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Plant Tissue Culture and Applied Plant
Biotechnology
Plant Tissue Culture and Applied Plant
Biotechnology

Dr. Manoj Kumar Singh

CENTRUM PRESS
NEW DELHI-110002 (INDIA)
CENTRUM PRESS
H.O.: 4360/4, Ansari Road, Daryaganj,
New Delhi-110002 (India)
Tel.: 23278000, 23261597, 23255577, 23286875
B.O.: No. 1015, Ist Main Road, BSK IIIrd Stage
IIIrd Phase, IIIrd Block, Bengaluru-560 085 (India)
Tel.: 080-41723429
Email: centrumpress@gmail.com
Visit us at: www.centrumpress.com

Plant Tissue Culture and Applied Plant Biotechnology

© Reserved

First Edition, 2017

ISBN 978-93-5084-929-3

PRINTED IN INDIA

Printed at Replika Press Pvt. Ltd., Sonipat, Haryana

Contents

Preface
1. Introduction
2. Plant Biotechnology
3. Plant Improvement Technology
4. Plant Pathology
5. Plant Transformation
6. Applications of Cell Biology
7. Plant Production and Technological Applications
8. Embryo Development in Plant
9. Genetical Improvement Techniques
10. DNA Maker and its Applications
Bibliography
Index
Preface

Biotechnology is broadly defined as any technique that uses live organisms viz. bacteria,
viruses, fungi, yeast, animal cells, plant cells etc. to make or modify a product, to improve
plants or animals or to engineer micro-organisms for specific uses. It encompasses genetic
engineering, inclusive of enzyme and protein engineering plant and animal tissue culture
technology, biosensors for biological monitoring, bioprocess and fermentation technology.
Biotechnology is essentially and interdisciplinary are consisting of biochemistry, molecular
chemistry, molecular and microbiology, genetics and immunology etc. it is concerned with
upgradation of quality and also utilization of livestock and resources for the well being of
both animals and plants.
Modern biotechnology holds considerable promise to meet challenges in agricultural
production. It makes use of life sciences, chemical sciences and engineering sciences in
achieving and improving the technological applications of the capabilities of the living
organism of their derivates to make products of value to man and society. It is used in living
systems to develop commercial processes and products which also includes the techniques of
Recombinant DNA, gene transfer, embryo manipulation, plant regeneration, cell culture,
monoclonal antibodies and bio-processed engineering. These techniques can transform ideas
into practical applications. Biotechnology can be used to develop safer vaccines against viral
and bacterial diseases. It also offers new ideas and techniques applicable to agriculture and
also develops a better understanding of living systems of our environment and ourselves. It
has a tremendous potential fir improving crop production, animal agriculture and bio-
processing. New approaches in biotechnology can develop high yielding and more nutritious
crop varieties, improve resistance to disease and also reduce the need for fertilizer and other
expensive agricultural chemicals. It could also improve forestry and its products, fibre crops
and chemical feedstocks. Biotechnology helps to isolate the gene, study its function and
regulation, modify the gene and reintroduce it into its natural host of another organism. It help
unlocking the secrets of diseases resistance, regulates growth and development or
manipulates communication among cells and among other organisms. It is a comparatively
new technique and is used in the field of agriculture and horticulture. This mainly involves
manipulation in the genetic code (which includes processes like gene transfer), tissue culture,
monoclonal antibody preparation protoplast fusion. These processes help in increasing yield,
producing better quality products both in plants and animals, increasing resistance to pests
and herbicides, micro propagation in several crops etc. are some of the advantages of using
biotechnological methods.
This book will be an important resource guide for agriculture and biotechnology students
and professionals in their study and job.
— Dr. Manoj Kumar Singh
 Chapter 1: Introduction
All present forms of life are the product of three factors:
• mutation, the fundamental source of heritable variation,
• environmental factors, which influence the selection of those mutations that survive
and reproduce, and
• time, during which the genotype and environment constantly interact and evolutionary
change is realized.
Genetic variation found in nature does not represent the original spectrum of spontaneous
mutations. Rather, this is the result of genotypes recombining in populations and continuously
interacting with environmental forces. Green plants are the ultimate source of resources
required for human life, food, clothing, and energy requirements. Prehistoric people, who
depended on their skills as hunters, drew upon abundant natural vegetation to collect
nutritious and nonpoisonous fruits, seeds, tubers, and other foods. As human populations
increased, greater and safer supplies of food had to be found, and gradually production
systems based on plant domestication were developed.
The domestication of crops historically has been influenced by ecological and
agricultural conditions, as well as by food gathering preferences. Genotypes that have
adapted to a wide range of climatic and edaphic conditions typically have been selected for
cultivation. The achievement of higher yielding crops facilitated population growth, sedentary
settlements, and further development. Which crops were domesticated depended not only on
the number of seeds or the size of fruits, but also on taste, palatability, and other factors. Only
a small fraction of the world’s approximately 200 000 plant species have been found suitable
for domestication; humans have used about 3000 of these for food, fibre, spices, etc., with
200 ultimately domesticated as crops.
Today, only 15-20 of these are food crops of major importance. The means of developing
new plant varieties for cultivation and use by humans has come to be called plant breeding.
Early on, it primarily involved selection, the choice between good and bad plants. People
learned not to eat all the “best fruit” but to plant the seed from some of them. Genetics became
a fundamental science of plant breeding after the Moravian monk J.G. Mendel discovered the
laws of heredity in the mid-19th century. Plant breeding further advanced when the
methodology of hybridization was developed. Its aim was to combine various desirable
properties of many plants in one plant, instead of just choosing between good and bad plants.
This method, often supplemented by germplasm derived from induced mutation, has become
the most common one for breeding plants through sexual reproduction.
However, some crops—including bananas, apples, cassava, and sugar cane—reproduce
vegetatively, especially those that are fully sterile without seeds. For this important group,
alternative approaches had to be developed, namely techniques of manipulation with somatic
tissue: mutation breeding and biotechnology.
Mutation Breeding
Plant breeding requires genetic variation of useful traits for crop improvement. Often,
however, desired variation is lacking. Mutagenic agents, such as radiation and certain
chemicals, then can be used to induce mutations and generate genetic variations from which
desired mutants may be selected.
Mutation induction has become a proven way of creating variation within a crop variety.
It offers the possibility of inducing desired attributes that either cannot be found in nature or
have been lost during evolution. When no gene, or genes, for resistance to a particular
disease, or for tolerance to stress, can be found in the available gene pool, plant breeders
have no obvious alternative but to attempt mutation induction. Treatment with mutagens alters
genes or breaks chromosomes. Gene mutations occur naturally as errors in deoxyribonucleic
acid (DNA) replication. Most of these errors are repaired, but some may pass the next cell
division to become established in the plant offspring as spontaneous mutations. Although
mutations observed in a particular gene are rare, there are probably 100 000 genes in a cell
of a higher plant. This means that every plant may carry one or more spontaneous mutations
into the next generation. Gene mutations without phenotypic (visible) expressions are usually
not recognized. Consequently, genetic variation appears rather limited, and scientists have to
resort to mutation induction. There are no other economic ways of altering genes, except to
wait a long time for spontaneous mutations to occur.
Artificial induction of mutations by ionizing radiation dates back to the beginning of the
20th century. But it took about 30 years to prove that such changes could be used in plant
breeding. Initial attempts to induce mutations in plants mostly used X-rays: later, at the dawn
of the “Atomic Age”, gamma and neutron radiation were employed as these types of ionizing
radiations became readily available from newly established nuclear research centres.
Major efforts were devoted during this initial phase of mutation induction to define
optimal treatment conditions for reproducibility. Research focused on changing “random”
mutation induction into a more directed mutagenesis to obtain more desirable and
economically useful mutations. However, it did not lead to the desired alterations in the
mutant spectrum. Limitations were the concomitant increase of plant injury with increasing
radiation dose and the low frequency of economically useful mutations. This led scientists to
search for potentially better mutagens. As a result, new methods of radiation treatment, as
well as chemical agents with mutagenic properties, were found.

