Conservation of Curcuma Caesia by in Vitro Techniques Wahengbam R

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Conservation of Curcuma caesia by in vitro techniques Wahengbam R. C. Singh1, Heigrujam B. Singh1, Shamurailatpam Surodhani Devi1, Wahengbam N. Singh2, Nongmaithem M. Singh3, Yendrembam P. Devi3 1DBT-Biotech Hub, Department of Biotechnology, S. Kula Women’s College, Nambol,2 DBT- Biotech Hub, K. P. College, Hiyangthang, Manipur,3DBT-Biotech Hub, Pettigrew College, Ukhrul, Manipur

Received: 5th April 2015, Accepted: 12th April 2015, Published: 1st July 2015

Abstract Black turmeric or black zedoary (Curcuma This study was conducted to develop an efficient caesia) is a perennial herb with bluish-black protocol for mass propagation of Curcuma caesia rhizome, native to North-East and Central India. L. Explants from underground rhizomes were The rhizome of black turmeric has a high economic cultured on Murashige and Skoog (MS) medium importance owing to its putative medicinal and it is a slow propagating species. Multiplication properties. In west Bengal, the rhizome of the plant and callus induction starting from the rhizome buds is used in Kali Puja, and hence the plant is called and shoot tips of Curcuma caesia in MS medium Kali haldi. By etymology, Kali is the feminine supplemented with NAA; 0.9 mg/l with kinetin; 0.9 form of Kala, which means black color and hence mg/l or NAA; 1.2 mg/l with 6-Benzylaminopurine the plant is termed as black turmeric in English. (BAP; 1.9 mg/l) was the optimum for rapid clonal This species has been regarded as endangered by propagation of Curcuma caesia. Callus induction the central forest department of India due to was conducted with 2,4-dichlorophenoxy-acetic biopiracy. acid (2,4-D; 2.7-3.4 mg/l). Regeneration of plantlets from a callus was successfully conducted Medicinal Uses: in MS medium supplemented with a combination The rhizomes are used as a rubeficient to rub the of 2.5 mg/l BAP and 1.0 mg/l NAA for body after taking a Turkish bath. In Bengal, it is multiplication at 260C under 24 h photoperiod. The used in the fresh state-turmeric. The rhizomes of best shoots length was obtained on the medium the herb are often used by the Baiga, Sahariya, containing 2.0 mg/l of BAP and 2.0 mg/l NAA. Agariya, Gond, Korku, and other tribal Thus, combined effects of BAP and NAA communities of Mandla, Balaghat, Chhindwara, improved significantly the shoot growth and Anooppur, and Dindori district of Madhya Pradesh proliferation. MS medium supplemented with a state for the treatment of pneumonia, cough, and combination of 4.0 mg/l BAP and 2 mg/l IAA gave cold in children, and for fever and asthma in adults. the highest number of roots. The proliferated shoots Fresh rhizomes are crushed and applied as a paste were green and healthy in appearance. Finally, on forehead for relief from migraine or applied on healthy and complete plants with well developed the body for sprains and bruises. The rhizomes are roots were hardened, acclimatized and planted in claimed to have a property of acting against the field successfully with a survival rate of 80%. leukoderma, epilepsy, cancer and HIV/AIDS. Cultivation and harvest Key words: Micropropagation, Explant, in vitro, Curcuma caesia, BAP, 2,4-D, NAA, Kinetin. Cultivation: The cultivation and harvest practices are similar to Introduction that of common turmeric which is used in recipes. Micropropagation is the art and science of plant In the fields, the rhizomes are washed thoroughly multiplication in vitro. The process includes many and are placed in a wide mouthed cauldron. The steps--stock plant care, explant selection and water is poured in the cauldron such that the sterilization, media manipulation to obtain rhizomes are completely sunk. The caouldron is proliferation, rooting, acclimation, and growing on covered with a lid, and the rhizomes are boiled for of liners. Micropropagation is used to multiply about 30 minutes until foam oozes out with strong novel plants, such as those that have been odour. The rhizomes are taken out whilst the water genetically modified or bred through conventional is reduced to one-third of its content and when they plant breeding methods. turn soft with their inner portion decolorized from Frederick Campion Steward was the person who blue to dark or pale brown. The rhizomes are then discovered and pioneered micropropagation and dried in hot sun for 10 to 15 days until they are plant tissue culture in the late 1950s and early hardened. These dried rhizomes are then packed for 1960s. marketing.

