UINVERSITY OF NATURAL RESOURCE AND ENVIRONMENT

STA 322.4 AGRICULTURAL BIOTECHNOLOGY

The Role of Tissue Culture in Crop Improvement

Robert Plak Pawilnga

Plant Breeding and Biotechnology

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TABLE OF CONTENTS PAGE

1.0 INTRODUCTION 3

2.0 PLANT TISSUE CULTURE TECHNQUES AND

THEIR APPLICATION 4

2.1 Embryo Culture 5

2.2 Meristem culture 6
2.3 Shoot Culture 7
2.4 Callus Culture 7
2.5 Cell Suspension Culture 10
2.5.1 Somaclonal variation 10
2.5.2 In vitro selection 11
2.6 Protoplast Culture 12
2.7 Anther/Ovule Culture 13

3.0 TISSUE CULTURE AND GENETIC ENGINEERING 14

3.1 Tissue Culture Systems 14

3.1.1 Cell suspension cultures 14
3.1.2 Embryogenic callus 15
3.1.3 Protoplast cultures 15

3.2 Transformation Systems 15

3.2.1 Agrobacterium-mediated genetic transformation 15
3.2.2 Particle bombardment 16
3.2.3 Protoplast electroporation 16

4.0 TISSUE CULTURE AND DISEASE ELIMINATION 16

4.1 Bacterial and Fungal Contamination 17

4.2 Virus infection in plants 17
4.2.1 Detection of viral diseases 17
4.2.2 Elimination of viruses 20
4.3 Perspective of disease freeness 21
4.4 Meristem Culture 21
4.5 Callus Culture 21
4.6 Other In Vitro Methods 22
4.7 Thermotherapy 22
4.8 Chemotherapy 23

5.0 CONCLUSION 23

REFERENCE 24

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 The Role of Tissue Culture in Crop Improvement *
1.0 INTRODUCTION

Plant tissue culture involves the culture of all types of plant cells (including
protoplast), tissues and organs (including embryos) under aseptic conditions.
Plant tissue culture has an important role to play in the manipulation of plants for
improved agronomic performance. It is an integral part of molecular approaches
to plant improvement and acts as an intermediary whereby advances made by
the molecular biologists in gene isolation and modifications are transferred to
cultured plant cells. The transformed plants that are regenerated in culture can
then be evaluated by the geneticist and plant breeder. Tissues of many plants
species are difficult to culture and require optimisation of in vitro growing
conditions. Therefore, there continues to be an ongoing need for extensive work
in the field of basic tissue culture methods for many crop plants before any
practical utilisation of molecular biology approaches in agriculture can be
achieved.

This bulletin is intended to highlight those tissue culture techniques that are
currently being applied to plant propagation and improvement. The role of tissue
culture and the advantages and disadvantages of particular tissue culture
techniques are also discussed. While many of the techniques are well
established, this document provides tangible evidence of the role tissue culture
has played and will continue to play in crop improvement and in the development
of the agricultural industries at large. The areas under review include: (1)
embryo culture, (2) meristem culture, (3) shoot culture, (4) callus culture, (5) cell
suspension culture, (6) protoplast culture and (7) anther/ovule culture. Problems
and limitations of each of the techniques are also discussed.

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2.0 PLANT TISSUE CULTURE TECHNIQUES AND THEIR APPLICATION

Plant tissue culture can be defined as the culture of all types of plant cells
(including protoplast), tissues and organs (including embryos) under aseptic
conditions. There is appreciable overlap between different techniques so that
success in one research area is very much dependent on success in other areas
(i.e. a system for reliably regenerating plants from callus cultures would appear
to be a pre-requisite if plants are to be obtained from protoplasts or if successful
gene transfer at the cell level is to be exploited at the whole plant level). The
tissue culture techniques and applications reviewed include: (1) embryo culture,
(2) meristem culture, (3) shoot culture, (4) callus culture, (5) cell suspension
culture, (6) protoplast culture, and (7) anther/ovule culture. These techniques
and their uses are summarized in Table 1.

Table 1: Tissue culture techniques and their uses in crop improvement

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Type of Culture Uses
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1) Embryo culture - shortening of breeding cycles
- overcoming incompatibility

2) Meristem culture - eliminating pathogens

- mass cloning of plants
- germplasm collection
- cyropreservation

3) Shoot culture - mass cloning of plants

- germplasm collection

4) Callus culture and - cloning of plants

5) Cell suspension culture - creation of genetic variants
- elimination of pathogens
- source of protoplast
- production of 20 metabolites

6) Protoplast culture - somatic hybridization

- transformation of plants

7) Anther culture - production of homozygotes

- mutation induction
----------------------------------------------------------------------------------------

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 2.1 Embryo Culture

Embryo culture was one of the first tissue culture techniques to be applied to
plant breeding. It involves the removal of the embryo from the seed and
subsequent growth in vitro until the developing plant can be transplanted to soil
and grown to maturity. Usually, late-stage embryos are used for embryo culture
but attempts have been made to culture early-stage embryos (i.e. globular and,
heart-shaped stages). Late-stage embryos can usually be cultured on simple
nutrient media but as more immature embryos are cultured, the hormone and
growth factor requirements become more specific and success is less frequent.

The starting point for all plant breeding programmes is genetic variation. Without
genetic variation for any particular character, advance is impossible. Within most
economically-important plant species, there are certain traits for which it is
difficult or impossible to identify genetic variation. This phenomenon is seen for
many characters of agronomic importance, such as pest and disease resistance.
Often, wild or domesticated related species will have such genes, which are of
interest to the geneticist/plant breeder.

