Micropropagation basics

1. By – Dr. Mafatlal M. Kher
Micropropagation
Date & Time : Wednesday, 08 September 2021
Semester : V
Program : B.Sc. Biotechnology
School : School of Science
Subject code : BSBO502 (Unit I)
1

2. Clonal propagation: True to type plants
 Clonal propagation of plants, which refers to multiplication of genetically identical
individuals by asexual methods of regeneration from somatic tissues or organs.
 It is a common practice in horticulture and forestry to preserve the desirable
characters of selected genotypes or varieties.
2

3. Clonal propagation: True to type plants
Traditionally, it is achieved by
 Cuttings,
 Layering,
 Splitting,
 Root suckers,
 Grafting and so on.
3

4. Why not seed?
 Due to heterozygous (in case of cross pollinated plant species) condition it is
difficult to obtained true to type plants.
 Bamboos can also propagated using seeds, but flowering is very rare in nature (70
years), hence limited numbers of seeds are produced.
 Plantlets produced using seed germination methods are generally infected with
seed born pathogen.
 Issues with seed viability (embryo maturity, seed dormancy etc)
4

5. Why micropropagation?
Limitations of traditional methods
 Time consuming, large space is required and limited plants can be produced
starting from a single individual.
 Very 0.5 to 1 meter of plant tissues are required to initiate vegetative propagation.
 The rate of multiplication is much very low.
 Vegetative propagation, however plantlet produced via vegetative propagation
carries diseased from stock plant
 Limited number of plants are produced via vegetative propagation for e.g. 5-7 root
suckers per year or 10-15 twigs suitable for vegetative propagation.
5

6. Why Micropropagation?
 Cultures are started
with very small peace
of plant material i.e.
explant (Hence, term
micropropagation is
used).
 Starting with 100
traditional cuttings;
able to produce
70,000 annual
clones. Start with
200 tissue culture
vials; produce 2
million annual
clones.
6

7. Why Micropropagation?
 In a relatively short time and
space a large number of plants
can be produced starting from
a single individual.
 Limited space is required to
maintain large number of
plants.
 Uses 1/10 the space of
traditional cloning. Two million
annual clones could be
produced in less than 3000
square feet.
 More suitable for long distance
transport.
7

8. Why Micropropagation?
8
Cryopreservation:
 1000 plus mother cultivars
could be stored inside a
refrigerator sized cyro bank
with no care or maintenance
for months, sometimes over
a year

9. Cryopreservation
9
Image source: Center for Plant Conservation National
Headquarters 15600 San Pasqual Valley Rd. Escondido, CA
92027-7000
Extra information

10. Why Micropropagation?: Cryopreservation
10
Extra information

11. Why Micropropagation?
 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.
 If the plants are derived from virus-
free stock they remain protected from
re-infection, and the micropropagated
plants can be exported with little
quarantine problem.
11

12. Why Micropropagation?
 The rate of multiplication in vitro is often
much faster than the in vivo methods of
vegetative propagation because in cultures
it is possible to manipulate the nutrient and
growth regulator levels, temperature, and
light more effectively.
• 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.
12

13. Why Micropropagation?
 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.
 It is applicable to many such genotypes for which in vivo vegetative propagation is
difficult or impossible.
 It goes on round the year.
 In vitro production can be better planned by storing the cultures at low temperature
in the season of low market demand, and
 Micropropagated plants may acquire new desirable traits, such as bushy habit of
ornamental plants and increased number of runners in strawberry.
13

14. Micropropagation technique
Micropropagation is a well-defined multistep process. It comprises of five steps, each
with its specific requirements and problems (Bhojwani and Razdan 1996):
 Stage 0: It is the preparatory stage to provide quality explants;
 Stage 1: Initiation of aseptic cultures;
 Stage 2: Multiplication;
 Stage 3: Rooting of in vitro formed shoots; and
 Stage 4: Transfer of plants to greenhouse or field conditions (transplantation)
14

15. Micropropagation technique
Stage 0: Preparatory Stage
 This step was initially introduced to check the contamination problem, especially
of fungi, but now it is considered to be important for the success of the
establishment and eventual multiplication at Stages 1 and 2, respectively.
 Any measure(s) taken to improve the quality of the parent plant, whether involving
its hygiene or physiological status, are included in this Stage.
 Contamination is one of the serious problems at Stage 1, and for successful
establishment of aseptic cultures, the hygiene of the mother plants is very
important.
 Generally, the explants derived from the plants maintained in green- house yield
better results.
15

