Morphological effects of induced polyploidy in Dendrobium nobile Lindl. (Orchidaceae)

Efeitos morfológicos da poliplodia induzida em Dendrobium nobile Lindl. (Orchidaceae)

ARTICLE

Morphological effects of induced polyploidy in Dendrobium nobile Lindl. (Orchidaceae)

Efeitos morfológicos da poliplodia induzida em Dendrobium nobile Lindl. (Orchidaceae)

Mívia Rosa de Medeiros VichiatoI, ^*; Marcelo Vichiato^I; Moacir Pasqual^II; Filipe Almendagna Rodrigues^II; Daniel Melo de Castro^III

^IPrefeitura Municipal de Belo Horizonte, SecretariaMunicipal de Meio Ambiente, Avenida Afonso Pena, 4000, Cruzeiro, 30.130-009, Belo Horizonte, MG, Brazil

^IIUniversidade Federal de Lavras, Departamento de Agricultura, C. P. 3037, 37.200-000, Lavras, MG, Brazil

^IIIUniversidade Federal do Recôncavo da Bahia, Centro deCiências Agrárias, Ambientais e Biológicas, Rua Rui Barbosa, 710, Centro, 44.380-000, Cruz das Almas, BA, Brazil

ABSTRACT

In general, polyploidy in plants causes an increase in the size ofvegetative structures. This work aimed to compare diploid plants (2n = 2 x =38) and induced tetraploid plants (2n = 4 x = 76) of Dendrobium nobile Lindl. through the evaluation of themorphological characteristics of flowers, leaves and pseudobulbs. We evaluatedthe parameters height and diameter of pseudobulbs; width and length of leavesand flowers (sepals, petals and labella); and diameter of flowers. The inducedpolyploidization resulted in the increasing the number of internodes (19.9%)and floral pieces, with greater height of the flower (4.5%) and width of thelip (18.5%) decrease in the number of flowers per pseudobulb (40.9%) and thediameter of the pseudobulb (64.9%) and delayed flowering.

Key words: Orchid, chromosome doubling, colchicine, tetraploidy

RESUMO

A poliploidia nas plantas ocasiona, geralmente, um aumento em tamanho dasestruturas vegetativas. Este trabalho objetivou comparar plantas diploides(2n=2x=38) e tetraploides induzidos (2n=4x=76) de Dendrobium nobile Lindl., mediante avaliação das características morfológicas deflores, folhas e pseudobulbos. Foram avaliados a altura e o diâmetro dospseudobulbos e a largura e o comprimento das folhas e flores (comprimento desépalas, de pétalas, de labelo e diâmetro da flor). A poliploidização induzidaem Dendrobium nobile L. resultou no aumento do número de entrenós (19, 9%)e das peças florais, com maior altura da flor (4, 5%) e largura do labelo(18, 5%), na diminuição do número de flores por pseudobulbo (40, 85%) e dodiâmetro do pseudobulbo (64, 9%) e no retardo na floração.

Palavras-chave: Orquídea, duplicação cromossômica, colchicina, tetraploidia

INTRODUCTION

Dendrobium nobile Lindl. (olhode boneca), native herbaceous epiphyte of Southeast Asian of cultivationwidespread in scientific circles, is considered the most orchid produced andmarketed both in Brazil and in the world, occupying a prominent position in themarket potted plants (Vichiato et al. 2008, Vilela et al. 2010).

The considerable interest in the genus Dendrobium is due to its widegeographic distribution, growth in different habitats, cultivation and relativelysimple, which is mainly due to the large floristic value of their hybrids andsignificant volume of trade (Vichiato et al. 2007, Vichiato et al. 2008). Thevalue of D. nobile depends on the sales of specific visual patternstransferred by genetic inheritance, such as color, size and shape of theflower. Breeding programs for Dendrobium aim at obtaining plants for the marketwith desirable characteristics, for example, pseudobulbs with high vigor, upright flowers, a large number of flowers and variability of colors and shapesas well as an extended flowering season (Faria et al. 2009). In Brazil, however, there have been few studies on breeding orchids due to the long cycleof plants in the Dendrobium genus, which leads to an average floweringdevelopment of three to four years.

