In vitro regeneration protocol development via callus formation from stem explant of tomato

The experiment was conducted on in vitro regeneration of tomato at the Plant Biotechnology Laboratory, Department of Horticulture, Patuakhali Science and Technology University, Patuakhali. The objective was to develop an efficient regeneration protocol in tomato through callus induction for subsequent plantlet regeneration. Seeds were inoculated on MS medium where germination rate was 78.4%. The stems of in vitrocultured seedlings were used as explants. Different concentrations and combinations of growth regulators were added to MS medium to observe their efficacy on callus induction, shoot initiation and root formation. Stem explants cultured on MS medium fortified with 2 mg/L BAPgave the highest number of shoots (3.0) at 45 DAC. Among the concentrations of PGRs, 0.25 mg/L IAA produced the highest length (4.064 cm) of plantlets, number (5.0) of leaves and fresh weight (0.663 g) of plantlets with the stem explants at 45 DAC. The concentration of 0.5 mg/L IAA produced the highest number (21.00) of roots/plantlet, length (7.676 cm) of roots at 45 DAC, from the same explants. The highest survival rate of in vitro regenerated plantlets in the pot was 70.00 % with the stem explants. The results of the current study showed significant increase in the growth of callus of Solanumlycopersicon Mill. Indicating a good efficiency of the optimized media composition and the experimental model used in comparison to other studies of similar nature.


Introduction
Tomato (Solanumlycopersicon Mill.) belonging to the family Solanaceae, is one of the most popular, important and nutritious vegetables in the world.Tomato is considered as the second most popular and highly nutritive vegetable crop after potato (Mamidala and Nanna, 2011) and is a model species for introduction of agronomically important genes into dicotyledonous crop plants (Wing et al., 1994).It is one of the most important protective foods as it possesses appreciable quantities of vitamins and minerals and sometimes rightly referred to as the poor men's orange (Devi et al., 2008).Hundred grams of edible parts of tomato contains 0.9 g protein, 0.1 g fat, 0.7 g fibre, 3.5 g carbohydrates, 15-20 calorie energy, 500-1500 IU vitamin A, 0.1 mg thiamine, 0.02 mg riboflavin, 0.6 mg niacin, 20-25 mg vitamin C, 6-9 mg calcium and 0.1-0.3mg iron (Uddinet al., 2004).Tomato is also an excellent source of lycopene (approximately 20-50 mg /100g of fruit weight), a powerful antioxidant in the carotenoid family which protects humans from free radicals that degrade many parts of the body; lycopene is also known to prevent cancer (Rao and Agarwal, 2000).Tomato is cultivated all over Bangladesh due to its adaptability to wide range of soil and climate (Ahamedet al., 1995).The technique of plant tissue culture has been emerged as a new and powerful tool for crop improvement like potato (Shahriyaret al., 2015).The plant regeneration in tomato is genotype, explant, growth regulator and medium dependent.Many kinds of plant growth regulators are used with varying concentrations for tomato regeneration.Different types of cytokinin and auxin combinations could induce callus induction in tomato from different source of explants (Mohamed et al., 2010).Furthermore, the hormones auxins and cytokinins have a multitude of complex interactions, which control plant development.The hormonal balance between auxins and cytokinins can regulate the formation of roots, shoots and callus tissue in vitro.There are, however, considerable difficulties in predicting the effects of plant growth regulators.This is because of the great differences in culture response between species, cultivars and even on the type of tissue in which the interaction occurs.In vitro regeneration of tomato is a subject of research because of the commercial value of the crop and its potential of amenability to further improvement.Efficient plantlet regeneration in tomato was reported from meristems (Mirghis et al., 1995), leaf (Ajenifujah-Solebo et al., 2012), stems (Liu et al., 2003), anthers, root (Singh and Bezei, 2002), shoot tip (Selvi and Khader, 1993) and hypocotyls (Mamidala and Nanna, 2011) in other countries.But very little studies are attempted in Bangladesh on protocol development for high frequency plant regeneration of tomato.Tomato seed production program in Bangladesh is practiced with imported virus free seeds which are expensive.It is possible to bring down the cost of production by developing virus free seeds through tissue culture.Moreover, maintenance of valuable germplasm in disease free conditions may be obtained by meristem culture.But a standard tissue culture technique for tomato with suitable explants and plant growth regulators in Bangladesh is yet to be established.On the above mentioned perspective, the present study was undertaken to develop a suitable protocol for in vitro regeneration of tomato plantlets via callus formation of stem explants.

