Introduction to Biotechnology by Navaneetha Krishnan J

1. ABT 301 Plant Biotechnology (2+1)
Lecture 1 : Plant tissue culture: Concepts,
history and scope

2. Main branches of the course (syllabus)
Plant
Biotechnology
Plant Tissue Culture
Recombinant DNA
Technology and Genetic
Engineering
Molecular Markers &
Molecular Breeding
Molecular Biology

3. Unit IBasics of Plant Tissue Culture
3
Plant tissue culture: Concepts, history and scope - Media and
Culture Conditions - Sterilization techniques- Regeneration
methods - morphogenesis, organogenesis and embryogenesis -
culture types - callus culture and cell suspension culture; shoot tip
and meristem tip culture; anther and pollen culture; ovule and
embryo culture

4. Unit IIApplied Plant Tissue Culture
4
Micro-propagation - banana and ornamental plants; National
certification and Quality management of TC plants- Applications of
organ culture - Meristem tip culture (virus free plants) and anther
culture (doubled haploids)- Protoplast isolation and fusion-
somaclonal variation- synthetic seeds - secondary metabolite
production- invitro germplasm conservation

5. UNIT III Basic Molecular Biology
5
Genome organization- prokaryotes vs eukaryotes- Central dogma of
life - Structure of nucleic acids - DNA replication, amino acids and
their classification- genetic codes- transcription, translation and
protein synthesis- Structure of a gene, regulation of gene
expression, Operon concept- basic techniques in molecular biology-
Blotting techniques- Polymerase chain reaction- DNA sequencing
methods.

6. Unit IV Recombinant DNA Technology and Genetic
Transformation
6
DNA manipulation enzymes: Polymerases, restriction endonucleases and
ligases - Different types of vectors: plasmids, phagemids, cosmids, BAC -
Construction of recombinant DNA molecules- Bacterial transformation - Direct
and indirect gene transfer methods in plants: microinjection, electroporation,
particle bombardment, Agrobacterium mediated method - Tissue specific
promoters, selectable and scorable markers, reporter genes- Molecular
analysis of transgenic plants – Transgenic plants: herbicide, pest and disease
resistant, abiotic stress resistant, nutritional enhancement and traits for
improved quality- Detection of GMOs – regulations and biosafety

7. Unit V Molecular Marker Technology and Molecular Breeding
7
● DNA markers - hybridization based markers (RFLP) - PCR based
markers: RAPD, SSR, AFLP, and SNPs - DNA fingerprinting of crop
varieties – Development of mapping populations- linkage and QTL
analysis- principles, methods and applications of Marker Assisted
Selection in crop improvement- Applications of Plant Genomics
and genome databases

8. Biotechnology – Definition, History, and
Scope, Importance of Biotechnology in Crop
Improvement
8

9. How technology, Science and engineering are related?
9

10. What isBiotechnology?
10
Biotechnology is technology that utilizes biological systems, living
organisms or parts of this to develop or create different products
Yeast-free
Golden rice

11. Biotechnology- approaches
11

12. Branches of Biotechnology
12
Animal Biotechnology
- transgenic animals, in vitro fertilization of egg
Medical Biotechnology
- Disease diagnosis, drug and hormone making
Industrial Biotechnology
- acetic acid, citric acid, acetone, glycerine,etc.,
Environmental Biotechnology
- detoxification of waste and industrial effluents, treatment of
sewagewater
Plant Biotechnology
- combination of tissue culture, genetic engineering and molecular
marker technology

14. •1663 – First recorded description of living cells by Robert Hooke.
•1677 – Antonie van Leeuwenhoek discovers and
describes bacteria and protozoa.
•1798 – Edward Jenner uses first viral vaccine to inoculate a child
from smallpox.
•1838 – Protein discovered, named and recorded by Gerardus Johannes
Mulder and Jöns Jacob Berzelius.
•1862 – Louis Pasteur discovers the bacterial origin of fermentation.
•1863 – Gregor Mendel discovers the laws of inheritance.
•1864 – Antonin Prandtl invents first centrifuge to separate cream from milk.
•1869 – Friedrich Miescher identifies DNA in the sperm of a trout.
Biotechnology milestones 16th – 20th century

15. •1877 – Robert Koch develops a technique for staining bacteria for
identification.
•1878 – Walther Flemming discovers chromatin leading to the discovery
of chromosomes.
•1881 – Louis Pasteur develops vaccines against bacteria that
cause cholera and anthrax in chickens.
•1885 – Louis Pasteur and Emile Roux develop the first rabies vaccine and
use it on Joseph Meister.
•1919 – Károly Ereky, a Hungarian agricultural engineer, first uses the word
biotechnology.
•1928 – Alexander Fleming notices that a certain mould could stop the
duplication of bacteria, leading to the first antibiotic: penicillin.

