meristem,embryo,ovule,anther,emdryo rescue, somaclonal variations in c

Information about meristem,embryo,ovule,anther,emdryo rescue, somaclonal variations in c

Published on July 19, 2014

Author: nagreddy33



PowerPoint Presentation: WEL COME PowerPoint Presentation: Term Paper On Meristem culture, embryo, anther, ovule culture, embryo rescue technology, somaclonal variation, their role in improvement of horticulture crops in general and fruit crops in particular Submitted by: P. Nagireddy, Ph.D (Horti) RHD-13/03 College of Horticulture, Rajendranagar Submitted to: Dr. Hamidunnisa Begum, Principal Scientist (Horticulture), Agriculture Research Institute, Rajendranagar MERISTEM CULTURE: A meristem is the tissue in all plants consisting of undifferentiated cells (meristematic cells) and found in zones of the plant where growth can take place. The term “meristem” was first used by Karl Wilhelm von Nägeli (1817-1891). It is derived from the Greek word “ merizein ”, meaning to divide in recognition of its inherent function. Meristematic cells are analogous in function to stem cells in animals, are incompletely or not at all differentiated, and are capable of continued cellular division (youthful). The apical meristem, or growing tip, is a completely undifferentiated meristematic tissue found in the buds and growing tips of roots in plants. Its main function is to begin growth of new cells in young seedlings at the tips of roots and shoots (forming buds, among other things).size -0.25-0.30mm(length)and 0.1mm( dia ). MERISTEM CULTURE Apical Meristem: Apical Meristem PowerPoint Presentation: The Apical meristem can be shoot, root or of floral origin Shoot Meristem Culture - The first application of meristem culture was to obtain virus free plants of dahalias . In 1952, Morel and Martin isolated 100 µm long shoot meristems and cultured them to obtain virus free shoots. Since then the technique of meristem culture has been greatly refined and used for obtaining plants free from viruses, viroids , mycoplasma and even fungi and bacteria in a range of crops. In India, some valuable clones of potato, sugarcane, etc. have been freed from virus infections through meristem culture. Care must be taken to remove the apical meristem with as little surrounding tissue as possible to minimise the chances of virus particles being present in the explant. Meristem culture –an overview: Meristem culture –an overview Factors affecting meristem culture:: Factors affecting meristem culture: Size of explant (inversely proportional). Physiological condition of explant (should be taken from actively growing region). Season of culture (imp. for plants which display periodic growth). Culture medium (auxin & cytokinin are used,NAA most effective). Storage condition (light incubation). Methods of virus elemination: Thermotherapy- hot water/ hot air(temp.-30˚C to 40˚C) Cryotherapy-low temp.(5˚C) . Chemotherapy-(malachite green,thiouracil,acetylsalicyclic acid). Virizole,vidarbine,cyclohexamide,actinomycin -D-more effective. Other methods like somatic cell hybridization,somaclonal variation also used. Methods of virus elemination Production of virus free crysanthemums : Crysanthemum marifolium plants were rescued from CVB by using meristem culture aided with thermo & chemotherapy . Meristem tip (0.3-1.0) along with 2-3leaf primordia of virus infected plant ↓ Sterilization &establishment in MS media ↓ Maintained at a photoperiod of 16h,temp-20±2˚C,humidity-70-80% ↓ shoot development after 7-8 weeks ↓ subculturing in1/2 MS media +6gm Agar+IBA (0.05gm/l)for rooting Production of virus free crysanthemums PowerPoint Presentation: ↓ Incubation at 38°C,16h photoperiod in a thermotherapy chamber ↓ Transferred to potting mixture ↓ kept in hardening chamber(temp.