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Biochemistry & Analytical Biochemistry
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Rapid In-Vitro Regeneration of an Important Medicinal and an Ornamental Plant (Catharanthus roseus L)

Runa Rashmi1* and Trivedi MP2

1Department of Botany and Biotechnology, Patna Science College, Patna University, Patna, Bihar, India

2Department of Botany, Patna Science College, Patna University, Patna, Bihar, India

*Corresponding Author:
Runa Rashmi
Department of Botany and Biotechnology
Patna Science College
Patna University
Patna, Bihar-800001, India
Tel: 9308209075
E-mail- [email protected]

Received Date: September 30, 2015; Accepted Date: November 26, 2015; Published Date: November 30, 2015

Citation: Rashmi R, Trivedi MP (2015) Rapid In-Vitro Regeneration of an Important Medicinal and an Ornamental Plant (Catharanthus roseus L). Biochem Anal Biochem 4:227. doi:10.4172/2161-1009.1000227

Copyright: © 2015 Rashmi R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Catharanthus roseus (L.) is an important alkaloid-yielding medicinal and ornamental plant belonging to family Apocynaceae. It comprises of total 8 species. The genus Catharanthus is well reported for producing biologically active terpenoid indole alkaloids (TIAs) with over 130 compounds isolated and identified. This plant produces secondary metabolites called alkaloids which have a great medicinal importance such as circulatory ailments (antihypertensives), ajmalicine to treat diabetes, malaria, menorrhagia, Hodgkin’s disease. This plant is grown commercially for its medicinal and ornamental uses in India. In view of these facts the study was conducted for micropropagation of Catharanthus roseus MS media supplemented with different concentrations (0.5–10.0 mg/l) of NAA, 2, 4-D, BAP and KIN were used singly and in combination. Among all the growth hormones 2, 4-D was the best for callus induction (85% in stem and 87% in leaf) and in combination 2, 4-D and BAP (65% in stem and 81% in leaf). Day of callus induction started from 13th to 37th day. This variation is due to the differences in culture conditions and the age of explants. In single combination BAP was the best for shooting (79%) and in double combinations BAP (1.5 mg/l) and NAA (0.5 mg/l) were the best for shooting (85%). Higher induction of root (91%) was observed at NAA (2 mg/l) and in double combinations BAP (1.5 mg/l) and NAA (0.5 mg/l) were the best for rooting (89%). Regenerated plants after hardenings were transferred to soil and they showed 77% survival. The protocol was optimized by manipulations of different plant hormones for enhanced multiplication. Protocol explained here provides a rapid plant regeneration system which could be used for the commercial purposes.

Keywords

Catharanthus roseus; Callus culture; Micropropagation; Plant growth regulators

Introduction

Catharanthus roseus (L.) is an important alkaloid-yielding medicinal and ornamental plant belonging to family Apocynaceae [1]. It comprises of total 8 species. The genus Catharanthus is well reported for producing biologically active terpenoid indole alkaloids (TIAs) with over 130 compounds isolated and identified [2,3]. These alkaloids are used to treat diabetes, malaria, menorrhagia, Hodgkin’s disease etc. C. roseus has potent secondary metabolism responsible for monoterpenoid glucosides and other terpenoid compounds, steroids, phenolics, flavanoids, anthocyanins and 130 terpenoid indole alkaloids (TIAs) [4,5]. The widely used anticancer TIA drugs vinblastine and vincristine [6] are semisythesized from their natural precursor’s vindoline and catharanthine that are obtained from C. roseus root and shoot organs. Ajmalicine, a cardiac tonic TIA drug is also extracted from C. roseus roots [6]. The high cost of production of pharmaceutically important TIAs is related to their low level of accumulation in C. roseus organs. Thus, to increase the concentrations of TIAs in plant organs is another important objective of genetic work on C. roseus. As a promising alternative to produce plant secondary metabolites, plant tissue culture technology has many advantages over traditional field cultivation and chemical synthesis, particularly for many natural compounds that are either derived from slow growing plants or difficult to be synthesized with chemical methods [2,7]. Catharanthus has been cultured in vitro to make it available in large amount by various workers [8-11].