The purpose of embryo culture, in most applications, is to recover plants from

their embryos during attempts at wide hybridisation by sexual crosses between
distantly related plants. The incompatibility between these plants, in many cases,
is caused by breakdown of the endosperm which nourishes the developing
embryo. By ‘rescuing’ the embryo and growing it on an appropriate medium, a
plant can be grown to maturity. Embryo culture is also used to break dormancy in
seeds, thereby shortening the breeding cycle by months or even years. It can
also be used when important seed lots have lost viability during storage and
have poor germination. The primary reason for attempting wide crosses is the
transfer of desired traits (e.g. disease resistance, stress tolerance etc) from
distantly related species to cultivated varieties. Embryo culture has been applied
successfully with interspecific crosses in cotton, tomato, barley, rice, cabbage,
melons, beans, jute etc). It has also been successful in some intergeneric
crosses such as barley x rye, wheat x rye and wheat x Elyrnus (Wagih, 1996).

The culture of unfertilised ovules and ovaries with subsequent fertilisation in vitro
is another approach to the problem of wide hybridisation. With further
understanding of the process of fertilisation in plants, and of the factors that
influence incompatibility, these techniques may have greater application in the
future.

In some inter-specific systems, pollen will not germinate, or will abort before
fertilization takes place (e.g. rice), or if fertilization takes place, there may be
some incompatibility resulting in seed abortion (e.g. oilseed rape, mungbean)
before maturity, or the development of seed with non-viable embryos. Hence,
barriers to hybridization can be pre-fertilization or post-fertilization. Successful
hybridization involves the following processes: (1) pollen germination, (2) pollen

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tube growth, (3) fertilization, (4) embryo and endosperm development (5), and
seed maturation. Each step may be a barrier to the formation of viable seed.

To overcome this problem, there are three possible solutions, all of which involve
the use of tissue culture techniques. The simplest, and often most successful
involves embryo rescue or use of in vitro pollination and fertilization to overcome
pre-fertilization incompatibility.

In vitro fertilization is used to overcome self-incompatibility for producing wide

hybrids of crops such as cotton, tobacco and maize. To successfully use such
methodology it is essential to identify the stage of floral development when
viable, obtain contaminant-free pollen, and to establish in vitro conditions under
which the pollen will germinate (dependent on many factors such as the medium,
phytohormones, pollen dosage, temperature, humidity etc.). A great deal of
experimentation is necessary to optimize this system in most species.

2.2 Meristem Culture

Meristem culture involves the removal of the axillary meristem or the apical
meristem with two or three leaf primordia and subsequent culture on nutrient
media. These meristems are domes of actively dividing cells. Crop plants that
are infected with pathogens result not only in subsequent reduction in their
vigour, quality and yield but also constitute a barrier to international exchange of
germplasm. Unlike fungal and bacterial diseases of plants, viral diseases are
persistent and are not curable with any commercially available treatment. In
plants, endogenous contaminants do not easily invade or rapidly multiply in the
apical meristem and often resulting in the formation of disease-free plants. When
combined with micropropagation techniques, large numbers of disease-free
plants may be produced from meristematic explants. Auxiliary-buds, however,
are normally contaminated and require substantial cleaning and eradication of
contaminants (Hartmann and Kester, 1983).

Viruses are generally unevenly distributed throughout the infected plant. The
apical meristems are frequently devoid of viral particles or contain very low viral
concentrations, and the titre increases with distance from the meristem tip. The
suggested reasons for the escape of the meristem from virus invasion are: A)
viruses move readily in a plant body through the vascular system which is absent
in the meristem, B) mitosis in the meristem cells compete with virus
multiplication, C) a virus “inactivating system” has highest activity in the meristem
and other regions, D) there is a high auxin level in the meristem which may
inhibit virus multiplication and E) tissue injury caused by excision of the meristem
may result in virus degradation.

This technique has been used for rapid production and conservation of wide
range of species including banana, yam, plantain, oil palm, sweet potato, potato,
and cassava and production of virus-free plants such as sugarcane, potato,

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garlic, taro, etc. To achieve successful viral elimination from plants a number of
important steps have to be developed: A) methodologies must be available for
identifying infected plants, B) tissue culture techniques must be developed
(aseptic meristem or bud-culture) for elimination of the virus and C) methods
should be developed for the rapid and reliable diagnosis (i.e. Enzyme-Linked
Immune Sorbent Assay - ELISA or with use of Polymerase Chain Reaction-
PCR) to ensure the tissues have become virus-free.

2.3 Shoot Culture

Shoot culture can be considered as an extension of the more traditional methods

of plant propagation. Its aim is the rapid clonal multiplication of superior
genotypes of pest and disease-free plants. Shoots tips or nodes with buds from
a desired plant are placed on an appropriate culture medium under specific
growth conditions to enhance production of axillary shoots and roots. Subculture
of the buds and shoots is repeated until many plants are produced all having the
genetic characteristics of the original plant.

In many instances conventional propagation is a slow process during which

disease and pest problems can limit production. Micropropagation offers the
potential to produce thousands, or even millions, of plants per annum. In many
cases the rapid increase serves as a valuable initial boost for the establishment
of large populations prior to multiplication by conventional means.

Micropropagation also provides a means of germplasm storage for maintenance

of disease-free stock. Space requirements are small and plants can be grown in
a controlled environment free of the vagaries of weather, diseases and pests,
which constantly threaten field gene banks. Conditions can also be created in
which the growth rate is slowed to minimise subculture requirements. When new
stock is required, subculture can be resumed under optimal growth conditions.
An extension of this approach has been the cryopreservation of meristems
whereby germplasm may be stored indefinitely in liquid nitrogen.

2.4 Callus Culture

Callus is basically a non-organised tumour tissue, which usually arises on

wounds of differentiated tissues and organs when cultured. It is possible with
many different plant species to produce a callus through hormone action and
then to grow this further on a new medium. In exceptional conditions, and
sometimes spontaneously, the regeneration of adventitious organs and/or
embryos can occur from a callus. In a liquid medium a callus may form
aggregates or individual cells.

