New Pineapple Somaclonal Variants : P 3 R 5 and Dwarf

The Food and Agriculture Organization has highlighted pineapple as one of the most important tropical fruits. Since classical pineapple breeding is difficult, biotechnology has emerged as an attractive instrument. We obtained two new pineapple somaclonal variants derived from in vitro culture of cv. Red Spanish Pinar: P3R5 and Dwarf. The AFLP analysis revealed an existing genetic distance. So far 44 phenotype indicators selected due to their relation to a wide range of important agricultural, morphological and physiological processes have been evaluated. P3R5 differed from the donor in 19 variables (19/44; 43.18%), while Dwarf varied in 31 indicators (31/44; 70.45%). The number of shoots was significantly different among the three plant materials. Dwarf showed two shoots per plant while P3R5 and the donor did not form any shoots. We also observed that water use efficiency, chlorophyll b concentration, total chlorophyll concentration, transpiration rate, chlorophyll a concentration, thickness of leaf photosynthetic parenchyma, fruit mass with crown, content of free phenolics and superoxide dismutase specific activity were also very different among the three plant materials. The Euclidean distances of each somaclonal variant to the donor plant material taking into consideration the genotype (AFLP) and the phenotype evaluations were also calculated. Regarding the genotype information, P3R5 is separated from cv. Red Spanish Pinar by 2.83 units of Euclidean distance, and Dwarf by 3.00 units. However, the phenotype indicators revealed higher differences: 3.74 in P3R5 and 4.71 in Dwarf. To our knowledge, this is the first report of a comprehensive analysis of pineapple somaclonal variants.


Introduction
Pineapple belongs to the Bromeliaceae family and is one of the most economically important tropical fruit.The interest for its production is due to the high cost that reaches in the fresh fruit and industrialized markets, its great food value, its pleasant taste and its beauty for commercialization.The worldwide production in 2008 was 19.16 million tons [1].Because of this, several research groups are developing basic and applied studies to create new varieties with better agronomic performance.Pineapple breeding using naturally occurring genetic variation and conventional methods has succeeded in several countries.
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 [24].Larkin and Scowcroft [25] were the first researchers to demonstrate and coin the term somaclonal variation.Variations may pre-exist in the natural population of plants from field collection or genebank or it may develop as a result of tissue culture conditions [26].
Pineapple somaclonal variations have been previously characterized [38][39][40][41][42].However, all these studies showed only few characters and were not studied in detail as only the number of leaves per plant; the number of thorns per leaf and leaf color were reported.
The present study culturing in vitro pineapple axillary buds with naphthalene acetic acid and 6-benziladenine for micropropagation; kinetin to induce callus formation; and indole-3-butyric acid and gibberellic acid for plant regeneration, was carried out to check possible genetic alterations in the plants produced.These growth regulators were the only putative mutagenic agents we used.
We obtained two new pineapple somaclonal variants derived from in vitro culture of cv.Red Spanish Pinar [43,44].This paper shows a broad genotypic and phenotypic analysis of P3R5 and Dwarf somaclonal variants.

Plant Material, Media and Culture Conditions
Fifty pineapple buds (cv.Red Spanish Pinar, donor) were collected from field-grown plants and cultured following the protocol described by Daquinta and Benegas [45].

Multiplication Culture Medium
Shoots were transferred to the multiplication culture medium (as described above except: 9.3 μM 6-benziladenine and 1.6 μM naphthalene-acetic acid).They were subcultured at 42 d intervals for 168 d.Twenty-four thousand seven hundred and sixty-eight shoots were then obtained.

Hardening and Field Conditions
For ex vitro hardening, plantlets were placed in plastic trays containing 82 cm 3 of a mixture zeolite + filter cake (1:1).Microject automated irrigations for 25 s every 30 min were applied.Plantlets were kept under a photosynthetic photon flux density of 458 μmol•m -2 •s -1 .Standard phytosanitary controls were applied.After hardening of in vitro-plantlets, 387 plantlets were transferred to the field environment and asexually propagated for two generations (30 months).The donor cultivar was used as a control.Two phenotype variants were then identified: P3R5 and Dwarf.A more detailed study was carried out to compare these two variants with the donor plant (cv.Red Spanish Pinar, plant material never cultured in vitro).
The experiment was developed in the Field Experimental Station at the Bioplant Centre.A random block design was implemented (80 plants/clone).Field management was performed according to instructions recommended by the Cuban Ministry for Agriculture.The most important pineapple phenotypic traits were recorded during 18 months.The agricultural evaluations were made in field conditions.

