Domain: Plant Tissue Culture & Vegetative Propagation

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Domain Track: Plant Tissue Culture and Vegetative Propagation

Domain Track: Plant Tissue Culture and Vegetative Propagation

Teacher

Dr. Pushpalatha Ganesh

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Track Total Credit (T-P-P): 9-9-11

Domain Track Objectives:

To impart and encourage students to get inculcate and accustomed to the industry-academia propagation of horticulture plants at large scale.

Domain Track Learning Outcome:

  • Hands-on knowledge acquisition to produce large scale GM-free commercially important horticulture plants.

Course Division:

Track Courses (3-6-0)

  1. Technologies of Plant Tissue Culture (1-2-0).
  2. Biotransformation using plant cells, organ cultures and enzyme systems (1-2-0).
  3. Vegetative Propagation – Methods and Techniques (1-2-0)

 

Practice Courses (0-9-0)

  1. Commercial Plant Tissue Culture (0-3-0).
  2. Applied Plant Tissue Culture (0-3-0).
  3. Techniques for conservation of Plant Genetic Resources (0-3-0)

 

AELP Project (0-0-11)

  1. Commercial production of horticultural crops like Banana.
  2. In vitro micro-propagation of banana and hardening.
  3. Protocol standardization for in vitro micro-propagation of horticulture crops like Bamboo, Gerbera, Orchids, etc.
  4. Propagation of ornamental crops through cuttings.
  5. Propagation of annual flower crops through seeds.
  6. Propagation of fruit crops through layering.
  7. Plant genomic DNA isolation, PCR amplification and its assessment.
  8. Development of Doubled Haploid and F2 generation mapping populations in maize for the identification of QTLs for seed yield components in maize.

Course Outcome (CO) - Program Outcome (PO) Mapping:

COs Course Outcome Mapping COs with POs
CO1 Imparting molecular knowledge to approach and analyze the in-vitro micro-propagated plants through virus-indexing and genetic fidelity. PO1, PO2, PO5
CO2 Entrepreneurship based hands-on knowledge acquisition for students to produce large scale commercially important GM-free horticulture plants. PO1, PO2, PO3, PO4, PO6

PO1: Knowledge Enrichment: Hands-on experience of molecular approach on plants, basic concepts, disciplinary approaches to maintain hygienic conditions and propagation of plants at large-scale.

PO2: Professional Development: Learning the principles and set the everlasting-systematic professionalism.

PO3: Entrepreneurship Skills: Managerial (financial, administration with technical expertise) aspects gets inculcated in students during the process of production of the plants in the lab and reaching the same to farmer’s in the land.

PO4: Extension & Team Work: Practical field experience for students to communicate directly with farmers with live demonstration.

PO5: Educational Leadership: Networking academia-industry to address the emerging climate-based challenges in agriculture.

PO6: Integrity & Uniqueness: Commitment, systematic and timely execution of the tasks, application oriented research and production.

1. Technologies of Plant Tissue Culture (1-2-0)

Introduction to Laboratory organization: Tools and techniques, methods of sterilization. Laboratory contaminants- it’s control and measures.

Media and Culture Preparation: Role of Micro and macro nutrients, Vitamins and carbon source in tissue culture, Media preparation- pH, Temperature, Solidifying agents, Slant Preparations etc. Maintenance of cultures, Environmental Conditions, explants characteristics.

Culture techniques: Explants selection, sterilization and inoculation; Various media preparations; MS, B5, SH PC L-2; Callus and cell suspension culture.

Initiation of Cultures: Induction and growth parameters; Culture initiation, Callus culture., Micro-propagation through various explants (Leaf, Stem, Axillary bud, Tuber, Corms and Bulbills).

In-vitro Fertilization: Role of Ovary and ovule in in-vitro Fertilization in production of agricultural and horticultural crops. Techniques and significance of Androgenesis and Gynogenesis (ovary, ovule, egg, synergids culture).

