A Protocol for Rapid Clonal Propagation and Microrhizome Production of Curcuma caesia Roxb. (Zingiberaceae): A Critically Endangerd Medicinal Plant of North East India 

Curcuma caesia Roxb. (Zingiberaceae), perennial aromatic herb with high medicinal potentialities is under the category of Critically Endangerd species as per the IUCN Red List of threatened species (Das and Saikia, 2019). It is commonly known as ‘kolahaldhi’(Black turmeric) in Assam. This plant has diverse medicinal potentialities (Abubakar and Pudake, 2014).The rhizome is pungent, fragrant, heating, appetizer, antihelmintic, alexiteric and also claimed to be ethno medicinally useful in asthma, tuberculous glands of the neck, enlargement of the spleen etc (Thingujam and Nayak, 2019). The rhizomes are also used in the treatment of smooth muscle relaxant activity (Arulmozhi et al., 2006) haemorrhoids, leprosy, cancer, epilepsy, menstrual disorder, Alzheimer’s disease (Sasikumar, 2005, Behar et al., 2013).
       
The indiscriminate exploitation of the species from natural habitat  leads to make  this species under the threat of extinction (Mannangatti and Narayanasamy, 2008). Moreover, Zingiberaceae plants are most frequently infected with various pathogens like Pythium species causing rhizome rot disease, Colletotrichum species causing the leaf spot etc. resulting reduction of the yield and the quality of the product and even causing the death of the plant (Dixit et al., 2000). To overcome, the limitation of conventional method of propagation in vitro propagation is the only method for conservation and large scale production of this endangered genotype in order to prevent their potential extinction.
       
Although in vitro plant propagation has been accomplished in other Curcuma species (Khumaida et al., 2019) however, little work has been reported on Curcuma caesia (Shahinozzaman et al., 2013).
       
Moreover, some work has been reported in other species of Curcuma for in vitro formation of storage organs such as micro rhizomes (Cheethaparambil et al., 2014) but not reported in Curcuma caesia till date. As these kinds of propagules can be directly transferred to the field without any acclimatization and hardening procedures it facilitate for easy transportation and mass scale production. The present investigation was carried out to establish a standard protocol for in vitro multiplication of Curcuma caesia and to study the factors affecting for micro rhizome induction of the species.
       
GC-MS analysis for essential oil and RAPD pattern of Curcuma caesia of both conventionally propagated and tissue cultured plant material also helped for qualitative analysis of the plantlets.

Rhizomes of C. caesia Roxb. collected and grown in the experimental garden of Department of Botany, Gauhati University, Guwahati. Dormant rhizome axillary buds were excised from the clean rhizome, which were used as the source of explants. Freshly collected rhizomes were cleaned with running tap water and immature buds were excised with sharp blade and washed with detergent (Tween-20, 0.1% v/v) for 15 min and subsequently rinsed with clean water. Explants were then surface sterilized in disinfectant (0.1% HgCl₂ to which two - three drops of Tween-20 were added) for 10 minutes after that rinsed five-six times with sterile distilled water and aseptically cultured on shoot induction medium to obtain contamination free cultures. MS(Murashige and Skoog, 1962) basal media supplemented with 3% sucrose and 2.2g/l Gelrite modified with various concentrations of N6-Benzyl-aminopurine (BAP 0.05 to 3.0 mg/l) in combination with Kinetin (Kn; 0.5- 2.0 mg/l), Indole-3-acetic acid (IAA; 0.5-1.0 mg/l), a-naphthaleneacetic acid (NAA; 0.5-1.0 mg/l) and 2, 4-D (0.5-4.0 mg/l) were used for establishment of the culture. Laboratory reagent-grade sucrose was replaced by locally available commercial sugar as carbon source for reducing the cost of the media.
       
