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High frequency direct shoot regeneration from corm axillary buds and rapid clonal propagation of taro, Colocasia esculenta var. esculenta (L.) Schott (Araceae)
plan ience ELSEVIER
Plant Science 104 (1994) 93-100
High frequency direct shoot regeneration from corm axillary buds and rapid clonal propagation of taro, Colocasia esculenta var. esculenta (L.) Schott (Araceae) Rosemary C.O. Chng, Chong-Jin Goh* Tissue Culture Laboratory, Department of Botany, National University of Singapore, Lower Kent Ridge Road, Singapore 0511, Singapore Received 30 June 1994; revision received 12 October 1994; accepted 12 October 1994
Abstract
High frequency direct shoot regeneration from corm axillary bud explants of Colocasia esculenta var. esculenta was achieved through enhanced axillary bud development. Plantlets with well-developed shoot and root systems were obtained after 4 weeks culture on Murashige and Skoog (MS) medium supplemented with 20 g/1 sucrose and solidified with 2.4 g/l gelrite. The rate of successful initiation was 76% of total corm axillary buds cultured. In addition, a rapid method for direct regeneration of over 80 shoots per corm in 8 weeks was achieved through thin section culture of tissue-cultured-taro corm on medium containing 50 #M BA. Regenerated shoots developed roots readily when transferred to hormone-free medium. Plantlets were ready for potting in 4 weeks and yielded 100% successful acclimatisation rate. The developed protocols can be employed for taro germplasm conservation, exchange and mass propagation for cultivation.
Keywords: Colocasia esculenta var. esculenta (L.) Schott; Plant regeneration; Thin section culture; Germplasm conservation; Taro
1. Introduction
Colocasia esculenta (L.) Schott (Araceae), commonly known as taro, is taxonomically classified into two different varieties, Coiocasia esculenta var. antiquorum and Colocasia esculenta var. Abbreviations: BA, 6-benzyladenine; BM, basal MS medium; CAB, corm axillary buds; MS, Murashige and Skoog Medium; NAA, l-naphthaleneacetic acid; SAB, shoot axillary buds; TC, taro corm cube; TE, taro extract. * Corresponding author.
esculenta, with hundreds of cultivars [1]. Colocasia esculenta var. antiquorum is triploid (2n = 3x) with 42 chromosomes whereas Colocasia esculenta var. esculenta is diploid (2n = 2x) with 28 chromosomes [2]. The latter is more widely preferred by consumers. Taro is one of 4 major root and tuber crops that form the staple diet of people in the Asia-Pacific and African regions. Compared with cassava, sweet potato and yam, taro is known to contain the highest concentrations of protein, minerals and vitamins [31.
0168-9452/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSD1 0168-9452(94)04008-5
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Taro is affected by viral diseases [4] which decrease the yield. Being vegetatively propagated and extremely heterogeneous as a group, conservation of taro germplasm is an urgent task. However, taro germplasm is not actively exchanged due to the difficulty of ensuring virus-free material. Hence recommendations by the International Board for Plant Genetic Resources (IBPGR) are for the exchange of in vitro materials only [5]. Furthermore, taro seeds are only viable for about 30 days after harvest [6] and the in vitro conservation of taro is thus necessary. Although much research on the tissue culture of taro had been reported during the past two decades, most of the success was achieved with the variety Colocasia esculenta var. antiquorum [7-11 ]. Methods developed for the tissue culture of Colocasia esculenta var. esculenta, however, were less consistent and plantlet regeneration rate was low until recent reports by Yam et al. [12-15] with the use of taro extract. However, regeneration through callus is subjected to somaclonal variations [16] and mutation in taro has been reported [17]. Hence, protocols that would minimize genetic changes during micropropagation need to be developed [10]. All previous reports of successful taro culture used shoot tips or axillary bud explants from shoot tips as starting material [7-151 and none used the more easily available and accessible axillary buds of the storage corm. We have developed a simple and highly regenerative direct shoot development method using axillary buds from both shoot tips and corms. In addition, a thin section culture protocol for mass propagation of in vitro plantlets was also developed. 2. Materials and methods
2.1. Plant materials Corms of Colocasia esculenta var. esculenta of Malaysian cultivar 'Pasang' were obtained from farms in Johor, Malaysia. Each corm, complete with shoot tip, weighed approximately 1 kg fresh weight. 2.2. Preparation of explants The corms were first scrubbed in 2% commercial
bleach Clorox solution and detergent. Dead leaf tissues and mud encasing the corms were removed. Shoot tips (30 nun) with 3-5 whorls of tightly bound petiolar bases and 10 mm of corm tissues were excised. Undamaged corm axillary buds (CAB, Fig. 1) located on the nodes of corms were isolated. The shoot tips and CAB were then washed under running water for 30 min before surface sterilization.
2.3. Sterilization and culture of corm axillary buds Between 15 and 20 buds were placed in a sterile 200-ml screw cap bottle containing 20% (v/v) ethanol for 5 min followed by 50% (v/v) Clorox with two drops of Tween 20 for 3 min and finally 25% (v/v) Clorox for 15 min. The bottles were shaken vigorously at each stage and the sterilant completely drained before the addition of fresh solution. This was followed with three rinses of sterile distilled water. The sterilized buds were blotted dry on sterile filter paper and the outer scale leaves of the buds and 1 mm of their apices removed. Adjoining corm tissues were also trimmed to 1 mm in thickness. Explants were then placed on basal medium in 16mm Petri dishes to test for contamination. After 3 days, clean buds were transferred into 150 mm × 15 mm glass culture tubes containing 15 ml of culture media. 2.4. Sterilization and culture of shoot tips and axillary buds Excised shoot tips were sprayed with 95% ethanol before surface sterilization in 50% (v/v) Clorox with two drops of Tween 20 for 20 min and three rinses with sterile distilled water. The petiolar bases were peeled and the exposed shoot axillary bud (SAB, Fig. 1) on each node was excised together with 1 mm of corm tissues and cultured. Apical domes with one or two leaf primodia and 2 mm of corm tissues were cultured whole or cut into two or four vertical sections and cultured. 2.5. Thin section culture Four-week-old in vitro plantlets initiated on basal medium (BM), averaging 100 mm in height were used for further propagation. Mini-corms, 4 - 6 mm in diameter and 8-10 mm in length were
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excised with 1 mm of shoot tip and cut transversely into 1.5-2 mm thick sections. These sections were then cultured in GA7 containers (Magenta Corps, Chicago, IL) with 40 ml of BM supplemented with various concentrations of BA.
