Impact of Colchicine Treatment on Sorghum bicolor BT×623  

Murali Kannan M , Jeevanandam Vanitha , Shuye Jiang , Srinivasan Ramachandran
Temasek Life Sciences Laboratory, 1 Research Link, the National University of Singapore, 117604, Singapore
Author    Correspondence author
Molecular Plant Breeding, 2013, Vol. 4, No. 15   doi: 10.5376/mpb.2013.04.0015
Received: 15 Apr., 2013    Accepted: 07 May, 2013    Published: 23 May, 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Murali et al., 2013, Impact of Colchicine Treatment on Sorghum bicolor BT×623, Molecular Plant Breeding, Vol.4, No.15 128-135 (doi: 10.5376/mpb.2013.04.0015)

Abstract

Colchicine is an alkaloid from Colchicum autumnale, which is widely used for the induction of polyploidy plants. No highly efficient method has been reported for the development of polyploidy plants in the grain sorghum BT×623. In this study, the sorghum seeds were treated with colchicine at two different concentrations (0.1% and 0.2%) for 24, 48 and 72 hours (hr). The impact of colchicine treatment on morphological, physiological, histological variations as well as on sucrose synthase gene expression was investigated. Some significant variations by comparing the control and colchicine treated plants were observed. The colchicine treated plants showed longer leaf length and stronger root system but exhibited smaller panicle size. Enlarged cells and some deformities by histology observation were also observed. The density of stomata decreased and its length increased with more opened guard cells in colchicine treated plants when compared with the control plants. Gene expression analysis showed that higher expression abundance was detected for most of genes encoding sucrose synthases in some colchicine treated plants and only one gene exhibited lower level of transcription abundance.

Keywords
Colchicine; Gene expression; Seeds; Sorghum bicolor

Colchicine is an alkaloid which obtained from Colchicum autumnale of angiosperm and belongs to the family Colchicaceae. Colchicine binds specifically to tublins to prevent polymerization of microtubules and to induce polyploidy cells. Colchicine is also considered as a mutagenic agent since it hinders the spindle formation. It may induce artificial polyploidy in plants and various reports have been reported to show the artificial polyploidy in various plant species and various families (Meyers and Levin, 2006; Otto, 2007). These artificial polyploidial effects by colchicines may induce some changes in morphological, cytological, histological and even in gene expression level.

Sorghum is a monocot C4 plant belonging to the family Poaceae where most of the cultivars are from the species Sorghum bicolor. As the fifth crop, sorghum is now cultivated in all the tropical and subtropical countries, providing an important food and animal feed, On the other hand, sweet sorghum is also considered to be a biofuel crop of growing importance, and in the US it has been considered to be the second crop used for ethanol production. The phenomenon of polyploid occurs in nature and is frequently observed in some plant families (Stebbins, 1949). Up to 30%~80% of plant species have been reported to be polyploid, and many diploid species might be originated from ancient polyploidy (paleopolyploidy) in their genomes (Meyers and Levin, 2006; Otto 2007; Rieseberg and Willis, 2007). Polyploidization may result in changes in gene dosage, the reunion of divergent gene regulatory hierarchies, chromosomal rearrangements, and epigenetic remodeling (Osborn et al., 2003; Chen and Ni, 2006; Chen, 2007). Many of these changes may contribute to novel variation or morphologies and some of them may lead to reproductive isolation and speciation (Comai, 2005; Rieseberg and Willis, 2007)in plant breeding. Besides, considerable varieties were developed from polyploidy breeding where an example is the development of triticale (Mergoum and Gómez- Macpherson, 2004) in wheat Triticumturgidum and rye Secale cereale. The initial hybrids are sterile and could not be propagated. After polyploidization, the hybrid becomes fertile and can thus be further propagated. The polyploid triticale combines the high yield and quality of grains from wheat as well as the disease and environmental tolerance from rye. Evidence showed that allopolyploids grew bigger and stronger than their parents and could be used for better use and exploitation of increased biomass and yield for food, feed and biofuels (Chen, 2010). However, in order to face severe challenges of rapid population growth, reduced crop land area and increased energy requirement, it is necessary to develop new sorghum cultivars with higher grain or biomass yield. The reports on the effect of colchcine treatment on Sorghum bicolor are very limited and this is highly needed for the standardization of the protocol for colchicine mediated polyploidy induction which may provide an altenative way for sorghum breeding. There is no data in the past literature describing the impacts of colchicine treatment on Sorghum bicolor to explore the posiblities of polyploidy induction. The present study report on the impact of colchicine treatment on various characteristics including morp- hology, cytology, histology and gene expression of sucrose synthase (SuSy) genes on Sorghum bicolor.
1 Results and Discussion
1.1 Morphological observations
Rosette formations in leaves were observed in the initial stage of the colchicine treated plants (Figure 1) which is not observed in the control plants (Figure 1). This type of rosette formation was also reported by Franzke and Ross (1963) in genus Sorghum after two to three weeks of germination. The percentage of germination of colchicine treated seeds was lower when compared to the control plants. Rosette formation of seedlings was observed in both the concentration of colchicine irrespective of time intervals. This may be due to the stress created by the colchicine treatment.