The pH of the media was adjusted to 5.8 and autoclaved at 121°C with 15 lbs pressure for 15 minutes. Excised buds were inoculated into culture media and incubated at 25±2°C under 16 hours of photoperiod. Most of the cultures sprouted within 7-12 days of inoculation and the emerged shoots (1.0-2.0 cm) were sub cultured in the same media for further multiplication. A total of 20 explants were used for each treatments and the number of shoots per explant was recorded after four weeks of inoculation. Each set of experiment was repeated thrice.
       
The in vitro established clumps of microshoots were sub cultured at 15 days interval for 5 cycles. Fully grown shoots were separated and transferred to rooting media containing Indole-3-acetic acid (IAA; 0.005 - 2.5 mgl^-1), a - naphthalene acetic acid (NAA; 0.005 - 2.5 mgl^-1), 2% and 3% sucrose (market sugar). Rooted shoots were transferred to ½ strength MS basal medium without growth regulator for 10 days. The plantlets were washed thoroughly with sterile distilled water and then transferred to potting mixture (soilrite and compost, 10: 1, v/v) and kept in the growth chamber for 7 days. The environment of the growth chamber was maintained initially at 80% R.H., 24°C temperature, 12 hrs photo-period and gradually adjusted with the slight alteration of the environment to reach as similar to the outside. Finally, in vitro acclimatized plantlets were transferred to the soil in the earthen pots and ultimately planted in the nursery for hardening.
 
Microrhizome Induction
 
After separating the fully grown shoots originated from the rhizome the micro shoots were divided into small parts and again transferred to fresh medium devoid of growth hormones and cultured for another four weeks to avoid carryover effects of growth regulators and then cultured in freshly prepared medium for micro rhizome induction. Aseptic in vitro grown shoots approximately 4-5 cm long were derived from the established culture of C. caesia were used as explants for induction of micro rhizomes. MS medium supplemented with BAP (0.5 to 5.0 mgl^-1), Kn(0.5-5.0 mgl^-1), NAA(0.5 to 1 mgl^-1) and IAA(0.5 to 1 mgl^-1) along with sucrose (3%, 6% and 9%) was used for the study of micro rhizome induction and cultures were incubated at 25±2°C. Varying photoperiods (16 hrs, 8 hrs, 4 hrs and 0 hr dark) were tried to test their effect on micro-rhizome formation. Each treatment was done on 15 replicates and repeated for 3 times. Data on the percentage of rhizome formation in each replication, rhizome weight and number of buds per rhizome were recorded after 45 days of culture. The micro rhizomes were harvested aseptically and were repeatedly washed in running tap water and air-dried prior to storage in soil. Micro rhizomes were then stored in poly bags containing sterile sand. Poly bags were kept in net house (30°C) and growth initiation percentages of these micro rhizomes were also recorded.
       
The recorded data was subjected to statistical analysis by using one way ANOVA using Duncan’s Multiple Range Test (DMRT; Duncan, 1955). The bio-active compounds of the rhizome of conventionally propagated and tissue cultured plants were studied by GC-MS analysis according to standard methods with slight modifications.
       
The protocol of Doyle and Doyle (1987) was followed for isolation and purification of genomic DNA from both micro propagated and field grown mother plants. The protocol for RAPD analysis was adapted from that of Williams et al., (1990) and Panda et al., (2007) to study the banding pattern of DNA.

       
The effect of 1.0 mgl^-1 BAP with 0.25 mgl^-1 2, 4-D on adventitious shoot proliferation was observed in C. caesia (Table 1). It was observed that the inclusion of 1.0 mgl^-1 BAP and 0.25 mgl^-1 2,4-D in the culture medium led to the highest number of shoot per explant (23.80±0.51) and shoot length (5.44±0.04 cm) as compared to that of other treatments within 8 weeks of culture (Table 1, Plate e-h). A maximum of (84.40±1.24%) of shoots shows multiplication under this combination of medium.
 

       
The synergistic effect of BAP (1.0 mgl^-1) in combination with 2,4-D (0.25 mgl^-1) on promotion of C. caesia shoot cultures is in agreement with the observations of other workers in Curcuma longa (Zapata et al., 2003) and in Zingiber officinale (Khatun et al., 2003).
       