Materials and Methods
The present research work was conducted at the Plant Biotechnology Laboratory, Department of Horticulture, Patuakhali Science and Technology University.The seeds were collected from the Regional Horticulture Research Station (RHRS), Lebukhali, Patuakhali.Winter variety of BARI tomato-14 was used as the plant material.Stem from in vitro grown tomato plants were cultured on MS medium and used as explants.P H of the medium was adjusted to 5.8 with 0.1 N NaOH or 0.1 N HCI.All the media were autoclaved for 20 minutes with 15 psi at 121°C.Collected tomato seeds were washed in tap water and surface sterilized with 70% ethanol for five minutes with vigorous shaking followed by washing with sterile distilled water, surface disinfected with sodium hypochlorite 5.25% for 10 minutes and rinsed 4-5 times with sterile water.Then they were washed with Tween 20 for 2-3 minutes and rinsed with sterilized water till the foam was completely removed.The surface sterilized seeds were then allowed to soak overnight to break dormancy.Then the seeds were placed in test tubes containing 10 ml MS medium and later transferred to growth room at 25 ± 1°C temperature under 16 hours photoperiod with a light intensity 1500 lux and relative humidity 60-70%.Sterilized seeds were placed onto seed germination medium in test tubes.In each test tube, 4 seeds were inoculated.The culture was then incubated in incubation room till the germination of seeds.It was noticed that seeds started growing in dark and later they were transferred to light.Thirty days old seedlings were used as the source of explants.

Callus proliferation
The seedlings raised in vitro culture were used as the source of stem explants.Stemparts were placed on the sterile culture medium with various concentrations and combinations of BAP (1, 2, 3 mg/L) and NAA (.25, .5 mg/L) and subsequent fresh weight and dry weight of the callus and changes in colors were recorded visually after 15, 30 and 45 DAC.

Dry weight of the callus
The calli were kept in an oven (Model no.: NIIVE FN-400) for drying for 72 hours at 50 0 C after taking fresh weights.After 72 hours, dried calli were weighed and the means were calculated.

Subculture of the callus for shoot regeneration
When the calli turned into green to yellow color, those were removed aseptically.The pieces were again cultured on freshly prepared medium supplemented with 0, 0.5, 1, 2 and 3 mg/L BAP for shoot induction from callus and subsequent fresh weight and number of shoot were recorded after15, 30 and 45 DAC.

Number of shoots /plantlet
The number of shoots emerged in each cultured bottle was calculated by counting the number of shoots emerged.The data were recorded at 15 days of interval up to 45 days of culture and the means were calculated.

In vitro plantlet regeneration with leaves
Initially 1.5 cm of plantlets were transferred to ½ strength MS media containing 0.0, 0.1, 0.25, 0.50, 1.0 mg/L IAA and average length of plantlets and number of leaves per plantlet were counted at 15, 30 and 45 DAC.

Subculture of the shoots for root induction
The sub cultured calli continued to proliferate and differentiated into shoots.When these shoots grew about 2-3 cm in length, those were rescued aseptically from the bottle and were separated from each other and again cultured on freshly prepared half strength MS medium containing 0.0, 0.1, 0.25, 0.50, 1.0 mg/L IAA supplements for root induction.Number of roots/plantlet formed was recorded at 15 days interval up to 45 days of culture and the means were recorded.The length of root was also measured at 45 days of culture using a scale and the mean values of data provided the root length.After 45 days of inoculation, the fresh weight (g) of plantlet was taken with the electric balance.Plantlets of the 5-7 cm length with well-developed roots were removed from culture vessel with the forceps and transferred into a small earthen pot containing garden soil, sand and well rotten cowdung at the ratio of 1:2:1.The pots were kept in the laboratory and irrigated regularly at an interval of 2 days.The plantlets established within 5 to 7 days and the polythene bags were removed.