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Materials and Methods simultaneously whereas, roots induction was lowest Fresh rhizomes of Black turmeric (Curcuma (4.50) for explants cultured on MS medium caesia) were collected from different districts of containing higher concentrations of BAP alone (5 Manipur. mg/l) and highest (17.10) in medium containing combination of 5 mg/l of BAP and 2 mg/l of NAA Sample preparation and cultural conditions (Table 1). Such type of simultaneous production of Fresh rhizomes of Black turmeric (Curcuma shoot and roots were reported earlier for Z. caesia) were collected from different districts of zerumbet by Stanly and Keng (2007) and on other Manipur. The explants were washed thoroughly Zingiberaceae species (Balachandran et al., 1990; under running tap water to remove adhering soil Chan and Thong, 2004; Bharalee et al., 2005; particles. To control the microbial contamination, a Yusuf et al., 2011). This study suggests that the use composite approach of washing the explants first of combination medium will shorten the time for with 60% Clorox and addition of 6 to 7 drops of plant regeneration. When BAP was used alone, the Tween 20 for 30 min was employed. Subsequently, maximum number of shoots (3.6) was obtained the explants were thoroughly washed once with from explants on MS medium with 5 mg/l BAP. sterile distilled water. Sterilized explants were then With an increase in the concentration of BAP, the dissected to remove one layer of leaf sheaths under number and length of shoots per explant increased aseptic conditions. Then, the excised explants were (Table 1). immersed again in 20% Clorox with the addition of 6 to 7 drops of Tween 20 for another 15 min and Therefore, it appears that a higher concentration of thoroughly washed seven times with sterilized BAP would have a positive effect on in vitro shoot distilled water. Outer layer were multiplication of Curcuma caesia. The proliferated removed aseptically and explants of 1 mm were shoots were green and healthy in appearance. The inoculated on basal Murashige and Skoog (MS) role of BAP in shoots proliferation has been medium supplemented with combination of KIN reported in other Zingiberaceae species (Ikeda and (0.9 mg/l), BAP (1-3 mg/l), NAA (0.5-1.6 mg/l), Tambe, 1989; Balachandran et al., 1990; Smith and and Ads (100 mg/l) for culture establishment. The Hamil, 1996; Rout et al., 2001; Panda et al., 2007; sucrose amount in the media was 30 gm/lt and agar Mohanty et al., 2011; Abdelmageed et al., 0.8% was used as basal. All experiments were 2011).The presence of BAP and NAA in the conducted in five replicas for each treatment. The medium markedly improved the number of pH of the medium was adjusted to 5.7 before proliferating shoots. Besides the number of shoots adding agar and was autoclaved at 1210C and 105 induced, BAP and NAA accelerated mean shoot kg/cm2 of pressure for 20 minutes. All the cultures length considerably (Table 1). The highest shoot were incubated at 25 ± 10C under white fluorescent multiplication was found in the medium containing light with 50μ mole m-2 s-2 light intensity during a 5.0 mg/l BAP + 2.0 mg/l NAA and also 3.0 mg/l photoperiod of 24 h light and dark cycles. The in BAP + 0.5 mg/l NAA, which produced nearly 5.65 vitro derived shoots were cultured on medium [MS shoots per explant, whereas, the longest shoots +KIN (0.9 mg/l) + BAP (2.5-3.5 mg/l) + NAA (1-2 length (9.64) were obtained on the media mg/l)] for proliferation and multiplication. Further containing 1.0 mg/l of BAP + 2.0 mg/l of NAA and these plantlets were cultured on rooting media. 5.0 mg/l of BAP + 2.0 mg/l of NAA, respectively (Table 1). It was observed that cytokinin was Plantlets with well developed roots were removed required in optimal quantity for shoot proliferation from the medium, then washed thoroughly under in some species of Zingiberaceae, but inclusion of running tap water to remove adhering solid MS low concentration of auxins along with cytokinin medium, and transplanted to plastic pots containing triggered the rate of shoot proliferation (Rout and sterilized peat moss soil and kept in a 50% shaded Das, 1997; Sharma and Singh, 1997). net house. The plants were covered by a transparent perforated polyethylene bags in order to maintain a With regards to number of leaves per explant, MS high humidity and to avoid plant dehydration by medium containing 5.0 mg/l BAP + 2.0 mg/l water loss. The plants were frequently watered to produced significantly the highest number of leaves keep high level of humidity. The polyethylene bags per explants (6.65) (Table 2), which indicate that were then removed after 7 days and the plantlets cytokinin alone or in combination with auxin (BAP were acclimatized for another three weeks. and NAA) had a significant response on the number of leaves per explant of this species. Results and Discussion Acclimatization of plantlets can be considered as The explants of Curcuma caesia cultured on MS one of the most important phase in tissue culture medium supplemented with different techniques. In this investigation, the mortality rate concentrations of BAP alone (0.0, 1.0, 3.0 and 5.0 of the plantlets with well developed roots that were mg/l) or in combination with NAA (0.0, 0.5, 1.0 acclimatized and hardened was low. On average, and 2.0 mg/l) produced both shoots and roots, 80% of in vitro transferred plantlets survived in

