Plant Cell and Tissue Culture

Mochammad Roviq
References:
Karl-Hermann Neumann , Ashwani
Kumar and Jafargholi Imani. 2009. Plant
Cell and Tissue Culture - A Tool in
Biotechnology. Springer.
Le BuiVan. 2009. Plant Biotechnology
Vietnam Open Course Ware. University of
Science
The term of tissue culture
 Tissue culture is the culture and maintenance of
plant cells or organs in sterile, nutritionally and
environmentally supportive conditions (in vitro).
 Tissue culture produces clones, in which all
product cells have the same genotype (unless
affected by mutation during culture).
 It has applications in research and commerce. In
commercial settings, tissue culture is primarily
used for plant propagation and is often referred
to as micropropagation.
Three Fundamental Abilities of Plants
• Totipotency
The potential or inherent capacity of a plant cell to develop into
an entire plant if suitably stimulated.
It implies that all the information necessary for growth and
reproduction of the organism is contained in the cell
• Dedifferentiation
Capacity of mature cells to return to meristematic condition and
development of a new growing point, follow by redifferentiation
which is the ability to reorganize into new organ
• Competency
The endogenous potential of a given cells or tissue to develop in a
particular way
Totipotency
 Unlike an animal cell, a plant cell, even one that highly
maturated and differentiated, retains the ability to change a
meristematic state and differentiate into a whole plant if it
has retained an intact membrane system and a viable nucleus.
 1902 Haberlandt raised the totipotentiality concept of plant
totipotency in his Book “Kulturversuche mit isolierten
Pflanzenzellen” (Theoretically all plant cells are able to give
rise to a complete plant)
 Totipotency or Totipotent: The capacity of a cell (or a group
of cells) to give rise to an entire organism.
The aims of using cell and tissue cultures
 Using cell and tissue cultures, at least in basic studies,
aims at a better understanding of biochemical,
physiological, and anatomical reactions of selected cell
material to specified factors under controlled conditions,
with the hope of gaining insight into the life of the intact
plant also in its natural environment.
• Crop Improvement
• Seed Production – Plant Propagation Technique
• Genetic material conservation
The domain cell and tissue culture
Seed culture
 Increasing efficiency of
germination of seeds that are
difficult to germinate in vivo
 Precocious germination by
application of plant growth
regulators
 Production of clean seedlings for
explants or meristem culture
 In vitro selection
 Embryo culture
• Rescue embryos (embryo
rescue) from wide crosses where
fertilization occurred, but
embryo development did not
occur
• Production of plants from
embryos developed by non-
sexual methods (haploid
production)
• Overcoming embryo abortion
due to incompatibility barriers
• Overcoming seed dormancy and
self-sterility of seeds
• Shortening of breeding cycle
Ovary or ovule culture
 Production of haploid plants
 A common explant for the
initiation of somatic embryogenic
cultures
 Overcoming abortion of embryos
of wide hybrids at very early stages
of development due to
incompatibility barriers
 In vitro fertilization for the
production of distant hybrids
avoiding style and stigmatic
incompatibility that inhibits pollen
germination and pollen tube
growth
Anther and microspore culture
 Production of haploid
plants
 Production of
homozygous diploid lines
through chromosome
doubling, thus reducing
the time required to
produce inbred lines
 Uncovering mutations or
recessive phenotypes
In vitro
pollination

 Production of hybrids
difficult to produce by
embryo rescue
Organ culture
 Any plant organ can serve as an explant to initiate cultures
 Shoot apical meristem culture
 Production of virus free
germplasm
 Mass production of desirable
genotypes
 Facilitation of exchange
between locations (production
of clean material)
 Cryopreservation (cold
storage) or in vitro
conservation of germplasm
 Somatic embryogenesis
 One major path of
regeneration
 Mass multiplication
 Production of
artificial seeds
 As source material
for embryogenic
protoplasts
 Amenable to
mechanization and
for bioreactors
Organogenesis
 One major path of regeneration
 Mass multiplication
 Conservation of germplasm at either normal
or sub-zero temperatures
Callus Cultures
 In some instances it is
necessary to go through a
callus phase prior to
regeneration via somatic
embryogenesis or
organogenesis
 For generation of useful
somaclonal variants (genetic or
epigenetic)
 As a source of protoplasts and
suspension cultures
 For production of metabolites
 Used in in vitro selection
In vitro
mutagenesis

 Induction of polyploidy
 Introduction of genetic
variability
 Chemical bases and
radiation bases
 Protoplast isolation, culture and fusion
 Combining genomes to
produce somatic hybrids,
asymmetric hybrids or
cybrids
 Production of organelle
recombinants
 Transfer of cytoplasmic
male sterility
 In vitro flowering