Plant Biotechnology
Breeding for improved plant cultivars is based on two principles: genetic variation and
selection. The process is extremely labourious and time consuming with high inputs of
intellectual and manual work. However, the development of plant cell and tissue culture over
the last 20 years has made it possible to transfer part of the breeding work from field to
laboratoryconditions. Extensive research has resulted in new areas of plant breeding, namely
“plant biotechnology” and “genetic engineering”. They are based on cellular totipotency or
the ability to regenerate whole, flowering plants from isolated organs (meristems). Pieces of
tissue, individual cells and protoplasts. The isolated plant parts are aseptically grown in test
tubes on artificial media. For many developing countries, breeding crops for tolerance to
salinity and acidity in soils is of high priority. Current breeding strategies (including mutation
induction and in vitro selection) have clearly been successful in incorporating degrees of
tolerance in different species. The use of genetic engineering for creating environmental
stress-resistant plants will depend on the identification of specific genes which contribute to
the adaptation to specific stress environments.
In tropical countries, agriculture practices have maintained the yield level of different
crops through “intercropping” instead of by increased monocrop cultivation. Breeding crops
for multiple functions— such as biomass production, improved soil and water practice, and
composting —is a desirable support of sustainable agriculture in developing countries. The
mixed planting of a main crop with specific cover crops (e.g. forage legumes or grasses)
minimizes the use of herbicides.
Role of the Seibersorf Laboratories
The Plant Breeding Unit of the IAEA Laboratories at Seibersdorf was set up in the mid-
1960s to support the Joint FAO/IAEA Division’s programme of genetic crop improvement.
Nuclear techniques in plant breeding are developed and transferred to countries by research
and development in mutation breeding and related biotechnological techniques, training
scientists from developing countries, and providing irradiation services and technical advice.
Initial research in the Plant Breeding Unit focused on the development of mutation induction
methods with ionizing radiation and chemical mutagens. The aim was to achieve high
mutagenic efficiency, i.e., a high frequency of desirable mutations at minimal plant injury and
the highest possible reproducibility. This required a definition of radiation source
characteristics in terms of dose homogeneity and precise assessment of absorbed dose in
biological targets by appropriate dosimetry. Irradiation of seeds with gamma rays and
neutrons was commonly done, given the ease of handling, the simple standardization of
factors which modify radiation sensitivity, and good reproducibility.
The establishment of methods for controlling oxygen-dependent effects in the
radiobiological response to electromagnetic radiation was a major achievement. The
Laboratory actively contributed to standardizing neutron irradiation of seeds in nuclear
reactors by developing special facilities for this purpose. These were known as SNIP, for
Standard Neutron Irradiation Facility for swimming-pool-type reactors; and as USIF, for
Uranium Shielded Irradiation Facility for Triga-type reactors. This research was the basis for
the IAEA Laboratories’ worldwide seed irradiation service using fast and thermal neutrons at
a high-dose precision and reproducibility of induced effects. Moreover, efficient and accurate
treatments of seeds with chemical mutagens, mostly alkylating agents and azides, were
developed with the aid of isotope-labelled compounds and compared with mutation induction
by ionizing radiation. The Unit has undertaken supportive research on mutation breeding in
cereals, pulse crops, industrial crops, and vegetatively propagated crops.
As each crop species has a variable reproductive capacity (number of progenies per
plant) and a specific system of reproduction (self- or crosspollinated sexual reproduction or
asexual propagation), a universal breeding approach cannot be developed and species-
specific procedures have to be applied. Most vegetatively or asexually propagated species
are difficult to improve genetically by conventional cross- and mutation breeding methods.
These breeding problems can be more easily resolved by using biotechnology in combination
with mutation induction, and the Unit initiated in vitro mutation breeding activities during the
mid-1980s. Several tropical food crops of great importance to the food security of
developing countries were chosen as the main focus of R&D and training activities in
biotechnological plant breeding at the IAEA Laboratories.
Research and Development Activities
The Unit provides focused support to the FAO/IAEA’s co-ordinated research and
technical co-operation programmes. Assistance is provided to numerous projects in terms of
expertise for building facilities for plant tissue culture and mutagenic treatment, for quality
control of dosimetry of mutagenic irradiation, and for the development and transfer of nuclear
technologies for plant improvement.
Ongoing R&D includes the application of nuclear methods and associated advanced
techniques, such as in vitro culture and molecular genetics, to improve the production of a
wide range of crops through mutation breeding. The development of biotechnological
methods for breeding vegetatively propagated crop plants of major importance in developing
countries has a high priority.
Currently, the following R&D areas are being pursued:
Somaclonal and mutagen induced variation: Systematic studies are being conducted to
compare the genetic variation caused by tissue culture (somaclonal) variation with that
induced by irradiation and chemical agents. Genetic variation is being studied among maize
plants derived from in vitro cultured material via somatic embryogenesis. This is being done
to assess the nature of somaclonal and induced variation and its potential for use in practical
breeding.
Mutation induction and breeding technology for banana and plantain: Low genetic
variation and sterility handicap genetic improvement of banana and plantains (Musa spp.) by
conventional breeding techniques. Shoot-tip culture and in vitro plant regeneration are being
investigated for use in mutation induction and mutant selection. Somatic embryogenesis and
plant regeneration from cell suspensions of Musa are used to develop somatic cell
manipulation procedures for banana and plantain breeding. Methods of screening such plants
for resistance to Panama disease are studied in tissue culture, and biochemical markers
(peroxidase) are applied for the identification of tolerant genotypes. DNA markers are used
for identifying mutants and characterizing cultivars and species of Musa. Mutant clones
identified at the Seibersdorf Laboratories are tested in the field in tropical countries.
Mutation breeding to improve the tolerance to environmental stress of Azolla:
Azolla is a small aquatic fern that lives in symbiotic relationship with the nitrogen-fixing
cyanobacterium Anabaena. Under suitable field conditions Azolla can double its weight
every 3-5 days. The Azolla-Anabaena symbiotic system provides green manure for flooded
crops, particularly rice. Induced mutagenesis has produced Azolla variants tolerant to high
salinity, toxic aluminium levels, and/ or to herbicides.
Tolerant plants are being investigated under field conditions to confirm that heritable
changes cause the increased tolerance to environmental stress.
Methods of mutation induction and breeding of tropical root and tuber crops
(cassava and yam): Cassava and yam are among the most important staple food crops of the
lowland tropics. Mutation breeding technology is being developed to increase variation in
plant stature, cyanide content, disease, and pest resistance. In vitro techniques are used for
the propagation of healthy plants and improved clones. Somatic embryogenesis is being
developed for cassava and yam improvement through in vitro mutagenesis and later on by
somatic cell manipulation. Mutant and polyploid clones are prepared for field testing in
Member States.
Tissue culture in cocoa as a system for more efficient mutation breeding: Attempts
to breed cocoa for disease resistance have yielded very limited success. A major constraint is
that little variation exists in currently available cultivars. Somatic embryogenesis is being
developed for propagation of desirable genotypes and, through in vitro mutagenesis and
pollen mutagenesis, is being applied for induction of virus-resistant cocoa trees in Ghana.
Plant breeding research at the Seibersdorf Laboratories is directly problem- and
clientoriented. Many positive results of scientific work have been achieved by junior
scientists from developing countries during their assignments under the IAEA’s fellowship
training programme. Local cultivars and genetic material from tropical countries are brought
to the Seibersdorf Laboratories, transferred to tissue culture conditions and used for
experimental work. Protocols and techniques that are specifically developed for a crop and a
particular genotype are then directly used in national programmes. Additionally, breeding
material originating from mutant lines and clones which are ready for field testing are
dispatched from Seibersdorf to developing Member States in support of their breeding
programmes.
Training of Plant Breeders
Training in plant breeding represents the most active component of technology transfer at
the Seibersdorf Laboratories. For 20 years the Plant Breeding Unit has supported the
Agency’s fellowship programme and organized interregional training courses. Training
activities are closely connected with R&D efforts on crop plant improvement and the
application of nuclear techniques in breeding. During a period of three to twelve months,
fellows usually work with radiation or chemical induced mutagenesis in plant species
cultivated in their home countries. Whenever possible, training of small groups of two-to-five
fellows is organized for solving common problems. The experiments are individually
designed to assure that laboratory techniques and results will be directly applicable upon
return to the home institute.
As a result of their work, fellows have produced numerous scientific publications in
internationally recognized journals and symposia proceedings. Very often, as continuation of a
fellowship in Seibersdorf, fellows participate in co-ordinated research and technical co-
operation projects of the IAEA.
The FAO/IAEA Interregional Training Course on “Induction and Use of Mutations in
Plant Breeding” has been held at the Seibersdorf Laboratories since 1982. Twenty
participants from different Member States of FAO and IAEA are admitted annually to this
intensive training course that usually lasts 6 to 8 weeks. Through lectures, laboratory
exercises, field experiment evaluations, seminars, and excursions, participants are made
aware of the latest advanced mutation techniques and biotechnological and molecular biology
methods for crop improvement. Special training is given in the safe handling of radiation
sources, radioisotopes, and particularly hazardous mutagenic chemicals. At the end of each
course, participants are able to discuss and evaluate the potential role of induced mutations
and advanced biotechnologies in their national breeding programmes for specific crop
improvement of cereals, legumes, oil crops, forages, vegetables, fruits, root and tuber crops,
palms, rubber, and other plants.
Support for National Programmes
A radiation treatment service is provided at no cost to FAO and IAEA Member States to
foster the application of nuclear techniques in crop improvement programmes and to render
direct support to plant breeders in developing countries. Mutagenic treatment is applied to
seeds, corms, tubers, scions, cuttings, and tissue cultures (“in vitro materials”) with precise
doses of gamma and fast neutron radiation. The doses are carefully calibrated to assure
reproducible effects. Users of the service are requested to report on the objectives of the
applied mutation breeding project and to provide an adequate material (population size) to
ensure a high probability for mutation induction of desired characters.
Moreover, a prior radiosensitivity test in a greenhouse is frequently performed to assess
useful radiation doses for the great variety of biological samples in mutation breeding. The
treated materials are dispatched with a detailed irradiation protocol and with the request to
report on the induced radiation effects in the first and second mutation generation. This
feedback is required to improve radiosensitivity estimates of species and cultivars from
different environments.
Over the last 25 years, the Unit has provided radiation services on more than 20 000
samples from the majority of Member States from the FAO and IAEA. Most of these were
seed samples which were irradiated with cobalt-60 gamma rays. Recently, however, requests
for mutagen treatment of in vitro materials and for fast neutrons have become more frequent.
This reflects the increasing importance of biotechnology and molecular genetics in plant
improvement programmes.
Less than 80 mutant varieties were officially released before the start of irradiation
services. Over the past quarter century, more than 1500 cultivars of crop plants and
ornamentals with significantly improved attributes — increased yield, improved quality,
higher market value, disease resistance, and/or stress tolerance —have been released. Some
of these mutant varieties were derived from radiation services provided by the Seibersdorf
Laboratory.