Polyploidy can produce desirable characteristics, which translate intoincreased floral parts, succulence degree, color intensification, durabilityand resistance of flowers. In the Dendrobium genus, autopolyploidyresults in increased floral pieces with larger sizes and widths of petalsand/or sepals as well as greater durability of flowering, greater fertility anda decreased number of flowers per pseudobulb (Chaicharoen and Aejew 1981, Chaicharoen 1995, McConnell and Kamemoto 1993, Ketsa et al. 2001, Dao-Long etal. 2012). Moreover, autopolyploidy results in a slow growth of the plants(Chaicharoen and Aejew 1981, Vichiato et al. 2007) as well as a greater widthand thickness of leaves (Chaicharoen and Aejew 1981, Dao-Long et al. 2012).Furthermore, there is a significant correlation between ploidy level and plantheight of Dendrobium (Dao-Long et al. 2012).

The aim of this study was to evaluate the morphological effects ofinduced polyploidy in Dendrobium nobile Lindl. through the comparison offlowers, leaves and pseudobulbs of diploid (2n = 2x = 38) and inducedtetraploid (2n = 4x = 76) plants.

MATERIAL AND METHODS

Local

The research was conducted in a greenhouse equipped with shading (providing 50%shading) by coverage with black polyethylene film (150 microns) in themunicipality of Igarapé (lat 20º 04' 13” S, long 44º 18' 06” W and alt 786 masl), Minas Gerais. The region has a humid subtropical climate (Köppen ClimateClassification-Geiger: Cwa) and is mountainous.

Genetic material

Diploid D.nobile plants (2n = 38 chromosomes – witnesses and the treatments did notoccur in polyploidization) and induced tetraploid plants (2n = 4x = 76chromosomes) derived from the work of Vichiato (2007) were used, and theseplants were cultivated in polyethylene pots (500 cm³) containingcoconut fiber substrate that were maintained on countertops.

Polyploidization

D.nobile diploid (2n= 38 chromosomes) plants with an average height of 5.0 cm and 3 leaves weretrimmed so that the remaining roots were 1.0-1.5 cm in length. The plants werecompletely immersed in an aqueous colchicine solution in 5 L plasticcontainers. Constant air bubbling achieved with domestic aquarium aeratingpumps was used to prevent plant damage caused by oxygen depletion. Tween-80(0.01%) was added to increase the colchicine efficiency (Vichiato et al. 2007).

The plastic containers were stored at room temperature and protected from light.After the colchicine treatment, the plants were washed thoroughly in runningwater for 20 minutes and in distilled water for 5 minutes to remove excesscolchicine (Vichiato et al. 2007). The plants were then properly identified andplanted in black polyethylene pots (volume of 500 cm³) containingfern fiber as substrate. The plants were transferred to the greenhouse and seton metal benches where they remained for seven months.

Irrigation was performed three times a week on average according to the conditions of the substratebeing moist and were fertilized bi-weekly with 2 mL of Biofert Plus® fertilizerper plant at a concentration of 5.0 mL L^-1 through foliar spray(Vichiato et al. 2007).

Thedetermination of ploidy level was performed by counting chromosomes sevenmonths after the beginning of the experiment when polyploidization was induced.An increased number of induced D. nobile tetraploid plants was obtainedby immersion of the plants in a solution of 0.1% colchicine for 96 hours.

Cytogenetic analysis

The determination of ploidy level was performed by counting chromosomes sevenmonths after the beginning of the experiment when polyploidy was induced.One-centimeter apices were extracted from 5 roots of D. nobile plants and pretreated in ice water with a temperature close to 0 ºC for 24 hours.After the pretreatment, the roots were fixed in a freshly prepared solution ofacetic acid:chloroform:95% ethanol (1:3:6) at 4 ºC for 24 hours and transferred to 70% ethanol where they remained stored at 4ºC in a refrigerator. Subsequently, the materialwas subjected to conventional crushing techniques and was washed three times indeionized water for a period of 10 minutes. Cell wall hydrolysis was performedusing 1 N HCl for 30 to 40 minutes depending on the thickness of the root in awater bath at 60 ºC. After this stage, the roots were immersed in deionized water for 1 minute for a chilled interruption ofthe hydrolysis reaction and were subsequently subjected to deionized water atroom temperature (Vichiato et al. 2007).