Cultured explants consist of differentiated cells, which may then undergo de-
differentiation under the influence of plant growth regulators (PGR) present in the
nutrient media. The exogenous regulator requirement depends strongly on the

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genotype and endogenous regulator content. With most species auxin is
required (Tisserat, 1985) while with others auxin and cytokinin, or cytokinin alone,
are required. Other factors such as carbohydrate source and concentration,
media additives (casein hydrolysate, malt extract, coconut milk etc.) are
important for good callus formation.

When embryos are generated on the callus surface or in liquid medium this is
referred to as somatic embryogenesis. Somatic embryos have a great similarity
with zygotic embryos. Callus cells which go on to form embryos are often small in
size, with a dense cytoplasm, large nuclei, prominent nucleoli, small vacuoles
and many starch grains. The development of an embryo from the embryogenic
cells is usually accomplished by the absence of auxin in the medium. Somatic
embryogenesis as a means of propagation is seldom used, however the
technique is used to produce mutant plants or somaclones which may have
some value in future breeding programs.

Somatic embryos can be produced from cells growing in suspension, thereby

making possible batch culture techniques, which can be scaled-up with minimum
handling costs. The multiplication rate in some plants (e.g. carrots, tobacco,
potato, celery) is very high and in the case of carrots, celery and tomato the
embryos have been encapsulated and treated as artificial seeds. Commercial
companies have patented a process of producing synthetic seeds by
encapsulating embryos in a biodegradable polymer.

Somatic embryos formed in cultures have generally been called “embryoids”, but
other names such as “accessory embryos”, “adventitious embryos”, and
“supernumerary embryos” are also found in the literature. Up to now, over 30
families have been used in experiments on somatic embryogenesis. In carrot,
somatic embryos are initiated in callus from superficial clumps of cells associated
with highly vacuolated cells which do not take part in embryogenesis, and the
embryoid-forming cells are characterized by their dense cytoplasmic content,
large starch grains and relatively large nucleus. These phenomena are very
similar to those found in Saccharum officinarum. Staining techniques reveal that
the carrot embryogenic cells are capable of passing through the same sequence
as zygotic embryo formation (globular stage, heart stage and torpedo stage
(Fig.1).

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Fig. 1. A schematic representation of the sequential stages of somatic embryo development.
Somatic embryos may develop from single cells or from a small group of cells. Repeated cell
divisions lead to the production of a group of cells that develop into an organised structure known
as a ‘globular-stage embryo’. Further development results in heart- and torpedostage embryos,
from which plants can be regenerated. Zygotic embryos undergo a fundamentally similar
development through the globular (which is formed after the 16- cell stage), heart and torpedo
stages. Polarity is established early in embryo development. Signs of tissue differentiation become
apparent at the globular stage and apical meristems are apparent in heart-stage embryos.

(A) Single cell stage (D) heart-shaped stage

(B) Small cell aggregate (E) torpedo stage
(C) Globular stage

Embroids derived from cell cultures have many distinctive features, some of
which are useful and may be of great value in the future. An embryoid is a
complete plant in itself. It has both shoot and root, and little effort is required to
induce the seed to germinate and grow. In contrast, an organogenetic plant,
which produces only a shoot at the early stages, needs considerable work in
changing the media to suit each stage of development before it becomes a
plantlet. If synchronization of growth can be readily controlled, somatic
embryogenesis will be amenable to mechanical handling, since it usually
produces a large number of embryoids per volume of medium which float freely.

Somatic embryogenesis is preferred to organogenesis as a method for

regenerating plants from cell culture, especially in conjunction with in vitro
selection and anther culture. The main reason is that the whole plant develops
from the somatic embryo and only requires growth to maturity. When
organogenesis occurs, shoots or roots develop from callus and induction of the
complementary structures frequently requires different culture requirements and
media formulations. This can sometimes be achieved only with difficulty or not at
all.

Although callus production is relatively easy for most plant species, somatic
embryogenesis and regeneration of plants from such unorganised cells, (callus
and cell suspensions), is usually a difficult process. In addition, although it is

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recommended that somatic embryogenesis is to be used for the rapid clonal,
multiplication of an important plant, there are numerous reports that show
generally regeneration from callus and cell suspensions may lead to genetic
variation in regenerated plants. Therefore, some caution must be exercised in
the use of these techniques as cloning methods.

Some secondary metabolites normally found in vitro are reported to be absent in

actively dividing cells in callus or suspension culture. Through mutation
selection, one might obtain such chemicals, or even new chemicals which never
appear in vivo. Alternatively, the synthesis of such a metabolite might begin
again in early embryogenesis so that the mature embryo ends up containing a
high level of it. The discovery of this possibility seems certain to lead to the
future improvement of techniques for obtaining secondary metabolites in vitro.

2.5 Cell Suspension Culture

Most suspension cultures are obtained by transfer of callus clumps to agitated

liquid medium of the same composition as that used for callus growth. Agitation
rates on orbital shakers should be in the range of 30-150 rpm with an orbital
motion stroke of 2-4 cm. At the first subculture into fresh medium, large clumps
of initial inoculum should be removed to allow smaller clumps to grow. For each
cell culture there is also a minimum inoculum size below which the culture will
not grow. The lag phase of the culture increases in length as the inoculum size
decreases towards the minimum level. Cell numbers in finely divided
suspensions can be determined using a haemocytometer.

Plant cell suspension cultures provide the scientist with a relatively homogenous
population of cells, readily accessible to exogenously applied chemicals and
growing under defined aseptic conditions. Cell suspension cultures are widely
used as model systems for studying pathways of secondary metabolism, gene
expression, as well as being a useful stage for in vitro selection and gene
transfer.