Farmers today are attracted towards small to large scale production of medicinal and aromatic plants for augmenting their income [12]. They are cultivating Ashwagandha, Surpgandha and Brahmi commercially but the problem is poor marketing avenues. The requirement is linking of farmers with industry [13-15]. In order to provide enough pant material for commercial exploitation, the cultivation of these plants using conventional method is not sufficient. Thus mass propagation through in vitro culture to get alkaloids and to make them available throughout the year is an urgent necessity. There are lots of works done separately on these plants but still there is no any common protocol.

This work aimed at development of a more efficient common protocol for callus induction, whole plant production, transfer of in vitro grown plant to soil and further studies of the above mentioned plants by optimizing the growth regulators such as auxins and cytokinins.

In view of these facts the study was conducted for the development of an efficient protocol for in vitro multiplication of this plant by optimizing the growth regulators such as auxins and cytokinins.

Materials and Methods

Collection of explant

Explants stem (nodal) and leaf of Catharanthus roseus were collected from the medicinal plant garden of Patna Science College under Patna University Patna, India (Figure 1). The plants were identified, confirmed and authenticated by Dr. M. P. Trivedi, Associate Prof. in Botany of Patna Science College, Patna. After authentication of this plant, in vitro culture studies were carried out.

biochemistry-analytical-biochemistry-whole-plant

Figure 1: Whole plant of Catharanthus roseus.

Surface sterilization and culture media

Explants – leaf and stem, washed thoroughly with running tap water for 30 minutes and then dipped for 15 sec. in 70% ethanol after that submerged in a disinfectant calcium hypochlorite (0.5%) for 25 minutes. Tween 80 added to the above solution to improve contact between tissue and disinfectant. Explants removed from disinfectant and washed 5 times in sterile distilled water. Explants blotted on filter paper in 5 replicates in Laminar Air Flow before placing it on Murashige and Skoog (MS) media.

Standard procedure was followed for the preparation of media with slight variations [6]. The pH of the media was adjusted to 5.8 and heat resistant growth regulators (NAA, 2, 4-D, BAP and KIN) were added to the media prior to sterilization done at 15 lbs/in for 15 min at 121oC. All media were solidified with 8 g/l agar. After autoclaving further work done under Laminar Air Flow.

Callusing

For callus induction juvenile stem (nodal) and leaf about 5 mm in length were aseptically prepared and were implanted vertically on MS medium prepared with specific concentrations of hormones. Culture of stem and leaf explants were initially incubated under darkness in a culture chamber at 25°C for callus induction.

Shoot regeneration on callus

The callus was cut into small pieces when it was observed in entire explants. Each piece of callus was transferred to MS media having same growth hormones in similar composition and concentration as for callus induction. Subsequently, calli were incubated under a 16/8 h (light/dark) photoperiod with light fluorescent lighting at an intensity of 60 μE m-5 s1 on a constant temperature as of callusing (25°C) for shooting. Sub culturing was done after every 15th day.

Root regeneration and acclimatization

For initiation of roots the 5-7 weeks old shoots 3.5 cm in length were cultured on MS media having same growth hormones in similar composition, concentration and incubation as for shooting.

The complete rooted plantlets 75 days old were washed to free them of agar and dipped in 0.2% bavistin fungicide for 10 minutes to protect from fungal attack in near future. These plants were potted in small plastic pots containing sterilized soil. The plantlets were covered with polythene bags to maintain high humidity. These were acclimatized at 28°C less than 16 hours, photoperiod and watered regularly. After 3-4 weeks the polythene bags were removed and established plantlets were transplanted in earthen pots in a green house with watering at 2-3 days intervals.

Results

All the experiments were carried in triplicates and the mean value was recorded (Figure 2).

biochemistry-analytical-biochemistry-triplicates-growth-hormones

Figure 2: Explants in triplicates on MS media with growth hormones for callus induction.

