This presentation is related to the application of plant tissue culture techniques in various sectors, and it also highlights the advantages and limitations of plant tissue culture

1. By – Dr. Mafatlal M. Kher
Unit: 2 Plant tissue culture: Applications
Date & Time : Monday, 02 August 2021
Semester : VI
Program : B.Sc. Biotechnology
School : School of Science
Subject : BSC5BT04

2. Vegetative propagation, however plantlet produced via vegetative
propagation carries diseased from stock plant 01
Limited number of plants are produced via vegetative
propagation.
02
Bamboos can also propagated using seeds, but flowering
is very rare in nature, hence limited numbers of seeds are
produced
03
Plantlets produced using seed germination methods are
generally infected with seed born pathogen
04
Natural
method
of
Cultivation
2

3. Tissue culture raised plants are more suitable for long
distance transport
Starting with 100 traditional cuttings; able to
produce 70,000 annual clones. Start with 200
tissue culture vials; produce 2 million annual
clones.
Uses 1/10 the space of traditional cloning. Two million
annual clones could be produced in less than 3000
square feet.
1000 mother cultivars could be stored inside a
refrigerator with no care or maintenance for months,
sometimes over a year
Propagation is significantly more efficient
3

4. Applications of Plant Tissue Culture
techniques
4

5. 5

6. A Success story of Taiwan’s Orchid Industry
6

7. Micropropagation
 Micropropagation is the rapid vegetative propagation of plants under in vitro
conditions of high light intensity, controlled temperature, and a defined
nutrient medium.
• Production of true to type plants.
• Some plants produce few (if any) viable seeds.
• Clonal progeny are highly uniform in all characters.
• Outcrossing plants produce highly variable progeny as in case of seed
derived plants.
• Plants may have extended juvenile period.
• Cloning allows for combining genotypes in one plant.
• Seeds may have lengthy and complex dormancies.
• Applications: Commercial scale propagation of Banana, Pomegranate,
Pineapple, Tectona grandis, Rose, Gerbera, Orchids
7

8. Somaclonal variation
 It is cheaper than other methods of genetic manipulation
and does not require ‘containment’ procedures.
 Tissue culture systems are available for more plant
species than can be manipulated by somatic
hybridization and transformation at the present time.
 It is not necessary to have identified the genetic basis of
the trait, or indeed, in the case of transformation, to have
isolated and cloned it.
 Novel variants have been reported among somaclones,
and evidences indicate that both the frequency and
distribution of genetic recombination events can be
altered by passage though tissue culture.
 This implies that variation may be generated from
different locations of the genome than those, which are
accessible to conventional and mutation breeding.
 Growth regulators affect the cell cycle and consequently
contribute to the causes of somaclonal variation. A high
concentration of 2,4-D (2,4-dichlorophenoxy acetic acid)
has been implicated in the increase in chromosomal
instability
8

9. Somaclonal variation
 The source for most breeding material begins with mutations, whether the
mutation occurs in a modern cultivar, a landrace, a plant accession, a wild
related species, or in an unrelated organism
 Total sources of variation:
 Mutation, Hybridization, Polyploidy
9

10. Somaclonal variation and mutation breeding
Somaclonal variation is a general phenomenon of all plant
regeneration systems that involve a callus phase for e.g.
2,4 D, Dicamba and Piclorma is extensively utilized for
callus culture.
There are two general types of Somaclonal Variation:
 Heritable, genetic changes (alter the DNA)
 Stable, but non-heritable changes (alter gene expression,
e.g. epigenetic)
10
186 Days Half life of 2,4- D
 2 mg/l 2,4-D is added in the medium
 1 mg/l (186)
 0.5 mg/l (186)
 0.25 mg/l (186)
 0.125 mg/l (186)
 744 days required to from 2 mg to 0.125
 0.1 mg/l 2,4-D also able to cause
somaclonal variation in Cotton: Plant Cell
Rep (2008) 27:1303–1316
2,4-D Picloram
Dicamba
2,4-D, Picloram and Dicamba are synthetic auxin
and extensively utilized as a Herbicide

