Plant Biotechnology Course: BT427 PDF

10/23/24 Plant Biotechnology Course code: BT427 Dr. Reda Gaafar Professor of Plant Molecular Biology and Biotechnology 1 Concepts of Biotechnology...

10/23/24 Plant Biotechnology Course code: BT427 Dr. Reda Gaafar Professor of Plant Molecular Biology and Biotechnology 1 Concepts of Biotechnology Biotechnology was first coined in 1919 by Karl Ereky which means products are produced from raw materials with the aid of living organisms. Biotechnology is NOT new. Man has been manipulating living things to solve problems and improve his way of life. Plants and animals were selectively bred and microorganisms were used to make food items such as beverages, cheese and bread. Biotechnology is currently being used in many areas including agriculture, bioremediation, food processing and energy production. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 2 Concepts of Biotechnology Definition Biotechnology- Bio means life and technology means the application of knowledge for practical use i.e., The use of living organisms to make or improve a product. Other definitions for the term Biotechnology Ø The use of living organisms to solve problems or make useful products. ØThe use of cells and biological molecules to solve problems or make useful products. Biological molecules include DNA, RNA and proteins. ØThe commercial application of living organisms or their products, which involves the deliberate manipulation of their DNA molecules. ØMake a living cell to perform a specific task in a predictable and controllable way. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 3 1  10/23/24 Concepts of Biotechnology Stages of Biotechnology Development: Ancient Biotechnology - 8000-4000 B.C Early history as related to food and shelter; includes domestication Classical Biotechnology - 2000 B.C.; 1800-1900 AD Built on ancient biotechnology; fermentation promoted food production and medicine 1900-1953: Genetics 1953 - 1976: DNA research, science explodes Modern biotechnology – 1977 Modern biotechnology enables an organism to produce a totally new product which the organism does not or can not produce normally through the incorporation of the technology of Genetic Engineering Totipotency of plant cell, different aspects of Plant Tissue Culture Techniques, cell fusion techniques along with the use of rDNA technology has developed a new era of Plant biotechnology Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 4 Biotechnology is a collection of various technologies that enable us to improve crop yield and food quality in agriculture and to produce a broader array of products in industries. Synthesis of Any one scientific and technology will technical be applied to a knowledge number of from many industries to academic produce an disciplines has even broader produced the array of set of enabling products technologies we call biotechnology Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 5 Various Technologies and Their Uses Genetic Engineering (Recombinant DNA) Techniques A gene coding for a desired product is transferred to another organism for production of useful proteins, hormones, vaccines, enzymes, etc. by microorganisms, plant cells or animal cell cultures. is essential for four reasons: large scale production of desired product; supply of natural source may be limited; the purity of naturally available material is doubtful; is the cost of the natural product may be higher. Protein Engineering Technology * Improve existing/create novel proteins to make useful products Antisense or RNAi Technology * Block or decrease the production of certain proteins Cell and Tissue Culture Technology * Grow cells/tissues under laboratory conditions to produce an entire organism, or to produce new products Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 6 2  10/23/24 High-throughput Technologies (the -omics) Transcriptomics (e.g. microarray expression profiling) Proteomics (e.g. structures/modifications/interactions of proteins) Proteins are responsible for an endless number of tasks within the cell. Proteome: complete set of proteins in a cell. Proteomics: the study of protein structure and function and what every protein in the cell is doing. The proteome is highly dynamic and it changes from time to time in response to different environmental stimuli. The goal of proteomics is to understand how the structure and function of proteins allow them to do what they do, what they interact with and how they contribute to life processes. Metabolomics (e.g. metabolite profiling, chemical fingerprinting, flux analysis) Metabolomics is one of the newest ‘omics’ sciences. The metabolome refers to the complete set of low molecular weight compounds in a sample. These compounds are the substrates and by products of enzymatic reactions and have a direct effect on the phenotype of the cell. Thus, metabolomics aims at determining a sample’s profile of these compounds at a specified time under specific environmental conditions. Transgenomics (e.g. knock-out, knock-in, gene tagging, mutagenesis) Translational genomics combines genome and transcriptome-wide studies in humans and model systems Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 7 Metabolic Network DNA Genomics RNA Transcriptomics Proteins Proteomics Metabolites Metabolomics Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 8 Plant Biotechnology /Agricultural Biotechnology A process to produce a genetically