Protoplasma DOI 10.1007/s00709-013-0545-4
SHORT COMMUNICATION
Nuclear DNA content of Pongamia pinnata L. and genome size stability of in vitro-regenerated plantlets Rimjhim Roy Choudhury & Supriyo Basak & Aadi Moolam Ramesh & Latha Rangan
Received: 18 June 2013 / Accepted: 20 August 2013 # Springer-Verlag Wien 2013
Abstract Pongamia pinnata L. is a multipurpose versatile legume that is well known as a prospective feedstock biodiesel species. However, to date, there has been little genomic research aimed at the exploitation of the biotechnological potential of this species. Genetic characterization of any plant is a challenging task when there is no information about the genome size and organization of the species. Therefore, the genome size of P. pinnata was estimated by flow cytometry with respect to two standards (Zea mays and Pisum sativum ), and compared with that of in vitro-raised plants (nodal segment, in vitro-rooted plantlets and acclimatized in vitro plants) to study the potential effect of somaclonal variation on genome size. This method can be used to support the establishment of true-to-type plants to encourage afforestation programs. Modified propidium iodide/hypotonic citrate buffer was used for isolation of the intact nuclei. The 2C DNA value of this species was estimated to be 2.51±0.01 pg. Statistically, there was no significant difference in the DNA content of the in vitro-grown plants and mother plant at α =0.05. As a result of the low genome size of P. pinnata , a species that has adapted itself to a wide range of edaphic and ecological condition, we can now proceed for its next generation sequencing and genomic diversity studies.
Handling Editor: Peter Nick Electronic supplementary material The online version of this article (doi:10.1007/s00709-013-0545-4) contains supplementary material, which is available to authorized users. R. R. Choudhury : S. Basak : A. M. Ramesh : L. Rangan (*) Department of Biotechnology, Indian Institute of Technology Guwahati, Assam 781 039, India e-mail:
[email protected] L. Rangan e-mail:
[email protected]
Keywords Biodiesel . Flow cytometry . Genome size . In vitro culture . Pongamia pinnata Abbreviations AVM Acclimatized in vitro plants CPT Candidate plus tree CV Coefficient of variation FCM Flow Cytometry IRP In vitro-rooted plants NGPP North Guwahati Pongamia pinnata
Introduction Pongamia pinnata (L.) Pierre also called as Derris indica or Millettia pinnata is a monotypic genus and grows abundantly along the coasts and riverbanks in India and Myanmar. It is a nitrogen-fixing, wild, perennial, leguminous tree generally known for its biodiesel properties with the potential for high oil seed production and the added benefit of the ability to grow on marginal land. The tree can grow on unproductive land and is adaptable to wide agroclimatic conditions. Besides, the oilyielding capacity, its multipurpose benefits as a provider of green manure and medicine, and its role in agroforestry make it a potential candidate for large-scale plantation on marginal lands (Scott et al. 2008). Pongamia has also been used as a folk medicinal plant, particularly in the Ayurvedic and Siddha systems of medicine (Kesari et al. 2009a). The plant is used as an anti-inflammatory, anti-lipidoxative and antihyperglycaemic agent (Punitha and Manoharan 2006) and to treat gastric ulcers (Singh et al. 1997), and rheumatic arthritis (Ballal 2005). It has also been used for the discovery of novel cancer chemopreventive agents (Carcache-Blanco et al. 2003), to treat wounds caused by fish poison and also to improve mothers’ health after child birth (Shibuya and Kitagawa 1996).
R.R. Choudhury et al.
