Abstract

Okra (Abelmoschus esculanthus L.) is a valuable vegetable crop member of Malvaceae family commonly known as lady’s finger, 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 seed powder. India is a leading country in okra production throughout the world with 5.5 million tonnes. 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, as well as its yield, is highly affected by various environmental factors including biotic and abiotic stresses which cause huge economic loss. The improvement of genetic engineering and plant transformation makes possible to transfer resistant genes from bacteria and plants into valuable crops for their effective growth and yield under stress conditions. Despite, belonging 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. esculanthus. Two different in vitro regeneration experiments were organized by means of okra green cultivar “Xiang Fu”. Using in-direct regeneration protocol by modified MS medium with reduced nitrogen levels were examined for friable Embryogenic callus, with favoring the shoot induction from cotyledon and hypocotyl explants. Using direct regeneration protocol, the effect of silver nitrate (AgNo₃) in combination with cytokinins for multiple shoot development from cotyledonary node explants was studied.

1. This study was carried out to test the effect of reducing nitrogen level in MS medium on Embryogenic callus formation and high-efficiency regeneration system in okra. In-direct in vitro tissue culture system was developed for “Xiang Fu” okra green cultivar by using hypocotyl and cotyledon as explants. MS medium containing different levels of inorganic-nitrogen (KNO₃ and NH4NO3 with 1:2 ratios, 30, 40, 50 and 60mMl^-1) along with the combination of auxin and cytokinins were tested. Callus were induced from both explant while, MS medium supplemented with 1.0 mgl^-1 BAP + 1.5mgl^-1 2, 4-D induced highest callus frequency (71%) in hypocotyl as compared to cytokinins (58%) on a medium containing 1.5mgl^-1 NAA + 0.5mgl^-1 BAP. Modified MS medium with 40 mM of total nitrogen level showed the best results amongst the tested nitrogen levels.  The highest shoot induction frequency (66.5%) was found with 3.5 numbers of shoots per calli in hypocotyl on a medium supplemented with 1.5mgl^-1 KIN + 0.5mg.L^-1 IBA at 40 mM of total nitrogen level. On the other hand, high nitrogen level 60 mM produced only 0.9 mean numbers of shoots per calli with 9% shoot induction frequency having a compact and whitish callus in cotyledon explant. Hypocotyls remained the best explant for callus induction (71%) and shoot regeneration (66.5%). In relative comparison to standard MS medium, results demonstrated that using an MS medium with less concentration of nitrogen reprogram the callus structures, forming the Embryogenic calli with high shoot induction. Highest rooting frequency 79% was achieved on half strength MS medium supplemented with 2.0mgl^-1 IBA and 100mgl^-1 activated charcoal. The plantlets were shifted to sterile plastic pots containing vermiculite and garden soil (1:1) with sufficient moisture for efficient acclimatization under the growth chamber and grow normally with 85% survival rate. The current study provides a contribution towards the development of an efficient regeneration and transformation system in okra (Abelmoschus esculanthus L.).

2. The regeneration frequency of okra (Abelmoschus esculanthus L.) is largely depends on its genetic structure and its recalcitrant nature. Direct regeneration has the potential to improve the phyto-pharming genetics under the influence of internal and external factors, used for genetic transformation. In present study, an efficient regeneration system in okra through shoot multiplication by cotyledonary node explants was developed. Furthermore, the influences of silver nitrate (AgNO₃) in combination with cytokinins such as 6-benzyl amino purine (BAP) and Kinetin (KIN) on shoot multiplication were also tested. The highest shoot bud regeneration frequency (75%) was achieved on MS medium supplemented with 2.0mgl^-1 BAP. Whereas, highest shoot multiplication and proliferation frequency (85%) with 6.75 number of shoots per explants having 3.75 cm length was achieved on MS medium comprising of 1.5mgl^-1 BAP + 1.5mgl^-1 KIN + 3.0mgl^-1 AgNO₃. On the other hand, excluding AgNO₃ the combination of 1.5mgl^-1 BAP + 1.5mgl^-1 KIN developed a maximum 2.3 number of shoots per explant with 3.75 cm length having the (53%) shoot multiplication frequency. Best results for shoot elongation 5.60cm were achieved on MS medium supplemented with 1.5mgl^-1 BAP + 1.5mgl^-1GA₃ (gibberellic acid). Highest root formation (83%) with 5.20 numbers of roots per shoots having 4.8 cm length was achieved on half strength MS medium + 1.0mgl^-1 IBA (Indole 3-butiric acid) + 200mgl^-1 AC (Activated charcoal). Rooted plantlets were acclimatized in plastic cups containing vermiculite and garden soil (1:1) with appropriate moisture contents under a plant growth chamber at 25±2 ⁰C and (70%) relative humidity with (87%) survival rate. Moreover, the whole protocol from seedling establishing to regenerated plants was completed within 3.5 to 4.0months. This rapid and reproducible protocol with high-frequency regeneration through shoots multiplication in okra (Abelmoschus esculents L.), will be beneficial tool for the further genetic transformation.