-25±2°C,16h photoperiod,80% humidity ↓ virus indexing Addition of 5-Bromouracil , 2- thiouracil , Acyclovir to the rooting media was done at diff. conc. to test their activity against CVB. 2- thiouracil gave max. no. of virus free plants at 40 gdm 3 conc. Least effective was 5-Bromouracil (gave only 10%virus free plants) A freshly excised meristem tip from an axillary bud of the potato Solanum tuberosum. The two smallest emergent leaf primordia are present. Scale bar represents 50 μM. : A freshly excised meristem tip from an axillary bud of the potato Solanum tuberosum . The two smallest emergent leaf primordia are present. Scale bar represents 50 μ M . Exploitation of meristem culture: Meristem culture has been used in eradication of banana virus(BMV,BBTV) from infected banana plants and production of certified banana plant. Elimination of viruses(PPV,PNRSV)through themotherapy and meristem tip culture in nectarine. Production of virus free crysanthemums . Pathogens other than virus like fungi,bacteria,mycoplasmacould also be got rid off. Baker &Philips(1962)obtained carnation plants free from fusarium roseum f.cerialis . Tramier (1965)obtained gladiolus plants free from oxysporium f.gladioli . Exploitation of meristem culture Pros n cons of meristem culture: ADVANTAGES; Lack of vascular tissue. Meristem have high metabolic activity. High auxin conc. DISADVANTAGES; Isolation is difficult. Low survival rate & regeneration time for explants may be long(about 8 months for potato explant). Removal of explant causes a setback in the growth of mother plant. Pros n cons of meristem culture PowerPoint Presentation: Anther/Pollen culture Method to produce haploid plants Spontaneous occurrence in low frequency Induction by physical and/or chemical treatment Chromosome elimination following interspecific hybridization PowerPoint Presentation: Haploids are very valuable in plant breeding for several reasons Since they carry only one allele of each gene, mutations and recessive characteristics are expressed in the plant. Plants with lethal genes are eliminated from the gene pool. Can produce homozygous diploid or polyploid plants - valuable in breeding Shorten the time for inbreeding for production of superior hybrids genotypes. Value of Haploids in Breeding ANTHER CULTURE: ANTHER CULTURE Anther culture is a technique by which the developing anthers at a precise and critical stage are excised aseptically from unopened flower bud and are cultured on a nutrient medium where the microspores within the cultured anther develop into callus tissue or embryoids that give rise to haploid plantlets either though organogenesis or embryogenesis. Pollen or microspore culture is an in vitro technique by which the pollen grains preferably at the uninucleated stage ,are squeezed out aseptically from the intact anther and then cultured on nutrient medium where the microspores, without producing male gametes , develop into haploid embryoids or callus tissue that give rise to haploid plantlets by embryogenesis or organogenesis. POLLEN CULTURE ANDROGENESIS: ANDROGENESIS Androgenesis is the in vitro development of haploid plants originating from totipotent pollen grains through a series of cell division and differentiation. It is of two types. ANDROGENESIS: ANDROGENESIS Direct androgeneis:- The microspores behaves like a zygote and undergoes chance to form enbryoid which ultimately give rise to a plantlet. Indirect Androgenesis :- The microspores divide repeatedly to form a callus tissue which differentiates into haploid plantlets. PowerPoint Presentation: The production of haploid plants exploiting the totipotency of microspore . In this process the normal development and function of the pollen cell to become a male gamete is stopped and is diverted forcely to a new metabolic pathway for vegetative cell division . PRINCIPLE OF ANTHER AND POLLEN CULTURE FACTOR INFLUENCING ANTHER CULTURE: Genotype of donor plants:- The genotype of the donor plant plays a significant role in determining the frequency of pollen production. Example :- Horedum of each genotype differs with respect to androgenic response in anther culture. Anther wall factor:- The anther wall provide the nourishment in the development of isolated pollen of a number of species. There are reports that glutamine alone or in combination with serine and myoinositol could replace the anther wall factor for isolated cultures. FACTOR INFLUENCING ANTHER CULTURE PowerPoint Presentation: CULTURE MEDIUM:- The anther culture medium requirements vary with genotype and probably the age of the anther as well as condition under which donor plants are grown. In corporation of activated charcol into the medium has stimulated the induction of androgenesis . The iron in the medium plays