Plant Hormones
Plant hormones (also known as phytohormones) are chemicals that regulate plant growth,
which, in the UK, are termed ‘plant growth substances’. Plant hormones are signal molecules
produced within the plant, and occur in extremely low concentrations. Hormones regulate
cellular processes in targeted cells locally and when moved to other locations, in other
locations of the plant. Hormones also determine the formation of flowers, stems, leaves, the
shedding of leaves, and the development and ripening of fruit. Plants, unlike animals, lack
glands that produce and secrete hormones, instead each cell is capable of producing
hormones. Plant hormones shape the plant, affecting seed growth, time of flowering, the sex of
flowers, senescence of leaves and fruits. They affect which tissues grow upward and which
grow downward, leaf formation and stem growth, fruit development and ripening, plant
longevity, and even plant death. Hormones are vital to plant growth and lacking them, plants
would be mostly a mass of undifferentiated cells.
Characteristics
 The word hormone is derived from Greek, meaning ‘set in motion.’ Plant hormones affect
gene expression and transcription levels, cellular division, and growth. They are naturally
produced within plants, though very similar chemicals are produced by fungi and bacteria that
can also affect plant growth. A large number of related chemical compounds are synthesized
by humans, they are used to regulate the growth of cultivated plants, weeds, and in vitro-
grown plants and plant cells; these manmade compounds are called Plant Growth Regulators
or PGRs for short. Early in the study of plant hormones, “phytohormone” was the commonly
used term, but its use is less widely applied now. Plant hormones are not nutrients, but
chemicals that in small amounts promote and influence the growth, development, and
differentiation of cells and tissues. The biosynthesis of plant hormones within plant tissues is
often diffuse and not always localized. Plants lack glands to produce and store hormones,
because, unlike animals, which have two circulatory systems (lymphatic and cardiovascular)
powered by a heart that moves fluids around the body, plants use more passive means to
move chemicals around the plant. Plants utilize simple chemicals as hormones, which move
more easily through the plant’s tissues. They are often produced and used on a local basis
within the plant body, plant cells even produce hormones that affect different regions of the
cell producing the hormone.
Hormones are transported within the plant by utilizing four types of movements. For
localized movement, cytoplasmic streaming within cells and slow diffusion of ions and
molecules between cells are utilized. Vascular tissues are used to move hormones from one
part of the plant to another; these include sieve tubes that move sugars from the leaves to the
roots and flowers, and xylem that moves water and mineral solutes from the roots to the
foliage.
Not all plant cells respond to hormones, but those cells that do are programmed to
respond at specific points in their growth cycle. The greatest effects occur at specific stages
during the cell’s life, with diminished effects occurring before or after this period. Plants
need hormones at very specific times during plant growth and at specific locations. They also
need to disengage the effects that hormones have when they are no longer needed. The
production of hormones occurs very often at sites of active growth within the meristems,
before cells have fully differentiated. After production they are sometimes moved to other
parts of the plant where they cause an immediate effect or they can be stored in cells to be
released later. Plants use different pathways to regulate internal hormone quantities and
moderate their effects; they can regulate the amount of chemicals used to biosynthesize
hormones. They can store them in cells, inactivate them, or cannibalise already-formed
hormones by conjugating them with carbohydrates, amino acids or peptides. Plants can also
break down hormones chemically, effectively destroying them. Plant hormones frequently
regulate the concentrations of other plant hormones. Plants also move hormones around the
plant diluting their concentrations. The concentration of hormones required for plant
responses are very low (10-6 to 10-5 mol/L). Because of these low concentrations, it has been
very difficult to study plant hormones, and only since the late 1970s have scientists been able
to start piecing together their effects and relationships to plant physiology. Much of the early
work on plant hormones involved studying plants that were genetically deficient in one or
involved the use of tissue-cultured plants grown in vitro that were subjected to differing
ratios of hormones, and the resultant growth compared. The earliest scientific observation
and study dates to the 1880s; the determination and observation of plant hormones and their
identification was spread-out over the next 70 years.
Classes of Plant Hormones
In general, it is accepted that there are five major classes of plant hormones, some of
which are made up of many different chemicals that can vary in structure from one plant to the
next. The chemicals are each grouped together into one of these classes based on their
structural similarities and on their effects on plant physiology. Other plant hormones and
growth regulators are not easily grouped into these classes; they exist naturally or are
synthesized by humans or other organisms, including chemicals that inhibit plant growth or
interrupt the physiological processes within plants. Each class has positive as well as
inhibitory functions, and most often work in tandem with each other, with varying ratios of
one or more interplaying to affect growth regulation.
The five major classes are:
Abscisic Acid
Abscisic acid also called ABA, was discovered and researched under two different
names before its chemical properties were fully known, it was called dormin and abscicin II.
Once it was determined that the two latter compounds were the same; it was named abscisic
acid. The name “abscisic acid” was given because it was found in high concentrations in
newly abscissed or freshly fallen leaves. This class of PGR is composed of one chemical
compound normally produced in the leaves of plants, originating from chloroplasts,
especially when plants are under stress. In general, it acts as an inhibitory chemical
compound that affects bud growth, seed and bud dormancy. It mediates changes within the
apical meristem causing bud dormancy and the alteration of the last set of leaves into
protective bud covers. Since it was found in freshly abscissed leaves, it was thought to play a
role in the processes of natural leaf drop but further research has disproven this. In plant
species from temperate parts of the world it plays a role in leaf and seed dormancy by
inhibiting growth, but, as it is dissipated from seeds or buds, growth begins. In other plants,
as ABA levels decrease, growth then commences as gibberellin levels increase. Without
ABA, buds and seeds would start to grow during warm periods in winter and be killed when
it froze again. Since ABA dissipates slowly from the tissues and its effects take time to be
offset by other plant hormones, there is a delay in physiological pathways that provide some
protection from premature growth. It accumulates within seeds during fruit maturation,
preventing seed germination within the fruit, or seed germination before winter. Abscisic
acid’s effects are degraded within plant tissues during cold temperatures or by its removal by
water washing in out of the tissues, releasing the seeds and buds from dormancy. In plants
under water stress, ABA plays a role in closing the stomata. Soon after plants are water-
stressed and the roots are deficient in water, a signal moves up to the leaves, causing the
formation of ABA precursors there, which then move to the roots.
The roots then release ABA, which is translocated to the foliage through the vascular
system and modulates the potassium and sodium uptake within the guard cells, which then
lose turgidity, closing the stomata. ABA exists in all parts of the plant and its concentration
within any tissue seems to mediate its effects and function as a hormone; its degradation, or
more properly catabolism, within the plant affects metabolic reactions and cellular growth
and production of other hormones.
 Plants start life as a seed with high ABA levels, just before the seed germinates ABA
levels decrease; during germination and early growth of the seedling, ABA levels decrease
even more. As plants begin to produce shoots with fully functional leaves - ABA levels begin
to increase, slowing down cellular growth in more “mature” areas of the plant. Stress from
water or predation affects ABA production and catabolism rates, mediating another cascade
of effects that trigger specific responses from targeted cells. Scientists are still piecing
together the complex interactions and effects of this and other phytohormones.
Auxins
Auxins are compounds that positively influence cell enlargement, bud formation and root
initiation. They also promote the production of other hormones and in conjunction with
cytokinins, they control the growth of stems, roots, and fruits, and convert stems into flowers.
Auxins were the first class of growth regulators discovered. They affect cell elongation by
altering cell wall plasticity. Auxins decrease in light and increase where it is dark. They
stimulate cambium cells to divide and in stems cause secondary xylem to differentiate. Auxins
act to inhibit the growth of buds lower down the stems (apical dominance), and also to
promote lateral and adventitious root development and growth. Leaf abscission is initiated by
the growing point of a plant ceasing to produce auxins. Auxins in seeds regulate specific
protein synthesis, as they develop within the flower after pollination, causing the flower to
develop a fruit to contain the developing seeds. Auxins are toxic to plants in large
concentrations; they are most toxic to dicots and less so to monocots. Because of this
property, synthetic auxin herbicides including 2,4-D and 2,4,5-T have been developed and
used for weed control. Auxins, especially 1-Naphthaleneacetic acid (NAA) and Indole-3-
butyric acid (IBA), are also commonly applied to stimulate root growth when taking cuttings
of plants. The most common auxin found in plants is indoleacetic acid or IAA. The
correlation of auxins and cytokinins in the plants is a constant (A/C = const.).
Cytokinins
Cytokinins or CKs are a group of chemicals that influence cell division and shoot
formation. They were called kinins in the past when the first cytokinins were isolated from
yeast cells. They also help delay senescence or the aging of tissues, are responsible for
mediating auxin transport throughout the plant, and affect internodal length and leaf growth.
They have a highly synergistic effect in concert with auxins and the ratios of these two groups
of plant hormones affect most major growth periods during a plant’s lifetime. Cytokinins
counter the apical dominance induced by auxins; they in conjunction with ethylene promote
abscission of leaves, flower parts and fruits. The correlation of auxins and cytokinins in the
plants is a constant (A/ C = const.).
Ethylene
Ethylene is a gas that forms through the Yang Cycle from the breakdown of methionine,
which is in all cells. Ethylene has very limited solubility in water and does not accumulate
within the cell but diffuses out of the cell and escapes out of the plant. Its effectiveness as a
plant hormone is dependent on its rate of production versus its rate of escaping into the
atmosphere. Ethylene is produced at a faster rate in rapidly growing and dividing cells,
especially in darkness. New growth and newly germinated seedlings produce more ethylene
than can escape the plant, which leads to elevated amounts of ethylene, inhibiting leaf
expansion. As the new shoot is exposed to light, reactions by phytochrome in the plant’s cells
produce a signal for ethylene production to decrease, allowing leaf expansion. Ethylene
affects cell growth and cell shape; when a growing shoot hits an obstacle while underground,
ethylene production greatly increases, preventing cell elongation and causing the stem to
swell.
The resulting thicker stem can exert more pressure against the object impeding its path to
the surface. If the shoot does not reach the surface and the ethylene stimulus becomes
prolonged, it affects the stems natural geotropic response, which is to grow upright, allowing
it to grow around an object. Studies seem to indicate that ethylene affects stem diameter and
height: When stems of trees are subjected to wind, causing lateral stress, greater ethylene
production occurs, resulting in thicker, more sturdy tree trunks and branches. Ethylene affects
fruit-ripening: Normally, when the seeds are mature, ethylene production increases and
builds-up within the fruit, resulting in a climacteric event just before seed dispersal. The
nuclear protein Ethylene Insensitive (EIN2) is regulated by ethylene production, and, in turn,
regulates other hormones including ABA and stress hormones.
Gibberellins
Gibberellins, or GAs, include a large range of chemicals that are produced naturally
within plants and by fungi. They were first discovered when Japanese researchers, including
Eiichi Kurosawa, noticed a chemical produced by a fungus called Gibberella fujikuroi that
produced abnormal growth in rice plants. Gibberellins are important in seed germination,
affecting enzyme production that mobilizes food production used for growth of new cells.
This is done by modulating chromosomal transcription. In grain (rice, wheat, corn, etc.)
seeds, a layer of cells called the aleurone layer wraps around the endosperm tissue.
Absoption of water by the seed causes production of GA. The GA is transported to the
aleurone layer, which responds by producing enzymes that break down stored food reserves
within the endosperm, which are utilized by the growing seedling.
GAs produce bolting of rosette-forming plants, increasing internodal length. They
promote flowering, cellular division, and in seeds growth after germination. Gibberellins
also reverse the inhibition of shoot growth and dormancy induced by ABA.
Other Known Hormones
Other identified plant growth regulators include:
• Brassinosteroids, are a class of polyhydroxysteroids, a group of plant growth
regulators. Brassinosteroids have been recognized as a sixth class of plant hormones
which stimulate cell elongation and division, gravitropism, resistance to stress and
xylem differentiation. They inhibit root growth and leaf abscission. Brassinolide was
the first identified brassinosteroid and was isolated from organic extracts of rapeseed
(Brassica napus) pollen in 1970.
• Salicylic acid - activates genes in some plants that produce chemicals that aid in the
defence against pathogenic invaders.
• Jasmonates - are produced from fatty acids and seem to promote the production of
defence proteins that are used to fend off invading organisms. They are believed to
also have a role in seed germination, and affect the storage of protein in seeds, and
 seem to affect root growth.
• Plant peptide hormones - encompasses all small secreted peptides that are involved in
cell-to-cell signalling. These small peptide hormones play crucial roles in plant
growth and development, including defence mechanisms, the control of cell division
and expansion, and pollen self-incompatibility .
• Polyamines - are strongly basic molecules with low molecular weight that have been
found in all organisms studied thus far. They are essential for plant growth and
development and affect the process of mitosis and meiosis.
• Nitric oxide (NO) - serves as signal in hormonal and defence responses.
• Strigolactones, implicated in the inhibition of shoot branching.
• Karrikins, a group of plant growth regulators found in the smoke of burning plant
material that have the ability to stimulate the germination of seeds