Figure 1 Rosette leaf variation of colchicine treated plants

Colchicine treated seeds showed significant variations in root formation in initial stage when compared to normal control plants. Variations were observed in both the concentrations of colchicine at 48 and 72 hr treatment. The colchicine induce seedlings produced an elongated roots and root hairs when compared to the control plants which produced only a single root. When the concentration and treatment time interval increased the root elongation also increased (Figure 2). There was no significant difference observed at the lowest concentration and time intervals like 0.1%, 0.2% colchicine for 24 hours of treatment. Prolonged treatment and higher concentration of colchicine may influence the endogenous auxin level which enhances the root formation in treated seeds. Observed variations in the inflorescence nature in control and colchicine treated plants, inflorescences were smaller in length and the numbers of seeds per spikes were also reduced in colchicine treated plants (Figure 3).


Figure 2 Rooting system in colchicine treated plants
 

Figure 3 Panicle variation of colchicine treated plants

1.2 Physiological observation
The density of stomata decreases in the colchicine treated plants as the time intervals of colchicine treatment increases (Figure 4). No difference was observed in the different concentration of the same time intervals like 0.1% and 0.2% colchicine for 24 h, 48 h and 72 h. The colchicine treated plants have a decrease in density of stomata when compared to controls.


Figure 4 Stomatal density variation of colchicine treated plants
  
On observing the individual stomatal length using cryo SEM, there was a significant increase in the On Observing the individual stomatal length using length of stomata as the treatment time interval increased (Figure 5). There was also an increase in the opening of guard cells in the colchicine treated leaf samples which was not observed in controls (Figure 5). There was no significant changes were observed in 0.1% and 0.2% colchicine at the same time intervals. The present observation supports the observation made by earlier researchers (Ye et al., 2010; Omidbaigi et al., 2010).


Figure 5 Stomatal length variation of colchicine treated plants
1.3 Histological analysis
The nature of difference in cell morphology between control and colchine treated seedlings is presented in Figure 6. There were some significant difference observed in the cell morphology between the control and colchicine treated seedlings. Some difference in the cell size and arrangement was noticed at 0.2% colchicine treated sample (72 h) when compared to the control plants.There was no much difference found in the other time intervals at 2 different concentrations.


Figure 6 Histological observation of stems and roots from both BT×623 and plants developed from colchicine treated seeds
The cell size and width between layers of the tissues decreased in treating when compared to the control samples (Figure 6 A~Figure 6 D). This may be due to the effect of colchicine, where the cells become larger which in turn leads to the decrease in width between the layers of tissues in treated samples. These cells were also enlarged in colchicine treatment when compared to the control. Some cell disruption was also found in the epidermal layers of the colchicine treated roots (Figure 6 E~Figure 6 H).