a very important role for the induction of haploids . Potato extracts ,coconut milk and growth regulators like auxin and cytokininare used for anther and pollen culture. FACTOR INFLUENCING ANTHER CULTURE PowerPoint Presentation: Stage of microspores:- In most of the cases anther are most productive when cultured at the uninucleate microspore stage. Example ,barely, wheat , rice etc. Anther of some species give the best response if pollen is cultured at first mitosis or later stage Example:-Datura ,tobacco. FACTOR INFLUENCING ANTHER CULTURE PowerPoint Presentation: Effect of temperature :- Temperature enhance the induction frequency of microspore androgensis . The low temperature treatment to anther or flower bud enhance the haploid formation. The low temperature effects the number of factors such as dissolution of microtubules lowering of absicisic acid maintenance of higher ratio of viable pollen capable of embryognesis . FACTOR INFLUENCING ANTHER CULTURE PowerPoint Presentation: PHYSIOLOGICAL STATUS OF DONAR PLANT:- Physiological status of donor plant such as water stress nitrogen requirement and age of donor plant highly effect the pollen embryogenesis. Plants starved of nitrogen may give more responsive anthers compared to those that are well fed with nitrogenous fertilizers. FACTOR INFLUENCING ANTHER CULTURE METHOD OF ANTER AND POLLEN CULTURE: METHOD OF ANTER AND POLLEN CULTURE PowerPoint Presentation: During anther culture there is always the possibility that somatic cells of the anther that are diploid will also respond to the culture condition and so produce unwanted diploid calli or plantlets. Sometimes the development of microspores inside the anther may be interrupted due to growth inhibiting substances leaking out of the anther wall in contact with nutrient medium. ADVANTAGE OF POLLEN CULTURE OVER ANTHER CULTURE PowerPoint Presentation: (1)Utility of anther and pollen culture for basic research:- (a) cytogenetic studies. (b)Study of genetic recombination in higher plants. (c) Study of mode of differentiation from single cell to hole organism. (d) Study of factor controlling pollen embryogenesis of higher plants. (e) Formation of double haploid that are homozygous and fertile. IMPORTANCE OF POLLEN AND ANTHER CULTURE PowerPoint Presentation: Anther and pollen culture are use for mutation study. Example :- Nitrate reductae mutants are reported in Nicotiana tabacum. Anther and pollen culture use for plant breeding and crop improvement. Anther culture are use to obtain the alkaloid Example :- Homozygous recombination Hyoscyamus niger having higher alkaloid content is obtain by anther culture. Haploid are use in molecular biology and genetic engineering. Example:- Haploid tissue of Arbidopsis and lycopersicon have been used for the transfer and expression of three genes from Escherchia coli.... IMPORTANCE OF POLLEN AND ANTHER CULTURE PowerPoint Presentation: Embryo culture, sometimes called embryo rescue, is an in vitro technique that has been used to save the hybrid products of fertilization. No offspring does not mean no fertilization took place. Many times formed embryo dies at an early stage due to known or unknown causes. Known as post-fertilization barriers. EMBRYO RESCUE PowerPoint Presentation: The term “embryo rescue” refers to a number of in vitro techniques whose purpose is to promote the development of an inherently weak, immature or hybrid embryos into a viable plant. PowerPoint Presentation: Embryo abortion occurs due to the endosperm fails to develop properly (Hub and wang, 1986). In interspecific crosses, intergeneric crosses, and crosses between diploids and tetraploids the endosperm often develops poorly or not at all. Need of embryo rescue PowerPoint Presentation: General technique of embryo rescue Mostly embryos are located in the sterile environment of the ovule and surface sterilization of embryos is not necessary. Direct disinfection of embryos is needed if seed coats are cracked or if endophytic pathogens exist ex. Corn ( zea mays L.), And dogwood ( cornus spp. L.) Seeds. Small embryos require the use of micro dissecting tools and a dissecting microscope to excise without injury. Embryos are easily damaged when the seed coat is cut; desiccation avoided.