Low-Cost Alternatives for Accelerating the Plant Cell Suspension Culture of Momordica charantia L.

Karthikeyan Subbarayan, Rajagopal Kalyanaraman and Shobana Vijayanand

Dept. of Biotechnology, School of Life Sciences, VELS University, Velan Nagar, Pallavaram, Chennai- 600 0117, Tamil Nadu, India

ABSTRACT:

The carbon source in plant cell suspension culture is a very important factor for growth and development. In the present investigation the role of different carbon sources such as analytical grade sucrose (Hi-Media, India), commercial grade sugar, sugarcubes and jaggery (both purchased from local market) were studied in cell growth of Momordica charantia in an effort to reduce the cost instead of using high cost carbon source. Studies have revealed that significant improvement was observed in cell growth on a medium containing sugarcubes. More callus could be induced successfully from leaf and shoot tissues when cultured on MS medium supplemented with two combinations of growth regulators influence (Kin+IAA and BA+IAA). More fresh weight and the best friability of callus was obtained from leaf explants on MS medium containing 1.0 mgl^-1 IAA and 0.5 mgl^-1Kin with semi-solid medium (0.6% agar). The cell suspension culture promoted the best cell growth in medium containing sugarcubes with biomass of 8.245 g fresh weight and 0.298 g dry weight in 24 days of inoculation. This is the first report using four different carbon sources in plant cell suspension cultures. The results showed that out of four carbon sources tested, sugarcubes could be the best source of carbon and it potentially reduces the cost of using costly carbon sources in cell suspension cultures.

KEYWORDS: callus, carbon source, kinetin, sugarcubes,

INTRODUCTION:

Micropropagation and Cell culture technology have been widely applied for the production of large number of economically important plants and secondary metabolites. Despite advancements in synthetic chemistry, we still depend upon biological sources for a number of secondary metabolites including pharmaceuticals¹. Commercialization of a large number of such technologies has been hampered by high production cost. The cost of components of tissue culture medium has been another concern for most commercial laboratories². Agar and sucrose are the chief constituents which play a significant role in cost of production. Attempts have been made for the use of cheaper alternatives of agar³and sucrose^4-5. Commercial grade sugar could replace analytical grade sucrose, with no significant change in the frequency of shoot formation in banana⁶. Plant cell cultures are typically grown as cell suspension cultures in liquid medium without agar or as callus cultures on solid medium. Momordica charantia L., native of Indo-Malayan region is an important pharmaceutical medicinal plant of Cucurbitaceae family, due to the high content of biologically active substances. In various systems of traditional medicines, M. charantia, named bitter melon, is used for several aliments: anti-diabetic, jaundice, contraceptive, piles, pneumonia, abdominal pain and anti-cancerous^7-10.

Table 1. Callus formation from different explants of M. charantia after 6 weeks culturing on MS medium supplemented with different concentrations of BAP/Kin (0.0–2.0mgl^-1) and IAA (0.0–4.0mgl^-1) with 3.0% Sucrose AR grade and 0.8% Agar

Explant	Hormone concentration (mgl^-1)	Fresh weight (g) ±SE	Average percentage (%) ± S.E. (texture)
Explant	Hormone concentration (mgl^-1)	Fresh weight (g) ±SE	Compact	Friable
Leaf	0.5 IAA	1.55 ± 0.04	31.6 ± 0.32	68.4 ± 0.28
	1.0 IAA	2.05 ± 0.11	10.8 ± 0.38	89.2 ± 0.22
	1.0 IAA +0.5 Kin	2.24 ± 0.08	7.2 ± 0.28	92.8 ± 0.26
	1.0 IAA +1.0 Kin	1.95 ± 0.14	14.8 ± 0.26	85.2 ± 0.21
	1.0 IAA +0.5 BAP	1.92 ± 0.09	13.6 ± 0.36	86.4 ± 0.32
Stem	0.5 IAA	1.36 ± 0.13	38.4 ± 0.34	61.6 ± 0.34
	1.0 IAA	1.88 ± 0.08	33.6 ± 0.31	66.4 ± 0.23
	1.0 IAA +0.5 Kin	1.92 ± 0.15	82.4 ± 0.34	17.6 ± 0.31
	1.0 IAA +1.0 Kin	1.76 ± 0.06	82.8 ± 0.41	17.2 ± 0.24
	1.0 IAA +0.5 BAP	1.78 ± 0.12	73.2 ± 0.35	26.8 ± 0.25

The fruits of bitter gourd contain very high amount of vitamins A and C, iron and minerals¹⁰. The aim of the present investigation was to test low cost alternatives of sucrose in cell suspension culture. Till date, there is hardly any report on the usage of commercial grade in plant cell suspension cultures.