11. Mutation breeding
 1927: Muller produced mutations in fruit flies using x-rays
 1928: Stadler produced mutations in barley
 Mutation breeding became a bandwagon for about 10 years (first claim to
“replace breeders”)
Today there are three groups of breeders:
 Mutation breeding is useless, we can accomplish the same thing with
conventional methods
 Mutation breeding will produce a breakthrough given enough effort
 Mutation breeding is a tool, useful to meet specific objectives
11

12. Inducing mutations
Physical Mutagens (irradiation)
 Neutrons, Alpha rays: Densely ionizing (“Cannon balls”), mostly
chromosome aberrations
 Gamma, Beta, X-rays: Sparsely ionizing (“Bullets”), chromosome
aberrations & point mutations
 UV radiation: Non-ionizing, cause point mutations (if any), low penetrating
Chemical Mutagens (carcinogens)
 Many different chemicals: Most are highly toxic, usually result in point
mutations
Callus Growth in Tissue Culture
 Somaclonal variation (can be combined with other agents)
 Can screen large number of individual cells
 Chromosomal aberrations, point mutations
 Also: Uncover genetic variation in source plant
12

13. Traditional Mutation Breeding Procedures
 Treat seed with mutagen (irradiation or chemical)
 Target: 50% kill
 Grow-out M1 plants (some call this M0)
 Evaluation for dominant mutations possible, but most are recessive, so →
 Grow-out M2 plants
 Evaluate for recessive mutations
 Expect segregation
 Progeny test selected, putative mutants
 Prove mutation is stable, heritable
13

14. Somaclonal Breeding Procedures
 Use plant cultures as starting material
 Idea is to target single cells in multi-cellular culture
 Usually suspension culture, but callus culture can work (want as much contact
with selective agent as possible)
 Optional: apply physical or chemical mutagen
 Apply selection pressure to culture
 Target: very high kill rate, you want very few cells to survive, so long as selection
is effective
 Regenerate whole plants from surviving cells
14

15. Somaclonal/Mutation Breeding
 Advantages
 Screen very high populations (cell based)
 Can apply selection to single cells
 Disadvantages
 Many mutations are non-heritable
 Requires dominant mutation (or double recessive mutation); most mutations are
recessive
 Can avoid this constraint by not applying selection pressure in culture, but you loose the
advantage of high through-put screening – have to grow out all regenerated plants, produce
seed, and evaluate the M2
 How can you avoid this problem?
15

16. 16
Somaclonal/Mutation Breeding

17. Successes of Somaclonal/Mutation Breeding
Herbicide Resistance and Tolerance
 Resistance: able to break-down or metabolize the herbicide – introduce a new
enzyme to metabolize the herbicide
 Tolerance: able to grow in the presence of the herbicide – either ↑ the target enzyme
or altered form of enzyme
 Most successful application of somaclonal breeding have been herbicide tolerance
 Glyphosate resistant tomato, tobacco, soybean (GOX enzyme)
 Glyphosate tolerant petunia, carrot, tobacco and tomato (elevated EPSP (enolpyruvyl shikimate phosphate
synthase))
 But not as effective as altered EPSP enzyme (bacterial sources)
 Imazaquin (Sceptor) tolerant maize
 Theoretically possible for any enzyme-targeted herbicide – it’s relatively easy to
change a single enzyme by changing a single gene
17

18. Other Targets for Somaclonal Variation
 Specific amino acid accumulators
 Screen for specific amino acid production
 e.g. Lysine in cereals
 Abiotic stress tolerance
 Add or subject cultures to selection agent
 e.g.: salt tolerance, temperature stresses, etc…
 Disease resistance
 Add toxin or culture filtrate to growth media
 Examples shown on next slide →
18