( Rang an, 1984). If liquid endosperm surrounds the embryo change in the pressure injure the embryonic tissue. (Hu and wang, 1986). PowerPoint Presentation: OVULE CULTURE Very young or small-seeded species . PowerPoint Presentation: The ovary is surface sterilized and the ovules removed and placed into culture. One technique, ovule perforation , requires making small holes in each ovule just prior to its placement on the culture medium. These perforations, which should be made with care not to damage embryos. Method PowerPoint Presentation: Ovule support systems 1. The filter paper support system: Culturing ovules on top of filter paper placed over liquid medium . 2. The vermiculite support technique: Placing ovules side down into a sterile vermiculite/liquid media mixture (vermiculite support). OVARY CULTURE : OVARY CULTURE Factors effecting embryo culture: Factors effecting embryo culture Mineral salts : Mineral salts Salts Species K + , Ca 2+ (high), NH 4 + (low) Casella monnier medium 1978) NH 4 + Barley, Datura stramonium and D. Tatula NO 3 - Jute Vitamins Not always essential. May even inhibit normal morphogenesis . Culture medium: Culture medium Media Scientist Murashige and Skoog (MS) media (Murashige and Skoog, 1962) Gamborg’s B-5 media (Gamborg et al ., 1968) White’s media White’s (1934) ph os phate with Glutamine, A lanine, and five other amino acids. (Cameron-Mills and Duffus, 1977) Energy source. Maintaining suitable osmotic potential. Mature embryos 2% to 3% sucrose, immature embryos grow better at 8% to 12%. Sucrose PowerPoint Presentation: Plant Growth Regulators Effect ABA with NH 4 + (ammonical nitrate) Prevents precocious germination of barley immature embryos. Gibberellins Barley immature embryos. Low concentrations of Auxins Somatic embryo induction stimulated. Exogenous Auxins Not effect plant embryo growth in vitro (Norstog, 1979). Cytokinins Not sole hormone butpromote growth with some auxins (Veen, 1963) Medium: Plant Growth Regulators PowerPoint Presentation: Plant sp. Optimal pH Ref. Capsella 5.4 – 7.5 Rijven (1952) Datura tatula (early heart-shaped embryos) 5.0-7.5 Matsubara (1962) Rice (8 day old embryos) 5 and 9 Sapre (1963) Barley (immature embryos) 4.9 Norstog and Smith (1963) PH of Medium Darkness for the first 1 to 2 weeks of culture generally, then transferred to light to allow chlorophyll formation. A high range 25 to 30 0 c is used (Narayanaswamy and Norstog, 1964). Lilium , require a lower temperature, i.e., 17 0 C, and others require a cold treatment of 4 0 C to break dormancy (Pierik, 1987). Temperature and Light PowerPoint Presentation: Amino Acid Use Scientist Glutamine Mostly used for cultured embryo growth. Monnier, 1978. Asparagine Enhance embryo growth. Hannig, 1904 Casein hydrolysate Widely used to stimulate growth. Burkholder, 1948 Proline, serine, and glutamine Can replace cassin hydrosate. ================ Usually it begins in the late heart-shaped embryo stage (raghavan, 1976). Less (2–3%) sucrose required in media. For interspecific hybrids, it may be useful to develop media that can nurture embryos of one or both parental species. The growth-promoting factor in the coconut milk was referred to as “embryo factor.” B) The Autotrophic Phase PowerPoint Presentation: Developmental stage Length of embryo ( m ) Nutritional requirements Early globular 20-60 Unknown for embryos < 40 m Late globular 61-80 Micronutrient salts + vitamins + 2% sucrose + hormones (IAA) Heart-shaped 81-450 Micronutrient salts + vitamins + 2% sucrose Torpedo-shaped 45-700 Macronutrient salts + vitamins + 2% sucrose Walking-stick-shaped and mature embryos 700 & larger Macronutrient salts + 2% sucrose Utilization of embryo rescue 1. Obtaining rare hybrids : Utilization of embryo rescue 1. Obtaining rare hybrids Cross between 2 species. Scientist Lilium henryi x L. regale Skirm (1942) L. speciosum-album x L. auratum (embryo-endosperm incompatibility) Emsweller and Uhring (1962) L. esculentum cv. VENT x L. peruvianum cv LA 1283-4 (embryo – callus culture approach) Thomas and Pratt (1981) L. esculentum x L. chilense (embryo – callus culture approach) Scott and Stevens L. esculentum x Solanum lycopersicoides (embryo – callus culture approach) Scott and Stevens Utilization of embryo rescue Obtaining rare hybrids : Utilization of embryo rescue Obtaining rare hybrids Cross between 2 species. Product Scientist O. sativa x O. australiensis, O. officinalis, O. brachyantha 38-77%, 46-80%, 31-80% Jena and Khush (1984) Tropical japonicas x O. officinalis, O. australiensis, O. grandiglumis, O. punctata Hybrid plants Abdullah and Somantri Cultivated rice x 8 wild rice sp. F 1 s and BC 1 plants Brar, Elloran and Khush V. mungo x V. radiata Hybrid plants Gosal and Bajaj (1983), Verma and Singh (1986) Utilization of embryo rescue : Utilization of embryo rescue PowerPoint Presentation: Utilization of embryo rescue 3).Overcoming seed dormancy Utilization of embryo rescue : Seeds of horticultural crops have long Dormancy periods, which may be reduced through embryo culture Iris 2-3 years 1 year Sunflower . 120-150 days 60-75 days. 4).Shortening the breeding cycle Utilization of embryo rescue PowerPoint Presentation: Utilization of embryo rescue 6. Rapid seed viability test Germination of excised embryos of a batch of seeds better than staining methods. Tetrazolium stain. PowerPoint Presentation: 7) . Transfer of disease resistance Utilization of embryo rescue Agrawal (2006) introduced downy mildew resistance in Thompson Seedless and Flame Seedless (grape cv.s ) through embryo rescue technique. PowerPoint Presentation: Arka Manik x Arka Manik (4x) (2x) Micro-cuttings Seedless watermelon (3x) Embryo rescue Micro-cuttings Clonal propagation (10 million plants a year) Utilization of embryo rescue 8). Development of Seedless watermelon PowerPoint Presentation: Sterile seeds not germinate under appropriate conditions e.g. peach, cherry, plum. Sterility may be due to incomplete embryo development, resulting embryo abortion. In some crosses, the transport of water and nutrients to the immature embryo is cut off too soon resulting in abortion, of the embryo. Ex. : Macapuno coconuts. Utilization of embryo rescue 9) Embryo rescue in early ripening stone fruits PowerPoint Presentation: 10.) Clonal Micropropagation Complete plantlets in vitro formed in long-leaved pine ( Pinus palustris ) through embryo culture (Sommer et al. 1975). Followed by plantlet formation and their clonal micropropagation from embryos of P. elliottii, P. radiata, P. regida, P. monticola, P. taeda, P. virginiana and P. sabiniana. In vitro rescue of immature avocado (Persea americana Mill.) embryos: In vitro rescue of immature avocado ( Persea americana Mill .) embryos Romero et al (2006) Effect of embryo developmental stage on germination on solid M1 medium was not significantly affected by embryo developmental stage.: Effect of embryo developmental stage on germination on solid M1 medium was not significantly affected by embryo developmental stage. Embryo size (mm ) Germination (%) 4.0 5.48 10.5 10.17 16.5 11.76 20.5 5.26 25.5 0 30.5 0 40.0 0 4.0 5.48 10.5 10.17 Germinated embryos of smaller sizes (4–10.5 mm) developed only shoots while complete germination (development of shoot and root) was only observed in 20.5 mm long embryos. Romero et al (2006) Basic Features of Somaclonal Variations: Genetic variations in plants that have been produced by plant tissue culture and can be detected as genetic or phenotypic traits. Somaclonal Variation Basic Features of Somaclonal Variations Variations for Karyotype, isozyme characteristics and morphology in somaclones may also observed. Calliclone (clones of callus), mericlone (clones of meristem) and protoclone (clones of Protoplast) were produced. Generally heritable mutation and persist in plant population even after plantation into the field Mechanism of Somaclonal Variations: Mechanism of Somaclonal Variations Genetic (Heritable Variations) Pre-existing variations in the somatic cells of explant Caused by mutations and other DNA changes Occur at high frequency Epigenetic (Non-heritable Variations) Variations generated during tissue culture Caused by temporary phenotypic changes Occur at low frequency PowerPoint Presentation: Callus Tissue Organogenesis Regenerated plants Hardening and Selfing Somaclonal Variants Steps involved in induction and selection of Somaclonal Variations PowerPoint Presentation: Causes of Somaclonal Variations Physiological Cause Genetic Cause Biochemical Cause Physiological Cause: Physiological Cause Exposure