Low-Cost Media for In-vitro Callus Development in

Catharanthus roseus (L) G. Don

 

Saumya S¹, Shalini S¹, S. Leelavathy¹, P. Deepa Sankar²*

¹School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore - 632 014, Tamil Nadu, India.

²VIT School of Agricultural Innovations and Advanced Learning (VAIAL), Vellore Institute of Technology, Vellore - 632 014, Tamil Nadu, India.

 

ABSTRACT:

Objective: The study focuses on formulating and validating low-cost media for in vitro callus induction and development in Catharanthus roseus. Methods: Percentage response, nature and mass of in vitro grown C. roseus callus was recorded for the conventional MS media with casein hydrolysate (1g/L) and three low-cost media supplemented with casein hydrolysate (1g/L). The effect of two hormonal combinations, viz., 1.5mg/L 2,4-D + 1mg/L Kin and 1mg/L BAP + 1mg/L NAA had been studied for callus development. Results: Two media combinations including MS (urea replacing NH₄NO₃) + table sugar (30g/L) + casein hydrolysate (1 g/L) + Food grade agar (FGA, 7g/L) and MS + table sugar (30g/L) + casein hydrolysate (1g/L) + sago (80g/L) was observed to take less number of days to callus induction, 11 and 12 days respectively, than the conventional MS media + casein hydrolysate (1g/L) + gelrite (2.2g/L) with 2,4-D (1.5mg/L) + Kin (1mg/L), which took 13 days to callus induction. MS (urea replacing NH₄NO₃) + table sugar (30g/L) + casein hydrolysate (1g/L) + FGA (7g/L) with 2,4-D (1.5mg/L) + Kin (1mg/L) gave rise to high callus mass, averaging to 12.351g, at the end of 4^th subculture and also, high percentage of cost reduction per liter of media was observed compared to conventional MS media with casein hydrolysate. Conclusion: The present study indicated MS (urea replacing NH₄NO₃) + casein hydrolysate (1g/L) + FGA (7g/L) with 2,4-D (1.5mg/L) + Kin (1mg/L) as a better media for developing and proliferating C. roseus callus In vitro.

 

 

 

INTRODUCTION:

A wide range of secondary metabolites are produced by plants, which are used for the treatment of cancer^1,2. Catharanthus roseus (Linnaeus) G. Don is one such perennial tropical plant, which belongs to the family Apocynaceae³, also commonly known as Madagascar periwinkle⁴. It produces a wide range of indole alkaloids which are known to have anti-cancer⁵, anti-septic⁶, anti-viral, anti-tumor, anti-fungal, anti-hypertensive, anti-fertility, anti-malarial, anti-oxidant, anti-mitotic and anti-diabetic properties⁷.

 

C. roseus is capable of producing more than 100 indole alkaloids in various sections of the plant⁸, such as vinblastine, vincristine, ajmalicine etc.,⁹. C. roseus has been the subject of various studies due to its valuable pharmaceutical properties¹⁰. Vincristine and vinblastine are compounds that are produced in very small quantity, about 0.0003% from 2.56% total alkaloid in C. roseus¹¹. The huge commercial value of these compounds as chemotherapy drugs, agrochemicals and biopesticides, needs industrialization but a large amount of biomass and an inefficient extraction process are being a disadvantage. An alternative approach is to produce these secondary metabolites In vitro through callus culturing by inducing stress response but the relative high cost of production poses a great disadvantage. Components like gelling agents, sucrose and growth regulators make the technique expensive. Hence for large scale application of tissue culture, there is a need to reduce cost in terms of the materials used.

 

MATERIALS AND METHODS:

Explant surface sterilization:

The leaf explants were collected from C. roseus plant grown at Vellore Institute of Technology, Vellore. The plant was identified through its taxonomic characters and a voucher specimen was maintained. The leaves were subjected to running tap water and then treated with 0.1% bavistin, 3 drops of Tween 20 for 30 mins and 15 mins respectively. The semi sterilized explants were then taken to the LAF chamber, where it was further treated with 70% ethanol and 0.1% HgCl₂, each for 3 mins. In between the treatments, the explants were washed with autoclaved Milli-Q water. The sterilized explants were then cut into small bits and inoculated into the media with varying growth hormones.