modified plant by removing genetic information from an organism, manipulating it in the laboratory and then transferring it into a plant to change certain of its characteristics. In a nutshell, it’s the manipulation of plants for the benefit of mankind. The plants are mainly manipulated for two major objectives: A. Crop improvement Herbicide tolerance Pest resistance Drought tolerance Nitrogen fixing ability Acidity and salinity tolerance Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 9 3  10/23/24 B. Nutritional value of crops Improving food quality and safety. Healthier cooking oils by decreasing the conc. of saturated fatty acids in vegetable oils. Functional foods: foods containing significant levels of biologically active components that impart health benefits. Various technologies applied in plant biotechnology includes: Genetic Engineering/Recombinant DNA Technology Tissue culture (in vitro propagation technology, in vitro production of secondary metabolites, applications of somaclonal variations) Application of nanobiotechnology in agriculture Various “omics” technologies including phenomics, genomics, proteomics, metabolomics RNA interference technology and miRNA-mediated regulation of biotic and abiotic stress responses in plants, Genome editing technology and its probable applications. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 10 Course Content Chapter 1: In vitro production of medicinal compounds from endangered and commercially important medicinal plants Chapter 2: Double haploid production and its applications in crop Improvement Chapter 3: Encapsulation technology: An assessment of its role in in vitro conservation of medicinal and threatened plant species Chapter 4: Somaclonal variation in improvement of agricultural crops Chapter 5: Transgenic implications for biotic and abiotic stress tolerance in agricultural crops Chapter 6: Genomics in crop improvement Chapter 7: RNA interference technology Chapter 8: miRNA-mediated regulation of biotic and abiotic stress responses in plants Chapter 9: Genome engineering to target traits Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 11 ‫ــ ت‬ ‫د‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫دا‬ - ‫ا د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫ا‬/ ‫ا‬ ‫ا ل‬ ‫ا را‬ ‫ا‬ ‫ا را‬ ‫ا‬ ‫ا‬ ‫ا‬ / ‫أ‬ ‫ر‬ ‫ا‬ ‫ر‬ ‫ا‬/ ‫ا‬ ‫ا‬ ‫ا‬ ‫ا‬ 0.0 0.0 ‫ر‬ ‫ا‬ ‫س‬ ‫ا‬ 0.0 0.0 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا‬ ‫ا‬ 362 1 ‫ا‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ ‫ا ا‬ ‫اّ ء‬ 5001 2 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ ‫ا‬ 5002 3 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ب‬ ‫ا‬ ‫ا‬ 5003 4 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ ‫ا‬ 5004 5 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ ‫اء‬ ‫ا‬ 5006 6 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ ‫ءا‬ ‫ا‬ 500 ‫ا‬ ‫ا‬ ‫ا‬ 12 ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ ‫ء‬ ‫ا‬ 500 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا ا‬ ‫ا ء‬ 500 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫ا ا‬ ‫ا ء‬ 5010 10 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ 5011 11 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫أ‬ ‫ا‬ ‫ا‬ 5012 12 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫إ ا‬ ‫ا‬ 5013 13 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫دا‬ ‫ا‬ 5014 14 ‫ا‬ 4 ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ ‫د‬ 5015 15 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ 5016 16 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ 501 1 ‫ا‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ 501 1 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫د ا ا‬ ‫ا‬ 501 1 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ ‫ء‬ 5020 20 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫ا‬ 5021 21 ‫ا‬ ‫ا‬ ‫ا‬ ‫ت‬ ‫د‬ ‫ا‬ ‫ا ا‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫د‬ 5022 22 ‫ا‬  10/23/24 Chapter 1: In vitro production of medicinal compounds from endangered and commercially important medicinal plants Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 13 Importance of medicinal plants Traditional and Modern Medicine: 25% of modern medicines and 60% of anticancer medicines are obtained from natural resources. Economic and Industrial Significance: Herbal medicines are less expensive and safer than synthetic or modern drugs. Medicinal compounds are mainly secondary metabolites. Primary metabolites: are essential for plant growth and development. include carbohydrates, lipids, proteins, and nucleic acids. Secondary metabolites: play role in defense, signaling, and chemical adaptations to environmental stresses. categorized as terpenoids (derived from acetyl coenzyme A or glycolysis cycle intermediates), phenolics (aromatic rings bearing a hydroxyl functional group), and alkaloids (nitrogen containing compounds). Majority of herbal collections are obtained from their natural habitat and only 10% are contributed by the cultivated medicinal plants. Dwindling populations of medicinal plants in wild. Conservation concerns and biotechnological solutions cell and tissue culture, hairy root culture, elicitor application, precursor feeding, and metabolic engineering. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 14 Tissue culture tools for production of desirable medicinal compounds Biotechnological tools offer valuable alternatives for production of desirable medicinal compounds by enhancing their biosynthesis and accumulation. Cell and Tissue Culture Techniques Various valuable medicinal compounds are biosynthesized in: callus or suspension cultures (widely utilized for production of secondary metabolites), while some metabolites are produced more in organized structures like shoots, roots, glands, or somatic embryos. Biosynthesis of bioactive compounds under in vitro conditions is dependent on various factors: culture media composition (carbon source, macronutrients, micronutrients, other organic compounds and plant growth hormones), pH of media, explant or inoculum concentration, and other suitable environmental conditions like light, temperature, aeration, and agitation. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 15 5  10/23/24 Production of bioactive plant secondary metabolites through in vitro technologies Wawrosch and Zotchev (2021) Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 16 Advantages of Cell/Tissue Culture Techniques Biosynthesis of secondary metabolites under in vitro conditions is more reliable, simpler, and predictable. process of extraction of valuable phytochemicals from in vitro grown cell suspensions or tissue culture plants is fast and effective. independent of environmental factors, seasonal variations, pest and microbial diseases, geographical location constraints. production time is less and cost effective due to minimal labor involved. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 17 Tissue culture tools for production of desirable medicinal compounds Hairy Root Culture Hairy roots are comprised of differentiated transformed roots which are produced by infection caused by Agrobacterium rhizogenes (soil-inhabiting gram-negative bacteria) T-DNA (transfer DNA) located in root-inducing (Ri) plasmid of this pathogen is transferred and integrated into the genome of host plant, thereby leading to formation of hairy roots at the wounded site. Different genes present in the T-DNA are encoded, and as a result, auxin and cytokinins are produced that stimulate the production of HR-like outgrowths from the wounded regions. Gantait and Mukherjee 2021 Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 18 6  10/23/24 Cellular mechanism of Agrobacterium rhizogenes– mediated transformation of the host cell Transformation is effected owing to the presence of different genes (rol genes, i.e. rol A, rol B and rol C) in the plasmid of the bacteria. Gantait and Mukherjee 2021 Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 19 Types of Agrobacterium plasmids Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 20 T-DNA segment penetrates the plant cell nucleus and integrates randomly into the genome. Expression of the natural genes on the T-DNA results in the synthesis of gene products that direct the observed morphological changes such as tumor inducing plasmid (Ti plasmid) or root inducing plasmid (Ri plasmid) Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 21 7  10/23/24 In vitro protocol for hairy root culture establishment using Agrobacterium rhizogenes and regeneration of transformed plantlet Gantait and Mukherjee 2021 Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 22 Advantages of Hairy Root Culture Technique proliferating roots could biosynthesize bioactive compounds throughout the year without the effect of seasonal variations. high productivity of secondary metabolites, this technique has become popular tool to produce same medicinal compounds corresponding to the wild type roots. Therefore, hairy roots provide an imperative alternative to natural plant material for biosynthesizing many valuable medicinal compounds. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 23 Elicitors and Precursor Feeding for Enhancement of Medicinal Compounds Production Elicitors act as the signal molecules, are capable of inducing or enhancing the production of specific secondary metabolites by initiating defense or stress related responses. Biotic elicitors are partially purified extracts of biological origin like fungus, bacteria, yeast, or the plant itself. Abiotic elicitors are substances which are of non-biological origin and are categorized into: Physical (UV rays, light, and temperature), chemical (heavy metal salts, osmotic stress, and antibiotics), and hormonal factors (jasmonates, methyl jasmonic acid, salicylic acid, acetyl salicylic acid, abscisic acid, etc.). Elicitors have been extensively used in cell/organ and hairy root cultures of different plant species. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 24 8  10/23/24 Nanoparticles Mediated Secondary Metabolites Production Nanoparticles are 1-100 nm sized particles. can interfere with various signaling pathways and can modulate secondary metabolite production in plants Example: 3.9-fold enhanced artemisinin content by silver nanoparticle treatment in Artemisia annua L. hairy roots. Nanoparticles have potential to increase secondary metabolite production in plants. Molecules 2022, 27, x FOR PEER REVIEW 6 of 20 Deeper understanding is still required to fully exploit this technique to produce commercially important medicinal compounds. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 25 Figure 4. EDX spectrum of ZnONPs synthesized using oat biomass. Weight % of Zn, 69.9%; O, 30.1%. Atomic % of Zn, 36.2%; O, 63.77%. A microscopic analysis was performed to visualize the surface structure (size and- shape) of the synthesized ZnONPs. The size and shape of ZnONPs are demonstrated in Molecules 2022, 27, 579 Figure 5a. This image revealed that the formed nanoparticles were nanocrystalline hex- 6 of 19 agonal in shape with a size distribution of around 100 nm. The SEM image demonstrat- ed that the clusters of ZnONPs were nearly hexagonal with rough surface (Figure 5b). Figure 5. (a) TEM and (b) SEM images of ZnONPs synthesized using oat biomass. Figure 5. (a) TEM and (b) SEM images of ZnONPs synthesized using oat biomass. (a) TEM and (b) SEM images of ZnONPs synthesized using oat 2.2. Thermal Stability of Biosynthesized ZnONPs 2.2. Thermal Stability of Biosynthesized biomass. ZnONPs The thermal behavior of the biosynthesized ZnONPs using oat biomass extract was The thermal behavior of the biosynthesized ZnONPs using oat biomass extract was investigated Dr. Redainvestigated through the TGA/DSC mode. As the synthesis of ZnONPs was carried out Gaafar through the TGA/DSC mode. As the synthesis of ZnONPs Professor was carried of Molecular Biology out and Biotechnology at 80 °C, the effect of temperature variation on the stability of the formed metal oxide at 80 C, the effect of temperature variation on the stability of the formed metal oxide 26 nanoparticles was examined. Figure 6a displays the thermogram behavior of nanoparticles was examined. Figure 6a displays the thermogram behavior of ZnONPs ZnONPs in the temperature range 80-700 °C. The obtained results show that the nominal in the temperature range 80–700 C. The obtained results show that the nominal overall overall loss of biosynthesized ZnONPs was around 9.5% indicating the significant ther- loss of biosynthesized ZnONPs was around 9.5% indicating the significant thermal stability mal stability of the sample. Moreover, it was reported that the zinc nitrate precursor that of the sample. Moreover, it was reported that the zinc nitrate precursor that had been used had been used in the synthesis process was completely converted to thermal stable Molecules 2022, 27, x FOR PEER REVIEW in the synthesis process was completely converted to thermal stable ZnONPs.7 of The 20 ZnONPs. The thermogram displayed three remarkable regions of weight losses and thermogram displayed three remarkable regions of weight losses and diffraction scanning diffraction scanning calorimetry (DSC) was applied to interpret these regions (Figure calorimetry (DSC) was applied to interpret these regions (Figure 6b). 6b). Metabolic Engineering Metabolic engineering is optimization of cellular processes and metabolic routes in an organism to improve secondary metabolites production Carbon flux towards the targeted product can be increased by: overexpression of selected genes or by knocking down/out the key genes of competitive pathways. Recombinant DNA tools such as RNA interference (RNAi), antisense RNA, co-suppression, dsRNA Figure Figure 6. 6. (a) (a) Thermogram Thermogram ofofZnONPs ZnONPsatatthe thetemperature temperature range range 80-700C°C 80–700 and and (b)(b) diffraction diffraction scan- scanning ning mediated gene silencing, etc. can be used to calorimetry calorimetry graph graph of ZnONPs of ZnONPs synthesized synthesized with with oat biomass oat biomass extract. extract. upregulate The recorded peaks The metabolic peaks that thatappeared pathways appearedatat140.5, 140.5,230.1 230.1 ofand370.3 and desired 370.3C °C were products related were to the related to loss the loss and of moisture obscuring and volatile of moisture the and volatile keyfrom components genes components from responsible particles surface particles (2.1 w% surface for (2.1loss), conversion w% loss), conver-of sion production Zn(OH) of 2 to complete of Zn(OH) the complete from to 2 complete degradation the complete unwanted calcined ZnONPs (4.5 calcined of organic degradation ZnONPs metabolites. matters of organic w% loss) and production of ZnONPs from (4.5 (2.9 w% matters w% loss) (2.9 loss). and production No other w% loss). peakpeak No other of ZnONPs was detected was de- at 700 atC, tected proving 700 the low-temperature °C, proving calcination the low-temperature of ZnONPs calcination of ZnONPs at 400at 400 C. According °C. Accord- to Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology ing to the obtained results, it can be concluded that the bioactive compounds in the oat biomass extract mainly had reduction abilities, with a minimum chelation interaction. 27 The reduction of zinc nitrate to Zn2+ ions by the bioactive components was performed in the first stage of synthesis with low chelation of a Zn-bioactive component complex. These bio-complex products were further degraded to ZnO by a further oxidation pro- cess of Zn2+ to ZnO to establish the proper formation of metallic ZnONPs from zinc ni- trate. 2.3. Antibacterial Activity ZnONPs have been reported for the advancement of next-generation nano- antibiotics against various pathogens to avoid multidrug resistance [52,53]. These metal oxide nanostructures exhibit extraordinary physicochemical features, such as (crystallin- 9 ity, porosity, particle size, and shape). According to these properties, ZnONPs pos- sess an immense antimicrobial potential versus several pathogens, including Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa [55–58]. The antibacterial effect of green synthesized ZnONPs with oat biomass was studied and the outcomes confirm that ZnONPs exhibited excellent dose-dependent manner an- tibacterial potential (Table 1). The calculated inhibition zones were found as E. coli (16 mm), P. aeruginosa (17 mm), S. aureus (12 mm) and B. subtilis (11 mm) for ZnONPs (Fig-  10/23/24 Metabolic Engineering Artemisinin and artemisinic acids were enhanced by overexpressing jasmonic acid responsive AP2/ERF transcription factors in Artemisia annua L. Overexpression of transcription elements like ORCA2 or ORCA3 in cell suspension/hairy roots cultures of C. roseus has enhanced the biosynthesis of different medicinal compounds like ajmalicine, catharanthine, serpentine, and tryptamine. CRISPR genome editing system has playing role in manipulating metabolic pathways to biosynthesize desired medicinal compounds. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 28 Chapter 2: Double haploid production and its applications in crop Improvement Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 29 Haploids are defined as plants with gametophytic chromosome number (n) Doubled haploids (DHs) are plants derived from haploids and doubled artificially to form homozygous diploids (2n) Haploids have been produced in a variety of plant species using a variety of methods (in vitro and in situ): Microspores (pollen grains) culture (in vitro) Anther culture Ovary/Ovules culture Techniques using irradiation of pollens or interspecific crosses (chromosome elimination following interspecific hybridization). (in situ) Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 30 10  mal. Therefore, haploids contain only the which can either be released directly chromosome set found after meiosis to farmers as cultivars or used arch, what’s next? in male (sperm cells) or female (egg as genitors (inbred lines) for the be learned about cells) gametes. This chromosome production of hybrid seeds. The ur, camouflage, set ‘n’ corresponds to only half of primary advantage of DH plants is this, both field and the chromosome set found in the to possess a phenotypic stability eded, but the former fertilization product (zygote) and other due to the fact that all alleles are in arative studies somatic cells. Depending on whether a homozygous state. In short, DH must be encouraged. nating animal in the the single set of chromosomes comes from the maternal or paternal side, technology increases the efficiency of plant breeding. 10/23/24 eat moment. the plant is referred to as maternal haploid and paternal haploid, What are the different methods out more? respectively. to produce haploid plants? The el, L., Chichery, M.-P., numerous methods to obtain haploid ry, R. (2004). Rapid ing in adult cuttlefish, What is a doubled haploid plants can be classified into two m. Behav. 68, 1291–1298. (DH) plant? In a DH plant, the categories (Figure 1). Firstly, in hery, R., and Dickel, L. ing, new evidence from the chromosome set of a haploid plant vitro methods are based on the cinalis. Biol. Lett. 2, 345–347. has been doubled spontaneously or culture of haploid cells and their el, L., and Mather, J.A. artificially. Chromosome doubling is differentiation into haploid embryos Cognition: Advances in ote Group. (Cambridge: Overview of doubled haploid necessary since haploid plants are and ultimately haploid plants. Both ty Press), p. 263. phalopod dynamic iol. 17, R400–R404. generally frail, have reduced organ size and are not fertile. The most technology male (microspores or pollen) and female haploid cells (megaspores or enger, J.B. (1996). commonly used chemical agent to ovules) are used, depending on the our. (Cambridge: Cambridge render haploid plantlets diploid is responsiveness of the cells in a given 256. Cuttlefish use startle displays, colchicine, which blocks cell division species. Secondly, in situ methods e predators. Anim. Behav. 77, Parts of flowers without blocking chromosome make use of particular pollination , H., Mori, A., and que arm-flapping behavior of sh, Sepia pharaonis: putative crab. J. Ethol. 35, 307–311. Microspore/anther In vitro alopod behaviour: skin culture In vitro R684–R685. culture , A.-S., and Shashar, Ovule/ovary culture Colchicine erpolation for contour treatment uropean cuttlefish (Sepia se in dynamic camouflage. , 2386–2390. Chromosome Interspecific cross Embryo Haploid plants doubling rescue In situ Normandie, UMR 6552 (in vitro) (weak and sterile) DH (2n) Pollen treatments Rennes 1 Ethos, Doubled haploid plants Humaine Equipe = Pure homozygotes Inducer lines itive des Céphalopodes Current Biology planade de la Paix, x 5, France. 2School of 2017 Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology sity of Sussex, Brighton Figure 1. Overview of doubled haploid technology. First different methods are used to create haploid plants. Then chromosome doubling on haploid [email protected] 31 plantlets results in perfect homozygous plants named doubled haploid (DH) plants. Current Biology 27, R1089–R1107, October 23, 2017 © 2017 Elsevier Ltd. R1095 Induction of genetic variability for use in plant breeding and crop improvement programs. Microspores are the prime targets for mutagenic treatment with: Chemicals such as (EMS) or sodium azide physical mutagens such as gamma rays, X-rays, and UV rays DH has been developed in at least 200 plant species and is widely used in Brassicas and cereals, including wheat, barley, rice, and maize. Once haploid plants become available, their genome must be doubled to produce fertile DH lines. Use of DH is key strategies for agricultural crop improvement with desirable trait such as quality, crop yield, and resistance to environmental stresses. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 32 Normal life cycle of angiosperm plant 78 showing haploid and diploid stages A. K. Mishra et al. Fig. 4.1 Normal life cycle of angiosperm plant showing Zygote Fertilization haploid and diploid stages Haploid (n) Diploid (2n) Embryo Egg Pollen grain Anther Sporophyte Ovary In flowering plants, sexual reproduction is characterized by a unique biological Fig. 4.2 Steps involvedprocess in named double fertilization (consists of two separate fusion events haploid production via anther Suitable parent plant selection culture between male and female gametes) Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 33 Selection of flower bud Microspore development stage determination Surface sterilization of flower buds 11 Inoculation of anther on appropriate medium Regeneration of anther on appropriate medium  10/23/24 Current Biology Comparison of double fertilization and in Magazine vivo gynogenesis in maize ‘Normal’ double fertilization In vivo gynogenesis Non-inducer pollen Inducer pollen Vegetative nucleus Vegetative nucleus Endosperm (3n) Endosperm (3n) 2 Sperm cells (n) 2 Sperm cells (n) Central Central cell cell (2n) (2n) ? Egg cell Zygote Embryo Egg cell Reprogammed Embryo (n) (2n) (2n) (n) egg cell (n) (n) Fertilization Mature Fertilization Mature Gametophytes Gametophytes products seed products seed Current Biology Central cell is fertilized by one male Only central cell is fertilized normally by Figure 2. Comparison of double fertilization and in vivo gynogenesis in maize. gamete In flowering plants,(n). sexual reproduction is characterized by a unique biological process a male namedgamete. double fertilization, which consists of two separate Egg isbetween events fusion fertilized by female male and one male gamete gametes (left). This double fertilization leadsegg to a cell develops diploid embryo andinto a haploid triploid endospermembryo that represent the two major seed components. In the case of fertilization with pollen from a maize ‘inducer’ line (right), double fertilization is impaired, resulting in seeds and containing becomes a haploid embryodiploid. with only the maternal genome (gynogenesis). lacking the paternal genome. 78 Dr. techniques Reda Gaafar using irradiated pollen, A. K. Mishra doubleet fertilization. al. It consists of two Professor of Molecular Biology NLD/MTL/ZmPLA1 and Biotechnology is specifi cally inter-specific crosses or so-called parallel fusion events between male expressed in male gametes and ‘inducer lines’. and female gametes (Figure 2). The encodes a patatin-like phospholipase Fig. 4.1 Normal life cycle of angiosperm plant showing haploid and diploid stages 34 What is a haploid inducer line? Fertilization haploid egg cell is fertilized by one Zygote haploid male gamete and becomes A localized at the plasma membrane of the male germ unit. The predicted Haploid inducer lines are routinely used the diploid embryo. At the same time, truncated protein is not detectable in plant breeding for maize only, and the diploid nucleus of the central cell in inducer lines and loses its thus represent an exception. Maize is fertilized by the second haploid male plasma membrane anchorage in Haploid (n) haploid inducer lines all derive from gamete of the same pollen tube to a heterologous system. How the Diploid (2n) a particular genotype discovered in form a seed nutritive tissue, the triploid biochemical function of NLD/MTL/ the 1950s that possesses the ability endosperm Embryo (Figure 2). Pollination by a ZmPLA1 relates to its inducing Egg Pollen grain to induce the development of haploid maize inducer line results in an atypical capacity is still unresolved, and embryos on a maize line of interest fertilization event in which only the either a structural or a signaling upon pollination with the inducer pollen. central cell is fertilized normally by a function has been hypothesized. The pollen from the inducer line triggers male gamete, and the egg cell develops Whereas loss of NLD/MTL/ZmPLA1 is Steps involved in haploid production via the development of the egg cell into an embryo containingAntheronly a haploid into a haploid embryo lacking the paternal genome (Figure 2). Note that Sporophyte sufficient to trigger haploid induction, quantitative trait locus (QTL) analysis anther culture maternal genome. This process is after pollination by a maize inducer demonstrated that additional, called in vivo gynogenesis (Figure 2). line, only about 10% of the developing currently unknown players take part Recently, haploid inducerOvary lines have seeds contain a haploid embryo, the in the process and influence the also been created in Arabidopsis remaining 90% are normal diploid efficiency of haploid induction. thaliana, Brassica juncea and maize embryos. by the use of engineered centromeric What future improvements are Fig. 4.2 Steps involved in histoneSuitable 3 (CENH3) variants. What are the molecular players needed for DH technology? In haploid production via anther parent plantHowever, selection this haploid induction method has behind in situ haploid induction plant breeding, and apart from culture not been reported in plant breeding in maize? The inducing capacity maize, the production of DH plants programs so far. of inducer lines has been recently requires at least an in vitro-based tracked to a 4 bp insertion at the process (Figure 1), the success of How doesSelection of flower in