New Plant Regeneration System from Nodal Segments Developed

Efficient plant regeneration system has been developed from the nodal segments of chrysanthemum (Chrysanthemum morifolium L). Nodal segments, after being sterilized with 1.0% mercuric chloride for three minutes, were inoculated in Murashige and Skoog (MS) media with varied concentrations of indole acetic acid (IAA), benzylaminopurine (BAP) and their combinations. Different parameters including shoot initiation percentage, average number of shoots per explant, length of shoots (cm), number of leaves per shoot and number of nodes per shoot were studied during the course of study.

Intermediate level (0.3 mg/l) of IAA exceeded all the other concentrations of IAA by producing 80.0 % shoot initiation, an average of 4.0 shoots per explants, 5.1 cm long shoots, 11.3 leaves and 5.6 nodes per shoot, when used alone. Similarly, intermediate level of BAP (1.0 mg/l) showed its supremacy over all the other concentrations as it produced 100% shoot initiation, 4.9 shoots per explant, 5.8 cm long shoots, 13.4 leaves and 6.3 nodes per shoot, when used alone. When the combination of different concentrations of IAA and BAP were used, significant results regarding the regeneration of chrysanthemum plantlets were also achieved.

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MS media supplemented with lower concentrations of IAA (0.1 and 0.2 mg/l) along with intermediate levels of BAP (1.0 and 2.0 mg/l) had a favorable effect on the regeneration of chrysanthemum plantlets using nodal segments of chrysanthemum, as compared to other concentrations and combinations. Satisfactory rooting response was obtained in half strength MS media supplemented with 0.2 mg/l indole butyric acid (IBA), followed by 0.2 mg/l naphthalene acetic acid (NAA) and IAA, respectively. Chrysanthemum commonly known as Gul-e-Daudi or Autumn Queen belongs to the family Compositeae (Asteraceae).

It is highly valued as a cut flower worldwide with its diverse floral types and colors. It is globally an important cut flower and pot plant species usually cultivated by vegetative cuttings. It is generally propagated using suckers and terminal cuttings. This approach, however, is inadequate to attain fast multiplication rate, as these conventional propagating methods are very slow, 10 | P a g e time consuming and tiring. Secondly, cuttings obtained repeatedly from mother plants may be subjected to any virus infection and degeneration, thereby increasing production costs. These problems have been solved by applying micro propagation methods, which are routinely applied to the clonal propagation of a variety of horticultural plants including Chrysanthemum. Micro propagation and other in vitro techniques have been used for plans which present particular problems in conventional horticulture. Chebet reported the use of biotechnological approaches to improve horticultural crop production. The regeneration of plants from tissue culture is an important and essential component of biotechnological research. High frequency regeneration of plants from the in vitro cultured tissue is a pre-requisite for successful application of tissue culture techniques for crop improvement. It is possible now to obtain a large number of plants from one explant in vitro. A decade ago, the protocols for rapid true to type, disease-free propagation has been developed in chrysanthemum through bud/shoot proliferation. In tissue culture, the use of plant growth regulators plays a pivotal role in influencing different plant processes comprising mostly of growth, differentiation and development for example, culture establishment, shoot initiation, callogenesis, embryogenesis, rooting, etc. Pierik stated that cytokinin are often used to stimulate growth and development, Kin and benzyl aminopurine (BAP) being in common use. Although the presence of a cytokinin is almost always advantageous, and is often all that is required, optimum rates of shoot initiation generally occur with the combinations of auxins and cytokinin. The presence of auxin in defined combinations with cytokinin in the culture medium is also necessary to obtain adventitious shoot formation. Presence of BAP in the culture medium was necessary for the shoot regeneration, although concentrations higher than 4.44 uM reduced the shoot regeneration frequency. Waseem inoculated chrysanthemum nodal segments in Murashige and Skoog (MS) media supplemented with different concentrations of indole acetic acid (IAA), naphthalene acetic acid (NAA) and indole butyric acid (IBA) and reported that 0.3 mg/l IAA, 0.5 mg/l NAA and 0.3 mg/l IBA showed their superiority over all their other respective concentrations, when used alone. Therefore, the attempts were made to determine the effect of different growth regulators on the shoot proliferation and rooting of chrysanthemum plantlets using nodal segments explant. 