 

Culture media:

Different combinations of MS basal media¹² and growth factors supplemented with casein hydrolysate (1g/L)¹³ and citric acid (60mg/L) was prepared (Table 1). In one of the media combination, the conventional ammonium nitrate (1.65g/L) had been replaced with urea (0.5g/L), as the former is flammable. Two hormonal combinations including 1mg/L BAP + 1mg/L NAA and 1.5mg/L 2,4-D + 1mg/L Kin, had been studied for callus development as they had been reported to be the best responsive media¹³. The pH was set at 5.8 prior to sterilizing the media at 121ºC under 15 psi.

 

Table 1: Media combinations prepared for callus induction and development of C. roseus

S. No.	Media combination	Sugar source	Growth factor		Gelling agent
			a	b
1	MS + casein hydrolysate (1 g/L) + citric acid (60 mg/L)	Lab grade sucrose (30 g/L)	2,4-D (1.5 mg/L) + Kin (1 mg/L)	BAP (1 mg/L) + NAA (1 mg/L)	Gelrite (2.2g/L)
2	MS + casein hydrolysate (1 g/L) + citric acid (60 mg/L)	Table sugar (30 g/L)	2,4-D (1.5 mg/L) + Kin (1 mg/L)	BAP (1 mg/L) + NAA (1 mg/L)	Food grade agar (7g/L)
3	MS + casein hydrolysate (1 g/L) + citric acid (60 mg/L)	Table sugar (30 g/L)	2,4-D (1.5 mg/L) + Kin (1 mg/L)	BAP (1 mg/L) + NAA (1 mg/L)	Sago (80g/L)
4	MS (0.5 g/L urea replacing 1.65 g/L NH4NO3) + casein hydrolysate (1 g/L) + citric acid (60 mg/L)	Table sugar (30 g/L)	2,4-D (1.5 mg/L) + Kin (1 mg/L)	BAP (1 mg/L) + NAA (1 mg/L)	Food grade agar (7g/L)

 

Callus initiation:

The explants after surface sterilization was inoculated in MS media supplemented with varied growth hormonal combinations as mentioned above. The cultures were maintained in dark condition and the room temperature was maintained at 24 ± 1°C. The date of initiation of callogenesis and percentage of response to callus induction were recorded in 20 culture bottles. The best responsive MS media was chosen and considered for further experiments.

 

Callus culture:

Thirty days old callus obtained from best responsive media were subcultured in the MS media supplemented with the same hormonal combinations as that of the parent culture. Four subsequent passages at 20 days of interval, were done using the similar media.

 

RESULTS AND DISCUSSION:

Callus initiation and proliferation:

The different MS media combinations were examined to determine the effective low-cost media for in vitro callus induction and proliferation (Table 2). The media combination 3a and 4a took less number of days to callus induction, 11 and 12 days respectively, than the control media combination 1a, which took 13 days to callus induction. The media combinations with BAP (1mg/L) + NAA (1mg/L) took more number of days to callus induction and the percentage response was low compared to the media combinations with 2,4-D (1.5 mg/L) + Kin (1mg/L), which is in consonance with the results reported by Rukhama Haq¹⁴ and Taha¹⁵.

 

Combination 3 showed good callusing initially and the texture was friable cum compact but it showed low callus mass generation as the usage of sago as gelling agent gave rise to loose medium which could not sustain the callus on the surface of the media (Fig 1). With subculturing, part of the callus got immersed into the medium which gave rise to poor callus mass at the end of the 4^th subculture. Based on the least number of days to callus induction, three media combinations viz., 1, 3 and 4 were chosen for further subculturing.