This is followed by the MS media modified with BAP (1.0 mgl^-1) and NAA (0.5 mgl^-1) showed (81.20±1.58%) shoot multiplication (Table 1, Plate b). However, Balachandran et al., (1990) and Panda (2007) had reported maximum shoot formation of Curcuma longa in the MS medium supplemented with BAP (3 mgl^-1). In this study a comparatively lower response was recorded when BAP was added alone in the medium which indicates that the addition of 2, 4-D in the culture medium results in high morphogenetic ability of the cultures of C. caesia. However, higher concentration of BAP is found to be inhibitory with or without the supplements of auxin. Stanley and Keng (2007) also support the same result in Zingiber zerumbet. Higher concentration of BAP (5.0 mgl^-1) with NAA (2.0 mgl^-1) showed callusing of the explants with fewer numbers of shoots (2.00±0.00 shoot/explants). In such cultures shoots were stunted with a mean shoot length of 2.30±0.08 cm. However, the higher concentrations of BAP reduced the frequency of shoot induction which is in agreement with the studies in Zingiber officinale (Kambaska and Santilata 2009) and in Kaempferia rotunda (Bejoy et al., 2006).
       
In the present study, BAP showed better results as compared to Kn (Table 1). But higher concentration of BAP and Kn exhibited negative effect on shoot regeneration in C. caesia. It has also been reported earlier that higher concentration of plant growth regulators was not suitable for in vitro culture of Zingiberaceae species (Stanly and Keng, 2007). The results of the present study confirm the importance of the PGR in the dedifferentiation process of C. caesia. Although positive response was observed in the control culture (without PGR) but it was very low compared to the other treatments. It has been observed that market sugar also effective for shoot multiplication of C. caesia.
       
Splitting and removal of induced shoots and transferring 2 to 3 shoots per culture favoured higher number of shoots than that of dividing into small parts. Moreover, retention of the explants with proliferated shoots in the initiation medium for more than 8 weeks resulted in decline as indicated by shoot necrosis and premature leaf browning. Shoot tip necrosis was also observed in few treatments with higher concentrations of cytokinins. BAP not only enhanced shoot growth but also supported root induction (Khatun et al., 2003). Shoot multiplication accompanied by simultaneous rooting of the shoots was seen after third sub culturing on PGR-free media. Spontaneous rooting was also recorded on the MS basal media supplemented with BAP along with IAA or NAA like other Zingibers, viz. turmeric and ginger (Chan and Thong, 2004), Alpinia galanga (Borthakur et al., 1999) and in Coccinia indica (Borah et al., 2019). Although the rhizome bud of C. caesia produced shoots and roots simultaneously on most of the media tested for shoot multiplication, however to develop a healthy root system, fully grown shoots (4-6 cm) after removing all the roots were transferred to MS medium supplemented with BAP, IAA or NAA. MS (half strength) medium enriched with NAA (1.5 mgl^-1) and reduced level of sucrose (20 gl^-1) responded for (91.00±0.32%) rooting in C. caesia, which produced a mean of (14.00±0.45) number of roots per shoot within a short period of (10.00±0.32) days of culture (Table 2). 
 

       
The rooted plantlets of C. caesia obtained from in vitro propagation were sequentially acclimatized (hardened). After hardening the plantlets were transferred in field conditions.
 
