Recurring Somaclonal Variation as a Factor of Intra-Specific Diversity Observed in Dioscorea alata L.

Transcription

1 Trop. Agr. Develop. 56(2):71-79,2012 Recurring Somaclonal Variation as a Factor of Intra-Specific Diversity Observed in Dioscorea alata L. P. K. BABIL 1, *,a, Daisuke YASUHARA 2, Keisuke SAKAGUCHI 3, Tsukasa IWASHINA 3,4, Kenji IRIE 1, Hironobu SHIWACHI 1, Hidekazu TOYOHARA 1 and Hiroshi FUJIMAKI 5 1 Graduate School of Agricultural Sciences, Tokyo University of Agriculture, Tokyo, , Japan 2 Miyoshi Agritech Co., Ltd, Yamanashi, Japan 3 United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, , Japan 4 Department of Botany, National Museum of Nature and Science, Tsukuba, , Japan 5 Research Fellow, National Agriculture and Food Research Organization, Tsukuba, Japan Abstract Somaclonal variations in pigmentation occurred repeatedly during the clone propagation of the landrace Okinawa A of water yam, Dioscorea alata L. An initial variation appeared when clone propagation of Okinawa A started in Reverting variants, closely resembling the original type appeared several times during the cloning of the initial variants. In 2009, a second variant without pigmentation emerged from the initial one. Every observed trait of the original type and the three types of variants was ordinarily transmitted to later generations during clone propagation by tuber. Genealogical tracing of clone pedigrees and analyses of variance in leaf and stoma traits indicated that these variants might be caused by tissue chimeras resulting from changes in the number or structure of chromosomes. However, the possibility that transposable elements were involved could not be excluded. The recurring somaclonal variations are considered to affect the genetic diversity within the species and to provide useful genetic resources for improvement of water yam. Key words: Anthocyanin, Clonal propagation, Genealogical tracing, Tissue chimera, Variance analysis Introduction Water yam (Dioscorea alata L.) is a monocot belonging to the family Dioscoreaceae. It is cultivated as a staple tuber crop in tropical Asia and Africa. It is also processed for confectionery and brewing products in the sub-tropical regions of Asia, including Taiwan and Japan. Water yam is sexually sterile and is conventionally cultivated by clone propagation with tubers (Coursey, 1967). Nevertheless, a wide range of phenotypic diversity is observed within the species (Toyohara et al.,1996; Lebot et al., 1998). Tubers, for instance, vary in shape and can be cylindrical, oval-oblong, flattened, roundish, or irregular, and leaves exhibit variations in size, shape, and pigmentation. In addition to the intra-specific diversity of phenotypes, water yam is known to exhibit different levels of ploidy, including diploid (2x = 40), triploid (3x = 60), and tetraploid (4x = 80) forms within the species (Abraham and Nair, 1991; Gamiette et al., 1999; Egesi et Communicated by M. Takagaki Received Oct. 19, 2011 Accepted Mar. 12, 2012 * Corresponding author babilp@yahoo.com a Present address: Tropical Agriculture Research Front (TARF), Japan International Research Center for Agricultural Sciences (JIRCAS), , Maezato-Kawarabaru, Ishigaki, Okinawa , Japan al., 2002; Arnau et al., 2009; Babil et al., 2010; Obidiegwu et al., 2010). Babil et al. (2010) reported that three levels of ploidy, diploid, triploid, and tetraploid, were identified in a landrace collection of water yam from Myanmar. They disclosed that the ploidy variations considerably affected the intra-specific diversity of leaf and stoma phenotypes. However, the mechanisms generating such intra-specific diversity, as observed in water yam, remain to be elucidated. Generally speaking, sexual reproduction is considered to generate extensive intra-specific diversity. However, the sexual reproduction of water yam is hampered by factors such as rare flowering and non-synchronization of male and female flowers. Thus, somaclonal variation that occurs during clone propagation, is considered to exert a conspicuous effect on genetic diversity in water yam. In the present study, somaclonal variations recurring in a water yam landrace Okinawa A collected from the southernmost island of Japan were investigated to determine the factor or factors causing the somaclonal variations. Materials and Methods Genealogical tracing of somaclonal variations recurring in Okinawa A Twenty landraces of water yam cultivated and

