C H A P T E R
11
Micropropagation
Saurabh Bhatia, Kiran Sharma
O U T L I N E
11.1 Introduction 361 11.4.2 Stage I: Aseptic Culture
Establishment 365
11.2 Types of Explants Used
11.4.3 Stage II: Multiplication
in Micropropagation 362
of the Explants 365
11.2.1 Meristem Culture 362
11.4.4 Stage III: Germination
11.2.2 Shoot-Tip Culture 363
of Somatic Embryo
11.2.3 Seed Culture 364
or Rooting of Regenerated
11.2.4 Single-Node Culture 364
Shoots 366
11.3 Methods of Plantlet Production 11.4.5 Stage IV: Hardening 366
Used in Micropropagation 364
11.5 Economic Significance 366
11.3.1 Direct Organogenesis 364
11.3.2 Indirect Organogenesis 364 11.6 Applications and Merits
11.3.3 Axillary Bud Method 364 of Micropropagation Over
Conventional Plant
11.4 Stages Involved
Breeding 367
in Micropropagation 365
11.4.1 Stage 0: Stock/Elite References 367
Plants Selection 365
11.1 INTRODUCTION
The method for obtaining large numbers of clonal explants in a short duration is called
micropropagation. It can also refer to use of a tissue culture technique for clonal propaga-
tion of plants. This can be feasible in a short space of time. Micropropagation methods were
used in 1960 by George Morel for the production of orchid plants at a commercial level [1].
Generally sexual and asexual methods are used for propagation of plants. Sexual methods of
propagation involve pollen grains to fertilize eggs and seeds that are produced at a high degree
Modern Applications of Plant Biotechnology in Pharmaceutical Sciences. [Link]
Copyright © 2015 Elsevier Inc. All rights reserved.
361
362 11. Micropropagation
of heterogeneity. In vegetative propagation or asexual methods mitotic division of plant parts
is used for the production of new varieties. In this case, both the new plants and parent plants
are genetically identical to each other. Clonal propagation refers to the multiplication of ge-
netically identical copies of plants. A clone is an individual plant obtained by sexual methods.
Cutting, budding, and grafting are the usual techniques used in vegetative propagation and
together this is known as in vivo clonal propagation of plants. In vivo clonal propagation is
mostly expensive, difficult, and unsuccessful. A better alternative approach for this purpose
is tissue culture [2].
The commonly used explants in micropropagation for initiation of the culture are meristem,
shoot tip, and axillary buds. The shoot apical portion consists of a group of actively dividing
meristematic cells at meristem and shoot tip. Shoot tip and meristem were clearly defined by
Cutter in 1965. The terminal portion of the shoot that contains actively dividing meristematic
cell mass is the meristem or apical meristem and its length is about 100–250 mm [3].
The terminal meristematic portion with one to three leaf primordia measures about 100–
500 mm in length and is referred to as shoot tip and shoot apex. Shoot apex is sufficient explant
for in vitro clonal propagation, but for the production of disease-free varieties of plants as in
meristem cultures, it is important to excise the meristem without any surrounding tissue. The
axile portion of the node contains axillary buds, which are made up of meristematic cells and
can also be used as explants. Based on the physiological state of the plant they may either be
in an active or a dormant state. The axillary bud method was once employed for carnation but
is now rarely used. Large-scale micropropagation of gerbera and strawberry is widely done
using the axillary method [4]. Several strategies that are currently used in micropropagation
and the major steps involved are highlighted in Fig. 11.1.
11.2 TYPES OF EXPLANTS USED IN MICROPROPAGATION
11.2.1 Meristem Culture
Meristem culture is used for shoot apical meristem culture in vitro. Meristem culture was
developed by Morel and Martin in 1952 for rivers eliminating from Dahlia [5]. Orchid Cym-
bidium was micropropagated using meristem culture by Morel in 1965 [6]. An already existing
shoot meristem grows in the meristem culture and adventitious roots regenerate from these
shoots. In the shoot tip beyond the youngest leaf lies the primordium meristem. It measures
up to 250 mm in length and 100 mm in diameter. In addition to the apical meristem one or
three leaf primordia would be present in a shoot tip of 100–500 nm. When virus elimination
is the objective, to obtain disease-free plants, shoot tips of up to 10 mm are used. For rapid
clonal propagation, a shoot-tip culture is followed in which (5–10 mm) explants are used.
Hence, the majority of meristem culture are essentially shoot-tip cultures. Various sized nodal
explants are also employed for rapid clonal propagation [7].
The size of the shoot tip used for culture is not important when the main objective is mi-
cropropagation. But when the objective is to obtain virus-free stock (or stock free from other
pathogens) it is necessary for the excision of the apical meristem to be done with a minimum
of the surrounding tissue. The shoot tip may be cut into fine pieces in order to obtain more
than one plantlet from each shoot tip. Pieces of curd (the inflorescence) are used in some spe-
cies such as cauliflower. Shoot tips or tissue pieces bearing buds of such stems may be used
11.2 Types of explants used in micropropagation 363
FIGURE 11.1 Illustration of the micropropagation process highlighting methods and major stages.
for those plants having an underground stem. Explants taken from actively growing plants
early in the growing season are the most suitable [8].