3. In conclusion, it can be concluded that this study has contributed new useful information on in vitro regeneration and high-frequency shoot multiplication of okra A.esculanthus. 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 (AgNO₃) also proved beneficial for high-frequency shoot multiplication from cotyledonary node explants of okra. Here, the developed regeneration protocols present a valuable tool for the effective mass propagation and gene transformation of A.esculanthus as well as these protocols can be used for other recalcitrant plant species such as tea, Chinese cabbage, peanut, cotton and brassica.

Keywords: Abelmoschus esculanthus L., silver nitrate, nitrogen, shoot multiplication, growth regulators and modified medium

General Introduction

 

1. Background of okra

Okra (Abelmoschus esculanthus L.) is one of the most widely known and utilized species of the Malvaceae family and commonly known as lady’s finger. It is an important vegetable crop due to its tender pods as a vegetable for human consumption, fibers for industries and cultivated in tropical and sub-tropical parts of several countries (Narendran et al., 2013a). Previously okra was included in Hibiscus genus but latterly it was designated to genus Abelmoschus. In Asia among Abelmoschus genus fruit of Abelmoschus esculentus specie is most common and frequently used as a vegetable (Fong et al., 2011; Venkataravanappa et al., 2011). More than 99% of okra cultivation is entirely done in emerging countries of Africa and Asia with very low production, among  which India ranks first in the world with production of 5.5 million tonnes on an area of 0.48 million hectares, followed by Nigeria, Sudan, Mali and Pakistan (FAOSTAT, 2016). Its center of origin is uncertain but according to literature it is believed that the center of diversity and origin of okra is Western Africa and then cultivated in North Africa to Arabia and 12^th century BC in India (Kumar et al., 2013). Okra is known by different local names in different places but the use of okra in English became popular during late 18^th century  which derives from Niger-Congo languages (Gemede et al., 2016). It is a warm weather crop (25-30 ⁰C), sensitive to low temperature, drought conditions, frost and water logging and the cultivars varied in various countries (Goneli et al., 2010). Okra is allopolyploid (2n = 72 -144), having a series of polyploidy with basal n = 12 are reported.  (Datta and Naug, 1968). It belongs to the Malvaceae family which includes other economical important crops such e.g. cotton (Gossypium hirsutum), Cocoa (Theobroma cacoa) and Durian (Durio zibethinus). The okra fruits (pods) can be classified depending upon the cultivars and matures after 5-10 days of flowering and mostly harvested 2-4 days of flowering for valuable marketability, while older pods are  generally fibrous and non-acceptable for market (Düzyaman and Vural, 2001).

Okra is multipurpose crop, not only grown for its tender pods as vegetable but other parts also used such as flowers, leaves, stems, particularly seeds which are rich source of oil and protein (Yonas et al., 2014). Immature pods used for pickle, salads, stews and soups. Seeds used as a coffee substitute (Çalışır et al., 2005) , protein (15-20%) and new edible oil source (15-30%) source (Gemede et al., 2015a). It is an essential constitute for balanced food owing to its amino-acid structure and dietary fibers which are rich in tryptophan and lysine. Okra is rich in vitamins, calcium, potassium, sodium, and other minerals and  its regular consumption prevents hepatitis, cancer, ulcers and diabetes (Adetuyi et al., 2011). Okra mucilage used as plasma replacement or blood volumn expander. It also contains a number of health promoting antioxidants and flavonoids including vitamin A, B, C and K, as well as  anthocyanin and carotenoids (Fan et al., 2014). Okra also used in industry as confectionary, fiber source from stem used for jute, paper and textiles, roots and stem used as cleaning agent during brown-sugar preparation (Kumar et al., 2013).

