Study On Factors Affecting In Vitro Regeneration System Of Okra (Abelmoschus Esculentus L.)

Okra(Abelmoschus esculentus L.)is a valuable vegetable crop of Malvaceae family commonly known as lady’s finger.It is grown in sub-tropical as well as the tropical region of several countries both for its edible immature pods for human consumptions as a vegetable,industrial use as fiber and seeds for powder.India is a leading country in okra throughout the world with 5.5 million tonnes of production.It is an important source of a balanced human diet containing vitamins,protein,minerals and provides significant compounds which prevent cancer,diabetes,ulcers and hepatitis.Recently,the presence of health beneficial phytochemicals and phenolic compounds in its succulent pods and in powdered seeds has been reported.However,okra growth and yield are highly affected by various environmental factors including biotic and abiotic stresses which cause huge economic losses.The improvement of genetic engineering and plant transformation has made it possible to transfer resistant genes from bacteria and plants into valuable crops for their effective growth and yield under stress conditions.Despite,belongings to the Malvaceae family,okra is the most recalcitrant crop having poor in vitro regeneration system amongst the main reasons.Therefore,the fundamental objective of this study was to develop an efficient in vitro regeneration system for A.esculentus.Two different in vitro regeneration experiments were organized by means of okra green cultivar "Xiang Fu".Using in-direct regeneration process with modified MS medium having reduced nitrogen levels were examined for friable embryogenic callus and shoot induction from cotyledon and hypocotyl explants.Using direct regeneration protocol,the effect of silver nitrate(AgNo3)in combination with plant growth regulators for multiple shoot development from cotyledonary node explants was studied.1.This study was carried out to test the effect of reduced nitrogen levels in MS medium on embryogenic callus formation and high-efficiency regeneration system of A.esculentus.In-direct in vitro tissue culture system was developed for "Xiang Fu" green cultivar of A.esculentus by using hypocotyl and cotyledon explants.MS medium containing different levels of inorganic-nitrogen(KNO3 and NH4NO3 with 1:2 ratios 30,40,50 and 60 mM)along with the combination of plant growth regulators were tested.MS medium supplemented with 1.0 mgL-1 BAP +1.5 mgL-1 2,4-D induced highest callus frequency 71%in hypocotyl,while cytokinins induced 58%calluses on a medium containing 1.5 mgL-1 NAA + 0.5 mgL-1 BAP.Maximum shoot induction frequency 66.5%were found with 3.5 numbers of shoots per calli in hypocotyl on a medium containing 1.5 mgL-1 KIN + 0.5 mgL-1 IBA at 40 mM of total nitrogen level.In a medium containing 30 mM of total nitrogen and 1.5 mgL-1 BAP + 0.5 mgL’1 NAA induced 49.50%shoots with 2.95 numbers of shoots per calli in cotyledon.High nitrogen level 60 mM showed a lowest response in cotyledon with 0.92 mean numbers of shoots per calli and 9.5%shoot induction frequency.Maximum rooting frequency 79%was achieved on 1/2 strength MS medium supplemented with 2.0 mgL-1 IB A +100 mgL-1 AC.The plantlets were shifted to plastic pots containing vermiculite and garden soil(1:1 ratio)than grows normally with 85%survival rate after acclimatization.Modified MS medium with 40 mM of total nitrogen level showed the best result amongst the tested nitrogen levels and hypocotyl remained the best explant.In relative comparison to standard MS medium,results demonstrated that using an MS medium with a modified concentration of nitrogen reprogram the callus structures and forming the embryogenic calli with high shoot induction rate.The current study provides a contribution towards an efficient regeneration and transformation system of A.esculentus.2.In the present study,an efficient regeneration system of okra through shoot multiplication by cotyledonary node explants was achieved.Furthermore,the influences of silver nitrate(AgN03)in combination with cytokinins such as 6-benzyl amino purine(BAP)and Kinetin(KIN)on shoot multiplication were also tested.Maximum shoot regeneration frequency 75%was achieved on MS + 2.0mgL-1 BAP.Whereas highest shoot multiplication frequency 85%with 6.75 number of shoots per explants having 3.75 cm shoot length was achieved on MS medium comprising of 1.5mgL-1 BAP + 1.5 mgL-1 KIN + 3.0 mgL-1 AgNO3,on the other hand,a medium containing 1.5 mgL-1 BAP + 1.5 mgL-1 KIN developed a maximum 2.3 number of shoots per explant with 3.75 cm shoot length having 53%shoot multiplication frequency without AgNo3.The best result for shoot elongation 80%with 5.60 cm shoot length was achieved on MS medium supplemented with 1.5 mgL-1 BAP + 1.5 mgL-1 GA3(gibberellic acid).Highest root formation(83%)with 5.20 numbers of roots per shoot having 4.8 cm of root length was achieved on 1/2 strength MS + 1.0 mgL-1 IBA(Indole 3-butiric acid)+ 200 mgL-1 AC(Activated charcoal).Rooted plantlets were acclimatized in plastic cups containing vermiculite and garden soil(1:1 ratio)with 87%survival rate.Moreover,the whole protocol from seedling establishing to regenerated plants was completed within 3.5 to 4.0 months.This rapid and reproducible protocol with high-frequency regeneration through shoots multiplication in A.esculentus will be a beneficial tool for the further genetic transformation.In conclusion,it can be concluded that this study has contributed new useful information on in vitro regeneration and high-frequency shoot multiplication of A.esculentus.Differences in vitro regeneration frequency could be due to the nutrient medium and the explant sources.Furthermore,reduction of nitrogen level(40 mM)in MS medium proved beneficial for the formation of embryogenic callus with high-frequency regeneration from hypocotyl explants while standard nitrogen(60 mM)concentration of MS medium produced compact whitish callus with a low rate of regeneration frequency.We concluded that to overcome the compact whitish callus with low regeneration frequency the explants should be inoculated initially on standard MS medium supplemented with different plant growth regulators and then should be transferred to modified MS medium for friable embryogenic callus formation with high regeneration frequency.In addition,use of silver nitrate(AgN03)also proved beneficial for high-frequency of shoot multiplication from cotyledonary node explants of A.esculentus.The developed regeneration protocols present a valuable tool for the effective mass propagation and gene transformation of A.esculentus as well as these protocols can be used for other recalcitrant plant species.