Micropropagation and secondary metabolites of Sclerocarya birrea.

Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.Sclerocarya birrea (marula, Anacardiaceae) is a highly-valued indigenous tree in most parts of sub-Saharan Africa because of its medicinal and nutritional properties. The marula tree is adapted to the semi-arid conditions that characterise most parts of sub-Saharan Africa and renders them unsuitable for conventional crop agriculture. The unique nutritional properties of marula and its high tolerance to dry conditions provide opportunities for its development into a plantation crop. On the other hand, the demand for marula plant parts, mainly the bark and roots as medicinal remedies, poses a great threat to wild populations. In the long term, the growing demand of marula products in the food, pharmaceutical and cosmetic industries will not be sustainable from wild populations alone. Plant tissue culture technologies can be useful for in vitro manipulation and mass propagation of the plant in the process of domestication and conservation. The aims of the project were to determine the optimum conditions for seed germination, in vitro propagation and plant regeneration, and to evaluate the potential bioactivity of secondary metabolites from its renewable plant parts as an alternative option in the conservation of S. birrea. An ex vitro seed germination study indicated that after-ripening and cold stratification are critical factors. Cold stratification (5 °C) of marula nuts for 14 days improved germination (65%) as compared to non-stratified nuts (32%). Direct shoot organogenesis was achieved from leaf explants through the induction of nodular meristemoids on Murashige and Skoog (MS) (1962) medium and woody plant medium (WPM) supplemented with 6-benzyladenine (BA) in combination with naphthalene acetic acid (NAA), indole-3-butryric acid (IBA) and indole-3-acetic acid (IAA). Induction of nodular meristemoids from 86% of the leaf cultures was achieved on a MS medium with 4.0 ìM BA and 1.0 ìM NAA. High levels (78–100%) of induction were also achieved on WPM with different concentrations of BA (1.0–4.0 ìM) and IBA (1.0–4.0 ìM). The highest conversion of nodular meristemoids into shoots on MS initiation medium was only 22% for 4.0 ìM BA and 1.0 ìM NAA. This was improved to 62% when nodular clusters were cultured in MS liquid medium. Histological studies revealed high numbers of unipolar meristematic buds developing from globular nodules. These embryo-like structures have in the past been mistaken for true somatic embryos. The initiation of high numbers of nodular meristemoids per explant provides potential for automated large-scale clonal propagation in bioreactors, in vitro phytochemical production and the development of synthetic seed technology, similar to somatic embryogenesis. Plant regeneration through nodule culture has potential for application in mass micropropagation and plant breeding of S. birrea. Adventitious shoot and root induction are important phases in micropropagation. Plant growth regulators play an important role in these developmental processes, and the type and concentration used have major influences on the eventual organogenic pathway. Three auxins (IAA, IBA and NAA) and four aromatic cytokinins (6-benzyladenine, meta-topolin, meta-topolin riboside, and meta-methoxytopolin riboside) were evaluated for their potential to induce adventitious shoot and root formation in S. birrea shoots, hypocotyls and epicotyls. Among the evaluated cytokinins, the highest adventitious shoot induction (62%) was achieved on MS medium supplemented with meta-topolin (8.0 ìM). The lowest adventitious shoot induction (2.5%) was obtained on MS basal medium containing 2.0 ìM meta-methoxytopolin riboside. The highest adventitious shoot induction for hypocotyls was 55% on MS medium supplemented with 8.0 ìM meta-topolin. For the tested auxins, IBA induced adventitious rooting in 91% of shoots at a concentration of 4.0 ìM after 8 weeks in culture. However, the in vitro rooted plants only survived for two weeks when transferred ex vitro. A temperature of 25 °C and 16-h photoperiod were optimum for adventitious root induction. Stomatal density (number per mm2) on the abaxial leaf surfaces was higher for the 16-h photoperiod treatment (206.6 ± 15.28) compared to that for a 24-h photoperiod (134.6 ± 12.98). Normal mature stomata with kidney-shaped guard cells and an outer ledge over the stomatal pore were observed for in vitro plants growing under a 16-h photoperiod. Total phenolic content, proanthocyanidins, gallotannins, flavonoids, and antioxidant activities of S. birrea methanolic extracts were evaluated using in vitro bioassays. Methanolic extracts of the young stem bark and leaves contained high levels of these phytochemicals. Sclerocarya birrea young stem extracts contained the highest levels of total phenolics (14.15 ± 0.03 mg GAE g-1), flavonoids (1219.39 ± 16.62 ìg CE g-1) and gallotannins (246.12 ± 3.76 ìg GAE g-1). Sclerocarya birrea leaf extracts had the highest concentration of proanthocyanidins (1.25%). The EC50 values of the extracts in the DPPH free radical scavenging assay ranged from 5.028 to 6.921 ìg ml-1, compared to ascorbic acid (6.868 ìg ml-1). A dose-dependent linear curve was obtained for all extracts in the ferric-reducing power assay. All the extracts exhibited high antioxidant activity comparable to butylated hydroxytoluene based on the rate of â-carotene bleaching (89.6 to 93.9%). Sclerocarya birrea provides a source of secondary metabolites which have potent antioxidant properties and may be beneficial to the health of consumers. Sclerocarya birrea young stem and leaf ethanolic extracts exhibited high bioactivity (MIC < 1.0 mg ml-1) against both Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria. The highest activity (MIC = 0.098 mg ml-1 and total activity = 1609.1 ml g-1) was recorded for young stem extracts against B. subtilis. The highest activity (MIC = 1.56 mg ml-1 and MFC = 1.56 mg ml-1) in the antifungal assay against Candida albicans was observed for young stem ethanolic extracts. Sclerocarya birrea extracts had moderate acetylcholinesterase (AChE) inhibition activity. The dichloromethane (DCM) and methanol (MeOH) fractions exhibited dose-dependent acetylcholinesterase inhibitory activity. The highest AChE inhibitory activities were from leaves (DCM fraction, IC50 = 0.1053 mg ml-1) and young stems (MeOH fraction, IC50 = 0.478 mg ml-1). High inhibitory activity against cyclooxygenase (COX-1 and COX-2) enzymes was observed. All extracts and fractions showed high COX-1 enzyme inhibition (90.7-100%). Petroleum ether (PE) and dichloromethane fractions also exhibited high inhibition against COX-2 enzyme (77.7-92.6%). The pharmacological activities observed suggest that S. birrea renewable plant parts (leaves and young stems) provide a substantial source of medicinal secondary metabolites. Based on these results, plant part substitution can be a practical conservation strategy for this species

