Abstract:
A CASE OF PLAGIARISM
(Case No. 09302011)
Mark Guiltinan, Professor of Plant Molecular Biology from Penn State University pointed out a plagiarism in a paper published in Biotechnology Volume 9, Number 3, 355-361, 2010.
On the receipt of the letter from Mark Guiltinan, the case forwarded to the Ethics Committee of the Science Alert. As per the report of the Ethics Committee, article entitled "Callus Induction and Somatic Embryogenesis in Five Cacao (Theobroma cacao L.) Genotypes in Ghana" authored by J.N. Buah, published in Biotechnology Volume 9, Number 3, 355-361, 2010 contains substantial sections of text that have been taken verbatim from earlier publication without clear and unambiguous attribution.
Science Alert considers misappropriation of intellectual property and duplication of text from other authors or publications without clear and unambiguous attribution totally unacceptable.
Plagiarism is a violation of copyright and a serious breach of scientific ethics. The Editors-in-Chief and Publisher agreed to officially retract this article.
Science Alert is highly thankful to Mark Guiltinan, Professor of Plant Molecular Biology from Penn State University for pointing out this plagiarism.
Detail of article from which text has been copied by J.N. Buah:
Zhijian Li, Abdoulaye Traore, Siela Maximova and Mark J. Guiltinan, 1998. Somatic Embryogenesis and plant regeneration from floral explants of Cacao (Theobroma Cacao L.) using thidiazuron. In Vitro Cell. Dev. Biol. -- Plant 34: 293-299.
INTRODUCTION
Cocoa, Theobroma cacao L. is a tropical perennial tree, endemic to lowland rainforests and has been domesticated since pre-columbian times (Hurst et al., 2002). The crop has been cultivated in Ghana since, 1887 and has since become the primary cash crop for the country. According to statistics, approximately 2.76 million metric tons of dried cocoa seeds were produced annually with an export value of $3.86 billion (Wakeling, 1996). In Ghana, the crop contributes about 30% of the nation’s export revenue. Ghana was the world’s leading producer of cocoa in the 1990 accounting for 30-40% of the global market (Bateman, 1990). This position was however lost because yields started to decline. One of the factors that accounted for the decline was trees that gave low yields due to their genetic traits. This was due to the fact that farmers used seeds from their previous harvest to establish new farms and this led to segregation over the years. Edwin and Masters (2005) have said that, using recently released varieties is associated with at least 42% higher yields and genetic improvement accounts to some extent for yield.
Genetic improvement of cocoa has however been hampered by the narrow genetic base and the long term breeding cycle of the crop (Edwin and Masters, 2005). The vegetative clonal propagation of superior cocoa genotypes has long been recognized as a potential means to increase cocoa production (Wood and Lass, 1987). However, progress in the development of improved methods for vegetative propagation of cocoa has been slow. Currently, cocoa trees are reproduced primarily by seeds and plagiotropic cuttings. The seeds are usually produced through open pollination and this leads to a highly heterozygous genetic plants.
Although, efforts have been made to develop organogenesis-based propagation methods, cocoa has remained recalcitrant to in vitro shoot regeneration and plant propagation methods (Orchard et al., 1979; Flynn et al., 1990; Figueira and Janick, 1995). Early attempts to develop a somatic embryogenesis-based system for cocoa propagation focused on direct embryogenesis from immature zygotic embryos (Esan, 1975; Pence, 1989). Although, somatic embryos were obtained from zygotic embryo-derived tissues, the conversion or germination of these somatic embryos into viable seedlings was problematic (Wang and Janick, 1984). More recently, efforts were made to induce somatic embryos from floral and nucellar somatic tissues (Sondahl et al., 1989; Alemanno et al., 1996, 1997). In spite of the current progress in this area, the reported efficiencies of so matic embryogenesis and plant regeneration obtained remain low. Furthermore, the practical utilization of this technology for clonal propagation remains hindered by an inability to induce somatic embryogenesis from a majority of elite cocoa genotypes. For these applications to be technically and economically feasible, it is essential to optimize the system variables to obtain high multiplication rates of quality somatic embryos. This is in view of the fact that different cocoa genotypes react differently to different callus-inducing hormones (Traore and Guiltinan, 2006). Thidiazuron has recently received attention as a hormone that is capable of inducing somatic embryogenesis in many tree crops.
This study therefore seeks to evaluate the callusing ability of some elite cocoa genotypes in Ghana, using different concentrations of Thidiazuron.