Statistical analyses
Data collected on different parameters under study were statistically analyzed to ascertain the significance of the experimental results.The Analysis of Variance was performed and means were compared by Least Significant Difference (LSD) test for interpretation of results.The significance of the difference between the pair of means was evaluated using MSTAT-C computer package programs.

Results
An experiment was conducted to assess the performance of the stem explants of tomato for callus induction and plantlet regeneration.

Seed germination
After surface sterilization, seeds were inoculated on plain MS medium.The germination rate was 78.4 wherein 2.4% seeds were contaminated and remaining was unable to grow.

Callus proliferation from explants
The effects of different concentrations and combinations of plant growth regulators (PGRs) in MS medium for stemexplants of tomato (var.BARI tomato-14) for callus proliferation was observed.

Fresh weight of callus
The fresh weights of calli were recorded at 15, 30 and 45 days after culture (DAC) of stem explants.The maximum fresh weights of calli was 0.638, 1.305 and 2.061g produced by stem explants at 15, 30 and 45 DAC at 3 mg/L BAP + 0.25 mg/L NAA (Table 1).The fresh weights of calli varied significantly due to different concentrations and combinations of PGR at all observing dates.The minimum fresh weights of calli (0.373, 0.971 and 1.268 g) were produced in control (hormone free medium) at 15, 30 and 45 DAC, respectively.

Dry weight of callus
The dry weights of calli varied significantly due to different concentrations and combinations of PGRs.The stem explants cultured on the MS medium containing 2 mg/L BAP + 0.25 mg/L NAA produced the maximum dry weight (0.1957 g) of callus and the minimum dry weight (0.2050 g) of callus was in control (hormone free medium).

Changes in colors of explants
After inoculation of explants to culture media, the color changes of stem segments were observed gradually with the advancement of culture period.The extent of color change was recorded at l5, 30 and 45 DAC (Figure 1).After 45 days of inoculation, the stem explants became yellow at 1 mg/L BAP + 0.5 mg/L NAA, 2 mg/L BAP + 0.25 mg/L NAA and 3 mg/L BAP + 0.25 mg/L NAA treatment and became green at 1 mg/L BAP + 0.25 mg/L NAA, 2 mg/L BAP + 0.5 mg/L NAA and 3 mg/L BAP + 0.5 mg/L NAA combinations of PGRs and the explants became brown at control (Table 2).

Shoot induction from callus
The effects of different concentrations and combinations of BAP on stem explant of tomato for shoot induction were observed.

Fresh weight of callus with shoots
The highest fresh weights of calli with shoots were 0.5810, 1.410 and 2.173 g obtained from stem explants at 15, 30 and 45 DAC, respectively at the hormonal concentration of 2 mg/L BAP (Table 3).There was significant difference among the different concentrations and combinations of PGRs in MS medium in respect of fresh weight of callus with shoots at all sampling dates.The minimum fresh weights of calli with shoots 0.3630, 0.752 and 1.259 g were produced from control (hormone free medium) at 15, 30 and 45 DAC, respectively.

Number of shoots
The maximum number of shoots were 2 and 3.0produced by stem explants at 30 and 45 DAC, respectively at 2.0 mg/L BAP (Figure 2).In the present work, the number of shoots gradually increased with the advancement of culture duration in all hormonal treatments.The increasing of BAP concentration up to 2 mg/L caused the number of shoots to continue developing, but it fell down in presence of BAP (3 mg/L) that indicates the toxic effect of growth regulators due to their accumulation.

Length of the plantlets
The lengths of the plantlet were recorded at 15, 30 and 45 DAC.The tallest plantlet 0.6210, 1.39 and 4.064 cm from stem explants at 15, 30 and 45 DAC, respectively at 0.25 mg/L IAA (Table 4).The smallest plantlets were recorded at control (hormone free medium).

Number of leaves/plantlet
The highest numbers of leaves (3.5, 4.25 and 5.0) were produced from stem explants at 15, 30 and 45 DAC, respectively on the half strength medium supplemented with 0.25 mg/L IAA and the lowest number of leaves 2.333, 2.50 and 3.25 was for the hormone free medium (control) at 15, 30 and 45 DAC, respectively.

