Research Article

Study of Dichlorophenoxyacetic Acid and 6-Benzylaminopurine Effects on Callus Development in Cucurbita moschata  

Saba Riaz1 , Athar Hussian Shah1 , Saif-ul- Malook2 , Qurban Ali3
1 Department of Botany, GC University Lahore, Pakistan
2 Kunming Institute of Botany, University of Chinese Academy of Sciences, China
3 Centre of Excellence in Molecular Biology, University of the Punjab Lahore, Pakistan
Author    Correspondence author
Molecular Plant Breeding, 2016, Vol. 7, No. 3   doi: 10.5376/mpb.2016.07.0003
Received: 07 Nov., 2015    Accepted: 22 Dec., 2015    Published: 02 Jan., 2016
© 2016 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:

Saba Riaz, Athar Hussian Shah, Saif-ul-Malook, and Qurban Ali, 2016, Study of Dichlorophenoxyacetic acid and 6-Benzylaminopurine effects on callus development in Cucurbita moschata, Molecular Plant Breeding, 7(03): 1-12 (doi: 10.5376/mpb.2016.07.0003)

Abstract

Cucurbita moschata is an important and popular vegetable, used throughout the world due to its taste, nutritional and medicinal values. Plant growth hormones play an important role in the growth and development of crop plants. Callus development of Cucurbita moschata was accessed produced from cotyledon, leaf, hypocotyls and root under the application of auxins dichlorophenoxyacetic acid (2,4-D) and cytokinins 6-Benzylaminopurine (BAP) with different concentrations. It was found that 1.5mg/L BAP+1.5mg/L 2,4-D and 0.5mg/L BAP+1.5mg/L 2,4-D combination was best for callus development from cotyledon, as both gave a callus index of 300 but the time for callusing was 9 and 10 days respectively vs. 2,4-D and BAP (3 mg/L) for callus induction by leaves, 2mg/L BAP+1.5mg/L 2,4-D for callus induction from hypocotyl and from root the combination of 1.5mg/L BAP +3mg/L 2,4-D produced the best results. It was suggested form the results that the combination of optimum concentrations (1.5-3mg/L) of BAP and 2, 4-D may be useful for developing higher callus mass in short time.

Keywords
Cucurbita moschata; Callus; Dichlorophenoxyacetic acid; 6-Benzylaminopurine; Plant growth regulators

Introduction
Cucurbita moschata, contains numerous cultivars of pumpkin and winter squash. It belongs to Cucurbitaceae family and is cultivated in warm areas of all over the world.  It is grown not only as a food crop but also for animal food. Butternut, cushaw crookneck and winter, are most popular cultivars of this family. Cucurbita moschata was initiated in Central America and Mexico, and is extensively cultivated in US (Kocabas et al., 1999). Theplants are frost-intolerant annuals with bald stem and climbing nature growing up to 3 meters. The fruit of Cucurbita moschata is big with extensive range of shapes and seed are 16-20 mm long. The name Cucurbita derives from the Latinword which means gourd or pumpkin, and moschata means “musk-scented”, derived from Italian word and literary it means plants bearing squash having elongated recurved necks and hard rinds. Plants have been used in folk medicine widely and maximum number of traditional medications were derived as of nearby growing wild plants which are locally available. The usages of the plants was hoarded by trial and error and approved to generations verbally. Global markets are rotating to plants as a basis of elements in healthy foods. C. moschata has wide range of pharmaceutical uses meanwhile centuries. Seeds are heated and eaten to kill caterpillars and other parasites and used as a diuretic, a research from the flowers used to treat smallpox and measles and C. moschata seeds are from time to time used as a natural worming agent for goats and sheep by farmers. The pharmacological and chemical properties of C. moschata extracts from its stalks, fruits and seeds have been examined. These researches confirmed that C. moschata has wide-ranging bioactivities, such as anti-diabetes, hepatoprotection, anti-cancer, and anti-obesity properties (Magdeleine et al., 2011). It is used as a medicinal plant for bladder problems and prostate, and as an anthelmintic, anti-emetic and galactogogue. It have wonderful medicinal properties such as anti-HIV, anti-diarrhoeal, anti-diabetic, antibacterial, laxative, carminative, anxiolytic, anti-pyretic, antioxidant, anthelmintic, anti-tuberculosis, and purgative (Zhang et al., 2012). Plant tissue culture seems to be a possible option for the enhancement of the Cucurbita moschata by means of improvement in secondary metabolites. In vitro plant regeneration practice appropriate for secondary metabolite production is the main step in this approach. Plant tissue culture practice among vegetative proliferation practices may successfully be useful among many gourd species of the Cucurbitaceae family. Callus culture in plant tissue culture might give certain benefits over outdated approaches of propagation of secondary metabolites of Cucurbita moschata. In Cucurbitaceae, regeneration of plants in vitro had reported in Cucurbita pepo L. (Ananthakrishnan et al., 2007), Cucurbita maxima L., Cucumis melo L. and Cucumis sativus L. (Abrie and Staden, 2001) Cucurbita moschata (Chee, 1991; Gonsalves et al., 1995; Kintzios et al., 2002; Leljak-Levani et al., 2004) and Riaz et al. (2015).
 