Potential Medical Applications

Plant stress hormones activate cellular responses, including cell death, to diverse stress
situations in plants. Researchers have found that some plant stress hormones share the ability
to adversely affect human cancer cells . For example, sodium salicylate has been found to
suppress proliferation of lymphoblastic leukemia, prostate, breast, and melanoma human
cancer cells. Jasmonic acid, a plant stress hormone that belongs to the jasmonate family,
induced death in lymphoblastic leukemia cells. Methyl jasmonate has been found to induce
cell death in a number of cancer cell lines.
Hormones and Plant Propagation
Synthetic plant hormones or PGRs are commonly used in a number of different
techniques involving plant propagation from cuttings, grafting, micropropagation, and tissue
culture. The propagation of plants by cuttings of fully developed leaves, stems, or roots is
performed by gardeners utilizing auxin as a rooting compound applied to the cut surface; the
auxins are taken into the plant and promote root initiation. In grafting, auxin promotes callus
tissue formation, which joins the surfaces of the graft together. In micropropagation, different
PGRs are used to promote multiplication and then rooting of new plantlets. In the tissue-
culturing of plant cells, PGRs are used to produce callus growth, multiplication, and rooting.
Seed Dormancy
Plant hormones affect seed germinations and dormancy by affecting different parts of the
seed. Embryo dormancy is characterized by a high ABA/GA ratio, whereas the seed has a
high ABA sensitivity and low GA sensitivity. To release the seed from this type of dormancy
and initiate seed germination, an alteration in hormone biosynthesis and degradation towards
a low ABA/GA ratio, along with a decrease in ABA sensitivity and an increase in GA
sensitivity needs to occur.
ABA controls embryo dormancy, and GA embryo germination. Seed coat dormancy
involves the mechanical restriction of the seed coat, this along with a low embryo growth
potential, effectively produces seed dormancy. GA releases this dormancy by increasing the
embryo growth potential, and/or weakening the seed coat so the radical of the seedling can
break through the seed coat.
Different types of seed coats can be made up of living or dead cells and both types can
be influenced by hormones; those composed of living cells are acted upon after seed
formation while the seed coats composed of dead cells can be influenced by hormones during
the formation of the seed coat. ABA affects testa or seed coat growth characteristics,
including thickness, and effects the GA-mediated embryo growth potential. These conditions
and effects occur during the formation of the seed, often in response to environmental
conditions. Hormones also mediate endosperm dormancy: Endosperm in most seeds is
composed of living tissue that can actively respond to hormones generated by the embryo.
The endosperm often acts as a barrier to seed germination, playing a part in seed coat
dormancy or in the germination process. Living cells respond to and also affect the ABA/ GA
ratio, and mediate cellular sensitivity; GA thus increases the embryo growth potential and can
promote endosperm weakening. GA also affects both ABA-independent and ABA-inhibiting
processes within the endosperm.

Cell Suspension Culture

There are two basic systems for growing cells in culture, as monolayers on an artificial
substrate (i.e., adherent culture) or free-floating in the culture medium (suspension culture).
Characteristics of Each Culture Type
The majority of the cells derived from vertebrates, with the exception of hematopoietic
cell lines and a few others, are anchorage-dependent and have to be cultured on a suitable
substrate that is specifically treated to allow cell adhesion and spreading (i.e., tissue-culture
treated). However, many cell lines can also be adapted for suspension culture. Similarly,
most of the commercially available insect cell lines grow well in monolayer or suspension
culture.
Cells that are cultured in suspension can be maintained in culture flasks that are not
tissue-culture treated, but as the culture volume to surface area is increased beyond which
adequate gas exchange is hindered (usually 0.2 – 0.5 mL/cm2), the medium requires agitation.
This agitation is usually achieved with a magnetic stirrer or rotating spinner flasks.