2 72 Trop. Agr. Develop. 56(2)2012 maintained at Tokyo University of Agriculture were used in the present investigation (Table 1). Clone propagation was started in Five clones (5 plants per clone) derived from separate seed tubers were grown for each landrace. For clone propagation, seed tuber sets of approximately 50 g were planted in plastic bags filled with vermiculite. In early May 2000, sprouting seed tuber sets were planted at a spacing of 1 m between rows and 50 cm between plants in the field of the Setagaya campus of Tokyo University of Agriculture (latitude N and longitude E). Pigmentation on the stem, wing, petiole, wing of petiole, fully developed leaf, young leaf, leaf base, and leaf axil was surveyed based on the descriptor lists for yams (IITA and IPGRI, 1997). Five healthy plants per clone of each accession were sampled to examine the pigmentation. Among the 20 water yam accessions, somaclonal variation for pigmentation in epigeal portions appeared in a landrace named Okinawa A. The lineages of the somaclonal variants together with the original type were traced for 9 consecutive generations from 2000 through Pigmentation patterns of fully developed and developing young leaves of Okinawa A were utilized for criteria to distinguish somaclonal variants. Comparison with the original type (Fig. 1-a) revealed that, the initial variant (Fig. 1-b) exhibited more purplish leaves and that the revertant (Fig. 1-c) looked very similar to the original type. Variance analyses of leaf and stoma morphological traits associated with somaclonal variation Seventeen clones consisting of 197 plants derived from the original type, the initial and the reverting variants were planted in the field at a spacing of 1 m between rows and 50 cm between plants in late May of Five fully developed leaves at the middle growth stage were sampled for measuring the size and shape of leaf and stoma. The observed five leaf traits included total length, maximum width, blade length, depth of sinus, and width of sinus. To measure the length, width, and density of stoma, SUMP (Suzuki s Universal Micro Printing Method) printing samples were taken from the central portion of the reverse sides of the leaf blades of each plant. Five stomata were randomly sampled within a single microscope field and measured under a 400x magnification. The variance analyses with one-way classification were carried out based on individual plant means separately for the eight traits. Total variance was divided into three components, namely, the between-group variance (three groups of the original type, the initial and the reverting variants), the within-group variance, and the error variance. The statistical significance of the variance component between groups was tested against the pooled variance component within a group and the error variance. Morphological traits and pigmentation of the second variants appearing de novo in 2009 were compared with those of the initial variant obtained in A tuber set with twin shoots expressing the characteristics of the initial and the second variants was planted in a plastic pot to grow for 6 months in a greenhouse at the Setagaya campus in Tokyo (same area as in the first experiment). Tubers formed on the twin shoots were planted in 2010 to investigate such leaf traits as total length, maximum width, depth of sinus, and width of sinus. Anthocyanin analyses of somaclonal variants Anthocyanin analyses of the original type, the initial, the reverting and the second variants were conducted in 2011 using HPLC. For these analyses, seed tuber sets of approximately 50 g were planted in Table 1 Water yam accessions used for clone propagation Name of Landrace Collection site Name of Landrace Collection site Sulka (M) PNG * Malaysia-A Malaysia Nani niyaka PNG Malaysia-B Malaysia Lisiteya PNG Jamaica-1 Jamaica Nani abukyanbai PNG Jamaica-3 Jamaica Wasunamisunjya PNG T-1 Thailand Obukosumbori PNG T-3 Thailand Tombonani PNG Arata Japan Basmi PNG Kagoshima Japan Ambuting PNG Kagoshima-daigaku Japan Abamina PNG Okinawa-A Japan Note *: Papua New Guinea