Practice: Tools and techniques, methods of sterilization. Laboratory contaminants, Media preparation- pH, Temperature, Solidifying agents, Slant Preparations, Explants selection, sterilization and inoculation.

2. Biotransformation using plant cells, organ cultures and enzyme systems (1-2-0)


Organ Culture:
Anther, Pollen, Embryo and Endosperm culture, Hairy Root Culture and their applications. Organogenesis and Somatic embryogenesis- Techniques and Applications.

Protoplast Culture: Protoplast-Isolation regeneration and Viability test, Somatic hybridization and methods of protoplast fusion- chemical, Viral, electrofusion. Practical application of somatic hybridization and cybridization.

Crop Improvement: Somaclonal variation, its genetic basis and application in crop improvement. Cell/callus line selection for resistance to herbicide, stress and diseases. Role of tissue culture in rapid clonal propagation, production of pathogen - free plants and “synthetic seeds”.

Transgenics: Transgenic plants for crop improvement (dicot and Monocot including Maize, Rice, Wheat, Cotton, Brinjal etc. Resistance to herbicide, insecticide, virus and other diseases, Flavr savr tomato, barnase and barstar, etc). Transgenic plants for molecular farming.

Plant transformation: Methods of gene transfer in plants. Agrobacterium and CaMV mediated gene transfer; direct gene transfer using PEG, micro injection, electroporation, microprojectile (biolistics) method, liposome mediated DNA delivery; Transposons as vectors.

Practice: Anther, Pollen, Embryo and Endosperm culture, Hairy Root Culture and their applications. Organogenesis and Somatic embryogenesis- Techniques, Protoplast-Isolation regeneration and Viability test, methods of protoplast fusion- chemical, Viral, electrofusion. Synthetic seed preparation, callus line propagation and selection for abiotic/biotic stress tolerance, Agrobacterium gene transfer.

3. Vegetative Propagation – Methods and Techniques (1-2-0)

 

Vegetative Propagation I: Introduction, Reasons to propagate by cuttings, Types -softwood, hardwood, semi hardwood, herbaceous, Stem Cuttings, Tip, heel, nodal, basal Leaf and Leaf-bud cuttings, Cane cuttings, Root Cuttings, Bulb Cuttings, Hormone Treatments for Cutting Propagation, Other Cutting Treatments; basal wounding, anti-transpirants, fungicides, disinfectants, mycorrhiza, etc. Artificial Light for Propagation, Cutting Propagation Efficiency, Rockwool Propagation.

Vegetative Propagation II: Care of stock plants, Layering, Division, Terminology and Managing Watering.

Vegetative Propagation III: Terminology, Budding and grafting, Reasons for Grafting, how a Graft forms, Grafting Techniques; Types of Grafts, What Plant to Graft on What Grafting Materials, Root Grafting, Bench Grafting, Soft Tissue Grafting.

Propagation Structures and Materials: Growing in a Greenhouse, Growing Structures: Types of Greenhouses, Cold Frames, Shadehouses, Propagating equipment -Heaters, Bottom Heat, Misting, Light Control, Benches etc., Managing a Greenhouse.

Practice: Preparation of different types of cuttings for ornamental crops and vegetable crops i.e., Croton, Acalypha, Araelia, Diffenbachia, Coleus, Sanseveria, Chrysanthemum, Tuberose Ivy gourd, Pointed gourd, water spinach, Drum stick, Elephant foot yam, Colocasia and curry leaf.

Practice of different types of grafting and budding methods for fruit and ornamental crops i.e., Mango, Rose, Sapota, Citrus Spp. Practice of different types of layering for fruit and ornamental crops i.e., Bougainvilllea, Guava, Litchi, Pomegranate and lime.