Explants of meristem are then placed on Murashige and Skoog’s (MS) medium, which is
considered to be an effective medium for the majority of species. Lower salt concentration
is suitable for some species. Fungicides (bavistin) or antibiotics (chloramphenicol/strepto-
mycin) can be added to the medium during growth to remove the endophytic contamination.
Similarly, meristem culture follows steps similar to micropropagation: (i) initiation of culture,
(ii) shoot multiplication, (iii) rooting of the developed shoots, and (iv) transfer of the plantlets
to the pots or soil [9].
11.2.2 Shoot-Tip Culture
Explants/shoot tips consist of shoot apical meristem, unexpanded leaves at various devel-
opment stages, and a number of leaf primordia about 1 cm in length. In shoot-tip culture the
explants are inoculated in cytokinin-supplemented media. Suppression of apical dominance
is caused by cytokinin and a highly branched shoot system formation is facilitated. Then ma-
nipulation of the shootlets is done in the rooting medium to develop plantlets [10].
364 11. Micropropagation
11.2.3 Seed Culture
The seed culture technique for micropropagation is effective for many dicots and monocots
as multiple shoots can be obtained from an embryo instead of single seedlings from a seed [11].
11.2.4 Single-Node Culture
Mature plants or seedlings containing single nodes can be used as explants as nodes con-
tain axillary buds, which when treated with cytokinin in in vitro conditions can lead to pro-
duction of branches. In rooting medium, such axillary shoots produce roots, thus forming a
number of plantlets [12].
11.3 METHODS OF PLANTLET PRODUCTION USED
IN MICROPROPAGATION
11.3.1 Direct Organogenesis
When axillary or apical meristems are difficult to obtain in culture, the direct organogenesis
method may prove to be useful for micropropagation. Any tissue of explants in this method
will produce shoots directly, e.g., root, cotyledon, and leaf may be used as the explants and
these produce plantlets on inoculation. Explant cells produce plantlets by directly undergo-
ing organogenesis [13].
11.3.2 Indirect Organogenesis
At the cut edges of the explants callus is formed that is known as dedifferentiation callus.
Organization of such a callus is done to give a new shoot. Manipulation of phytohormones is
required for indirect organogenesis [14].
11.3.3 Axillary Bud Method
In the axillary bud method, isolation of a shoot tip is achieved from which the axils of leaves
develop axillary buds under the effect of high concentrations of cytokinin. Apical dominance
is suppressed under the effect of high cytokinin concentration and permits the development
of axillary buds. If a number of side shoots or axillary shoots have been formed on the shoot
tip then their inoculation may be done on the fresh medium containing cytokinin. Often the
axillary method is used in combination with the single node method. Certain points must be
kept in mind concerning axillary shoot formation [15,16]:
• Often the ratio used in the axillary bud method between cytokinin and auxin is 10:1.
• Concentration of cytokinin is proportional to the developmental stage of the material/
culture. Adult material requires more cytokinin than juvenile material.
• Most plants do not react as well to kinetin as they do to 6-benzyladenine. The
requirement of cytokinin is therefore variable and must be adjusted according a
particular plant species or cultivar.
11.4 Stages involved in micropropagation 365
• Sometimes it is advisable to allow the development of the shoot tip prior to increasing
cytokinin concentration in order to induce axillary shoot formation.
• The apical meristem must be removed or killed if development of axillary shoot is
unsatisfactory.
• Axillary shoot formation is sometimes promoted by the liquid medium.
• Several substituted pyridylphenylurea compounds and thidiazuron stimulate axillary
branching in wide range of species.
11.4 STAGES INVOLVED IN MICROPROPAGATION
The various steps or stages involved in the complex process of in vitro clonal propagation
or micropropagation are summarized in four stages. The four stages of overall micropropaga-
tion were recognized in 1978 by Murashige [17]. Stages I to III were considered under in vitro
conditions and stage IV under greenhouse conditions. Stage 0 was introduced in the micro-
propagation system by Debergh and Mane in 1981 [18].
11.4.1 Stage 0: Stock/Elite Plants Selection
The first step in micropropagation is the selection of stock or elite plants having desirable
characters for their multiplication on a large scale. After selecting the stock/elite plants they
are maintained in controlled environmental conditions of low humidity, irrigation, and with-
out any systemic microbial infection for a period of 3 months for culture initiation.
11.4.2 Stage I: Aseptic Culture Establishment
In this stage, explants selected in Stage 0 are prepared for inoculation after treatment with
suitable sterilizing agent and established on a well-defined culture medium. The axillary bud,
shoot tip, and meristem are surface sterilized by using various chemicals such as 5% sodium
hypochlorite, 0.1% mercuric chloride, or 70% alcohol with various contact times either in
combination or alone depending upon the level of surface contamination. After surface steril-
ization the explants are inoculated onto the MS medium (or any other suitable basal medium)
supplemented with various growth regulators, vitamins, and sucrose. For micropropagation,
culture medium containing cytokinins (1–3 mg/L 6-benzyl amino purine) with less concen-
tration of auxin (indole-3-acetic acid) was used. The auxin 2,4-dichlorophenoxyacetic acid is
good because it suppresses organogenesis and stimulates formation of callus. The cultures
are incubated at a 16-h photoperiod at 3000–10,000 flux light intensity.