16. Micropropagation technique
Stage 0: Preparatory Stage
 Stage 0 may also include manipulation of parameters such as light and
temperature regimes under which the mother plants are maintained
 The application of growth regulators, which could influence the response of
explants at the later stages.
16

17. Micropropagation technique
Stage 1: Initiation of Cultures
 The aim of this stage is to establish aseptic cultures of the plant to be
micropropagated using suitable explants.
 Although 100 % infection-free cultures are difficult to obtain but reproducibly high
percentage of aseptic cultures are desirable for a satisfactory micropropagation
protocol.
 The success at this stage is dependent on the choice of the right explant,
proper sterilization procedure, and preventing any hypersensitivity reactions
of the explants.
17

18. Micropropagation technique
Stage 1: Initiation of Cultures
 Explant:
 The choice of explant is mainly dependent upon the mode of regeneration and
multiplication desired and aim of the study.
 The explant most commonly used for micropropagation is either apical bud or nodal
segments with at least one axillary bud.
 If the aim is virus elimination one goes for meristem tip culture. Otherwise, meristem tip
culture should be avoided because of its poor survival and complex culture requirements.
 The choice of explants increases when the aim is de novo regeneration of shoots or
somatic embryogenesis. Direct or indirect adventitious bud formation can be obtained
from root, stem, leaf, or nucellus explants.
 Young immature zygotic embryos have been used for obtaining somatic embryogenesis in
cereals, legumes, and tree species.
 Nucellus is the explant of choice for obtaining somatic embryos in mango and citrus. The
only tissue from the adult plants of cashew that could be used for micropropagation is the
nucellus.
18

19. Micropropagation technique
Stage 1: Initiation of Cultures
 Browning:
 The explants from many plants, especially tree species and some horticultural crops,
release phenols, which upon oxidation form quinones and turns the medium black that
could be toxic to the tissue.
19

20. Micropropagation technique
Stage 2: Multiplication
 The success of micropropagation protocol is largely dependent on the efficiency of
this stage.
 Shoot multiplication is achieved through: (i) regeneration from callus, (ii) direct
adventitious bud formation from the explant, and (iii) forced axillary branching.
 Each of these methods has its own advantages and disadvantages.
 The choice of explant is mainly dependent upon the mode of regeneration and
multiplication desired and aim of the study.
20

21. Micropropagation technique
Stage 2: Multiplication
Regeneration from callus,
 Plant cells from almost all parts of a plant are capable of forming callus under
suitable culture conditions, and the callus can be induced to regenerate plants via
organogenesis or somatic embryogenesis.
 Both these methods have the potential for producing a large number of plants, the
latter being much more efficient.
21

22. Micropropagation technique
Stage 2: Multiplication
Regeneration from callus
Advantages of somatic embryogenesis
 Somatic embryogenesis is a highly efficient process capable of producing large
numbers of embryos that can germinate as zygotic embryos to form plants with a
primary root system.
 This is in contrast to a separate rooting step required for the de novo formed
shoots. Moreover, once the protocol for somatic embryogenesis is standardized,
the process is amenable to greater degree of control and scaling up for mass
production in bioreactors and automation
22

23. Micropropagation technique
Stage 2: Multiplication
Regeneration from callus, somatic embryogenesis continue…..
Despite the many advantages, an efficient and reproducible protocol for somatic
embryogenesis is not available for many species.
Why? (Limitations of somatic embryogenesis)
 Synchronization of embryogenic cultures is not easy to achieve and, often the
conversion of embryos into plants has been very poor due to morphological and
physiological abnormalities.
 The major disadvantage of somatic embryogenesis is an intervening callus phase
which can induce variability. Therefore, regeneration from callus is not the
preferred method for mass clonal multiplication.
 Yet, in vitro propagation of palms, such as coconut, oil palm, and date palm, has
been possible only through adventive embryony in callus cultures derived from
inflorescence segments or shoot tips from offshoots.
23

24. Micropropagation technique
Stage 2: Multiplication
Adventitious bud formation
 The shoot buds formed directly from any part of the plant other than apical or
axillary bud is termed adventitious bud.
 Strictly speaking, de novo regenerated shoot buds from callus are also
adventitious in nature.
 Many horticultural plants exhibit natural capacity to form adventitious shoots from
leaf pieces (Begonia, Saintpaulia) and root cuttings (blackberry, raspberry), which
have been exploited for clonal propagation of these plants.
24