MATERIALS AND METHODS
Unopened immature flower buds about 4 to 5 mm long from five different genotypes of field grown cocoa plants were collected in November 2008 from the Cocoa Research Institute of Ghana, Tafo. The collection was done early in the morning. The explants preparation protocol used by Da Silva et al. (2008), Young et al. (2003) was followed. Immature flower buds were surfaced sterilized by immersion in 70% (v/v) ethanol for 1 min, 30 sec, followed by 20 min in 2.5% (v/v) sodium hypochlorite solution containing 0.1% (v/v) Tween 20 and then five rinses in sterile distilled water.
Explants were prepared by briefly blotting the immature flowers with sterile paper towels and slicing them perpendicular to their longitudinal axis at a position 1/3 of the flower length from the base, using a sterile scapel blade. Staminodes were extracted from the upper part of the flower bud and placed on the culture medium. Stamonides were preferred to petals because in previous works, petal explants had performed poorly compared to the staminodes.
Chemicals for medium preparation were purchased from Sigma Chemical Co., St. Louis, MO or Fisher Scientific, Pittsburg, PA. Staminodes were first cultured on Primary Callus Growth (PCG) medium, that contained DKW basal salts as described by Li et al. (1998) supplemented with 250.0 mg glutamine L-1, 200 mg myo-inositol L-1, 2.0 mg thiamine HCL L-1, 1.0 mg nicotinic acid L-1, 2.0 mg glycine L-1, 20.0 g glucose L-1, 9 μM 2,4-D, various concentrations of Thidiazuron (TDZ) and 2.0 g Phytagel L-1. Plastic petri dishes (100x15 mm) containing 30 mL of medium were used as culture vessels. All culture media were adjusted to a pH of 5.8 with 1 M KOH and autoclaved at 121°C for 20 min.
Cultures were maintained in the dark at 25°C for 14 days. Various concentrations (0.0, 25.6, 45.5, 116.8, 227.4 nM) of TDZ in combination with 9 uM 2,4-D in the primary callus growth medium were evaluated for their ability to stimulate callus growth and somatic embryo production with five cocoa genotypes. Each treatment contained 25 staminodes per culture vessel, with five replicate vessels.
After 14 days on PCG medium, explants were transferred onto Secondary Callus Growth (SCG) medium and maintained for another 14 days under the culture conditions described above. The SCG medium was composed of basal salts of the low salt McCown’s woody plant medium (Lloyd and McCown, 1980), Sigma m-6774, (Gamborg, 1966) Sigma G-1019, 20 g sucrose/L, 9 uM 2,4-D/L, 1.4 uM kinetin L-1, 50.0 mL coconut water L-1 and phytagel 2.2 g L-1. Somatic embryos were induced by transfer of floral tissue-derived calli onto petri dishes containing 30 mL of Embryo Development (ED) medium. Embryo Development (ED) medium was composed of DKW basal salts, 100.0 mg myo-inositol L-1, 2.0 mg thiamine HC L-1, 1.0 mg nicotinic acid L-1, 2.0 mg glycine L-1, 30.0 g sucrose and 1.0 g L-1 glucose.
Cultures were maintained in the dark at 25°C and sub cultured at 14 days interval. Somatic embryos at the torpedo-shaped stage of development were separated from callus and cultured further on ED medium under similar conditions.
The percentage of embryo-producing staminodes over the total number of cultured explants representing the frequency of embryogenesis, the average number of embryos produced from each responsive staminode were determined 2 months after culture initiation. Data were analyzed and graphs plotted using Excel software.
RESULTS
There was an enlargement of the staminode explants cultured on the Primary Callus Growth (PCG) medium which was followed by the development of compact callus over the explants. Frequently, globular callus clusters appeared over the surface of the explants.
| Fig. 1: | Mean fresh weight (mg) of staminode explants of different cocoa genotypes cultured on PCG medium supplemented with different concentrations of TDZ for 14 days, at 25°C |
Thidiazuron (TDZ) showed evidence of its ability in cell growth stimulation and callus induction. There were fresh weight increases of the cultured staminodes from the five genotypes of cocoa within 14 days after culture initiation (Fig. 1).
Staminode explants cultured on PCG medium without TDZ expanded slightly and generated smaller amount of callus at the cut sites. In contrast, staminode explants of all five cocoa genotypes increased significantly in fresh weight and produced compact callus in most instances when cultured on PCG medium supplemented with TDZ. It was observed that the highest fresh weight figures for all the five genotypes of cocoa tested, were obtained from staminodes which were cultured on medium supplemented with 25.6 nM L-1 and 45.5 nM L-1 TDZ, respectively.
Staminodes of the genotype IMC23 cultured on 25.6 nM L-1 TDZ had a fresh weight of 25.3 mg, which was statistically insignificant but slightly higher than those cultured on a TDZ concentration of 45.4 nM L-1, with a fresh weight value of 21.6 mg. This trend declined with increased TDZ concentration for all the five genotypes. Three of the genotypes, IMC23, Atila 7 and Tei 3 had high fresh weight values and the differences among them was not significant statistically.