Okra is highly affected by a number of biotic and abiotic stresses which affects growth, quality yield and cause huge economic losses due to the lack of resistance in cultivated species (Ali et al., 2000). The cultivated okra is mostly affected by okra leaf curl disease (OLCD), yellow vein mosaic virus (YVMV), okra enation leaf curl disease (OELCD) associated by whitefly (Bemiscia tabaci) a vector of   Begomovirus (Venkataravanappa et al., 2014). The most harmful insect pests including fruit borer (Helicoverpa armigera), shoot borer (Earias vittela), white flies (Bemiscia tabaci) and green peach aphid (Myzus persicae) although, the okra production is also affected by the aphids, fungi such as Erysiphe cichoracearum, Sphaerotheca fuliginea, Fusarium oxysporum (Dhingra et al., 2008). A-biotic factors include extreme in temperature, soil properties (salinity, acidity) and drought (Pendre et al., 2012).

Okra takes longer time to make a new cultivar through conventional breeding method to increase its resistance against the above mentioned biotic and abiotic stresses. The okra having large number of chromosomes 2n =130 with quite unique genome (Nwangburuka et al., 2011) and  have aggressive  movement  of chromosome during metaphase in wide hybridization.  Agriculture Biotechnology has the potential to increase stress tolerance, improve the nutrients efficiency and enhance the productivity in order to empower the farmers in term of productivity and profitability (Hansen et al., 2011; L Hefferon, 2010). The use of transformation system can make progress in okra genetic engineering and its genomic research whereas; an efficient tissue culture system is the pre requirement for genetic transformation that permits the regeneration and transformation of transgenic plants. As belonging to the Malvaceae family including cotton and okra are highly recalcitrant crop for tissue culture system. Till date, few reports had made progress in okra tissue culture and transformation. An- efficient tissue culture system will be beneficial for crop genetic transformation.

1.2 Plant tissue culture

Plant tissue cultures is the aseptic culture of tissues, cells, organs or protoplast under well-defined chemically and physically in vitro conditions and are very important technique for both basic and applied studies as well as in commercial application (Thorpe, 2007). Tissue culture developed during 1960s and it’s explore conditions promotes the genetic engineering and cell division in in vitro conditions. It depends on the totipotency of plant cells to regenerate from small starting material called as “explant” to complete plantlet. Plant tissue culture also depends on the plant species, nutrient medium, explant type taken from shoot tips, root or shoot, lateral bud, leaf of the mother plant and culture conditions. However, there is an important role of environmental conditions which affects the appearance and genotype of tissue cultured cells or organs. Application of traditional methods for controlling, chemical and physical conditions is inadequate and time consuming for mass propagation. Modern technology using different engineering system such as robotic, automatic and computerized has become popular for micropropagation to provide an optimal environmental condition on large scale. Tissue culture propagation is generally practiced in three steps, a) initiation and sterilization of material, b) composition of medium used for culturing, c) environmental condition in cultured room and in the vessels (Bhatia et al., 2015). In vitro media generally consist of macronutrients, micronutrients, amino acids, vitamins, solidifying agents and growth regulators (Saad and Elshahed, 2012).

Explant in tissue culture goes through the initiation, multiplication and rooting steps to become complete plantlets able to grow in the field conditions after the acclimatization in green house. Tissue culture is a valuable technique which makes us able to produce uniform and true to type plants, enhance the plant propagation rate, reduce labor cost and time consuming and higher yield with good quality production (George et al., 2008b). This technology has become a standard technique for modern biotechnology and it can be seen in different areas especially in five main zones where in vitro tissue culture are recently functional including, in plant fundamental physiology, metabolic manufacturing of fine chemicals, conservation of sensitive species, propagation on large scale and genetic modification  (Loyola-Vargas, 2012). Tissue culture produces a wide range of genetic variation in plants which can be integrated for breeding programs. In addition, plant tissue culture is a widespread part of plant biotechnology, by use of tissue culture mutants with desired agronomic traits can be incorporated in short period of time (Jain, 2001).

1.3 Role of nutrient media composition in plant tissue culture

The successful application of plant tissue culture depends on the nature of explant, culture medium used and the optimum growth of cell or tissues for numerous plants depends on their nutrition supplies. This helps to select a suitable medium to fulfill the nutritional requirements of designated explants. Furthermore tissues from different portions of plant also vary for their nutrition supply for an optimum growth e.g. Whites medium for roots, Gautheret for callus. Basic medium which are mostly used including Murashiage and Skoog (MS) medium,

Nitsch and Nitsch (NN) medium, Gamborg (B₅) medium, Linsmaier and Skoog (LS) mediums. Plant tissue culture medium contains the essential macronutrients such as nitrogen (N), potassium (K), phosphorus (P), calcium (Ca), sulphur (S) magnesium (Mg) in addition to oxygen (O), hydrogen (H) and carbon (C) (Saad and Elshahed, 2012). He et al. (1989) reported that for the induction of Embryogenic callus need an optimum level of nutrient medium.