Callusing

Effects of different concentrations of auxins and cytokinins singly on callus induction-

MS media supplemented with different concentrations (0.5–10.0 mg/l) of 1-naphthaleneacetic acid (NAA) showed stimulatory effects on callus induction. Maximum callusing response (75% in stem and 79% in leaf) was recorded at 1.5 mg/l of NAA. At 0.5 mg/l the callusing response was recorded less and it increased up to 2 mg/l. At 2.5 mg/l onward callusing response was reduced and found minimum at 5 mg/l. At 10 mg/l no callusing or growth was observed. It was observed that the higher concentration of NAA in media had an inhibitory effect on callus proliferation.

2, 4-Dichlorophenoxyacetic acid (2, 4-D) with different concentrations (0.5-10 mg/l) showed stimulatory effects on callus induction (Table 1 and Figure 3). Maximum callusing response (85% in stem and 87% in leaf) was noted at 2.5 mg/l.

Comsition of media 2,4-Dmg/l Catharanthus roseus
Stem Leaf
% of callusinduction degree of callusing day of callus induction % of callus Induction degree of callusing day of callus induction
0.5 - - - - - -
1 - - - - - -
1.5 - - - - - -
2 - - - 73 +++ 15
2.5 85 +++ 13 87 +++ 14
3 35 + 17 47 + 17
4 23 + 19 25 + 19
5 17 + 21 21 + 23
10 No callusing - - No callusing - -

Table 1: Callus induction on stem (nodal) and leaf explants on MS medium under the influence of different concentrations of 2, 4-D mg/l and KIN mg/l separately (0.5, 1, 1.5, 2, 2.5, 3, 4, 5 and 10).

biochemistry-analytical-biochemistry-callus-induction-fortified

Figure 3: Callus induction of MS fortified with 2, 4-D (2.5 mg/l).

No callus formation was observed on stem and leaf explants inoculated on MS media supplemented with 0.5 mg/l to 10 mg/l of Kinetin (KIN) .

With 6- benzylaminopurine (BAP) maximum callusing response (59% in stem and 63% in leaf) was noted at 2.5 mg/l. Lower concentrations of BAP (0.5 mg/l to 1.5 mg/l) were unable to induce callusing and higher concentrations of BAP (10 mg/l) in media had an inhibitory effect on callus induction.

Effects of different concentrations and combinations of growth hormones on leaf and stem callus induction

2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 6-benzylaminopurine (BAP) with different concentrations (0.5-10 mg/l) showed stimulatory effects on callus induction. Maximum callusing response (59% in stem and 64% in leaf) was recorded at BAP 1 mg/l and 2, 4-D 2 mg/l. At 3 mg/l of BAP and 2, 4-D 1 mg/l swelling of callus was observed. At 5 mg/l to 10 mg/l of BAP and 2, 4-D no callusing or growth was observed.

MS media supplemented with different concentrations (0.5–10.0 mg/l) of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) showed stimulatory effects on callus induction (Table 2). Maximum callusing response (78% in stem and 81% in leaf) was recorded at 0.5 mg/l and 1 mg/l for BAP and NAA respectively.

Composition of mediamg/l Catharanthus roseus
Leaf  Stem
% of callusInduction degree of callusing day of callus induction % of callusInduction degree of callusing day of callus induction
MS - - - - - -
BAP NAA            
0.1 0.1 - - - - - -
0.5 0.5 21 + 20 - - -
1 0.1 25 + 20 15 + 23
1.5 0.5 65 ++ 17 20 + 19
2 0.1 69 +++ 14 60 ++ 17
0.1 1 75 +++ 14 67 ++ 17
0.5 1 81 +++ 15 78 +++ 15
1 1 77 +++ 15 71 +++ 15
1.5 1 73 +++ 15 65 ++ 17
2 1 70 +++ 14 85 ++++ 13
1 2 75 +++ 14 70 +++ 15
3 2 91 ++++ 14 60 ++ 17
5 4 67 ++ - No growth - -
10 5 swelling - - Swelling - -

Table 2: Callus induction on stem (nodal) and leaf explants on MS medium supplemented with different concentrations and combinations of BAP and NAA mg/l (0.5, 1, 1.5, 2, 2.5, 3, 4, 5 and 10).

