Balwinder Singh, Amritpal Kaur and Jaspreet Singh

Received: April 09, 2011;   Accepted: May 20, 2011;   Published: November 15, 2011

Balwinder Singh, Amritpal Kaur and Jaspreet Singh, 2011. Evaluation of Natural Exudate Gum from Sterculia urens as Gelling Agent in Culture Media for In vitro Regeneration of Rough Lemon (Citrus jambhiri Lush.) Shoot Tips. Journal of Biological Sciences, 11: 374-380.

DOI: 10.3923/jbs.2011.374.380

URL: https://scialert.net/abstract/?doi=jbs.2011.374.380

MATERIALS AND METHODS

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Source of explant and gum: Rough lemon plant selected at Govt. Nursery, Department of Horticulture (Punjab), Attari, Amritsar, India was used as a source of explant for raising shoot tip cultures. The bud flushes were induced by defoliating and tipping some shoot branches of a healthy plant in February 2009. These bud flushes were harvested after 10-15 days of tipping and transported to lab in moist bags. Sterculia gum in the form of lumps (Fig. 1a) was purchased from Girijan Cooperative Corporation Ltd., Visakhapatnam, India and stored in airtight polypropylene jars. Gum lumps were powdered using high speed mechanical blender (Bajaj, India) and sieved through 150 size mesh before use.

Explant sterilization and inoculation: Bud flushes (2-3 cm) collected from the healthy rough lemon plants were washed with detergent to remove any external microbes and soil particles. After this they were washed thoroughly under running tap water so as to remove the detergent completely. The sterilization of the explant was done under aseptic conditions in a laminar flow hood. The explants were disinfected with 0.05% aqueous solution of mercuric chloride (HgCl₂) for 4 min and then rinsed three times with sterilized double-distilled water to remove any traces of disinfectant_. Shoot tips (0.5 cm) were excised inside a laminar flow cabinet with the help of sterilized sharp fine scalpel blade.

Shoot regeneration: Sterilized shoot tips of rough lemon were cultured on MS medium (Murashige and Skoog, 1962) gelled with sterculia gum and agar (HiMedia, India) either alone or in combination.

Fig. 1(a-f):

In vitro propagation of rough lemon (Citrus jambhiri Lush): (a) Intact lumps of sterculia gum (b) In vitro regeneration of shoot tip on agar (8 g L-1) gelled medium containing BAP 1.5 mg L-1, KN 0.5 mg L-1 and NAA 0.5 mg L-1 (c) In vitro regeneration of shoot tip on sterculia gum (30 g L-1) gelled medium containing BAP 1.5 mg L-1, KN 0.5 mg L-1 and NAA 0.5 mg L-1. (d) In vitro root regeneration on agar (8 g L-1) gelled medium containing IBA (2 mg L-1) (e) Complete plantlet development after 45 days of culture on sterculia gum (30 g L-1) gelled medium containing IBA (2 mg L-1) (f) Plant development after transplantation of regenerated plantlets into a sand/soil mixture

Initially, the shoot tips were inoculated on MS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar. Different concentrations of BAP, KN and NAA in combinations were tried in agar gelled media to find out the best suitable medium for shoot regeneration (Table 1). The media were adjusted to pH 5.6 with 1N NaOH and autoclaved at 121°C and 15 lb in^-2 pressure for 20 min. After finding out the best suitable concentration of plant growth regulator in MS medium, the sterculia gum (20, 25 and 30 g L^-1 ) alone and in combination with agar (2, 4 and 6 g L^-1) was tested as gelling agent. MS medium supplemented with suitable plant growth regulator and gelled with 8 g L^-1agar served as control in gum gelled experiments. For preparing sterculia gum gelled media, overnight soaked gum and sucrose were mixed with other constituents of the medium and the volume was raised to the required level on a magnetic stirrer before adjusting the pH to 5.6. For all the experiments, 20 mL of culture medium was dispensed in 25x150 mm culture tubes which were closed with cotton plugs. All cultures were maintained at 26±1°C with a luminous intensity of 40 μmole/m²/sec and 16 h photoperiod. For each treatment percentage of cultures showing shoot regeneration were recorded 30 days after initial culturing.

Rooting of regenerated shoot: For rooting, the regenerated shoots were cultured on MS medium supplemented with various concentrations (0.5-2.5 mg L^-1) of Indole-3-Acetic Acid (IAA), Naphthalene Acetic Acid (NAA) and Indole-3-Butyric Acid (IBA) along with 3% (w/v) sucrose. Initially, 8 g L^-1 agar was used as gelling agent to find out the best suitable medium for rooting. After finding out the best suitable concentration of plant growth regulator in MS medium, the sterculia gum (25 and 30 g L^-1) alone and in combination with agar (2, 4 and 6 g L^-1) was used as gelling agent. A total of 48 regenerated shoots were inoculated for individual treatment and the experiment was repeated 3 times. The percentage of cultures showing root regeneration was recorded after 30 days of inoculation.