16. •1933 – Hybrid corn is commercialized.
•1942 – Penicillin is mass-produced in microbes for the first time.
•1951 – Artificial insemination of livestock is accomplished using frozen
semen.
•1953 – James D. Watson and Francis Crick describe the structure of DNA.
•1968 - Meselson and Yuan – Restriction endonuclease – a class of
enzyme involved in cleaving DNA
•1970 - Kelly and smith – first restriction endonuclease from Haemophilus
influenzae Rd. it was later purified and named HindII.
•1972 – The DNA composition of chimpanzees and gorillas is discovered to
be 99% similar to that of humans.
•1973 – Stanley Norman Cohen and Herbert Boyer perform the first
successful recombinant DNA experiment, using bacterial genes.
•1975 – Method for producing monoclonal antibodies developed
by Köhler and César Milstein.

17. •1980 – The U.S. patent for gene cloning is awarded to Cohen and Boyer.
•1982 – Humulin, Genentech's human insulin drug produced by genetically
engineered bacteria for the treatment of diabetes, is the first
biotech drug to be approved by the Food and Drug Administration.
•1983 – The Polymerase Chain Reaction (PCR) technique is conceived.
•1990 – First federally approved gene therapy treatment is performed
successfully on a young girl who suffered from an immune disorder.
•1994 – The United States Food and Drug Administration approves the first
GM food: the "Flavr Savr" tomato.
•1997 – British scientists, led by Ian Wilmut from the Roslin Institute, report
cloning Dolly the sheep using DNA from two adult sheep cells.

18. •1999 – Discovery of the gene responsible for developing cystic fibrosis.
•2000 – Completion of a "rough draft" of the human genome in the Human
Genome Project.
•2001 – Celera Genomics and the Human Genome Project create a draft of
the human genome sequence. It is published by Science and
Nature Magazine.
•2002 – Rice becomes the first crop to have its genome decoded.
•2003 – The Human Genome Project is completed, providing information
on the locations and sequence of human genes on all
46 chromosomes.
• 2006 - FDA approves the recombinant vaccine Gardasil®, the first
vaccine developed against human papillomavirus (HPV), an
infection implicated in cervical and throat cancers, and the first
preventative cancer vaccine.

19. • 2009: Global biotech crop acreage reaches 330 million acres.
• 2010: Dr. J. Craig Venter announces completion of “synthetic life” by
transplanting synthetic genome capable of self-replication into a
recipient bacterial cell.
• 2011: Trachea derived from stem cells transplanted into human recipient.
• 2013: Two research teams announced a fast and precise new method for editing
snippets of the genetic code. The so-called CRISPR system takes advantage
of a defense strategy used by bacteria.
• 2013: Doctors announced that a baby born with HIV had been cured of the disease.
• 2019: Scientists report, for the first time, the use of the CRISPR technology to
edit human genes to treat cancer patients with whom
standard treatments were not successful.

20. 20

21. 21

22. 22

23. Plant Tissue culture
Invitro cultivation of plant cell or tissue under aseptic (sterile condition) and
controlled environmental condition, in liquid or on semisolid well defined
medium for the production of primary and secondary metabolites or to
regenerate plant.
The culture condition include proper supply of nutrient, medium, pH,
adequate temperature, proper gaseous and liquid environment.

24. History of Plant TissueCulture
Schleiden (1838) and Schwann (1839) – concept of celltheory
German botanist Gottlieb Haberlandt (1902) - He first attempted to culture
plant tissues ‘in vitro’. He used cells from palisade tissues of leaves, cells from
pith, epidermis and epidermal hairs of various plants for culture in media -
containing Knop’s solution, asparagine, peptone and sucrose. cells remained
alive for up to 1 month, increased in size, accumulated starch but failed to
divide. He is the Father of plant tissue culture.
Problem : selection of tissues, growth hormones and culture conditions
Totipotency - The ability of a plant cell develop into a complete/whole plant
(coined by Morgan in 1901)