The fresh weight of staminodes cultured on the highest TDZ concentration (227.4 nM L-1) for all the five genotypes, were statistically not different from the fresh weight obtained on the control medium.
The Thidiazuron concentration in the PCG medium significantly affected the percentage of explants that developed somatic embryos. Percentage callus induction for the five genotypes ranged from 100% on a TDZ concentration of 25.6 nM L-1 to 7.2% on a TDZ free medium (Fig. 2).
| Fig. 2: | Effect of different concentrations of TDZ (nM) in the PCG medium on percentage mean responsive staminodes from different cocoa genotypes cultured for two months at 25°C |
Percentage callus induction was optimal on medium supplemented with a TDZ concentration of 25.6 nM/L. This was so for all the five cocoa genotypes but these were not statistically different from the percentage callusing for the five genotypes cultured on a TDZ concentration of 45.5 nM L-1. Percentage callusing values for staminodes cultured on a TDZ concentration of 25.6 nM L-1 and 45.5 nM L-1 for all the genotypes were however significantly different from the rest of the treatments.
Among the three genotypes IMC23, Tei 3 and Atila 7 that gave appreciable callusing percentage, Tei 3 had the highest of 100% callusing followed by IMC23 and Atila 7 which had 96.7 and 82.4%, respectively. The differences between Tei3 and IMC23 were however not significant statistically.
It was noted that IMC23 cultured on TDZ concentration of 25.6 nM L-1 had the highest fresh weight among the five cocoa genotypes tested but in terms of percentage callusing, Tei 3 was superior. Again, in terms of fresh weight, staminode explants cultured on PCG medium without thidiazuron and those cultured on medium supplemented with highest level of TDZ (227.4 nM L-1) were not statistically different. However, in terms of percentage callusing, staminodes cultured on medium without TDZ were statistically superior to those cultured on the two higher TDZ concentrations. For example, Atila7 cultured on TDZ free medium had a callusing percentage of 40.1 whilst the staminodes on a TDZ concentration of 116.8 and 227.4 nM L-1 had values of 15.0 and 9.3%, respectively.
| Fig. 3: | Effect of different concentrations of TDZ in the PCG medium on the mean number of Somatic embryos from staminode explants cultured on ED medium for 2 months at 25°C |
Somatic embryogenesis for all the five cocoa genotypes were reduced when explants were cultured on PCG medium containing more than 25.6 nM L-1 of Thidiazuron (Fig. 3).
More embryos were formed from staminode explants cultured on medium supplemented with 25.6 nM L-1 TDZ and among the five genotypes, IMC23 had the highest number of staminodes (110.0 ) forming embryos, followed by Atila 7 and Tei 3 which had 101.0 and 94.0, respectively, even though the difference among the three top genotypes was not statistically significant. However, the number of embryos obtained from staminodes cultured on a TDZ concentration of 25.6 and 45.5 nM L-1 were statistically different from all the other treatments.
Percentage callusing was higher on the TDZ free medium than those cultured on the two highest concentrations of TDZ however, in terms of the number of embryos, staminodes cultured on medium with TDZ concentrations of 116.8 and 447.4 nM L-1 had significantly more embryos than staminodes on medium without thidiazuron.
DISCUSSION
An important step for the successful clonal propagation of plants through somatic embryogenesis is the availability of a culture procedure that would ensure somatic embryo production. Several previous reports showed the production of somatic embryos from various somatic tissues of cocoa but the efficiency of these procedures was low (Sondahl et al., 1989, 1993). The reported rates of primary somatic embryo production from the culture of a large number of petal and nucellar explants were 4.3 and 2.0%, respectively (Sondahl et al., 1993). In some other work by Alemanno et al. (1996), only five out of twenty-five tested cocoa genotypes were capable of producing somatic embryos while the rest remained unresponsive and failed to produce any embryos.
In previous studies on somatic embryogenesis in cocoa, MS medium, Murashige and Skoog (1962) was usually used as the main source of inorganic nutrients (Pence, 1989; Figueira and Janick, 1993). Work done by the cocoa research institute previously, using MS medium supplemented with NAA and kinetin, often resulted in reduced growth, rapid senescence and eventually tissue necrosis (data not shown). DKW medium for the in vitro propagation of woody perennial species, provide high concentration of Calcium, Sulphur and Magnesium than the MS medium. These elements are essential for cell differentiation and somatic embryogenesis (Pedroso et al., 1996).
The use of Thidiazuron-supplemented DKW medium in this study, stimulated the rapid growth of embryogenic callus and the development of somatic embryos.