Similar observations were also reported in canaberry by Dermen et al (1944). In the report, they specified about the increase in cell size of the polyploid plants. Behera et al (1974) also observed this type of colchicine induced deformities in Amaranthuscausatus and discussed about the deformities in the histology of colchicine treated leaf.
1.4 Changes in gene expression level between diploid and tetraploid plants
To investigate the difference in gene expression between these colchicine treated and control plants, we carried out quantitative real-time RT-PCR (qRT-PCR) to analyze the expression of sucrose synthase genes. Total of 7 genes encoding sucrose synthases was identified by HMM (Hidden Markov Model) searches (see Materials and methods). These genes were named as SuSy1 to SuSy7. They were listed in Figure 7 A. The qRT-PCR was then carried out using total RNA samples from plants developed from colchicine treated seeds. On the basis of the data from qRT-PCR analysis (Figure 7 B~Figure 7 H), 4 genes were detected 4 genes (SuSy1, SuSy2, SuSy3 and SuSy6) with up-regulation after colchicines treatment by statistic analysis . The data suggested that expression level for some genes after colchicines treatment have been increased when compared with control plants. However, decreased expression level was also observed in colchicines treated plants for the gene SuSy5. However, significant expression signal has been detected in diploid plants (Figure 3 F). There was no difference detected in their expression abundance of SuSy and SuSy7 between control and colchine treated plants (Figure 6 E; Figure 6 H).


Figure 7 Comparative expression analysis of SuSy genes between diploid and tetraploid sorghum lines
In general, the differential transcript abundance has been observed between the control and colchicine treated plants for some genes, which may partially be due to the changes in gene dosage. Besides, the probability of observing a value of t greater (6.32) in absolute value than 3.182 is exactly 0.05. It reveals that the treatment affects the four characteristics viz. rosset formation, rooting nature, panicle and stomatal change of the plant.
In this study, various phenotypic and physiological variations have been observed in the colchicines treated sorghum. New phenotypes often arise with polyploid formation and insight mechanisms include changes in gene expression through increased variation in dosage-regulated gene expression, altered regulatory interactions, and rapid genetic and epigenetic changes (Osborn et al., 2003). To preliminarily explore the changes in gene expression after colchicines treatment in sorghum, one gene family was selected for such analysis (Figure 7). The family contains 7 members with different expression patterns in diploid plants. Our expression data showed that the increased gene dosage not only enhanced their expression abundance but also could down-regulated their expression. A similar result has been reported in other plants and polyploidy has considerable effects on duplicate gene expression, including silencing and up- or down-regulation of these genes (Adams and Wendel, 2005). The effect of colchicine on phenotype variations is not yet understood well and further study should be carried out to better explore its mechanisms for the artificial polyploidy induction in Sorghum bicolor.
 