Number of roots/plantlet
Number of roots/plantlet was recorded at 15, 30 and 45 DAC.The highest numbers of roots (13.5, 17.75 and 21.00) were produced from stemex plants at 15, 30 and 45 DAC, respectively on ½ MS medium supplemented with 0.5 mg/L IAA(Table 5).The lowest numbers of roots were observed on ½ MS medium supplemented with 0.1 mg/L IAA.No root formation was observed in control treatment at 15, 30 and 45 DAC.

Length of roots
The results revealed that the explants differed significantly in respect of root length.Stem explants produced the longest root (7.676 cm) at 45 DAC on 0.5 mg/L in ½ MS medium.The shortest root (5.001 cm) was observed on ½ MS medium supplemented with 0.1 mg/L IAA.No root was formed in control (Figure 3).

Fresh weight of plantlets
The highest fresh weight of plantlet was 0.6630 g produced from stem explants at 45 DAC on 0.25 mg/L IAA in ½ MS medium.The fresh weights of plantlets were significantly influenced by the application of IAA.Oppositely, the lowest weight 0.2480 g was produced by control (hormone free medium) at 45 DAC.

Survival rate of regenerated plants
The regenerated plantlets were transferred into pots, which were filled with soil, sand and cowdung (1: 2: 1).The percentage of survival rate of regenerated plants from stemwas 70 (Figure 4).

Callus proliferation from explants
The effects of different concentrations and combinations of plant growth regulators (PGRs) in MS medium for stem explants of tomato (var.BARI tomato-14) for callus proliferation was observed.

Fresh weight of callus
As the maximum and the minimum fresh weight of calli of stem explants were2.061g and 1.579 g at 45 DAC at 2 mg/L BAP + 0.25 mg/L NAA and control respectively.Liu et al. (2003) reported similar results while working with leaf and stem explants with 3 mg/L BAP + 0.25 mg/L NAA.The fresh weights of calli varied significantly due to different concentrations and combinations of PGR at all observing dates.The minimum fresh weights of calli (0.3730, 0.971 and 1.268 g) were produced in control (hormone free medium) at 15, 30 and 45 DAC, respectively.Kayum (2004) observed the best callus formation of tomato with the same concentrations and combinations of PGRs.These findings also support the results of Harish et al. (2010) while working with leaf disc, stem and hypocotyl of six tomato cultivars (Sindhu, Shalimar, CO 3 , PKM, Vaishnavi and Ruchikar) with 0.5 mg/L NAA + 2 mg/L BAP.

Dry weight of callus
The dry weights of calli varied significantly due to different concentrations and combinations of PGRs.The stem explants cultured on the MS medium containing 3 mg/L BAP + 0.25 mg/L NAA produced the maximum (.2270 g) dry weight of callus.Capote et al.,(2000) reported similar results while working with leaf tissue and stem segments of different cultivars with BAP + NAA combinations of PGRs.

Changes of color in explants
After inoculation of explants to culture media, the stem segments showed light yellow appearance at the first sight and gradually became grey and then yellow.The color changes were observed gradually with the advancement of culture period.The results appeared that the color change of inoculated explants also showed clear variation due to different PGRs treatments.Harish et al., (2010) also observed the color change of tomato explants while they worked with tomato for regeneration.The findings of his results support the present experiment.

Fresh weight of callus with shoots
The highest fresh weight of calli with shoots was 2.173 g obtained from stem explants at 45 DAC at the hormonal concentration of 2 mg/L BAP.There was significant difference among the different concentrations and combinations of PGRs in MS medium in respect of fresh weight of callus with shoots at all sampling dates.The minimum fresh weight of calli with shoots was 1.257 g produced from control at 45 DAC.Ugandhar et al., (2012) reported similar results in MS medium supplemented with 2 mg/L BAP.