Different concentrations (0. 5–10.0 mg/l) of 1-naphthaleneacetic acid (NAA) and KIN showed stimulatory effects on callus induction. Maximum callusing response (69% in stem and 70% in leaf) was recorded at KIN 1 mg/l and NAA 2 mg/l. At 2.5 mg/l to 10 mg/l of KIN and NAA no callusing or growth was observed.

in vitro Shoot Regeneration

All the experiments were carried in triplicates and the mean value was recorded.

Effects of different concentrations of auxins and cytokinins singly on shoot regeneration

MS media supplemented with different concentrations (2–5.0 mg/l) of 6-benzylaminopurine (BAP) maximum shooting response (79%) was noted at 2.5 mg/l (Table 3). Lower concentrations of BAP (2 mg/l ) was having low value to induce shooting and higher concentrations of BAP (up to 5 mg/l) in media had lower effect on shooting. Days of shoot generation started from 21th to 37th days.

Composition of mediamg/l Catharanthus roseus
BAP % of Shooting degree of shooting day of shoot induction
2 73 +++ 23
2.5 79 +++ 21
3 65 ++ 25
4 37 + 27
5 21 + 31

Table 3: Shoot regeneration on MS medium under the influence of different concentrations of BAP mg/l (2, 2.5, 3, 4 and 5).

No shoot formation was observed of callus on MS media supplemented with 0.5 mg/l to 10 mg/l of 1-naphthaleneacetic acid (NAA) and 2, 4-Dichlorophenoxyacetic acid (2, 4-D).

Effects of different concentrations and combinations of growth hormones on shoot regeneration

2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 6-benzylaminopurine (BAP) with different concentration (0.1-2.5 mg/l) showed stimulatory effects on shoot regeneration (Table 4). Maximum shooting response (65%) was recorded at BAP 1.5 mg/l and 2, 4-D 1.5 mg/l. Day of shoot generation started from 25th to 39th day.

Composition of mediamg/l Catharanthus roseus
BAP 2,4-D % of Shooting degree of shooting day of shoot induction
0.1 2 29 + 39
0.5 2 41 + 33
1 1.5 55 ++ 28
1 2 47 + 35
1.5 1.5 67 ++ 27
1.5 2 51 ++ 31
1.5 2.5 53 ++ 29
2.5 1 70 +++ 25
2.5 2 35 + 37

Table 4: Shoot regeneration on MS medium supplemented with different concentrations and combinations of BAP and 2, 4-D mg/l (0.1 to 2.5).

MS media supplemented with different concentrations (0.1–3.0 mg/l) of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) showed stimulatory effects on shoot regeneration (Table 5 and Figure 4). Maximum shooting response (85%) was recorded at BAP 1.5 mg/l and NAA 0.5 mg/l. Days of shoot generation started from 19th to 39th day.

Composition of mediamg/l Catharanthus roseus
BAP NAA % of Shooting degree of shooting day of shoot induction
0.5 0.5 75 +++ 21
1 0.1 67 ++ 25
1.5 0.5 85 ++++ 19
2 0.1 77 +++ 25
0.1 1 41 + 29
0.5 1 25 + 35
1 1 35 + 31
1.5 1 71 +++ 23
2 1 59 ++ 33
1 2 23 + 39
3 2 25 + 37

Table 5 : Shoot regeneration on MS medium supplemented with different concentrations and combinations of BAP and NAA mg/l (0.1, 1, 1.5, 2, 2.5 and 3).

biochemistry-analytical-biochemistry-shoot-regeneration-fortified

Figure 4: Shoot regeneration of Catharanthus roseus on MS fortified with BAP (1.5 mg/l) and NAA (0.5 mg/l).

in vitro Root Regeneration

All the experiments were carried in triplicates and the mean value was recorded.