Practice Courses

  1. Commercial Plant Tissue Culture (0-3-0)

Micropropagation and its Applications; Types, Stages, Establishment of propagated plants, micropropagation for large scale multiplication of crop plants, forest trees, medicinal plants and ornamentals. Germplasm preservation- Definition, Importance and Methods, In-situ and Ex-situ conservation, Centers of germplasm preservation in India. Hardening: Hardening stages, Role of Polyhouse, Net House, Compost, Chemical fertilizer, Cocopit, Soil in hardening. Floriculture – commercial floriculture – Production of cut flowers and home floriculture. Disease and pest control in gardening- Fungicides and pesticides. Plant growing problems and their control, cold house storage. Commercial Horticulture: Propagation of Horticultural crops by tissue culture techniques such as Banana, Sugarcane, Papaya, Mango and some Medicinal and Aromatic plants.

 

  1. Applied Plant Tissue Culture (0-3-0)

Preparation of various Tissue culture media (MS, B5, Gamber’s etc.), Effect of Growth Hormones on organogenesis: a) Shoot differentiation, b) Callus differentiation, Root differentiation. Single cell culture. Shoot tip, auxillary bud, Meristem culture. Propagation of horticultural. Propagation of Medicinal plants. Propagation of Aromatic plants. Hardening methods and their utility. Agrobacterium mediated gene transfer. Protoplast fusion by PEG. Preparation of artificial seeds. Liposome mediated gene transfer. Effect of Auxins and Gibberllins on seed germination. Studies on enzyme amylase. Effect of temperature and alcohol on permeability of cell membrane. Test for starch, Glucose, Tannins and Alkaloids. Isolation of DNA and RNA from Plants. Amplification of plant DNA by using random primer. Determination of specific activity of the enzyme. Separation of DNA by agarose gel electrophoresis.

 

  1. Techniques for conservation of Plant Genetic Resources (0-3-0)

Plant genetic resources- Different kinds of PGR basic, derived and molecular, core collections, principles of germplasm characterization, DNA fingerprinting and plant barcodes, germplasm evaluation, maintenance and regeneration, In-situ and ex-situ methods of conservation, cryopreservation of genetic materials, gene banks and cryobanks.

 

Session Plan: Plant tissue culture and vegetative propagation

  1. Technologies of Plant Tissue Culture (1-2-0)

Session 1.1

Theory:

Basics of Plant tissue culture and tools and techniques involved.

https://www.youtube.com/watch?v=0lzOjd9Izn8

Practice:

Demonstration of the usage of tools and techniques in Plant tissue culture.

https://www.youtube.com/watch?v=KjhriNbNRn0

Session 1.2

Theory:

Sterilization and its importance in tissue culture. Control and measure of contamination.

https://www.youtube.com/watch?v=Bh-ytzY5uVY

Practice:

Demonstration of different kinds of sterilization techniques.

https://www.youtube.com/watch?v=KCwzw-i0Ya0

Session 1.3

Theory:

Composition of plant growth media, micro and macro-nutrients, vitamins, and carbon sources.

https://www.youtube.com/watch?v=_inV2jzu1MQ

Practice:

Demonstration of different kinds of plant growth media preaparation.

https://www.youtube.com/watch?v=rhrC_Lrqgac

Session 1.4

Theory:

Role of temperature, pH, and solidifying agents in growth media.

Practice:

The sterilizing and incubation technique of media.

https://www.youtube.com/watch?v=7vXsl0ufcKU

Session 1.5

Theory:

Preparation of various media; MS, B5, SH PC L-2. Explant selection and sterilization.

https://www.youtube.com/watch?v=eMv_PMNPYMc

https://www.youtube.com/watch?v=lqLu8W-hik0&t=79s

Practice:

Media preparation following standardize protocols.

https://www.youtube.com/watch?v=ArneRUolgeA

Session 1.6

Theory:

Explant selection, callus culture, cell suspension culture

https://www.youtube.com/watch?v=6IGt7aXRmsw&t=108s

Practice:

Demonstrating explant inoculation techniques.

