In Vitro Cell. Dev. Biol.—Plant 38:617–622, November–December 2002 q 2002 Society for In Vitro Biology 1054-5476/02 $10.00+0.00

DOI: 10.1079/IVP2002319

GENETIC ANALYSIS OF VARIATION IN MICROPROPAGATED PLANTS OF MELIA AZEDARACH L. S. E. OLMOS1, G. LAVIA1, M. DI RENZO2, L. MROGINSKI1,

AND

V. ECHENIQUE3*

1

Facultad de Ciencias Agrarias, Universidad Nacional del Nordeste, Sgto. Cabral 2131. 3400, Corrientes, Argentina Facultad de Agronomı´a y Veterinaria, Universidad Nacional de Rı´o Cuarto, Agencia n 3, 5800, Rı´o Cuarto, Argentina 3 CONICET and Departamento de Agronomı´a, Universidad Nacional del Sur, San Andre´s 800, 8000 Bahı´a Blanca, Buenos Aires, Argentina 2

(Received 23 November 2001; accepted 23 April 2002; editor M. A. O’Connell)

Summary Plants were regenerated by shoot multiplication from four clones of Melia azedarach L. during 12 mo. of subculturing. One hundred and one of these plants were examined by randomly amplified polymorphic DNA analysis. All regenerated plants showed at least one polymorphism. However, no chromosome number alterations were observed. The pattern of variation obtained by principal coordinated analysis showed a random distribution of variation among regenerated plants and their controls, indicating that genetic alterations were not cumulative during in vitro culture. Similar results were found using Shannon’s index, which revealed that 50% of the observed diversity resided among plants coming from the same subculture generation. This high intraclonal variation does not provide a clear scenario for predicting the amount of culture time required to preserve genetic fidelity in commercially micropropagated M. azedarach plants. Our work suggests that other mechanisms, such as chimerism, contribute to intraclonal heterogeneity in vitro. Key words: somaclonal variation; RAPD; diversity analysis; chromosome counting.

insufficient, with morphological and cytogenetical techniques being valuable complementary tools (Fourre´ et al., 1997). Randomly amplified polymorphic DNA (RAPD) is an effective molecular technique for screening for genomic alterations among tissue culture-derived plants (Valle`s et al., 1993; Heinze and Schmidt, 1995; Gallego et al., 1997; Shoyama et al., 1997). In the case of dicotyledonous woody plants there are few reports of RAPD analysis being applied to monitor genomic changes (Heinze and Schmidt, 1995; Klopfenstein and Kerl, 1995; Gallego et al., 1997; Hashmi et al., 1997; Shoyama et al., 1997). Instead, this method has been applied extensively to evaluate natural genetic diversity among plants (Deng et al., 1995; Farooq et al., 1995; Gallois et al., 1998; Lim et al., 1999). Recently, this technique was questioned because of inconsistencies between laboratories. However, results can be very reliable and informative when the technique is carefully applied, using a set of samples subjected to standardized procedures. In addition, each primer results in the amplification of about 10 scorable genetic markers, and many primers are currently available for screening (Henry et al., 1998). Chromosome evaluation is another approach that has been used successfully to study genetic abnormalities among tissue culturederived plants (Straus, 1954; Murata and Orton, 1982; Maluszynska and Schweizer, 1999). Most studies indicate that the common chromosomal abnormalities found are polyploidy, aneuploidy and breakages at heterochromatic regions (Fourre´ et al., 1997; Al-Zahim et al., 1999). RAPD analysis and chromosome evaluation used together can consistently reveal genomic and gene mutations appearing during in vitro culture. Melia azedarach L. cv. ‘Gigantea’ (Persian lilac) is a species from the Himalayan region naturalized in many subtropical areas around