2.0. Media and culture conditions Basal medium (BM) consisted of Murashige and Skoog (MS, Ref. [18]) salts enriched with 20 g/1 sucrose and solidified with 2.4 g/l gelrite. Benzyladenine (BA) was added before autoclaving. Taro extract (TE) was prepared by boiling 600 g of taro corm in distilled water for 5 rain and simmered for 1 h [11]. The cooled supernatant (made up to 1 1) was then fdtered twice through Whatman No. 1 filter paper. The extract (25 ml/1) was added to the medium as required. In another treatment, fresh taro corm cube (TC) of 10 × 10 × 10 mm (approx. 1.2 g fresh weight), was added to 15 ml of the medium in each culture tube before autoclaving. The pH of all media was adjusted to 5.7-5.8 prior to autoclaving at 120°C under a pressure of 1.2 kgtcm2 for 20 min. Cultures were incubated at 26 ± 2"C under a 16-h/day photoperiod provided by cool-white fluorescent light with a photon flux density of 18 ~E/mZ/s at culture level. 2.7. Statistical analysis All experiments were repeated at least twice. Results for CAB explants (Table 1) were pooled from three separate experiments. Those of SAB explants (Table 2) and thin section culture (Table 3) were pooled from two separate experiments. Results are presented as means ± S.D. Data were subjected to one way analysis of variance (ANOVA) using the Duncan-Waller multiple range test at c~ = 0.05 using the Statistical Analysis Systems [19]. 3. Remits
3.1. Axillary bud culture In preliminary experiments, buds that were cultured with less than 1 mm of corm tissues did not develop and corm tissues more than 2 mm thick led to excessive corm exudate buildup that inhibited normal bud development. Subsequently, all bud explants were isolated with 1 mm corm tissues. On average, 12 CAB and three SAB were iso-
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lated for culture from a single mature corm. Out of a total of 475 CAB and 157 SAB isolated, 363 CAB and 124 SAB were clean cultures 3 days after initiation. Most of the cultures (about 80% for both the CAB and SAB) later developed into plantlets with complete shoot and root systems at the end of 4 weeks on BM. In CAB cultures, the basal portion of the creamcolored buds started to swell and greening of buds took place 3 days after initiation. New leaves and roots emerged after 5-7 days. By the end of 4 weeks, most of the cultured buds formed welldeveloped plantlets with 3-5 new leaves with main petiole lengths measuring about 100 mm, 5-10 roots and corms of 4-6 mm in diameter and 8-10 mm in length (Table 1). Small secondary shoots with petiole lengths ranging from 2 to 20 mm were also observed in some of the cultures. Growth and development of newly initiated shoots and their subsequent regenerant were highly asynchronous. In the presence of BA, multiple shoots were formed from explants. These shoots were observed to develop from pre-existing bud primodia between the scales of the buds. Regenerated multiple shoots on medium containing 25 #M of BA, averaging 35 mm in height were, however, much shorter than those developed from CAB grown on basal medium after 2 weeks of culture (Fig. 2). Rhizogenesis was inhibited in the presence of high BA levels (Table 1) but rooting readily occurred once the shoots were transferred to hormone-free medium. Optimal shoot formation was obtained with 25 t~M BA although there was no obvious correlation between shoot regeneration and BA concentration. Except for the greening of some of the cultures, neither leaf nor root development was observed for cultures grown in 75 ~M BA at the end of 4 weeks (Table 1). There was no significant beneficial effect when explants were cultured in the presence of TE (Table 1, Fig. 2). However, in medium with 25 t~M BA, TE appeared to cause a reduction in shoot regeneration although it stimulated rhizogenesis. For cultures grown with TC, only about 50% survived (Table 1) and growth and development of new shoots were stunted (Fig. 2). Growth of SAB cultures was slow when cultured on BM (Table 2). Not all buds turned green at the
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end of 3 days and many did not develop further, especially for those younger buds located close to the apex (Fig. 3). For the buds that developed, the emergence of leaves and roots occurred after 2
weeks of culture. Plantlets were generally small with a single new leaf, 1 or 2 roots, 40-60 mm in height and corm diameter of 4 - 6 mm after 4 weeks of culture (Table 2).
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Table 1 Effects of benzyladenine (BA), taro extract (TE) and taro cube (TC) on the morphogenic responses of taro corm axillary bud explants at the end of 4 weeks of culture Culture medium
BM BM BM BM BM BM BM BM BM BM BM BM
+ + + + + + + + + + +
10 20 25 30 40 50 60 75 T] TE TC
LM BA LM BA ,M BA LM BA ~M BA ~M BA LM BA LM BA + 25 I~M BA 25 ttM BA
Number of explants
24 24 24 20 24 24 24 24 20 20 20 20
Number of cultures with shoots
roots
20 15 19 18 18 18 14 16 0 18 20 11
19 13 I1 3 9 4 0 1 0 18 14 3
Number of shoots per culture
Diameter of corm (mm)
Length of petiole (mm)
!.2.4.- 0.5 c 3.4 4- 2.6 bc 3.4 4- 2.5 bc 6.3 4- 5.0 a 4.0 4- 3.2 a~ 5.5 4- 3.7 ab 4.7 4- 2.6 abe 4.8 4- 3.8 ab 0d 1.8 4- 2.3 c 3.5 ..'- 1.7 bc 2.9 4- 0.5 Ix:
3.28 2.45 2.75 3.45 2.65 2.44 3.16 2.47 0d 3.59 2.43 5.28
61.7.4- 44.9 a 18.1 4- 20.1 b 17.0.4.. 16.4 bc 18.1 .4- 16.0 b 13.1 4- 11.2 bc 12.7 q- 12.0 bc 13.9 ± 11.2 bc 10.9 4- 8.7 ¢ 0d 103.7 4- 25.3 a 13.0 -t- 10.6 bc 21.3 4- 4.0 b
44444444-
1.42 bc 1.54 bc 1.67 c 2.87 ab 1.81 bc 1.62 bc 1.86 bc 1.65 c
4- 2.70 a 4- 1.30 c 4- 0.72 a
Results are expressed as means 4- S.D. Means were calculated from measurements of all shoots > 1 m m in height. In each column, the means followed by the same letter are not significantly different as indicated by the D u n c a n - W a l l e r multiple range test at a = 0.05.