https://www.youtube.com/watch?v=C0OrG8yvEpA

Session 1.7

Theory:

Culture initiation under various growth parameters

https://www.youtube.com/watch?v=AlbnKShDnBA

Practice:

Maintenance of incubated cultures in the growth period.

https://www.youtube.com/watch?v=a3Sg8hsRYu4

Session 1.8

Theory:

Micro-propagation using various plant parts such as stem, leaf, tubers, bulbils etc.

https://www.youtube.com/watch?v=IXM7UmvLKQ4

Practice:

Inoculation of different plant parts as explant.

https://www.youtube.com/watch?v=mnmDkvNAcfM

Session 1.9

Theory:

In-vitro fertilization in plants.

https://www.youtube.com/watch?v=9jemh1QkEcE

Practice:

Demonstration of in-vitro fertilization in different agricultural and horticultural crops.

https://www.youtube.com/watch?v=9jemh1QkEcE&t=39s

Session 1.10

Theory:

Androgenesis and gynogenesis

https://www.youtube.com/watch?v=phCeuu-2IgE

Practice:

Techniques for ovary, ovule, synergid, anther cultures.

https://www.youtube.com/watch?v=GTiNsjnty1o

  1. Biotransformation using plant cells, organ cultures and enzyme systems (1-2-0)

Session 2.1

Theory:

Organ culture and its significance.

https://youtu.be/FNEeX3TJhZI

Practice:

Inoculation of various organs as explant for culture.

https://youtu.be/C0OrG8yvEpA

Session 2.2

Theory:

Somatic embryogenesis and its application.

https://youtu.be/cNFFqa3I4Hg

Practice:

Somatic embryo generation

https://youtu.be/C9gfNY6F3SQ

Session 2.3

Theory:

Applications of endosperm culture, hairy -root culture.

https://youtu.be/ifqgVPVqXZs

Practice:

Comparison of different cultures and their significance in commercial scale production. Demonstration on Endosperm culture.

https://youtu.be/fZHwO92Rv7U

Session 2.4

Theory:

Illustration on Protoplast culture

https://youtu.be/JG2kP7rrVwg

Practice:

Demonstration on Isolation of protoplast and viability test

https://youtu.be/vQzFoO3Pebw

Session 2.5

Theory:

Illustration on Somatic hybridization methodology and protoplast fusion

https://youtu.be/eYoqr43Ka5Q

Practice:

Demonstration of chemical, viral, electrofusion methods of protoplast fusion and somatic hybridization

https://youtu.be/utlKKvqxbWw

Session 2.6

Theory:

Illustration on soma clonal variation, its genetic basis and importance in crop improvement

https://youtu.be/yt79ji3Qcx4

Practice:

Demonstration on Soma clonal variation creation in plants and practical crop improvement examples

https://youtu.be/1wsyYkwRfck

Session 2.7

Theory:

Illustration on Cell/callus line selection for resistance to herbicide, stress and diseases.

https://youtu.be/x2b0Qks6Z6U

Practice:

Practical explanation of callus formation, selection of callus, and practical applications in field

https://youtu.be/j17wdeIfMYs

Session 2.8

Theory:

Illustration on Rapid clonal propagation, production of pathogen- free plants and illustration on synthetic seeds

https://youtu.be/nhvPUQPUvR4

Practice:

Demonstration on clonal propagation and practical breeding plot visit for synthetic seed production

https://youtu.be/LPDX7M52ft4

Session 2.9

Theory:

Introduction to transgenics, Importance of transgenic plants in crop improvement in quality, crop improvement through transgenics in rice, maize, wheat, cotton. Methods of gene transfer in plants. Agrobacterium and CaMV mediated gene transfer

https://youtu.be/riQz6U-YUm0

Practice:

Demonstration of transgenic protocols in a laboratory, tools used in transgenics, (basic to advanced), safety precautions in handling the tools

https://youtu.be/z3m-u-6ZF1g

Session 2.10

Theory:

Concept of plants acquiring resistance, to herbicides, diseases, virus.

https://youtu.be/fxzrDA-8GT0

Practice:

Agrobacterium gene transfer standardized protocol and practical demonstration.

https://youtu.be/f1_ctQSUEaE

 

  1. Vegetative Propagation – Methods and Techniques (1-2-0)

Session 3.1

Theory:

Introduction to propagation and its importance in commercial horticultural production

https://youtu.be/JNPo1IfGdW0

Practice:

Exposure to green house, visit to nearest nurseries

https://youtu.be/GNezSZNfZpI

Session 3.2

Theory:

Types of stem cuttings (Tip, heel, nodal, basal Leaf and Leaf-bud cuttings, Cane cuttings, Root Cuttings, Bulb Cuttings.

https://youtu.be/RUVE9Vpncis

Practice:

Preparation of different types of cuttings for ornamental crops and vegetable crops i.e., Croton, Acalypha, Araelia, Diffenbachia, Coleus, Sanseveria, Chrysanthemum, Tuberose Ivy gourd, Pointed gourd, water spinach, Drum stick, Elephant foot yam, Colocasia and curry leaf.

https://youtu.be/csqoteE430c

https://youtu.be/CboErsaUAXk

Session 3.3

Theory:

Hormone Treatments for Cutting Propagation, dosage and standard procedures

https://youtu.be/Te0BBpb8phA

Practice: Demonstration on hormone applications

https://youtu.be/TTLgTIipmA8

Session 3.4

Theory:

Artificial Light for PropagationCutting Propagation Efficiency, Rockwool Propagation.

https://youtu.be/Su326qUNtlo

 Practice: Demonstration on propagation and procedures for making cuttings

https://youtu.be/WHiv1OvXGcI

 Session 3.5

Theory:

Illustration on Cutting Treatments; basal wounding, anti-transpirants, fungicides, disinfectants, mycorrhiza

https://youtu.be/LnVIENLKO60

Practice:

Demonstration on cutting treatments, basal wounding, application of anti transpirants, fungicides, disinfectants.

https://youtu.be/LnVIENLKO60

Session 3.6

Theory:

Care of stock plants, Layering, Division, Terminology and Managing Watering.

Practice:

Demonstration on layering and water management,

Practice of different types of layering for fruit and ornamental crops i.e., Bougainvilllea, Guava, Litchi, Pomegranate and lime.

https://youtu.be/RHJZazLMvDo

Session 3.7

Theory:

Terminology, Budding and grafting, Reasons for Grafting, how a Graft forms

https://youtu.be/Gt587ABrrl4

Practice:

Practical application on grafting, application of grafting in commercial scale production with live demonstrations in nurseries

https://youtu.be/2UUtwUhI0RM

Session 3.8

Theory:

Grafting Techniques; Types of Grafts, What Plant to Graft on What Grafting Materials, Root Grafting, Bench Grafting, Soft Tissue Grafting.

https://youtu.be/tFWvZzMv2Io

Practical:

Grafting in laboratory premises and aquatinting with the technique

https://youtu.be/tFWvZzMv2Io

Session 3.9

Theory:

Introduction to greenhouses types and uses of different green houses

https://youtu.be/JdL8PHOHGec

Practical:

Growing in a Greenhouse, Growing Structures: Types of Greenhouses, Cold Frames, Shadehouses, Propagating equipment -Heaters, Bottom Heat, Misting, Light Control, Benches

https://youtu.be/JdL8PHOHGec

Session 3.10

Theory:

Practical advantages of vegetative propagation, its use in commercial scale production and opportunities as entrepreneur

https://youtu.be/_STJfubKx28

Practical:

Visit to best available commercial nurseries and understanding the outcomes of the course