One of the advantages to induce polyploidy plants for breeding is that some of polyploids can significantly increase their grain/fruit size and yield (Randolph, 1941; Guzy et al., 1989; Milan, 2008), which could be used for crop breeding to improve grain/fruit yield. However, the giant grain by polyploidization is the species/genotype-dependent. In this study, colchicine treated plants showed stronger root systems but exhibited smaller panicle size and grain yield (Figure 2; Figure 3). Thus, more experiments should be carried out to examine if the grain yield can be improved by polyploidization using different sorghum species or genotypes for colchicine treatments. Other advantages for polyploidy breeding include fixing heterosis and producing a higher biomass yield, which can be used for breeding biofeul plants. Studies reported that polyploid plants exhibited higher photosynthetic rate (Warner and Edwards, 1993) and higher biomass yield could be obtained by polyploidization (Guzy et al., 1989; Lavania et al., 2012). On the other hand, polyploidization can also be used for fixing some kind of heterosis (Bansal et al., 2012).
So our study reveals the cytohistological and morphological changes which take place during the colchicine treatment. This data will be useful for the further application in the production of polyploid Sorghum bicolor plants by artificial polyploid induction using colchicine.
2 Materials and Methods
2.1 Direct potting method for polyploid induction using colchicine treatment of Sorghum bicolor seeds
BTx623 is derived from the sexual cross between BTx3197 and SC170-6 and was released by the Texas Agricultural Experiment Station (https://billrooney.tamu.edu/research/files/germplasm/Tx643%20-Tx645%20Sorghum%20Inbreds%20Website.pdf). BT×623 is a maintainer line used for three-line hybrid sorghum production. It is a grain sorghum but its stem is also sweet. So, BT×623 was used for colchicine mediated polyploidy induction. Seeds were surface sterilized with commercial detergents (Microsterile, 4% Chloro ) for about 5 minutes followed by 30% H2O2 for 2-3 minutes, 50% Clorox for 10 minutes, 0.1% HgCl2 for 5 minutes in aseptic conditions. The sterilized seeds were washed with sterile distilled water after every treatment. They were then finally dried using filter papers. The seeds were treated with colchicine at two different concentrations of 0.1% and 0.2% for about 16 h, 24 h, 48 h and 72 h. Treated seeds were washed with distilled water at respective time intervals and dried using filter paper completely to avoid contamination. The dried seeds were directly potted into pots containing potting soil.
2.2 Plant morphology observation
Plant morphological investigation was carried out during the whole growth period.The potted seeds were observed at regular intervals and the observation (changes in the plant morphology) has been recorded.
2.3 Physiological changes
Various physiological parameters were examined between the control plants and colchicine treated plants viz.stomatal density and stomatal length.
2.4 Stomatal density and length
The stomatal density and length as as physiological parameter was examined. Fresh leaves of control and colchicine treated plants were taken and xylene was applied to the lower epidermis and kept for drying. After drying the lower epidermis was peeled off with forceps and was stained with tryphan blue (Sigma, USA) for visualization of stomatal density under Nikon eclipse 80i light microscope under 20 times of magnification. The fresh leaves were used for the analysis. The difference in stomata length between the colchicine treated and control plants was observed by Cryo SEM analysis. The sample holder was slightly immersed in liquid nitrogen (beware that the sample should not completely immersed in liquid nitrogen) and the sample holder with the sample was placed inside the Cryo SEM (Jeol JSM-6360LV) stage for observation.
2.5 Genome-wide identification of genes encoding sucrose synthases in sorghum
To investigate the effects of colchicine on gene expression, genes encoding sucrose synthases was selected because these genes might contribute to biomass yield and their gene family size is in the middle level. SuSy genes were selected when their encoding proteins contained a Sucrose_synth domain with the Pfam accession number PF00862. All sorghum protein sequences were downloaded and annotated by Paterson et al (2009) from the website http://www.phytozome.net/sorghum. We then built a Hidden Markov Model (HMM) profile using HMMER 2.3.2 (http://hmmer.janelia.org/) with default values using seed domain members from multiple species. Using the profile HMMs, the annotated protein database were scanned to search for candidate sucrose synthases. The putative sucrose synthases were identified by confirming the presence of the domain Sucrose_synth using the Pfam database (http://pfam.sanger.ac.uk/).
2.6 Expression profiling of SuSy genes in sorghum
To examine the difference in expression profiling of the SuSy gene family between control and colchicines treated sorghum lines, quantitative Reverse Transcription PCR (qRT-PCR) was carried out. Total RNA samples were extracted from 14 days old leaves in both control and colchicine treated plants. Total RNA samples were prepared using the RNeasy Plant Mini Kit (Qiagen). The first-strand cDNA was synthesized using an Invitrogen kit. The real-time PCR analyses were carried out using Applied Biosystems (AB) 7900HT Fast Real-Time PCR system 384 well formats according to the description in our previous description (Jiang et al. 2007). All primers used for qRT-PCR were designed by Applied Biosystems Primer Express® software. The designed primer sets were then subjected to the phytozome sorghum database (http://www.phytozome.net/sorghum) for a BLAST search to eliminate the non-specific primers. All primer sequences were listed in Table 1.


Table 1 Primer sequences used in this study
A paired t-test was used for comparison of two different observations where treatment was applied to the same group of plants. The impact of treatment on various characteristics of Sorghum bicolor has been explained.