Addition of BA into culture medium enhanced growth and development of SAB (Fig. 3). The buds swelled and turned green within the first few days of culture. They later formed enlarged corms up to 12 mm in diameter and exhibited accelerated shoot growth (Table 2). After 4 weeks of culture, weU-developed plantlets with 4 or 5 leaves with main petiole lengths averaging 100 mm were obtained, especially from cultures with 25 ~M BA (Table 2). However, rhizogenesis was inhibited during the first 2 weeks (Fig. 3). A small number of very young buds did not develop beyond the
greening and swelling stages, but produced numerous tiny shoots after 6 weeks. The presence of TE or TC did not significantly enhance shoot regeneration (Table 2, Fig. 3). 3.2. Shoot tip culture Shoot tips cultured on BM showed only some swelling. On BM supplemented with 25 t~M BA, the apical dome of the shoot tip turned green after 1 week and swelling of the petiolar bases with enhanced anthocyanin pigmentation was observed. For shoot tips that were cultured whole,
Fig. I. Field grown corm of Colocasia esculenta var. esculenta cv. Pasang showing corm axillary bud (thick arrow) and exposed shoot axillary bud (thin arrow). Scale bar, 4 cm. Fig. 2. Corm axillary bud culture after 2 weeks on: A, BM; B, BM + TE; C, BM + TC; D, BM + 25 ~M BA. Note multiple shoot formation in D. Scale in cm. Fig. 3. Shoot axillary bud culture after 2 weeks on: A, BM; B, BM + TE; C, BM + TC; D, BM + 25 #M BA. Note enlarged basal corm formation in D. Scale in cm. Fig. 4. Four-week-old plantlet regenerated from corm thin section culture on BM. Plantlet initiated from the axiilary meristem of original explant (arrow). Scale in cm. Fig. 5. In vitro plantlet 4 months after transplant to soil mix in 200-mm pot. Scale in cm. Fig. 6. In vitro plantlets 4 months after transplant to soil showing healthy shoot, corm and root development, and developing runner (thick arrow). The plantlet at left had much of the lateral roots trimmed to show developing corm with axillary bud (thin arrow).
R.CO. Chng, C-£ Gob~Plant Sci. 104 (1994) 93-100
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Table 2 Effects of benzyladenine (BA), taro extract (TE) and taro cube (TC) on the morphogenic responses of taro shoot axillary bud explants at the end of 4 weeks of culture Culture medium
BM B M + 10#M BA BM + 20/~M BA BM + 25 ~M BA BM + 30 ~M BA BM + 40/~M BA BM + 50 #M BA B M + 6 0 #M BA BM + 75 ~tM BA BM + TE BM + TE + 25 #M BA BM + TC + 25 ttM BA
Number of explants
7 7 7 10 7 7 7 7 7 10 7 10
Number of cultures with shoots
roots
5 7 6 9 6 4 4 5 0 9 6 6
3 5 3 7 3 1 1 2 0 9 6 3
Number of shoots per culture
Diameter of corm (ram)
Length of petiole (ram)
1.0 1.1 1.5 1.4 1.7 2.0 1.3 3.4 0c i.1 1.5 2.0
4.40 4- 1.14fs 5.064- 1.32efg 7.10 4- 3.30 abe 9.62 4- 3.40 a 4.90 4- 2.37 def 5.06 4- 2.51 bcd 7.80 4- 3.35 b°d 2.76 4- 2.31s Oh 5.35 4. 1.67 cdef 8.22 4. 2.39 ab 5.58 4- 4.40 bcde
37.2 4- 20.8 cd 51.24-36.3 cd 33.8 4- 12.0 bcd 85.8 4- 26.8 a 36.4 4- 24.3 bc 25.0 4- 18.3cd 29.8 4- 14.7 co 15.3 4- 18.4d Oh 51.2 4. 36.4 ab 84.4 4- 33.5 a 77.3 4- 25.6 ab
4- 0.0 b 4-0.4 b 4- 1.2 ab 4- 0.9 ab 4- 0.8 ab 4- 0.8 ab 4- 0.5 ab
:t: 3.4 a 4- 0.3 b 4- 0.8 ab 4- 2.0 ab
Results are expressed as means 4- S.D. Means were calculated from measurements of all shoots > I mm in height. In each column, the means followed by the same letter are not significantly different as indicated by the Duncan-Waller multiple range test at ct = 0.05.
rooting occurred within 1 week followed by the formation of new leaves. For those that were sectioned, a single shoot emerged from the cut surface of the explant 4 weeks later. Rooting occurred after shoot development and a well-formed plantlet was obtained after 8 weeks of culture. Survival of the explant was also dependent on explant size at isolation. Of the 20 replicates per
TaMe 3 Multiple shoot formation from thin sections of in vitro corms at the end of 8 weeks of culture on MS media supplemented with BA BA 0tM)
Number of corms cultured*
Number of corms with shoots
Number of shoots per corm
0 10 25 50 60
20 20 20 20 20
20 20 20 20 20
5.2 9.9 36.0 81.2 1.0
4- 1.9 d 4- 4.3 c 4- 6.2 b 4- 2.5 a 4- 0.0 e
*Each corm was cut into four sections. Results are expressed as means 4- S.D. Mean calculated from measurements of all shoots > 1 nun in height. In each column, the means followed by the same letter are not significantly different as indicated by the Duncan-Waller multiple range test at ¢x = 0.05.
treatment cultured on BM with 25 #M BA, new shoots developed in 90% of the tips that were halved compared to only 25% of those that were cut into 4 pieces. 3.3. Thin section culture Transverse thin sections of in vitro corms were cultured on BM as well as BA-enriched media (Table 3). On BM, 1 or 2 new shoots per section developed during the first week and rooting occurred shortly thereafter. Each mini-corm yielded at least four thin sections and an average of five plantlets per corm was obtained from thin section culture at the end of 8 weeks (Table 3). New shoots were observed to develop from axillary buds on the nodes of the sections (Fig. 4). For cultures on media containing BA, multiple secondary shoot formation from nodes of newly formed shoots was common after the second week. The number of shoots formed increased with increasing BA concentrations up to 50 #M but decreased sharply at 60 #M BA. After 8 weeks, thin sections cultured on medium with 50 ~M BA produced a mean of 81 shoots per four sections of single corm through enhanced axillary bud growth. However, the growth of shoots was much slower in high BA media than in BM. It took
R.CO. Chng, C-J. Gob~Plant Sci. 104 (1994) 93-100
another 4 weeks on BM for mini-corms to reach the size suitable for further thin section culture. For cultures with 60/tM BA, only the shoot tip sections survived after 8 weeks of culture. Some of these shoot tips later produced highly faciated multiple shoots. However, when these faciated shoots were subcultured onto BM, rooting and normal shoot development occurred after 3 days. 3.4. Plant establishment Tissue-cultured plantlets were ready for potting 4 weeks after whole bud culture and 12 weeks after microprogation from thin section. Acclimatization was not necessary other than the trimming of existing leaves and roots. Survival rates of 100% were obtained and no morphological variations were detected in any of the regenerated plantlets (Fig. 5). All plants exhibited healthy and normal shoot, root and corm development, with many of the plants producing side shoots and runners, after 3 months of transplant into pots with soil mix (Fig. 6).