19. Disease Resistant Success using Somaclonal
Variation
Crop Pathogen Toxin
Alfalfa Colletotrichum sp. Culture filtrate
Banana Fusarium sp. Fusaric acid
Coffee Colletotrichum sp. Partially purified culture filtrate
Maize Helminthosporium maydis T-toxin
Oat* Helminthosporium victoriae Victorin
Oilseed Rape* Phoma lingam Culture filtrate
Peach Xanthomonas sp. Culture filtrate
Potato** Phytophthora infestans Culture filtrate
Rice* Xanthomonas oryzae Culture filtrate
Sugarcane** Helminthosporium sp. Culture filtrate
Sugarcane** Helminthosporium sachari Partially purified HS toxin
Tobacco* Psedomonas tabaci Methionine-sulfoximine
Tobacco* Alternaria alternata Partially purified toxin
*Shown to be heritable through sexual propagation
**Shown to be stable through vegetative propagation
19

20. Requirements for Somaclonal/Mutation Breeding
 Effective screening procedure
 Most mutations are deleterious
 With fruit fly, the ratio is ~800:1 deleterious to beneficial
 Most mutations are recessive
 Must screen M2 or later generations
 Consider using heterozygous plants?
 Haploid plants seem a reasonable alternative if possible
 Very large populations are required to identify desired mutation:
 Can you afford to identify marginal traits with replicates & statistics? Estimate: ~10,000 plants
for single gene mutant
 Clear Objective
 Can’t expect to just plant things out and see what happens; relates to having an
effective screen
 This may be why so many early experiments failed
20

21. Questions with Mutation Breeding
 Do artificial mutations differ from natural ones?
 Most people agree that they are, since any induced mutation can be found in
nature, if you look long enough & hard enough
 If this is true, then any mutation found in nature can be induced by mutation
breeding
 Is it worthwhile, given the time & expense?
Still require conventional breeding to incorporate new variability into
crop plants (will not replace plant breeders)
Not subject to regulatory requirements (or consumer attitudes) of
genetically engineered plants
21

22. Production of
Haploids-
Polyploids via
Anther Culture /
Ovule Culture

23. Androgenesis/Anther culture/Microspore/Pollen
(Haploids)
 Haploid: For recessive trait breeding.
 Traditional breeding methods are slow and take 10–15 years for cultivar development.
 Another disadvantage is inefficiency of selection in early generations because of
heterozygosity.
These two disadvantages can be over come by double haploids, and more elite crosses
can be evaluated and selected within less time.
Polyploidy:
 According to "Genetics: A Conceptual Approach," the volume of each cell in a plant often
seems to be correlated with the volume of its nucleus.
 The larger the genome, the larger the nucleus, and hence the larger the cell.
 Many polyploid plants have bigger cells and are bigger overall than their diploid relatives.
Sometimes plant breeders have taken advantage of this phenomenon in their quest to
breed plants with larger leaves and fruits.
23

24. Haploid Plant Production
 Embryo rescue of interspecific crosses
 Creation of alloploids (e.g. triticale)
 Bulbosum method
 Anther culture/Microspore culture
 Culturing of Anthers or Pollen grains (microspores)
 Derive a mature plant from a single microspore
 Ovule culture
 Culturing of unfertilized ovules (macrospores)
 Sometimes “trick” ovule into thinking it has been fertilized
24

25. Bulbosum Method of Haploid Production
 This was once more efficient than microspore culture in creating haploid barley
 Now, with an improved culture media (sucrose replaced by maltose), microspore
culture is much more efficient (~2000 plants per 100 anthers)
Hordeum
vulgare
Barley
2n = 2X = 14
Hordeum
bulbosum
Wild relative
2n = 2X = 14
Haploid Barley
2n = X = 7
H. Bulbosum
chromosomes
eliminated
X
Embryo Rescue
↓
25

26. Features of anther/microspore culture
26

27. Anther/Microspore Culture Factors
 Genotype
 As with all tissue culture techniques
 Growth of mother plant
 Usually requires optimum growing conditions
 Correct stage of pollen development
 Need to be able to switch pollen development from gametogenesis to
embryogenesis
 Pretreatment of anthers
 Cold or heat have both been effective
 Culture media
 Additives, Agar vs. ‘Floating’
27