References
Aleza P., Juárez J., Ollitrault P., and Navarro L., 2009, Production of tetraploid plants of non apomictic citrus genotypes, Plant Cell Reports, 28: 1837-1846 http://dx.doi.org/10.1007/s00299-009-0783-2
Allum J.F., Bringloe D.H., and Roberts A.V., 2007, Chromosome doubling in a Rosa rugosaThunb.hybrid by exposure of in vitro nodes to oryzalin: the effects of node length, oryzalin concentration and exposure time, Plant Cell Reports, 26: 1977-1984
http://dx.doi.org/10.1007/s00299-007-0411-y
Ascough G.D., Van Standen J., and Erwin J.E., 2008, Effectiveness of colchicine and oryzalin at inducing polyploidy in Watsonialepida NE Brown, HortScience, 43: 2248-2251
Bansal P., Banga S., and Banga S.S., 2012, Heterosis as investigated in terms of polyploidy and genetic diversity using designed Brassica junceaamphiploid and its progenitor diploid species, PLoS One, 7: e29607 http://dx.doi.org/10.1371/journal.pone.0029607
Behera B., Tripathy A., and Patnaik S.N., 1974, Histological analysis of colchicine-induced deformities and cytochimerasin Amaranthuscaudatusand A.dubius, Journal of heredity, 65: 179-184
Breslavetz L., 1939, Polyploids in rye induced by X-rays.Akad.Nauk. (Doklady) N.S. Moscow, SSSR 22: 354-357
Chen Z.J., 2007, Genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids, Annual Review of Plant Biology, 58: 377-406
http://dx.doi.org/10.1146/annurev.arplant.58.032806.103835
Chen Z.J., 2010, Molecular mechanisms of polyploidy and hybrid vigor, Trends in Plant Science, 15: 57-71
http://dx.doi.org/10.1016/j.tplants.2009.12.003
Chen Z.J., and Ni Z., 2006, Mechanisms of genomic rearrangements and gene expression changes in plant polyploids, Bioessays, 28: 240-252
http://dx.doi.org/10.1002/bies.20374
Chin T.C., 1946, The cytology of polyploid sorghum. American Journal of Botany, 33: 611-614 http://dx.doi.org/10.2307/2437403
Comai L., 2005, The advantages and disadvantages of being polyploidy, Nature Reviews Genetics, 6: 836-846
http://dx.doi.org/10.1038/nrg1711
Dermen H., 1940, Colchicine polyploidy and technique, The Botanical Review, 6: 599-635
http://dx.doi.org/10.1007/BF02919557
Dermen H., and Bain H.F., 1944, A general cytohistological study of colchicine polyploidy in cranberry, American Journal of Botany, 31: 451-463
http://dx.doi.org/10.2307/2437405
Franzke C.J., and Ross J.G., 1963, Colchicine induced variants in sorghum, Journal of heredity, 54:221-228
Głowacka K., Jeżowski S., and Kaczmarek Z., 2010, In vitro induction of polyploidy by colchicine treatment of shoots and preliminary characterization of induced polyploids in two Miscanthus species, Industrial Crops and Products, 32: 88-96
http://dx.doi.org/10.1016/j.indcrop.2010.03.009
Guzy M.R., Ehdaie B., and Waibes J.G., 1989, Yield and its components in diploid, tetraploid and hexaploidwheats in diverse environments, Annals of Botany, 64: 635-642
Hess D., and Bayer D, 1974, The effect of trifluralin on the ultrastructure of dividing cells of the root meristem of cotton (Gossypiumhirsutum L. "Acala 4-42'), Journal of Cell Science, 15: 429-441
Jiang S.Y, Bachmann D., La H., Ma Z., Venkatesh P.N., Ramamoorthy R., and Ramachandran S., 2007, Ds insertion mutagenesis as an efficient tool to produce diverse variations for rice breeding, Plant Molecular Biology, 65: 385-402
http://dx.doi.org/10.1007/s11103-007-9233-0
Lavania U.C., Srivastava S., Lavania S., Basu S., Misra N.K., and Mukai Y., 2012, Autopolyploidy differentially influences body size in plants, but facilitates enhanced accumulation of secondary metabolites, causing increased cytosine methylation, The Plant Journal, 71: 539-549
http://dx.doi.org/10.1111/j.1365-313X.2012.05006.x
