Micropropagation of Madagascar periwinkle (Catharanthus roseus)

1. WELCOME

2. Presented by,
Mr. Wani Aditya Balasaheb
A
Presentation on,
Micropropagation of Madagascar periwinkle (Catharanthus roseus)
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3. CONTENT
⮚ Introduction and Objective
⮚ Materials and Methods
⮚ Results of the programme
⮚ Outcome of the programme
⮚ Summary of the programme
⮚ References
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4. ⮚ Catharanthus roseus (commonly Known as Madagascar periwinkle,
Periwinkle, Vinca, Sadabahar, Sadaphuli), Catharanthus roseus (L.) G.
Don belongs to the family Apocynaceae (Pereira et al., 2010).
⮚ Origin-Madagascar, Africa (Pereira et al., 2010).
⮚ It is an important medicinal plant of Apocynaceae containing abundant
useful alkaloids, used in diabetes, blood pressure, asthma, constipation,
cancer and menstrual problems (Das S et al., 2017).
⮚ Catharanthus roseus is an evergreen sub herb or herbaceous plant
(Hogan., 2003).
⮚ The chromosome number of all species of the genus Catharanthus is
2n=16 (Mendioro et al, 2005).
INTRODUCTION
Fig no.1 Periwinkle
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5. ⮚ In Madagascar, The bitter and astringent leaves are used as vomitive, roots used as hemostatic and
toothache remedies
⮚ Varieties:
Based on flower colour
1. Pink colour flowered – rosea (contains more of alkaloids)
2. White colour flowered – alba
3. White with pink or yellow ring in the orifice region – occellata
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6. 1. Catharanthus roseus plant is being exploited on a large scale on commercial basis for its
medicinal property (valuable alkaloids like vincristine, vinblastine act as anti-cancer and
agmalicine act as anti-hypertensive compound).
2. Conventionally, it is being propagated through seed, vegetative splits, stem cuttings, where the
success is around only 70% and when propagated through seeds, the germination is only 30%.
3. Due to low productivity and high production costs of alkaloids by cultures of Catharanthus
roseus, non-conventional methods have to be employed for better production.
4. Tissue culture techniques are being widely used to produce uniform quality, disease free plants
at a faster rate within a limited space.
NEED OF STUDY
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7. OBJECTIVE
⮚ To standardize in vitro regeneration protocol for Madagascar periwinkle (Catharanthus
roseus).
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8. The present study was carried out at Plant Tissue Culture laboratory under the Department of
Plant Biotechnology.
A. MATERIALS
1. Plant source : Healthy plants were collected from the Papaya nursery, Nashik, 422012.
2. Explant preparation : Nodal segments (1.5-2cm) of Madagascar periwinkle were selected.
3. Media preparation : MS medium (Murashige and Skoog, 1962) was prepared using different
nutrients.
4. Plant growth regulator : Used different concentration of plant growth regulators i.e. BAP with
combination of IAA, IBA and NAA .
5. Sterilants : 0.5 % Tween 20, 0.5 % Bavistin, 0.1% HgCl2 and 70% Ethanol was used.
MATERIALS AND METHODS
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9. 6. Instruments used
• Autoclave
• Laminar air flow
• Refrigerator
• pH meter
• Weighing balance
• Micropipette
7. Miscellaneous
• Forceps
• Scalpels and Blades
• Cotton and blotting paper
• Hand gloves
• Labels and marker
• Paraffin
8. Glassware & Plasticware
• Measuring cylinder
• Micro tips
• Culture bottles
• Conical flasks
• Beakers
9. Culture Room condition
• Temp (25 ± 2oC )
• Light intensity (1000-2000 lux)
• Photoperiod (16/8 hours)
• Humidity (60-70%)
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10. Table No. 1: Composition of Murashige and Skoog (MS) media
B. METHODS
1. Preparation of Media :
Preparation of MS media with appropriate amount of stock solutions of macronutrients,
micronutrients, growth regulators, vitamins, iron source, carbon source i.e sucrose (3%), required
concentrations of plant growth regulators with other supplementary substance like , activated
charcoal, etc. and finally 0.8% agar was added.
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Sr. No. Components Quantity Strength Final volume
01 Macro stock 50 ml 20 X
Makeup
volume up to
1000 ml
02 Micro stock 10 ml 100 X
03 Vitamins stock 10 ml 100 X
04 Fe-EDTA stock 10 ml 100 X
05 Sucrose 30 gm/l 3%
06 Inositol 0.1 gm/l 0.1 %
07 Agar 8 gm/l 0.8 %
Maintain pH at 5.7
10