The extraction and fragmentation of the meristem were performed under astereoscopic microscope using a blade assembly by smashing in 45% acetic acid.The samples were stained with Giemsa solution with 3% phosphate buffer (pH 6.8)for 10 minutes, and the samples were mounted on cover slips with an Entellan®.The samples were imaged using an OlympusBX 60 microscope under bright field illumination with a 100X objective (oilimmersion) (Vichiato et al. 2007).

Morphological analysis

Diploid D.nobile plants (2n = 38 chromosomes - witnesses and the treatments did notoccur in polyploidization) and induced tetraploid D. nobile plants (2n =4x = 76 chromosomes) were used seven years after the induction ofpolyploidization, and these plants were cultivated in polyethylene pots (500 cm³)containing coconut fiber substrate and maintained on countertops.

Irrigation was performed threetimes a week on average according to the conditions of the substrate beingmoist and were fertilized bi-weekly with 2 mL of Biofert Plus® fertilizer perplant at a concentration of 5.0 mL L^-1 through foliar spray.

Acompletely randomized design was used with two treatments and 32 repetitionswith one plant per plot totaling 64 plots. The diploid and induced tetraploid D.nobile plants were assessed at flowering for the following variables:frequency of flowering, number of flowers per pseudobulb, flower size and lipsize (measured with a caliper and expressed in centimeters). The followingvegetative characteristics were measured: shoot length (measured with a rulergraduated in millimeters and expressed in inches), pseudobulb diameter(measured with a caliper and expressed in millimeters) and leaf number.

The results of these assessments were submitted to analysis of variance, and themeans were compared by Tukey's test at 5% probability. From these coefficients, we used the analysis of simple correlation between the variable sizes of the flowersand lips.

RESULTS AND DISCUSSION

Changes in flowering and floral morphologyof D. nobile plants

The first flowering of D. nobile diploid plants with normal flowers occurredtwo years after the conclusion of the polyploidization experiment. Neptune (1984)also found that this species produces flowers in the second year of growth.

Five years after the experimental induction of polyploidy, the D. nobile tetraploid plants showed the first flowering with few flowers that hadmalformed petals, sepals and/or lips. Anurita and Girjesh (2007) also found adelay in flowering in polyploid plants of Beijo-de-frade (Impatiensbalsamina).

The delay in flowering of induced tetraploid D. nobile plants may haveresulted from a gene dosage doubling. The immediate effect of polyploidy wasmorphophysiological with increased size of the cells due to a greater nuclearvolume. Accordingly, these cells consume more energy and require more time forthe duplication of DNA, which leads to a reduction of cell division during development, causing a delay in the mitotic cycle and life cycles as expressed in lowbiomass production per unit time (Takamura and Miyajima 1996, Vichiato et al.2007, Jadrná et al. 2010).

Seven years after the conclusion of the polyploidization experiment, the inducedtetraploid D. nobile plants visually had normal flowering. In assessingthe frequency of flowering plants, the percentage of diploid and tetraploidflowering plants was 96 and 71.875%, respectively.

The number of flowers per pseudobulb also varied among the different ploidy levels.Diploid plants had a higher average number of flowers per pseudobulb (6.6)differing significantly from the tetraploid plants, which had 4.7 flowers perpseudobulb (decrease of 40.85%). The decrease in the number of flowers perpseudobulb in D. nobile polyploid plants has also been confirmed byChaicharoen and Aejew (1981) and Chaicharoen (1995).

The diploid and tetraploid plants had flowers with an average duration of 11 days, which was the same duration of wild D. nobile flowers (Faria et al.2009). In this study, the tetraploid plants had greater flower heights (4.54%)and lip widths (18.46%) when compared to diploid flowers (Figure 1 and Table 1). All variables related to floral morphology showed low variability for thecoefficient of variation values representing a low degree of variability, whichindicated a more homogeneous distribution of variable values around the mean. Thus, concludedthat the effect of chromosome doubling in D. nobile floral morphologywas verified.

Regarding the flower and lip sizes, induced tetraploid plants showed the highest meanvalues ofheight and width of the flower and lip differing significantly from the diploidplants. These data indicated that increasing the width and height of the flowerimplies an increase in lip size. The positive correlation between the flowerlength and width is important because orchids are appreciated for the formationof an equilateral triangle by both rounded sepals and petals.

The increase in the D. nobile floral piece sizes caused by polyploidizationhas also been observed by Chaicharoen and Aejew (1981), McConnell and Kamemoto(1993), Chaicharoen (1995), Ketsa et al. (2001) and Dao-Long et al. (2012).

