In Vitro Cell.Dev.Biol.—Plant DOI 10.1007/s11627-011-9342-y
REPORT
Morphological and physiological characterization of two new pineapple somaclones derived from in vitro culture Guillermo Pérez & Andrew Mbogholi & Fernando Sagarra & Carlos Aragón & Justo González & Miriam Isidrón & José Carlos Lorenzo
Received: 2 June 2010 / Accepted: 4 January 2011 / Editor: D. T. Tomes # The Society for In Vitro Biology 2011
Abstract We previously reported a partial agricultural and amplified fragment length polymorphism (AFLP) characterization of two new pineapple somaclones (P3R5 and Dwarf) derived from in vitro culture of the donor cv. Red Spanish Pinar. Both somaclonal variants showed different AFLP banding patterns compared to the donor cultivar, although they were separated by less than 0.09 U of genetic distance. The present report shows data of various indicators of morphology and physiology of P3R5 and Dwarf D leaves. The stoma diameter, number of stomata per square millimiter, diameter of leaf vascular tissue, thickness of the leaf aquiferous parenchyma, and thickness of the leaf photosynthetic parenchyma were measured. The photosynthetic rate, the transpiration rate, the water use efficiency, the internal leaf CO2 concentration, and the chlorophyll pigment contents were recorded as well. Between the somaclonal variant P3R5 and the donor genotype, statistically significant differences were recorded in all indicators with the exception of the stoma diameter and the photosynthetic rate. Comparing the somaclonal variant Dwarf and the cv. Red Spanish Pinar (donor), statistically significant differences were also recorded in all parameters except in the stoma diameter and in the transpiration rate. This investigation was performed to demonstrate that small changes in the pineapple DNA may result in relevant phenotypic modifications. G. Pérez (*) : A. Mbogholi : F. Sagarra : C. Aragón : J. González : M. Isidrón : J. C. Lorenzo Laboratory for Plant Breeding, Centro de Bioplantas, Universidad de Ciego de Ávila, Ciego de Ávila 69450, Cuba e-mail:
[email protected] URL: www.bioplantas.cu
Keywords Ananas comosus (L.) Merr. . Phenotype variation . Leaf morphology . Physiology
Introduction Pineapple belongs to the Bromeliaceae family and is one of the most economically important tropical fruits. The worldwide production in 2008 was 19.16 million tons (Food and Agriculture Organization of the United Nations (FAOSTAT) 2010). Because of this, several research groups are developing basic and applied studies to create new varieties with better agronomic performance. As classical pineapple breeding is extremely laborious and time consuming (Cabot and Lacoevilhe 1990), biotechnology is an attractive tool for improving elite clones (Sripaoraya et al. 2001; Espinosa et al. 2002; Botella and Fairbairn 2005; Sripaoraya et al. 2006; Carlier et al. 2007; Yabor et al. 2009). Genetic variation is very important in crop improvement and forms the basis of development of new varieties. Somaclonal variation is a valuable tool in plant breeding wherein variation in tissue culture regenerated plants from somatic cells can be used in the development of crops with novel traits (Cardoza and Stewart 2004). Larkin and Scowcroft (1981) were the first researchers to demonstrate and coin the term somaclonal variation. Variations may preexist in the natural population of plants from field collection or genebank or it may develop de novo as a result of tissue culture conditions (Castillo et al. 2010). In recent years, a number of studies have measured, through molecular markers, the extent of somaclonal variation in plants (Aversano et al. 2009). Lack of polymorphisms associated with in vitro regeneration was reported in tomato (Smulders et al. 1995), Norway spruce
PÉREZ ET AL.