11 | P a g e 1.2: OXIDATIVE STRESS IN PLANT TISSUE CULTURE Higher plants are sessile therefore are continuously exposed to different environmental stress factors, such as drought, salinity, heavy metals, nutritional disorders, radiation without any protection. Most of these stresses produce certain common effects on plants, like induced oxidative stress by overproduction of reactive oxygen species (ROS), besides their own specific effects. Thus, plants have developed their own specific response(s) against each of these stresses as well as cross-stress response(s). Investigating these responses is difficult under field conditions, but plant tissue culture techniques are performed under aseptic and controlled environmental conditions. These advantages of plant tissue culture allow various opportunities for researcher to study the unique and complex responses of plants against environmental stresses. ROS have inevitably been factors for aerobic life since the introduction of molecular oxygen (O2) into our atmosphere by O2-evolving photosynthetic organisms. ROS can simply be described highly reactive and partially reduced-oxygen forms. ROS, including the superoxide radical (O2™), singlet oxygen (1O2), hydroxyl radical (OH™), hydroperoxyl radical (HO2™), hydrogen peroxide (H2O2) like that, are produced not only during metabolic pathway in several compartments of plants, including chloroplasts, mitochondria, peroxisomes, plasma membrane, apoplast, endoplasmic reticulum, and cell-wall but also as a result of induced environmental stress factors. When exposing of environmental stress factors, ROS levels can dramatically increase and this increase, in the later stage, leads to oxidative stress. Oxidative stress is defined a serious imbalance between the production of ROS and antioxidant defense and this situation can cause damage to cellular macromolecules, including proteins, lipids, carbohydrates and DNA. Under steady-state conditions, the ROS are scavenged by various antioxidant defense systems: both enzymatic antioxidant (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; glutathione reductase, GR; monodehydroascorbate reductase, MDHAR; dehydroascorbate reductase, DHAR; glutathione peroxidase, GPX; guaiacol peroxidase, POX and glutathione-S- transferase, GST) and non-enzymatic (ascorbate, glutathione, carotenoids, phenolic compounds, proline, glycine betaine, sugar, and polyamines) defense systems. Plant tissue culture techniques are used to grow plants under aseptic and controlled environment for the purpose of both commercial (like mass production) and scientific (like germplasm preservation, plant breeding, physiological, and genetic) studies. Two of these application areas are important to study ROS homeostasis in plants. The first one of these techniques is used as a model to induce 12 | P a g e oxidative stress under controlled conditions via different stressor agents for researching in vitro screening in plants against abiotic stress, studying and observing morphological, physiological and biochemical changes in both unorganized cellular (i.e. suspension cultures and callus cultures) and organized tissue (i.e. axillary shoot, shoot tip, mature embryo, whole plant) levels .Additionally, plant tissue culture techniques also allow opportunities for the researcher to improve plants against abiotic stress factors with the in vitro selection method. The purpose of this study is to compile the recent studies about ROS and oxidative stress, how to maintain ROS homeostasis in plants, plant tissue culture, the effects of induced-oxidative stress on antioxidant defense system in plant tissue culture and antioxidant defense systems of in vitro selected-plant against abiotic stresses. 