28. Ovule Culture for Haploid Production
 Essentially the same as embryo culture
 Difference is an unfertilized ovule instead of a fertilized embryo
 Effective for crops that do not yet have an efficient microspore culture
system
 e.g.: melon, onion
 In the case of melon, you have to “trick” the fruit into developing by using irradiated
pollen, then x-ray the immature seed to find developed ovules
28

29. What do you do with the haploid?
 Weak, sterile plant
 Usually want to double the chromosomes, creating a dihaploid plant with
normal growth & fertility
 Chromosomes can be doubled by
 Colchicine treatment
 Spontaneous doubling
 Tends to occur in all haploids at varying levels
 Many systems rely on it, using visual observation to detect spontaneous dihaploids
 Can be confirmed using flow cytometry
29

30. Uses of Hapliods in Breeding
Creation of allopolyploids
as previously described
Production of homozygous diploids (dihaploids)
Detection and selection for (or against) recessive alleles
Specific examples on next slide →
30

31. Specific Examples of DH uses
 Evaluate fixed progeny from an F1
 Can evaluate for recessive & quantitative traits
 Requires very large dihaploid population, since no prior selection
 May be effective if you can screen some qualitative traits early
 For creating permanent F2 family for molecular marker development
 For fixing inbred lines (novel use?)
 Create a few dihaploid plants from a new inbred prior to going to Foundation Seed (allows you to
uncover unseen off-types)
 For eliminating inbreeding depression (theoretical)
 If you can select against deleterious genes in culture, and screen very large populations, you may
be able to eliminate or reduce inbreeding depression
 e.g.: inbreeding depression has been reduced to manageable level in maize through about 50+
years of breeding; this may reduce that time to a few years for a crop like onion or alfalfa
31

32. Applications: Germplasm conservation and
cryopreservation
 Cryopreservation plays a vital role in
the long-term in vitro conservation of
essential biological material and
genetic resources.
 It involves the storage of in vitro cells
or tissues in liquid nitrogen that results
in cryo-injury on the exposure of
tissues to physical and chemical
stresses.
 Successful cryopreservation is often
ascertained by cell and tissue survival
and the ability to re-grow or regenerate
into complete plants or form new
colonies
32
Image source: https://link.springer.com/chapter/10.1007/978-3-319-22521-0_3

33. Germplasm Preservation
 Extension of micropropagation techniques
 Two methods:
1. Slow growth techniques
o e.g.: ↓ Temp., ↓ Light, media supplements (osmotic inhibitors, growth retardants), tissue
dehydration, etc…
o Medium-term storage (1 to 4 years)
2. Cryopreservation
o Ultra low temperatures
o Stops cell division & metabolic processes
o Very long-term (indefinite?)
o Details to follow on next two slides →
33

34. Cryopreservation Requirements
 Preculturing
 Usually a rapid growth rate to create cells with small vacuoles and low water
content
 Cryoprotection
 Cryoprotectant (Glycerol, DMSO/dimetil sulfoksida, PEG) to protect against
ice damage and alter the form of ice crystals
 Freezing
The most critical phase; one of two methods:
 Slow freezing allows for cytoplasmic dehydration
 Quick freezing results in fast intercellular freezing with little dehydration
34

35. Cryopreservation Requirements
 Storage
 Usually in liquid nitrogen (-196oC) to avoid changes in ice crystals that occur
above -100oC
 Thawing
 Usually rapid thawing to avoid damage from ice crystal growth
 Recovery (don’t forget you have to get a plant)
 Thawed cells must be washed of cryoprotectants and nursed back to normal
growth
 Avoid callus production to maintain genetic stability
35

36. Cryopreservation Steps
 Selection
 Excision of plant tissues or organs
 Culture of source material
 Select healthy cultures
 Apply cryo-protectants
 Pre-growth treatments
 Cooling/freezing
 Storage
 Warming & thawing
 Recovery growth
 Viability testing
 Post-thawing
36

37. Applications: Synthetic seed production
 Will ensure abundant supply of desired
plant species.
 Synthetic seeds would also be a channel
for new plant lines produced through
biotechnological advances to be delivered
directly to greenhouse and field.
 High volume propagation potential of
somatic embryos combined with formation
of synthetic seeds for low-cost delivery
would open new vistas for clonal
propagation in several commercially
important crop species. Germplasm
exchange and cryopreservation.
37