Plant Material and DNA Extraction
In the second generation, in order to perform the AFLP characterization, young leaves of the donor genotype (cv.Red Spanish Pinar) and the two variants (P3R5 and Dwarf) were collected.Samples were stored at -20˚C until DNA extraction.Extraction started from 250 mg fresh mass that were finely grounded in liquid nitrogen.Extraction buffer (650 μl) was then added.It included: Tris-Cl (pH 7.5, 50 mmol•l -1 ), ethylene-diamine-tetraacetic acid (20 mmol•l -1 ), sodium chloride (0.3 mmol•l -1 ), sarcosil (2%), sodium dodecyl sulfate (0.5%) and urea (4.8 mmol•l -1 ).A mixture (650 μl) of phenol:chloroform: isoamyl alcohol (25:24:1, v:v:v) was added.Samples were centrifuged for 15 min at 12,000 rpm at room temperature.Supernatants were collected and the pellets, discarded.Isopropanol (0.8 volumes) was added.Samples were shortly shaken in a vortex and incubated for 10 min at room temperature.Samples were centrifuged for 10 min at 12,000 rpm.Supernatant was discarded and the pellet was washed with ethanol (70%).DNA was dried (vacuum) and dissolved in 50 μl water supplemented with 10 μg•ml -1 RNase A. DNA integrity and purity was checked by electrophoresis in agarose gels (0.8%) stained with ethidium bromide.Concentration was estimated visually by comparison with standards (100 -1000 ng•μl -1 ).Concentrations of DNA samples were adjusted to 500 ng•μl -1 .

AFLP Analysis
AFLP technique was carried out [48].The digestion of genome DNA, the pre-amplification with a selective base and the selective amplification was performed [47].
Autoradiographs were analyzed visually to build a dicotomic numerical matrix: the number one was assigned when the band was present while zero was assigned when absent.We disregarded all weak and low peak AFLP bands.The matrix was processed with the NTSYSpc software [49].The simple matching index was used to create a matrix of similarity.From this matrix, a matrix of genetic distance was obtained.The UPGMA (Unweighted Pair Group Method with Arithmetic Averages) method was used to generate a dendogram.

Morphological, Physiological and Biochemical Determination
In a subsequent procedure, the three plant materials were transferred to the Pineapple Germplasm Bank at the Bioplant Centre in a random block design.Plants grew for 6 months and then D leaves [50] of Red Spanish Pinar (donor), P3R5 and Dwarf were collected.Ten plants per genotype were studied (one leaf per plant).The stoma diameter, number of stomata per mm 2 , diameter of leaf vascular tissue, thickness of the leaf aquiferous parenchyma, and thickness of the leaf photosynthetic parenchyma were measured [51].The photosynthetic rate, the transpiration rate, the water use efficiency, and the internal leaf CO 2 concentration were recorded using a Portable CIRAS-2 Photosynthesis System (Europe, PP Systems, UK); covering with the leaf, the whole area of the cuvette (PLC6, 2.5 cm 2 ).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˚C -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 [52].Extraction was carried out with 5.0 ml acetone (80%, v:v).The samples were centrifuged (12,000 rpm, 4˚C, 15 min), supernatants collected and absorbances at 647 and 664 nm were recorded.

Data Analysis
The Statistical Package for Social Sciences (Version 8.0 for Windows, SPSS Inc.) was used to perform One-Way ANOVA and Tukey tests (P ≤ 0.05).The Euclidean distances of each somaclonal variant to the donor plant material were calculated.Data were standardized to vary from 0 to 1 [60].

Agricultural and AFLP Characterization
Only two plant materials were found to be "solid" somaclonal variants after studying three vegetative generations (P3R5 and Dwarf) and the AFLP analysis, which represents 0.52% (2 somaclonal variants/387 plants transferred to the field).The dendogram generated with the AFLP information revealed an existing genetic distance among the somaclonal variants and the donor plant [43].The genetic distances among the three plant materials are not too significant.However, as they have different banding patterns, they are different at the genetic level.
The agricultural characterization of the third vegetative generation in Tables 1(a)-(b) and Figure 1 shows that, in comparison with the donor plant (cv.Red Spanish Pinar), the variant P3R5 showed differences in the number of slips and suckers, and in the presence of thorns in the leaves and in the fruit crowns.The somaclonal variant Dwarf was different from the donor plant in regard to the plant height; the peduncle diameter; the number of shoots, slips and suckers; the fruit mass with crown; the number of eyes in the fruit; the fruit height and diameter; the leaf color; the plant architecture; the length of plant generation cycle; and the fruit color and shape (Table 1(a), agricultural characterization of the third vegetative generation).

Morphological, Physiological and Biochemical Characterization
The morphological, physiological and biochemical characterization of D leaves in Overall coefficient of variation = (Standard deviation/Average) × 100.To calculate this coefficient, average values of the donor plant material, P3R5 and Dwarf were considered.The higher differences among the three plant materials compared, the higher the overall coefficient of variation.

Plant materials
Overall coefficients of variation recorded in each phenotype indicator (%)  1).
Changes in the above mentioned morphological, physiological and biochemical indicators have been frequently studied when plants have been submitted to different sources of stress.Reference [61], recorded the optimization of CO 2 gain through stomatal aperture while minimizing water loss in rice.The effects of flooding and drought stress on citrus seedlings physiology were measured [62].The response of cucumber seedlings to drought stress also were measured [63].However, to our knowledge, the effects of somaclonal variation on plant physiology have not been deeply studied.Further studies are required to elucidate the mechanisms that explain the differences observed in P3R5 and Dwarf somaclonal variants.