1 Materials and Methods 
Seeds of Cucurbita moschata collected from the Seed Certification department, Lahore and stored at 10°C. Seeds of Cucurbita moschata were germinated in in vitro aseptic conditions. Seeds were subjected to surface sterilization with 0.1% aqueous HgCl2 and thoroughly rinsed 5-6 times with autoclaved distilled water under sterilized conditions in laminar air flow cabinet which was sterilized by spraying alcohol (spirit) and Ultra violet rays treatment for 15 minutes. For the germination of seeds, culture vessels were used. Cotton pads soaked with 15 mL distilled water was used and placed in the culture jar. The culture vessels were tightly sealed and autoclaved. After autoclaving the culture vessels, shifted to laminar air flow cabinet. Three seeds per culture jar were used for seed germination response. After that culture jars were placed in growth chamber having 25 ± 3°C temperature for seed germination of Cucurbita moschata.MS medium (Murashige and Skoog, 1962) with different plant growth regulators (PGRs) were used for explants culturing. MS medium was prepared by mixing organic and inorganic compounds from stock solutions in calculated amount from stocks. Tasaki (1985) scheme was followed for making concentrated stock solutions.
 
1.1 Tasaki scheme of stock solution preparation:
 
 

 

1.2 Plant growth regulators (PGRs)
PGRs added essentially in MS media as given below.
AUXINS:
2,4-D (dichlorophenoxyacetic acid)
 
1.3 Cytokinins
BAP (6-Benzylaminopurine)
 
1.4 Preparation of PGRs stock solutions
2,4-Dichlorophenoxyacetic acid 1.0mg/mL:100mg 2,4-D was dissolved in 10mL ethyl alcohol with distilled water addition gradually to make the solution clear. Stock volume was raised upto 100mL and refrigerated.
 
6-Benzylaminopurine 1.0mg/mL:
100mg BAP was dissolved in 10mL 0.1N HCl by heating. Stock volume was raised upto 100mL using warm distilled water in a measuring cylinder and refrigerated.
 
1.5 Scheme for combinations of PGRs

 

 

 

2 Results and Discussion
Analysis of varianceof all combinations having copious amount of callus with 300 callus index was considered to be most effective in thirty six combinations. The results of these values were subjected to statistical analysis; one way ANOVA and DMRT was applied and result were significant at p ≤ 0.05 (Appendices 1-4). The optimum value of cotyledon callus for days to callusing was more significant.
 