MATERIALS AND METHODS:

Standardization of Hormone concentration for callus induction:

M. charantia plants collected from the herbal garden of the VELS University, Chennai, India. Fresh healthy plants were used as explants for the establishment of callus culture. The explants were first washed with detergent several times and rinsed under running tap water. They were then immersed in 70% (v/v) ethanol for five seconds before surface-disinfection in 0.1 % HgCl₂ solution under continuous agitation for 2 min. They were subsequently rinsed three times with sterile distilled water and then surface-disinfected again with 20 % (v/v) with the addition of three drops of Tween-20 (polyoxyethylene sorbitan monolaurate) for 20min. After three rinses with sterile distilled water, they were cultured in Murashige and Skoog basal medium (MS) supplemented with 30 gl^-1Analytical grade sucrose (Hi-Media, India) and 7.5 gl^-1agar (Hi-Media, India) for 4 weeks. A total of 16 combinations of IAA and BAP/Kin were tested for each type of explant.

Effect of carbon source and agar concentrations for callus induction:

Fresh explants were surface sterilized in the same manner as described in the previous section. The leaf explants were inoculated in MS medium supplemented with 1.0 mgl^-1IAA and 0.5 mgl^-1Kin, the best growth regulator combination determined for callus induction that is, standard induction (SI) medium. Different carbon sources such as analytical grade sucrose (Hi-Media, India), commercial sugar, sugarcubes and jaggery (both purchased from local market) were added to medium at 3% concentration. With the efficient carbon source detected, experiments were also conducted with semi-solid medium (0.6% agar). The experiment was repeated three times. The average fresh weight of callus formed from each medium was recorded after 6 weeks. The number of explants that produce compact type or friable type of callus was also determined.

Effect of carbon source for cell culture establishment and study of the growth kinetics of the cell culture:

Friable callus induced from leaf explants were inoculated into 100 ml Erlenmeyer flasks containing 20 ml of standard induction (SI) medium with sugar cubes. Analytical grade sucrose was taken as control. All the cultures were placed in a culture room with temperature regulated at 25±2°C and maintained under 24 h light provided with cool white fluorescent lamps at 3000 lux. All the cell cultures were placed on a rotary shaker with a speed of 125 rpm. The fresh weight of the cell biomass was determined from each flask. Three culture flasks were taken randomly every 3 days over a 27 days period to determine the cell growth pattern and the optimum inoculum density for best cell growth. The cells were harvested by filtering the cell suspension cultures through filter paper (Whatman No. 1, diameter 90 mm) using a filter funnel (90 mm) connected to a vacuum pump. The average fresh weights of the cell biomass from three cell suspension culture flasks were taken every 3 days over a 30 days period. The dry weights of harvested cell were recorded after 10 days of air-drying until a constant weight was obtained.

RESULTS:

Standardization of Hormone concentration for callus induction:

Our preliminary test indicated that the leaf explants of M. charantia showed good response towards friable callus formation when cultured on MS medium supplemented with 1.0 mgl^-1IAA plus 0.5 mgl^-1Kin. Formation of compact callus was observed in case of shoot explants. Since friable callus is finely dispersed in suspension cultures, it was used for subsequent experiments. Thus, this study indicated that the type of explants did influence the formation of callus in M. charantia in agreement with many other studies^11,12. Based on the fresh weight of callus formed from each type of explant in 6 weeks, the leaf explants were found to promote the most callus tissue than stem explants. In Table 1, some of the data from this study was described that is, only the best five media for the induction of callus for each type of explant. The results showed that MS medium supplemented with 1.0 mgl^-1 IAA plus 0.5 mgl^-1Kin induced the most calluses growth (2.24 g) from leaf explants in 6 weeks (Table 1). For the leaf explants, culture medium without IAA resulted in less friable callus.

Most of the leaf explants (92.6%) that were cultured on MS+1.0 mgl^-1IAA+0.5 mgl^-1Kin produced friable callus, hence it was considered as the best medium for callus induction from the leaf explants. But this medium was not the best medium for the induction of friable callus using stem explants, 1.92 g of compact callus was produced instead (Table 1).

Effect of carbon source and agar concentrations for callus induction:

The standard induction (SI) medium containing 3.0% AR grade sucrose and 0.8% agar was used as control for callus induction from leaf explants. All the carbon sources (AR grade sucrose, commercial sugar, sugarcubes and jaggery) at their 3.0% concentration evoked almost similar response during 1^st subculture. However, a marked difference was noticed when the cultures were maintained for three consecutive passages on same fresh medium. The type of carbon source and number of subculture showed a significant interaction. A higher induction rate than the control was obtained on medium containing sugarcubes. The other carbon sources showed a decline in the rate of induction; at least one is by jaggery (Table 2). But the callus cultures on all the four carbon sources were apparently green without any sign of deterioration even after three passages. Reducing agar concentration with semi-solid agar (0.6%) in the SI medium did not cause any reduction in induction rate. Interestingly, a combination of semi-solid medium with sugarcubes caused increment in the rate of induction (Table 3). Both semi-solid medium and sugarcubes showed no adverse effect on the callus cultures. Hence, based on biomass and friable type of callus formed from leaf explants on MS medium supplemented with 1.0 mgl^-1 IAA+0.5 mgl^-1 Kin (SI medium) with 3.0% AR grade sucrose/sugarcubers in semi-solid medium was found to be the most suitable culture medium for the production of callus that could be used as material source for the preparation of cell suspension culture of M. charantia.