2.5.1 Somaclonal variation

As it is for the callus, the greatest disadvantage of some cell suspension culture
systems is the chance that genetic variation and mutation may occur. When
heritable genetic variation is observed in plants, which have been regenerated, in
vitro, then the term somaclonal variation is used. The occurrence of mutations is
not easy to ascertain. Various factors have been attributed to its origin, for
example cytoplasmic or nuclear mutations, polyploidy or other chromosomal
abnormalities in vivo or in vitro, or possibly transposable elements. It has been
suggested that somaclonal variation may be a way of generating useful genetic
variation and if selection for desired traits was performed in vitro somaclonal
variation may be a powerful tool for the production of tolerance to environmental

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stresses. In recent years researchers have directed increasing effort towards the
isolation of mutant from plant tissue cultures as a result of somaclonal variation.

Amongst the cereals, somaclonal variation has been studied in detail in several
species, e.g. wheat, maize, sorghum, and rice. In rice, alteration of
characteristics such as height, fertility, seed production, salt tolerance and
submergence tolerance have been reported. In some studies the rate of
somaclonal variation has been promoted by the use of ionizing irradiation
(gamma, x-rays etc.) or chemicals (ethylmethane-sulphonate, colchicine).

2.5.2 In vitro selection

In vitro selection is a useful tool in identifying plants, which are resistant or

tolerant to stresses produced by phytotoxins from pathogens, herbicides, cold
temperature, and aluminium, manganese and salt toxicity. In vitro selection
usually involves subjecting a population of cells to a suitable selection pressure,
recovering any variant lines which have developed resistance or tolerance to the
stress, and then regenerating plants from the selected cells. This approach
presumes that tolerance operating at the unorganised cellular level can act, to
some degree of effectiveness, in the whole plant. If the tolerance has a genetic
basis then the trait can be transferred to other plants.

While straight-forward in theory there are a number of technical obstacles that

must be overcomed in producing new plants through in vitro selection. In the first
instance it is important that there is a reproducible system for regenerating large
numbers of plants from unstressed cells, as the selecting agent frequently
reduces the ability of the cell to regenerate plants. Tolerance to the stress should
operate at both the cellular and whole plant level so that there is a greater
chance recovering desirable plants. Unfortunately, many of the agriculturally
important traits are multi-genic and depend on the structural and physiological
integrity of the whole plant. Traditional breeding and selection, at the whole plant
level, has been successful in incorporating many of these traits into cultivated
varieties. In vitro selection will complement these traditional methods and should
be of most use where no natural resistance is found within a species, or where
conventional breeding practices are difficult within a species.

Selection with cultured cells may yield a large number of mutants. Selection for
resistance is the most straight forward method for mutant selection, whereby
resistant cells in a large population can be selected by their ability to grow in the
presence of an inhibitor while the sensitive cells do not. The method can be very
powerful since millions of cells can usually be screened quite easily. Research
has been conducted to select many cell lines resistant to herbicides and
attempts have been made to select for salt and drought tolerant cells.

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The inhibitor concentration used in the selection experiments, will, of course,
depend on the sensitivity of the plant cell line under study. Therefore, a dose-
response curve with respect to growth inhibition must be performed for each
selection agent of interest.

Selection of inhibitor-resistant plant cells from calli is probably one of the least
complex methods. Both one-step selections, and multi-step selections have
been accomplished. In this procedure small uniform sized callus pieces (25-250
mg fresh wt) are placed on to fresh agar medium supplemented with a lethal,
sublethal or borderline lethal concentration of the inhibitor. Upon incubation,
resistant cells will usually be observed as a sectoring of vigorous growth.
Selection of resistant cell lines from cell or protoplast preparations offers several
advantages. Protoplasts and cell cultures can be prepared directly from whole
plant tissue and, more importantly, the clonal origin of cell lines arising from
selections using individual cells is more assured than with callus cultures.

Although most changes are deleterious or of no commercial value, there are

some instances where somaclonal variation has produced agriculturally useful
changes in the progeny [e.g. sugarcane - increase in cane and sugar yield, and
resistance to eye-spot disease, potato - improvement of tuber shape, colour and
uniformity, and late blight resistance, tomato - increased solids, resistance to
Fusarium.

Stability of the altered phenotype is an important consideration. For instance,

eye-spot disease resistance in sugarcane induced through somaclonal variation
was lost after 10 years of asexual propagation, indicating epigenetic variation.
Stable genetic changes can also be induced through somaclonal variation and
be inherited in a Mendelian fashion, as indicated from work with tomato. Studies
such as these indicate that once new variants are identified they need to be
rigorously field tested in replicated plots to ascertain genetic stability.

One of the limitations of mutation breeding is the number of individuals that must
be examined before those with desirable characteristic are identified. If a
selection pressure can be applied to a large population of somatic cells to
uncover a desired phenotype (i.e. in vitro selection) then the number of plants
that eventually require field evaluation would be decreased and more direction
built into a program.

2.6 Protoplast Culture

Protoplast culture is the sterile isolation of the cell protoplast with the goal of
genetically modifying the cell. The technique has been used for a number of
different cell modification techniques including protoplast fusion and direct DNA
transfer (passive uptake, electroporation, microinjection and microprojectile
bombardment, etc.).

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Plant protoplast are single cell which have had the cell wall removed by
mechanical and/or enzymatic methods, and are bound only by the plasma
membrane. The fact that protoplasts have no cell wall means they are useful for
a great number of protocols, particularly for those regarding genetic
manipulations.

Regeneration of plants from protoplasts is generally more difficult to accomplish

than from cell and callus cultures. This difficulty has limited the widespread
application of protoplasts in somatic hybridisation and gene transfer studies.
This has been particularly true with sugarcane and cereals, which have been one
of the most recalcitrant group of crop plants to culture because of problems with
developing a reproducible method of establishing cultures capable of
regeneration. Nevertheless, successful regeneration of whole plants from
protoplasts of an increasing number of plant species is possible (e.g. potatoes,
carrots, tobacco, sugarcane) and the list of such plants can be expected to grow.