2.1 Callus induction and development
Effect of different concentrations of BAP and 2,4-D for callus developed from cotyledon of C. moschata
The effect of range of different combinations of BAP and 2,4-D on cotyledon for callus induction was studied (Table 1). Different parameters were studied i.e. callus colour with texture, duration of callus initiation, and callus index. PGR (plant growth regulators) strength ranged from 0.5 to 3mg/L for both with a variable of 0.5. PGR Combinations were developed by using the strength mentioned.  Nearly all combinations of BAP and 2, 4-D produce callus with callus index 300 except few. Copious callus produced with brown colour in MS medium +1.5mg/L BAP and 0.5mg/L 2,4-D. Initially callus colour found, was white and then turns to brown which was granular in texture. Callus formation started from margins and a mass of cells was produced in 14 days. 2mg/L BAP +0.5mg/L 2,4-D enhanced production of the callus and formed light brown colour callus in 13 days with granular callus texture. Green callus was produced by 3mg/L +1mg/L 2,4-D within 10 days with copious amount, with granular form. Brown callus found with combination of 0.5mg/L BAP +1.5mg/L 2,4-D which was compact in nature callus percentage response was 100 (Figure 1). Copious amount of callus has been produced by 1mg/L BAP +1.5mg/L 2,4-D with compact yellow green callus. Duration for callus initiation was 10 days. 1.5mg/L BAP +1.5mg/L 2,4-D formed whitish yellow callus in 9 days. Medium combination with 1.5mg/L BAP +1.5mg/L 2,4-D produced more callus than any other combination of phytohormones (Figure 2). 2mg/L BAP +1.5mg/L 2,4-D combination also produced mass of yellow callus. Yellow brown callus was produced in the combination of 2.5mg/L BAP +1.5mg/L 2,4-D in 14 days and also formed copious amount of callus. Moderate amount of callus produced in Conc. of 1.5mg/L BAP +2mg/L 2,4-D in 17 days, light yellow and granular callus formed. Callus induction was found 67% and the callus index was 133. Results observed with 3mg/L BAP +2.5mg/L 2,4-D were as like 2.5mg/L BAP + 2.5mg/L 2,4-D. The calluses with both combinations were light yellow and compact. Whitish brown callus has been formed by the effect of 0.5mg/L BAP +3mg/L 2,4-D and 100% explants produced callus. 1mg/L BAP +3mg/L 2,4-D formed 100% yellow callus. 1.5mg/L BAP +3mg/L 2,4-D formed moderate amount of callus. 2mg/L BAP + 3mg/L 2,4-D produced yellow callus in 19 days. 2.5mg/L BAP +3mg/L 2,4-D showed copious amount callus in 15 days, callus was friable and yellow in colour (Figure 3).

 

 

Table 1 Effect of different concentrations of BAP and 2,4-D for callus developed from cotyledon of C. moschata

Note: All these values are sum means of three parallel replicates in which ± indicates standard error among the values, which differ significantly at p ≤ 0.05. The optimum value of Duncan for days to callusing is significant of these results in terms of statistical analysis.

  

 

Figure 1 Brown and compact callus developed from cotyledon of C. moschata in medium MS + 0.5 mg/L BAP and 1.5 mg/L 2,4-D (22 days old) (1x)

  

 

Figure 2 Whitish Yellow and granular callus developed from cotyledon of C. moschata in medium MS + 1.5 mg/L BAP and 1.5 mg/L 2,4-D (20 days old) (1x)

 

 

Figure 3 Yellow and friable callus developed from cotyledon of C. moschata in medium MS + 2.5 mg/L BAP and 3 mg/L 2,4-D (10 days old) (1x)

  

Marta et al. (2009) produced Cucurbita moschata cultivars which are profitable in the regeneration of plants and fruits. In this report reproducible technique for somatic embryogenesis used and tested genotype and source of explants using different PGRs (2,4-D, BAP and 2,4,5-T) concentrations. Embryogenic calli with friable texture was produced by zygotic embryo (56%) and cotyledon (70%) of Cucurbita moschata culture on 0.5mg/L and 2.5mg/L 2,4-D. Cucurbita moschata pure cultivars produced by embryogenic calli (75%) having calli percentage frequency ranged from 5-34%. The results are in accordance with present study. Usman et al., (2011) reported highest response of callus in leaves on medium of 2mg/L 2,4-D, NAA+BAP (1.5mg/L) respectively. Cotyledon showed maximum response (77%) on 4mg/L BAP+0.75mg/L NAA. Highest level of 2,4-D in medium which was 5 mg/L induced embryogenic response and formed embryos. The findings of Leeet et al., (2003), Garcia-Sogo (1990) and Lou andKako (1994) from cotyledons matched with these results.
 