4. Discussion In our present study, direct shoot regeneration was successfully achieved through the culture of CAB on basal MS medium. These shoots produced mini-corms suitable for thin section culture in 4-5 weeks. Each mini-corm subsequently regenerated on average five shoots per corm on BM at the end of 8 weeks (Table 3). Hence, on 8week cycles, about 15 000 plantlets can be obtained from a single corm 1 year after initiation. This protocol, without the use of any plant growth regulators (thereby minimizing the risk of somaclonal variation), is thus recommended for the in vitro conservation of taro germplasm. Multiple direct shoot formation from CAB occurred in the presence of BA, especially at 25/~M BA (Table 1). Miui-corms from these shoots (together with those initiated on BM) can be used for thin section culture on medium with 50 ~,M BA (Table 3). Regenerated shoots (in 8 weeks) transferred to BM were in turn ready for thin section culture after 4 weeks. Considering the 12week cycle to produce 80 plantlets, mass propagation of up to 40 million plantlets can be obtained
99
from a single corm in 1 year. This compares favourably with a previous report of 100 000 plants per year through callus culture [10]. It is also much faster than previously described protocols for taro [8,10,12-15]. In our experiments, auxin was excluded since mutants have been reported from media containing as low as 0.1 rag/1 NAA [10]. Indeed, no morphologicial variations were detected in regenerated plantlets, even amongst those regenerated from BA treatment. Thus, the present direct shoot regeneration system, by-passing the callus phase, may produce more true-to-type clonal plants. Shoot-tip explants regenerated single shoots. Axillary buds from shoot tips regenerated fewer shoots than those from storage corms. Nevertheless, these regenerated shoots could also be used for thin section cultures for mass propagation. However, the greater success with the use of CAB and the easy availability of these buds indicate that CAB is the choice of starting material. Although the possible deleterious effects of using sterilants directly on axillary bud explants have been reported [14], it was not apparent in the case of CAB explants in the present study. Previous reports stressed the requirement of taro extract for successful culture [12-15]. In our system, the beneficial effect of TE was, at most, marginal. However, the explants were cultured with 1 mm of the corm tissues. The use of larger explants thus simplified and circumvented the need for TE. It was possible that the larger explant size also helped in culture establishment and initial multiple shoot regeneration. The mini-corms produced by regenerated plantlets can be used, after aseptic removal of culture medium, directly for germplasm exchange. They are easy to handle and can survive for a period of 5 weeks, longer than the period commonly experienced during trans-shipment of plant materials. Thus, the present protocols developed for direct shoot regeneration and thin section culture can be employed for germplasm conservation, exchange and mass propagation for cultivation.
Acknowledgements This research was supported in part by the International Board for Plant Genetic Resources
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(IBPGR), Rome, Italy. We would like to thank Ir. D.H. van Sloten, Assistant Director General, and Dr J.M.M. Engels, Group Leader of Germplasm Maintenance and Use, International Plant Genetic Resources Institute (previously IBPGR), for their support for R. Chng to carry out this collaborative project while being employed as the Head of the IBPGR Seed Handling Unit for the Asia-Pacific region, in the Botany Department, National University of Singapore. References [I] J.W. Pursglove, Tropical Crops, Monocotyledons, Longnmn Group Ltd., London, 1972, pp. 61-69. [2] D.L. Plucknett, Taxonomy of the genus Colocasia, in: J.K. Wang (Ed.), Taro, A Review of Colocasia esculenta and its Potential, University of Hawaii Press, 1983, pp. 14-19. [3] FAO, Food Composition Table for Use in East Asia, Food and Agricultural Organisation of the United Nations, Rome, 1972. [4] J.J. Ooka, Taro diseases, in: J.K. Wang (Ed.), Taro, A Review of Coiocasia esculenta and its Potential, University of Hawaii Press, 1983, pp. 141-147. [5] F.W. Zettler, G.V.H. Jackson and E.A. Frison (Eds.), FAOflBPGR Technical Guidelines for the Safe Movement of Edible Aroid Germplasm, Food and Agricultural Organisation of the United Nations, Rome/International Board for Plant Genetic Resources, Rome, 1989, pp. 1-24. [6] K. Kikuta, L.D. Whitney and G.K. Parris, Seeds and seedlings of the taro, Colocasia esculenta. Am. J. Bot., 25 (1938) 186-188. [7] M.O. Mapes and W.J. Cable, Mericloning of taro (Colocasia esculenta L.), Hawaii Agric. Exp. Station, J. Series No. 1649, 1972. [8] G.V.H. Jackson, E.A. Ball and J. Arditti, Rapid propagation of taro (Colocasia esculenta (L.) Schott) using tissue culture techniques, Regional meeting on the
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production of root crops, Technical Paper No. 174, South Pacific Comm., Noumea, New Caledonia, 1977, pp. 114-117. M.M. Abo El-Nil and F.W. Zettler, Callus initiation and organ differentiation from shoot tip culture of Colocasia esculenta. Plant Sci. Lett., 6 (1976) 401-408. M. Morishita, Taro (Colocasia esculenta Schott.), in: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry 6, Crops II, Springer-Verlag, Berlin 1988, pp. 322-338. S. Sabapathy and H. Nair, In vitro propagation of taro with spermine, arginine and ornithine, I. Plantlet regeneration from primary shoot apices and axillary buds. Plant Cell Rep., il (1992) 290-294. T.W. Yam, E.L. Webb and J. Arditti, Callus formation and plantlet development from axillary buds of taro. Planta, 180 (1990) 458-460. T.W. Yam, G.I. Hsu and J. Arditti, Plant regeneration in vitro of South Pacific taro (Colocasia esculenta var. esculenta cv. Akalomale, Araceae). Plant Cell Rep., 9 (1990) 229-232. T.W. Yam, J.L.P. Young, K.P.L. Fan and J. Arditti, Induction of callus from axillary buds of taro (Colocasia esculenta vat. esculenta, Araceae) and subsequent plantlet regeneration. Plant Cell Rep., 9 (1990) 459-462. T.W. Yam, S. Ichihashi and J. Arditti, Callus growth and plantlet regeneration in taro, Colocasia esculenta vat. esculenta (L.) Schott (Araceae). Ann. Bot., 67 (1991) 317-323. P.J. Larkins and W.R. Seowcroft, Somaclonal variation - - a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet., 60 (1981) 197-214. L.P. Nyman, C.J. Gonzales and J. Arditti, In vitro selection for salt tolerance of taro (Colocasia esculenta vat. antiquorum). Ann. Bot., 51 (1983) 229-236. T. Murashige and F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant., 15 (1962) 473-497. SAS, SAS/STAT Users Guide, 4th edn, Vol I and II, Version 6, SAS Institute Inc. Cary, NC, 1982.