Meristem Culture
Culture of meristem tips were started first by Morel and co-workers in the nineteen
fifties with the intention of obatining virus -free plants from those already infected . In 1960
he tried this methods in a virus infected Cymbidiuma and succeeded in ontaining a aplantling
free of infection. Following this, Wimber (1963) ,Hamilton (1964) and Morel (1964)
developed methods by which a single detached meristem could be cultured , divided into
several bits and each bit subcultured by repeating this several times over , an incredibly large
number of plantings could be obtained from a single protocorn in one year.
Meristems are dissected out with the help of a sharp razor-blade , the operation being
carried out under a microscope. It should be 500 in length and about the same in diameter .
These tiny bits of meristems are then sterilised with 2 % calcium hypochlorite solution and
placed in a liquid nutrient medium in a flask under sterile conditions. The flask is then placed
on a rotary shaker and rotated at a given speed. This is to prevent formation of any polarity to
the developing meristem . Light equivalent to 200 foot candles and a temperature of 22 C are
maintained throughout . Within a week the meristems will turn green . It starts developing
several lumbs of tissue . At this stage the protocorn is taken out , washed and cut up into as
many pieces as there are balls of tissue . Each bit is dipped in a sterilant and put in a tube
containing fresh liquid medium and agitated . The procedure is repeated until as many
plantings as required are produced . The tiny balls of tissue are then transferred to normal
nutrient agar medium and akept static as in seedling culture. Each ball of tissue , after
reaching 4 mm in length differentiates into an individual plant.
The technique of propagation by meristem culture provided a great boost to the orchid
industry . Rare plants of special qualities which could not be propagated by seeds due to
genetic factors , could be multiplied easily and that too at a fantastic rate. This naturally
brought down the prices of these orchids which were otherwise too expensive for most
people to possess. Precious plants which fell victims to the incurable virus diseases also
could be salvaged .
The only disadvantage with propagation by meristem culture is that removal of meristem
causes temporary set-back in the growth of the mother plant. To avoid this ,parts of seeds due
to genetic factors , could be multiplied easily and that too at a fantastic rate . This naturally
brought down the prices of these orchids which were otherwise too expensive for most
people to possess. Precious plants which fell victims to the incurable virus diseases also
could be salvaged. The only disadvantage with propagation by meristem culture is that
removal of meristem causes temporary set-back in the growth of the mother plant. To avoid
this , parts of the plant other than the apical meristem , have been tried in tissue culture
experiments .Churchill, Arditti and Ball (1971) conducted experiments with leaf-tips and
reported that the excised leaf-tips of Epidendrum and Laeliocattleya produced calli when put
in liquid culture medium and roated . Upon being transferred to agar media these calli
produced plantings. One of the more recent development in vegetative multiplication of
orchids is the use of cytokinis for promoting dormant buds to develop into young plants or
‘Keikis. This has been well described in an article by Brasch and kocsis (1980) from the
McMaster University , Hamilton , Ontario , Canada . Experiments are in progress on several
genera , but the greatest success so far has been achieved in Phalaenopsis , which has a
mopodial habit. While the mericloning technique is wiedly used by large establishmets like
commercial nurseries , for the amateur growing a few plants and desiring to increase them ,
the discovery of “ Keiki grow “ , and its availability are of great interest . Orchid “ keiki Fix
“ formulated by N.F.Hass and marketted by H.G. Hees , 99 a kilin Ride, workingham , Berks ,
RG11 3 PD, U. K is reported to be equally effective on the dormant buds on Phalaenopsis
inflorescence stalk .

Diseases and Pests

Likey other group of cultivated plants, orchids also are prone to a number of diseases
caused by various agents like viruses , fungi, bacteria, insect pests etc. All bacterial , fungal
and virus diseases are infectious and unless checked in time, the disease will spread from
plant to plant. Except for the viruses , effective preventives and remedies have been
discovered for these ailments and if detected in time, they could successfully be eliminated
before serious damage is done to the plant.
Preventive measures should be followed strictly and according to rules. Most fungi and
bacteria thrive under damp and soggy conditions, because invariably the spores need
moisture for germination . Hence overcrowding should be avoided in orchid houses and
facilities provided for free air circulation . Debris should not be allowed to accumulate in the
house or in its near vicinity . Watering , spraying , fogging etc. should be done early in the day
so that by night ,when usually the temperature comes down , teh plants may remain completely
dry and relatively impervious to attack by fungi or bacteria .
The grower has to be always on the alert for the symptoms of disease in plants . They
should be examined daily so that even the slightest symptom is not overlooked and immediate
steps are taken in order to check the spread of the disease.

Virus Diseases
Viruses and the diseases they produce are the most difficult to tackle , since our
knowledge about them is far from complete, and whatever little information we have is often
confusing . This is because viruses are not specific or reliable in the reactions they produce
in different species of plants, e.g.. Cymbidium Mosaic Virus produces mosaic mottle on
leaves of Cymbidium, whereas in Cattleya it produces necrotic spots (dead spots ), which is
entirely different in appearance from the mosaic mottle. The reverse situation may also occur
in infected plants , i.e. two different viruses producing two different diseases , may induce the
same lesions when they occur in the same plant. Another peculiarity of the virus disease is
that, when the infection is mild, the virus may remain inside the plant for years together,
without any visible symptoms, but always to be reckoned with as a dangerous sources of
fresh infections. At other times the infection may be so virulent that the plants are killed
within a few weeks. Altogether about 32 virus diseases are known to occur in orchids
(Jensen , 1959) . But this does not necessarily mean that orchids are susceptible to thirty two
different kinds of viruses . The same virus may produce more than one disease in different
species of orchids . Among orchid viruses , Cymbidium Mosaic Virus (CyMV) ,
Odontoglossum Ring Spot Virus ( ORsV) and Dendrobium nobile Mosaic Virus are reported
to infect non-orchidaceous plants as well.
CyMV, Tobacco Mosaic Virus-O (TMV-O) and ORsV are the most common among
orchid viruses. Most of the cultivated genera are susceptible to these three viruses. CyMV is
a flexuous rod 18m in diameter and 500 m in length as shown by Gold (1950), Gold and
Jensen (1951) and Jensen and Gold (1955). TMV-O is related to ,but not the same as , the
Tobacco Mosaic Virus which infects also the potato plant. It is a straight short road about 300
m in length . According to Kado et al. (1968) it is serologically the same as the TMV has a
wide range of hosts whereas TMV-O infects only a limited number of plants. ORsV was
identified by Paul and Wetter (1965) as a rigid short rod. According to Kado, this virus is the
same as TMV-O (Northen , 1970).