2.2 Effect of different concentrations of BAP and 2,4-D for callus developed from leaf of C. moschata
Nearly all combinations of BAP and 2, 4-D produce callus with callus index 300 except few. Initially the callus colour was white and then turns to brown which was granular in texture. Callus formation started from margins.  0.5mg/L BAP +0.5mg/L 2,4-D formed copious callus in yellow colour developed in 18 days. Copious greenish brown callus developed in Conc. of 1mg/L BAP +0.5mg/L 2,4-D in 16 days. Combination of 2mg/L BAP +0.5mg/L 2,4-D showed less production of mass of cells and formed light yellow colour callus in 12 days. Medium of 2.5mg/L BAP +0.5mg/L 2,4-D produced granular yellow callus in 19 days and callus induction percent was resulted 100%. 3 mg/L BAP +0.5mg/L 2,4-D had produced copious amount of callus with green colour (Figure 4). Copious yellow green compact callus produced with Conc. of 1mg/L BAP +1 mg/L2,4-D in 11 days. Yellow green callus was produced by 3mg/L +1mg/L 2,4-D in 15 days with callus index 33 and compact callus texture. Brown and granular copious amount of callus was produced in 1.5mg/L BAP +2mg/L 2,4-D in 11 days. Yellow callus was produced with 2.5mg/L BAP +2mg/L 2,4-D with induction percentage of 100% in 19 days. 3mg/L BAP +2mg/L 2,4-D formed copious light brown colour callus in 21 days. In combination 0.5mg/L BAP +2.5mg/L 2,4-D gave rise to yellow brown callus which was compact in nature. 1.5mg/L BAP +3mg/L 2,4-D formed callus in copious amount. 2.5mg/L BAP +3mg/L 2,4-D formed granular light brown copious callus in 10 days (Figure 5).

 

 

Figure 4 Green and granular callus developed from leaf of C. moschata in medium MS + 3 mg/L BAP and 0.5 mg/L 2,4-D (16 days old) (1x)

 

 

Figure 5 Light brown and compact callus developed from leaf of C. moschata in medium MS + 2.5 mg/L BAP and 3 mg/L 2,4-D (12 days old) (1x)

   

3mg/L BAP +3mg/L2,4-D produced yellow brown colour callus which was compact in texture (Table 2). Rakha et al., (2012) reported similar results and suggested that the combination may be used for the development of haploids plant through embryo, anther or ovule culture. The results are also in accordance with the findings reported by Mahzabin et al., (2008); Ali et al., (2014) and Jahangir et al., (2014). These findings were contrary to the study of Punja et al., (1990). Tissert et al., 1995 and Elfahmi et al., 2011 in which callus cultures have been established using plant growth hormones 2, 4-D, BAP and kinetin.

 

 

Table 2 Effect of different concentrations of BAP and 2,4-D for callus developed from leaf of C. moschata

Note: All these values are sum means of three parallel replicates in which ± indicates standard error among the values, which differ significantly at p ≤ 0.05. The optimum value of Duncan for days to callusing is significant of these results in terms of statistical analysis.

  

2.2 Effect of different concentrations of BAP and 2, 4-D for callus developed from hypocotyl of C. moschata
Nearly all combinations of BAP and 2, 4-D produce callus with callus index 300 except few (Table 3). Copious amount of callus formed with 0.5mg/L BAP +0.5mg/L 2,4-D in yellow colour in 11 days. Copious light brown callus was produced in 1mg/L BAP + 0.5 mg/L 2,4-D induced callus in 14 days. Copious callus observed with yellow green colour in 1.5mg/L BAP +0.5mg/L 2,4-D. The effect of Conc. of 2mg/L BAP+0.5mg/L 2,4-D produced copious amount of compact callus and formed light yellow colour callus in 10 days. 2.5mg/L BAP+0.5mg/L 2,4-D produced compact yellow brown colour callus in 15days and callus induction percent was 67% whereas callus index was 133.