In spite of its numerous favorable attributes, not even half of its full potential has been realized. To ensure that Pongamia emerges in the near future as a species that maximizes its potential for sustainable oil production, directed crop improvement program is essential. The fact that Pongamia has adapted itself to a wide range of edaphic and ecological conditions suggests that there exists considerable amount of genetic variability to be exploited for potential realization. Knowledge of genome size is more than a practical necessity in large-scale sequencing programs; it can also be of use in understanding other key features including structure, organization, and composition (Gregory 2005), which are the bottlenecks in the genetic characterization of Pongamia. To increase the biodiesel production, it is important to have superior genotypes of P. pinnata bearing high oil-yielding seeds. Earlier studies in systematic characterization and seed oil analysis in P. pinnata enabled us to tag high-yielding genotypes from natural populations occurring in North Guwahati that can be included in programs aimed at the genetic improvement of the species (Kesari et al. 2008). Characterization and selection of candidate plus trees (CPTs) is essential for the improvement of this species, in addition to experiments on controlled crossing among selected genotypes (Kesari et al. 2008, 2009a, b). A plus tree is an individual tree of a species possessing superior morphological and reproductive characters than other individuals of the same species. To meet the future demands for biodiesel, establishing extensive plantations comprising high-yielding lines through in vitro techniques will offer the best possibility towards obtaining lines of homogeneous clones (Kesari et al. 2012). The inherent problem associated with commercially valuable tree species of P. pinnata is to establish good axenic cultures that are genetically identical to the donor plant. However, the incidences of somaclonal variation amongst progeny of one parental line, arising as a direct consequence of in vitro culture of plant cells, tissues or organs can seriously limit the broader utility of in vitro systems (Larkin and Scowcroft 1981; Sultana et al. 2005). Gross changes such as a variation in ploidy level, number of chromosomes, and structural changes represent major alterations to the genome and they are often generated during in vitro proliferation and differentiation (Neelakandan and Wang 2012). Frequency of variation increases as the number of multiplication cycles increases. It is well documented that genome size of in vitro culture can be altered through changes either in the chromosome number or in the ploidy level. Therefore, to ascertain plant genome size stability following in vitro culture, nuclear DNA content estimation of in vitro-raised plants and its comparison with mother plant is crucial. Flow cytometry (FCM) provides a rapid, accurate, and simple means to determine nuclear DNA contents (C value) within plant homogenates (Galbraith 2009), analyzing cell cycles (Bergounioux and Brown 1990), determining chromosome
karyotypes, sorting cells and chromosomes, and characterizing other cellular parameters (Rayburn et al. 1989, 1992; Fuchs and Pauls 1992; Liu et al. 1997). This technique has also proven to be suitable for routine large-scale studies of ploidy level in plants growing in the field and in green house as well as in different plant materials cultured in vitro.
Materials and methods Plant material and culture Earlier characterized mature CPT of P. pinnata (NGPP 46) obtained from Sila Forest Range, North Guwahati, Assam (latitude 26º14′6″N and longitude 91º41′28″E) was the starting material. Acclimatized in vitro plants (AVP) from CPT were obtained using nodal segments raised from seedlings of P. pinnata as described previously (Kesari et al. 2012) and analyzed after a 4-year cultivation period in the open field. Simultaneously, in vitro-rooted plantlets (IRPs) of the CPT were sampled at the end of the rhizogenesis phase, immediately before transplanting to ex vitro condition. In each of the case, estimation of nuclear DNA content was carried out on young and tender leaf tissues. DNA content of nodal segments (NS) that were the starting material for micro propagation was also estimated. Leaf tissues of pea (Pisum sativum cv. Ctirad, 2C=9.09 pg) and corn (Zea mays cv. CE-777=5.43 pg) were used as internal reference plant standards. Seeds and the corresponding genome size of internal reference standards were kindly provided by Doležel, Institute of Experimental Botany, Academy of Sciences of the Czech Republic. Isolation buffer For this study, an optimized nuclei isolation method was used with leaf tissues of P. pinnata. The standard nuclei isolation buffer used for most species did not result in accepted histograms. The most probable reason for this was the presence of phenolic compounds in the cytosol of Pongamia cells. Phytochemical investigation of P. pinnata indicated the presence of prenylated flavonoids in abundance, in addition to phenolic compounds which increased the complexity of the cytosolic extract, thereby interfering with the staining of the isolated nuclei using propidium Iodide (PI). In preliminary experiments, five different procedures for the extraction and staining of the cells were tested: Galbraith’s buffer (Galbraith et al. 1983), LB01 (Doležel et al. 1989), propidium iodide/ hypotonic citrate (Krishan 1975), general purpose buffer (Loureiro et al. 2007a), and woody plant buffer (Loureiro et al. 2007b), respectively. Among all, HPI (hypotonic propidium iodide) was preferred because it gave clear histograms with minimum nuclear disruption and background noise.