1.4 Influence of nitrogen and its sources in vitro

Successful plant tissue culture based on various factors however the chemical composition of the nutrient medium to provide adequate nutrients is important, which may accomplish by optimization of salt or ion based experiments. Nitrogen is an important macronutrient for plant growth and development. There is a great effect of nitrogen on morphogenesis and development of tissue culture. Accessibility of nitrogen and its form also has great influence. The availability of nitrogen in growth medium strongly influenced the responses of plant tissue in terms of proteins, secondary metabolites, organic acids and hormones (Preece, 1995). In plant tissue culture medium nitrogen is mostly used in the form of KNO₃and NH₄NO₃ (Ramage and Williams, 2002). Organic nitrogen as amino acids including glutamine, proline and hydro lysates may also be added to the medium which do not have inorganic nitrogen, whereas the ammonium (NH₄⁺) and Nitrate (NO₃^–) are the two major components of inorganic nitrogen sources, used in typical plant tissue culture growth media (Niedz and Evens, 2008). NO₃^– alone as nitrogen source has not been found successful whereas reduced source of nitrogen i.e. NH₄⁺ is essential for optimal tissue culture. Several studies resulted that the growth of a cultured tissue is probably possible on a medium comprising of NH₄⁺ as exclusive nitrogen source Krebs’s cycle of dicarboxylic acid also existing in the medium (Gamborg and Shyluk, 1970). Using NH₄⁺ alone as nitrogen source were found to be successful for cell of Tobacco cell growth (Behrend and Mateles, 1976), and  latterly  work was done on wild-carrot  (Dougall and Verma, 1978).

Combination of NH₄⁺ and NO₃^– is considered a perfect source of nitrogen for tissue culture medium; their ionic concentration affects the pH. Uptake of NH4⁺ and NO₃^– forms are associated with the medium pH, as nitrate needs acidic pH however medium moves towards alkalinity, while ammonium uptakes then moving the medium towards acidic and thus inhibited the further uptake of ammonium. Obviously in  an un-buffered medium the frequency of nitrogen uptake rely on the occurrence of both ions. The effects of various nitrogen form associated with pH factor have focused on the cell growth and embryogenesis studies by use of un-buffered medium (Sathyanarayana and Blake, 1994).

1.5 Influence of reduced nitrogen in vitro conditions

Plant roots rarely encounter the deficiency of nitrogen as long as bacteria oxidized the feasible sources in natural conditions. Whereas in comparison, in tissue culture condition, if NH4⁺ or other nitrogen sources reduced then plant take up and utilize it. Reduced nitrogen in vitro gives an important advantage to plant because a) conversation of nitrate to ammonium required energy which plant can save, b) high ammonium ion causing the toxicity and increase the ethylene level also, c) due to high concentration of ammonium causes the un-buffered medium leading to the  stunted shoot growth. (Mott et al., 1985) reported that the plant culture growth may also effected by the high concentration of both ions NH₄⁺ and NO₃^–, and the toxicity  is due to the high concentration of NH₄⁺ ion, not by the depressed pH. The toxic effects of NH₄⁺ion may avoided by the conversation of ammonium ion to the amino acids which takes place in two ways as describes in (Fig. 1), in which the most significant is under regular condition through which, glutamate is synthesis from glutamine by the action of glutamate synthetase (GOGAT) and glutamine synthetase (GS) enzymes. If there is an excess amount of NH₄⁺ ions, the reaction between NH₄⁺ ions and α-ketoglutaric acid has great importance. Availability of α-ketoglutaric acid limits the assimilation and detoxification of NH₄⁺ ions which may be able to increase, by the addition of one or more acids that are intermediates in the Krebs’ cycle (tricarboxylic acid) and their accumulation may promote the growth of some media having excess level of ammonium ions.