 This can be done in some instances but I am not sure there are
any practical applications
Micrografting
 Overcoming graft incompatibility
 Rapid mass propagation of elite scions by grafting onto
rootstocks that have desirable traits like resistance to soil-
borne pathogens and diseases
 To allow survival of difficult to root shoots
 Development of virus free plants
Genetic transformation
 Many different explants can be used, depending on the plant
species and its favored method of regeneration as well as the
method of transformation
 Introduction of foreign DNA to generate novel (and typically
desirable) genetic combinations
 Used to study the function of genes
Pembagian kultur
Istilah Deskripsi
1. Kultur organ Kultur yang diinisiasi dari bagian-bagian tanaman
(organ culture) seperti; ujung akar, pucuk aksilar, ujung pucuk
(meristem dengan beberapa primordia daun), dan
embrio sebagai bagian dari biji
2. Kultur kalus Kultur sekumpulan sel yang tidak terorganisir, hanya sel-
(callus culture) sel parenkim yang berasal dari berbagai bahan awal
3. Kultur suspensi Kuktur sel bebas atau agregat sel kecil dalam media cair
(suspension dengan pengocokan. Pada umumnya kultur suspensi
culture) diinisiasikan dari kalus
Pembagian kultur
Istilah Deskripsi
4. Kultur protoplasma Sel-sel muda yang diinisiasi dalam media cair kemudian
dihilangkan dinding sel-nya dengan menggunakan enzim.
Protoplasma kemudian dibiarkan membelah diri dan
membentuk dinding kembali pada media padat. Kultur
protiplasma digunakan untuk hibrididasi somatik (fusi dua
protoplasma baik intraspesifik maupun interspesifik
4. Kultur haploid Kultur yang berasal dari bagian reproduktif tanaman yaitu:
(mikrospora/anther) kepala sari dan tepung sarinya. Diharapkan yang tumbuh dan
beregenerasi adalah; tepung sari sehingga diperoleh kultur
haploid. Apabila secara khsusus yang dipakai sebagai bahan awal
adalah tepung sari, maka kultur tersebut disebut kultur
mikrospora. Kultur anther adalah kultur yang diinisiasi dari
seluruh kepala sari
What conditions do plant cells need
to multiply in vitro?
Tissue culture has several critical requirements:
 Appropriate tissue (some tissues culture better than others)
 A suitable growth medium containing energy sources and
inorganic salts to supply cell growth needs. This can be liquid
or semisolid
 Aseptic (sterile) conditions, as microorganisms grow much
more quickly than plant and animal tissue and can overrun a
culture.
 Growth regulators - in plants, both auxins & cytokinins.
 Frequent subculturing to ensure adequate nutrition and to
avoid the build-up of waste metabolites
Appropriate tissue (Explant)
 Explants: Cell, tissue or organ of a plant that is used to start
in vitro cultures. Many different explants can be used for
tissue culture, but axillary buds and meristems are most
commonly used.
 The explants must be sterilized to remove microbial
contaminants. This is usually done by chemical surface
sterilization of the explants with an agent such as bleach at a
concentration and for a duration that will kill or remove
pathogens without injuring the plant cells beyond recovery.
 Plant source (axillary buds, meristems Leaves, stems,
roots, hypocotyl…)

 Surface sterilization of explants

Young flower stalk of Vertiver sp Leaf explants of Stevia sp

polyphenolics
Many plants are rich in polyphenolics
 After tissue injury during dissection, such compounds will be
oxidized by polyphenol oxidases → tissue turn brown/black
 Phenolic products inhibit enzyme activities and may kill the
explants
Methods to overcome browning:
 adding antioxidants [ascorbic acid, citric acid, PVP
(polyvinylpyrrolidone), dithiothreitol], activated charcoal or
presoaking explants in antioxidant
 incubating the initial period of culturing in reduced light/darkness
 frequently transfer into fresh medium
The appearance of phenolic
compound and death tissues
Nutrition medium
 When an explant is isolated, it is no longer able to receive
nutrients or hormones from the plant, and these must be
provided to allow growth in vitro.
 The composition of the nutrient medium is for the most part
similar, although the exact components and quantities will
vary for different species and purpose of culture.
 Types and amounts of hormones vary greatly. In addition, the
culture must be provided with the ability to excrete the
waste products of cell metabolism.
 This is accomplished by culturing on or in a defined culture
medium which is periodically replenished.
 A nutrient medium is defined by its mineral salt
composition, carbon source, vitamins, plant growth
regulators and other organic supplements.
 pH determines many important aspects of the structure and
activity of biological macromolecules. Optimum pH of 5.0-
6.0 tends to fall during autoclaving and growth.
Mineral salts
Function of nutrients in plant growth
Carbon sources and vitamins
 Sucrose or glucose (sometimes fructose), concentration 2-
5%
 Most media contain myo-inositol, which improves cell
growth
An absolute requirement for vitamin B1 (thiamine)
• Growth is also improved by the addition of nicotinic acid and
vitamin B6 (pyridoxine)
• Some media contain pantothenic acid, biotin, folic acid, p-
amino benzoic acid, choline chloride, riboflavine and
ascorbic acid (C-vitamin)
Thank you
 Thank you