of culture to plant growth regulators. Culture conditions PowerPoint Presentation: Genetic Cause Change in chromosome number Euploidy : Changes chromosome Sets Aneuploidy: Changes in parts of chromosome Sets Polyploidy: Organisms with more than two chromosome sets Monoploidy : Organism with one chromasomes set Change in chromosome structure Deletion Inversion Duplication Translocation PowerPoint Presentation: 3. Gene Mutation Tansition Transversion Insertion Deletion 4. Plasmagene Mutation 5. Transposable element activation Genetic Cause PowerPoint Presentation: 6. DNA sequence Change in DNA Detection of altered fragment size by using Restriction enzyme Change in Protein Loss or gain in protein band Alteration in level of specific protein Methylation of DNA Methylation inactivates transcription process. Genetic Cause PowerPoint Presentation: Lack of photosynthetic ability due to alteration in carbon metabolism Biosynthesis of starch via carotenoid pathway Nitrogen metabolism Antibiotic resistance. Biochemical Cause PowerPoint Presentation: Analysis of morphological characters Qualitative characters: Plant height, maturity date, flowering date and leaf size Quantitative characters: yield of flower, seeds and wax contents in different plant parts Variant detection by cytological Studies Staining of meristematic tissues like root tip, leaf tip with feulgen and acetocarmine provide the number and morphology of chromosomes. Variant detection by DNA contents Cytophotometer detection of feulgen stained nuclei can be used to measure the DNA contents Detection and Isolation of Somaclonal Variants PowerPoint Presentation: 4. Variant detection by gel electrophoresis Change in concentration of enzymes, proteins and hemical products like pigments, alkaloids and amino acids can be detected by their electrophoretic pattern 5. Detection of disease resistance variant Pathogen or toxin responsible for disease resistance can be used as selection agent during culture. 6. Detection of herbicide resistance variant Plantlets generated by the addition of herbicide to the cell culture system can be used as herbicide resistance plant. Detection and Isolation of Somaclonal Variants PowerPoint Presentation: 7. Detection of environmental stress tolerant variant Selection of high salt tolerant cell lines in tobacco Selection of water-logging and drought resistance cell lines in tomato Selection of temperature stress tolerant in cell lines in pear. Selection of mineral toxicities tolerant in sorghum plant (mainly for aluminium toxicity) Detection and Isolation of Somaclonal Variants PowerPoint Presentation: Help in crop improvement Creation of additional genetic varitions Increased and improved production of secondary metabolites Selection of plants resistant to various toxins, herbicides, high salt concentration and mineral toxicity Suitable for breeding of tree species Advantages of Somaclonal Variations PowerPoint Presentation: Improvement of existing clonal cultures sugarcane – selections for higher yield & disease resistance potatoes – yield & disease resistance improved geraniums (esp. scented varieties) woody ornamentals (e.g., Paulownia – selection for leaf variegation Applications to crop improvement PowerPoint Presentation: A serious disadvantage occurs in operations which require clonal uniformity, as in the horticulture and forestry industries where tissue culture is employed for rapid propagation of elite genotypes Sometime leads to undesirable results Selected variants are random and genetically unstable Require extensive and extended field trials Not suitable for complex agronomic traits like yield, quality etc. May develop variants with pleiotropic effects which are not true. Disadvantages of Somaclonal Variations Some examples of somaclonal variants possessing improved traits in crops : Some examples of somaclonal variants possessing improved traits in crops Potato Resistance to Fusarium oxysporium Resistance to Phytophtora infestans Sugarcane Resistance to eyespot , fiji desease and downy mildew Sweet potato Darker and stable skin colour Wheat Resistance to Helminthosporium maydis , tolerance to heat stress

Related presentations

Other presentations created by nagreddy33

nutrient uptake
15. 07. 2014

nutrient uptake