Standardization protocol of medicinal coleus (Coleus forskohlii) for rapid regeneration under in vitro conditions and its effective use in bio-pharming

Introduction

Coleus forskohlii is a medicinal plant which is a perennial that grows up to 45-60 cm tall and aromatic in nature. Leaves are usually pubescent, narrowed into petioles which are 7.5 to 12.5 cm in length and 3 to 5cm in width. Nodes are often hairy and have four angled stems that are branched. Inflorescence is a raceme, which is 15-30 cm in length and the flowers are stout, 2 to 2.5 cm in size, usually perfect and calyx hairy inside. Calyx is broadly ovate. The blue or lilac corolla is bilabiate. Lower lobes are concave and elongated so they can enclose the essential organs. The stigma is two lobed and ovary is four parted and the flower is crosspollinated by wind or insects (Prajapati et al., 2003) [6] . Coleus forskohlii can be used as a plant system to produce recombinant proteins because of its short duration of 6 to 7 months and it can be grown in moderate climate with light, medium and drained soil and produce enough biomass of about 400 to 600 kg dry wt /acre (Hegde et al., 2009) [3] . Callus induction and proliferation systems are known to be very useful for the genetic improvement of Coleus forskohlii which is an endangered multipurpose medicinal plant that has widespread applications. Mersinger et al. (1988) [5] successfully obtained dark green to yellow green callus from C. forskohlii when placed on Gamborg's B5 medium supplemented with 0.5 mg L -1 2, 4-D, and 0.2 mg L -1 kinetin. Callus cultures were also initiated on MS media containing NAA (2 mg/l) and BAP (1 mg/I) using 6-to 8-mm-long hypocotyl segments from 30-day-old aseptically grown seedlings (Sen et al., 1991) [11] .

The best medium for callus induction in C. forskohlii was reported by Tefera (1998) [13] , which contained MS medium supplemented with 1.0 mg L -1 indole-3-acetic acid (IAA) and 1.5 mg L -1 benzyl adenine purine (BAP) for the formation of a green compact callus. The highest rate of shoot multiplication, i.e., more than 150 shoots per callus, was produced in the MS medium supplemented with 4.6 mm kinetin and 0.54 mm NAA from the leaf-derived callus of C. forskohlii (Reddy et al., 2001) [8] . Recombinant proteins are primarily produced from bacteria cells, insect and cultures of mammalian cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms (Qiang and Huafang 2014) [16] . Biopharming is the use of plants for the production of heterologous proteins using genetic engineering techniques. It is widely used for scientific and medical purposes, including on an industrial scale. Plant systems are a promising direction of development of protein production technology (Fischer et al., 2015 and Loos et al., 2014) [1,2] . A wide variety of bacterial to complex multi subunit glycosylated human proteins can be produced in plants (Vyacheslavova et al., 2012) [14] . Plant systems provide the stable inherited expression of a foreign gene by transforming the nuclear DNA and fast highly effective production of the desired protein by extra chromosomal amplicons (Kawaka et al., 2017) [4] . Plants also provide a cost-effective and safe platform for vaccine development due to ease in scalability and a low risk of contamination with endotoxins or human pathogens. Hence this production system we can expect to convince the sceptic and can become approved for the use of emergency response therapeutics and vaccines in humans (Waheed et al., 2016) [15] . Coleus is suitable for transformation as there is an abundant protein accumulation in leaves, further it can be easily propagated using cuttings and hence easy to mass propagate with the additional advantage of horizontal gene transfer since they do not produce any pollen.

Materials and Methods

The study pertaining to the research on "Standardization protocol of medicinal coleus (Coleus forskohlii) for rapid regeneration under in vitro conditions and its effective use in bio-pharming"was carried out in the Department of Plant Biotechnology, University of Agricultural Sciences, GKVK, Bengaluru. This research was carried to find out the optimal concentration of growth regulators for producing callus and multiple shoots from leaf explant of C. forskohlii. The following six treatments were formulated using MS as a basal media with varying concentrations of 6benzylaminopurine (BAP) and Indole-3-acetic acid (IAA). Each treatment had three replications. The treatments are as follows. T 1: MS basal medium with 4 mg L -1 BAP+0.4mgL -1 IAA T 2: MS basal medium with 5 mg L -1 BAP + 0.4 mg L -1 IAA T 3: MS basal medium with 6 mg L -1 BAP + 0.4 mg L -1 IAA T 4: MS basal medium with 7 mg L -1 BAP + 0.4 mg L -1 IAA T 5: MS basal medium with 8 mg L -1 BAP + 0.4 mg L -1 IAA T 6: MS basal medium with 9 mg L -1 BAP + 0.4 mg L -1 IAA Observation: The various parameters recorded for observations were 1. No. of explants responding 2. Average number of days taken for callus initiation 3. Callus intensity, color and texture. 4. Average number of days taken for shoot initiation 5. Average number of days taken for root initiation 6. Average number of shoots per explant 7. Average number of roots per explant Preparation of explants 1. The fresh young leaves of C. forskohlii were collected from the green house and kept under running tap water overnight to remove the surface contaminants. 2. The leaves were then treated with 1 percent bavistin for 2 hr, rinsed with sterile water several times and transferred into sterile laminar airflow chamber. 3. The leaves were treated with 0.1 percent HgCl 2 thrice for 10 sec. inside the laminar airflow chamber. 4. The treated leaves were thoroughly washed 3-4 times with sterile distilled water. 5. The treated leaves were blot dried and were cut into pieces of 1 cm 2 -1.25 cm 2 size by using scalpel. 6. The explants were then placed on tissue culture bottles containing MS media with different concentrations of growth hormones. 7. The bottles were then placed in the culture room by maintaining temperature at 25 °C and 16 h photoperiod with 65 percent of relative humidity.