2.7 Anther/Ovule Culture

Anther (or ovule) culture is the sterile isolation of the anthers (or ovules) and
development of a haploid callus culture in vitro. The technique is useful for a)
the production of haploids for the rapid production of homozygotes and b) the
easy selection of mutant forms. Natural haploids of higher plants are rare and
restricted to a few species (e.g. tobacco, cotton, maize, rice) Geneticists and
plant breeders have therefore sought dependable methods for the production of
haploid plants for the purpose of obtaining mutants at a much higher frequency
than from diploids, and also for producing homozygous breeding lines after
chromosome doubling.

Anther culture is one means of producing haploid plants. The technique involves
the removal of anthers and/or immature pollen from a plant and placing them on
a suitable culture medium which will induce regeneration from the haploid tissue
of the microspores or pollen. The culture of unfertilised ovules is another method
of obtaining haploids. The chromosomes of the resultant plants are then doubled
to the normal diploid state using colchicine. Chromosome doubling can also
occur spontaneously.

Haploid cells are useful in in vitro selection schemes and have been used
successfully to produce plants resistant to various metabolic inhibitors,
environmental stresses, herbicides and phytopathotoxins. Also, apart from
fundamental genetic studies with homozygous and isogenic lines, homozygous
plants are important in the production of F1 hybrids which are of increasing
importance in agriculture. Anther culture offers the possibility of obtaining
homozygous plants in a matter of months rather than the years of inbreeding
required by traditional breeding methods.

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Although the potential uses of haploids are apparent, the use of anther culture
for the routine production of haploids has been limited. Most progress has been
with the Solanaceous species but with many other agriculturally important plants,
such as cereals and legumes anther culture has been more difficult. Even when
production of haploids is possible, it is usually at a low frequency (1-10% of the
anthers cultured) and requires a considerable investment in time and labour to
generate the numbers needed for critical evaluation in a breeding program.
Another difficulty lies in distinguishing plants that regenerate from diploid somatic
tissue from spontaneously doubled haploids. In spite of these difficulties,
progress is being made and the routine production of haploids from a wider
range of species is increasing each year.
Many factors will affect the ability of anthers to form callus, embryoids or shoots.
Briefly, these include:

a. Donor plant physiology (e.g. photoperiod, temperature, nutrient);

b. Development stage of pollen;
c. Anther/bud pre-treatment (e.g. cold, dark);
d. Basal medium composition (e.g. maltose for barley);
e. Phytohormone balance;
f. Planting density;
g. Culture environment (e.g. light, photoperiod, temperature, etc), and
h. Donor genotype - this is often the most important.

3.0 TISSUE CULTURE AND GENETIC ENGINEERING

The availability of a good in vitro system is essential for genetic transformation.

The main characteristic of such a system is that a normal true-to-type plant can
be regenerated from the explant material at a high frequency. The tissue culture
techniques of potential are: (i) cell suspensions, (ii) embryogenic callus and (iii)
protoplast cultures.

3.1 Tissue Culture Systems

3.1.1 Cell suspension cultures

Homogeneous embryogenic cell suspension cultures are the material of choice

for genetic transformation because most cells are totipotent and regeneration
proceeds through somatic embryogenesis. As such, low risk of chimerism is
expected during the selection of transgenic plants. The main bottleneck is the
difficult and time-consuming initiative process as well as the maintenance of
embryogenic cell suspensions.

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 3.1.2 Embryogenic callus

The embryogenic callus obtained on immature male inflorescences or

proliferating meristems also produces numerous immature somatic embryos at
the globular stage. These embryos can be multiplied in a temporary immersion
system where a multiplication can be obtained through adventitious somatic
embryogenesis.

3.1.3 Protoplast cultures

Embryogenic cell suspensions are the material of choice for the isolation of
regenerable protoplasts in many crops, specially monocot. A high protoplast
yield is obtained after enzymatic digestion of the cell wall. Plant regeneration
through somatic embryogenesis can be obtained after culture on feeder layers or
using high plating densities. The main obstacle to a wide application of
protoplast cultures for genetic transformation is that highly embryogenic cell
suspensions are required to obtain regenerable protoplasts.

3.2 Transformation Systems

Two categories of transformation systems can be distinguished. The first

comprises the indirect transformation systems of which Agrobacterium-mediated
transformation is the most important one. Agrobacterium tumefaciens is a natural
soil-borne bacterium, which has the far-reaching ability to transport a piece of
DNA, situated on a tumour-inducing plasmid, into the nucleus of a plant cell.
Direct gene transfer techniques fall into the second group, which comprises,
among other techniques, particle bombardment and protoplast electroporation.

Transferring cytoplasm from one genotype to another (possibly interspecific

transfer) can give rise to cytoplasmic male sterility, which is very useful for hybrid
breeding programmes. The method for transferring organelles is fusion of
irradiated protoplasts. The levels of irradiation are sufficient to inactivate the
nucleus, but not the organellar genomes (mitochondrial and chloroplast).

3.2.1 Agrobacterium-mediated genetic transformation

Numerous systems were developed for the Agrobacterium-mediated

transformation of in vitro tissues culture systems of many crops. Firstly, tissues
are bombarded with naked gold particles to induce a wound response. After a 3-
day recovery period, meristems are cultured for 30 minutes with the
Agrobacterium strain LBA4404 in the presence of acetosyringone. Following
inoculation, the plant tissue is transferred to non-selective medium and cultured
for 3 days in the dark, again in the presence of acetosyringone. After two cycles
on selective medium, transgenic plants can be regenerated.