Practice Courses

  1. Commercial Plant Tissue Culture (0-3-0)

Practice 1

Micropropagation in plants

https://www.youtube.com/watch?v=IXM7UmvLKQ4&t=2s

Practice 2

Germplasm preservation techniques

https://www.youtube.com/watch?v=BCPtquhW0_k

Practice 3

Hardening of tissue cultured plants; Steps and processes.

https://www.youtube.com/watch?v=ydhbS7Bo9qw

Practice 4

Floriculture; Disease and pest management in the garden

https://www.youtube.com/watch?v=bIkip6cvcrw

Practice 5

Commercial horticulture: Propagation of horticultural plants by using PTC technique.

https://www.youtube.com/watch?v=ZA_QuiIkikI&t=12s

  1. Applied Plant Tissue Culture (0-3-0)

Practice 1

Preparation of various plant tissue culture growth media.

https://www.youtube.com/watch?v=9vnYznz5CoA

Practice 2

Effect of growth hormones in organogenesis; Callus culture, meristem culture, single cell culture.

https://www.youtube.com/watch?v=0Oy9ijxf5us

Practice 3

Methods of gene transfer: agrobacterium-mediated gene transfer, Liposome mediated gene transfer

https://www.youtube.com/watch?v=riQz6U-YUm0

https://www.youtube.com/watch?v=WIfiwoC55bg

Practice 4

Isolation of DNA and RNA from plant samples

https://www.youtube.com/watch?v=_J_tB9PKNGI&t=49s

https://www.youtube.com/watch?v=94ei5s16rVI

Practice 5

Amplification of genomic DNA using PCR technology; Gel-electrophoresis technique for separating DNA fragments.

https://www.youtube.com/watch?v=172DTI1C38Y

https://www.youtube.com/watch?v=wXiiTW3pflM

  1. Techniques for conservation of Plant Genetic Resources (0-3-0)

Practice 1

PGR conservation techniques; derived and molecular

https://www.youtube.com/watch?v=gCcU6laStcw

Practice 2

DNA fingerprinting: methods and techniques.

https://www.youtube.com/watch?v=7onjVBsQwQ8

Practice 3

Plant barcoding by using molecular techniques

https://www.youtube.com/watch?v=8QT163ampy8

Practice 4

Ex-situ and in-situ methods of PGR conservation.

https://www.youtube.com/watch?v=XXXTY4IxHrg

Practice 5

Cryopreservation: Methods and techniques

https://www.youtube.com/watch?v=MYlSj-gCx6g

 

List of Projects to be done under the Domain

  • In vitro micro-propagation of banana and hardening.
  • Standardization of protocol for in vitro micro-propagation of horticulture crops.
  • DNA isolation, amplification, and gel electrophoresis of plant sample.
  • Phenotyping and genotyping of rice varieties for abiotic stress tolerance.
  • Genome-wide identification study and expression analysis of various genes under abiotic stress conditions.
  • Genome-wide association studies (GWAS) for abiotic stress tolerance in field crops.

Job Offers:

  1. Seed Breeders in Seed Companies (DuPont, NuzeVedu Seeds, Monsanto, Advanta, etc.)
  2. Horticulturists – Nurseries, NGO’s, Agriculture Set-up, Entrepreneurs, MNC Companies (Indo-Amercian Seeds, Kaveri Seeds, Infosys, Syngenta, Biocon, etc.)
  3. Scientists (Any research organizations)
  4. Research Technician (Any research organizations)
  5. Higher Education (M.Sc.-Horti, Biotech, Plant Molecular Biology, Breeder, etc.)

Our Main Teachers

Dr. Pushpalatha Ganesh, Professor & Head in the Department of Plant Biotechnology, M. S. Swaminathan School of Agriculture, Centurion University of Technology and Management, Paralakhemundi, Odisha, India. She possesses more than 11 years of research and teaching experience in Agricultural Biotechnology and Crop Physiology at several national level organizations including DRR, Hyderabad and NRCPB, IARI, […]