Hardening and acclimatization: The well-developed plantlets were washed with water in order to remove adhering agar and transferred to autoclaved plastic pots containing a mixture of garden soil, sand and vermiculite in the ratio of 3:1:1. Hardening of potted plantlets was accomplished in a culture room set at 26±1°C, 16 h day-length (40 μmole/m²/ sec) and covered with polyethylene bags to maintain high humidity. After 12-15 days, polyethylene bags were removed initially for a short duration (15-30 min) daily for about one week. Gradually, the daily exposure time was increased by 30 min for each day. Polyethylene bags were completely removed after 20 days; subsequently the plantlets were transferred to the earthen pots containing only garden soil and kept in the polyhouse for one month and there-after transferred to the field conditions.

Statistical analysis: For each treatment, 48 culture tubes were inoculated and the experiments were repeated thrice. The data pertaining to the effect of gelling agent and plant growth regulators on shoot and root regeneration were subjected to one way Analysis of Variance (ANOVA) and the differences among means (p = 0.05) were compared by High-range Statistical Domain (HSD) using Tukey’s test (Meyers and Grossen, 1974).

RESULTS

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Shoot regeneration: Shoot tips of rough lemon cultured on agar gelled (8 g L^-1) MS medium supplemented with different concentrations of plant hormones (BAP, KN and NAA) showed signs of shoot regeneration after 6-8 days of inoculation. The percentage of cultures showing shoot formation was recorded after 30 days of inoculation (Table 1). Among different concentrations of plant hormones tested, maximum percentage of cultures showing shoot formation (63.19%) was observed with MS medium containing BAP 1.5. KN 0.5 and NAA 0.5 mg L^-1 (Fig. 1a). A further increase in concentration of BAP (2.0-3.0 mg L^-1) resulted in a decrease in percent shoot regeneration. Different concentrations (0.5-2.0 mg L^-1) of BAP or KN alone in combination with NAA (1.0 mg L^-1) were not effective for shoot regeneration from shoot tip explants. The effect of different concentrations of sterculia gum and agar, alone and in combination, on shoot regeneration in MS medium supplemented with BAP 1.5, KN 0.5 and NAA 0.5 mg L^-1 is shown in Table 2. The shoot regeneration response on media gelled with 25 g L^-1 sterculia gum was less (49.99%) than those of 30 g L^-1 (61.11%). The culture tubes containing gum gelled medium at a concentration of 20 g L^-1 often resulted in sinking of the shoot tips explant to subsurface levels. Such submerged explants also responded positively indicating that sufficient aeration was available in gum gelled media. The media gelled with sterculia gum was as equally transparent as liquid medium (Fig. 1c). Regenerated shoots had yellow green leaves on gum gelled medium as compared to dark green leaves on agar containing medium. Addition of agar (2 g L^-1) along with gum (25 g L^-1) increased the firmness of the media but not showed significant improvement in shooting response. Shoot regeneration response on gum gelled media along with all the concentrations of agar was less than those of control (agar 8 g L^-1). Among all the concentration of gum tested the maximum shoot regeneration was observed with 30 g L^-1 gum gelled media.

Root regeneration: Regenerated shoots were transferred to the rooting medium gelled with agar (8 g L^-1). The effect of different concentrations of NAA, IBA and IAA (0.5-2.5 mg L^-1) on root regeneration after 30 days of inoculation is shown in Table 3. The initiation of roots took place after 2 weeks of inoculation. In MS medium supplemented with IBA 2 mg L^-1, maximum of 56.94% cultures showed root regeneration (Fig. 1d). A decrease or increase in concentration of IBA resulted in decrease in rooting response. Among IAA and NAA, the later was more effective with a maximum of 35.41% cultures showing root regeneration at 2 mg L^-1 concentration.

Table 1:	Response of shoot tips on agar (8 g L-1) gelled MS medium containing different concentrations of plant hormones
Data shown are Mean±SE of three experiments. Each experiment consisted of 48 replicates. *Significant at p≤0.05. Values followed by the same letter with in a column are not significantly different using HSD multiple comparison test. Observations recorded after 30 days of inoculation

Table 2:	Response of shoot tips on medium (MS medium + BAP 1.5 mg L-1+KN 0.5 mg L-1 + NAA 0.5 mg L-1) gelled with sterculia gum and agar either alone or in combination
Data shown are Mean±SE of three experiments. Each experiment consisted of 48 replicates. *Significant at p≤0.05. Values followed by the same letter with in a column are not significantly different using HSD multiple comparison test. Observations recorded after 30 days of inoculation

The effect of different concentrations of sterculia gum and agar, alone and in combination, on root regeneration in MS medium supplemented with IBA (2 mg L^-1) is shown in Table 4. Rooting response on media gelled with 25 g L^-1 sterculia gum was less (53.46%) than those of 30 g L^-1 (56.24%). Addition of agar (2, 4 and 6 g L^-1) along with sterculia gum (25 and 30 g L^-1) in media resulted in decrease in percentage of cultures showing root formation. The quantitative response obtained on media fortified with gum and agar was not significantly different. The growth and morphology of regenerated roots were similar on both the media.