Plant Science 104 (1994) 93-100
High frequency direct shoot regeneration from corm axillary buds and rapid clonal propagation of taro, Colocasia esculenta var. esculenta (L.) Schott (Araceae) Rosemary C.O. Chng, Chong-Jin Goh* Tissue Culture Laboratory, Department of Botany, National University of Singapore, Lower Kent Ridge Road, Singapore 0511, Singapore Received 30 June 1994; revision received 12 October 1994; accepted 12 October 1994
Abstract
High frequency direct shoot regeneration from corm axillary bud explants of Colocasia esculenta var. esculenta was achieved through enhanced axillary bud development. Plantlets with well-developed shoot and root systems were obtained after 4 weeks culture on Murashige and Skoog (MS) medium supplemented with 20 g/1 sucrose and solidified with 2.4 g/l gelrite. The rate of successful initiation was 76% of total corm axillary buds cultured. In addition, a rapid method for direct regeneration of over 80 shoots per corm in 8 weeks was achieved through thin section culture of tissue-cultured-taro corm on medium containing 50 #M BA. Regenerated shoots developed roots readily when transferred to hormone-free medium. Plantlets were ready for potting in 4 weeks and yielded 100% successful acclimatisation rate. The developed protocols can be employed for taro germplasm conservation, exchange and mass propagation for cultivation.
Keywords: Colocasia esculenta var. esculenta (L.) Schott; Plant regeneration; Thin section culture; Germplasm conservation; Taro
1. Introduction
Colocasia esculenta (L.) Schott (Araceae), commonly known as taro, is taxonomically classified into two different varieties, Coiocasia esculenta var. antiquorum and Colocasia esculenta var. Abbreviations: BA, 6-benzyladenine; BM, basal MS medium; CAB, corm axillary buds; MS, Murashige and Skoog Medium; NAA, l-naphthaleneacetic acid; SAB, shoot axillary buds; TC, taro corm cube; TE, taro extract. * Corresponding author.
esculenta, with hundreds of cultivars [1]. Colocasia esculenta var. antiquorum is triploid (2n = 3x) with 42 chromosomes whereas Colocasia esculenta var. esculenta is diploid (2n = 2x) with 28 chromosomes [2]. The latter is more widely preferred by consumers. Taro is one of 4 major root and tuber crops that form the staple diet of people in the Asia-Pacific and African regions. Compared with cassava, sweet potato and yam, taro is known to contain the highest concentrations of protein, minerals and vitamins [31.
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Taro is affected by viral diseases [4] which decrease the yield. Being vegetatively propagated and extremely heterogeneous as a group, conservation of taro germplasm is an urgent task. However, taro germplasm is not actively exchanged due to the difficulty of ensuring virus-free material. Hence recommendations by the International Board for Plant Genetic Resources (IBPGR) are for the exchange of in vitro materials only [5]. Furthermore, taro seeds are only viable for about 30 days after harvest [6] and the in vitro conservation of taro is thus necessary. Although much research on the tissue culture of taro had been reported during the past two decades, most of the success was achieved with the variety Colocasia esculenta var. antiquorum [7-11 ]. Methods developed for the tissue culture of Colocasia esculenta var. esculenta, however, were less consistent and plantlet regeneration rate was low until recent reports by Yam et al. [12-15] with the use of taro extract. However, regeneration through callus is subjected to somaclonal variations [16] and mutation in taro has been reported [17]. Hence, protocols that would minimize genetic changes during micropropagation need to be developed [10]. All previous reports of successful taro culture used shoot tips or axillary bud explants from shoot tips as starting material [7-151 and none used the more easily available and accessible axillary buds of the storage corm. We have developed a simple and highly regenerative direct shoot development method using axillary buds from both shoot tips and corms. In addition, a thin section culture protocol for mass propagation of in vitro plantlets was also developed. 2. Materials and methods
2.1. Plant materials Corms of Colocasia esculenta var. esculenta of Malaysian cultivar 'Pasang' were obtained from farms in Johor, Malaysia. Each corm, complete with shoot tip, weighed approximately 1 kg fresh weight. 2.2. Preparation of explants The corms were first scrubbed in 2% commercial
bleach Clorox solution and detergent. Dead leaf tissues and mud encasing the corms were removed. Shoot tips (30 nun) with 3-5 whorls of tightly bound petiolar bases and 10 mm of corm tissues were excised. Undamaged corm axillary buds (CAB, Fig. 1) located on the nodes of corms were isolated. The shoot tips and CAB were then washed under running water for 30 min before surface sterilization.