Liu G., Li Z., and Bao M., 2007, Colchicine- induced chromosome doubling in plantusacerifolia and its effect on plant morphology, Euphytica, 157: 145-154
http://dx.doi.org/10.1007/s10681-007-9406-6
Omidbaigi R., Mirzaee M., Hassani M.E., and SedghiMoghadamc M., 2010, Induction and identification of polyploidy in basil (Ocimumbasilicum L.) medicinal plant by colchicine treatment, International Journal of Plant Production, 4: 87-98
Omidbaigi R., Yavari S., Hassani M.E., and Yavari S., 2010, Induction of autotetraploid in Dragohead (Dracocephalaum moldavica L.) by colchicine treatment, Journal of Fruit and Ornamental Plant Research, 18: 23-35
Osborn T.C., Pires J.C., Birchler J.A., Auger D.L., Chen Z.J., Lee H.S., Comai L., Madlung A., Doerge R.W., Colot V., and Martienssen R.A., 2003, Understanding mechanisms of novel gene expression in polyploids, Trends in Genetics, 19: 141-147
http://dx.doi.org/10.1016/S0168-9525(03)00015-5
Paterson A.H., Bowers J.E., Bruggmann R., Dubchak I., Grimwood J., Gundlach H., Haberer G., Hellsten U., Mitros T., Poliakov A., Schmutz J., Spannagl M., Tang H., Wang X., Wicker T., Bharti A.K., Chapman J., Feltus F.A., Gowik U., Grigoriev I.V., Lyons E., Maher C.A., Martis M., Narechania A., Otillar R.P., Penning B.W., Salamov A.A., Wang Y., Zhang L., Carpita N.C., Freeling M., Gingle A.R., Hash C.T., Keller B., Klein P., Kresovich S., McCann M.C., Ming R., Peterson D.G., Mehboob-ur-Rahman Ware, D., Westhoff P., Mayer K.F., Messing J., and Rokhsar D.S., 2009, The Sorghum bicolor genome and the diversification of grasses, Nature, 457: 551-556
http://dx.doi.org/10.1038/nature07723
Ranney T,G., 2006, Polyploidy: from evolution to new plant development, Combined Proceedings of the International Plant Propagators Society, 56: 604-607
Rieseberg L.H., and Willis J.H., 2007, Plant speciation, Science, 317: 910-914
http://dx.doi.org/10.1126/science.1137729
Sakhanokho H.F., Rajasekaran K., and Kelley R.Y., 2009, Induced polyploidy in diploid ornamental ginger (Hedychiummuluense) using colchicine and oryzalin, HortScience, 44: 1809-1814
Sarathum S., Hegele M., Tantiviwat S., and Nanakorn M., 2010, Effect of concentration and duration of colchicine treatment on polyploidy induction in Dendrobiumscabrilingue L, European Journal of Horticultural Science, 75: S123-S127
Siddiqui S.H., and Marwat K.B., 1983, Cytomorphological effects of colchicine on wheat (Triricum aestivum), Pakistan Journal of Agricultural Research, 4: 120-125
Stebbins G.L., 1949, The evolutionary significance of natural and artificial polyploids in the family gramineae, Hereditas, 35: 461-485
http://dx.doi.org/10.1111/j.1601-5223.1949.tb03355.x
Warner D.A., and Edwards G.E., 1993, Effects of polyploidy on photosynthesis, Photosynthesis Research, 35: 135-147
http://dx.doi.org/10.1007/BF00014744
Ye Y.M., Tong J., Shi X.P., Yuan W., and Li G.R., 2010, Morphological and cytological studies of diploid and colchicine induced tetraploid lines of crape myrtle (Lagerstroemia indica L.), Scientia Horticulturae, 124: 95-101
http://dx.doi.org/10.1016/j.scienta.2009.12.016
Yemets A.I., and Blume Y.B., 2008, Progress in plant polyploidization based on antimicrotubular drugs, Open Horticulture Journal, 1: 15-20
http://dx.doi.org/10.2174/1874840600801010015
Yu Z., Haage K., Streit V.E., Gierl A., and Ruiz R.A., 2009, A large number of tetraploid Arabidopsis thaliana lines, generated by a rapid strategy, reveal high stability of neo-tetraploids during consecutive generations, Theoretical and Applied Genetics, 118: 1107-1119
http://dx.doi.org/10.1007/s00122-009-0966-9

Zlesak D.C., Thill C.A., and Anderson N.O., 2005, Trifluralin-mediated polyploidization of Rosa chinensis minima (Sims) Voss seedlings, Euphytica, 141: 281-290
http://dx.doi.org/10.1007/s10681-005-7512-x