If a media with having only NO₃^– ions as nitrogen source cause the disadvantage of hyperhydricity in shoots, furthermore the hyperhydricity also occurs due to the highly reduce or elimination of  NH₄⁺ ions from the medium because in the existence of NH₄⁺ ions leads to promote protein and amino acids synthesis  towards the carbohydrates compounds. Reduced nitrogen seems to be valuable for, at least two procedures involving the cell division a) during the cell wall formation, which is operational only when the ammonium ions are existing in the medium beside these glutamine does not a substitute of ammonium ions, b) the action of growth regulators, several reports described that ammonium sulphate has not been found a good source of NH₄⁺ ion in the presence same NO₃^– ions. Feasible reasons are the formation of acidic medium in the presence of ammonium sulphate (George et al., 2008a).

1.6 Silver nitrate (AgNO₃) a growth promoter

Successful genetic engineering depends on several factors including an effective tissue culture system. Media composition plays an important role in regeneration efficiency. Shoot and root development plays an important role to realize the potential of tissue culture for the development of plants. Silver ions in AgNO₃ form, present an important role for embryogenesis, shooting and root induction. Silver thiosulphate also used in various tissue cultures (Bais et al., 2001).  Ethylene effects the callus development, shoot induction and embryogenesis. Kumar et al. (1998) development and growth of cells cultured in vitro rely on existence of ethylene and phytohormones in culture environment. Inhibition of ethylene biosynthesis is a good alternative to improve the adventitious bud induction. Different evidence’s showed that the morphogenic response of adventitious bud induction can be improved by inhibition of ethylene biosynthesis and gaseous environment in the culture medium (Pua, 1999). Silver Nitrate (AgNO₃) has been reported, a potential ethylene inhibitor during in vitro culture in various economically important plant species (Aylin Ozudogru et al., 2005; Jakubowicz et al., 2010; Kumar et al., 2016; Mookkan and Andy, 2014; Venkatachalam et al., 2017; Zhang et al., 2001). Silver nitrate is a potent shoot multiplication enhancer whose use has been reported  in various economically important plants such as Vigna mungo (Mookkan and Andy, 2014), Gossypium hirsutum (Kumar et al., 2016), Arachis hypogaea (Aylin Ozudogru et al., 2005), Cucumis sativus (Venkatachalam et al., 2018), Catharanthus roseus (Panigrahi et al., 2017), Prosopis cineraria (Venkatachalam et al., 2017)

1.7 Tissue culture in okra

Okra is a member of Malvaceae family, are quite recalcitrant to regeneration which makes it inadequate for producing a transgenic plant (Cook and Brown III, 1995).  In vitro tissue culture and okra regeneration by using shoot tips and nodes was reported by (Mangat and Roy, 1986). Later on, regeneration of okra, by using different explants has been reported by the different researcher (Anisuzzaman et al., 2010; Ganesan et al., 2007; Irshad et al., 2017; Kabir et al., 2008a; Manickavasagam et al., 2015). Several factors effects the okra regeneration including, nutrient medium, genotypes, culture conditions, phenolic substances from explants to cultured medium, browning of callus, compact and non Embryogenic callus induction with various texture and colour (Anisuzzaman et al., 2010; Cook and Brown III, 1995; Irshad et al., 2017; Narendran et al., 2013a). Amongst those factors, compact and non-Embryogenic callus with a low frequency of shoot induction are well-known problems which harmfully effects okra regeneration. Formation of Friable Embryogenic callus (FEC) and compact non-Embryogenic callus significantly affected by the culture medium, type, and concentration of nitrogen and phosphorus sources (Elkonin et al., 1995; Elkonin and Pakhomova, 2000). Direct regeneration has the potential to improve the phyto-pharming genetics under the influence of internal and external factors, used for genetic transformation. Shoot multiplication by direct organogenesis is an alternative way of regeneration, which takes less time, has lower somaclonal variability and higher efficiency index than indirect regeneration system (Juturu et al., 2015). Additionally, it has the ability to overcome the limitation of genotype requirement in transformation system. So there was a need to establish an efficient protocol for in vitro regeneration of okra which will be beneficial for further crop improvement.