38. Applications: Somatic Hybridization using
Protoplasts
 Development of hybrid plants through the fusion of somatic protoplasts
of two different plant species/varieties
 Created by degrading the cell wall using enzymes
 Very fragile, can’t pipette
 Protoplasts can be induced to fuse with one another:
 Electrofusion: A high frequency AC field is applied between 2 electrodes immersed in the suspension of
protoplasts- this induces charges on the protoplasts and causes them to arrange themselves in lines
between the electrodes. They are then subject to a high voltage discharge which causes them membranes to
fuse where they are in contact.
 Polyethylene glycol (PEG): causes agglutination of many types of small particles, including
protoplasts which fuse when centrifuged in its presence
 Addition of calcium ions at high pH values
38

39. Somatic hybridization technique
1. isolation of protoplast
2. Fusion of the protoplasts of desired species/varieties
3. Identification and Selection of somatic hybrid cells
4. Culture of the hybrid cells
5. Regeneration of hybrid plants
39

40. Isolation of Protoplast
(Separation of protoplasts from plant tissue)
1. Mechanical Method 2. Enzymatic Method
40

41. Mechanical Method
Plant Tissue
Collection of protoplasm
Cells Plasmolysis
Microscope Observation of cells
Cutting cell wall with knife
Release of protoplasm
41

42. Mechanical Method
Used for vacuolated cells like onion bulb scale, radish and beet root tissues
Low yield of protoplast
Laborious and tedious process
Low protoplast viability
42

43. Leaf sterlization, removal of
epidermis
Plasmolysed
cells
Plasmolysed
cells
Pectinase +cellulase Pectinase
Protoplasm released
Release of
isolated cells
cellulase
Protoplasm
released
Isolated
Protoplasm
Enzymatic Method
43

44. Enzymatic Method
Used for variety of tissues and organs including leaves, petioles, fruits,
roots, coleoptiles, hypocotyls, stem, shoot apices, embryo microspores
 Mesophyll tissue - most suitable source
 High yield of protoplast
 Easy to perform
 More protoplast viability
44

45. Protoplast Fusion
(Fusion of protoplasts of two different genomes)
1. Spontaneous Fusion 2. Induced Fusion
Intraspecific Intergeneric Electrofusion
Mechanical
Fusion
Chemofusion
45

46. Uses for Protoplast Fusion
 Combine two complete genomes
 Another way to create allopolyploids
 Partial genome transfer
 Exchange single or few traits between species
 May or may not require ionizing radiation
 Genetic engineering
 Micro-injection, electroporation, Agrobacterium
 Transfer of organelles
 Unique to protoplast fusion
 The transfer of mitochondria and/or chloroplasts between species
46

47. Protoplast Fusion
Spontaneous Fusion: Protoplast fuse spontaneously during isolation process mainly due
to physical contact
 Intraspecific produce homokaryones
 Intergeneric have no importance
Induced Fusion: Chemical or Mechanical or Electrofusion
 Chemofusion- fusion induced by chemicals i.e. fusogens
 PEG
 NaNo3
 Ca 2+ ions
 Polyvinyl alcohol
 Mechanical Fusion- Physical fusion of protoplasts under microscope by using
micromanipulator and perfusion micropipette.
 Electrofusion- Fusion induced by electrical stimulation. Fusion of protoplasts is induced
by the application of high strength electric field (100kv m-1) for few microsecond.
47

48. Possible Result of Fusion of Two Genetically
Different Protoplasts
= chloroplast
= mitochondria
= nucleus
Fusion
heterokaryon
cybrid cybrid
hybrid
hybrid
48

49. Identifying Desired Fusions
 Complementation selection
 Can be done if each parent has a different selectable marker (e.g. antibiotic or
herbicide resistance), then the fusion product should have both markers
 Fluorescence-activated cell sorters
 First label cells with different fluorescent markers; fusion product should have
both markers
 Mechanical isolation
 Tedious, but often works when you start with different cell types
 Mass culture
 Basically, no selection; just regenerate everything and then screen for desired
traits
49