11. 2. Collection of plant sample:
A healthy, mature and vigoursely growing Madagascar periwinkle
(Source Explant) plant was collected from Papaya nursery, Nashik.
3. Collection of explants:
Nodal explants (1-1.5 cm) were collected from healthy and
vigorously growing mother stock plants.
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Fig. no. 3. Plant material
Fig no. 4: Explant source

12. Nodal explants were hold under running tap water for 30 min.
Explants were soaked in (0.1)% Tween 20 and (0.5)% Bavistin for 20 min
respectively and rinsed with autoclaved distilled water at least 3 times.
0.1% HgCl2 solution treatment was given for 5 min under
Laminar Air Flow cabinet and rinsed with distilled water for 3-4 times.
Explants were treated with 70% ethanol for 5 sec.
Finally wash the explants with autoclaved distilled water
for 3-4 time.
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4. Explant Sterilization :
Fig no. 5: Bavistin (0.8%) treatment
Fig no. 6: Mercuric Chloride (0.1%)
treatment

13. Sterilized nodal segments were cut with the help of sterilized blade.
Nodal segments were aseptically inoculated on media having different plant
growth hormones concentration BA (3.0 - 4.5 mg/l).
Cultures were kept in growth room(16/8 hr photoperiod, temp 25 ± 2ᴼC , light
intensity-1000- 2000 lux).
5. Inoculation of Nodal Explant:
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14. Plate no.1: Explants inoculated on cultured medium
A) Inoculation of explants on MS medium
B) Inoculated explants in culture bottles on MS + PGR’s
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A B

15. The surface sterilized explants were inoculated on MS medium supplemented with various
concentration BA (3.5-4.0)mg/l
Cultures were kept in growth room
After 30-35 days of inoculation, sprouted buds were transferred on MS medium with BA
(4.0)mg/l for multiplication
6. Shoot Initiation and Elongation
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16. 7. Root initiation :
The elongated shoots were excised and cultured on MS media supplemented with of plant growth hormones.
The culture will be incubated at 25±2°C with 16 hour of light and 8 hour of dark photoperiod. The root growth
will record within four weeks of the culture
Table No. 2: Composition of Different Concentration of IAA and IBA with MS media for Root Induction
Bottle No. Media Concentration
1. MS+ IAA (4.0 mg/l)
2. 0.5 MS+ IAA (4.0 mg/l)
3. MS+ IBA (3.0 mg/l)
4. 0.5 MS+ IBA (3.0 mg/l)
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17. 8. Hardening:
The in vitro rooted plantlets will be held with sterile distilled water to remove the traces of agar
The plantlets will be transferred to plastic pots containing a mixture of sterilized garden soil and compost
in the ratio of 1:1 and then transferred to the greenhouse for hardening after 10 days.
The potted plants will be maintained in a greenhouse
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18. 14-11-2022 Wani Aditya Balasaheb 18
Sr. No. Month Work Plan
1. May-2022
1stweek Registration of VII semester
2ndweek
Searching for different research papers related to Madagascar periwinkle (Catharanthus
roseus).
3rdweek
Reading of research papers, literature, reviews and preparing the outline of research work
(ORW)
4thweek
Collection of plant from nursery, collection of required materials (glassware and chemicals) at
laboratory
2. June-2022
1stweek
Preparation of macronutrient, micronutrient, hormone, vitamin stock and
Fe-EDTA.
2ndweek Preparation of media with different concentrations of hormones.
3rdweek
Explant sterilization with chemical sterilization methods, inoculation of explants on media
containing different concentrations of Hormones for shooting.
4thweek Incubation of culture bottles in growth room.

19. 3. July-2022
1stweek Checking and observation of inoculated explants on shooting media.
2ndweek Subculturing of explants for multiplication of shoots.
3rdweek
Incubation of culture in growth room and simultaneously cleaning of contaminated cultures
and discarding them.
4thweek
Checking and observation of inoculated explants on multiplication media.
Observation and simultaneously washing and cleaning of contaminated cultures.
4.
August-
2022
1stweek
Preparation of rooting media and transfer of well shooted plants to rooting media.
Incubation of culture in growth room for root initiation.
2ndweek
Analysis of root initiation.
Observation of root initiation and washing of contaminated cultures.
3rdweek Primary hardening of rooted plants in plastic pots containing sterile soil and cocopeat.
4thweek Secondary hardening of plants.
5.
September-
2022
1stweek Data interpretation and tabulation of the result. Thesis writing and compilation of all data.
2ndweek Correction of final report presentation, PowerPoint presentation of the final project report.