1.3: ROLE OF CADAVERINE IN OXIDATIVE STRESS ALLEVIATION Initially identified as a lysine decomposition product in organic matter, cadaverine, or 1,5- pentanediamine, is found ubiquitously in the environment. Cadaverine, from the word, cadaver, is often associated with decaying matter and is one of the components that gives carrion its distinctive smell. Cadaverine functions in a multitude of cellular processes critical to living organisms. In Escherichia coli, cadaverine is used to mediate acid stress, and the deathly odour of cadaverine provides behavioural cues to animals. In plants, it has been reported to contribute to plant growth and development, cell signalling, stress response, and insect defence. The regulation of these diverse processes is critical for plant fitness in natural ecosystems, and also for healthy crop production. This minireview highlights contributions to the understanding of cadaverine's functional role in plant development and environmental response by focusing on cadaverine's biosynthesis and metabolism, its impact on plant growth and development, its potential contribution to plant-microbe interactions, and its role in stress response. 1.3.1: CADAVERINE MAY CONTRIUBUT TO ENVIRONMENTAL STRESS RESPONSE As with other polyamines, cadaverine has been implicated in stress response. However, there is a dichotomy between cadaverine acting as a stress protectant or exacerbating stress damage. Cadaverine has been reported to facilitate seed germination and seedling growth under environmental stress. For instance, mustard seeds exposed to salt, lead or cadmium displayed increased germination rate when 13 | P a g e treated with cadaverine, suggesting a role for this diamine in stress mitigation. Similarly, in barley, exogenous cadaverine promoted seed germination and seedling growth in the presence of salt. Cadaverine was reported to accumulate in the tissues of several plant species in response to a wide variety of environmental stimuli. For instance, in the common ice plant (Mesembryanthemum crystallinum L.), cadaverine accumulated in response to heat shock, salt stress, and exogenous ethylene treatment. Furthermore, local application of heat shock to either shoots or roots promoted cadaverine accretion in distal organs, suggesting transport throughout the plant. Similarly, pepper plants (Capsicum annuum L.) were shown to accumulate cadaverine and putrescine in leaves, and spermidine and spermine in roots, upon exposure to drought conditions. In leaves, polyamines may contribute some protective effect against water-deficient conditions by inhibiting potassium influx into guard cells, thereby inducing stomatal closure and reducing water loss. While the previous studies reported cadaverine-induced stress mitigation, an experiment with Arabidopsis thaliana suggested induction of stress hypersensitivity. In this experiment, seedlings were pre-treated with cadaverine for 1 week, and then moved to media containing 150 mM NaCl for another week. Cadaverine-pre-treated seedlings displayed a hypersensitive response to salt despite an obvious accumulation of spermine, a polyamine previously associated with salt-stress mitigation. This result was interpreted to suggest that increased spermine levels, may not be sufficient for salt-stress mitigation. Instead, an increase in spermine catabolic products may be required. The previous discussion nicely illustrates a major complication in the study of cadaverine's role in plant stress response: many environmental stimuli, such as cold, salt, and drought stresses, also influence the expression of putrescine-derived polyamine biosynthetic enzymes. Furthermore, cadaverine is also known to influence the accumulation of putrescine-derived polyamines in plant tissues. Therefore, the contribution of cadaverine to plant stress response cannot be assessed in isolation. Instead, it will be important to carefully examine its impact in relation to that of putrescine- derived polyamines under the same conditions. Identification and characterization of additional polyamine response mutants will undoubtedly help in this difficult endeavour. To further assess cadaverine's role in plant-stress response, it will be critical to elucidate the pathways that lead to its biosynthesis, conjugation, transport and catabolism in control and stressful conditions. 14 | P a g e In species lacking clear LDC genes, such as Arabidopsis and rice, it will be necessary to investigate possible alternative pathway(s) for stress-induced cadaverine synthesis or uptake. In this regard, cadaverine delivery by rhizosphere and phyllosphere microbes should be considered. A more global analysis of the types of cadaverine-producing microbes associated with various plant species would be useful, as would an evaluation of the amount of cadaverine they deliver to the plant under diverse conditions. Furthermore, characterization of plant stress-response in the presence of cadaverine- defective microbial mutants should help demonstrate a role for microbial-derived cadaverine in stress mitigation.