(Fourré et al. 1997), oil palm (Rival et al. 1998), begonia (Bouman and De Klerk 2001), almond (Martins et al. 2004), and potato (Barandalla et al. 2006; Sharma et al. 2007) using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) markers. By contrast, major differences were found in alfalfa (Piccioni et al. 1997), in Codonopsis lanceolata (Guo et al. 2006), and in wild pear (Palombi et al. 2007) using RAPD and ISSR markers. Pineapple somaclonal variations have been previously characterized by Wakasa (1977, 1989), Dewald et al. (1988), Lii et al. (1989), and Feuser et al. (2003). However, all these studies showed only a few characteristics, and the studies were not carried out in detail. The number of leaves per plant, number of thorns per leaf, and leaf color were reported. We previously published a partial agricultural and AFLP characterization of two new pineapple somaclones derived from in vitro culture of cv. Red Spanish Pinar (Pérez et al. 2009). The differences shown by the variant P3R5 were number of slips and suckers, number of thorns in leaves and in fruit crowns. The somaclonal variant Dwarf and the donor plant were antithetic in regard to plant height; peduncle diameter; number of shoots, slips, and suckers; fruit mass with crown; number of eyes in fruit; fruit height and diameter; leaf color; plant architecture; length of plant generation cycle; and fruit color and shape. Both somaclonal variants showed different AFLP banding patterns in comparison with the donor cultivar, though separated by less than 0.09 U of genetic distance. In this study, we show a detailed phenotypic and physiological analysis of two somaclonal variants that are currently evaluated as improved genotypes in our institute.
Materials and Methods Pineapple D leaves (Py et al. 1987) of Red Spanish Pinar (donor), P3R5, and Dwarf were collected from the Experimental Field Station at the Bioplant Centre Ciego de Avila, Cuba. The plants were grown for 6 mo in the field after being planted in a random block design; ten plants per genotype were studied (one leaf per plant). According to Johansen (1940), the stoma diameter, number of stomata per square millimeter, diameter of leaf vascular tissue, thickness of the leaf aquiferous parenchyma, and thickness of the leaf photosynthetic parenchyma were measured. The photosynthetic rate, the transpiration rate, the water use efficiency, and the internal leaf CO2 concentration were recorded using a Portable CIRAS-2 Photosynthesis System (Europe, PP Systems, Norfolk, UK)—covering with
the leaf, the whole area of the cuvette (PLC6, 2.5 cm2). The carbon dioxide concentration and the relative humidity of the air entering the cuvette were 375 μmol mol−1 and 80% respectively, under environmental temperature (25–27°C). Prior to obtaining the experimental data, we measured the maximum light intensity at which photosynthesis was stable which was attained at 600 μmol m−2 s−1. To determine the levels of chlorophyll pigments (a, b, total), leaves were thinly grounded in liquid nitrogen. Evaluations were made as recommended by Porra (2002). Extraction was carried out with 5.0 ml acetone (80%, v/v). Samples were centrifuged (12 100 g, 4°C, 15 min), supernatants were collected, and absorbances at 647 and 664 nm were recorded. The Statistical Package for Social Sciences (Version 8.0 for Windows, SPSS Inc., New York, NY) was used to perform one-way ANOVA and Tukey HSD tests (p=0.05).