50. Example of Protoplast Fusion
 Male sterility introduced into cabbage by making a cross with radish (as the female)
 embryo rescue employed to recover plants
 Cabbage phenotypes were recovered that contained the radish cytoplasm and were
male sterile due to radish genes in the mitochondria
 Unfortunately, the chloroplasts did not perform well in cabbage, and seedlings
became chlorotic at lower temperatures (where most cabbage is grown)
 Protoplast fusion between male sterile cabbage and normal cabbage was done, and
cybrids were selected that contained the radish mitochondria and the cabbage
chloroplast
 Current procedure is to irradiate the cytoplasmic donor to eliminate nuclear DNA –
routinely used in the industry to re-create male sterile brassica crops
50

51. Applications: Protoplast culture Cybrids
 Cybridization is the fusion of the nucleus from one parent and the
cytoplasm of both parents.
 The product formed is called a cybrid and the process is known as
cybridization.
 In cybridization one protoplast contributes to the cytoplasm while the other
contributes to the nucleus alone.
51

52. Applications: Plant cell cultures for secondary
metabolite production
 Secondary metabolites are defined as bioactive compounds having pharmacological or
toxicological effects on living organisms.
 They are organic compounds typically with a molecular weight less than 3000 Daltons that are
produced by plants, microbes and fungi.
 They are produced within the plants besides the primary biosynthetic and metabolic routes for
compounds associated with plant growth and development, and are regarded as products of
biochemical “side tracks” in the plant cells.
 Several of them are found to hold various types of important functions in the living plants such as
protection, attraction or signalling.
 They are accumulated in specific tissues and structures such as vacuoles, specialized glands,
trichomes, roots etc.
 Their biosynthesis occurs through phenylpropanoid, mevalonate, 2-C-methyl-d-eryth- ritol-4-
phosphate, amino acid, glucose, acetate-malonate or combined pathways.
 Production of these compounds is affected by genotype, plant physiology, climate, environmental
conditions, and pathogens among others.
52

53. Why plants?
53

54. 54

55. Why Plants
 Mom’s conventional wisdom of eating fruits and vegetables to lead a healthy
life has evolved with scientific, fact-finding research.
 From prehistoric times, people have used plants for food (as sources of
proteins, carbohydrates, fats, vitamins, and minerals) and as medicines for
treating a variety of illnesses.
 Human knowledge so far encompasses the existence of at least 270,000 plant
species, and researchers believe that more than 400,000 plant species exist
worldwide.
 About 75% of the world’s population relies on plants for traditional medicine.
 Plants remain a major source of pharmaceuticals and fine chemicals.
 On the order of 40% or more of the pharmaceutical products are already
derived or at least partially derived from plants.
55

56. Why Plants
 Despite advancements in synthetic chemistry, we still depend upon biological
sources for a number of secondary metabolites including pharmaceuticals.
Their complex structural features are difficult to synthesize.
 Many of them are unique to the plant kingdom and are not produced by
microbes or animals.
 However, with the advancement of transgenic research, it is possible to
produce compounds and molecules, which were also not originally synthesized
in plants.
 Where natural sources are scarce and chemical synthesis is unfeasible,
another potential strategy for the production of valuable metabolites is the in
vitro culture of plant cells/tissues that produce the desired compound naturally.
56

57. Production of bioactive
compounds
57
Why?

58. 58

59. Production of bioactive compounds: WHY?
 Destruction of natural habitats and technical difficulties in cultivation: Although
plants are renewable resources, some species are becoming more difficult to
obtain in sufficient amounts to meet increasing demands. For example, in
Korea wild mountain ginseng (Panax ginseng) is highly prized for its medicinal
properties, largely mediated by ginsenosides. This has resulted in a massive
reduction of wild P. ginseng populations. As a result a singe plant might sell for
many thousands of dollars.
 For example, it is claimed that the demand for paclitaxel, a potent anticancer
compound, could endanger forests of Taxus brevifolia because of the low
paclitaxel (trade name:Taxol) content (40–100 mg/kg of bark) in and slow
growth of the trees.
 Some of the these compounds found in low quantities.
 The yield of plant secondary metabolites from natural sources can be highly
variable depending on the source plant, location, season of harvest and the
prevailing environmental conditions.
59