Artificial Photosynthesis

Photosynthesis is one of the most important reactions on Earth, and it is a scientific field that is intrinsically interdisciplinary, with many research groups examining it. We could learn many strategies from photosynthesis and can apply these strategies in artificial photosynthesis. Artificial photosynthesis is a research field that attempts to replicate the natural process of photosynthesis. The goal of artificial photosynthesis is to use the energy of the sun to make different useful material or high-energy chemicals for energy production. This book is aimed at providing fundamental and applied aspects of artificial photosynthesis. In each section, important topics in the subject are discussed and reviewed by experts

Development of biotechnological tools for the in vitro propagation of elite trees for the Basque Country forest

278 p.Located in the North of Spain, Basque Country has edaphoclimatic conditions that have allowedthe establishment and development of an important forestry sector at European level. In thissense, 54.8% of the territory is dedicated to natural and cultivated forests. The most cultivatedspecies in the Basque territory is Pinus radiata, whose first report of forest plantationestablishment dates back to 1897. From that year to the present, its cultivation has increased inthe Basque Country, reporting for the year 2022 the largest total area (25.9 %) and the highestforest productivity (80-85% of the annual cuts).Pinus radiata, has a natural distribution from the central coast of California to Mexico. Theversatility and quality of the wood, together with the rapid growth of the species, has allowed itsplantations to extended to countries such as Australia, Chile, New Zealand, South Africa andSpain. However, in recent years P. radiata in the Basque Country has been affected by severaldiseases caused mainly by fungi: Pine canker caused by Fusarium circinatum, Dothistromaneedle blight caused by Dothistroma septosporum and Dothistroma pini, and brown spot needleblight caused by Lecanosticta acicula. This last disease has affected 3,300 ha in Gipuzkoa,Basque Country. In addition, P. radiata plantations in the Basque Country have decreased from123,921 ha (2016) to 102,488 ha (2022), a decrease that coincides with a historical outbreak ofthe needle blight during the years 2018-2019. For all of the abovementioned reasons, andtogether with the increase in the world population that causes a strong demand for timber andnon-timber products, the forestry sector and HAZI (www.hazi.eus) proposed a list of candidatespecies to be used, compatible with the demand of the forestry sector, as well as socially andculturally accepted.Using the list of possible alternative species, the following were selected for this study:Cryptomeria japonica (Thunb. ex L.f.) D. Don, Pinus ponderosa (P. Lawson and C. Lawson),Sequoia sempervirens ( D.Don). Endl and apparently tolerant genotypes of P. radiata.However, these species display desirable characteristics for clonal propagation at maturity,when traditional vegetative propagation and rooting are unfeasible. Therefore, the developmentof alternative methods of propagation, such as in vitro propagation, for these species isnecessary. In vitro propagation through tissue culture could be performed mainly byorganogenesis or somatic embryogenesis; those techniques combined with traditional onescould be efficient for the vegetative propagation of the abovementioned alternative species. Inaddition, these methods allow to improve the quality, yield and health of commercially valuableforest species. In this context, organogenesis in species of the Pinaceae family is generallyrestricted to zygotic embryos, part of them or young seedlings as initial source of explant, andattempts to clone selected adult individuals have been feasible but have faced severaldifficulties. In this regard, in our laboratory at Neiker, several in vitro propagation protocolshave been successfully developed for Pinus pinaster Ait, Pinus sylvestris L., Pinus pinea L.,Pinus radiata or Pinus halepensis among others.Taking into account all this information, the general hypothesis of this work is that themodification of the physico-chemical environment in the micropropagation process caninfluence the regeneration efficiency of elite trees of forest species such as C. japonica, P.ponderosa and S. sempervirens to be considered as an alternative to P. radiata in the BasqueCountry. The second hypothesis to be corroborated is that adult P. radiata trees with apparenttolerance to pine needle blight can be used as a source of material for in vitro propagation.In this context, the objective of the first chapter of the thesis was to analyze the influence ofcollection dates and different concentrations of 6-benzyladenine (BA) in the shoot