1.8 Project aims and objectives

Fruits and vegetables are important diverse group of plants which supplies balanced food in the form of dietary fibers, vitamins, minerals, phytochemicals source of antioxidant, anti-inflammatory agents and phytoestrogens. Due to increasing world’s population there is a great demand of  diversification in crop plants in order to meet the requirement of nutritionally balanced food (HUGHES, 2009). But a number of biotic and a-biotic factors affect the quality, growth and production of crop plants. Biotechnology has the potential to increase stress tolerance, improve the nutrients efficiency and enhance the productivity in order to maintain productivity and profitability (Hansen et al., 2011; L Hefferon, 2010).  In okra conventional breeding is difficult to attain its genetic improvement and advisable to have a regeneration system for the development of a resistant cultivar to survive in biotic and abiotic stresses. The use of transformation system can make progress in okra genetic engineering and its genomic research whereas; an efficient tissue culture system is the requirement for genetic transformation that permits the regeneration and transformation of transgenic plants. A variety of plant secondary metabolites are used for the production of various pharmaceutical compounds. Tissue culture system deliver continues, dependent and renewable source of beneficial plant pharmaceuticals. Alkaloids are the main secondary metabolites which plays important role in different medicinal application. Due to growing in commercial market, tissue culture methods could be used for the improved production of alkaloids via genetic transformation and through somaclonal variation (Ahmad et al., 2013). Okra has antioxidant and medicinal importance, so tissue culture system is a pre-requirement to produce a compound with more significance for human consumption. In addition, for the development of a new cultivar with desired genetic makeup an efficient in vitro tissue culture is essential.

Therefore, the goal of this study was to optimized culture conditions for successful improvement and efficient regeneration of in vitro okra. This goal was achieved through following objectives:

1. Establishment of an efficient protocol for in vitro regeneration of okra.
2. Improving the regeneration efficiency of in vitro culture establishment of okra by modification of the standard MS basal medium.
3. Establishment of an efficient micropropagation system for in vitro regeneration of okra plantlets and acclimatization protocol for the in vitro plantlets.

Research directed on the basis of above mentioned objectives are reported in the following chapters.

Chapter 2 reported in vitro regeneration of okra through callus; with modified basal MS nutrient medium by altering the total nitrogen. Results indicated that nutrient medium plays an important role in controlling the callus organogenesis, shoot induction and are dependent on explant type and plant growth regulators combination.

Chapter 3 observed the effect of growth enhancer on culture establishment and multiple shoot regeneration from cotyledonary explants of okra. Results indicated the positive effects of silver nitrate (AgNO₃) in combination with growth regulators enhanced the multiple shoot induction from cotyledonary node explants.

Highlights

• Cotyledon and hypocotyl explants were excised from 10-12 days in vitro raised seedling for in vitro regeneration.
•  Reduced the nitrogen level in MS medium results the formation of Embryogenic callus and enhance the regeneration frequency.
• Hypocotyl showes the excellent  performance in direct in vitro regeneration.
• Plant growth regulator, modified MS medium and explant types plays an important role in in vitro response of okra culture.
• By incorporation of activated charcoal along with auxin improved the in vitro routing protocol.

Abstract. In this study, we have developed an efficient protocol with reduced availability of nitrogen in the MS medium, favored for the friable Embryogenic callus (FEC) formation by inhibition of whitish compact, non- Embryogenic callus growth and promoted the shoot induction of hypocotyl and cotyledon explants in okra. Different levels of inorganic –nitrogen (KNO₃ and NH₄NO₃ with 1:2 ratio, 30-60 mM) in MS medium were tested, along with the combination of auxin and cytokinins, to evaluate their ability to Friable Embryogenic callus (FEC) formation with the promotion of shoot induction. Hypocotyl produced the highest callus induction (71%) on medium 1.0 mgl^-1BAP and 1.5mgl^-1 2, 4-D as compared to cotyledon (58%), on medium 1.5mgl^-1 NAA and 0.5mgl^-1 BAP. Results show that hypocotyl has more potential for callus induction than cotyledon explant. Among the tested modified MS medium with four levels of total nitrogen, 1.5mgl^-1 KIN and 0.5mg.l^-1 IBA induced (66.5%) shoots in hypocotyl with 3.5 numbers of shoots per calli at 40 mM of total nitrogen level, whereas high nitrogen level of nitrogen (60 mM), induced only (9%) shoots, in cotyledon explant with 0.9 mean numbers of shoots per calli. In relative comparison to MS medium, results demonstrated that using a medium with reducing nutrients (less concentration of nitrogen) reprogram the compact non-FEC to FEC with high shoot induction. The healthy and strong roots were obtained on media included 2.0mgl^-1 IBA and 100mgl^-1AC. Rooted plantlets were successfully acclimatized and then grow normally. These findings were more efficient in inducing the shoot induction than the previous protocol. The current study provide a contribution towards the development of an efficient regeneration and transformation strategy in Okra (Abelmoschus esculanthus L.).

 

Keywords:Abelmoschus esculanthus, nitrogen, growth regulators and medium optimization.