Table 3:	Rooting response of regenerated shoots in agar gelled MS medium containing different concentrations of plant hormones
Data shown are Mean±SE of three experiments. Each experiment consisted of 48 replicates. *Significant at p≤0.05. Values followed by the same letter with in a column are not significantly different using HSD multiple comparison test. Observations recorded after 30 days of inoculation

Table 4:	Rooting response of regenerated shoots on MS medium supplemented with IBA (2 mg L-1) and gelled with sterculia gum and agar either alone or in combination
Data shown are Mean±SE of three experiments. Each experiment consisted of 48 replicates. *Significant at p≤0.05. Values followed by the same letter with in a column are not significantly different using HSD multiple comparison test. Observations recorded after 30 days of inoculation

The elongation of roots was much better on gum-gelled medium than on agar medium. The plantlets responded positively in gum gelled medium and there was no softening of the medium even after 45 days of culture (Fig. 1e). The plantlets, thus developed where transferred to pots containing a mixture of garden soil, sand and vermiculite for acclimatization (Fig. 1f) which showed 64 % survival rate after transfer to field conditions.

DISCUSSION

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In the present study plantlets were regenerated via direct organogenesis from in vitro cultured shoot tips of rough lemon (C. jambhiri Lush). Direct organogenesis does not involve intervening callus formation and plants produced by direct organogenesis may exhibit greater genetic stability than those produced from callus (Khan et al., 2009). Plantlets were regenerated on sterculia gum and agar gelled medium supplemented with various combinations of cytokinin and auxin. Shoot formation can easily be induced from shoot tips of rough lemon plant on MS medium supplemented with BAP, KN and NAA and subsequently rooted on IBA-containing medium. Role of cytokinin in shoot regeneration has been studied in many plant species (Usman et al., 2005; Zhang et al., 2008) and was considered as an exclusive element for shoot formation. NAA along with BAP and KN was found to be beneficial for direct organogenesis from shoot tips explants. These results are in conformity with some of the earlier studies on citrus (Liu et al., 2000; Ghorbel et al., 2000; Guo et al., 2002) and pineapple (Mercier et al., 2003) where low concentration of NAA along with cytokinins was found to be effective for organogenesis. It was noted that by increasing the concentration of cytokinins the regeneration response was decreased which may be due to toxic effects of high concentration of the hormone (Wang et al., 2007). Among different plant growth regulators tried for rooting, IBA was found to give better results as compared to NAA and IAA. Similar results have been reported by Carimi and De Pasquale (2003) and Perez-Tornero et al. (2010) in Citrus species. On the contrary, there are some reports indicating NAA to be better rooting hormone for Citrus species (Chakravarty and Goswami, 1999; Usman et al., 2005).

Agar is the most widely used gelling agent for plant tissue culture media. The properties of agar like stability, clarity, non-toxic nature and resistance to metabolism during culture make it a gelling agent of choice (Henderson and Kinnersley, 1988). During last two decades, there has been an increase in the efforts to look for cost effective suitable substitutes (Zimmerman et al., 1995; Babbar and Jain, 1998; Jain and Babbar, 2002; Babbar et al., 2005). In this report, effort has been made to evaluate sterculia gum as gelling agent in plant tissue culture media. Sterculia gum is a dried tree exudate from Sterculia urens growing in the regions of Central and Northern India. Sterculia gum gelled medium (30 g L^-1) was found to be effective for shoot organogenesis and rooting. It was observed that the initial response for organogenesis was better on agar gelled medium as cultures establish on this medium grow faster as compared to sterculia gum gelled medium. But once the cultures establish themselves on gum gelled medium they exhibit normal growth and proliferation. Sterculia gum forms more transparent medium as compared to agar, therefore, it can be used as gelling agent of choice for the experiments that require regular observations of the cells, tissues or organs growing inside the medium. Moreover it is highly cost effective gelling agent as its price in India is about one-fourth of the agar. Therefore, the cost of commercial micropropagation can be reduced if less expensive alternatives are used. The quality of sterculia gum varies with climatic conditions of plant like moisture, temperature, soil composition and use of herbicides, which may limit its use as standard gelling agent. However, if demand for its use as gelling agent arises, efforts can be made to motivate suppliers to supply good quality gum in branded packs.

CONCLUSION

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Agar represents one of the most expensive used media components. The present study describes possibility of using sterculia gum in place of agar as gelling agent to reduce production cost of tissue culture raised plants. Sterculia gum forms a transparent medium which supported regeneration response comparable to agar.

ACKNOWLEDGMENTS

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The authors are thankful to the University Grants Commissions, New Delhi (India) for providing financial assistance in the form of major research project to author at Department of Biotechnology, Khalsa College Amritsar. Thanks are due to Mr. K.P Rao, State Project Advisor (Mktg), Indira Kranthi Pathan, Department of Rural Development, Govt. of Andhra Pardesh for procuring and supplying sterculia gum from Girijan Cooperative Corporation Ltd. Visakhapatnam, India.