suggest that higher concentrations of sugar (8 & 10%) and BAP (22 & 22 µM) in M2 and M3 media had the beneficial effect on in vitro formation of corm in 100% taro cultures. On the other hand, however, the maximum number of shoots (5.8 & 7.8 shoots/culture) with the maximum shoot length (8.5 & 10.5 cm) were regenerated on M1 medium as compared to shoots regenerated on M2 (3.8 shoots/culture; 3 & 3.5 cm) and M3 media (3.6 shoots/culture; 3 7 3.4 cm) in both 3- and 6-month-old cultures (Table 1). Moreover, the number of shoots and their length increased with the increase in age of taro cultures on M1 medium; while no significant effect of age was observed on M2 and M3 media. Also, the number of regenerated shoots and their length remained same on M2 and M3 media.

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Fig. 1 (A-GHA) In vitro corm formation on media M2 and M3, arrows indicate the corms; Shoot regeneration on MI medium in 6- month-old cultures. (B) Close-up of corm as-in M2 of (A). (C) 15-month-old cultures conserved through corm formation. @) Plants regenerated from in vitro-formed corms growing in field. (E) 4-month-old uprooted plants developed from in vitro-formed corms, showing normal development of natural corms in field. (F) Close-up of normal leaf and vein pattern in plants as in (E). (G) Close-up of corms showing secondary corms (arrow), buds and roots in plants as in (E).

HUSSAIN & TYAGI: CORM INDUCTION AND GENETIC STABILITY IN IN VITRO PLANTS OF TARO

Table l--Zn vitro corm formation in taro (IC 420791) on MS medium containing varied concentrations of sucrose and BAP

Age of cultures

Medium*

- - -

3 months 6 months No. of corms1 No. of shoots/ Shoot length No. of corms/ No. of shoots1 Shoot length

culture culture culture culture (cm)

*MI = MS (3% Sucrose) + 2.2 pM BAP + 0.6 pM NAA + 0.8% agar M1 = MS (8% Sucrose) + 22 pM BAP + 0.6 pM NAA + 0.8% agar M3 = MS (10% Sucrose) + 22 yM BAP + 0.6 pM NAA + 0.8% agar The values are means of each experiment; SE in parentheses Superscripts with same letter do not differ significantly (pl0.05)

In the present study, 3.8 and 3.6 corms/culture) were obtained from single shoot tip explant on M2 and M3 media containing 8 and '10% sucrose, respectively and 22 pM BAP; whereas, only one corm/culture in liquid medium containing high sucrose (8-10%) and cytokinin was reported earlier". Ex-ogenous growth substances, including sucrose, constituted a key factor in the rocess of storage organ formation in taro", 29 potato and c o ~ o ~ a m ' ~ . High sucrose levels are needed for the initiation of storage organs, as they constitute the main component of the stored food. In ptatoZ6 and taro1', corm formation was stimulated by the presence of a cytokinin in the culture medium, whereas this phenomenon is favoured by auxin and inhibited by cytokinin in yam27. Sucrose and BAP concentrations have also affected the growth and corm induction in taro cultures in the present study.

In vitro corm induction was recorded in 100% cultures on Mz and Mg media. These cultures were stored on M2 and M3 media without subculturing further. The rooting was not a constraint as profuse rooting was observed in 100% shoots regenerated with or without corms on parent media. With increased age of cultures, thickness of root also increased (Fig. 1C). Some 60% cultures with corms survived up to 15 months on M2 and M3 media, as new shoots were periodically emerging; whereas, the older shoots necrosed and died after every 4-5 months from initiation of corm-forming cultures. However, shoot cultures on MI could not survive beyond 6 months as there was no emergence of new shoots and such cultures became chlorotic and eventually died.

interval could be increased from 6 to 15 months in taro pig. 1C). Induction of in vitro storage organs has been successfully used to increase the conservation period in

yams14.", and cocoyam15. In vitro conservation of taro has so far been reported at low temperature (4°C) up to 10 months''. Conservation of germplasm at low temperature, particularly in tropical countries like India, is a costly method on long-term basis, where electric power failure or shortage is a major problem. Following the present protocol the taro cultures could be conserved up to 15 months at ambient culture room temperature (25S0C), which would decrease the cost of conservation of taro germplasm.

All the transplanted plantlets with corms (100%) survived in the pots as well as in the field (Fig. ID). The plants grew normally in the field and produced 2 or 3 secondary corms (Fig. 1E-G). However, the transplanted blantlets without corms could not survive in the pots and in the field.

Genetic Stability Analyses Morphological Markers

Uniformity manifested in all the 12 qualitative morphological traits related to leaf, petiole, corm and root among 8 regenerated (Ro) plants tested (Table 2; Figs 1 E-G). Data on 10 qualitative morphological traits exhibited the viability and functionality of Ro plants grown in the field (data not shown). Therefore, no significant variation was recorded among the plantlets regenerated with in vitro-formed corms on morphological basis and plants were apparently uniforI?l.