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 3.2.2 Particle bombardment

In this system, 1 m tungsten or gold particles coated with plasmid DNA which
contain the gene(s) of interest are bombarded into regenerable plant cells. This
will result in a mixture of non-hit cells, lethally damaged cells and cells where
foreign DNA has entered. Besides commercially available bombardment
equipment based on electric discharge or helium pressure, home-made helium-
driven particle guns are now frequently manufactured. Numerous parameters
can be varied to optimize the bombardment system. The precipitation method
used to attach the DNA to microparticles is of the utmost importance.

3.2.3 Protoplast electroporation

DNA introduction by electroporation, a technique commonly used in bacterial

transformation, has also been applied for plant cells devoid of their cell wall.
Short electrical pulses create micropores in the plasmalemma through which the
DNA can enter the protoplasm.

Certain parameters were found to be essential in obtaining high transient

expression frequencies: (i) electroporation buffer (the presence of chloride
seems to be detrimental), (ii) polyethylene glycol treatment, and (iii) the use of a
heat shock before electroporation. Owing to high expression levels in
protoplasts, this system can be utilized to compare the strength of various
promoters.

4.0 TISSUE CULTURE AND DISEASE ELIMINATION

In micropropagation, the health status of the donor mother plant and of the
plants multiplied from it are among the most critical factors, which determine the
success of a tissue culture operation. The indexing of the mother plants for
freedom from viral, bacterial, and fungal diseases is a normal procedure in large-
scale plant propagation through tissue culture. Plants not originating from
pathogen-tested material must be screened for the presence of viruses.
Laboratories, which do not have in-house facilities to carry out plant indexing,
should obtain their indexed stock plants from organizations such as the
secretariat of the South Pacific Commission germplasm bank, overseas
laboratories specialized in indexing of different plants, agricultural universities or
privately owned certified germplasm repositories that routinely produce such
plant material. Batches of micropropagated plants should be tested for freedom
from diseases either in-house or by other laboratories. ELISA has been the most
effective method for virus and pathogen detection in plants. Polymerase chain
reaction (PCR) and nucleic acid hybridisation are more sensitive than ELISA,
and can detect pathogens in extremely low amounts. The elimination of most

16
viruses can be achieved by a combination of apical meristem culture and
thermotherapy.

4.1 Bacterial and Fungal Contamination

While most of the fungal and bacterial diseases are eliminated during surface
sterilization and culture, viruses and viroids survive through successive
multiplication if the mother plant is infected (Sessitsch et al., 2002). Even
cultures from seeds may carry viroids and bacterial endophytes, in certain cases
also viruses. Bacteria may be controlled under in vitro conditions to some extent
using certain bacteriostatic agents. Nevertheless, such chemical treatments with
e.g. ‘plant protection mixture’ (ppm) or commercial antibiotics even when applied
on long term do not fully eliminate microorganisms.

4.2 Virus infection in plants

Viruses are obligate parasites, and for their multiplication, they use the metabolic
machinery of the cells, and inhibit plant growth. Although, many viruses do not
produce disease symptoms, they adversely affect plant metabolism and increase
progressively with repeated vegetative propagation (Wang and Hu, 1980). Most
viruses infect only a limited number of species, but a few have a wide host range
(Mathews, 1991). Many plant viruses are transmitted through vectors such as
insects, nematodes, bacteria, and fungi. Propagation from vegetative parts,
grafting and sap can transmit viruses from one plant to another. The damage
from viruses depends on their concentration in the tissues and spread within the
plants. The greater the concentration of the virus, the greater the debilitating
effect observed on plant growth. Many varieties of vegetatively propagated crops
decline in performance with viral accumulation and must be discarded. Even
though the viruses do not produce visible symptoms, the plants have reduced
yield and poor quality. The removal of specific viruses by meristem-tip culture
has been reported to lead to a dramatic yield increase (Murashige, 1980), and
recently in sweet potato (Gao et al., 2000) and rejuvenation of plant varieties that
are vegetatively propagated. This is a unique contribution of meristem-culture not
achievable by any other techniques.

4.2.1 Detection of viral diseases

Plants not originating from pathogen-tested material must be screened for the
presence of viruses. Procedures to detect the presence of viruses include visual
examination for viral symptoms, infection tests on indicator plants, serological
tests, electron microscopy, and direct detection of RNA using molecular
techniques. Internationally approved and recognized detection systems include
serological and molecular laboratory assays, and indicator hosts in the
greenhouse.

17
Indexing with indicator plants

Prior to the advent of antigen-antibody reaction detection systems, the earlier

method of detecting virus relied upon indicator plants. For example, in potato,
viruses A, X, and Y can be detected by rubbing the juice of the infected plant on
the leaves of indicator plants such as Chenopodium spp., and Nicotiana spp.
The development of necrotic spots on the leaves is a typical symptom indicating
the presence of the viruses. Another example is sweet potato, where its wild
relative, Brazilian Morning Glory (Ipomaea setosa) is still used as the indicator
host. Similarly, in many fruit trees, the appropriate rootstocks are used for
indexing of various virus strains (Arthofer, 2001). The detection of plant diseases
has changed radically in the last 20 years. A number of indicator plant based
tests have been replaced with specific Enzyme Linked Immuno Sorbent Assays
(ELISA). Nevertheless, indexing with indicator plants is a low cost and highly
effective method to detect many viruses.

ELISA test

ELISA has been the most effective method for virus and pathogen detection in
plants. Each virus has a unique protein coat. Hence they can be detected by
using antibodies, which are usually linked to enzymes for reporting the affinity
binding via colour signal from the used enzyme substrate. The double antibody
sandwich (DAS) method in a 96-well microtitre plate (Clark and Adams, 1977) is
still widely used. Various alternative formats, substrates, and antibody binding
modifications have been developed over the years to increase specific sensitivity
(Agdia, 2002; Bioreba, 2002). The most common method nowadays uses
“biotinylated” virus-specific antibodies to improve their affinity to the respective
virus surface thus improving the signal. The use of monoclonal antibodies has
been so far restricted to certain potato and barley viruses, because of their high
cost and inability to detect the range of strains in several diseases.