60. Production of bioactive compounds: WHY?
 They typically possess highly complex structures, consequently it is often difficult
to synthesize these compounds on an industrial scale.
 For many natural chemicals it is possible to synthesize alternatives from
petroleum, coal, or both. But its has limitations like cost and pollution. For
example, after extensive research a total synthesis procedure was
independently established for the key anti-cancer drug, paclitaxel by Holton et al.
(1994). However, industrial production of paclitaxel via this method was not
commercially viable. At the moment, Python Biotech is the largest producer of
paclitaxel by plant cell culture.
 These outcomes have driven efforts towards the development of renewable and
environmentally friendly production processes.
60

61. Production of bioactive
compounds from plants
61
How?

62. Aspects of biotechnological approaches to plant-derived
bioactive compounds
 Plant cell tissue and organ cultures
 Cell culture
 Shoot culture
 Root culture
 Scale-up of cultures
 Transgenic plants/organisms
 Metabolic engineering
 Heterologous expression
 Molecular farming.
 Micropropagation of medicinal plants
 Endangered plants
 High-yielding varieties
 Metabolically engineered plants.
 Newer sources
 Algae
 Other photosynthetic marine forms
62

63. State-of-the-art technological platform for plant cell culture
63

64. Strategies to enhance production of bioactive
compounds in plant cell cultures
 Optimization of biosynthesis by culture conditions.
 Production in differentiated tissues.
 Obtaining efficient cell lines for growth.
 Selection of high-producing cell lines.
 Precursor feeding and biotransformation.
 Elicitation and stress induced production.
 Screening of high-growth cell line to produce metabolites of interest.
 Mutation of cells.
 Amenability to media alterations for higher yields.
 Immobilization of cells to enhance yields of extracellular metabolites and to facilitate biotransformations.
 Use of elicitors to enhance productivity in a short period of time.
 Permeation of metabolites to facilitate downstream processing.
 Adsorption of the metabolites to partition the products from the medium and to overcome feedback inhibition.
 Scale-up of cell cultures in suitable bioreactors.
64

65. Background for production of bioactive compounds
The production of plant secondary metabolites by means of large-scale culture
of plant cells in bioreactors is technically feasible. The economy of such a
production is the major bottleneck. For some costly products it is feasible, but
unfortunately some of the most interesting products are only in very small
amounts or not all produced in plant cell cultures.
In case of phytoalexins, elicitation will lead to high production. But for many
of the compounds of interest the production is not inducible by elicitors.
The culture of differentiated cells, such as (hairy) root or shoot cultures, is an
alternative, but is hampered by problems in scaling up of such cultures. Hairy
root cultures follow a definite growth pattern, however, the metabolite
production may not be growth related. Production of certain secondary
metabolites requires participation of roots and leaves. A solution to this
problem is the root-shoot co-culture using hairy roots.
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66. Metabolic engineering
 Metabolic engineering offers new perspectives for improving the production of compounds of interest.
 This approach can be used to improve production in the cell culture, in the plant itself or even production in
other plant species or organisms.
 Studies on the production of terpenoid indole alkaloids have shown that the overexpression of single genes of
the pathway may lead for some enzymes to an increased production of the direct product, but not necessarily to
an increased alkaloid production.
 On the other hand feeding of such transgenic cultures with early precursors showed an enormous capacity for
producing alkaloids, which is not utilized without feeding precursors.
 Overexpression of regulatory genes results in the upregulation of a series of enzymes in the alkaloid pathway,
but not to an improved flux through the pathway, but feeding loganin (iridoid glycosides) does result in
increased alkaloid production if compared with wild-type cells.
 Indole alkaloids could be produced in hairy root cultures of Weigelia by overexpression of tryptophan
decarboxylase and strictosidine synthase.
 Alkaloids could be produced in transgenic yeast overexpressing strictosidine synthase and strictosidine
glucosidase growing on medium made out the juice of Symphoricarpus albus berries to which tryptamine is
added.
 Metabolic engineering thus seems a promising approach to improve the production of a cell factory. 66