Introduction In vitro techniques have proven to be useful for the cloning of selected woody plant genotypes (Park et al., 1993, 1994; Dunstan et al., 1995; Gupta and Grob, 1995; Merkle et al., 1997) producing plants theoretically identical to the original material. However, this technology carries with it the possibility of inducing genetic and/or epigenetic modifications widely known as somaclonal variation (Larkin and Scowcroft, 1981). Among mechanisms causing somaclonal variation are activation of transposable elements (McClintock, 1984; Hirochika et al., 1996; Ozeki et al., 1997), DNA hypomethylation (Jaligot et al., 2000), genome adaptation to different regulatory microenvironments (Bogani et al., 1996), and the presence of hot spots of DNA instability (Linacero et al., 2000). Phenotypic changes associated with genetic alterations were reported among tissue culture-derived plants of several species from Pelargonium, Ananas, Phalaenopsis, Arachis, Musa, and Saintpaulia genera (Cassels et al., 1997; Das and Bhowmik, 1997; Chen et al., 1998; Eapen et al., 1998; Grajal-Martı´n et al., 1998; Paek and Hahn, 1999). Identifying variants during the early stages of plant development is essential to avoid propagation of mutant plants in species with extensive growing periods, like forest and fruit trees (Rival et al., 1998; Jaligot et al., 2000). The complexity of somaclonal variation requires the use of several approaches so that plants can be correctly evaluated. Use of molecular markers alone to assess genetic stability in vitro is

*Author to whom correspondence should be addressed. Email [email protected]

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the world (Pennington et al., 1981; Mabberley, 1984). This species has useful properties for timber production, as well as therapeutic and insecticidal application. Cultivar ‘Gigantea’, which shows vigorous growth, is planted on eroded lands of Argentina to produce valuable furniture wood (Mangieri, 1979; Cozzo, 1994). Because of the high phenotypic diversity observed in plants grown from seeds, attempts have been made to micropropagate selected genotypes using axillary buds as explants (Domecq, 1988; Ahmad et al., 1990; Thakur et al., 1998). These reports demonstrated the promise of axillary bud enhancement and shoot proliferation for commercial multiplication. Consequently, this enterprise has been carried on for several years in Argentina. The advantage of this system is the relative ease of establishing explants, especially when they are taken from rejuvenated trees. High proliferation rates of multiple shoots are obtained by the inclusion of 6-benzylaminopurine (BA) as a cytokinin in the culture media. In addition, this procedure allows a high percentage of plants to be recovered after rooting and acclimatization. Growth regulators are important components of the tissue culture process, being critical for growth and genome stability. For instance, Thakur et al. (1998) reported the positive effect of BA on the leaf development and shoot elongation of M. azedarach, whereas callogenesis was minimized by using 0.22– 2.22 mM BA. Reports of somaclonal variation in woody plants were well reviewed by Ahuja (1998). The need for using standardized evaluation protocols and assessing organs such as meristems, which undergo direct differentiation with minimal callus formation, to minimize the risk of somaclonal variation in clonal propagation, was emphasized. Hence, the histological nature of the explants and the regeneration system through which they are obtained become essential to deploying clonal propagation. However, previous studies reporting the micropropagation of Melia have not described the nature of the multiple shoots regenerated by using buds as a primary source of explants. On the other hand, long-term cultured plants of Melia obtained using the technique developed by Domecq (1988) often produce phenotypically altered plants displaying chlorosis, fasciation and hyperhydricity, which can be associated with in vitro cultureinduced somaclonal variation. The study of clonal fidelity of such materials at a genetic level would provide useful information for clonal propagation of this species. The aim of this work was to assess the genetic stability of micropropagated clones of Melia azedarach L. ‘Gigantea’ obtained through several rounds of in vitro culture using RAPD fingerprinting and chromosome analysis. Materials and Methods Plant material. In order to reproduce the micropropagation system currently used for commercial purposes, a sequential series of subcultures from elite seed-derived materials was initiated. This elite population was prepared by selecting plant genotypes based on their desirable agronomic performance, obtained from a breeding orchard at Puerto Victoria, Misiones, Argentina. So far, this population has been used as a primary source for commercial clonal and seed-derived plantations. The contribution of genome-dependent variation to the total variation was studied by selecting four genotypes from a bulk of approximately 300 fruits as explant donors. Seeds were germinated under greenhouse conditions. Shoot tips from 6 mo.-old-plants were taken and surface-disinfested by immersion in 70% ethanol for 3 min, then 20% NaOCl solution (2% active Cl) with two drops of Tween 20w for 20 min, followed by three rinses with

