Amaranthin Production Through Cell suspension Cultures of Celosia Cristata

Abstract

OBJECTIVES Colours make food items attractive and add to their acceptability and palatability. Natural colours are short in supply so synthetic colours are used to meet over increasing demands. It has, however, been observed that many of the synthetic colours are not safe for human consumption. Ingestion of synthetic, even if consumed in very small quantities over a long period, can cause cancer. There is great awareness among the consumers to replace such synthetic with safe colouring agents. Natural colours of fruits and vegetables have been consumed for husband years without any ill effect. These days the consumption of colours of natural origin is faster as compared to the artificial colours. Market for natural food colours have growth rate 6% compared with 10% for the artificial ones. Many methodologies to be developed for plant cell consumption culture and its evaluation for technical and economical feasibility. Through this technology direct production of pigments in amount equal or superior to natural colours from intact tissues of plant is quite possible. Moreover the production of colours from plant tissues can be carried out round the year whereas the growth of natural plants is seasonal. Rapid advancement in the field of cultured cells for production of specific secondary metabolites, flavours, pigments, drugs, agrochemicals etc. has made it one of the most rewarding and promising fields in plant biotechnology. EXAMINATION AND RESULTS To achieve the objective a through study was carried out and following experiments were carried out: Seeds of celosia cristata were collected from double hybrid plant and stored at low temperature. Hypocotyl and leaves were obtained aseptically by culturing celosia seeds in Ms Basal medium. Celosia seeds were sterilized with 95% ethanol and 0.1% HgCl2 solution. Hypocotyl and leaves need no surface sterilization as they were obtained aseptically. Murashige and Skoog’s medium supplemented with 30g/L sucrose as a source of carbon and solidified with agar was used for initiation and maintenance of celosia cristata cultures. pH of all media was adjusted at 5.7 Modified technique was used to prepare stock solution of 2,4-D, BAP, NAA All solutions were prepared with the help of magnetic stirrers. Details are given in the report. OBSERVATION PROCEDURE Cultural were frequently observed after inoculation and the following formula was adopted:

Cl = 100 n G ------------ N Where Cl = Callus index n = no. of explants per culture G = Mean callus growth index N = No. of replicates for qualification of callus All cultures were incubated at 26 ±1°C under 16 hours photo period at 3000 lux of cool white light by fluorescent tubes. Hypocotyl Callus Callus induction of hypocotyl explants from in vitro grown seedling of celosia cristata was observed after 25-30 days in media supplemented with 0.1-0.5 mg/2-4 D. It was found that: Of various concentrations tested. Best results were achieved in medium supplemented with 1 mg/1 of 2,4-D. Similar results were also observed at concentrated 2&3 mg/l of 2,4-D. Experiments showed that lower (0.1-0.5 mg) concentration of 2,4-D were not stimulatory to callusing. Similarly higher (4-5 mg/l) concentration were less stimulatory to callusing. Effect of 2,4-D in combination with KIN on callus induction showed relatively improved textures of calli. Higher concentration of 2,4-D and KIN enhanced the browning of cultures. Medium supplemented with NAA alone did not have any pronounced effect on the growth from hypocotyl explants of Celosia cristata. Of various concentration of NAA, best effects on growth was observed in medium supplemented with 4 mg/l (Cl = 180) The results of the combined effect of NAA and BAP on callusing of Celosia Cristata are best in medium supplemented with 1 mg/l each of NAA and BAP (Cl =320) Hypocotyl plant did not produce any significant results in media supplemented with lower auxin to higher cytokinin concentrations. Moderately growing proliferating calli changes from off-white to green colours (Cl 140-200) in media supplemented with higher auxin to lower cytokinin concentration. Stem Callus Of various concentration best results were obtained at the concentration 1.0 mg/l. Very low (0.1 mg/l) and higher (5-10 mg/l) concentrations did not induce any growth. BAP, on the whole, did not considerably enhance differentiation as mostly slow to moderate growth were observed. NAA also did not produce any significant results when incorporated in the media for callusing from stem explants. NAA and BAP when tested together, produced significant results. Compact greenish calli with frequent red pigmented zone were observed in MS medium supplemented with 1.0 mg/l each of NAA and BAP. Best results were obtained in media supplemented with 2.0 mg/l 2,4-D and 1.0 mg/l K/N (Cl – 240). Leaf Callus No callus induction was observed from leaf explant in media supplemented with lower of 2,4-D. Growth and proliferation improved significantly when KIN was added to the supplements of 2,4-D. Best results were achieved with 3.0 mg/l 2,4-D and 1.0 mg/l KIN. Best growth in leaf cultures was observed in medium supplemented with 5.0 mg/l NAA (Cl=180). Highest concentration of BAP, best growth was observed at concentration of 5.0 mg/l. Some browning was, however, observed in these cultured. Highly pigmented and rapid proliferating were achieved in the medium supplemented with 5 mg/l each of NAA and BAP (CL-520). Cultures Results of the experiment showed that pigmentation was prominent in callus lines initiated from hypocotyl explants though the quantity of pigmented mass in production to total biomass was less then the leaf calli. Best pigmentation among the hypocotyl lines was achieved in mediums supplemented with 2,4-D (1.0 mg/l) or NAA (1.0 mg/l) + BAP(1.0 mg/l). Leaf calli exhibited a tendency of both rapid growth and heavy pigmentation. Calli grown in medium supplemented with 5 mg/l each of NAA and BAP exhibited best results (++++). This callus line was labelled as PCSIR – CC 250. Calli grown in this above medium proliferated rapidly and red pigmented callus zones continued to increase during subcultivations. Supplementation of medium with coconut water reduced both callus growth and pigmentation. Results were same when sucrose concentration were reduced from 4 to 102%. Similar observations were also made when auxin and cytokinin of the parent medium were modified by first reducing and then eliminating them one by one. Reduction of nitrogen did not presented the loss of pigmentation. Growth and pigmentation in PCSIR-CC25D callus line improved. Better growth and pigmentation were recorded at 5 that at 7.5% sucrose concentration. Stability could not be induced in the pigments of PCSIR-CC25D callus line when other factors, namely, double strengthening the micro salts, supplementation of tyrosine addition of flower extract, addition of NaCl and variation in the medium pH were studied. Regeneration through Callus Celosia cristata calli induced and grown from leaf explant with 0.5 mg/l each of NAA and BAP or 7.5 mg/l BAP, regenerated short buds during third or fourth subculturing on the same medium. Extensive short regeneration from calli induced from the inter model explant and subsequent proliferation in the same culture was also observed in medium supplemented with 0.1 mg/l 2,4-D and 0.5 mg/l KIN during Cultivation. Red violent flower regenerated from leaf calli induced and sub cultivated on the same medium was rapidly noted in those grown in MS medium supplemented with 0.1 mg/l 2,4-D and 0.5 mg/l KIN. Extraction of Pigments Flowers of celosia cristata were socked overnight in distilled water, filtered and pigment powder was obtained by following techniques. Spray Drying Pigmented solutions was fed to the laboratory spray dryer. Operation was started when the inlet and doublet temperatures were 100°C + 55°C respectively. Compressed air pressure was maintained at 0.8-1 kg/cm.

