ABSTRACT. A regeneration system via somatic organogenesis from leaf callus was developed for common buckwheat (Fagopyrum esculentum Moench.).
Proceedings ofthe 9th International Symposium on Buckwheat, Prague 2004
Plant Regeneration via Shoot Organogenesis from Leaf Callus Culture of Common Buckwheat (Fagopyrum esculentum Moench.) 4
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Woo, S. H.t, M. Takaoka2, H. S. Kim 1, C. H. Park , T. Adachi and S. K. Jong
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} Dept. ofAgronomy, College ofAgriculture, Chungbuk National University, Chongju 361-763, Korea 2 Kamakura Women's College, !wase, Kamakura, Japan 3 Lab. ofPlant Genes and Physiology, College ofAgriculture, Osaka Prefecture Univ., Gakuen-cho, Sakai, Osaka, Japan 4 Division ofBiotechnology, Kangwon National University, Chunchon, Korea
ABSTRACT A regeneration system via somatic organogenesis from leaf callus was developed for common buckwheat (Fagopyrum esculentum Moench.). Best results were obtained on MS medium containing 2,4-D(2.0 mg/l), KIN(0.2 mg/l) and 3% sucrose. Friable callus was transferred to MS media containing BAP and KIN at varied concentrations for embryogenic callus induction. The optimum media was MS medium supplemented with 0.2 mg/l KIN, 2 or 3 mg/l BAP and 3% sucrose. Sucrose concentration of 3% or 6% did not show any significant effect on callus induction and embryogenesis. Whole plantlets were obtained when the embryogenic calli with somatic embryos and organized shoot primordia were transferred to 1/2 MS media with 3% sucrose. Regenerated plants after acclimatization were transferred to greenhouse and variations in both vegetative and floral characters were observed. This work successfully demonstrated the complete regeneration of plants via shoot organogenesis from leaf callus cultures of common buckwheat. This regeneration system may be valuable for genetic transformation and cell selection in common buckwheat. Keywords: common buckwheat (Fagopyrum esculentum Moench.), leaf explant, plant regeneration, somatic organogenesis Abbreviations: 2,4-D, 2,4-dichlorophenooxiaxetic acid; BAP, 6-benzylaminopurine; NAA, naphthaleneacetic acid; IBA, indolbutric acid; KIN, kinetin; IAA, indole-3-acetic acid INTRODUCTION At present, infonnations on tissue culture, transfonnation and protoplast fusion in buckwheat are rather limited and restricted to mainly on micro-propagation. Such results were due to the fact that most of the works on tissue culture in buckwheat remains empirical and tiresome. Fagopyrum species are diploid (2n=16) but tetraploid species occur either spontaneously or artificially. Buckwheat has been, for centuries, remained as a crop with low seed set due to its characters preventing the application of conventional breeding methods (KREFT 1983). The main obstacles in buckwheat breeding include its very strong sel£lcrossincompatibility and indertenninate growth and flowering habits. Modern biotechnology may provide means to address these problems in a novel way (NESKOVIC et al. 1995). Regenerations of buckwheat in vitro from explants such as hypocotyls (YAMANE 1974, LACHMANN AND ADACHI 1990, Woo et al. 1997), cotyledons (STREJOVIC AND NESKOVIC 1981, MILJUs-DJUKIE et al. 1992, WOO et al. 2000), immature inflorescence (TAKAHATA 1988), leaf segment (PARK et al. 1999) and anthers (ADACHI et al. 1989, BOHANEC et al. 1993) were reported. Somatic embryogenesis has been previously reported in cultures of immature embryos of common buckwheat (NESKOVIC et al. 1989, RUMYANTSEVA et al. 1989) and tartary buckwheat (RUMYANTSEVA et al. 1989, LACHMANN AND ADACHI 1990). The objective of this research was to develop plant regeneration system for common buckwheat for future application in genetic transfonnati on. In this paper, we report plant
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Proceedings ofthe 9th International Symposium on Buckwheat, Prague 2004
regeneration via shoot organogenesis from callus of leaf segments of the cultivated species Fagopyrum esculentum Moench. MATERIALS AND METHODS
Innennost leaves of common buckwheat (Fagopyrum esculentum) were collected from young plants and used as explants. The explants were surface-sterilized with a 0.1 % mercuric chloride for 5 minutes, rinsed thoroughly in sterilized distilled water and cut transversely into 3 mm long sections. After rinsed five times with autoclaved water, the explants were placed on moistened and sterilized filter papers in petri dishes and incubated at 25°C. The leaf segements were excised and placed on callus induction medium. Twelve combinations with different concentrations of auxin and cytokinins were tested for callus induction. Approximately equal number of explants were placed on medium containing MS (Murashige & Skoog, 1962) supplemented with myo-inositol (100 mg/I), sucrose (3% or 6%) and different concentrations of 2,4-D, NAA and KIN. pH was adjusted to 5.7 before autoclaving and the media were solidified with 0.8% agar. Cultures were incubated in dark and/or diffused light at 24°C and subcultured to fresh medium every three weeks. To induce somatic embryos, small fragments of friable callus were transferred to MS basal medium supplemented with BAP, KIN, lAA, 3% sucrose and 0.8% agar. The somatic embryos and miniature organogenic shoots were then transferred to MS and 112 MS hormone free medium at 16/8h (light/dark) photoperiod at 25°C. Plantlets with well developed root system were transplanted to organic soil mixture for hardening in growth chamber with 16/8h (light/dark) photoperiod at 25°C and 80% humidity for two weeks and them transferred to regular greenhouse. Data on vegetative and floral characters were collected. RESULTS