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potted soil and did not show any morphological of Curcuma caesia, species with a very important abnormalities. In conclusion, this protocol is a step medicinal value. forward towards improvement of the propagation

Table 1. Effect of growth regulators on in vitro multiplication of the number of shoots, shoot length and number of roots of Curcuma caesia grown on MS medium

Sl. No. PGRs (mg/l) No. of shoot Length of shoot No. of root 1 MSO 2.00e* 2.80g 4.50i 2 1.0 BAP 2.00e 3.30g 6.20h 3 3.0 BAP 3.30d 5.20f 11.10g 4 5.0 BAP 3.60cd 3.30g 4.30i 5 1.0 BAP + 0.5 NAA 4.00cd 7.57e 13.90d 6 1.0 BAP + 1.0 NAA 4.00cd 7.57e 13.70de 7 1.0 BAP + 2.0 NAA 4.20bc 9.64a 13.80de 8 3.0 BAP + 0.5 NAA 5.70a 9.20ab 12.80ef 9 3.0 BAP + 1.0 NAA 4.80ab 8.29cd 12.40fg 10 3.0 BAP + 2.0 NAA 5.10a 7.75de 15.85b 11 5.0 BAP + 0.5 NAA 5.30a 8.25cd 14.90c 12 5.0 BAP + 1.0 NAA 5.45a 8.82bc 16.10b 13 5.0 BAP + 2.0 NAA 5.65a 9.27ab 17.10a *Means followed by the same letter(s) within each column are not significantly different at P ≤ 0.05, according to Duncan’s multiple range test for each parameter. PGRs, plant growth regulator.

Table 2. Effect of growth regulators on in vitro multiplication of length of roots, number of rhizomes and number of leaves of Alpinia galanga grown on MS medium Sl. No. PGRs (mg/l) Length of Root No. of Rhizome No. Of Leaf 1 MSO 4.95ab* 1.10d 2.10g 2 1.0 BAP 5.40a 1.20d 2.85f 3 3.0 BAP 4.75bc 1.50c 3.30ef 4 5.0 BAP 2.30i 1.40c 3.40e 5 1.0 BAP + 0.5 NAA 3.25gh 1.50c 4.45cd 6 1.0 BAP + 1.0 NAA 3.75ef 2.05b 4.45d 7 1.0 BAP + 2.0 NAA 4.15de 2.10b 4.25d 8 3.0 BAP + 0.5 NAA 3.10h 2.05b 4.45cd 9 3.0 BAP + 1.0 NAA 3.57fg 2.10b 4.85bcd 10 3.0 BAP + 2.0 NAA 3.85def 2.05b 5.10bc 11 5.0 BAP + 0.5 NAA 3.90def 3.12a 5.25b 12 5.0 BAP + 1.0 NAA 4.10de 3.08a 5.50b 13 5.0 BAP + 2.0 NAA 4.25cd 3.07a 6.65a *Means followed by the same letter(s) within each column are not significantly different at P ≤ 0.05, according to Duncan’s multiple range test for each parameter

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