Simple correlation between floralvariables

Table 2 presents a matrix of simple correlations between floral morphology variables of D. nobile. By providing the correlation between several variables, thistype of analysis is an important indicator of the linearity of the relationshipresulting from the treatments. The simple correlations between variablesindicated that the variable groups were not independent. Specifically, thevariables related to tetraploid D. nobile floral morphology showedstrong correlations with the variables related to lip morphology (0.6<R<1). The correlation between the flower height (FH) and flower width(FW) showed the highest coefficient of linearity. Both the flower height andwidth showed a positive correlation with lip size and height (0.6862 and0.6766, respectively).

The only non-significant correlation was between flower width and lip width (p>0.05), which suggested that the amendments made by the flower width were notaccompanied by changes in the lip width with polyploidy induction.

Changes in vegetative growth

For the vegetative characteristics evaluated in the control and induced polyploid D.nobile plants, only significant differences (P<0.5) were found forpseudobulb diameter and internode number (Table 3). There were no significantdifferences between the diploid and induced tetraploid plants for the otherevaluated vegetative characteristics. This result may be due to the effect ofgigas, which are increased vegetative structures that are commonly found inorgans that have a highly specific growth pattern, such as flowers and seeds.Therefore, these differences are not always observed between plants withdifferent ploidy levels (Vichiato et al. 2007).

During the development of these plants, there was a tendency for diploidization orpolyploidization if the plants were diploid. It is common for a polyploid toundergo diploidization because two or more similar genomes start to behave asdiploid over time (Ramsey and Schemske 2002).

Diameter of the pseudobulbs

The pseudobulb diameter of the control plants was 64.86% higher than that of thetreated plants (Table 2). The pseudobulb of epiphytic orchids is the mainstorage organ for water, carbohydrates and mineral nutrients (Zimmerman 1990).As the volume of orchid pseudobulbs may vary during growth due to the transferof stored reserves (Vichiato et al. 2008), there was a significant correlationbetween the level of ploidy of the plants and the height of Dendrobium, which agreed with the study by Dao-Long et al. (2012). Thus, it can beconcluded that the diameter reduction of the tetraploid plant pseudobulbs wasdue to the induced gene dosage resulting in increased power consumption duringdevelopment and flowering because the flowers and seeds are organs that havehighly specific growth patterns (gigas effect). These increases in cellularenergy consumption likely affected the pseudobulbs as they are the storageorgans. Greater resistance and plant survival under conditions of water andnutrition stress results in better flower quality (Rech et al. 2010).

Number of internodes

The number of internodes was influenced by polyploidization (Table 2), and thetetraploid plants showed an increase of 19.86% in internode number compared tothe control plants. The internode number accompanies growth in plant heightbecause internodes directly participate in the process of division andelongation of meristematic cells (Machado et al. 2009). Even though there wasno significant difference in pseudobulb height between diploid and tetraploid D.nobile plants, it can be concluded that the addition of polyploidizationfavored the number of internodes.

The increased number of internodes (region between two nodes) proved to be amorphological variable that is economically viable because the orchid haslateral inflorescence starting on pseudobulb nodes forming several groups withtwo or three flowers each (Faria et al. 2009).

Advantages of polyploidy in D. nobile

For selection of ornamental plants with superior quality, the ornamental featurescolor, size and proportionality of flowers are considered (Cardoso 2010). Thetetraploid plants had larger flowers D. nobile with labella wider andmore rounded when compared with diploid flowers. Whereas the decrease in the number of flowersper pseudobulb of tetraploid plants may be offset by increased floral parts andthe number of internodes, polyploidy in D. nobile becomes interestingand economically viable, since it results in plants with ornamental qualitiessatisfactory and competitive market for the flower vase.

CONCLUSIONS

The induced polyploidization resulted in the following morphological effects a)increasing the number of internodes (19.9%) and floral pieces, with greaterheight of the flower (4.5%) and width of the lip (18.5%) b) decrease in thenumber of flowers per pseudobulb (40.8%) and the diameter of the pseudobulb(64.9%) and c) delayed flowering.

Received 26 March 2014

Accepted 14 April 2014

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Publication Dates

• Publication in this collection
  25 Nov 2014
• Date of issue
  Oct 2014

History

• Received
  26 Mar 2014
• Accepted
  14 Apr 2014