In In vitro Genebank (IVGB) at NBPGR, 131 RAPDandZSSR marker accessions of taro germplasm are being conserved in A total of 1 11 amplification bands, with an average the form of shoot cultures by periodic subculturing at frequency of 8.23 bands per primer were produced by 6-8 months interval for the last 5 years. By using the 13 RAPD primers (Table 3). The total number of present in vitro corm formation protocol, the subculture bands produced by an individual primer ranged from

INDIAN J BIOTECHNOL, OCTOBER 2006

Fig. 2-(A-E) RAPD amplification patterns obtained for plants developed from in vitro-formed c o r n (Ro), using primer OPW 17 (A), R 10 (B), OPC 1 (C), OPC 1 1 (D) and OPC 12 (E). (F & G) ISSR amplification patterns of abbe-mentioned plants, using primers ISSR 3 (F) and ISSR 5 (G). (laneL denotei for 1 Kb DNA ladder, lane M for mother clone DNA andknes 1 to 5 for individual %plants. Note the uniformity in banding pattern between M and Ro plants (1-5) in each profile)

4 in OPD 02 to 14 in OPW 15, with size ranging from 250 bp to 4000 bp in primer OPW 15. Of these 1 1 1 bands, only one (0.9%) was polymorphic which was produced by the primer OPA 10. The remaining 1 10 bands were observed to be m o n o m o ~ i c across all of the regenerated planlets with in vitro corms and their mother clone DNA (Table 3; Figs 2 A-E). RAPD markers have been effectively used to assess the

genetic stability of micropropagated plants of other tuber crops, for example, cassava2', sweet potato30 and yams31.

Earlier workers17s32 have suggested the use of both types of markers for better analysis of genetic stability of regenerants due to their amplifying ability in different regions of the genome. Therefore, ISSR markers were also tested to study the genetic stability

HUSSAIN & TYAGI: CORM INDUCTION AND GENETIC STABILITY IN IN VITRO PLANTS OF TARO

Table 2-Morphological analysis of qualitative traits of regenerated (Ro) plants with corms grown in field

Morphological traits Description

Predominant position (shape) of leaf Erect-apex down lamina surface Leaf blade margin Undulate Leaf blade colour Yellow-green Leaf main vein colour Green Leaf vein pattern Y shape Petiole colour

Top third portion Green Middle third portion Light green Basal third portion Whitish

Corm shape Dumb-bell Corm branching Branched Corm cortex colour Yellow Corm skin surface Fibrous Corm bud colour White Root colour White

Table 3-RAPD and ISSR markers used to analyze the genetic stability of regenerated plants with corms .,

Primer number No. of Size No. of sequence amplification range monomorp (5' ... 3') products (bp) hic bands

RAPD analysis

OPA 10 (GTGATCGCAG) OPC 01 (TTCGAGCCAG) OPC I I (AAAGCTGCGG) OPC 12 (TGTCATCCCC) OPC 14 (TGCGTGCITG) OPC 19 (GTTGCCAGCC) OPC 20 (AC'ITCGCCAC) OPD 02 (GGACCCAACC) OPD 10 (GGTCTACACC) OPW 15 (ACACCGGAAC) OPW I6 (CAGCCTACCA) OPW I7 (GTCCTGGGTT) R 10 (TGATGCCGCT) ISSR analysis

ISSR 01 (GA)9T ISSR 02 (GA)9AC ISSR 03 (GA) gAT ISSR 04 (ACC)6G ISSR 05 (GACA)4 ISSR 06 (GATA)4

of in vitro regenerated plants with corm. Six inter- simple sequence repeats (ISSR) primers, including tetra-, tri- and dinucleotide repeat motifs, were individually used to amplifL DNA. A total of 43 amplification products were produced, ranging in size from 250 bp in (GA)9T to 2000 bp in (GA)9AT and (GATA)4. All the six primers produced uniform

amplification profiles of DNA of regenerated plantlets and their mother clone (Table 3; Figs 2 F-G). The ISSR markers have been successfully used to analyze the genetic stability of in vitro regenerated plants of almond^'^ and somatic embryo derived plants of coffee33. On the basis of morphological, RAPD and ISSR markers tested, no significant difference was observed among the Ro plants with corm from shoot tip explants.

Thus, our results suggest that the culture conditions used for in vitro corm induction and conservation are optimal for taro to conserve the germplasm for 15 months. Therefore, the present protocol may cost- effectively be applied to conserve taro germplasm while maintaining the genetic stability and functionality of plants. Further possibility may also be explored to use this protocol for productions of disease-free planting materials. This will facilitate the national and international exchange of taro germplasms as in vitro-formed corms.

Acknowledgement \ .

The authors are grateful to Dr B S Dhillon, Director, NBPGR, New Delhi, for providing the facilities. The financial support provided under National Agricultural Technology Project (PB) is gratefully acknowledged.

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