Large scale testing based on screen-spot samples is also a routine procedure.

Such dot-immuno- binding assays are versatile and cheaper alternatives to
microtitre plate tests. Plant sap is directly spotted onto a pre-treated microporous
membrane (e.g. nitro-cellulose or polyvinylidene difluoride). The test kits can be
used directly in the field. Sample membranes can be dried and shipped to
another laboratory for processing. However, such systems are not useful for
viruses present in low concentrations in the plant sap.

ELISA has several limitations. First of all, a test is specific only to a given strain
of the pathogen. For a large group of ubiquitous viruses, which do not produce
disease symptoms or fatal consequences, ELISA kits have not been developed.
Thus, the strain specificity has severe limitations for comprehensive quality

18
control. Further, ELISA is less sensitive than PCR, and may fail to detect low
amounts of the virus in tissue-cultured plants. Whereas a positive ELISA is a
good indicator of the existence of microbes (but false positives may also occur),
a negative test does not ensure their absence. The use of Immuno-Tissue-
Printing allows the localization of viruses in tissues and therefore the
improvement of elimination strategies in vitro by visualizing the result of virus
removal (Fitch et al., 2001).

Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) is more sensitive than ELISA, and can
detect pathogens in extremely low amounts. PCR detects pathogens from their
genetic material, i.e. DNA or RNA. This technique uses a specific enzyme and
the respective genomic start code for a relevant section of the pathogen-DNA to
reproduce millions of copies from it. Therefore even minute quantities of the
pathogen DNA can be detected. Since most plant viruses are made of only RNA,
first a DNA copy of the RNA is made, whereas DNA from bacteria and
phytoplasmas may be amplified directly. Because the genetic material of the
pathogen is mixed with that of the plant, it is necessary to target only the genetic
material to be copied.

This is done by adding small pieces of DNA called primers which stick only to
borders of the region of genetic material that need to be copied. The primers
then direct the enzyme where to start and end amplification. During PCR, the
DNA is subjected to a series of hot, cold, and warm cycles in a thermocycler. Hot
cycles split DNA into single strands. Cold cycles allow the primers to attach to
the DNA. During warm cycles, the enzyme makes a copy of each piece of
primed DNA. If a phytoplasma is present, PCR produces a large quantity of a
specific piece of DNA.

By using gel electrophoresis, DNA pieces are separated in size, and the specific
piece of DNA is identified. If the critical concentration of a DNA particle is
sufficiently low and the sample tests negative, the plant material can be regarded
as practically free of the target microbe.

Once a test is standardized with an appropriate primer, and by a method, which

excludes inhibitors, a negative PCR test is a more reliable indicator of practical
freeness. The use of PCR drastically reduces the number of samples, which test
negative although they may carry the virus. Once proper probes are developed,
the nylon membrane spot test is a practical, rapid and accurate method for
diagnosis. PCR has the advantage that by determining “degenerate primers”, a
fairly large group of microbes such as viruses, bacteria and fungi can be
detected.

19
Nucleic acid Hybridization test

The presence of viruses can be detected from isolation and characterization of

double stranded RNA (ds-RNA) produced in most plants during viral replication.
Although non-specific presence of ds-RNA in a sample extract strongly suggests
viral infection, for further identification of the virus, the test has to be verified by
serological and other methods.

Viroids are much smaller than viruses, and are made of a circular piece of RNA
and do not have a coat protein. Viroids such as ‘Potato Spindle Tuber Viroid’ can
be detected by nucleic acid hybridization. The RNA of the virus "hybridizes" with
probe RNA. When the two single strands come together, they form a double
strand. To detect the viroid, RNA extracted from plant tissue is attached to a
microporous membrane. This bound RNA is then detected by a “probe”, which is
an RNA particle labeled with either 32 P or a chemical called DIG (digoxigenin).
If a viroid is present, the probe hybridizes with it; if not, the probe is lost when the
membrane is washed. DIG bioluminescence or radioactivity if present on the
membrane can be detected on an exposed film. Samples from known healthy
and known infected plants are used as controls. Large scale testing on nylon
screen-based spot samples is also a routine procedure.

4.2.2 Elimination of viruses

The elimination of viruses can be achieved by a combination of apical meristem

culture and thermotherapy. Meristem culture is the most commonly used method
to free plants from virus diseases. Virus-free plants have been obtained by
thermotherapy and meristem culture in several species (Morel and Martin, 1952;
Belkengren and Miller, 1962; Mullin et al., 1974; Boxus, 1976, (Gao et al., 2000).

The plant meristem is a zone of cells with intense divisions, situated in the
growing tip of stems and roots. The virus travels through the plant vascular
system, which is absent in the meristem. Moreover the cell-to-cell movement of
the viruses through plasmodesmata cannot keep up with the growth and
elongation of the apical-tip. The high metabolic activity of meristematic cells,
usually accompanied by an elevated endogenous auxin content in shoot apices
may also inhibit virus replication. Thus, the meristem is highly protected from
infection (Limasset et al., 1949). Based on this finding, meristem culture has
been extensively used to eliminate viruses, bacteria and fungi from plants. The
culture of meristems or alternatively small shoot tips, in combination with
enhanced cell division in vitro and/or thermal pre-treatment allows the elimination
of viruses in plants propagated from vegetative parts. If explants are too big, they
are likely to contain virus particles in the associated vascular tissue.