Acclimatization 1) Washing of rooted plants

After the proper development of roots, the plants were taken out carefully from the culture bottles. Then the plants were placed under gentle flow of tap water for 2 h to remove adhering agar completely. Plants were then treated with a fungicide bavistin 0.4 percent for half an hour to prevent fungal contamination and then were transferred to varying potting mixture.

2) Planting in potting mixture

Vermicompost and sand were mixed in a proportion of 1:1 and autoclaved. This was done for adequate drainage of surplus water in tiny plastic cups with holes at the bottom. The plants were removed from the MS medium and moved to the plastic cups with correct root orientation. The pot mixture was replenished with half a MS medium (diluted 100 times) until it was moist. Plants were then covered with poly bags having holes in them for air circulation to maintain high humidity. All plastic cups were kept at about 20-22 °C in the green house, which was maintained by cooling pads and fans. Plants were irrigated according to their requirements.

Results and Discussions Standardization of regeneration protocol of Coleus forskohlii using leaf explants

The standardization of regeneration protocol of Coleus forskohlii using leaf explants was conducted. The various concentration of growth hormones such as auxin (IAA) and cytokinin (BAP) was used and effects of these hormones were studied on callus initiation, shoot regeneration, and root regeneration. The results pertaining to this is given below.

Induction of callus

In vitro callus culture of Coleus forskohlii was established, which was prepared from leaf explants using MS media containing 0.4 mg L -1 of IAA with different concentrations of BAP (4 mg L -1 to 9 mg L -1 ). The callus texture obtained during this experiment was compact and fragile and the color of the callus was pale green to dark green. Significantly highest percent of callus was obtained in the combination containing 9 mg L -1 of BAP and 0.4 mg L -1 of IAA with 94 percent response (Table 1, Fig. 1 and Plate 1). Tefera (1998) [13] , reported the formation of green compact callus using leaf as explants on the MS medium supplemented with 1.0 mg L -1 IAA and 1.5 mg L -1 BAP. Sanjay Rao (2013) [10] , found that 6 mg L -1 BAP and 0.4 mg L -1 IAA was good for callus induction.

Shoot initiation

The mean number of shoots per callus differed significantly between the treatments. It was significantly highest in T 6 (11.16) (9 mg L -1 of BAP and 0.4 mg L -1 of IAA). In T 3 (6 mg L -1 of BAP and 0.4 mg L -1 of IAA) it was 9.00 which was significantly higher than that in T 4 (7.11) (7 mg L -1 of BAP and 0.4 mg L -1 of IAA) T 2 (4.53) (5 mg L -1 of BAP and 0.4 mg L -1 of IAA) T 5 (3.22) (8 mg L -1 of BAP and 0.4 mg L -1 of IAA) and T 1 (3.00) (4 mg L -1 of BAP and 0.4 mg L -1 of IAA). The percentage of shoot formation from callus ranged from 51.0 to 71.33 percent. Significantly high shoot formation of 71.33 percent was obtained for the combination of hormones containing 9mg L -1 of BAP and 0.4 mg L -1 of IAA (Table 2, Fig. 2, Fig. 3 and Plate 1). An effective multiplication of shoots from the callus was observed in MS medium supplemented with 2 mg L -1 BAP and 0.4 mg L -1 NAA (Sandesh, 2007) [9] . Sreedevi and Pullaiah (2013) [12] , cultured the nodal explants of C. forskohlii on MS medium supplemented with BAP (0.25 mg L -1 ) and Kin (0.25 mg L -1 ) resulting in average of 41 multiple shoots.

Rooting and Hardening

Rooting was obtained with the MS media containing different concentrations of BAP ranging from 4 mg L -1 to 9 mg L -1 and IAA 0.4 mg L -1 after sub culturing at the 60 th day. Rooting was significantly highest in T 6 (6.34) (9 mg L -1 of BAP and 0.4 mg L -1 of IAA). In T 2 (5mg L -1 of BAP and 0.4 mg L -1 of IAA) it was 5.44 which was significantly higher than that in T 4 (5.22) (7 mg L -1 of BAP and 0.4 mg L - 1 of IAA) T 3 (4.65) (6 mg L -1 of BAP and 0.4 mg L -1 of IAA) T 5 (3.8) (8 mg L -1 of BAP and 0.4 mg L -1 of IAA) and T 1 (3.33) (4 mg L -1 of BAP and 0.4 mg L -1 of IAA). The rooted plants were transferred into the pot mixture containing peat and sand in 1:1 ratio. The plants were covered with polythene bags containing holes for maintaining the relative humidity (Table 2 and Plate 2). Tefera (1998) [13] , reported that although IAA at 1mg L -1 was effective in inducing roots, normal rooting was also observed in hormone free medium. Reddy et al. (2001) [8] , cultured in vitro differentiated shoot lets of C. forskohlii measuring 3.5-4 cm in length, derived from the callus on MS medium supplemented with different concentration of IAA for rooting.

Conclusion

The standardization of regeneration protocol of Coleus forskohlii using leaf explants was conducted. The various concentration of growth hormones such as auxin (IAA) and cytokinin (BAP) was used and effects of these hormones were studied on callus initiation, shoot regeneration, and root regeneration. The callus texture obtained during this experiment was compact and fragile and the color of the callus was pale green to dark green. Significantly highest per-cent of callus, mean number of shoots per callus and the rooting was obtained in the combination containing 9 mg L -1 of BAP and 0.4 mg L -1 of IAA with 94 percent, 11.16, and 6.34 respectively.

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