 

 

Table 3 Effect of different concentrations of BAP and 2, 4-D for callus developed from hypocotyl of C.

Note: All these values are sum means of three parallel replicates in which ± indicates standard error among the values, which differ significantly at p ≤ 0.05. The optimum value of Duncan for days to callusing is insignificant of these results in terms of statistical analysis

 

Yellow callus turned to dark brown after sub-culturing. 3mg/L BAP+0.5mg/L 2,4-D induced the amount of copious yellow green callus. Copious yellow brown compact callus was produced in Conc. of 1mg/L BAP +1mg/L 2,4-D which was having 17 days. 1.5mg/L BAP +1mg/L 2,4-D formed yellow copious callus in 11 days. Copious amount of yellow green callus was observed in 2.5mg/L BAP and 1mg/L 2,4-D. Granular light brown callus was produced by 3mg/L BAP +1mg/L 2,4-D in 13 days. The concentration of 0.5mg/L BAP +1.5mg/L 2,4-D gave light yellow colour callus that was granular in texture, callus induction percent was 100%. Copious amount callus has been produced by 1mg/L BAP +1.5mg/L 2,4-D with yellow colour callus. The callus texture was granular. Duration for callus initiation was 12 days. 1.5mg/L BAP +1.5mg/L 2,4-D formed green callus in 13 days. 2mg/L BAP +1.5mg/L 2,4-D combination produced copious mass of callus of green brown colour (Figure 6). Conc. of 0.5mg/L BAP +2mg/L 2,4-D formed copious amount of callus and colour was whitish green. 1mg/L BAP +2mg/L 2,4-D formed brown colour of callus with 100% callus induction. Callus development occurred in 11 days (Figure 7). Moderate amount of callus was produced in the Conc. of 1.5mg/L BAP +2mg/L 2,4-D. callusing occurred in 13 days and yellow brown granular callus formed. Induction of callus was 67% and callus index found was 133. Callus induction with combination 2mg/L BAP +2mg/L 2,4-D was in copious amount. The callus was yellow and compact in 12 days. Light yellow callus were produced with 2.5 mg/L BAP +2mg/L 2,4-D. The callus response percentage was 100%, produced in 14 days. Copious amount of callus growth was found with Conc. of 1mg/L BAP +2.5mg/L 2,4-D that has callus yellow in colour. Yellow callus formed under effect of 0.5mg/L BAP +3mg/L 2,4-D and 100% explants produced callus. 1.5mg/L BAP+3mg/L 2,4-D formed less amount callus of yellow green colour. 2.5mg/L BAP +3mg/L 2,4-D found copious amount of callus in 11 days. Ashutosh et al., (2012) examined highest development of total alkaloid contented was found in and 1mg/L BAP +0.5mg/L 2, 4-D associated with other amalgamations. Everaldo and Anthony, (2001) reported that the use of BAP and 2, 4-D caused the improvement in the plant body weight and growth of the plants. Oswell et al., (2007); Ali et al., (2014); Qamar et al., (2014); Butt et al., (2015) reported similar results. Present results were in conformity with the results of Alsop et al., (1978) and Punja et al., (1990) in which callus induction from hypocotyl explants was induced.