Nuclear DNA content of Pongamia pinnata L.
Sample preparation Samples of the plant material for FCM analysis were prepared according to Krishan (1975) with some minor modifications. The selected plant tissue was chopped with a sharp razor blade in a plastic Petri dish with 2 ml of HPI isolation buffer (0.1 % (w/v) sodium citrate tribasic dehydrate (Sigma-Aldrich, India, cat. no. S4651), 2 mg/ml RNase A (Sigma-Aldrich UK, cat. no. P4875), 25 μg/ml PI (Sigma-Aldrich, USA, cat. no. P4170), and 0.3 % (v/v) IGEPAL CA- 630 (Sigma-Aldrich, USA, cat. no. I3021)). Antioxidants like PVP-40 (1 %) (Sigma-Aldrich, USA) and 1 % of β-mercaptoethanol (Sigma-Aldrich, France) were occasionally added to the buffer to minimize the negative effects of cytosolic compounds on PI fluorescence. The suspension was then filtered through 30 μm nylon mesh filter before analyzing it on a flow cytometer. Flow cytometric analysis A FACSCalibur flow cytometer equipped with a 15-mW 488nm-air-cooled argon-ion laser and Cell Quest Pro software (BD Bioscience, CA, USA) was used for the acquisition of multiple parameters. For each sample, 10,000 events (nuclei) were collected, and the resulting histograms were analyzed for mean fluorescent intensity (MFI), coefficient of variation (CV) and percentage of nuclei in each stage of cell cycle using FlowJo v.7.6.5 (FlowJo, TreeStarInc, Ashland, OR). A stepby-step gating procedure was followed to resolve the data and to measure the parameters of intact nuclei in a heterogeneous population (Supplementary Fig. 1). Doublets and clumps were eliminated by gating on fluorescence width and fluorescence area profiles. In this profile, an “interest zone” was defined such that only single intact nuclei were included in the fluorescence histogram. To reduce the level of debris and disintegrated nuclei, the nuclei were gated in PI fluorescence channel vs. SSC dot plots. In this diagram, the nuclei can clearly be identified by their defined fluorescence intensity/ scattered light pattern. Depending on the age of tissue analyzed, CV obtained typically lie in the range of 2–5 %. For accurate genome size estimation of P. pinnata genotype, nodal segments and artificially raised plants (five in vitro and five ex vitro), a total of three readings were taken on different days, and an average of all were reported. Nuclear DNA content in the unknown samples was determined by interpolation from the fluorescence signals generated from the known (Doležel and Bartoš 2005) with the following formula: 2CSample
G1 sample : ¼ 2CStandard G1 standard
Here, 2Csample is the 2C DNA amount of the investigated sample (pg), G1sample is the mean fluorescence intensity of the
G1 peak (2C) of the sample, 2Cstandard is the 2C DNA amount of the standard (Pisum sativum cv. Ctirad, 2C=9.09 pg; Zea mays cv. CE-777=5.43 pg), and G1standard is the standard G1 peak mean fluorescence intensity (2C). Conversion from picograms (pg) to base pairs (bp) was done (1 pg DNA=978 Mbp; Doležel et al. 2003). Test for the presence of inhibitors The presence of an inhibitor is confirmed if the mean peak position of reference standard is lower in the presence of the target sample (Price et al. 2000). In this study, we first tested for the presence of such inhibitors in the cytosol of Pongamia, by comparing the peak positions of pea nuclei that were separately processed and those that were simultaneously processed (co-chopped as one sample, with leaves layered on top of each other) with Pongamia. Preliminary trials were performed for establishing the concentration and combination of antioxidants that stabilize PI fluorescence in leaf samples. Test for inhibitors was repeated using Krishan’s buffer with the addition of 1 % (w/v) PVP-40. As P. pinnata still showed the inhibition effect on PI fluorescence, the concentration of PVP in the buffer was increased to 1.5 % (w/v). However, increasing the concentration of PVP disturbed the stability of nuclei and increased the background noise. Therefore PVP was again reduced to 1 % (w/v) and in addition 1 % (v/v) βmercaptoethanol was applied to further minimize the inhibition. Statistical analysis To compare the genome size of CPT, NS, IRP and AVP they were analyzed in triplicates. Replicate measurements carried out on different days to confirm the repeatability of the experiments. The data obtained during the experiments was analyzed using one-way ANOVA. Multiple mean comparisons were performed using Tukey’s HSD test. A linear regression analysis and a Pearson correlation were performed between the mean fluorescence intensity of test and standard nuclei. All statistical analyses were carried out using SPSS software (IBM Corporation, Somers, NY, USA).