11.4.3 Stage II: Multiplication of the Explants
Multiplication of explants takes a considerable time through regeneration of shoots from
explants. In some cases only single shoots develop from the apical shoots and such shoots are
used for excising a number of nodal explants. Further cytokinin-rich media are used to inocu-
late nodal explants for multiple shoot proliferation. Multiple shoots may also be produced
during this process by somatic embryogenesis or organogenesis directly on explants. Each
366 11. Micropropagation
explant produces five to six shoots in a period of 4 to 5 weeks. If all the plants survive, single
explants would produce 510–612 plants in 1 year.
11.4.4 Stage III: Germination of Somatic Embryo or Rooting
of Regenerated Shoots
Rooting in fresh medium is induced by multiple shoots produced during stage II. In-
oculation of individually separated shoots is done to the rooting medium (with auxins)
and in some cases rooting is directly induced in the soil in conditions of high moisture.
The rooted plants are highly sensitive and fragile to the moisture. In the case of somatic
embryos, germination is allowed to form the plantlets and then they are transferred to the
soil. The plantlets, after going through hardening process, are slowly transferred to
the soil. The hardening medium should be either pearlite, peat, or vermiculite, hold-
ing considerable moisture and maintained at high humidity conditions before it is trans-
ferred to the soil.
11.4.5 Stage IV: Hardening
In the hardening stage the plantlets of stage III are prepared for external soil conditions
from their in vitro conditions. This involves the plants becoming resistant to stress, mois-
ture, and disease resulting in the autotrophic nature of plants from the heterotrophic nature
in in vitro culture conditions. Protection must be given to plantlets from direct sunlight and
a decrease in relative humidity should be done over a period of time. Well-developed roots
are formed by the plantlets during this acclimatization period and cuticular wax is also
formed in the aerial tissues. After this the plantlets become suitable for transfer to the open
fields [19].
In some cases vitrification is observed under in vitro conditions where some species or
shoots appear brittle, glossy, and water soaked. Vitrification is due to abnormal functioning
of stomata, poorly developed vascular bundles, and abnormal wax quality resulting in loss
of plantlets. This condition can be overcome by use of growth retardants, bottom cooling of
culture tubes, addition of high concentration of agar (1%), and addition of hydrolysate com-
pounds [20].
11.5 ECONOMIC SIGNIFICANCE
Throughout the world interest in natural drugs is increasing. The market for flavors, phar-
maceuticals, fragrances, and colors derived from natural plants is worth several billion dol-
lars per year. Vincristine, vinblastine, colchicine, taxol, forskolin, artemisinin, saponins, etc.
are phytochemicals of interest in medicine and biology and it has been possible to produce
these phytochemical compounds using plant tissue culture techniques such as micropropaga-
tion in short periods. Hence, the advanced plant tissue culture techniques serve to satisfy the
ever-growing needs of the commercial market for herbal drugs [21].
REFERENCES 367
11.6 APPLICATIONS AND MERITS OF MICROPROPAGATION OVER
CONVENTIONAL PLANT BREEDING [22–24]
The various applications of micropropagation are:
• Plant tissue in small amounts is sufficient for the production of millions of clones in a
year using micropropagation. It would take a great deal of time to produce an equal
number of plants using conventional methods.
• The technique of micropropagation provides a good alternative for those plant species
that show resistance to practices of conventional bulk propagation.
• An alternative method of vegetative propagation for mass propagation is offered through
micropropagation. Plants in large numbers can be produced in a short period. Any
particular variety may be produced in large quantities and the time to develop new
varieties is reduced by 50%.
• Large amounts of plants can be maintained in small spaces. This helps to save
endangered species and the storage of germplasm.
• The micropropagation method produces plants free of diseases. Hence, disease-free
varieties are obtained through this technique by using meristem tip culture.
• Proliferation of in vitro stocks can be done at any time of the year. Also, a nursery can
produce fruit, ornamental, and tree species throughout the year.
• Increased yield of plants and increased vigor in floriculture species are achieved.
• Fast international exchange of plant material without the risk of disease introduction is
provided. The time required for quarantine is lessened by this method.
• The micropropagation technique is also useful for seed production in certain crops as the
requirement of genetic conservation to a high degree is important for seed production.
• Through somatic embryogenesis production of synthetic artificial seeds is becoming
popular nowadays.
With micropropagation having various advantages over conventional methods of propa-
gation, this method holds better scope and future for production of important plant-based
phytopharmaceuticals. Independent of availability of plants, micropropagation offers a lu-
crative alternative approach to conventional methods in producing controlled amounts of
biochemicals. Therefore, intense and continuous efforts in this field will direct controlled and
successful production of valuable, specific, and yet undiscovered plant chemicals.
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