  
 

Figure 6 Greenish brown and granular callus developed from hypoctyl of C. moschata in medium MS + 2 mg/L BAP and 1.5 mg/L 2,4-D (11 days old) (1x)

  

 

Figure 7 Brown and friable callus developed from hypocotyl of C. moschata in medium MS + 1 mg/L BAP and 2 mg/L 2,4-D (22 days old) (1x)

 

2.3 Effect of different concentrations of BAP and 2,4-D for callus developed from root of C. moschata
Effect of different combinations of BAP and 2,4-D on root for callus induction were studied. Nearly all combinations of BAP and 2, 4-D produce callus with callus index 300 except few (Table 4). Combination of 0.5mg/L BAP and 0.5mg/L 2,4-D produced yellow green colour callus with compact texture in 10 days. The callus index for this combination was 133. 1mg/L BAP and 0.5mg/L 2,4-D has given light yellow colour callus with granular texture in 11 days. Moderate amount of callus was produced. Less amount of  callus has been observed with whitish yellow colour in 1.5mg/L BAP +0.5mg/L 2,4-D. Conc. of 2mg/L BAP +0.5mg/L 2,4-D produced copious amount of callus and formed yellow colour callus in 14 days. The callus was granular in texture. 2.5mg/L BAP + 0.5 mg/L 2,4-D produced compact callus of yellow with brown shade. Callusing took place in 18 days, induction of callus was 100%. 3mg/L BAP +0.5mg/L 2,4-D induced copious callus amount. Callus colour was light yellow.Orange green callus formed with 0.5mg/L BAP +1mg/L 2,4-D which was granular in texture. Callus initiation duration was 10 days. The callus produced in lesser amount. Yellow green compact callus produced with 1mg/L BAP +1mg/L 2,4-D in 12 days, copious amount of callus produced. 1.5mg/L BAP+1mg/L 2,4-D formed yellow callus.

 

 

Table 4 Effect of different concentrations of BAP and 2,4-D for callus developed from root of C. moschata

Note: All these values are sum means of three parallel replicates in which ± indicates standard error among the values, which differ significantly at p ≤ 0.05. The optimum value of Duncan for days to callusing is significant of these results in terms of statistical analysis.

  

Concentration of 0.5mg/L BAP +1.5mg/L 2,4-D showed yellow and green callus that was compact in nature,  callus response was 100%. Lesser amount of callus was formed by 1mg/L BAP+1.5mg/L 2,4-D with light yellow colour callus. The callus texture was granular. Less amount of callus was produced in the combination of 1.5mg/L BAP +2mg/L 2,4-D. Callusing occurred in 14 days, light yellow and granular callus formed. Callus induction with combination 2mg/L BAP +2mg/L 2,4-D was copious in amount. The callus was yellow brown and compact. Days for callusing were 12. Combination of 1mg/L BAP +2.5mg/L 2,4-D formed yellow brown callus in copious amount (Figure 8). Yellow Green callus found in Conc. of 2mg/L BAP +2.5mg/L 2,4-D. Copious amount of callus growth observed. 2.5mg/L BAP+2.5mg/L 2,4-D formed copious amount of light yellow colour callus. Moderate amount of callus was found in combination of 3mg/L BAP+2.5mg/L 2,4-D. Callus colour was yellow and compact. Green callus formed under effect of 0.5mg/L BAP+3mg/L 2,4-D and 67% explants produced callus. 1mg/L BAP+3mg/L 2,4-D formed light yellow and compact colour with callus index 33. 1.5mg/L BAP+3mg/L 2,4-D formed copious amount of callus of light brown colour (Figure 9). 2mg/L BAP+3mg/L 2,4-D produced brown callus within 12 days (Figure 10). These findings were supported by observations of Gambley and Dodd (1990), produced callus from cucumber. Pavlov et al., (2007); Irshad et al., (2012); Butt et al., (2015) reported similar results.

 

 

Figure 8 Yellow Brown and compact callus developed from roots of C. moschata in medium MS + 1 mg/L BAP and 2.5 mg/L 2,4-D (10 days old) (1x)

  

 

Figure 9 Light brown and compact callus developed from root of C. moschata in medium MS + 1.5 mg/L BAP and 3 mg/L 2,4-D (12 days old) (1x)

  

 

Figure 10 Brown and compact callus developed from root of C. moschata in medium MS + 2 mg/L BAP and 3 mg/L 2,4-D (13 days old) (1x)

  

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Molecular Plant Breeding
• Volume 7
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