3 Babil et al.: Somaclonal variations in Dioscorea alata L. 73 Fig. 1 Pigmentation of young developing leaves of somaclonal variants of Okinawa A, the original type (a), the initial variant (b), and the revertant (c). plastic bags filled with vermiculite. In early May 2011, sprouting seed tuber sets were planted in plastic pots and grown in a greenhouse at the Setagaya campus in Tokyo. Three plants per clone and 2 leaves per plant were sampled for the flavonoid analyses. Tissue samples of 0.1 g were collected from each leaf and anthocyanins were extracted from a total of 0.2 g leaf tissue per plant in the 8:92 (v:v) solution of formic acid and methanol. Analyses were conducted using the Shimadzu HPLC system (Shimadzu, Japan) with ODS-4 Inertsil column (GL Science, USA). The eluent consisted of a 3:8:10:79 solution of phosphoric acid/acetic acid/acetonitrile/ water (v:v:v:v). Anthocyanidin, which was liberated by acid hydrolysis of crude extracts from the original type, the initial variant and the revertant, was identified by HPLC comparison with an authentic sample. Results Genealogical tracing of somaclonal variations recurring in Okinawa A Among 20 water yam accessions presently investigated, one of 5 clones of Okinawa A exhibited a different pigmentation of leaf from that of the other 4 clones. No variation in pigmentation was observed in any clone of the other 19 accessions. Clone cultivation of Fig. 3 Twin shoots with different pigmentation appearing from a single tuber set, suggesting the occurrence of a tissue chimera from which the initial (left) and the second (right, newly appearing) variants were generated. Fig. 4 Pigmentation in tubers of the initial (a) and the second variants (b).

4 74 Trop. Agr. Develop. 56(2)2012 Fig. 2 Clonal pedigree of a water yam accession Okinawa A.

5 Babil et al.: Somaclonal variations in Dioscorea alata L. 75 Okinawa A has been continued for 9 consecutive generations since a somaclonal variation initially appeared in A clone pedigree of Okinawa A from 2000 through 2008 is shown in Fig. 2. When clone cultivation of Okinawa A started in 2000, clone No.4 was distinctively recognized by its deeper color of leaf and stem than that of any other four clones (Fig. 1-a).This somaclonal variant was designated as intial variant (Fig. 1-b). Clones No.1 and No.2 of the original type and clone No.4 of the initial variant were grown in Their characteristics were normally transmitted to the following generation. Ten clones of the original type and five clones of the initial variant were grown in Another initial variant appeared from the clone family 2-5. The remaining clones exhibited a similar pattern of pigmentation to that of their ancestors. Ten clones of the original type and seven clones of the initial variant were grown in Among the seven clones of the initial variants, three clones 4-1-3, 4-5-2, and generated reverting variations (Fig. 1-c) closely resembling the original type. In the clone cultivation of 2004, most clones did not vary but two initial variant clones, and , generated the reverting type of variation in The other two initial variant clones, and , generated revertants again in 2006 (Fig. 2). Every clone remained unchanged in In 2009, a tuber set of the initial variant clone generated twin shoots, one with characteristics resembling those of the initial variant and the other with a characteristics of a new type of (second) variant without pigmentation on leaf and stem (Fig. 3). Pigmentation patterns of the original type, the initial, the reverting and the second variants were compared with each other in 2010 (Table 2). Pigmentation in young leaves and stems of the initial variants was deeper than that in the original type and the revertants. No pigment was observed on any part of the second variants, indicating that the second variant was entirely different from the initial one. Clone propagation of the second variants has continued for 3 generations since 2009 and every observed characteristic of the somaclonal variants was confirmed to be transmitted to the following generation. No pigment was observed on tubers of the second variant, while the initial variant had purple pigment on tuber (Fig. 4). Variance analyses of leaf and stoma morphological traits associated with somaclonal variation An example of variance analysis for total leaf length is shown in Table 3. Variance analyses for the other traits were conducted in the same manner. Summarized results of significance tests of variance components for leaf and stoma traits are presented in Table 4. For total leaf length, leaf blade length, depth of sinus, and width of sinus, the variance components between clone groups (between-group variances) were statistically significant at the 1% level, suggesting the presence of a significant genetic variation associated with the somaclonal variation. For observed stomatal traits, the betweengroup variances were statistically significant at the 1% level. Differences in variant group means for leaf and stoma traits are shown in Fig. 5 and Fig. 6. Statistical tests at the 5% level revealed that revertants tended to revert to the original type for total leaf length, leaf blade length, and depth of sinus. No significant difference was detected between means of the initial and the second variants, possibly due to insufficient data (data not shown). Anthocyanin analyses of somaclonal variants HPLC profiles of leaves from the original type of Okinawa A and those from three somaclonal variants are shown in Fig. 7. Cyanidin glycoside was detected in the original type, the initial and the reverting variants. The cyanidin glycoside peak was observed at the retention time of 7.5 min in the three clones. In addition to the peak at 7.5 min, the initial variant also showed another peak at 9.0 min unlike the revertant. Another peak was also found to correspond to cyanidin glycoside, since acid hydrolysis of the crude extract from the initial variant Table 2 Pigmentation patterns of the original type, the initial, the reverting and the second variants of Okinawa A Clone group Stem Wing Adaxial side of leaf Abaxial side of leaf Petiole Leaf base Axil of leaf Wing of Petiole Original phenotype ± + ± ± ± + + ± ± Initial variant ± ± + + ± + Revertant ± + ± ± ± + + ± ± Second variant Note: +: pigmentation observed; ±: some pigmentation observed; -: pigmentation not observed Young leaf

