الفهرس 
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Abstract
The developmental pathway leading to plant somatic embryogenesis (SE) is true demonstration of totipotency of plant cells. During this process, somatic cells, under appropriate conditions, divide and differentiate into embryos. This viewed developmental pathway was adopted for deciphering the intricate molecular regulatory mechanism (s) of the extracellular 24-epi application upshot to uncover its signaling pathway expressions (gene expression patters) during this process signaling applying rice(Oryza sativa L. cv. Sakah).The ontogeny of such in vitro developmental process was superintendent via convoying the process incidence morgho-histological, biochemical, physiological and molecular approaches were studied in such amazing integrated study.The establishment of an efficient protocol for callus induction is extremely important as a first step towards many molecular purposes. In this study, protocol for somatic embryogenesis and regeneration were tested in one rice genotype (Oryza sativa L. cv. Sakah) to evaluate 24-epi role during somatic embryogenesis in comparison with other hormones. Sterilized de-husked rice seeds as explants were inoculated on basal Murashige and Skoog (MS) medium supplemented with different types and concentrations of growth regulators alone and in combinations were tested in order to evaluate rice seed performance. Two auxin types : 2,4-dichloro-phenoxyacetic acid (2,4-D) , indole -3- acetic acid (IAA) at three levels (0.5,1,2.5 mg l-1)and two cytokinines: benzyl adenine (BA) and 6-furfuryl amino purine (Kn) at three levels (0.5, 1.0 and 1.5 mg l-1) and 24-epi at three levels ( 0.025,0.1 and 0.5 mg l-1) were used in this study. It was found that growth regulator type and concentration had a significant effect on the callus induction day, frequency, callus growth score, index and certain growth traits (fresh, dry weight and water content). The highest scored parameters of callus (day-5, 90 %, prolific (4), 360,303 mg,142 mg and 53 %) were obtained from mature embryo –derived callus cultured on MS medium supplemented with 0.1 mg l-1 24-epi in combination with 2 mg l-1 of 2,4-D(TC8)( enhanced BAK1gene expression in 24-epi signaling transduction pathway) compared to control (TC1, 2 mg l-1 of 2,4-D only)(day-5,70 %,good(3),210,224 mg,96 mg and 57 %).Same media were suitable enough to continue culture as initiated callus exhibited visible embryogenic appearance(nodular –globular).During this period 24-epi (regardless of the applied dose) enhanced cells differentiation (as accelerate dedifferention) especially when combined with 2 mg l-1 2,4-D(TC7-TC9).The most promising data were scored concerning TC8 cultivated tissues as after nine days (histological analysis investigate precocious meristemtic centers day-5) concrete globular embryos were visible with 70 % frequency, 4 prolific growth score and 280 growth index referred to control which exhibit visible globular at day-14,40 % frequency, medium growth score(2) and 80 index. Morpho-histological observation and light microscopy investigation were used to analyze different types of calli during early rice callogenesis. Three types of calli were observed ,embryogenic composed of compact, opaque, nodular and yellowish-white (creamy) or pale yellowish in color, non-embryogenic composed of soft, friable, translucent and yellow in color and rhizogenic with root-like structure. Histological observations of callus paraffin sections revealed that initiation position of mature embryo derived-callus in all treatments was observed from the more mitotically active epithelial cells of rice scutellum. Embryogenic callus occurred on the surface as well as in the deeper regions of the callus. 24-epi investments enhanced xylem differentiation revealed in the abundance of vessel tracts in the treated callus which was consisted of a high content of vessel elements. This may imply shortened time towards both dedifferention and differentiation resulted from 24-epi addition guarantee easily nutrients transportation via these vessel tracts for the nourishment of growing somatic embryos. In addition embryogenic cells (24-epi treated) had a high capability for cell division (enhanced CYCD3; 1 gene expression in 24-epi signaling transduction pathway) and continued to divide and produced somatic pro-embryos with a well–defined protoderm which could develop further through the typical globular, heart, torpedo and cotyledonary stages (TC8). Mature somatic embryos were transferred to regeneration medium supplemented with BA at 1mg l -1 concentration of green spots were recorded , turned into complete regeneration with different frequency percentage increased in TC8 (27 %) when compared with the control (12.5 %), but two treatments missed regeneration pathway TC2 and TC10.Naturally occurring plant hormones brassinosteroids can affect plant somatic embryogenesis by reprogramming cell metabolism state and regulating ROS production in cell but the underlying mechanisms of this scavenging activity by 24-epi are not well understood. Precocious changes in rice cells metabolism state accompanied with the acquisition of embryogenic competence were investigated. Rice embryo-derived calli analysis showed universal increase in soluble carbohydrates (total soluble sugars, reducing sugars, non-reducing, sucrose and starch) and in soluble proteins resulted from 24-epi addition to the culture media, peaked with globular stage differentiation, otherwise total soluble proteins levels were always lower than starch levels. Contrary TC2 (2, 4-D; 2.5 mg l-1) and in TC10 (2, 4-D; 2.5 + 24-epi; 0.025 mg l-1) application impacted both traits negatively. 24-epi (regardless of the applied dose) along with low 2, 4-D dose resulted in lower accumulation for GA3 compared with the control. While increasing the applied 2, 4-D dose lead to GA3 accumulation within rice tissues. With globular stage differentiation a (referred to each treatment time) sudden DROP in IAA levels with concomitant increment in ABA and TC befall referred to control. Present study allocate possible route between exogenous 24-epi and endogenous plant hormone (GA3, IAA, ABA and TC) during somatic embryogenesis may be through sucrose and starch metabolism and CYCD3.1 gene expression increment. To maintain balance of intracellular ROS content, plant possesses the antioxidant enzymatic and non-enzymatic system to manage the surplus ROS level. Harvested rice tissues cultured in TC2 medium exhibited significant conspicuous increment in hydrogen peroxide and MDA amounts escorted with SOD, APX and GR increment meanwhile CAT, POD (soluble and wall-bound), PPO and PAL decreased, likelihood negatively affect rice cells rate for normal dedifferentiation consequently cell impaired redox homeostasis negatively impacted cell cycle progression and final expected cell fate acquisition (failed regeneration). Contrary rice tissues analysis confirm 24-epi conspicuous role in commanding cell homeostasis redox state revealed as significant decrease in H2O2 and MDA levels. Monitoring (degrading and synthesis) H2O2 enzyme system in which probably CAT, POD, PPO, APX and PAL significant increment against SOD and GR retardation explain somatic embryos differentiation(globular stage need certain H2O2 levels and oxidation state) and maturation. Also, profound effects on ascorbate-glutathione cycle (APX and GR) via switching the ascorbate pools towards the oxidized forms compared to the control thereby lowering the AA/DHA ratio , such changes were ascribed to ascorbate peroxidase power along with glutathione reductase inhibition (after excluding TC10, missed regeneration pathway).
One-dimensional SDS -PAGE gel electrophoresis revealed that augmenting rice culture media with 24-epi (TC8) lead to precise changes in rice cells gene expression which was recorded as three unique proteins investigated during the early stages of cells totipotency expression (molecular weighs; 25, 30 and 38 KD).Native –PAGE for peroxidase and polyphenol oxidase isozymes showed one marker band (Rf 0.58) expressed at day-9 (globular –stage) in rice tissues under TC8 cultivation condition. Detected peroxidase isozymes Rf 0.37, 0.58 and 0.64 was previously recorded. PPO isozymes pattern revealed two marker bands one of them Rf 0.83 was previously detected. Also, another PPO isozymes expressed in rice Rf 0.65 and 0.58 was previously stated. However, number of banding patterns of polyphenol oxidase isozymes revealed eight bands with levels of polymorphism 100 % while peroxidase exhibit lesser number of isozymes patterns (seven) with levels of polymorphism 71.4 %.Changing in protein and peroxidase , polyphenol oxidase isozymes pattern and their activity fluctuations during different stages under different cultivation conditions explain their role in cell wall dynamic architecture changes required during cell growth and development.BAK1 gene was significantly expressed (up-regulated) in 24-epi treated sample (TC8) compared to control until day-9 expression significantly declined (down-regulated). While CYCD3 gene expression pattern analysis revealed significant increase in expression levels at day-9 were 2-fold change and at day-14 were 4-fold change compared to the control depleted 24-ep