Callus Culture
 The callus culture is a technique of tissue culture, it is usually carried out on solidified
gel medium in the presence of growth regulators and initiated by inoculation of small explants
or sections from established organ or other cultures (the inocula).
Callus Culture and Regeneration
Regeneration of plants by micropropagation of h vitro cultures can be achieved from
organ primordia existing in shoot tips and axillary bud explants. Alternatively, plants can be
regenerated from unorganized callus tissues derived from different explants by
dedifferentiation induced by exogenous growth regulators. Plant regeneration from calli is
possible by de novo organogenesis or somatic embryogenesis.
Callus cultures also facilitate the amplification of limiting plant material. In addition,
plant regeneration from calli permits the isolation of rare somaclonal variants which result
either from an existing genetic variability in somatic cells or from the induction of mutations,
chromosome aberrations, and epigenetic changes by the in vitro applied environmental
stimuli, including growth factors added to the cultured cells. In Arabidopsis thaliana, one of
the earliest studies on callus formation was conducted by Loewenberg, who grew seedlings
in a medium containing kinetin and parachlorophenoxyacetic acid. Subsequent studies of cell
culture and regeneration demonstrated that it is relatively easy to regenerate Arabidopsis from
callus cultures by many approaches. However, the efficiency of regeneration and the
proportion of somaclonal variants are considerably influenced by the ecotype and the source
of explants, as well as by the medium and growth regulators employed. Hypocotyl and root
explants thus provide excellent materials for callus initiation and regeneration in contrast to
stem and leaf explants.
The tissue culture protocols include callus induction in auxin containing media. Shoot
regeneration is induced by lowering the auxin content and increasing the cytokinin levels in
the media. The elongated shoots are usually transferred into root induction media, but even in
the absence of roots, flowering and seed setting can take place in test tubes in vitro. The
method described here utilizes root explants that can be cultured and efficiently regenerated
in large quantities. The described technique can be applied equally well for the Arabidopsis
ecotypes Columbia, C24, RLD, and Wassilewskija. Without changing the general protocol,
some slight alterations may be required for other ecotypes or mutants of Arabidopsis.
Materials
1. Dry seeds of Arabidopsis thaliana.
2. 10% Sodium hypochlorite solution containing 0.1% Triton X-100.
3. Eppendorf tubes.
4. Sterilized double-distilled water.
5. Culture tubes.
6. 9 cm Petri dishes.
7. Pair of forceps.
8. Scissors or scalpel blades.
9. Sterile 250-mL Erlenmeyer flasks with plugs.
 10. Rotary shaker set at 120 rpm.
11. Microfuge.
12. Basal medium (BM): Murashige and Skoog (MS) medium (6) or MS medium for
Arabidopsis (MSAR) medium (7) with 3% sucrose (pH 5.8) can equivalently be used.
13. 0.5X BM: BM consisting of half concentration of MS macroelements and 0.5%
sucrose.
14. MSAR I (Callus medium): Supplement BM with 0.5 mg/L 2,4— dichlorc~
phenoxyacetic acid (2,4-D), 2.0 mg/L indole3-acetic acid (IAA), 0.5 mgL 6-(y,y-
dimethylallylamino~purine riboside (IPAR), and gel using either 0.8% agar or 0.2%
gelrite.
15. MSAR I1 (Shoot medium): Supplement BM with 2.0 mg/L 6-(y,y-
dimethylallylamino)- purine riboside (IPAR), 0.05 mg/L a-naphtaleneacetic acid
(NAA), and either 0.8% agar or 0.2% gelrite.
16. MSAR 111 (root inducing medium): Supplement BM with 1.0 mg/L IAA, 0.2 mg/L
indole3-butyric acid (IBA), 0.2 mg/L 6-furfuryl-aminopurine (kinetin), and either
0.8% agar or 0.2% gelrite.
Methods
1. Surface sterilize 0.1 g (approx 5000) seeds in Eppendorf tubes by adding a 1 mL of
10% (v/v) solution of sodium hypochlorite containing 0.1% Triton X-100 as
surfactant and shaking for about 15 min.
2. Pellet the seeds by slow centrifugation in a microfuge for a few seconds and remove
the supernatant. Wash the seeds five times with 1 mL of sterile water.
3. Germinate the seeds in Petri dishes containing 0.8% agar gelled 0.5X BM medium
(BM medium containing half concentration of macroelements) using 16 h light and 8 h
dark period at 25°C.
4. Place 15-20 1-wk-old seedlings into 250-mL Erlenmeyer flasks containing approx 35
mL of liquid BM medium with 3% sucrose. 5. Place the flasks on a rotary shaker at
120 rpm using 16 h light and 8 h dark period at 25°C.
6. Harvest the roots of these seedlings after 15-20 d of growth.
7. Place the roots into a Petri dish, remove all liquid and cut the roots into small pieces.
8. Transfer root explants onto MSAR I plates and either cover them with aluminium foil
or place them in low light conditions.
9. After 3 wk, transfer the root-derived calli either to fresh MSAR I plates for
maintenance or to MSAR I1 plates for shoot regeneration.
10. After 2 wk, pick the regenerating shoots from the calli and transfer them into glass jars
with MSAR I1 medium for further elongational growth.
11. Shoots consisting of 4-6 leaves may be transferred further into MSAR 111 medium for
3-6 d to induce root development.
12. Transfer the elongated shoots (approx 2 cm) into culture tubes containing 0.5 BM agar
 medium with 0.5% sucrose for rooting, flowering, and setting seeds.
Notes
1. Stocks of growth regulators are prepared at 1 mg/mL concentration, filter sterilized,
and stored at 4OC. Growth regulators are added to autoclaved medium after it has
cooled to about 60°C.
2. Do not sterilize too many seeds in one Eppendorf tube, as they may not be easily
dried. In case seeds clump, use a sterile toothpick to break the clumps before plating
the seeds.
3. Calli can also be obtained from germinating seeds that are placed on MSAR I plates
after surface sterilization.
4. After 15-20 d of culture, 3-5 g (fresh weight) of roots should be produced in each
flask. The roots should be white, actively growing, and not yellow-brown or green.
5. At this stage, it is possible to regenerate and amplify shoots from the regenerated
leaves by cutting them into pieces and placing them on MSAR I1 Medium.
6. The culture tubes are capped with loose cotton to facilitate the aeration required for
seed setting and maturation. Take care not to place the tubes too close to the light
source because this will cause moisture condensation inside the tubes and result in a
low rate of fertilization.
7. Rooted plants can be transferred to soil after washing the roots with water containing
a fungicide, such as Benomyl (0.02%). The plants are gradually acclimated by
reducing the humidity of transfer chambers stepwise.

Somaclonal Variation
Somaclonal variation It is the term used to describe the variation seen in plants that have
been produced by plant tissue culture. Chromosomal rearrangements are an important source
of this variation.
Somaclonal variation is not restricted to, but is particularly common in plants
regenerated from callus. The variations can be genotypic or phenotypic, which in the latter
case can be either genetic or epigenetic in origin. Typical genetic alterations are: changes in
chromosome numbers (polyploidy and aneuploidy), chromosome structure (translocations,
deletions, insertions and duplications) and DNA sequence (base mutations). Typical
epigenetic related events are: gene amplification and gene methylation. If no visual,
morphogenic changes are apparent, other plant screening procedures must be applied. There
are both benefits and disadvantages to somaclonal variation. The phenomenon of high
variability in individuals from plant cell cultures or adventitious shoots has been named
somaclonal variation.
Benefits
The major likely benefit of somaclonal variation in plant is improvement. Somaclonal
variation leads to the creation of additional genetic variability. Characteristics for which
somaclonal mutants can be enriched during in vitro culture includes resistance to disease
pathotoxins, herbicides and tolerance to environmental or chemical stress, as well as for
increased production of secondary metabolites. Micropropagation can be carried out
throughout the year independent of the seasons.
Disadvantages
A serious disadvantage of somaclonal variation occurs in operations which require
clonal uniformity, as in the horticulture and forestry industries where tissue culture is
employed for rapid propagation of elite genotypes. Ways of reducing somaclonal variation:
Different steps can be used. It is well known that increasing numbers of subculture increases
the likelihood of somaclonal variation, so the number of subcultures in micropropagation
protocols should be kept to a minimum. Regular reinitiation of clones from new explants
might reduce variability over time. Another way of reducing somaclonal variation is to avoid
2,4-D in the culture medium, as this hormone is known to introduce variation. Vitrification
[hyperhydracity] may be a problem in some species. In case of forest trees, mature elite trees
can be identified and rapidly cloned by this technique. High production cost has limited the
application of this technique to more valuable ornamental crops and some fruit trees.

Plant Embryogenesis
Plant embryogenesis is the process that produces a plant embryo from a fertilised ovule
by asymmetric cell division and the differentiation of undifferentiated cells into tissues and
organs. It occurs during seed development, when the single-celled zygote undergoes a
programmed pattern of cell division resulting in a mature embryo. A similar process
continues during the plant’s life within the meristems of the stems and roots.
Seeds
Embryogenesis occurs naturally as a result of sexual fertilization and the formation of the
zygotic embryos. The embryo along with other cells from the motherplant develops into the
seed or the next generation, which, after germination, grows into a new plant. Embryogenesis
may be divided up into two phases, the first involves morphogenetic events which form the
basic cellular pattern for the development of the shoot-root body and the primary tissue
layers; it also programs the regions of meristematic tissue formation. The second phase, or
postembryonic development, involves the maturation of cells, which involves cell growth and
the storage of macromolecules (such as oils, starches and proteins) required as a ‘food and
energy supply’ during germination and seedling growth. Embryogenesis involves cell growth
and division, cell differentiation and programed cellular death. The zygotic embryo is formed
following double fertilisation of the ovule, giving rise to two distinct structures: the plant
embryo and the endosperm which together go on to develop into a seed. Seeds may also
develop without fertilization, which is referred to as apomixis. Plant cells can also be
induced to form embryos in plant tissue culture; such embryos are called somatic embryos.
Following fertilization, the zygote undergoes an asymmetrical cell division that gives rise to a
small apical cell, which becomes the embryo and a large basal cell (called the suspensor),
which functions to provide nutrients from the endosperm to the growing embryo. From the
eight cell stage (octant) onwards, the zygotic embryo shows clear embryo patterning, which
forms the main axis of polarity, and the linear formation of future structures. These structures
include the shoot meristem, cotyledons, hypocotyl, and the root and root meristem: they arise
from specific groups of cells as the young embryo divides and their formation has been shown
to be position-dependent. In the globular stage, the embryo develops radial patterning through
a series of cell divisions, with the outer layer of cells differentiating into the ‘protoderm.’ The
globular embryo can be thought of as two layers of inner cells with distinct developmental
fates; the apical layer will go on to produce cotyledons and shoot meristem, while the lower
layer produces the hypocotyl and root meristem. Bilateral symmetry is apparent from the heart
stage; provascular cells will also differentiate at this stage. In the subsequent torpedo and
cotyledonary stages of embryogenesis, the embryo completes its growth by elongating and
enlarging. In a dicot embryo, the hypophysis, which is the uppermost cell of the suspensor,
differentiates to form part of the root cap. Plant cells can also be induced to form embryos in
plant tissue culture; these embryos are called somatic embryos, which are used to generate
new plants from single cells.