20. RESULTS OF THE PROGRAMME
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Plate no.2: Shoot Initiation: (T0) MS + Control
(T1) MS + BA (3.0 mg/L)
(T2) MS + BA (3.5 mg/L)
(T3) MS + BA (4.0 mg/ L)
T1
T2 T3
T0

21. Table No. 3:Shoot initiation after days of MS media Containing different concentrations of BAP
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Treatment
(T)
MS+BAP
(mg/L)
No. Explant
Inoculated
No. of
explants
response
Shooting induction
Frequency(%)
Days to shoot
induction
T0 Control 5 1 20% 18-19
T1 3.0 5 4 80% 16-17
T2 3.5 5 2 40% 20-21
T3 4.0 5 3 60% 17-18

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Plate no 2 : Shoot Multiplication on MS medium
(T1) MS+BAP+NAA 4+2 mg/l
(T2) MS+BAP+NAA 4+3 mg/l
(T3) MS+BAP+NAA 4+4 mg/l
T1
T1
T1 T2 T3

23. Table No. 4 : Shoot Multiplication after days of MS media Containing different concentrations of BAP
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Treatment
MS+
BAP+NAA
(mg/L)
No.
of explants
transferred
No. of
explant
response
Days to shoot
Multiplication
T1 4.0 + 2.0 3 1 17-18
T2 4.0 + 3.0 3 2 15-16
T3 4.0 + 4.0 3 1 20-21

24. 1. An efficient and reproducible procedure for Micropropagation of Madagascar periwinkle (Catharanthus
roseus) is standardized up to the multiplication.
2. The sterilization technique was developed and standardized by using Bavistin (0.5%) for 20 min,
mercuric chloride (0.1 %) for 5 min and ethanol (70 %) for 1min.
3. MS medium supplemented with 4.0 mg/liter BAP + 3.0 mg/liter NAA induced Shoot Initiation and
multiplication with Rooting.
4. In the shoot proliferation nodal stem explants showed better response than other explants.
5. Initiation of in MS modified with the best growth observed and shooting in 17 days.
6. Multiplication of shoots is observed in multiplication media with different concentration of BAP and
NAA.
OUTCOME OF THE PROGRAMME
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25. SUMMARY OF THE PROGRAMME
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⮚ The present investigation aims “Micropropagation of Madagascar periwinkle (Catharanthus roseus)” and the
related work were carried out in Plant Tissue Culture Laboratory.
⮚ Demand for the healthy planting material of Madagascar periwinkle is very high.
⮚ As this herb, is an important medicinal plant, it is becoming a vulnerable species due to its low seed
germination percentage and poor seed viability. Productions of large numbers of Catharanthus roseus plants
were possible through in vitro micro-propagation techniques.
⮚ Propagation of Madagascar periwinkle through tissue culture is reliable solution to produced disease and
virus free planting material to the farmers.
⮚ In this study sterilization protocol were standardized.
⮚ Growth regulator’s play an important role on growth and development of Micropropagation of Madagascar
periwinkle shoot culture.

26. REFERENCES
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⮚ Das S. and Sharangi A. (2017). Madagascar periwinkle (Catharanthus roseus L.): Diverse medicinal and
therapeutic benefits to humankind. J. Pharmacogn. and Phytochem. 6(5): 1695-1701.
⮚ Adinpunya M., Khan B., Rawal S. and Mitra A. (1997). Photoautotrophic shoot cultures: an economical
alternative for the production of total alkaloid from Catharanthus roseus (L.) G. Don. Curr. Sci. 73(7):
608–609.
⮚ Pati P., Kaur J. and Singh P. (2010). A liquid culture system for shoot proliferation and analysis of
pharmaceutically active constituents of Catharanthus roseus (L.) G. Don. Plant Cell Tiss Organ Cult. 105:
299–307.
⮚Pereira D., Faria, J., Gaspar L., Ferreres F., Valentao P., Sottomayor M. and Andrade P. (2010). Exploiting
Catharanthus roseus roots: Source of antioxidants. Food Chem. 121: 56–61.
⮚ Sharma V., Kumar A., Kumar A. and Kumar S. (2020). An efficient in-vitro propagation protocol for
Catharanthus roseus (L.) Research J. Biotech. 15(1): 82-87.