Number of shoots
The maximum number of shoot was 3.0 produced by stem explants at 45 DAC at 2.0 mg/L BAP.In the present work, the number of shoots gradually increased with the advancement of culture duration in all hormonal treatments.The increasing of BAP concentration up to 2 mg/L caused the number of shoots to continue developing, but it fell down in presence of BAP (3 mg/L) that indicates the toxic effect of growth regulators due to their accumulation.These results are also similar with the findings of Mohamed et al., (2010) who had reported the highest number of shoots in MS medium supplemented with BAP (2 mg/L) and no adventitious shoots was noticed in the control (hormone free medium).

Length of the plantlets
The tallest plantlet was found as 4.064 cm from stem explants at 45 DAC at 0.25 mg/L IAA.The smallest plantlets were recorded at control (hormone free medium).

Number of leaves/plantlet
The highest number (5.0) of leaves per explant was produced from stem explants at 45 DAC on the half strength medium supplemented with 0.25 mg/L IAA and the lowest number of leaves was 2.5 for the hormone free medium (control) at 45 DAC.

Number of roots/plantlet
The highest number of roots 21.00was produced from stem explants at 45 DAC, on ½ MS medium supplemented with 0.5 mg/L IAA.Stem explants showed the most important organogenesis capacity in comparison to cotyledon explants (Majoul et al., 2007).The lowest number of roots was observed on ½ MS medium supplemented with 0.1 mg/L IAA.No root formation was observed in control treatment.Liu et al., (2003) reported that tomato initiated high rooting at the same concentration and produced thick and strong roots.Similarly the highest number of roots/shoot (22.1) was observed on ½ MS medium supplemented with IAA 0.5 mg/L (Osman et al., 2010).Devi et al., (2008) also reported that the best rooting in tomato was obtained on ½ MS medium.

Length of roots
The results revealed that the explants differed significantly in respect of root length.Stem explants produced the longest root (7.676 cm) at 45 DAC on 0.5 mg/L in ½ MS medium.The shortest root (5.001 cm) was observed on ½ MS medium supplemented with 0.1 mg/L IAA.No root was formed in control.This result dissimilar to the findings of Ishag et al., (2009), Osman et al., (2010) and Parmar et al. (2012) who had also observed the longest roots on ½ MS medium supplemented with IAA at 0.5 mg/L.

Fresh weight of plantlets
The highest fresh weight of plantlet was 0.6630 g produced from stem explants at 45 DAC on 0.25 mg/L IAA in ½ MS medium.The fresh weights of plantlets were significantly influenced by the application of IAA.Oppositely, the lowest weight 0.2480 g was produced in control at 45 DAC.

Survival rate of regenerated plants
The regenerated plantlets were transferred into pots, which were filled with soil, sand and cowdung (1: 2: 1).The percentage of survival rate of regenerated plants from stem was 70.

Conclusions
Results of these experiments show the influence and importance of growth regulators on the number of shoots and roots regenerated from tomato stem explants.The in-vitro morph genic responses of in-vitro cultured plant tissues are therefore affected by the different components of the culture media, especially by concentration of growth hormones.These responses are also dependent on cultivar and explants types.

Figure 3 .
Figure 3. Root initiation from stem explants of tomato in ½ MS + 0.25 mg/L IAA.

Figure 4 .
Figure 4. Establishment of tomato plantlets in pots containing a mixture of garden soil, sand and cowdung at the ratio of 1:2:1 from stem explants.

Table 1 . Effect of BAP on stem explants for callus proliferation at different DAC. Concentrations and combinations of PGRs Fresh weight (g) of explants inoculated Fresh weight (g) of callus at different DAC
, values having different letter (s) differ significantly at the 1% level of probability according to LSD. ** denotes significant at the 1% level of probability.

Table 3 . Interaction effects of stem and BAP on the fresh weight of callus with shoot and average number of shoots at different DAC.
, values having different letter(s) differ significantly at the 1% and 5% levels of probabilities according to LSD. **, *, ns denotes significant at the 1%, 5% level and non-significant, respectively.

Table 5 . Interaction effects of stem and IAA in ½ MS medium on the average no. of roots/plantlet, length of roots and fresh weight of plantlets at different DAC.
** In a column, values having different letter(s) differ significantly at the 1% level of probability according to LSD. ** denotes significant at the 1% level of probability.