| Okra(Abelmoschus esculentus L.)is an economically important vegetable crop grown worldwide for its edible immature pods and powdered seeds.More recently,presence of health beneficial phytochemicals and phenolic compounds has been reported in its succulent pods as well as in powdered seeds.However,okra plants are highly susceptible to various biotic and abiotic factors,which adversely affect the growth and yield of the crop and result in great economic losses.These problems could be addressed by genetic engineering methods to complement conventional breeding techniques.However,okra is one of the most recalcitrant crops for genetic modification with the lack of an efficient tissue culture system amongst the chief reasons.Therefore,the aim of this study was to develop an efficient in vitro regeneration system for A.esculentus and to gain more knowledge on how nitrogen concentration and light intensity affect the anthocyanin and other secondary antioxidants accumulation in callus cultures.Three different in vitro experiments were conducted via using two different okra cultivars,using green pod okra cultivar 'wufu' several parameter were studied to establish an efficient and reproducible tissue culture system,whereas,using red pod okra cultivar 'Hongjiao' several parameters for in vitro production of anthocyanin were investigated.During tissue culture work phenolic compounds were secreted from cut ends of hypocotyl,cotyledon and cotyledonary node explants to culture medium and hindered callus formation and culture establishment.Attempts to overcome this phenomenon such as supplementation of anti-browning additives like activated charcoal,citric acid,ascorbic acid and silver nitrate in culture medium were positive effect on callus formation and culture establishment.1.Using hypocotyl and cotyledon as explants,an in direct in vitro regeneration protocol was developed for 'wufu' okra cultivar.MS medium supplemented with 200 mg 1-1 activated charcoal in combination with 0.5 mg 1-1 2,4-D + 1.5 mg-1 BA was most effective to overcome phenolic secretion and efficient callus induction(67.67 ± 3.48%)from hypocotyl explants.On the other hand MS medium supplemented with 5 mg 1-1 each of citric and ascorbic acid in combination with 1.5 mg 1-1 NAA + 0.5 mg 1-1 BA was most effective to overcome phenolic secretion and efficient callus induction(30.00 ± 3.61%)from cotyledon explants.The combination of 2 mg-1 BAP and O.lmg 1-1 IBA in culture medium was effective for shoot induction.Hypocotyl remained the best explant for callus formation(67.67± 3.48%)and shoot regeneration(52.67 ± 3.61%).The rooting protocol was improved(76.67 ± 2.91%rooting response)by supplementation of 200 mg 1-1 activated charcoal and 2 mg 1-1 IBA in half-strength MS medium.Sterile soil pots holding vermiculate and sand(3:1)with sufficient moisture were found efficient for acclimatization(with 80%survival)of plantlets under growth chamber condition.2.Using cotyledonary node as explant source,a rapid and reproducible protocol for direct shoot regeneration was developed.Phenolic secretion from explants into culture medium was successfully overcome by supplementation of 15 mg 1-1 ascorbic acid into plant growth regulator(PGR)free MS medium.Addition of regeneration enhancers(Silver nitrate and Pluronic F-68)in culture medium was found effective treatments to enhance the multiple shoot production from established culture.Multiple shoot regeneration medium containing 0.5 mg 1-1 NAA + 1 mg 1-1 TDZ + 0.1%Pluronic F-68 produced highest number of shoots(a mean of 9-3 ± 0.9 shoots per cotyledonary node explant).Best shoot elongation(5.3 ± 0.9 cm)was obtained on medium containing 1 mg 1-1 BA + 0.1 mg 1-1 GA3.Rooting protocol was improved(82%frequency)with incorporation of 200 mg 1-1 activated charcoal and 1 mg 1-1 IBA in 1/2 MS medium.Plantlets acclimatization(with 85%survival)was carried out in plastic pots containing sterile vermiculite and sand(3:1)with suitable moister,inside a plant growth chamber at 25 ± 2? and 70%relative humidity.Moreover,the entire protocol,from seed germination to establishment of regenerated plants,was accomplished in only three and a half(3.5)months.3.Parameters were optimized for anthocyanin production in callus cultures of red pod okra cultivar 'Hongjiao'.Maximum callus biomass accumulation(3 g FW)was achieved when calluses were cultured on MS medium containing 60 mM nitrogen under 40 ?mol m-2 s-1 light intensity.In contrast,maximum values of total anthocyanin accumulation(TA;7.3 CV/g FW),total phenolic content(TP;12.07 mg/100 g FW),total flavonoid content(TF;2.47 ±0.15 mg/100 g FW),and total antioxidant activity(TAA;56.10 ?mol Trolox/g FW)were observed when calluses were cultured on MS medium containing 40 mM total nitrogen under 80 ?mol m-2 s-1 light intensity at 25 ± 2? and 70%relative humidity.In addition,positive correlations among anthocyanin accumulation,total phenolic content,flavonoid content,and antioxidant activity were observed.High performance liquid chromatography(HPLC)was performed for qualitative and quantity analysis of callus cultures.Most of the pigments from the callus extracts were identical with pod anthocyanins,and appeared on the ODS-column HPLC with lower concentration than the main pigments of the pod tissues.These findings indicate that callus cultures of red-pod okra represent a potential source of bioactive compounds with antioxidant properties for industrial applications.4.In conclusion this PhD project has contributed new knowledge on in vitro regeneration and anthocyanin induction in callus cultures of A.esculentus.Inclusion of anti-browning additives in culture medium proved beneficial to reduce phenolic compounds secretion from explants into the culture media and to improve culture establishment.Furthermore,manipulation of plant growth regulators should be taken into account depending on anti-browning additives and explant use during okra tissue culture.In addition,total nitrogen concentration of MS medium and light intensity proved important factors for anthocyanin accumulation in callus culture.We concluded that to overcome on phenolic exudation to culture medium,browning of explants and culture failure in okra tissue culture,explants should be incubated on anti-browning supplemented medium for initial culture,and then should be transferred to suitable culture medium without anti-browning additives.The regeneration protocols developed here represents a valuable tool for the rapid mass propagation and genetic transformation of A.esculentus.With further investigations,these developed protocols can be employed for mass propagation and molecular farming in other recalcitrant plant species including cotton,tea,chines cabbage,peanut and brassica.Nitrogen deprivation in MS medium and moderate high irradiation produces red colored calluses with higher anthocyanin content and antioxidant potential.Anthocyanin induction in callus cultures opens up new perspectives for producing health beneficial secondary metabolites from okra under in vitro conditions.This optimized protocol can serve as a tool for subsequent studies and in the future for getting enhanced extraction of bioactive compounds of interest from this plant as well as from other plant species for pharmaceutical and industrial use. |
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