6 76 Trop. Agr. Develop. 56(2)2012 Table 3 Example of variance analysis for total leaf length Variance component D.F. Sum of square Variance F-test*1 F-test *2 Between clones ** Between clone groups ** 3.65** Within a clone group ** Error Note: *1: Test against the error, *2: Test against the within a clonal group, ** indicate statistical significance at the 1% level Table 4 Statistical significance of variance components in analyses of variance for leaf and stoma traits with the prototype, the first and the restored variants Variance component F-value to the error variance Between clone groups F-value to the within a clone group Within a clone group F-value to the error variance Total leaf length (cm) 21.10** 7.15** 3.65** Leaf blade length (cm) 34.37** 6.81** 5.05** Leaf width (cm) 3.33NS 0.62NS 5.36** Depth of sinus (cm) 18.76** 4.18* 4.47** Width of sinus (cm) 1.95NS 0.50NS 3.92** Stoma length (µ) 70.27** 6.49** 10.82** Stoma width (µ) 16.90** 0.60** 28.28** Stoma density (number/sight) ** 19.61** 5.45** Note: * and ** indicate statistical significance at the 5% and 1% levels, NS: no-significance Fig. 5 Changes in mean values of leaf traits of the original type, the initial variant, and the revertant of Okinawa A. Note: Different letters indicate significant difference at the 5% level. Fig. 6 Changes in mean values of stoma traits of the original type, the initial variant, and the revertant of Okinawa A. Note: Different letters indicate significant difference at the 5% level.

7 Babil et al.: Somaclonal variations in Dioscorea alata L. 77 Fig. 7 HPLC profiles of formic acid/methanol extracts from leaves of the original type (a), the initial variant (b), the revertant (c), and the second variant (d). produced cyanidin alone. The cyanidin content of the initial variant was significantly higher than that of the original type and the revertants (Fig. 8). No anthocyanin was detected in the second variants. Discussion The clone propagation of 20 water yam accessions was started in Among 5 clones of a Japanese landrace Okinawa A, a clone exhibited a very different aspect of pigmentation from that of the other 4 clones. The clone was referred to as initial variant (Fig.1-b) with deeper coloring of leaf compared with the original type (Fig. 1-a). The reverting variant (Fig. 1-c) closely resembling the original type in terms of leaf color appeared several times during the cloning of the initial variant. In 2009, a second variant without pigmentation appeared from the initial variant (Fig. 3). Every phenotypic trait of the three types of variants as well as the original type seemed to be normally transmitted to later generations during clone propagation. Although colored portions of the initial variant were apparently similar to those of original type, the initial variant tended to display a deeper color than the original type. A common component of anthocyanin, cyanidin glycoside, was detected in the original type, the initial and the reverting variants. Another cyanidin glycoside was observed in the initial variant but not in Fig. 8 Areas of peaks detected in HPLC analyses of the original type, the initial variant, and the revertant of Okinawa A. Note: Different letters indicate significant difference at the 5% level. the revertant and the original type, indicating that the revertant had almost perfectly reverted to the original type for anthocyanin pigmentation. This somaclonal variation is considered to be caused by a genetic change either in the anthocyanin synthetic pathway or on its regulating system. Functions and variations of genes such as chalcone synthase involved in anthocyanin synthesis have been reported in several publications (Boss et al., 1996; Mano et al., 2007). A wide range of intra-specific diversity is observed in anthocyanin pigmentation of epigeal portions and tubers of water yam. Somaclonal variation appearing