25. Micropropagation technique
Stage 2: Multiplication
Adventitious bud formation (Advantages)
 The number of shoots per propagule can be considerably enhanced,
 Very small pieces of tissues that do not survive in vivo can form shoot buds in
cultures, and
 Many plants that do not exhibit adventitious shoot formation in nature do so in
vitro.
 Adventitious bud formation from leaf scales has been most exploited in the
Liliaceae and Iridaceae members. Using small segments of outer bulb scales
almost 100 bulblets could be obtained from a single scale of lily.
 This tremendous regeneration potentiality in lily has raised the technique to mass
production on a commercial scale in bioreactors.
 Genetic fidelity of plants obtained through direct adventitious shoot bud formation
is definitely greater as compared to those regenerated from callus.
25

26. Micropropagation technique
Stage 2: Multiplication
Forced axillary branching
 Normally, the buds present in the axil of leaves remain suppressed due to the well-known
phenomenon of apical dominance.
 Removal of the apical meristem stimulates the axillary bud to grow out into a shoot. In the
well- established horticultural practice of clonal propagation by stem cuttings, the new
plant develops from the axillary bud at the node.
 However, this process of clonal multiplication is very slow and is limited by the number of
cutting (10”–12” long with 2–3 nodes) that can be obtained from the mother plant.
 Exogenous application of growth regulators, particularly a cytokinin, can force the axillary
buds to grow even in the presence of terminal bud and increase the number of useable
flushes.
 This method of shoot multiplication is most reliable in terms of the genetic uniformity of
the micropropagated plants, and therefore is the most preferred method of in vitro clonal
propagation of plants.
 Moreover, clonal multiplication of commerical plants can be successfully achieved through
forced axillary branching.
26

27. Micropropagation technique
Stage 2: Multiplication
Forced axillary branching
 Normally, the buds present in the axil of leaves remain suppressed due to the well-known
phenomenon of apical dominance.
 Removal of the apical meristem stimulates the axillary bud to grow out into a shoot. In the
well- established horticultural practice of clonal propagation by stem cuttings, the new
plant develops from the axillary bud at the node.
 However, this process of clonal multiplication is very slow and is limited by the number of
cutting (10”–12” long with 2–3 nodes) that can be obtained from the mother plant.
 Exogenous application of growth regulators, particularly a cytokinin, can force the axillary
buds to grow even in the presence of terminal bud and increase the number of useable
flushes.
 This method of shoot multiplication is most reliable in terms of the genetic uniformity of
the micropropagated plants, and therefore is the most preferred method of in vitro clonal
propagation of plants.
 Moreover, clonal multiplication of commercial plants can be successfully achieved through
forced axillary branching.
27

28. Micropropagation technique
Stage 2: Multiplication
 Excess cytokinins or a wrong cytokinin used may cause epigenetic changes in the
plants obtained through forced axillary branching.
 Bushiness in the micropropagated Gerbera plants, which is accompanied by
excessive leaves, decrease in flower number, and short peduncle, has been
attributed to the use of excess BAP.
 Similarly, long exposures to culture conditions adversely affected the size and
shape of fruits in strawberry.
28

29. Micropropagation technique
Stage 3: Shoot Elongation and Rooting
 Somatic embryos are bipolar structures with root and shoot primordia and can,
therefore germinate to form complete plants.
 However, the shoots formed through regeneration from callus, direct adventitious
bud formation or forced axillary branching require an additional step of rooting for
complete plant formation.
 In some cases, the shoots formed in vitro by any of the three methods described
above, being continually exposed to cytokinin, may remain short and require an
intermediate elongation step before transfer to rooting medium.
 shoots to a suitable medium for rooting. Rooting is generally achieved by
transferring individual shoots (about 2 cm long) to a medium supplemented with a
suitable auxin.
29

30. Micropropagation technique
Stage 3: Shoot Elongation and Rooting
To cut down the cost of rooting of micropropagated shoots, many commercial companies
resort to in vivo/ex vitro rooting. For this, the micro- propagated shoots are treated as
microcuttings and planted directly in potting mix after treating the cut basal end with a
commercial rooting mix or an auxin solution. It has many advantages:
 The roots formed in vitro die after transplantation and the new roots are formed which
sustain the plant,
 The vascular connection between the in vitro formed roots and the shoot may not be well
developed,
 The in vitro formed roots lack root hairs that makes them less effective when transplanted,
 Transplantation is generally done by unskilled labor and the plants to be transferred being
very large, quite often the in vitro roots get damaged during this process, and
 Callusing at the junction of roots and shoot is often a problem faced with in vitro rooting.
 In vivo/ex vitro rooting not only cuts down the cost but also circumvents the above
problems associated with in vitro rooting.
30