Effects of different concentrations of auxins and cytokinins singly on root regeneration-

MS media supplemented with different concentrations (2–5.0 mg/l) of 1-naphthaleneacetic acid (NAA) maximum rooting response (91%), (Table 6). Lower concentrations of NAA (0.5- 1 mg/l ) was having low value to induce rooting and higher concentration of NAA (up to 5 mg/l) in media had lower effect on rooting. Day of root generation started from 09th to 17th day

Composition of mediamg/l Catharanthus roseus
NAA % of Rooting degree of Rooting day of Root induction
0.5 57 ++ 13
1 71 +++ 11
1.5 88 ++++ 09
2 91 ++++ 12
2.5 81 ++++ 13
3 75 +++ 14
4 63 ++ 15
5 41 + 17

Table 6: Root regeneration from shoot on MS medium under the influence of different concentrations of NAA mg/l (0.5, 1, 1.5, 2, 2.5, 3, 4, 5 and 10).

No root formation was observed on shoot inoculated on MS media supplemented with 0.5 mg/l to 10 mg/l of 6- benzylaminopurine (BAP), and 2, 4-Dichlorophenoxyacetic acid (2, 4-D).

Effects of different concentrations and combinations of growth hormones on Root regeneration-

2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 6-benzylaminopurine (BAP) with different concentrations (0.1-2.5 mg/l) showed stimulatory effects on root regeneration (Table 7). Maximum rooting response (64%) was recorded at BAP 1.5 mg/l and 2, 4-D 1.5 mg/l. Days of root generation started from 11th to 19th day.

Composition of mediamg/l Catharanthus roseus
BAP 2,4-D % of Rooting degree of Rooting day of Root induction
0.1 2 25 + 18
0.5 2 37 + 17
1 1.5 51 ++ 14
1 2 45 + 16
1.5 1.5 67 ++ 1;2
1.5 2 49 + 14
1.5 2.5 47 + 15
2.5 1 65 ++ 13
2.5 2 37 + 17

Table 7: Root regeneration from shoot on MS medium supplemented with different concentrations and combinations of BAP and 2, 4-D mg/l (0.1 to 2.5).

MS media supplemented with different concentrations (0.1–3.0 mg/l) of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) showed stimulatory effects on root regeneration (Table 8 and Figure 5). Maximum rooting (89%) was recorded at BAP 1.5 mg/l and NAA 0.5 mg/l. Days of root generation started from 09th to 18th day.

Composition of mediamg/l Catharanthus roseus
BAP NAA % of Rooting degree of Rooting day of Root induction
1 0.1 - - -
1.5 0.5 89 ++++ 09
2 0.1 57 ++ 14
0.1 1 77 +++ 12
0.5 1 79 +++ 11
1 1 75 +++ 13
1.5 1 77 +++ 12
2 1 79 +++ 11
1 2 57 ++ 14
3 2 37 + 15

Table 8: Root regeneration from shoot on MS medium supplemented with different concentrations and combinations of BAP and NAA mg/l (0.1, 1, 1.5, 2, 2.5 and 3).

biochemistry-analytical-biochemistry-root-regeneration-fortified

Figure 5: Root regeneration of Catharanthus roseus on MS fortified with BAP (1.5 mg/l) and NAA (0.5 mg/l).

The most essential requirement for the successful transplantation is to maintain the plants under a very high humidity (85%). To achieve it, fan cooling system was used and temperature was maintained at 28°C for the first 15 days. They were covered with clear plastic (Figure 6). Later some small holes were poked in the plastic for air circulation. In the next step they were transferred to the shade house (75%) with overhead sprinkler system for irrigation for next 15 days (Figure 7). Finally they were transferred to the open area for 13 days before transferring them to the field.

biochemistry-analytical-biochemistry-hardening-plants-plastic

Figure 6: Hardening- Plants covered with plastic.

biochemistry-analytical-biochemistry-hardening-plant-transfer

Figure 7: Hardening- Plant ready to transfer into soil.

Discussion

Callusing

Standard procedure was followed for the preparation of media [17]. In the present study, two explants leaf and nodal stem were used in which leaf explants were found the best for callus induction than stem, which is in accordance with the earlier findings [18]. MS media without any growth hormone was unable to induce callus [19]. Among all the growth hormones, 2, 4-D was the best for callus induction.

In the present work Kin alone could not induce callus [20]. In further experiments Kinetin (Kin) was supplemented to the MS media in combination with auxins (2, 4-D and NAA). It was observed that Kin had enhanced callus growth in the presence of auxins.