Results and Discussion The comparison of the somaclonal variant P3R5 and the donor genotype in Table 1 shows statistically significant differences in all indicators apart from the stoma diameter and the photosynthetic rate. Additionally, the differences between the somaclonal variant Dwarf and the cv. Red Spanish Pinar (donor) were also evident in all parameters, excluding the stoma diameter and in the transpiration rate. Compared to the donor plant, P3R5 somaclonal variant showed significant decreases in several aspects but mainly in the transpiration rate that only reached 28% of the rate in the donor (11.5 mmol H 2 O m − 2 s − 1 /41.6 mmol H2O m−2 s−1). Moreover, thickness of the leaf photosynthetic parenchyma in P3R5 merely represented 58% (33.6/ 57.6 μm). Significant increases were also recorded in P3R5 in comparison with cv. Red Spanish Pinar. The donor showed 39% of the water use efficiency evaluated in P3R5 (0.7 mmol CO2 mol−1 H2O/1.8 mmol CO2 mol−1 H2O), 60% of the internal leaf CO2 concentration (222.8 μmol CO2 mol−1/373.7 μmol CO2 mol−1), and about 49% of chlorophyll pigments contents (Table 1). Changes in the above-mentioned physiological indicators have been studied frequently when plants have been submitted to different sources of stress. Price et al. (2002) recorded the optimization of CO2 gain through stomatal aperture while minimizing water loss in rice. GarcíaSánchez et al. (2007) evaluated the effects of flooding and drought stress on citrus seedlings physiology. QingMing et al. (2008) measured the response of cucumber seedlings to drought stress. However, as far as we know, the effects of somaclonal variation on plant physiology have not been studied deeply. Further studies are required to
MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERIZATION Table 1. Morphological and physiological characterization of D leaves of two new pineapple somaclones Plant materials Cv. Red Spanish Pinar (donor)
Somaclonal variants P3R5
Dwarf
Stoma diameter (μm) * Number of stomata per mm2 *
28.2 ab 110.1 a
24.0 b 99.3 b
30.1 a 84.0 c
Diameter of leaf vascular tissue (μm) * Thickness of the leaf aquiferous parenchyma (μm) * Thickness of the leaf photosynthetic parenchyma (μm) * Photosynthetic rate (μmol CO2 m−2 s−1) * Transpiration rate (mmol H2O m−2 s−1) * Water use efficiency (mmol CO2 mol−1 H2O) * Internal leaf CO2 concentration (μmol CO2 mol−1) * Total chlorophyll concentration (mg g−1 fresh weight) * Chlorophyll a concentration (μg g−1 fresh weight) * Chlorophyll b concentration (μg g−1 fresh weight) *
38.1 a 119.1 a 57.6 a 20.2 b 41.6 a 0.7 b 222.8 c 17.8 b 10.7 b 6.5 b
32.1 b 86.4 b 33.6 b 19.0 b 11.5 b 1.8 a 373.7 a 35.5 a 21.3 a 14.2 a
17.2 c 43.8 c 19.9 c 21.7 a 45.7 a 0.5 c 253.4 b 11.7 c 7.7 c 4.0c
*p>0.05, results with the same letter are not statistically different (one-way ANOVA, Tukey HSD)
elucidate the mechanisms that explain the increases and decreases observed in P3R5 somaclonal variant. Dwarf somaclonal variant showed significant decreases compared to the donor plant material: 45% of the diameter of the leaf vascular tissue (17.2/38.1 μm), 37% of the thickness of the leaf aquiferous parenchyma (43.8/119.1 μm), and 35% of the thickness of the leaf photosynthetic parenchyma (19.9/57.6 μm) (Table 1). We previewed these results because Dwarf showed a statistically significant decrease in plant height in comparison with the donor. Dwarf plant architecture also changed from lightly wide (donor) to compact (Pérez et al. 2009). We observed that, according to AFLP analysis, P3R5 was separated from the donor cv. Red Spanish Pinar by only 0.05 U of genetic distance and Dwarf by 0.08 U (Pérez et al. 2009). While antecedently stated that these genetic distances are not too remarkable, Table 1 shows the contrary—that they are sufficient to cause a significant change in D leaf morphology and physiology. P3R5 and Dwarf variants also exhibited a number of differences at the phenotypic level in comparison with the donor (Table 2). We have used 32 indicators based on a wide range of horticultural and physiological traits. These data clearly show the various aspects where somaclonal variation can occur in pineapple. P3R5 differed from the donor in 14 variables (14/32; 43.7%) while Dwarf in 24 indicators (24/32; 75.0%). These figures support the impressive phenotypic effects of small genetic modifications caused by in vitro culture.