25. History of Plant TissueCulture
1904: Hanning - excised nearly mature embryos of crucifers and
successfully grew them into a plant on mineral salts and sugar
solutions. This is the first attempt in embryo culture
1922: Kotte and Robbins - suggested that shoot buds and root tips
could possibly be used to initiate in vitro cultures.
1925: Laibach – Development of interspecific embryo under invitro
condition in linum crop
1926: Went - first plant growth regulator discovery – Indole 3 acetic
acid (IAA)
1934: White – Established tomato roots culture – first synthetic
medium (sucrose, iron salts, thiamine, glycine, pyridoxine and nicotinic acid etc)

26. History of Plant TissueCulture
1939: Gautheret, Nobecourt and white – successfully established
callus culture from cambium tissue in carrot and tobacco (Using
Indoleacetic acid (IAA) and Bvitamins)
1943: White-The first book on plant tissue culture.
Callus

27. History of Plant Tissue Culture
1941: Van Overbeck - demonstrated for the first time the stimulatory
effect of coconut milk (embryo sac fluid) on embryo development and
callus formation in Datura.
1944: Skoog - demonstrated that in vitro cultures of tobacco can be
used to study adventitious shoot formation
1946: Ball - whole plant developed from shoot tips of Lupinus and
Tropaeolum.

28. History of Plant Tissue Culture
1952: Morel & Martin-developed the technique of meristem culture of
Dahlia to raise virus-free plants from infected individuals
1954: Muir et al. succeeded in inducing divisions in mechanically
isolated single cells (Tobacco) cultured in the presence of a nurse
tissue
1955: Miller et al. discovered the first Cytokinin (Kinetin) from
autoclaved herring sperm DNA
1957: Skoog and Miller - proposed the concept of hormonal control of
organ formation. High concentration of auxin promoted rooting
whereas proportionately more kinetin initiated bud or shoot formation

29. History of Plant Tissue Culture
1958: F.C. Steward and J. Reinert - independently, reported the
formation of embryos from somatic cells of carrot (somatic
embryogenesis).
1959: Gautheret - published first extensive hand book on Plant
Tissue Culture
1960: Morel - described a method for rapid in vitro clonal propagation
of orchids (micro-propagation).
1960: Cocking isolated plant protoplasts enzymatically from cultured
cells.

30. History of Plant TissueCulture
1962: Kanta et al. developed the technique of in vitro pollination;
viable seed formation by in vitro pollination of naked ovules.
1962: Murashige & Skoog – Developed MS medium (Most commonly
used) for tobacco.
1964: Guha and Maheshwari - produced the first androgenic
haploid plants of Datura by anther culture
1970: Power et al. - First reported chemical fusion of plant
protoplast

31. History of Plant TissueCulture
1971: Heinz and Mee reported somaclonal variation in the regenerants from
callus cultures of sugarcane.
1971: Takebe et al. achieved plant regeneration from isolated protoplasts of
tobacco.
1972: Carlson et al. produced the first somatic hybrids by the fusion of
isolated protoplasts of Nicotiana glauca and N. langsdorffii.
1973: Nitsch and Norreel- succeeded in producing haploid plants from
isolated microspore cultures of tobacco

32. History of Plant TissueCulture
1973: Nag and Street succeeded in regeneration of plants from carrot cells frozen in liquid
nitrogen (-196◦C)
1974: Kao et al. and Walin et al. introduced PEG as a versatile chemical for the fusion of
plant protoplasts.

33. 33
TEXTBOOK REFERENCES
• Elements of Biotechnology – P.K. Gupta, Rastogi Publications
• Plant Biotechnology- B.D. Singh, Kalyani Publishers
• Plant biotechnology : the genetic manipulation of plants – Adrian
Slater, Nigel W Scott, Mark R Fowler – Oxford University Press
• Gene Cloning and DNA Analysis: An Introduction – T.A. Brown –
Wiley-Blackwell Publishers
• Introduction To Plant Tissue Culture – M.K. Razdan - Oxford & IBH
publishing

34. Navaneetha Krishnan J
AP (Plant Biotechnology) – Joined KIA- April, 2022
• B.Sc (Ag) – APAC, Kalavai (2009-13)
• M.Sc (Biotechnology) - TNAU, Coimbatore (2013-15)
• Senior Research Fellow – IARI, Regional Station- Wellington, Nilgiris
(2015-16)
• Ph.D (Biotechnology) – Punjab Agricultural University, Ludhiana (2017-
22) – thesis submitted
My introduction…

35. 13