Enhancement of callus induction and regeneration efficiency from embryo cultures of Datura stramonium by adjusting carbon sources and concentrations

Carbon source in the medium is considered to be an essential component for the high production costs of callus and plantlets in tissue culture. We report here the establishment of an efficient tissue culture cycle (callus induction and plant regeneration) for Datura stramonium by adjusting carbon sources and concentrations. Embryo explants of D. stramonium L. were cultured in vitro with six different carbon sources (sucrose, glucose, fructose, galactose, maltose and lactose) and four concentrations (1, 2, 3 and 4%) for callus induction. After that, the best carbon sources for regeneration were investigated with four different carbon sources (sucrose, glucose, fructose and sorbitol) and four concentrations (1, 2, 3 and 4%). The best sugar for callus induction was 2% lactose. The medium containing 3% glucose shows the higher regeneration.


INTRODUCTION
The genus Datura is comprised of 15 species of annuals, trees and shrubs distributed over warm and temperate regions of the world (Chittenden, 1965).Previous studies have demonstrated that Datura is amenable to tissue culture (Sharma et al., 1993).Plant cell, tissue or organ culture normally requires the incorporation of a carbon source to the culture medium (Karhu, 1997).
In living plant cells, carbohydrates are necessary as a source of energy and a carbon substrate for biosynthesis.Continuous supply of carbohydrates to plants cultured in vitro is essential, since photosynthetic activity of in vitro grown tissues is usually reduced.These compounds are also necessary in media as osmotic agents.For all these reasons, sugars have a great potential effect on the physiology, growth and differentiation of cells (Gibson, 2000).Therefore, the optimal carbon source for callus induction and plant regeneration needs to be considered *Corresponding author.E-mail: amiri_s_m@yahoo.com.
Although, sucrose has been the carbohydrate of choice in the vast majority of work on in vitro callus and shoots induction and development in various species, it is not always the most effective carbon source for these purposes (Thompson and Thorpe, 1987).The substitution of the culture medium carbon source by osmotically active solutes has shown that sugars act as a carbon source and as osmotic regulators (Neto and Otoni, 2003).The carbon source osmotic contribution has an inverse relationship with carbon source concentration and in general, causes an initial increase followed by a reduction in the values of the assessed parameter.This reduction can be caused by an excessive osmotic contribution or by toxicity of the carbohydrate (Slesak et al., 2004).
Species-specific carbon sources and carbon concentrations for optimal growth rates have been discovered for various culture systems.It was found that different patterns of morphogenesis were attributable to the type of carbohydrate and its concentration (Romano et al., 1995).What happens to the absorbed carbon depends on the type of sugar in the medium.Blanc et al. (2002) reported that the morphological and biochemical changes reflected a specific physiological state as a consequence of the type of carbohydrate supplied in the medium.
This report, for the first time, details an assessment of the performance of embryos culture of Datura stramonium on a combination of media conditions, with varied carbon sources and concentrations.An efficient plant regeneration system allowing rapid callus induction and plant regeneration from mature Datura embryos by using carbohydrate is described.

Callus induction condition
Immature seeds of surface sterilized fruits were removed.The seed coat encasing the immature embryo was peeled away and the seed was squeezed hard using scalpel handle until the immature zygotic embryo was on the loose.Immature embryos were transferred to MS callus induction medium supplemented with 2 mgl -1 2,4dichlorophenoxyacetic (2,4-D).To test the effect of sugar types, different types of carbon sources, including sucrose, glucose, fructose, galactose, maltose and lactose at 3% (w/v) were added before autoclaving.After determining the best sugar type, the best concentration of sugars was examined at 1 to 4% (w/v) concentration.The experiment was designed in complete randomized design with four replications.All the cultures were incubated in the dark at 25 ± 2°C.

Regeneration condition
To investigate the best type of sugars for regeneration, four types of carbon source including sucrose, glucose, fructose and sorbitol at 3% (w/v) were added to B5 medium supplemented with 2 mgl -1 BAP and 1 mgl -1 NAA.After identifying the best type of sugar, various concentrations (1, 2, 3 or 4% w/v) were also tested.The experiment was performed with 5 replications.After calli culturing in test tubes, they were incubated at 25± 2°C under cool white fluorescent light (35 mol photons m-2 s-1) with 16 h photoperiod.The experiment was performed with 5 replications.PH of all media was regulated between 5.8 and 5.9 by using 1 N NaOH before autoclaving.

Statistical analysis
Analyses were carried out for these characters on transformed data (by using (X+0.5)transformation).The statistical analysis was carried out by using the SAS.Differences were determined by analysis of variance (ANOVA) and the differences between the means were compared by Duncan's multiple range tests using MSTAT-C computer program.