Plant growth and Buds

Embryonic tissue is made up of actively growing cells and the term is normally used to
describe the early formation of tissue in the first stages of growth. It can refer to different
stages of the sporophyte and gametophyte plant; including the growth of embryos in seedlings,
and to meristematic tissues, which are in a persistently embryonic state, to the growth of new
buds on stems. In both gymnosperms and angiosperms, the young plant contained in the seed,
begins as a developing egg-cell formed after fertilization (sometimes without fertilization in a
process called apomixis) and becomes a plant embryo. This embryonic condition also occurs
in the buds that form on stems. The buds have tissue that has differentiated but not grown into
complete structures. They can be in a resting state, lying dormant over winter or when
conditions are dry, and then commence growth when conditions become suitable. Before they
start growing into stem, leaves, or flowers, the buds are said to be in an embryonic state.
Somatic Embryogenesis
Somatic embryos are formed from plant cells that are not normally involved in the
development of embryos, i.e. ordinary plant tissue. No endosperm or seed coat is formed
around a somatic embryo. Applications of this process include: clonal propagation of
genetically uniform plant material; elimination of viruses; provision of source tissue for
genetic transformation; generation of whole plants from single cells called protoplasts;
development of synthetic seed technology. Cells derived from competent source tissue are
cultured to form an undifferentiated mass of cells called a callus. Plant growth regulators in
the tissue culture medium can be manipulated to induce callus formation and subsequently
changed to induce embryos to form from the callus. The ratio of different plant growth
regulators required to induce callus or embryo formation varies with the type of plant.
Asymmetrical cell division also seems to be important in the development of somatic
embryos, and while failure to form the suspensor cell is lethal to zygotic embryos, it is not
lethal for somatic embryos.

Somaclonal Variation for Disease Resistance

Most Brazilian wheat cultivars have a high yield potential but are susceptible to several
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alli no se la hauia dado, la qual
dezia en esta manera.

CARTA DE FLAMIANO Á
VASQUIRAN
Verdaderamente, Vasquiran, tus
cartas me desatinan porque
quando miro en ellas el
encarecimiento de tu daño me
parece grande, quando considero
la causa dél lo juzgo pequeño.
Pero en esta carta tuya postrera
he conocido en las cosas que me
escribes lo que te engañas, en
especial en quererte hazer ygual
en el martirio con Petrarca y
Garcisanchez. Si supiesses de
quan lexos vas errado,
maravillarte yas por cierto. Los
tiros de su combate muy lexos
hizieron los golpes de donde los
tuyos dan. De virgines y martires
ganaron ellos la palma si bien lo
miras, que no de confessores de
sus vitorias como tú hazes. Si
gozo ellos han hauido, en la
muerte lo habrian; que en la vida
nunca lo houieron. Mi dolor
sintieron e tu gozo ignoraron.
Claro está segun muestran las
liciones del uno e los sonetos del
otro, e quanto ambos escriuieron,
porque de ninguno dellos leemos
sino pesares en la vida, congoxas
y dolores en la muerte; desseos,
sospiros, ansias apassionadas,
cuydados e disfauores e
desesperados pensamientos;
quando quexando, quando
plañendo, quando pidiendo la
muerte, quando aborreciendo la
vida. Destos misterios dexaron
llenos de tinta sus papeles e de
lastimas su memoria, estos
hizieron sus vidas llenos de pena
e sus fines tan doloridos; con
estos que son los males do mis
males se engendran, con estos
que fueron martirizados como yo
lo soy; verdad es que de dias
vencieron como tú a quien de
amor y fe vencidos los tuvo e los
hizo viuir desseando la muerte
con mas razon que tú la desseas.
Assi que mira lo que por la boca
escriuiendo publicaron e
conoceras lo que en el alma
callando encubierto suffrieron, e
mira si hallarás en ellos vn dia de
victoria como tú plañes doze años
de gloria que dizes que perdiste.
Yo digo que los ganaste, mas
hate parecido a ti que la fortuna te
era obligada a tenerte queda la
rueda en la cumbre del plazer; yo
te prometo que si de sus bienes
no te houiera hecho tan contento,
que de sus males no fueras tan
quexoso sin razon, como estos e
yo lo somos. Tambien me
escriues como soñaste que viste
en vision tu alegria, tus placeres,
tu descanso, tu consentimiento, tu
esperança, tu memoria, tu
desseo; beato tú que primero las
gozaste en la vida y en la muerte
las ensueñas, yo te prometo que
avnque mi placer, ni mi alegria, ni
mi descanso, ni mi
contentamiento, ni mi esperança
yo los encontrasse a medio dia,
que no los conociesse pues que
nunca los vi; mi desseo y mi
memoria no me los cale soñar,
que velando me hazen soñar la
muerte sin dormir cada hora.
Tambien me escribes que viste á
Violina e te habló, e quexaste
dello, ¿qué te pudo hazer
viuiendo que muerta no te quiere
oluidar? No me alegraré yo de lo
que tú, que ni agora en vida ni
despues de mis dias acabados de
mi tuuo memoria ni terná, no digo
de verme que es impossible, mas
avn de pensar si soy en el mundo.
Contentate pues, recobra tu
juyzio, no des mas causa para
que las gentes te juzguen, no
corrompas la reputacion de tu
fama, ni el agudeza de tu ingenio
con tan flaca causa, dando lugar
a tu dolor que de pesar te haya de
tener tal que á ti pierdas e a mi no
ayudes, pues que vees que mi
vida penando se consume; sino te
voy a ver es por la necesidad que
tengo que a verme vengas. Lo
qual te pido que hagas tanto
caramente quanto rogartelo
puedo, porque avnque soledad
busques para tu descanso, la
compañia de mis sospiros te la
dará, e con la mucha confianza
que de ti tengo quedo con tu vista
esperando la respuesta glosando
esta cancion:

Sin remedio es mi herida

pues se cansa quando os veo
y en ausencia mi desseo
más dolor me da en la vida.
¿Qué remedio haurá en mi
pena
si veros fue causa della
y el dolor de mi querella
vuestra ausencia lo condena?
de suerte que no hay salida
para mi, ni yo la veo,
pues veros é mi desseo
son el cabo de mi vida.

LO QUE VASQUIRAN ORDENÓ

DESPUES DE LEYDA LA
CARTA, E COMO SE PARTIO
PARA NOPLESANO.
Otro dia Vasquiran despues de
leyda la carta de Flamiano, de
gran mañana se fue a caça de
ribera y lleuó a Felisel consigo, al
qual despues de hauer volado
una pieça del dia le dixo
tomandole aparte: Ya sabes,
Felisel, como tengo deliberado de
yr a ver a tu señor, porque pues
mis congoxas no bastan para
acabarme quiças las suyas lo
haran; quissiera tenerte comigo
para lleuarte por el camino para
mi descanso e no es cosa que
hazerse pueda por la necesidad
que Flamiano tiene de ti, en
especial con mi yda e tambien
porque no seria razon tomalle
impensado, assi que más eres
allá menester para seruir a
Flamiano que no acá para mi
plazer pues no le tengo, assi que
mañana te parte y darle has
aviso, e pues que yo allá sere tan
en breue, no le delibero escriuir
sino que solamente de mi parte le
digas que si su señora le ha
mostrado sospirar que consigo
aprendera bien á llorar; e assi
hablando se tornaron a Felernisa.
Otro dia Felisel se partió e llegado
que fue á Noplesano fizo saber a
Flamiano la venida de Vasquiran.
Sabido que Flamiano la houo
mandó aparejar dentro en su
posada vn aposento para
Vasquiran, el qual se contenia con
vn jardin que en la casa hauia el
qual mandó adereçar conforme a
la voluutad e vida del que en el
hauia de posar.

LO QUE VASQUIRAN HIZO

DESPUES DE PARTIDO
 FELISEL HASTA LLEGAR A
NOPLESANO
Partido Felisel, Vasquiran deliberó
de yr aquel camino por mar e
mandó fletar vna muy buena naue
de las que en el puerto hauia, e
mandó meter en ella las cosas
que hauia necessarias para el
camino, y embarcar la ropa e
caualgaduras que deliberaua
lleuar; e assi partia á su heredad
ante de embarcar por visitar la
sepultura de Violina. Llegado alli
vna tarde mandó sobre la tumba
pussiesen un titulo con esta letra:

Aqui yaze
todo el bien que mal me haze.

E assi mandó dar orden en todo

lo que en ausencia suya deuia
hazer assi en el concierto de la
casa como en los officios de la
capilla, e assi despidiendose a la
partida hizo esta cancion a la
sepultura:

Pues mi desastrada suerte

contigo no me consiente,
quiero ver si estando ausente
pudiesse hallar la muerte.
Lo que mi viuir querria
es no verse ya comigo
porque yo estando contigo
más contento viuiria,
e pues que veo qu'en verte
 mi pena descanso siente,
cierto so que estando ausente
no verna buscar la muerte.

Otro dia se tornó a Felernisa e

queriendo partirse para
Noplesano mandó poner sobre el
portal de su casa un titulo que
dezia:

Queda cerrada la puerta

que la muerte halló abierta.

Aquesta noche mandaron

embarcar sus servidores, él se
embarcó ante que fuesse de dia
por escusarse de la importunidad
de las visitaciones e de los que al
embarcar le houieran querido
acompañar, hauiendo empero
visitado algunas personas
principales a quien la raçon e
alguna obligacion le constriñia.
Pues siendo ya embarcado
queriendo la naue hazer vela ante
que amaneciese, hizo esta
cancion:

El morir vino a buscarme

para matar mi alegria,
e agora que yo querria
no me quiere por matarme.
El me vino a mi a buscar
teniendole aborrecido
e agora que yo le pido
no le halla mi pesar,
 assi que haurá de forçarme
a buscalle mi porfia
pues veo que se desuia
de mi para más matarme.