MS media fortified with 2, 4-D and BAP was found the best for callus induction [21]. Day of callus induction started from 17th to 37th day [22]. This variation observed in the present investigation may be attributed due to the difference in culture conditions and the age of explants.

Shoot generation

The callus was sub cultured in all BAP containing media differentiated into multiple shoots giving out an average of 5 shoots per piece of callus in MS +BAP (2.5 mg/l). The media with lower concentration of NAA further gave multiple shoots as well as roots [23]. Inductions of callus and plant regeneration are the most reliable tools to multiply the plants in a large scale [23]. In combination of NAA and BAP normally lower NAA and higher BAP concentration favoured the production of shoot [24]. MS media with BAP and NAA is most suitable for shooting in our case [25].

In our findings Shoot culture was initiated on MS medium containing BAP (0.5 mg/l) with NAA (0.5 mg/L). Maximum shoot proliferation was achieved in medium containing BAP (1.5 mg/L) with NAA (0.5 mg/L) (80%). Among all the growth hormones, in single combination BAP (2.5 mg/l) was the best for shoot induction (77% from callus). Day of shoot generation started from 19th to 39th day.

Root generation

Root generation was satisfactory with BAP and NAA. NAA was the best in all other hormones like 2,4-D and BAP for rooting in our result [26].

In our findings, root cultures were initiated on MS medium containing BAP (1 mg/l) with NAA (0.1 mg/L). Maximum root proliferation was achieved in media containing BAP (1.5 mg/L) with NAA (0.5 mg/L) (87% from shoot). Day of root generation started from 11th to 17th day.

Hardening

Plantlets after 81 days old in secondary hardening were ready for field transplantation. Regenerated plants after hardenings were transferred to soil and they showed 77% survival. The regenerated plants were morphologically similar to control plants.

Therefore, in the present investigation, a protocol for micropropagation of locally adapted population is successfully established which may be used for commercial application as a substitute to natural propagation through conventional methods.

Conclusion

Catharanthus roseus is an ornamental plant and has potent secondary metabolites, mono-terpenoid glucosides, steroids, phenolics, flavonoids, anthocyanines and terpenoid indole alkaloids. Anticancer TIA drugs vinblastine and vincristine have been semi-synthesized from their natural precursors.

For rapid in vitro propagation, eight different nutrient media were tried. These are basal MS, MS+NAA, MS+BAP, MS+2,4-D, MS+KIN, MS+BAP+NAA, MS+BAP+2,4-D and MS+KIN+NAA. None of the explants responded in basal MS medium. Leaf explants were the best for callus induction than stem. 2,4-D (2.5 mg/l) was adjudged the best for callus induction in stem (85%) and in leaf (87%). It was also noted that it had enhanced callus growth in the presence of auxin. MS media fortified with NAA (1 mg/l) and BAP (0.5 mg/l) was found the best for callus induction in stem (78%) and in leaf (81%). Callus induction started from 13th to 27th day.

For shooting lower NAA and higher BAP concentrations favoured the production of shoot. Maximum shoot proliferation (85% from callus) was achieved in medium containing BAP (1.5 mg/l) with NAA (0.5 mg/l). Day of shoot generation started from 19th to 39th day.

For initiation of root 5 to 7 weeks old shoot 3.5 cm in length were cultured on MS media having NAA, 2,4-D and BAP singly and in combination BAP+2,4-D and BAP+NAA in same concentration as for callus induction. Maximum root proliferation (89% from stem) was achieved in media containing BAP (1.5 mg/l) with NAA (0.5 mg/l). Day of root generation started from 09th to 18th day.

The complete rooted plantlets 75 days old were freed from agar and after treatment potted suitably. After 81 days plantlets were ready for transplantation and the survival rate was 77%. The plants were similar to mother plants.

Therefore, in the present investigation a protocol for micropropagation of locally adapted population is successfully established.

Acknowledgement

The authors are thankful to the Indian Institute of Bioinformatics and Biotechnology, Patna, especially to Sharda Charan Jha, Fazal Ahmad and Sadre Alam for providing infrastructure and technical support during experiments.

References

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