Results Presence of inhibitors The variation in the regression equation of MFI was y = 0.278x +127.13; R 2 =0.99 in the case of P. pinnata (test) and P. sativum (standard) whereas it was y =0.342x −41.77; R 2 =0.763 for P. pinnata (test) and Z. mays (standard) before the addition of the antioxidants. The regression between sample and standard fluorescence differed in terms of their slopes
R.R. Choudhury et al.
and did not go through the origin. Difference in nuclear DNA content of the above two sets was therefore demonstrated by the distance between MFI of standard and test nuclei clusters. The steeper slope (m =0.342) and negative intercept (c= −41.77) obtained by usage of Z. mays depicts the larger inhibition resulting in the coarseness in the coefficient of regression (R 2 =0.76) (Supplementary Fig. 2). Moreover, from the FCM analysis, there was a visible difference between the mean peak position of reference standards prepared separately and those prepared by co-chopping with Pongamia (Supplementary Fig. 3). This implies that the secondary compounds (alkaloids) present in Pongamia do inhibit PI staining of standard nuclear DNA. 1 % (w/v) PVP-40 and 1 % (v/v) βmercaptoethanol were added to the isolation buffer to minimize this inhibition. The quantitative shift in the MFI reduced and regression coefficient enhanced with addition of antioxidants during internal standardization process in our study (Ramesh et al., unpublished data).
DNA content of CPT and ploidy stability of P. pinnata plants grown in vitro Clearly defined histograms for accurate determination of 2C values were obtained following FCM analysis of intact leaf nuclei of P. pinnata CPT using the modified propidium iodide/hypotonic citrate buffer. According to the comparison of their G 0 /G 1 peak values, the genome size of CPT Pongamia was 0.27 times of diploid P. sativum and 0.45 times of that of Z. mays (Table 1). Therefore, it could be estimated that the relative 1C genome size of Pongamia, is 1,198 Mbp or 2.51 pg. Based on records from plant DNA Cvalues database (Bennett and Leitch 2010), the genome size of P. pinnata falls in the upper end of C-value distribution of its family– Fabaceae with 683 records ranging from 298 (Leucaena macrophylla ; Hartman et al. 2000) to 26797 (Vicia faba; Bennett et al. 1982) Mbp/1C. It falls somewhere in between the genome size of Acacia nilotica (1,174 Mbp; Mukerjee and Sharma 1993) and Lupinus texensis (1,193 Mbp; Price et al. 2005). The estimated DNA content of P. pinnata CPT was used for comparison with its in vitro-raised plants and in vitroraised field grown plants. Simultaneous analysis of nuclear DNA content of P. pinnata CPT, IRP, AVP and NS showed no significant difference at α = 0.05 (ANOVA: Standard P. sativum - F3,32 = 0.436 and P =0.729; Standard Z. mays F3,32 =0.850 and P =0.477) and were grouped together in the same group via Tukey’s HSD (Table 1, Fig. 1). In addition, simultaneous analysis of mixtures of plant homogenates of CPT with each of the in vitro-raised plants resulted in one single peak in the 2C range. This confirms that the little variation observed in the measurements is mainly due to sample handling and instrumental errors.