situ haploid bud induction end of the coding sequence of which remains highly species- and work in maize? All flowering plants a gene named NOT LIKE DAD genotype-dependent, as well as are characterized by a particular (NLD) / MATRILINEAL (MTL) / labor-intensive and time-consuming. way of sexual reproduction called ZmPHOSPHOLIPASE A1 (ZmPLA1). Moreover, the DH technology Microspore development stage determination R1096 Current Biology 27, R1089–R1107, October 23, 2017 Surface sterilization of flower buds Inoculation of anther on appropriate medium Regeneration of anther on appropriate medium in the tetrad stage. After that, surface sterilization Dr. Reda Gaafar protocol for flower buds should be Professor of Molecular Biology and Biotechnology applied for obtaining contamination free explants. Anthers/microspores are isolated with care and inoculated on appropriate regeneration medium. The regenerated 35 shoots are haploid plants (Fig. 4.2). Similarly, haploid plant production through ovary or ovule culture can be achieved. In this process, from donor plant, ovary or ovule will be taken out and proper developmental stage of ovary/ovule will be determined by histology. After confirmation of developmental stage of ovary/ microspore of apricot Embryogenic pollen-derived friable calli and embryos (Citrus) Direct microspore embryogenesis division of nucleus (Citrus, 3 months) in an olive Heart-shaped pollen-derived Microspore-derived plantlet embryo of of clementine Review by Germanà (2011) Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 36 12  10/23/24 Common steps for haploid production via ovary/ovule culture 4 Double Haploid Production and Its Applications in Crop Improvement 79 3 Fig. 4.3 Common steps for Healthy parent plant selection haploid production via ovary/ ovule culture Oary/ovule developmental stage determination by histology Select appropriate flower bud and surface sterilized Inoculation of ovary/ovule in suitable medium Incubation of ovary/ovule culture Regeneration of callus/embryo Haploid plant recovery Figure 1 Yesim et al. 2017 ovule, the suitable flower buds are selected and surface sterilized. Ovary/ovule Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology excised from surface sterilized flower buds is inoculated on suitable medium for regeneration/embryo which is derived from cultured ovule/ovary. This process of 37 regeneration is given in Fig. 4.3. Gerbera jamesonii 4.3 General Fate of Pollen Grains Embryogenesis in plants is a unique process and it can be initiated from a wide range of cells other than the zygote. Androgenesis refers to the development of embryos from anther or pollen grains (Touraev et al. 1997). Androgenesis represents an Doubled haploid production from unpollinated important tool for research in plant genetics and breeding, since androgenic embryos can germinate into homozygous double haploid plants. Androgenic development ovary culture of cucumber can be completed in characteristic phases: acquisition of embryogenic potential, initiation of cell division, and pattern formation. In flowering plants, the male reproductive processes occur in the stamens. During normal process of microsporo- genesis, the diploid cells (pollen mother cell/microspore mother cell) undergo meiosis and produce haploid microspores. Normally, the microspores (n) divide mitotically and differentiate into 2-or 3-celled male gametophytes. The principle of androgenesis is to shift the fate of the microspores from gametophytic to sporophytic development (Fig. 4.4). Microspores are uninucleate (single nucleus) and contain large vacuole. Further these pollen grains can follow any one of the following developmental process: (1) it can undergo mitosis and produce haploid mass of the tissues; (2) microspores Unpollinated can undergo symmetric divisions ovary culture which structures Embryo-like after Shoot formation Complete plantlet transplanted plantlet Sorntip et al (2017) 38 Production of Double Haploid Plants DH production includes two major steps: haploid induction and chromosome doubling. Chemical (colchicine) treatments for chromosome doubling may occur: at either in vitro culture stage by using media containing the chemical to directly generate DH embryoids or callus. at a later stage on the regenerated haploid plantlets. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 39 13  10/23/24 Applications of Double Haploid Plants DH technology enables significant shortening of time during production of pure lines. Complete homozygosity of DH lines offers a higher phenotype to genotype correlation, thereby facilitating better estimation of quantitative trait loci (QTL) effects in marker trait association studies. Important tool for exploring the genetic diversity, for maintenance of genetic resources, and introducing novel variation to magnify the genetic base of elite germplasm. Haploid Inducer Mediated Genome Editing (IMGE) is a recent application that enables direct genomic modification of commercial inbred lines and eliminates several costly and time-consuming steps when incorporating genome-edited traits into elite cultivars. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 40 Procedure of Haploid-Inducer Mediated Genome Editing (IMGE) in Maize IMGE utilizes a maize haploid inducer line carrying a CRISPR/Cas9 cassette targeting for a desired agronomic trait to pollinate an elite maize inbred line to generate genome-edited haploids in the elite maize background. Homozygous pure DH lines with the desired trait improvement could be generated within two generations. bypassing the lengthy procedure of repeated crossing and backcrossing used in conventional breeding for integrating a desirable trait into elite commercial backgrounds. Wang et al. 2019 Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 41 Applications of Double Haploid Plants Advantage of double haploid technique is based on: Quick method to obtain 100% pure inbred lines. The resulting inbred lines are highly homozygous. Rapid development of DH based inbred lines is useful in development of resistance against Aspergillus flavus, aflatoxin, insect and disease, drought and heat tolerance. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 42 14  10/23/24 Chapter 3: Encapsulation Technology: An assessment of its role in in vitro conservation of medicinal and threatened plant species Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 43 Loss of golbal biodiversity Plant biodiversity loss: Global climate change. Severe biotic and abiotic stresses. Destruction of natural habitats. Overexploitation of many economically important plants: rapid human population growth. high demand of plant-based products. More medicinally important species (at an alarming rate) have become a cause of serious concern and these species require an urgent attention for conservation, management and their further commercial utilization. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 44 Encapsulation-based Synthetic seed Technology In situ conservation method is insufficient to meet the challenges of conserving these threatened plant species. Advances in plant tissue culture-based biotechnological tools and techniques have paved a new way for the rapid propagation, conservation and management of rare, endangered and other commercially important plant species. Micropropagation coupled with encapsulation-based synthetic seed technology has emerged as a promising approach for ex situ conservation and germplasm distribution of elite threatened plant species. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 45 15  10/23/24 Encapsulation-based Synthetic seed Technology Application of encapsulation technology in conserving threatened as well as medicinal plants particularly those used in herbal pharmaceutical industry has been extensively studied in recent years. Synthetic seed-based, slow growth culture and cryopreservation technique has now been widely used to conserve many threatened plant species for short to medium and long-term storage. Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology 46 Advantages of synthetic seeds over 106 conventional seed bank M. K. Rai et al. Poor Seed viability and Unknown reproduc!ve Transfer of diseased Erosion of gene!c germina!on in many biology of many wild raw material resources plants medicinal plant Problems associated with conven!onal conserva!on method i.e. seed bank Synthe!c seed Advantages of synthe!c seed in in vitro conserva!on Large scale Diseased free Gene!c stability of Short to medium and propaga!on and germplasm exchange plant resources a"er long term conserva!on Conserva!on and distribu!on storage throughout year Dr. Reda Gaafar Professor of Molecular Biology and Biotechnology Fig. 5.1 Schematic illustration of advantages of synthetic seeds over conventional seed bank 47 The need of synthetic seed production and its advantages in medicinal plant is presented in Fig. 5.1. 5.2 Encapsulation Technology: A Resourceful Biotechnological Tool for Propagation, Conservation, and Germplasm Distribution Encapsulation technology-based production of synthetic seed or artificial seed involves artificially encapsulation of somatic embryos or other vegetative tissues such as shoot tips, nodal segments, axillary buds, protocorms, etc. for plant propa- gation, conservation and germplasm exchange or distribution (Rai et al. 2009; Sharma et al. 2013). Since its discovery in 1980s (Kitto and Janick 1982; Redenbaugh et al. 1984), encapsulation technology has been successfully employed for propagation and conservation of a number of commercially important plant species including agricultural crops, forest trees, fruits, medicinal, ornamental and orchids, etc. (reviewed by Gray et al. 1991; Attree and Fowke 1993; Standardi and Piccioni 1998; Ara et al. 2000; Rai et al. 2009; Reddy et al. 2012; Sharma et al. 2013; Gantait et al. 2015; Faisal and Alatar 2019). Due to bipolar nature and its ability to forms root and shoot in one step, somatic embryo is a choice of explant for encapsulation and synthetic seed production in most of the plant species, however development of synthetic seed using somatic embryos is restricted only in those 48 16  10/23/24 Concept of Synthetic Seed Artificial (synthetic) seed was first time given by Toshio Murashige (1978) as “an encapsulated single somatic embryo” Artificial (synthetic) seed technology involves the production of tissue culture derived somatic embryos encased in a protective coating. Criteria in the Designing of Synthetic Seed Three basic properties of botanical seeds have to be fulfilled: it must contain a propagule which later grows up as a plantlet (like zygotic embryo in botanical seed). it should contain a nutrient medium which serves as storage food for plant propagule (like endosperm or cotyledons in botanical seed). plant propagule should be covered by a hard protective layer for mechanical protect