Callus were fonned initially on the cut ends of the leaf segments on callus induction medium after 4 weeks. Callus covered almost all the whole explant. The callus formation was strongly dependent on the combination and concentration of the growth regulators supplied to the culture media as well as on the genotype. After 4 weeks, compact green calli were produced on media either KIN(O.2 mg/l) or KIN(0.2 mg/l). Of the various media tested, MS medium supplemented with KIN and 2,4-D were found to be ideal for callus induction and proliferation of tissue. Prolonged culture up to 4 weeks resulted in root fonnation. The optimum callus induction with minimum root induction was obtained on MS media supplemented with KIN(0.2 mg/l), 2,4-D(2.0 mg/I) and 3% sucrose. At higher levels of KIN(0.2 mg/I) and 2,4-0(2.0 mg/l), callusing was associated with suppressed rootlike structures. One subculture was made on this initiation medium after 3 weeks before transferring onto embryogenic callus induction medium. After callus induction, subculture was carried out on MS medium containing BAP, KIN and lAA at varied concentrations. The sucrose concentration was maintained at 3%. After 2 passages in the same media with an interval of 3 weeks, KIN at 0.5 mg/l and BAP at 2.0 mg/I and 4.0 mg/l produced callus with well organized nodular regions with high possibility of embryogenesis. The callus other than the embryogenic region turned brown, presumably due to excess exudation of phenolics. To maintain the embryogenic potential, embryogenic tissue were separated from the underlying translucent dark callus while subculturing onto fresh medium with KIN, 0.2 mg/l and varied concentrations of BAP. The mean number of embryogenic calli after planting the nodular calli ranged from 10% to 50% in different media combinations tested. The highest percent induction (50%) was observed on MS media supplemented with 2.0 mg/l BAP and 0.2 mg/l KIN (Fig 1. A, B). All the treatments were maintained under diffused light
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Proceedings of the
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International Symposium 011 Buckwheat, Prague 2004
conditions. The somatic embryoids were then transferred to hormone-free 1/2 MS medium with 3% sucrose, The embryos germinated and grew into plants (Fig 1. C). The frequency of regeneration varied with respect to the embryogenic callus induction media. The highest number of plants were obtained when the embryogenic callus induction media was MS supplemented with 2.0 mg/l BAP and 0.2 mg/l KIN and 3% sucrose. After 3 weeks in hormone free media, well rooted plantlets were separated, washed and transplanted to an organic soil mixture in small cups. After acclimation, the hardened plants were then transferred to large pots in greenhouse (Fig 1. D). Roots could be induced on all media tested. However, character, time of appearance and frequency of root formation depended on the types of medium used for root induction. Long, thin hairlike roots were observed either on half or full strength MS basal medium with 3 or 6% sucrose. The regenerated plants were rarely suitable for planting out. Early healthy rooting was obtained with 0.2 mg/l KIN or NAA and 3 or 6% sucrose, with NAA at 0.2 mg/l and 3% sucrose. DISCUSSION
This is the first report on the induction of somatic organogenesis from calli derived from leaf segments in common buckwheat. Regeneration via somatic organogenesis is desirable in several ways. Regeneration via somatic embryogenesis has few problems related to chimeric variation since it originates from a single-cell. Therefore, it is possible to recover natural genetic variation or artificial mutants. Regeneration with high frequency (30%) of Fagopyrum escuientum, a species not previously tested for response in tissue culture, was achieved on MS medium supplemented with BAP (2.0 mg/l) and KIN (0.2 mg/l). The somatic embryos appeared in clusters on the embryogenic callus. At cotyledonary stages, many embryos were very small and not conspicuous. Similar phenomenon was reported by Neskovic et ai. 1995. However, direct regeneration of shoots were also observed in a few cases with F. esculentum in this experiment. The leaf morphology and size indicated somaclonal variation among the regenerated plants. The effect of sucrose remains somewhat obscure, as the sucrose content in callus induction and later regeneration media had no significant effect on development of somatic embryos. This is in contrast to NESKOVTC et at. (1987) and LACHMANN (1991) who reported that the sucrose content was critical for development of somatic embryos in buckwheat. Species specificity could be one of the explanations. While F. escuientum is insensitive to sucrose, other species of Fagopyrum might be sensitive to sucrose. Shoot regeneration had been found in many buckwheat species (LACHMANN AND ADACHI 1990, NESKOVIC et ai. 1995). This variation was evident in our first experiments. Since explants were isolated from a mixed seedling population, all segments of one leaf formed shoots, whereas all segments of another leaf might not form shoots even in the same petri dish. Explants from all donor plants formed shoots but regeneration frequency and the number of shoots regenerated varied greatly among the seedings. This experiment demonstrated that the regeneration from seedling leaves of common buckwheat might be valuable for genetic transformation and cell line selection in common buckwheat. As seed production of common buckwheat (F. esculentum) is limited due to heterostyly, methods are being investigated to overcome this barrier by crossing between F. escuientum and F. homotropicum. Our attempt has resulted in homostyle self-pollinating type buckwheat (Woo et at. 1999). But plant regeneration system in F. escuientum is highly relevant as we can utilize tissue culture in protoplasts fusion to achieve the same objective. The leaf segments expressed their totipotency even after a prolonged callus phase as PARK et ai. (1999) has previously reported to show respectively 90% of callus induction and 5.6% of pant regeneration rates in leaf tissue culture. The regeneration was influenced by the growth regulators and high sucrose
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