Cabinet Dryer Pigment solution was placed on 55 steel tray in layer of 3mm thickness and was dried in cabinet dryer. IDENTIFICATION AND CHARACTERIZATION OF PIGMENTS Solubility : Solubility of dehydrated colour material was checked in various solvents. Colour reaction: Changes in colour of pigmented solution from both natural and callus culture in acidic and basic media was noted. Spectrophotometry: Pigmented solutions of both dehydrated material and callus culture was observed by addition of different compounds/salts as a stabilizer. Findings Red and violent pigments produced as a secondary metabolite by the explants were predominate in callus lines initiated from leaf explant. The quality of pigmented mass in proporation to total biomass was always maximum in leaf calli. Leaf calli exhibited a tendency of both rapid growth and heavy pigmentation. Red coloured callus line products from leaf explants on medium supplemented with 5 mg/l each of NAA and BAP was best in respect of pigmentation. This callus line labelled as PCSIR CC 25D proliferated rapidly in the medium and the red callus zones continued to increase during sub culturing. When ratio of red biomass to green biomass exceeded 60% red cell to 40% green cells, quick breakdown occurred and brown discolouration resulted thereof. Dehydrated pigment was found to be rapidly soluble in water and insoluble in organic solvents i.e. absolute ethanol (C2H5OH), acetate (CH3)2O, Chloroform CHCl3, ethylacetate C2H5COOCH3, hexane C6H6, methylene chloride (CH3Cl) and ether ( C2H5)2O. Stability of aqueous coloured solutions was affected by pH on addition if 1 N HCl – a distinct colour from pink to deep pink was observed whereas addition of 1 N NaOH changed the colour from pink to violet and the brown. The UV-Vis spectrum of pigmented solutions showed a maximum absorbance at 540 nm which is characteristic max. of betacyanin group. It was also observed that addition of NaCl salt improved the stability of natural colour solution. The colour extracted from the plant with 0.6% NaCl solution and spray dried resulted in a stable colour and maximum pigment yield. The colour was identified by IR spectroscopy – the spectrum showed the presence of doublet for > NH or OH at 3442 cm, - COOH at 2524 cm, alkene at 1632 cm C-0 stretch of ether at 1053 cm. For removing proteins and carbohydrates from natural colour a successful method has yet to be developed. The pigment isolated with 0.6% lactose solution was found to be highly hygroscopic and was difficult to collect.

CONCLUSION It is now established that synthetic colours are harmful for human health so every effort is made to replace them by natural colours. But there is a great difficulty in the procurement of natural colours as these are not available according to the required quantity. This study was aimed at developmental extraction and natural colours from callus and the flowers. It has now been proved that the colours of the two are quite identical in chemical as well as physical properties. This cultured cells of Celosia cristata can be exploited commercially round the year. Presently this technique is the only hope for producing natural products in abundance. The study has established also that the natural colours can be stabilized and isolated without using chemicals. There is an urgent need to develop tissue culture techniques to increase the production of natural colours.