67. One Last Role of Plant Tissue Culture
 Genetic engineering would not be possible without the development of
plant tissue
 Genetic engineering requires the regeneration of whole plants from single
cells
 Efficient regeneration systems are required for commercial success of
genetically engineered products
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68. Plant tissue culture
Advantage and limitations
68

69. Advantage of Plant tissue culture
 Cultures are started
with very small
peace of plant
material i.e. explant
(Hence, term
micropropagation is
used).
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70. Advantage of Plant tissue culture
 Limited space is required to maintain large number of plants.
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71. Advantage of Plant tissue culture
 Propagation is ideally carried out in
aseptic conditions (avoiding
contamination). Hence, once
aseptic culture has been
established, there should be no loss
through diseases, and the plantlets
finally produced should ideally free
from bacterial or fungal diseases.
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72. Advantage of Plant tissue culture
 Methods are available to free plants from specific virus diseases. Providing
these techniques are employed, or virus-tested material is used for initiating
cultures, certified virus-tested plants can be produced in large numbers.
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73. Advantage of Plant tissue culture
 A more flexible adjustment of factors
influencing vegetative regeneration is
possible such as nutrient and growth
regulator levels, light and temperature.
 Therefore, the rate of propagation is much
greater than in macropropagation and
many more plants can be produced in a
given time.
 This may enable newly selected varieties
to be made available quickly and widely,
and numerous plants to be produced in a
short time.
 The technique is very suitable for large-
scale quality plantlet production.
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74. Advantage of Plant tissue culture
 It may be possible to produce clones of some kinds of plants that are
otherwise slow and difficult (or even impossible) to propagate vegetatively.
 Plants may acquire a new temporary characteristic through
micropropagation which makes them more desirable to the grower than
conventionally-raised stock for e.g.:-
 A bushy habit (in ornamental pot plants) and
 Increased runner formation (strawberries) are two examples.
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75. Advantage of Plant tissue culture
 Production can be continued all the year round and is more independent of
seasonal changes.
 Less energy and space are required for propagation purposes and for the
maintenance of stock plants.
 Plant material needs little attention between subcultures and there is no
labour or materials requirement for watering, weeding, spraying etc.
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76. 76

77. Limitations of Plant tissue culture
 The setting up of plant tissue culture laboratory is very expensive including
its instruments like AC, Laminar, Autoclave, Aseptic area, Green house and
Polyhouse facilities etc. and reagents for e.g. plant growth
regulators/hormones.
 Advanced skills are required for their successful operation.
 A specialised and expensive production facility is needed; fairly specific
methods may be necessary to obtain optimum results from each species
and variety and, because present methods are labour intensive, the cost of
propagules is usually relatively high.
 Further consequences of using in vitro adaptations are although they may
be produced in large numbers, the plantlets obtained are initially small and
sometimes have undesirable characteristics
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78. Limitations of Plant tissue culture
 In order to survive in vitro, explants and cultures have to be grown on a
medium containing sucrose or some other carbon source.
 The plants derived from these cultures are not initially able to produce their
own requirement of organic matter by photosynthesis (i.e. they are not
autotrophic) and have to undergo a transitional period before they are
capable of independent growth.
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79. Limitations of Plant tissue culture
 As plants are raised within glass or plastic vessels in a high relative
humidity, and are not usually photosynthetically self-sufficient, the young
plantlets are more susceptible to water loss in an external environment.
 Plants may therefore have to be hardened in an atmosphere of slowly
decreasing humidity and increased light.
 If plants are produced via callus culture, the chances of producing
genetically aberrant/ somaclonal variation plants may be increased.
 If all plants are genetically similar, there is reduction in genetic diversity.
 If precautions were not taken, the whole stock many be contaminated or
infected.
79