Jain (2001) and Li et al. (2010) summarized that 22 cultivars had been released from somaclonal variation with improved traits, including yield; plant architecture; color; pest resistance; salt, heat, and freezing tolerance. Examples are “He Zu No. 8” wheat (Triticum aestivum L.) with high yield, “Yidan No. 6” maize (Zea mays L.) with improved grain quality, “CIMAP/bio-13” aromatic grass (Cymbopogon winterianus Jowitt) with increased oil yield and “DAMA” rice (Oryza sativa L.) with Picularia spp. resistance. However, considering that pineapple culture through in vitro-derived plants have been in practice for a long time (more than 20 yr in Cuba), we have only these two (P3R5 and Dwarf) variants that are stable. Thus, somaclonal variation in this crop should be considered a rare event. Chen et al. (2006) studied several Syngonium podophylum somaclonal variants within which small genetic differences and remarkable phenotype modifications were also observed. Similar results were recorded by Prado et al. (2007) in Actinidia deliciosa somaclonal variants. Somaclonal variation has been associated with changes in chromosome number and structure, point mutations, DNA methylation (Brown et al. 1993), transposon activation, deletion, genome rearrangement, polyploidy, or nucleotide substitution (Bhatia et al. 2005). However, not much has been published about the effects of somaclonal variation at morphological and physiological levels. At this point of our investigation, it is difficult to say which genes are involved in the morphological and physiological changes that were observed in this study.
Decreased with respect to the donor plant material
Increased with respect to the donor plant material
Other modifications with respect to the donor plant material
16 Indicators: plant height, peduncle 2 Indicators: presence of thorns in 2 Indicators: number of diameter, number of shoots, fruit leaves (*), presence of thorns in slips, number of suckers mass with crown, number of eyes fruit crowns (*) in the fruit, fruit height, fruit diameter, number of crowns in the fruit, fruit content of vitamin C, fruit acidity, plant generation cycle, leaf color (*), plant architecture (*), shape of fruit eyes (*), fruit color (*), fruit shape (*) 2 Indicators: stoma diameter, 5 Indicators: number of stomata 5 Indicators: water use efficiency, photosynthetic rate per square millimeter, diameter internal leaf CO2 concentration, total chlorophyll concentration, of leaf vascular tissue, thickness of the leaf aquiferous parenchyma, chlorophyll a concentration, thickness of the leaf photosynthetic chlorophyll b concentration parenchyma, transpiration rate 6 Indicators: number of crowns in the 7 Indicators: plant height, peduncle 3 Indicators: number of shoots, 4 Indicators: leaf color from fruit, fruit content of vitamin C, fruit diameter, fruit mass with crown, number of slips, number of greenish with red zones to acidity, presence of thorns in leaves number of eyes in the fruit, fruit suckers greenish (*), plant architecture (*), shape of fruit eyes (*), presence height, fruit diameter, plant from lightly wide to compact (*), of thorns in fruit crowns (*) generation cycle fruit color from red-orange to yellow-green (*), fruit shape from tonel to cylindrical block (*) 2 Indicators: stoma diameter, 8 Indicators: number of stomata 2 Indicators: photosynthetic rate, transpiration rate per square millimeter, diameter internal leaf CO2 concentration of leaf vascular tissue, thickness of the leaf aquiferous parenchyma, thickness of the leaf photosynthetic parenchyma, water use efficiency, total chlorophyll concentration, chlorophyll a concentration, chlorophyll b concentration
Not modified with respect to the donor plant material
Phenotype indicators
Classification supported by one-way ANOVA and Tukey HSD (p=0.05) but asterisks indicate qualitative analysis
Dwarf
Dwarf
P3R5
P3R5
Somaclonal variants
Table 2. Summary of phenotypic modifications of P3R5 and Dwarf somaclonal variants
Table 1
Pérez et al. (2009)
Table 1
Pérez et al. (2009)
Source of information
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MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERIZATION
Molecular studies are in progress in our laboratory in order to clarify the effects of these somaclonal variations. Acknowledgements This research was supported by the Cuban Ministry for Science, Technology, and the Environment. We are grateful to Julia Martínez, Alitza Iglesias, and Mayda Arzola for their excellent technical assistance. We thank Taletha Laudat for the professional language editing.
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