8 78 Trop. Agr. Develop. 56(2)2012 in clonal propagation is cosidered to expand intraspecific diversity for pigmentation. Hayashi et al. (2001) reported that pigmentation patterns of epigeal portions could be important markers for characterization of water yam accessions. However, frequent occurrence of somaclonal variation of pigmentation should be carefully considered. Three types of somaclonal variation recurred were observed during clone propagation of a water yam landrace Okinawa A. The initial variant and its revertant were recurrently generated, followed by the appearance of the second variant from the initial one. The revertant closely resembled the original type. Twin shoots with different characteristics from those of the initial and the second variants sprouted from a single tuber set. Based on these observation, the somaclonal variation observed in the clone propagation of Okinawa A is considered to be caused by tissue chimeras which might have arisen from any genetic factor including euploidy, aneuploidy, structural changes in chromosomes, or gene mutation. Euploidy and a single gene mutation, however, may be excluded because of the following reasons. First, flow-cytometric analyses revealed that all three types of variants as well as the original type had an identical level of diploidy with a chromosome number (2x = 40) (Babil et al, unpublished data). Second, variance analyses revealed that the somaclonal variations of pigmentation were accompanied by changes in various quantitative traits of leaf and stoma, suggesting that a whole chromosome or a section of chromosome (a linkage block) with a gene (or genes) affecting pigmentation was more probably involved rather than a single gene mutation. Therefore, the tissue chimeras are considered to have arisen from aneuploidy or a structural change of chromosome. Since different types of somaclonal variation were recurrently generated from a single clone,the involvement of a transposable element may not be excluded. Kobayashi et al. (2004) reported that a retro-transposon could induce a genic change in an anthocyanin pathway to inhibit pigmentation in grape. Epigenetic changes in gene expression should not be excluded as a factor of somaclonal variation. Miura et al. (2009) reported the occurrence of a spontaneous mutant, Epi-d1, in rice with a dwarf phenotype that could revert to the wild type when the DWARF1 gene was epigenetically silenced or activated. The Epi-d1 mutant could be chimeric, and could bear dwarf and normal tillers at the same time on one plant. While cell fates may have initially induced a dwarf or normal type, they could vary epigenetically in the developing plant. Epigenetic regulation of the gene could change in either direction, from an active state to a repressed one, and vice versa. A similar phenomenon was observed in the somaclonal variation of Okinawa A. Further studies should be conducted to determine what genetic factor causes such tissue chimeras as those observed in Okinawa A. In the case of the tuber of water yam, adventitious buds are formed from any part of the tuber when cut into sets. Somaclonal variation affected by ploidy, chromosome aberrations, gene mutations and epigenetic changes in gene expression likely occur rather frequently during the process of formation of adventitious buds. Such somaclonal variation as observed in Okinawa A may contribute to genetic diversity within water yam. Somaclonal variants could become valuable breeding materials for the improvement water yam which cannot be easily propagated sexually by seeds, but is clonally propagated by tuber. Acknowledgements The authors thank the late Prof. Dr. Atsushi Komamine for his advice and suggestions. Appreciation is also extended to Prof. Dr. Junzo Fujigaki, Tokyo University of Agriculture, for his guidance. References Abraham, K. and P.G. Nair Polyploidy and sterility in relation to sex in Dioscorea alata L. (Dioscoreaceae). Genet. 83: Arnau, G, A. Nemorin, E. Maledon and K. Abraham Revision of ploidy status of Dioscorea alata L. (Dioscoreaceae) by cytogenetic and microsatellite segregation analysis. Theor. Appl. Genet. 118: Babil, P.K., K. Irie, H. Shiwachi, Ye Tint Tun, H. Toyohara and H. Fujimaki Ploidy variation and their effects on leaf and stoma traits of water yam (Dioscorea alata L.) collected in Myanmar. Tropic. Agric. Dev. 54: Boss, K.P., D.Christopher and P.R. Simon Expression of anthocyanin biosynthesis pathway genes in red and white grapes. Plant Mol. 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