الفهرس 
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Abstract
The evergreen azalea (Rhododendron genus, Ericaceae family) contains diverse species and phenotypes which have a broad range of ornamental traits and bears various environmental conditions. These features make this plant necessary for landscape use, thus it needs a new methods of rapid propagation and novel breeding. The traditional method for propagation of most Rhododendron species is stem cuttings, but using in vitro micropropagation is significantly expanded in commercial production. However, the stimulation of the shoot organogenesis system, callus induction and adventitious root formation by using tissue culture methods of evergreen azalea plants is still important because of the diverse morphogenic potential of different genotypes. Efforts to establish effective protocols have been ongoing for several years. In this study, protocols for shoots and leaves organogenesis, somaclonal variation, callus induction and in vitro adventitious root formation in evergreen azalea were investigated.
1. Shoot organogenesis is affected by different cytokinin types. Two explants (shoot tip and nodal segments) are tested in two evergreen azalea cultivars (‘Zihudie’ and ‘Mingchao’). Nodal segment is effective as explants and can induce shoot organogenesis when cultured on Anderson medium supplemented with Zeatin (Z) in both azalea cultivars. Concerning the shoot elongation was suppress, the multiplied shoots were moved to a Anderson media fortified with 3 mg/L 2iP, and shoots elongated to 5-6 cm within 10 weeks. It is investigated the leaf organogenesis and callus formation through the application of Thidiazuron (TDZ) at different concentration. The results indicated that 1 mg/L of TDZ is effective on the leaves organogenesis in both cultivars, however, the highest regeneration survival is 86.66% and 85 %, respectively for the ‘Zihudie’ and ‘Mingchao’ cultivars. Contrary, the using TDZ in higher concentration (10 mg/L) reduce the regenerating explant ratios. Histological evaluations are conducted to achieve an understanding of morphological development during in vitro shoot organogenesis from in vitro- leaf explants. Leaf culture indicated that the cell division and mesophyll cell enlargement have occurred. Histological evaluation shows that shoot organogenesis of both cultivars occurs through the formation of embryo-like structures. Depend on the importance of balance of endogenous hormonal in successful of callus organogenesis. The results suggests callus samples taken from media supplemented with 0.5 mg/L TDZ have the highest contents of indole acetic acid (IAA), zeatin riboside (ZR), and isopentenyl adenosine (iPA), confirm that both auxins and cytokinins have been in a relationship with the regeneration ability of callus.
2. Formation of adventitious roots enhanced by indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA). The root development of two azalea cultivars (‘Zihudie’ and ‘Mingchao’) was investigated in 56 days, involved in the influence of two auxins types on biochemical changes and endogenous hormones. In this study, auxins (IBA and NAA) effectively enhanced adventitious root formation in evergreen azalea microshoots. However, the biochemical parameters (POD, IAAO, H2O2 and soluble protein) are stimulated in both cultivars by auxin treatments with peak increasable after 7 days of transferring to media, which corresponding to rooting induction phase. In addition, POD activity reduced at the end of the rooting phase, which involves in the expression period. The endogenous IAA levels increase during the first period of adventitious root formation, due to its high content are needed for root initiation. Also, the high content of IAA accompanies by low activity of IAAO during the most periods of root induction. Moreover, other endogenous hormones such as GA, iPA and ZR are promoted by the presence of IBA and NAA in media levels during the in vitro rooting period. It is found a decrease in iPA and ZR levels after 7 days of culture to media in both studied cultivars then they increased at last period of rooting which confirms the vital role of ZR in the formation of cortex root during rooting expression. The present results indicate that IBA and NAA are efficient on the formation of adventitious root through their positive effect on biochemical changes and endogenous hormones levels.
3. Humic acid (HA) could improve efficiency for rooting in vitro. Due to imposed restrictions on the use of chemicals, including plant hormones in recent years. As a natural biostimulator, the role of humic acid (HA) was evaluated on the rooting system in evergreen azalea cultivar ‘Zihudie’. The influence of humic acid (HA) on adventitious root formation is tested through its effect at different concentration on histological development, antioxidant enzyme changes and endogenous hormone. Using HA enhanced the morphological root character of microshoots especially at 1 and 2 mg/L. The histological analysis indicated that the first cell divisions are shown after 3 days of culture in the area of phloem adjacent to the cambium, identifiable root primordium with an oval shape and several cell layers are observed after 10 days in azalea microshoots. Besides the morphological and anatomical, HA application (1 and 2 mg/L) increased the endogenous IAA, GA, ZR and iPA hormone levels in treated microshoots, as it happened in prior auxin treatments. Moreover, the same HA concentration involves in the increase of antioxidant activities like peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), polyphenol oxidase (PPO) and total soluble protein. The results demonstrated that HA is effective for rooting in evergreen azaleas, and this effect was related to physiological and metabolic changes during adventitious root formation. Therefore, this application of HA during in vitro rooting may also reduce the losses that occurred due to poor quality of the root process or caused by auxin used in the rooting system.
4. Biochemical response and different genes expression corresponding to the rooting process were presented. To investigate the relationship between reactive oxygen species (ROS) changes, carbohydrates and phenolic compounds and formation of the adventitious root, the impact of effects of IBA, NAA and HA are studied in evergreen azalea cultivar ‘Zihudie’. It is important to understand the mechanisms of carbohydrates and phenolic compounds biosynthesis regulation as well as some related genes to help to develop vital strategies for enhancing adventitious root formation. The results showed that the NAA treatments mostly affected the physiological levels, metabolic changes and the phenolic compounds contents more than other treatments. The important role that played by (ROS), carbohydrates and phenolic compounds during in vitro azalea adventitious root growth. Fourteen genes associated with adventurous root formation are analyzed by quantitative RT-PCR. The obtained results indicated that HA treatment up-regulated the expression of POD1 gene in tested microshoots more than IBA and NAA. Six ARFs genes associated with rooting development are tested to elucidate the molecular mechanism of auxins and humic in azalea rooting by quantitative RT-PCR. The relationship between the expression of the IAA1, IAA9, IAA14, IAA26, AUX1 and TIR genes and root formation is evaluated.
These findings allow us to develop an understanding of the evergreen azalea organogenesis and callus formation to open the way for genetic engineering by transformation. Moreover, this information may provide a source for further study regarding improvement in micropropagation and adventitious root formation of Rhododendron plants. In addition, to improving our understanding of the regulatory mechanisms and molecular functions of different genes related to root development.