In vitro mass propagation, polyploidization, and elicitor-mediated enhancement of vincristine production in Catharanthus roseus (L.) G. Don
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Date
2023-12-20
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GENETICS AND PLANT BREEDING, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia – 741252
Abstract
Catharanthus roseus, often known as Madagascar periwinkle or periwinkle, is a species
of flowering and medicinal plant that belongs to the family Apocynaceae, having
chromosome number 2n=16. The two most common cultivars of C. roseus are "rosea"
with pink flowers and "alba" with white flowers. One of the major alkaloids, vincristine,
which has anti-cancerous activity, is present in very small amounts (around 0.0005% of
total alkaloids) in the leaves of C. roseus. Vincristine has proven to be a highly effective
drug in the chemotherapy of various kinds of cancer including both paediatric and adult
acute lymphoblastic leukaemia, neuroblastoma, Wilkins' tumour, Hodgkin's disease, and
reticulum cell sarcoma. Since vincristine is particularly demanded yet difficult to extract,
there is a continuing effort to improve the production of this dimeric alkaloid. Traditional
seed propagation limitations affecting secondary metabolite production include low
germination rates, low seed viability and vigour, etc. Moreover, spontaneous outcrossing
results in genetic changes that alter the quantity and quality of beneficial alkaloids
including vincristine. Taking all of these factors into account, the current study has been
conducted with the aim of developing an in vitro methodology for high vincristine
cultivar among alba and rosea, and further vincristine enhancement using
biotechnological approaches such as in vitro polyploidization and elicitation. HPTLC
analysis for the vincristine content of both cultivars showed that the C. roseus cv. rosea
had considerably higher vincristine (1025.69±0.76 μg g-1 DW) at three months of age
than the C. roseus cv. alba (939.23 ± 0.42 μg g-1 DW). The initial culture of C. roseus
cv. rosea was established by using apical shoot tips as explants and following an
effective surface disinfection method. For direct regeneration, the in vitro shoot tips were
inoculated on MS medium fortified with different types and doses of cytokinin alone, out
of which MS medium supplemented with 0.5 mg L-1 mT emerged as promising since it
showed the highest number of shoots (10.9±0.23) per inoculated shoot tip. However, the
addition of 0.1 mg L-1 NAA to the MS medium supplemented with 0.5 mg L-1 mT
increased the number of shoots (12.67±0.67) in the shoot proliferation experiment. The
maximum number (21.9±1.85) of roots with the maximum length (39.2±1.65 mm) were
reported on half-strength MS medium supplemented with 0.5 mg L-1 IBA. For indirect
regeneration, in vitro leaves were used as explants for callus induction. Among various
auxins used, the maximum response percentage (97.5±0.50%) to callus induction with
earliest (4.7±0.15 days) induction and highest biomass (159.8±0.59 mg fresh weight,
29.6±0.16 mg dry weight) was achieved on MS medium supplemented with 1 mg L-1
Picloram. Organogenic calli were found to be inducing a maximum number of shoots
(9.00±0.57) on MS medium supplemented with 1.5 mg L-1 BA in conjugation with 0.2
mg L-1 NAA, with a response percentage (52.66±1.52%). Among the various planting
substrates investigated in combination, cocopeat and vermiculite showed the highest
survival percentage (99.0±1.00%) in the acclimatisation experiment. The clonal fidelity
of in vitro-regenerated plantlets to the mother plant was demonstrated by the DNA
fingerprinting of the plantlets using ISSR primers. In order to induce in vitro
polyploidization, shoot tips from the already formed multiple shoot cultures were
immersed in different doses of colchicine solution for diverse amounts of time. The
maximum rate of polyploidy induction (40.00%) was achieved during a 12-hour
treatment with a 0.1% colchicine solution. The cytological study revealed that putative
polyploids had a combination of diploid and tetraploid cells, which was further supported
by FCM. Vincristine concentration was reported to be 1.49 times higher in colchicine-
induced mixoploid (2348.85±0.60 μg g-1 DW) acclimatised plants compared to in vitro
diploids (1577.22±0.67 μg g-1 DW) and about 2.29 times higher compared to the in vivo
mother plant after treatment with a 0.1% colchicine solution for 12 hours. On the
elicitor-supplemented media, the shoot tips from the multiple shoot culture and the
induced calli from the in vitro leaves were injected. After 12 weeks of culture, it was
shown that the shoot tip cultures on the medium supplemented with 50 mM AgNO3 had
greater vincristine levels (2228.72±0.92 μg g-1 DW) than the control (1178.59 ± 0.87 μg
g-1 DW), which is over 1.89 times higher than the control and 2.17 times higher than the
in vivo mother plant. The calli on 50 M SA supplemented medium were also shown to
have more vincristine (302.67 ± 1.45 μg g-1 DW) among elicited calli after 6 weeks of
culture, however the non-elicited calli did not exhibit a peak for vincristine in HPTLC
analysis. The findings of this study indicate great promise for a precise and effective in
vitro direct and indirect regeneration system, elicitor-mediated enhanced vincristine
production in C. roseus. Also, in vitro induction of polyploidy was attempted for the first
time in C. roseus, which resulted in mixoploids with higher vincristine content. These
findings can be employed as an addition to the current extraction techniques and as a
source of vincristine for the pharmaceutical sectors.