sterile distilled water. Meristems were excised and established in vitro according to Domecq (1988). Culture conditions. At least five meristems were established in a 400-ml flask containing 40 ml of MS (Murashige and Skoog, 1962) medium supplemented with sucrose (3%), 2.22 mM BA, 0.29 mM gibberellic acid (GA3), 0.25 mM indole-3-butyric acid (IBA), and 0.7% agar (Sigma, St. Louis, MO). Medium pH was adjusted to 5.8 prior to autoclaving at 1218C and 124 kPa for 20 min. For shoot multiplication, five nodal segments with four buds were cultured on MS medium supplemented with 2.22 mM BA and routinely subcultured every 5 wk. All fully expanded leaves were excised at each subculture. After five subcultures, the composition of the multiplication media was modified due to a high incidence of hyperhydricity and callogenesis at the shoot base. As a result, the BA concentration was reduced to 0.44 mM and IBA was omitted. All the material was cultured at 258C with 14 h light (150 mmol m22 s21). For each genotype, 11 subcultures were conducted from the primary regenerants (R-1) and the clonally propagated subsequent generations were designated as proposed by Ahuja (1998): R-2, R-3, R-4, R-5, R-6, R-7, R-8, R-9, R-10, and R-11. In most cases, at least three regenerating plants were chosen at random within flasks containing plants belonging to the same subculture generation. Shoots were rooted on MS medium supplemented with 9.84 mM IBA for 2 d, followed by subculture on basal MS medium. After 30 d of culturing, 101 of these plants were selected, harvested, and analyzed. DNA extraction and RAPD analysis. Genomic DNA was isolated from 0.1 g of frozen leaf tissue using the DNeasy Plant Mini Kit purchased from Qiagen (Hilden, Germany). DNA concentration was measured with a spectrophotometer (Shimadzu UV-2100, Tokyo, Japan). Ten 10-base oligonucleotide primers (series P and G OPP-01, 02, 03, 04, 06; OPG-02, 03, 04, 08, 10) from Operon Technologies (Alameda, USA) were used for PCR amplification. The PCR mix comprised 50 ng template DNA, 0.4 unit of Taq polymerase Bluew (5 U ml21) (Promega, Madison, USA) in 1 £ reaction buffer (20 mM Tris–HCl pH 8.0, 100 mM KCl, 0.1 mM EDTA, 1 mM DL dithiothreitol, 50% glycerol, 0.5% Tween 20w, and 0.5% Nonidet-P40w), 0.2 mM of primer, 100 mM of each dNTP (wPromega), and 2 mM MgCl2 in a total of 25 ml. The reaction mixtures were overlaid with 20 ml of mineral oil and amplified in a 9600 Perkin Elmer Cetus thermocycler (Perkin Elmer, Shelton, USA) programmed for 1 cycle of 5 min at 948C; 40 cycles of 1 min at 948C, 1 min at 368C and 2 min at 728C; 1 cycle at 728C for 7 min. The PCR products were subjected to electrophoresis in a 1.5% (w/v) Tris– acetate–EDTA agarose gel and DNA bands were analyzed by staining with ethidium bromide. Only the fragments that amplified consistently and were reproducible in a minimum of two replicated reactions were considered. A negative control reaction containing water in place of the genomic DNA was included each time for the all reactions. The presence/absence of consistent bands was recorded for preparing the initial data matrices. Cytological techniques. A total of 58 regenerated plants taken at random from subcultures R-8 to R-11 (8 –11 mo. of culturing) were used for the cytological study. Root tips were pretreated for 3 h at room temperature with 0.02% colchicine, fixed overnight in Farmer’s solution, ethanol/glacial acetic acid 3:1 (v/v), and stored in 70% ethanol at 48C. Hydrolysis was performed in 1 N HCl for 15 min at 608C, followed by washing with distilled water and staining with Feulgen. Chromosome numbers were determined using light microscopy and oil immersion at a magnification of 2200 £ . A minimum of three metaphase plates were scored for every root examined. Data analysis. The genetic diversity of regenerating plants was estimated using the Shannon–Weaver index H (Jain et al., 1980) defined as: n X k X ½pij log2 ðpij Þ=n H¼2 i¼1 j¼1

where H denotes the diversity associated with a given primer in a given population; pij are the frequencies of i markers of the jth primer; n is the number of RAPD markers generated by the jth primer; and k is the number of primers tested. The H standardized index varies between 0 (monomorphism) and 1 (highest polymorphism). Total diversity (HT) for each primer was calculated from genotypic frequencies taken from the whole collection tested. Clone diversity (HC) was calculated averaging H over all subcultures for each single clone, and the subculture diversity (HS) was obtained from the average diversity among all clones. Partitioning the diversity observed for each primer within and between subcultures and clones enabled us to compare the level of diversity detected

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by different primers. HS/HT is the component of diversity within subcultures, (HC –HS)/HT is the component among subcultures (within clones), and (HT–HC)/HT is the component among clones. Data sets were subjected to principal coordinated analysis using the NTSYS-PC software (Rohlf, 1998). Jaccard similarity coefficients were estimated by the similarity for qualitative data (SIMQUAL) function and aligned using the double center (DCENTER) option. Eigenvectors (EIGEN) were calculated from the transformed matrix using the square root scaling (SQRT-LAMDA) option, and finally a two-dimensional model was displayed.