2.3. Sterilization and culture of corm axillary buds Between 15 and 20 buds were placed in a sterile 200-ml screw cap bottle containing 20% (v/v) ethanol for 5 min followed by 50% (v/v) Clorox with two drops of Tween 20 for 3 min and finally 25% (v/v) Clorox for 15 min. The bottles were shaken vigorously at each stage and the sterilant completely drained before the addition of fresh solution. This was followed with three rinses of sterile distilled water. The sterilized buds were blotted dry on sterile filter paper and the outer scale leaves of the buds and 1 mm of their apices removed. Adjoining corm tissues were also trimmed to 1 mm in thickness. Explants were then placed on basal medium in 16mm Petri dishes to test for contamination. After 3 days, clean buds were transferred into 150 mm × 15 mm glass culture tubes containing 15 ml of culture media. 2.4. Sterilization and culture of shoot tips and axillary buds Excised shoot tips were sprayed with 95% ethanol before surface sterilization in 50% (v/v) Clorox with two drops of Tween 20 for 20 min and three rinses with sterile distilled water. The petiolar bases were peeled and the exposed shoot axillary bud (SAB, Fig. 1) on each node was excised together with 1 mm of corm tissues and cultured. Apical domes with one or two leaf primodia and 2 mm of corm tissues were cultured whole or cut into two or four vertical sections and cultured. 2.5. Thin section culture Four-week-old in vitro plantlets initiated on basal medium (BM), averaging 100 mm in height were used for further propagation. Mini-corms, 4 - 6 mm in diameter and 8-10 mm in length were
R.C.O. Chng, C.-J. Goh/Plant Sci. 104 (1994) 93-100
excised with 1 mm of shoot tip and cut transversely into 1.5-2 mm thick sections. These sections were then cultured in GA7 containers (Magenta Corps, Chicago, IL) with 40 ml of BM supplemented with various concentrations of BA.
2.0. Media and culture conditions Basal medium (BM) consisted of Murashige and Skoog (MS, Ref. [18]) salts enriched with 20 g/1 sucrose and solidified with 2.4 g/l gelrite. Benzyladenine (BA) was added before autoclaving. Taro extract (TE) was prepared by boiling 600 g of taro corm in distilled water for 5 rain and simmered for 1 h [11]. The cooled supernatant (made up to 1 1) was then fdtered twice through Whatman No. 1 filter paper. The extract (25 ml/1) was added to the medium as required. In another treatment, fresh taro corm cube (TC) of 10 × 10 × 10 mm (approx. 1.2 g fresh weight), was added to 15 ml of the medium in each culture tube before autoclaving. The pH of all media was adjusted to 5.7-5.8 prior to autoclaving at 120°C under a pressure of 1.2 kgtcm2 for 20 min. Cultures were incubated at 26 ± 2"C under a 16-h/day photoperiod provided by cool-white fluorescent light with a photon flux density of 18 ~E/mZ/s at culture level. 2.7. Statistical analysis All experiments were repeated at least twice. Results for CAB explants (Table 1) were pooled from three separate experiments. Those of SAB explants (Table 2) and thin section culture (Table 3) were pooled from two separate experiments. Results are presented as means ± S.D. Data were subjected to one way analysis of variance (ANOVA) using the Duncan-Waller multiple range test at c~ = 0.05 using the Statistical Analysis Systems [19]. 3. Remits
3.1. Axillary bud culture In preliminary experiments, buds that were cultured with less than 1 mm of corm tissues did not develop and corm tissues more than 2 mm thick led to excessive corm exudate buildup that inhibited normal bud development. Subsequently, all bud explants were isolated with 1 mm corm tissues. On average, 12 CAB and three SAB were iso-
95
lated for culture from a single mature corm. Out of a total of 475 CAB and 157 SAB isolated, 363 CAB and 124 SAB were clean cultures 3 days after initiation. Most of the cultures (about 80% for both the CAB and SAB) later developed into plantlets with complete shoot and root systems at the end of 4 weeks on BM. In CAB cultures, the basal portion of the creamcolored buds started to swell and greening of buds took place 3 days after initiation. New leaves and roots emerged after 5-7 days. By the end of 4 weeks, most of the cultured buds formed welldeveloped plantlets with 3-5 new leaves with main petiole lengths measuring about 100 mm, 5-10 roots and corms of 4-6 mm in diameter and 8-10 mm in length (Table 1). Small secondary shoots with petiole lengths ranging from 2 to 20 mm were also observed in some of the cultures. Growth and development of newly initiated shoots and their subsequent regenerant were highly asynchronous. In the presence of BA, multiple shoots were formed from explants. These shoots were observed to develop from pre-existing bud primodia between the scales of the buds. Regenerated multiple shoots on medium containing 25 #M of BA, averaging 35 mm in height were, however, much shorter than those developed from CAB grown on basal medium after 2 weeks of culture (Fig. 2). Rhizogenesis was inhibited in the presence of high BA levels (Table 1) but rooting readily occurred once the shoots were transferred to hormone-free medium. Optimal shoot formation was obtained with 25 t~M BA although there was no obvious correlation between shoot regeneration and BA concentration. Except for the greening of some of the cultures, neither leaf nor root development was observed for cultures grown in 75 ~M BA at the end of 4 weeks (Table 1). There was no significant beneficial effect when explants were cultured in the presence of TE (Table 1, Fig. 2). However, in medium with 25 t~M BA, TE appeared to cause a reduction in shoot regeneration although it stimulated rhizogenesis. For cultures grown with TC, only about 50% survived (Table 1) and growth and development of new shoots were stunted (Fig. 2). Growth of SAB cultures was slow when cultured on BM (Table 2). Not all buds turned green at the
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R.C.O. Chng, C.-J. Goh/Plant ScL 104 (1994) 93-100
end of 3 days and many did not develop further, especially for those younger buds located close to the apex (Fig. 3). For the buds that developed, the emergence of leaves and roots occurred after 2
weeks of culture. Plantlets were generally small with a single new leaf, 1 or 2 roots, 40-60 mm in height and corm diameter of 4 - 6 mm after 4 weeks of culture (Table 2).
R. C O. Chng, C-J. Gob~Plant $ci. 104 (1994) 93-100
97
Table 1 Effects of benzyladenine (BA), taro extract (TE) and taro cube (TC) on the morphogenic responses of taro corm axillary bud explants at the end of 4 weeks of culture Culture medium
BM BM BM BM BM BM BM BM BM BM BM BM
+ + + + + + + + + + +
10 20 25 30 40 50 60 75 T] TE TC
LM BA LM BA ,M BA LM BA ~M BA ~M BA LM BA LM BA + 25 I~M BA 25 ttM BA
Number of explants
24 24 24 20 24 24 24 24 20 20 20 20
Number of cultures with shoots
roots
20 15 19 18 18 18 14 16 0 18 20 11
19 13 I1 3 9 4 0 1 0 18 14 3
Number of shoots per culture
Diameter of corm (mm)
Length of petiole (mm)
!.2.4.- 0.5 c 3.4 4- 2.6 bc 3.4 4- 2.5 bc 6.3 4- 5.0 a 4.0 4- 3.2 a~ 5.5 4- 3.7 ab 4.7 4- 2.6 abe 4.8 4- 3.8 ab 0d 1.8 4- 2.3 c 3.5 ..'- 1.7 bc 2.9 4- 0.5 Ix:
3.28 2.45 2.75 3.45 2.65 2.44 3.16 2.47 0d 3.59 2.43 5.28
61.7.4- 44.9 a 18.1 4- 20.1 b 17.0.4.. 16.4 bc 18.1 .4- 16.0 b 13.1 4- 11.2 bc 12.7 q- 12.0 bc 13.9 ± 11.2 bc 10.9 4- 8.7 ¢ 0d 103.7 4- 25.3 a 13.0 -t- 10.6 bc 21.3 4- 4.0 b
44444444-
1.42 bc 1.54 bc 1.67 c 2.87 ab 1.81 bc 1.62 bc 1.86 bc 1.65 c
4- 2.70 a 4- 1.30 c 4- 0.72 a
Results are expressed as means 4- S.D. Means were calculated from measurements of all shoots > 1 m m in height. In each column, the means followed by the same letter are not significantly different as indicated by the D u n c a n - W a l l e r multiple range test at a = 0.05.