 

Table 2: Effect of MS media combinations on callus development in C. roseus

Media combination	Mean no. of days to callus induction	Percentage response	Callus proliferation scoring	Texture	Color
1a	13 days	100%	+++	Compact	White
1b	15 days	94.44%	++	Hard	White turned light green
2a	13 days	100%	+	Compact	White
2b	16 days	90%	+	Compact	White
3a	12 days	95%	++	Friable cum Compact	White
3b	13 days	94.44%	+	Friable cum Compact	White
4a	11 days	100%	+++	Compact	White
4b	13 days	100%	+	Hard	White turned light green

+++ High ++ Moderate + Low

 

Table 3: Mass of callus obtained at the end of 4^th subculture

 

 

Fig 1: Callus developed in C. roseus with growth factor 2,4-D (1.5 mg/L) + Kin (1mg/L), after 60 days A: Callus growth in MS + sucrose (30 g/L) + casein hydrolysate (1g/L) + gelrite (2.2g/L), B: MS + table sugar (30g/L) + casein hydrolysate (1 g/L) + sago (80g/L), C: MS (urea replacing NH₄NO₃) + table sugar (30 g/L) + casein hydrolysate (1g/L) + FGA (7 g/L)

 

Mass of callus:

At the end of the 4^th subculture, the mass of the callus of each media combination was measured (Table 3). Combination 4a gave rise to high callus mass, averaging to 12.351g. The control combination 1a gave 10.327g of callus. Other media combinations with BAP (1mg/L) + NAA (1mg/L) showed low mass of callus development. Combination 3a gave a low callus mass of 3.943g. Such low proliferation could be connected to the loose medium.

 

Cost analysis:

Without compromising on the quality of media, the cost of medium per litre has been worked out by using alternate carbon source¹⁶, agar¹⁷ etc. Cost analysis of the media combinations, revealed that 1L of the conventional MS media + casein + citric acid with gelrite costs Rs. 105.63. But when gelrite was substituted with FGA and sago and sucrose with table sugar, the cost of 1L media got reduced to Rs. 49.18 and Rs. 22.58 respectively. The MS media where NH₄NO₃ was replaced by urea, with FGA as the gelling agent costs Rs. 49.61 per liter of media. Compared to the conventional MS media, a high percentage of cost reduction was observed (Table 4).

 

 

Table 4: Cost analysis of varied MS media

	MS + LG sucrose (30 g/L) + casein hydrolysate (1 g/L) + citric acid (60 mg/L) + gelrite (2.2 g/L)	MS + table sugar (30 g/L) + casein hydrolysate (1 g/L) + citric acid (60 mg/L) + FGA (7 g/L)	MS (urea replacing NH4NO3) + table sugar (30 g/L) + casein hydrolysate (1 g/L) + citric acid (60 mg/L) + FGA (7 g/L)	MS + table sugar (30 g/L) + casein hydrolysate  (1 g/L) + citric acid (60 mg/L) + sago (80 g/L)
Cost of 1L media (INR)	105.63	49.18	49.61	22.58
Cost of media for 4 subcultures for 20 explants (INR)	422.52	196.72	198.44	90.32
% Cost reduction compared with conventional MS media	-	53.44	53.03	78.62

*LG – Lab Grade

 

CONCLUSION:

In the present investigation of using low-cost media for callus induction and development in C. roseus, it was found that MS (urea replacing NH₄NO₃) + casein hydrolysate (1g/L) + table sugar (30g/L) + FGA (7g/L) with 2,4-D (1.5mg/L) + Kin (1mg/L) gave high mass of callus, in accordance to the conventional MS media. Also, growth factor combination 2,4-D (1.5mg/L) + Kin (1 mg/L) led to early callus induction and good proliferation compared to BAP (1mg/L) + NAA (1 mg/L). Also, FGA has been observed to serve as a good solidification medium, which can sustain callus development. Through cost analysis, it is clear that there is significant reduction in cost of developed media per liter, compared to the conventional media with casein hydrolysate (1g/L) and gelrite (2.2g/L). Therefore, MS (urea replacing NH₄NO₃) + casein hydrolysate (1g/L) + table sugar (30g/L) + FGA (7g/L) with 2,4-D (1.5mg/L) + Kin (1mg/L) can be considered as a better media for developing and proliferating callus in C. roseus.

 

ACKNOWLEDGEMENT:

Our sincere thanks to Vellore Institute of Technology, Vellore for providing us with the support and facility.

 

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Received on 24.05.2020           Modified on 03.08.2020

Accepted on 04.09.2020         © RJPT All right reserved