Results and Discussion The 10 primers used in this study detected a total of 46 polymorphic bands shared in all the clones analyzed. The total diversity obtained by using these primers ranged from 0.62 to 0.89 with a mean value of 0.79 indicating a high level of variability. When that index was used to compare diversity within and among subcultures and clones, 50% of the diversity was detected among plants coming from the same subculture. The following 36% was attributed to diversity among subcultures (within clones), while the remaining 13% accounted for differences between genotypes selected as explant donors. In this study, all the regenerated plants showed at least one RAPD polymorphism. These results showed a high level of variation among micropropagated plants, which was higher than among the population used as explant donors. This suggests that other mechanisms, such as chimerism, contribute to variation. Chimeras are non-grafted plants with tissues representing two or more genotypes. There are reports showing preexisting variation present in tissue-derived explants related to chimerism (Cassells, 1998; Pe´rez Ponce et al., 1998). Genome-dependent somaclonal variation is particularly associated with polysomatic tissues (Cassells, 1998). The stability of chimeras is directly related to the plant regeneration system. Thus, plant regeneration, whether from a cell or a group of cells, will affect the chimeric constitution of the regenerated plants. In periclinal chimeras, at least one entire apical layer in the shoot apical meristem is genetically different from the other layers. Hence, periclinal chimeras can be maintained by propagating stem cuttings that possess axillary meristems, since axillary meristems almost always ‘inherit’ the same apical composition as the terminal apex from which they arose. Instead, adventitious shoots can result in the dissociation of chimeras into their component genotypes because these shoots are frequently derived either from single cells or from a group of genetically identical cells within the chimera (Tian and Marcotrigiano, 1993). In this manner, diplontic selection would operate through subculturing to obtain solid, non-chimeric mutants from adventitious buds. The high percentage of shoots regenerated by the protocol described here suggests that shoot production is a result of both axillary and adventitious shoot development. A principal coordinate analysis was used to express the association among regenerants in two dimensions. By this method, variation in regenerated plants seems to have occurred at random, resulting in a pattern in which regenerated plants were surrounding the control (donor plant) in all the clones analyzed (Fig. 1). Currently it is assumed that with long-term cultures more genetic and epigenetic changes are expected as a consequence of cumulative mutational events (Bogani et al., 1996). However, no comparative studies have been done to detect such changes to calculate variation. Although a few reports mention frequencies of RAPD polymorphisms, such as 0.05% in sugar beet (Munthali et al.,

FIG . 1. Principal coordinate analysis of regenerated plants of Melia azedarach L. derived from clone 1. The donor plant (C) showed similarity with regenerated plants originated from R-10 (10 mo. of culture) as well as from R-3 and R-4 (3 and 4 mo. in culture, respectively). Axes represent principal coordinates 1 and 2 accounting for the variance given by the corresponding eigenvectors.

FIG . 2. Values of diversity obtained from regenerated plants of four different clones of Melia azedarach L. by using subsequent subculturing (Rsuffix represents number of monthly subcultures). Bars indicate SE of the mean.

1996) and 0.63% in garlic (Al-Zahim et al., 1999), no studies differentiating the distribution of this diversity have been done. The principal coordinate analysis performed in this study provides a descriptive model, which demonstrates that genetic changes can be produced at any time during in vitro culture. A clear trend of variation with the time in culture was not observed, providing evidence against the hypothesis that variations accumulate by increasing time of culture (Fig. 2). On the other hand, decreased variability is expected as clonal multiplication cycles progress due to diplontic selection forces (Pe´rez Ponce et al., 1998). This process and mutational events induced by somaclonal variation would consequently interact in the opposite direction as multiplication cycles increase, contributing to the final phenotype constitution of the regenerated plants analyzed. The total diversity obtained for each clone of M. azedarach L. ‘Gigantea’ was partitioned to assess diversity distribution among regenerated plants according to the primers tested. The sets of primers used produce different total diversity indices. The highest