20
 4.3 Perspective of disease freeness

Despite the fact that many viruses may not show visible symptoms, their
presence can reduce the yield and quality of crops. Yield increases of up to
300% were reported following replacement of virus-infected stock with specific
pathogen-free plants (Murashige, 1980). In China, yield losses from virus
diseases of sweet potato (Feathery Mottle Virus, SPFMV, Latent Virus, SPLV
and Caulimo-Like Virus, SPCLV) used to be as much as 20%. After the
establishment of a virus elimination program, within a few years almost 10% area
of 6.6 million ha under sweet potato is now planted with virus-free planting
material (Gao et al., 2000).

There are no chemicals to cure virus-infected plants in the field. However, some
less virulent viruses can protect plants from infection of their more virulent strains
and create cross protection (Jones and Mullin, 1974). The phenomenon of cross
protection need not prevent the use of meristem culture. Under field conditions a
crop does not stay absolutely virus free even though derived from tissue culture
material. Even under tissue culture, total freeness may not be achieved. Many
symbiotic organisms contribute to the vigor and ability of the tissue-cultured
plants to perform well in the field. In the course of tissue culture, many plants are
made free from such beneficial microbes, e.g. symbiotic nitrogen fixing
endophytic bacteria, and mycorrhizae. There is no practical way to retain the
beneficial microorganisms during tissue culture. The deliberate re-infection of
propagules with selected strains can be a valid way of retrieving the benefits of
such microbes.

4.4 Meristem Culture

The pioneering work of meristem culture was undertaken by Morel and Martin
(1952), who first demonstrated the concept of regenerating virus-free plants from
meristem tips of virus-infected dahlias. Since this pioneering work, meristem
culture has been extensively used for the production of pathogen-free plants.
Although other tissue culture techniques (i.e. callus culture) have been
successfully used to eliminate viruses from plants, meristem culture remains the
most satisfactory technique. Virus-free plants have been produced from
asparagus, banana, cassava, cauliflower, gooseberry, pea, potato, raspberry,
strawberry, sweet potato, taro, coco, yams, and sugarcane. Although the
principle use of meristem culture has been for virus elimination, it can be used to
eliminate other systemic pathogens of fungi, bacteria and mycoplasma.

4.5 Callus Culture

Callus cultures have been used to produce disease-free plants from infected
donors. Explants taken from viral infected plants may themselves be virus-free
or the callus developed becomes virus-free with subsequent subculture. In
callus it appears that virus-free areas develop from ‘sectoring’ of the callus.

21
Subsequent regeneration of plantlets from these areas results in ‘chance
selection’ of virus-free plants.

It has been suggested that the virus-free areas arise because virus replication is
unable to keep pace with cell proliferation, and that kinetin-treated callus is more
likely to produce virus-free plants since cell division is accelerated. In contrast to
this some callus cultures have been shown to retain virus, without reduction, for
a period of 10 years.

4.6 Other In Vitro Methods

The occurrence of populations of uninfected cells even in severely infected

plants and the totipotency of plant cells in culture, has allowed for rapid
regeneration of virus-free plants. One of such method involves the rapid
regeneration of plants from leaf tissue and is particularly useful because it
minimize or eliminates callus formation and minimizes the chance of associated
somaclonal variation. The callus-inducing PGR, like 2,4-D, are also voided in
this method and shoot primordia are induced instead. A second method involves
the regeneration of plants from virus-free protoplasts. Virus-free plants have
also been produced form gametophytic tissues such as the nucellus and the
ovule.

4.7 Thermotherapy

The principle involved in heat therapy for virus eradication is that at elevated
temperatures many viruses in plant tissues are partially or completely inactivated
with little or no injury to the host tissues. Low temperatures have also been used
with some success. Generally, the appropriate heat treatment is thought to
either restrict virus multiplication or enhance vegetative growth of the plant tissue
resulting in the production of new virus-free shoot buds.

The methods of heat treatment include hot water, hot air, moist hot air or aerated
steam. Temperatures that are most commonly used are within the range of 50
to 55ºC for approximately 30 minutes to 3 hours. the major limitation of
thermotherapy for virus eradication has been that only small proportions of plants
survive the heat treatment. A long heat treatment is required if the tissue pieces
are large. Prolonged heat treatment of tissue may affect the growing points.

The application of heat treatment in conjunction with tissue culture is especially

advantageous when contamination is serious and deepset. The most common
technique is to use auxiliary buds of meristem tip cultures with the thermotherapy
treatment and there are a large number of published reports of this technique
being successful for chrysanthemum, carnation, strawberry, hop, sweet potato,
cassava, sugarcane, cow pea, tobacco and potato.

22
 4.8 Chemotherapy

Chemical treatment of plant viral diseases is in its infancy. Currently, there are
no good established examples of the practical control of plant viruses with
chemicals acting on the virus in the host. Most chemicals tested have been
shown to be phytotoxic and the virus remultiplies after the chemical treatment
ends.

One of the most promising group of antiviral chemicals tested so far are the
nucleoside analogues (Virazole, 2-Thiouracil, 6-Azauracil etc.) which upset the
nucleic acid metabolism of the virus. virazole is a broad-spectrum antiviral agent
which has been effective in eradicating viral particles in tobacco, however like
other agents it has been shown to be phytotoxic. Thiouracil has been used in
the media of several meristem tip culture systems and some virus-free plantlets
obtained.

5.0 CONCLUSION

Plant tissue culture offers exciting prospects for future improvements in crop
productivity. Combined with the powerful techniques of plant molecular biology
and integrated with well-established plant breeding practices, these closer
collaborative ties should strengthen our efforts in achieving a common goal, both
in research and commercial applications. Disease-free superior clones can be
regenerated through tissue culture techniques, which can be applied to all know
infectious agents but are especially valuable in elimination viruses and viroids
from vegetatively propagated crops. Producing clean planting material through in
vitro methods can significantly raise crop yield and quality.

23
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