Microrhizome Induction
 
Fully grown in vitro raised plantlets were trimmed aseptically and transferred to microrhizome induction medium. After 20-25 days of incubation in microrhizome induction medium containing various phytohormones and different level of sucrose, the swelling of shoot bases were observed, followed by appearance of microrhizomes at the base within 30-45 days of incubation (Plate k). It was observed that sucrose plays a significant role in the size and number of microrhizomes in C. caesia and it was observed that 3% sucrose  reduced both number and size of the microrhizomes even by increasing the concentration of BAP from the range of 0.5 to 5 mgl^-1 or by increasing or decreasing the duration of the photoperiod. In the present study 9% sucrose was found to be optimum for the production of microrhizomes on ½ strength MS basal medium supplemented with BAP (1.0 mgl^-1) after 4 weeks of culture. Healthy, large sized and maximum number (16.00±0.45) of microrhizomes with an average weight of (1.77±0.03) g were obtained in this combination  under 16 hours of photoperiod (Table-3, Plate-l). Both 6% and 9% sucrose gave good response in case of induction of healthy microrhizome in C. caesia. The results of the present investigation supports the report of Shirgurkar et al., (2001) and Nayak (2000) who obtained optimum micro-rhizome induction in Curcuma longa using 6-9% sucrose. The enhanced rate of in vitro organ formation with increasing concentration of sucrose may be attributed to the presence of high carbon energy in the form of sucrose since storage organs mostly store carbohydrates (Nayak, 2000).
 

 

       
BAP had significant effects on in vitro microrhizome induction of C. caesia (Table 3). Among different combinations of PGRs, 1.0 mgl^-1 BAP exhibited a higher per cent of healthy microrhizomes (73.80±0.37%) induction. Our studies agree with Nayak (2000) in C. aromatica Salisb. for enhancement of microrhizome production.
       
In the present study half strength of MS is found to be more suitable in comparison to full strength of MS salts producing a large number and comparatively larger sized rhizomes (Table 3, Plate l).  And the similar result was found by Shirgurkar et al., (2001) in turmeric. While Islam et al., (2004), Nayak (2000) and Sunitibala (2001) used full strength of MS basal medium for microrhizome induction in C. longa. Sharma and Singh (1995) also found full strength of MS basal medium along with 7.5% sucrose and 35.2 μM BA optimal for the production of in vitro microrhizome in ginger. But incubation under complete darkness reduced the number and size of the microrhizomes significantly. In the present study, Kn alone or in presence of NAA induces very small sized (0.67±0.00 g) microrhizomes. However, Sunitibala et al., (2001) reported that Kn (1.0 mgl^-1) is better suited for in vitro rhizome induction in C. longa L.
 
Microrhizome evaluation
 
In vitro produced microrhizomes were isolated after 60 days and harvested microrhizomes were directly transferred to the sand-soil bed for plantlet development. Plants developed from three different sizes of microrhizomes [0.5 - 1.0 cm (small), 1.1, 2.0 cm (medium) and > 2.0 cm (large)] (Table 4) were evaluated using various morphological characters. Germination, survival rate and morphological characters varied depending upon the sizes of microrhizomes. Plants regenerated from bigger microrhizomes were found to be more vigorous in terms of their shoot, root and leaf growth parameters (Table 4). Bigger microrhizomes were more competent and vigorous in comparison to smaller rhizomes as also reported earlier by other workers (Islam et al., 2004 and Shirgurkar et al., 2001). In the present study also a much higher survival rate was obtained because the average weight of microrhizomes was higher, which indicates that large sized microrhizomes is a key factor in achieving commercial success in microrhizome induction in Curcuma caesia. Microrhizomes produced in the present investigation were stored under moist conditions at room temperature and more than 80% of the sprouted microrhizomes developed shoots and roots two months after they had been successfully transferred to the field. Sharma and Singh (1995) studies also confirm the same result.
 

       
GC-MS analysis of essential oil extracted from tissue culture raised rhizomes revealed the presence of major compounds similar to that of the source (naturally propagated) plants i.e., 1, 8-cineole(35.3%), camphor (14.7%), beta-pinene (9.23%), bonieol(5.7%) and α- terpineol (3.3%) as the major compounds (Fig 1 and 2). From the present investigation, it was established that in Curcuma caesia, camphor is one of the specific component which gives intense camphoreous odour.
 

 

       
Even though micropropagated plants produced more leaves, grew faster and had more multiplication response than their vegetative counterparts, there was no significant variation in plant morphology and essential oil content as indicated by GC-MS study. Micropropagated plants of both the species were genetically uniform to donor plants, as indicated by random amplified polymorphic DNA (RAPD) analysis (Plate 2).