Discussion In this study, we report the 2C DNA content of P. pinnata CPT. DNA FCM requires preparation of suspensions of intact nuclei, which are stained using a DNA-specific fluorochrome prior to analysis. The buffer should protect nuclear DNA from degradation and provide an appropriate environment for specific and stoichiometric staining of nuclear DNA, including the minimization of negative effects of some cytosolic compounds on DNA staining (Loureiro et al. 2007b). Although various popular nuclear isolation buffer formulas are available, quantitative data on the performance of the most buffers has showed that none of them worked well with all species that represented different types of leaf tissues and different nuclear genome sizes, and researchers are encouraged to optimize protocols for their specific application (Bainard et al. 2010). It has been shown that buffer choice, staining period and PI concentration have statistically significant effect on the genome size estimates, altering the 1C values (Loureiro et al. 2007a; Greilhuber et al. 2007; Doležel et al. 2007). In order to maintain the perfect quality and good stability of the cell nuclei, we tested several buffers for preparation of the nuclei sample. The best results for the isolation of nuclei, which is used in the present work, were obtained with sodium citrate buffer (Krishan 1975) containing non-ionic detergent IGEPAL CA-630 to facilitate the release of nuclei from the cell and prevent nuclei clumping and attachment of debris. However, the method has its own limitation as it does not eliminate small cell fragments and organelles. There was also a high amount of variation in the results in nuclear DNA estimates. The solution was turning brown and formed precipitate even after filtration. This happens mainly due to the high amount of phenolics in the cytoplasm which is one of the main problems in the application of FCM to the biotechnological plant sciences (Greilhuber et al. 2007). Test for inhibitors (Supplementary Fig. 3) showed that there was a significant shift in PI fluorescence peaks of separately processed standard nuclei and those co-processed with P. pinnata , suggesting that saponins in P. pinnata inhibit PI intercalation or fluorescence. Abundance of cytosolic compounds was expected in the case of P. pinnata as it is a medicinal plant and its leaf extracts have traditionally been used to cure various diseases. Antioxidant β-mercaptoethanol and PVP40 (Polyvinyl pyrrolidine-40) were added to check whether the variation was due to the variable genome size of different plants or because of the interference of the cytosolic compounds. The presence of cytosolic compounds that can interfere with the binding of the fluorochrome to DNA is one such problem, which has been extensively researched (Noirot et al. 2005; Price et al. 2000). PVP and antioxidants have been used successfully to eliminate inhibitory compounds in many studies (Thiem and Sliwinska 2003; Meng and Finn 1999; Rupp et al. 2010). In addition, this buffer contains citric acid that
Nuclear DNA content of Pongamia pinnata L. Table 1 2C DNA content of P. pinnata estimated using P. sativum/Z. mays as an internal reference standard Tissue origin
P. sativum (Internal reference standard)
CPT In vitro-rooted plantlets Acclimatized in vitro plants Nodal segments
Z. mays (Internal reference standard)
N
P. pinnata DNA content (pg) (Mean ± S.E.*)
DNA index
Coefficient of variation
N
P. pinnata DNA content (pg) (Mean ± S.E.*)
DNA index
Coefficient of variation
1
2.51±0.017a
0.27
4.90
1
2.66±0.078a
0.45
4.95
5 5 1
a
0.28 0.28 0.27
4.12 4.70 4.50
5 5 1
2.65±0.005a 2.66±0.005a 2.66±0.006a
0.44 0.44 0.44
4.50 4.12 4.45
2.49±0.007 2.51±0.011a 2.49±0.023a
N = Number of plants analyzed (three replicate processing and measurements was noted for each plant) *Multiple-range test groupings (P