General Variation: Overall Coefficients of Variation and Euclidean Distances
The overall coefficients of variation in Table 1 indicate that the number of shoots was significantly different among the three plant materials (173.21%).Dwarf showed two shoots per plant while P3R5 and the donor did not form any shoot.We classified the overall coefficients of variation of the other phenotype indicators in three categories: less than 23%, between 23 and 46%, and over 46%.Then we observed that water use efficiency, chlorophyll b concentration, total chlorophyll concentration, transpiration rate, chlorophyll a concentration, thickness of the leaf photosynthetic parenchyma, fruit mass with crown, free phenolics content and superoxide dismutase specific activity were also very different among the three plant materials.However, other aldehyde content, malondialdehyde content, content of cell wall-linked phenolics, protein content, phosphoenol pyruvate carboxylase activity, phosphoenol pyruvate carboxylase specific activity, total content of phenolics, number of stomata per mm 2 , stoma diameter, photosynthetic rate, plant generation cycle, fruit content of vitamin C, number of crowns in the fruit and fruit acidity showed low variability.
Table 2 summarizes the phenotypic changes of P3R5 and Dwarf somaclonal variants with respect to the donor plant material.We have used 44 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 19 variables (19/44; 43.18%), while Dwarf in 31 indicators (31/44; 70.45%;Table 2).
Figure 2 shows the Euclidean distances of each somaclonal variant to the donor plant material taking into consideration the genotype (AFLP) and the phenotype evaluations.Regarding the genotype information, P3R5 is separated from cv.Red Spanish Pinar by 2.83 units of Euclidean distance, and Dwarf by 3.00 units.However, the phenotype indicators revealed bigger differences: 3.74 in P3R5 and 4.71 in Dwarf.These figures support the impressive effects on phenotype of small genetic modifications caused by in vitro culture.Authors studied several Syngonium podophylum somaclonal variants within which small genetic differences and significant phenotype modifications were also observed [64].Similar results were recorded in Actinidia deliciosa somaclonal variants [65].
References [66,67] summarized that 22 cultivars had been released from somaclonal variation with improved traits including yield; plant architecture; colour; pest resistance; salt, heat and freezing tolerance.However, considering that pineapple culture through in vitro derived plants is in practice for a long time (over 20 years in Cuba), we have only these two (P3R5 and Dwarf) variants that are stable and thus somaclonal variation in this crop should be considered a rare event.
Somaclonal variation has been associated with changes in chromosome number and structure, point mutations, DNA methylation [68], transposon activation, deletion, genome rearrangement, polyploidy, or nucleotide substitution [69].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.This paper differs from our previous one [43] largely in the addition of a large amount of phenotypic data.The additional data make it less likely that AFLP comparisons of the wild-type line and the two mutants will lead to tagging of the genes for thorn production and dwarf stature.On the other hand, AFLP-based gene tagging will require sexual hybridization between the two mutants.Due to the well documented pineapple self-incompatibility, we do not expect that the mutants set seed after pollination.In Ananas comosus (L.) Merr., the self-incompatibility is brought about by inhibition of pollen tube growth in the upper third of the style [70,71].It is gametophytically controlled by a single locus with multiple alleles [72].The self-rejection reaction is variable in intensity and generally stronger in the cultivated varieties, which is probably a result of the domestication process and selection for seedless fruits [73].Therefore, other strategies are in progress in our laboratory such as the linkage disequilibrium-based association mapping according to [74] to identify the genes for thorn production and dwarf stature.Studies about the sterility ratios of the new somaclonal variants, as well as, possibilities for somatic cell fusion are also being carried out.

Conclusions
We report here the identification of an agriculturally seful mutant (P3R5), with less thorny leaves and there-u fore, easier to manage in the field.Another mutant with ornamental value (Dwarf) was also obtained.Taking into consideration the phenotype, somaclonal variant Dwarf is more different from the donor plant than P3R5.The AFLP characterization supports these phenotype differences at genome level [43,44].As far as we know, this is the first report of a comprehensive analysis of pineapple somaclonal variants.

Figure 1 .
Figure 1.Pineapple material of Red Spanish Pinar (Donor), P3R5 and Dwarf somaclonal variants.(a) Plants at 6 months of growth under controlled environment; (b) D leaves of 6 month-old plants grown under the field environment; (c) Fruits just harvested in the field.

Table 1
(b) shows that, in omparison with the donor plant (cv.Red Spanish c

Table 1 . Phenotypic characterization of pineapple somaclonal variants. (a) Agricultural characterization of the third vegeta- tive generation; (b) Morphological, physiological and biochemical characterization of D leaves.
**

** Phenotype indicator Cv. Red Spanish Pinar (Donor) P3R5 Dwarf
*Results with the same letter are not statistically different (One Way ANOVA, Tukey HSD, P > 0.05).

Table 2 . Summary of phenotypic modifications of P3R5 and Dwarf somaclonal variants. Classification supported by One Way ANOVA and Tukey HSD (P = 0.05) but asterisks indicate qualitative analysis (Table 1).
* ); Fruit color from red-orange to yellow-green ( * ); Fruit shape from tonel to cylindrical-block ( * )