Table 3. Effect of different agar concentrations on callus induction from leaf explants of M. charantia grown on Standard Induction (SI) medium with 3.0% sucrose AR/sugarcubes

Standard Induction (SI) medium +different concentrations of agar (%)	Fresh weight (g) ±SE after subculture
	Sucrose AR	Sugarcubes
0.8	2.24 ± 0.08	2.42 ± 0.04
0.75	2.25 ± 0.07	2.45 ± 0.06
0.7	2.29 ± 0.12	2.46 ± 0.11
0.65	2.29 ± 0.05	2.49 ± 0.09
0.6	2.32 ± 0.14	2.52 ± 0.12

Effect of carbon source on cell culture establishment and the growth kinetics of the cell culture:

The cells of M. charantia were found to grow well in liquid MS medium that contained the same combination of plant growth regulators (1.0 mgl^-1 IAA+0.5 mgl^-1 Kin) as in solid /semi-solid medium for callus induction and maintenance. A higher induction rate than the control was obtained on medium containing sugarcubes. The results indicated that 0.75 g inoculum in 20 ml culture medium gave the best and typical growth kinetics. The culture reached the growth peak on the 24th day and declined after that. The maximum increase of cell biomass with sugarcubes was almost 8.245 g after 24 days of culture. AR grade sucrose produced only 7.825 g of cell biomass within the same period (Table 4). The lag phases were short (6 days) for both fresh weight and dried weight growth curve but the following log phase were different. Fresh weight of the cell culture reached its maximum biomass (7.985 g) with sugarcubes after 18 days of culture followed by the stationary growth phase. The maximum dry weight (0.255 g) of the culture occurred 15 days after inoculation followed by a gradual decline of growth.

Table 4. Cell biomass of M. charantia after 24 days culture in liquid MS medium supplemented with 1.0 mgl^-1IAA+0.5 mgl^-1Kin and 3.0% sucrose AR/sugarcubes

Carbon sources 3.0%	Cell biomass (g)
Carbon sources 3.0%	Fresh weight	Dry weight
Sucrose AR	7.985 ± 0.06	0.291 ± 0.07
Sugarcubes	8.245 ± 0.08	0.298 ± 0.06

DISCUSSION:

This study has demonstrated that best callus induction rate could be achieved on the medium containing sugarcubes as carbon sources, replacing analytical grade sucrose used in the control experiments. Promoting role of sugarcubes in tissue culture has been observed in Leucaena leucocephala, Vitex negundo and Achras sapota⁵. However, Use of LR grade sucrose and sugarcubes in Curcuma longa cultures was recommended¹³. Incorporation of jaggery in the medium during the present study was not useful. Similar results like this in micropropagation of Wrightia tomentosa⁵.

Cheaper alternatives of gelling agents used by various workers have suffered from one or the other drawback limiting their use on commercial scale. In the present study, semi-solid (0.6%) agar similar response as that of control which is 0.8% agar in standard induction medium at the same concentration. Combination of semi-solid medium with sugarcubes in SI medium in the present case has resulted in a significant increment in rate of callus induction as compared to other factors tested separately.

Botau et al¹⁴ have observed two cytochinines (BA+Kin) with IAA influenced the callus induction and polyphenols synthesis from leaf explants of M. charantia. Similar results were observed in this experiment with IAA and Kin combinations.

The present study has also demonstrated that the cost of plant cell culture of M. charanta could be reduced significantly by the incorporation of sugarcubes and semi-soild agar. The prepared cell suspension cultures will be used as the material source for the production of secondary metabolites study in the forthcoming research activity.

ACKNOWLEDGEMENT:

Authors are thankful to the Management of Vael’s Educational Trust, Chennai, Tamilnadu, India, for providing the infrastructure for the present study.

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Received on 11.06.2010

Accepted on 12.08.2010

Research J. Science and Tech. 2(3): May –June. 2010: 60-63

Standard Induction (SI) medium +different C-sources at 3% concentration	Fresh weight (g) ±SE
	I subculture	II subculture	III subculture	Mean ± SD
Sucrose AR	2.24 ± 0.05	2.28 ± 0.11	2.22 ± 0.12	2.25 ± 0.03
Sugarcubes	2.42 ± 0.04	2.38 ± 0.09	2.44 ± 0.08	2.41 ± 0.05
Commercial sugar	2.02 ± 0.07	1.97 ± 0.12	1.94 ± 0.11	2.98 ± 0.04
Jaggery	1.84 ± 0.09	1.81 ± 0.14	1.72 ± 0.13	1.79 ± 0.06