inductionphase, as well as the effect of different types of light on growth success of rooting. Also, theeffect of physico-chemical factors in the content of amino acids and carbohydrates in the shootsdeveloped in vitro was studied. Although reinvigorated shoots were obtained in both BA concentrations (22 or 44 ¿M), the highest BA concentration showed the best results in terms of shoot induction and number of shoots per explant when the explants were collected in the first week of February. In addition, in shoots developed in vitro a significantly higher differences were found in the different phases of the rooting process in the levels of fructose, glucose and sucrose and in the threonine and tyrosine contents.The objective in the second chapter was to optimize the micropropagation method of C. japonica using adult trees as a source of plant material. In a first experiment, the effect of different types of initial explants and three basal culture media on shoot induction was studied. In addition, two sucrose concentrations and two light treatments (LED versus fluorescent lights) were tested for rooting stage. In a second experiment, the effect of different growth regulators (6-benzylaminopurine, metatopolin and thidiazuron) on shoot induction and subsequent phases of the organogenesis process were analysed. As main results, the highest number of shoots was obtained when using basal explants longer than 1.5 cm of length grown in the QL culture medium (Quoirin and Lepoivre, 1977). The shoots produced a higher number of roots when grown under red LED lights. Finally, root induction was significantly higher in shoots previously induced with meta-topolin.The third chapter was focused on the analysis of the influence of different types of explants, basal culture media, cytokinins, auxins and light treatments on the in vitro propagation success in P. ponderosa. In this sense, whole zygotic embryos cultured in 44 ¿M BA performed better in terms of explant survival. For shoot organogenesis, whole zygotic embryos and half LP (LP medium, Quoirin and Lepoivre, 1977, modified by Aitken-Christie et al., 1988) macronutrients were selected. Regarding the light treatments applied, a significantly higher percentage of rootable shoots was obtained under blue LED light. However, the percentage of acclimatization was higher in shoots previously cultivated under fluorescent light. Furthermore, anatomical studies using optical and scanning microscopy showed that light treatments promoted differences in anatomical aspects in in vitro shoots; needles of plantlets exposed to red and blue LEDs showed less stomata compared with needles from plantlets exposed to fluorescent light.The objective in the fourth chapter was to optimize the micropropagation of adult elite trees of S. sempervirens. To accomplish this objective, the effect of different cytokinins (6-benzylaminopurine, meta-Topolin and Kinetin), and four types of explants in shoot induction were studied. At rooting stage, the effect of two types of auxins was evaluated: 1-naphthalene acetic acid, indole-3-butyric acid and a mixture of both. The best induction rate was obtained when apical explants longer than 1.5 cm were induced with 4.4 ¿M BA. Regarding the rooting, shoots previously induced in 4.4 ¿M BA and rooted in a culture medium with 50 ¿M 1-naphthalene acetic acid showed the best result. However, acclimatization was better when explants had been cultivated with Kinetin and rooted in a culture medium with indole-3-butyric acid.Our study demonstrated that in vitro culture method can be used as an alternative for the propagation of elite individuals of C. japonica, P. ponderosa, P. radiata and S. sempervirens. Furthermore, the success of in vitro propagation in these species is influenced by genetic factors, collection dates, and physicochemical conditions of the culture environment.Neiker Eusko Jaurlaritza UNA:Univerdiad Nacional de Costa Rica Inisefo

Plant Science

The book "Plant Science" consists of 12 chapters divided into three sections authored by many researchers from different parts of the Globe. Section-I: Plant and Environment, describes the relationship between plants and environment, particularly enumerating species-environment relationship and response of plants to different environmental stress conditions. Section-II: Plant-Microbe relation, embodies broadly on both positive and negative aspects of microbes on plants. Section-III: Plant Biotechnology, shed light on current biotechnological research to develop modern technology for producing biologicals and also increasing plant immunity in present environmental conditions. The book "Plant Science" will be helpful to a wide group peoples; readers, scientists, researchers and allied professionals. We recommend it to you; enjoy reading it, save the plant and save life