31. Micropropagation technique
Stage 4: Transplantation and Acclimatization
Background information
The ultimate success of micropropagation depends on the establishment of the plants in soil or potting mix.
 The plants grown in vitro are exposed to the artificial environment of the culture vial, which is characterized
by the culture medium rich in inorganic and organic nutrients, sucrose and growth regulators, high humidity,
low light, and poor gaseous exchange.
 Under these unnatural conditions, the plants are able to grow well but suffer from many morphological,
anatomical, cytological, and physiological abnormalities, which necessitate their careful acclimatization to
the in vivo/ ex vitro conditions.
 The two main abnormalities of these plants are heterotrophic mode of nutrition due to the culture medium
being rich in organic nutrients and poor control of water loss.
 Under high humidly of the culture vials the leaves show poor development of cuticle, scanty deposition of
wax, abnormally large stomata, which do not close even in response to ABA, high CO2 or dark treatment,
poor differentiation of the mesophyll tissue, which mainly comprised spongy parenchyma, and poorly
developed chloroplasts with low chlorophyll content and disorganized grana.
 The lack of cuticle and impaired structure and movements of the stomata cause excessive water loss on
trans- plantation, reducing their chances of survival. Therefore, the in vitro plants should be carefully
hardened (acclimatization) before transfer to field conditions.
31

32. Micropropagation technique
Stage 4: Transplantation and Acclimatization
Acclimatization
The principle governing acclimatization of the in vitro plants, habituated to grow under high humidity and low light
heterotrophic conditions is to make them grow under low humidity and high light autotrophic conditions. It takes about
4–6 weeks for hardening the in vitro plants, so that they can survive under normal conditions.
 The individual micropropagated plants are first taken out of the agar medium, the roots are washed to remove the
agar and individually planted in pots containing appropriate potting mix (peat, vermiculite, perlite, polystyrene
beads, or coco compost).
 The plants are irrigated with a mild nutritive solution such as Knop’s solution or one-fourth strength MS salt
solution.
In Research and teaching lab:
 The simplest method to maintain high humidity around the transplanted in vitro plants, often used in research and
teaching laboratories, is to cover them with plastic bags perforated with small holes for air circulation.
 The plants are maintained in shade or low light for about 15–20 days, and slowly acclimatized to low humidity
conditions by removing the polybags for few hours every day in the beginning and slowly increasing the time of
exposure until the plants are able to withstand complete removal of the cover.
 The plants during this phase are hardened to survive on inorganic nutrition and their photosynthetic machinery is
reactivated to become autotrophic. The plants are able to survive under field conditions only when new roots and
leaves are formed.
32

33. Micropropagation technique
Stage 4: Transplantation and Acclimatization
Acclimatization
In Commercial lab:
 The plants are transplanted into protrays having 96 holes, which are easy to handle for irrigation and transfer
purpose and drastically cut down on the labor for handling the plants.
 High humidity is maintained by fogging or misting, of which fogging, with extremely small droplets.
 In tropical countries high humidity, cooler temperatures and low light conditions are maintained in large polyhouses
fitted with heavy duty exhaust fans at one end and pads, constantly drenched with water, at the other end (fan and
pad system).
 Within the polyhouse, the plants in protrays are shifted from high humidity near the pad end to low humidity near
the fan end over a period of about 4 weeks.
 During this period, the plants are irrigated with high phosphorous containing nutritive solution to promote rooting,
and as the protrays are moved toward the fan the fertilization is changed to solution containing equal NPK.
Frequent sprays offungicides and insecticides are necessary as these conditions are conducive to the growth of
pests and pathogens. Alternately, the rooted plants could be par-
 tially hardened in vitro by reducing the humidity inside the culture vessel by gradually unscrew- ing the lid of the
culture jar over a period of 3–4 weeks. The humidity inside the culture vial could also be reduced by using
desiccants, use of culture vials with microporous closure to allow gaseous exchange and cooling the bottom of the
culture vessels.
33

34. Suggested reading
34