Effects of carbohydrates on callus induction
Carbohydrate type and concentration have been found to play important roles in different stages of the tissue culture processes.

Determine the best type of carbon source
The results of variance analysis related to effects of different carbon sources on frequency of callus were shown in Table 1.Based on this table, the effect of carbon sources on callus induction from embryo explants were significantly different at P < 0.01.From Figure 1, the highest fresh weight of callus was obtained in lactose.The lowest callus formation amount was observed in medium containing galactose.
Based on Table 2, it was known that various concentrations of lactose can significantly affect callus fresh weight (p < 0.01).From Figure 2, 2% lactose produced the most callus fresh weight.The lowest callus fresh weight was related to 4% concentration.

Effects of carbohydrates on regeneration
Regeneration took the form of indirect organogenesis and its frequency was strongly dependent on sugar supply.

Determine the best type of carbon source
The results of Table 3 indicated that sugar types influenced regeneration from embryo-derived calli significantly.Among different carbon sources, the medium containing glucose produced the more shoot buds (Figure 3).It was observed that there is no significant difference between sucrose and glucose.The lowest frequency of regeneration was related to fructose.

Determine the best concentration of carbon source
Analysis of variance (Table 4) showed that different glucose concentrations play important role in producing shoot from embryo-derived calli, so that different concentrations of glucose have influence were significant (p < 0.01).3% glucose allocated the highest regeneration to itself, while the lowest percentage of regeneration obtained at 1% glucose (Figure 4).

DISCUSSION
Experiments demonstrated that the addition of carbohydrates was essential, inducing callus and regeneration of embryo cultures of D. stramonium is affected both type and concentration of sugar in the culture medium (Figure 5).The best type and concentration of carbohydrate were 2% lactose and 3% glucose for callus induction and regeneration, respectively.
The disaccharide lactose has been detected in only a few plants.When added to tissue culture media, it has been found to induce the activity of -galactosidase enzyme which can be secreted into the medium (George    et al., 2008).Similar result were obtained by Chen and Dribnenki (2002) who reported that in comparison to sucrose, lactose was found to increase callus induction from Linum usitatissimum L. anthers for all three genotypes tested.In contrast, Jain et al. (1997) observed media with lactose, manitol or sorbitol supported limited growth or failed to induce any measurable increase in indica and japonica rice varieties callus fresh weight.Plant cell, tissue or organ culture normally requires a carbohydrate supply in order to satisfy energy demands.
Many studies have been conducted to define type and concentration of carbon source that allow culture establishment and development (Karhu, 1997).Al-Khateeb (2008) observed significant difference among the three different concentrations of carbohydrates for callus fresh weight and axillary shoot numbers.Our study indicated that the most callus fresh weight was observed at 2% lactose.This observation confirms data of Short and Warburton, (1987)      The results of the present study showed that the performance of shoot culture of D. stramonium is affected by both the type and concentration of sugar in the culture medium.In general, glucose was better for inducing shoot proliferation than other carbon sources.Similar results were obtained by Salvi et al. (2002) who reported that a significant increase in shoot length and number and length of roots was observed when glucose was added to the medium suggesting that glucose could be the best carbon source for in vitro multiplication of turmeric cv.'elite'.Glucose was the most effective carbon source for both axillary branching and adventitious shoot regeneration in in vitro beech cultures (Cuenca and Vieitez, 2000).
In contrast our results, Baskaran and Jayabalan (2005) suggested among the three carbon sources, sucrose proved to be better than fructose or glucose for shoot regeneration of Eclipta alba.Amutha et al. (2003) reported that glucose, fructose and maltose led to very poor shoot differentiation.They observed sucrose evoked maximum regeneration frequency and increased the number of regenerating shoots.Nowak et al. (2004) results suggested that sucrose was a better carbon source than glucose for organogenesis of 'W¸egierka Zwykła', even though at lower concentrations the efficiency of the sugars was comparable.

Figure 1 .
Figure 1.Effect of carbon source on callus fresh weight (g).

Figure 2 .
Figure 2. Effect of different lactose concentrations on callus fresh weight (g).

Figure 3 .
Figure 3. carbon source influence on percentage of regeneration.

Figure 4 .
Figure 4. Effect of different glucose concentrations on percentage of regeneration.

Table 1 .
Analysis of variance of carbon source effect on callus fresh weight (gr).
** is significant at 0.01 probability level.

Table 2 .
Variance analysis of different lactose concentration influence on callus fresh weight (gr).
** is significant at 0.01 probability level.

Table 3 .
Analysis of variance of carbon source influence on percentage of regeneration.
** is significant at 0.01 probability level.

Table 4 .
Variance analysis of different glucose concentration influence on percentage of regeneration.
** is significant at 0.01 probability level.