It has been demonstrated in this work that Thidiazuron (TDZ) has the potential of giving a breakthrough to the difficulties previously encountered with the five genotypes of cocoa used in this work, with respect to somatic embryogenesis. Thidiazuron was developed in 1976 as a cotton defoliant (Arndt et al., 1976) and its effect on the induction of leaf abscission was believed to be mediated by an increase in endogenous ethylene production (Suttle, 1985). Subsequent studies demonstrated that TDZ, a phenylurea derivative, possesses a strong cytokinin-like activity exceeding that of most other commonly used adenine-type cytokinins including Zeatin, Benzylaminopurone and kinetin possibly due to its capacity to stimulate endogenous cytokinin biosynthesis or alter endogenous cytokinin metabolism (Mok et al., 1982). Thidiazuron (TDZ) is highly resistant to degradation by cytokinin oxidase (Mok et al., 1987). Xue-Lin (2006) has also reported that TDZ has the unique property of mimicking both auxin and cytokinin effects on growth and differentiation of cultured plants. Recently, Murch et al. (1997), revealed that TDZ treatment could result in significant changes in tissue accumulation of minerals including Manganese, Iron, Copper, Calcium, Magnesium and Potassium and these elements are important in somatic embryogenesis making TDZ a good option for somatic embryogenesis.
Thidiazuron has been used to induce somatic embryogenesis, adventitious shoot formation and axillary shoot proliferation in numerous crop genera in including woody plant species (Huetteman and Preece, 1993; Lu, 1993). TDZ has however not been tested on cocoa genotypes in Ghana.
The results of this study has established that a TDZ concentration of 25.6 nM L-1 was optimal in inducing high somatic embryos (Fig. 3) from staminodes of the five cocoa genotypes. This concentration is about 400 fold less than that commonly used in other studies (Gill and Saxena, 1992; Gray et al., 1993; Lu, 1993) even though they worked on different plants. Our results have also indicated that TDZ concentrations higher than 45.5 nM L-1 reduced callus growth and embryo production and this became pronounced as the concentration exceeded 116.8 nM L-1 (Fig. 1, 3). The fresh weight of callus also followed a similar pattern. This finding is in line with what has been observed by Li et al. (1998) who also reported reduced callus growth of cocoa genotypes on similar TDZ concentrations. The reduced effect of high TDZ concentrations may be an indication of high sensitivity of cocoa tissues to elevated levels of ethylene that can be induced by TDZ ( Lu, 1993; Li et al., 1998).
In some other works however, callus has been induced in other plants including cocoa, on higher concentrations of TDZ. Kondamudi et al. (2010), Vila et al. (2003), Abd Elaleem et al. (2009) and Nakajima et al. (2000) have all reported somatic embryogenesis on higher concentrations of TDZ. Huetteman and Preece (1993) have also reported that high concentration of TDZ induces formation of somatic embryos but low concentrations induces shooting instead of callusing.
There were clear differences in the callusing abilities and the number of somatic embryos among the five genotypes (Fig. 1, 3). In terms of fresh weight of the callus, IMC23 and Atila7 had the highest fresh weight on medium supplemented with a TDZ concentration of 25.6 nM/L and the two genotypes also had more number of embryos compared to the rest of the genotypes.
Li et al. (1998) have reported differences in somatic embryogenesis among different cacao genotypes. Such differences have also been confirmed in other crops by Ombori et al. (2008), Ali et al. (2007), Zouzou et al. (2008) and Thawaro and Te-Chato (2009). The somatic embryo formation among different genotypes may be due to differences in sensitivity of plant tissues to callusing (Zouzou et al., 2008).
Even though the explants cultured on TDZ-free medium showed high percentage responsiveness to callusing (Fig. 2), compared to those on high TDZ concentrations (116.8 and 227.4 nM L-1), few somatic embryos were formed from explants cultured on the TDZ-free medium. This is an indication that Thidiazuron (TDZ) is plays a key role in producing somatic embryos from callus. This has been reported by Murch et al. (1997) who attributed it to TDZ’s ability to bring about changes in tissue accumulation of minerals such as magnesium and calcium that are necessary for the formation of somatic embryos. The efficiency of somatic embryo production achieved with the established procedure offers an opportunity for the practical use of somatic embryogenesis in the clonal propagation of cacao genotypes.
CONCLUSION
It is concluded that the use of Thidiazuron at a concentration of 25.6 nM L-1 in the primary callus growth medium enhances the induction of callus as well as optimizing the number of embryogenic callus formed form staminode explants. The efficiency of somatic embryogenesis achieved with the established procedure offers an avenue for the practical use of somatic embryogenesis for the clonal propagation of cacao genotypes.
ACKNOWLEDGMENT
The support of Cocoa Research Institute of Ghana in providing the plant materials, chemicals and the laboratory facilities for the work is much acknowledged.