Addition of BA into culture medium enhanced growth and development of SAB (Fig. 3). The buds swelled and turned green within the first few days of culture. They later formed enlarged corms up to 12 mm in diameter and exhibited accelerated shoot growth (Table 2). After 4 weeks of culture, weU-developed plantlets with 4 or 5 leaves with main petiole lengths averaging 100 mm were obtained, especially from cultures with 25 ~M BA (Table 2). However, rhizogenesis was inhibited during the first 2 weeks (Fig. 3). A small number of very young buds did not develop beyond the
greening and swelling stages, but produced numerous tiny shoots after 6 weeks. The presence of TE or TC did not significantly enhance shoot regeneration (Table 2, Fig. 3). 3.2. Shoot tip culture Shoot tips cultured on BM showed only some swelling. On BM supplemented with 25 t~M BA, the apical dome of the shoot tip turned green after 1 week and swelling of the petiolar bases with enhanced anthocyanin pigmentation was observed. For shoot tips that were cultured whole,
Fig. I. Field grown corm of Colocasia esculenta var. esculenta cv. Pasang showing corm axillary bud (thick arrow) and exposed shoot axillary bud (thin arrow). Scale bar, 4 cm. Fig. 2. Corm axillary bud culture after 2 weeks on: A, BM; B, BM + TE; C, BM + TC; D, BM + 25 ~M BA. Note multiple shoot formation in D. Scale in cm. Fig. 3. Shoot axillary bud culture after 2 weeks on: A, BM; B, BM + TE; C, BM + TC; D, BM + 25 #M BA. Note enlarged basal corm formation in D. Scale in cm. Fig. 4. Four-week-old plantlet regenerated from corm thin section culture on BM. Plantlet initiated from the axiilary meristem of original explant (arrow). Scale in cm. Fig. 5. In vitro plantlet 4 months after transplant to soil mix in 200-mm pot. Scale in cm. Fig. 6. In vitro plantlets 4 months after transplant to soil showing healthy shoot, corm and root development, and developing runner (thick arrow). The plantlet at left had much of the lateral roots trimmed to show developing corm with axillary bud (thin arrow).
R.CO. Chng, C-£ Gob~Plant Sci. 104 (1994) 93-100
98
Table 2 Effects of benzyladenine (BA), taro extract (TE) and taro cube (TC) on the morphogenic responses of taro shoot axillary bud explants at the end of 4 weeks of culture Culture medium
BM B M + 10#M BA BM + 20/~M BA BM + 25 ~M BA BM + 30 ~M BA BM + 40/~M BA BM + 50 #M BA B M + 6 0 #M BA BM + 75 ~tM BA BM + TE BM + TE + 25 #M BA BM + TC + 25 ttM BA
Number of explants
7 7 7 10 7 7 7 7 7 10 7 10
Number of cultures with shoots
roots
5 7 6 9 6 4 4 5 0 9 6 6
3 5 3 7 3 1 1 2 0 9 6 3
Number of shoots per culture
Diameter of corm (ram)
Length of petiole (ram)
1.0 1.1 1.5 1.4 1.7 2.0 1.3 3.4 0c i.1 1.5 2.0
4.40 4- 1.14fs 5.064- 1.32efg 7.10 4- 3.30 abe 9.62 4- 3.40 a 4.90 4- 2.37 def 5.06 4- 2.51 bcd 7.80 4- 3.35 b°d 2.76 4- 2.31s Oh 5.35 4. 1.67 cdef 8.22 4. 2.39 ab 5.58 4- 4.40 bcde
37.2 4- 20.8 cd 51.24-36.3 cd 33.8 4- 12.0 bcd 85.8 4- 26.8 a 36.4 4- 24.3 bc 25.0 4- 18.3cd 29.8 4- 14.7 co 15.3 4- 18.4d Oh 51.2 4. 36.4 ab 84.4 4- 33.5 a 77.3 4- 25.6 ab
4- 0.0 b 4-0.4 b 4- 1.2 ab 4- 0.9 ab 4- 0.8 ab 4- 0.8 ab 4- 0.5 ab
:t: 3.4 a 4- 0.3 b 4- 0.8 ab 4- 2.0 ab
Results are expressed as means 4- S.D. Means were calculated from measurements of all shoots > I mm in height. In each column, the means followed by the same letter are not significantly different as indicated by the Duncan-Waller multiple range test at ct = 0.05.
rooting occurred within 1 week followed by the formation of new leaves. For those that were sectioned, a single shoot emerged from the cut surface of the explant 4 weeks later. Rooting occurred after shoot development and a well-formed plantlet was obtained after 8 weeks of culture. Survival of the explant was also dependent on explant size at isolation. Of the 20 replicates per
TaMe 3 Multiple shoot formation from thin sections of in vitro corms at the end of 8 weeks of culture on MS media supplemented with BA BA 0tM)
Number of corms cultured*
Number of corms with shoots
Number of shoots per corm
0 10 25 50 60
20 20 20 20 20
20 20 20 20 20
5.2 9.9 36.0 81.2 1.0
4- 1.9 d 4- 4.3 c 4- 6.2 b 4- 2.5 a 4- 0.0 e
*Each corm was cut into four sections. Results are expressed as means 4- S.D. Mean calculated from measurements of all shoots > 1 nun in height. In each column, the means followed by the same letter are not significantly different as indicated by the Duncan-Waller multiple range test at ¢x = 0.05.