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PARTITIONING OF TOTAL DIVERSITY (1) WITHIN SUBCULTURES, (2) AMONG SUBCULTURES (WITHIN CLONES), AND (3) AMONG CLONES, ACCORDING TO THE PRIMER TESTED ON REGENERATED PLANTS OF MELIA AZEDARACH L Primers

HTa

HCb

HSc

HS/HT (1)

(HC-HS)/HT (2)

(HT-HC)/HT (3)

OPG-02 OPP-01 OPP-02 OPG-04 OPG-03 OPP-03 OPP-06 OPG-08 OPG-10 OPP-04 Mean

0.89 0.88 0.88 0.86 0.86 0.81 0.75 0.68 0.66 0.62 0.79

0.79 0.80 0.79 0.74 0.73 0.74 0.65 0.61 0.47 0.51 0.68

0.42 0.50 0.50 0.42 0.39 0.42 0.44 0.33 0.27 0.28 0.40

47.03 56.21 56.59 48.90 45.76 51.74 58.90 48.69 40.91 45.68 50.32

41.91 33.98 33.62 37.46 38.93 39.67 27.62 41.12 29.77 36.84 36.21

11.06 9.81 9.79 13.64 15.31 8.59 13.49 10.19 29.33 17.48 13.47

a

Total diversity indices. Clone diversity. c Subculture diversity. b

FIG . 3. Values of diversity obtained from regenerated plants of Melia azedarach L. according to the clone origin and primer tested.

indices were obtained with primers OPG-02, OPP-01, OPG-04 and OPG-03, and the lowest with OPP-04 and OPG-10 (Table 1). The partitioning of this diversity among clones also showed a differential pattern of variation according to the origin of the clone. The primer OPG-10 detected less variation in all clones, suggesting that its target genomic site could be less affected by changes during micropropagation (Fig. 3). On the other hand, primers OPP-03, OPG-04, OPG-02, OPP-02, and OPP-01 detected the highest level of variation in all clones tested. These results support the idea of hot spots of instability, being genotypically and environmentally dependent in a similar way as was shown by Linacero et al. (2000). Changes in cytosine residues as a consequence of hypermethylation (Kovarik et al., 1997) or hypomethylation processes (Jaligot et al., 2000) may have indirectly contributed to RAPD polymorphism by shifting the primer target region. The activation of retrotransposons could also produce a similar effect (Hirochika et al., 1996). Diversity values were affected by the genotype of the donor plant involved. The lowest mean values were obtained in the generation R-10 ð0:28 ^ 0:08Þ and R-2 ð0:31 ^ 0:06Þ; while the highest mean value ð0:47 ^ 0:03Þ was obtained in an intermediate generation

FIG . 4. Metaphase plate observed in a regenerated plant obtained from subculture R-9 ðbar ¼ 3 mMÞ:

(Fig. 2). The genetic stability of each clone was assessed, comparing the mean values of diversity among clones, with clones 10 and 12 being the most variable and clone 4 the most stable (Fig. 2). Cytological analysis of donor plants corroborated the 2n ¼ 28 chromosomes reported for M. azedarach (Styles and Vosa, 1971; Khosla and Styles, 1975; Datta and Samanta, 1977). Root tips of 58 regenerated plants showed the expected chromosome number (Fig. 4), inhibiting our ability to establish a correlation between chromosomal stability and high degree of RAPD polymorphism exhibited among regenerated plants. This was also observed in the previous study of Al-Zahim et al. (1999) in somaclonal variants of garlic. Point mutations do not necessarily lead to structural mutations. Our results demonstrate the relevance of testing in vitro culture procedures for variation before applying them for commercial purposes. The most polymorphic primers tested in this study could be useful tools for detection of genomic changes in the early stages of the large-scale micropropagation of M. azedarach. Because woody plants exhibit a long generative cycle with an extended vegetative phase, they are constantly exposed to changing environments over long periods of time, which may influence their developmental performance. Further studies are needed to test the stability of RAPD polymorphism through plant life cycles. Additional investigations showing associations among such polymorphisms detected in this study and the agronomic value of commercial clones might provide a useful tool for selecting true-types of in vitroderived plants at early stages of development. Acknowledgment This research was supported by a grant from FOMEC-Universidad Nacional del Nordeste of Argentina.

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