treatment cultured on BM with 25 #M BA, new shoots developed in 90% of the tips that were halved compared to only 25% of those that were cut into 4 pieces. 3.3. Thin section culture Transverse thin sections of in vitro corms were cultured on BM as well as BA-enriched media (Table 3). On BM, 1 or 2 new shoots per section developed during the first week and rooting occurred shortly thereafter. Each mini-corm yielded at least four thin sections and an average of five plantlets per corm was obtained from thin section culture at the end of 8 weeks (Table 3). New shoots were observed to develop from axillary buds on the nodes of the sections (Fig. 4). For cultures on media containing BA, multiple secondary shoot formation from nodes of newly formed shoots was common after the second week. The number of shoots formed increased with increasing BA concentrations up to 50 #M but decreased sharply at 60 #M BA. After 8 weeks, thin sections cultured on medium with 50 ~M BA produced a mean of 81 shoots per four sections of single corm through enhanced axillary bud growth. However, the growth of shoots was much slower in high BA media than in BM. It took
R.CO. Chng, C-J. Gob~Plant Sci. 104 (1994) 93-100
another 4 weeks on BM for mini-corms to reach the size suitable for further thin section culture. For cultures with 60/tM BA, only the shoot tip sections survived after 8 weeks of culture. Some of these shoot tips later produced highly faciated multiple shoots. However, when these faciated shoots were subcultured onto BM, rooting and normal shoot development occurred after 3 days. 3.4. Plant establishment Tissue-cultured plantlets were ready for potting 4 weeks after whole bud culture and 12 weeks after microprogation from thin section. Acclimatization was not necessary other than the trimming of existing leaves and roots. Survival rates of 100% were obtained and no morphological variations were detected in any of the regenerated plantlets (Fig. 5). All plants exhibited healthy and normal shoot, root and corm development, with many of the plants producing side shoots and runners, after 3 months of transplant into pots with soil mix (Fig. 6).
4. Discussion In our present study, direct shoot regeneration was successfully achieved through the culture of CAB on basal MS medium. These shoots produced mini-corms suitable for thin section culture in 4-5 weeks. Each mini-corm subsequently regenerated on average five shoots per corm on BM at the end of 8 weeks (Table 3). Hence, on 8week cycles, about 15 000 plantlets can be obtained from a single corm 1 year after initiation. This protocol, without the use of any plant growth regulators (thereby minimizing the risk of somaclonal variation), is thus recommended for the in vitro conservation of taro germplasm. Multiple direct shoot formation from CAB occurred in the presence of BA, especially at 25/~M BA (Table 1). Miui-corms from these shoots (together with those initiated on BM) can be used for thin section culture on medium with 50 ~,M BA (Table 3). Regenerated shoots (in 8 weeks) transferred to BM were in turn ready for thin section culture after 4 weeks. Considering the 12week cycle to produce 80 plantlets, mass propagation of up to 40 million plantlets can be obtained
99
from a single corm in 1 year. This compares favourably with a previous report of 100 000 plants per year through callus culture [10]. It is also much faster than previously described protocols for taro [8,10,12-15]. In our experiments, auxin was excluded since mutants have been reported from media containing as low as 0.1 rag/1 NAA [10]. Indeed, no morphologicial variations were detected in regenerated plantlets, even amongst those regenerated from BA treatment. Thus, the present direct shoot regeneration system, by-passing the callus phase, may produce more true-to-type clonal plants. Shoot-tip explants regenerated single shoots. Axillary buds from shoot tips regenerated fewer shoots than those from storage corms. Nevertheless, these regenerated shoots could also be used for thin section cultures for mass propagation. However, the greater success with the use of CAB and the easy availability of these buds indicate that CAB is the choice of starting material. Although the possible deleterious effects of using sterilants directly on axillary bud explants have been reported [14], it was not apparent in the case of CAB explants in the present study. Previous reports stressed the requirement of taro extract for successful culture [12-15]. In our system, the beneficial effect of TE was, at most, marginal. However, the explants were cultured with 1 mm of the corm tissues. The use of larger explants thus simplified and circumvented the need for TE. It was possible that the larger explant size also helped in culture establishment and initial multiple shoot regeneration. The mini-corms produced by regenerated plantlets can be used, after aseptic removal of culture medium, directly for germplasm exchange. They are easy to handle and can survive for a period of 5 weeks, longer than the period commonly experienced during trans-shipment of plant materials. Thus, the present protocols developed for direct shoot regeneration and thin section culture can be employed for germplasm conservation, exchange and mass propagation for cultivation.
Acknowledgements This research was supported in part by the International Board for Plant Genetic Resources
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(IBPGR), Rome, Italy. We would like to thank Ir. D.H. van Sloten, Assistant Director General, and Dr J.M.M. Engels, Group Leader of Germplasm Maintenance and Use, International Plant Genetic Resources Institute (previously IBPGR), for their support for R. Chng to carry out this collaborative project while being employed as the Head of the IBPGR Seed Handling Unit for the Asia-Pacific region, in the Botany Department, National University of Singapore. References [I] J.W. Pursglove, Tropical Crops, Monocotyledons, Longnmn Group Ltd., London, 1972, pp. 61-69. [2] D.L. Plucknett, Taxonomy of the genus Colocasia, in: J.K. Wang (Ed.), Taro, A Review of Colocasia esculenta and its Potential, University of Hawaii Press, 1983, pp. 14-19. [3] FAO, Food Composition Table for Use in East Asia, Food and Agricultural Organisation of the United Nations, Rome, 1972. [4] J.J. Ooka, Taro diseases, in: J.K. Wang (Ed.), Taro, A Review of Coiocasia esculenta and its Potential, University of Hawaii Press, 1983, pp. 141-147. [5] F.W. Zettler, G.V.H. Jackson and E.A. Frison (Eds.), FAOflBPGR Technical Guidelines for the Safe Movement of Edible Aroid Germplasm, Food and Agricultural Organisation of the United Nations, Rome/International Board for Plant Genetic Resources, Rome, 1989, pp. 1-24. [6] K. Kikuta, L.D. Whitney and G.K. Parris, Seeds and seedlings of the taro, Colocasia esculenta. Am. J. Bot., 25 (1938) 186-188. [7] M.O. Mapes and W.J. Cable, Mericloning of taro (Colocasia esculenta L.), Hawaii Agric. Exp. Station, J. Series No. 1649, 1972. [8] G.V.H. Jackson, E.A. Ball and J. Arditti, Rapid propagation of taro (Colocasia esculenta (L.) Schott) using tissue culture techniques, Regional meeting on the
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