Hecho que houo vela la naue, en

pocos dias fueron a vista de la
tierra de Noplesano, e por hauer
tenido algo el viento contrario
hallaronse algo baxos del puerto,
e no podiendole tomar acordaron
por aquella noche de surgir en
vna costa que está baxo de dicho
puerto a quarenta millas de
Noplesano, la qual es tan aspera
de rocas e peñas e alta montaña
que por muy pocas partes se
puede andar por ella a cauallo,
empero es muy poblada de
jardines e arboles de diuersas
maneras, en especial de
torongeros e sidras e limones e
toda diuersidad de rosas, e
muchas caserias assentadas por
lo alto de las rocas; e a la marina
hay algunos lugares e vna gentil
cibdad que ha nombre Malhaze
de donde toma el nombre la
costa. Pues assi llegados, la naue
surgió en vn reparo del viento que
venian muy cerca de tierra, en el
qual lugar, ya otra vez hauia
estado Vasquiran trayendo
consigo a Violina hauia mucho
tiempo. Pensar se puede lo que
Vasquiran sentiria viniendole a la
memoria, la qual le renouo
infinitos e tristes pensamientos los
quales le sacauan del coraçon
entrañables sospiros e infinitas
lagrimas, las quales porque mejor
e mas encobierto derramallas
podiesse, con una viuela en la
mano, de la nao se salio e
sentado sobre una roca muy alta
que la mar la batia, debaxo de vn
arbol començo a cantar esta
cancion:

No tardará la vitoria
de mi morir en llegar,
pues que yo vi este lugar
qu'era tan lleno de gloria
quanto agora de pesar.
Yo vi en toda esta riuera
mill arboles de alegria,
veola agora vazia
de plazer de tal manera
que me da la fantasia
qu'el dolor de su memoria
ya no dexará tardar
mi morir de no llegar
para darme tanta gloria
quanto m'a dado pesar.

Estando alli assi cantando e

pensando acordose que en aquel
mismo lugar hauia estado,
quando por alli passaron él e
Violina e otras señoras que en la
naue venian, toda vna tarde a la
sombra de aquel arbol jugando a
cartas e razonando, e hauian
cenado con mucho plazer
mirando la mar, e assi
acordandose dello començo a
cantar este villancico.

Di, lugar sin alegria,

¿quién te ha hecho sin plazer
que tú alegre solias ser?
¿Quién ha hecho tus
verdores
e tus rosas e tus flores
boluer todas en dolores
de pesares e tristuras,
quién assi t'a hecho ascuras
tus lumbres escurecer
que tú alegre solias ser?

Passada parte de la noche, ya

Vasquiran recogido en la naue,
con el viento de la tierra hizieron
vela e llegaron a hora de missa al
puerto de Noplesano. Mandó
Vasquiran que ninguna señal de
alegria la naue en la entrada
hiziesse de las que acostumbran
hazer. Sabido Flamiano por un
paje suyo que de unos corredores
de su casa vio la naue entrar, lo
que en la entrada hauia hecho,
penso lo que podía ser, e con
algunos caualleros mancebos que
con él se hallaron, sin más
esperar junto con ellos al puerto
se vino, e llegaron al tiempo que
la naue acabaua de surgir, e assi
todos apeados en vna barca en
ella entraron e hallaron a
Vasquiran que se queria
desembarcar. E assi se recibieron
con mucho amor e poca alegria.
Estando assi todos juntos
teniendo Flamiano a Vasquiran
abraçado, en nombre de todos
ellos le dixo: Vasquiran, a todos
estos caualleros amigos tuyos e
señores e hermanos mios que
aqui vienen o son venidos a verte,
no les duele menos tu pesar que
a mi; con tu vista se alegran tanto
como yo. Al qual él respondió:
Plega a Dios que a ti e a ellos
haga tan contentos con la vida,
como a mi con la muerte me
fazia. Al qual respondio el
marques Carlerin: Señor
Vasquiran, para las aduersidades
estremó Dios los animos de los
caualleros como vos, pues que no
es menos esfuerzo saber suffrir
cuerdamente que osar venzer
animosamente. Vasquiran le
respondio: Verdad es, señor
marques, lo que dezis, pero
tambien hizo Dios a los discretos
para saber sentir las perdidas,
como a los esforçados para
gozarse de las ganancias de las
vitorias, e no es menos virtuoso el
buen conocimiento que el buen
animo, ni vale menos la virtud por
saber bien doler, que saber bien
sofrir e osar bien resistir.
E assi razonando en muchas
otras cosas semejantes, salieron
de la naue, e todos juntos vinieron
a la posada de Flamiano donde
hallaron muchos caualleros que
los esperauan, e todos juntos alli
comieron hablando de muchas
cosas. E assi aquel dia passaron
en visitas de los que a ver
vinieron a Vasquiran y de muchos
señores que a visitar le embiaron.

LO QUE VASQUIRAN HIZO

DESPUES DE LLEGADO
Á NOPLESANO
Otro dia despues de hauer
comido, Vasquiran acordo de yr a
besar las manos a la señora
duquesa de Meliano e a Belisena,
e despues al visorey e al cardenal
de Brujas e a la señora princesa
de Salusana e a algunas otras
personas que sus estados e la
raçon lo requeria. E assi
acompañado de algunos
mancebos que con él e con
Flamiano se hallaron, hauiendolo
hecho saber a la señora duquesa
se fueron a su posada, y yendo
por el camino, Flamiano se llegó a
Vasquiran e le dixo: agora ymos
en lugar donde tú de tus males
serás consolado e yo de los mios
lastimado. Al qual respondio
Vasquiran: mas voy a oyr de
nueuo mis lastimas; tu vás a ver
lo que desseas; yo recibire pena
en lo que oyre; tú recibiras gloria
en lo que verás. Assi razonando
llegaron a la posada de la señora
duquesa, a la qual hallaron en
vna quadra con aquel atauio que
a tan gran señora siendo uiuda se
requeria, acompañada de la
señora Belisena su hija, con todas
las otras damas e dueñas de su
casa. E como las congoxas de los
lastimados con ver otros llagados
de su herida no pueden menos de
no alterar el dolor de las llagas,
alli hauiendo sido esta noble
señora vna de las que con más
raçon de la aduersa fortuna
quexarse deuia, uiendole perder
en poco tiempo el catolico abuelo,
la magestad del serenissimo
padre, el clarissimo hermano en
medio del triunfo mas prospero de
su gobierno reynando, e sobre
todo el ylustrissimo marido tan
tiranamente de su estado e
libertad con el heredero hijo
desposseidos, de manera que no
pudo menos la vista de Vasquiran
hazer que de mucho dolor su
memoria no lastimasse, e
verdaderamente ninguna de las
que viuen para ello mas raçon
tiene.
Pues assi llegados, hauiendo
Vasquiran besado las manos a la
señora duquesa, e a Belisena
hecho aquel acatamiento que se
deue hazer e a todas las otras
señoras e damas, despues de
todos sentados, la duquesa
començó de hablar en esta
manera.

LO QUE LA SEÑORA DUQUESA

HABLÓ A VASQUIRAN EN
PRESENCIA DE TODOS; E
LO QUE VASQUIRAN LE
RESPONDIO E ALLI PASSÓ.
Vasquiran, por vida de mi hija
Belisena qu'es la mas cara cosa
que la fortuna para mi consuelo
me ha dexado, que considerado
el valor e virtud e criança tuya, y
el amor e voluntad que al duque
mi señor, que haya santa gloria, e
a mi casa siempre te conoci tener,
sabido tu perdida tanto tu daño
me ha pessado, que con los mios
ygualmente me ha dado fatiga.
Esto te digo porque conozcas la
voluntad que te tengo, lo que
consolarte podria remitolo a ti
pues te sobra tanta discrecion
para ello quanto a mí me falta
consuelo para mis males.
Vasquiran le respondio: Harto,
señora, es grande mi desuentura
quando en tan alto lugar ha hecho
señal de compasion, mas yo doy
gracias a Dios que me ha hecho
tanto bien en satisffacion de tanto
mal qu'en tan noble señora como
vos e de tan agrauiados males
combatida mi daño haya tenido
cabida o lugar de doler; lo que yo
señora siempre desseo vuestro
seruicio Dios lo sabe; lo que en
vuestras perdidas yo he sentido
ha sido tanto que el dolor dellas
tenia ya en mí hecho el aposento
para quando las mias llegaron.
En esto y en otras cosas
hablando llegó el tiempo de
despedirse, en el que nunca
Flamiano los ojos apartó de
Belisena. Pues siendo de pies ya
de la duquesa despedidos,
Vasquiran se despidio de
Belisena a la qual dixo: señora,
Dios os haga tan contenta como
vos mereceys e yo desseo,
porque ensanche el mundo para
que sea vuestro y en que mi
pesar pueda caber. Al qual ella
respondio: Vasquiran, Dios os dé
aquel consuelo que con la vida se
puede alcançar, de manera que
tan alegre como agora triste
podays viuir muchos dias. E assi
la señora Yssiana se llegó a ellos
e muy baxo le dixo: señor
Vasquiran, esforçaos, que no
juzgo menos discrecion en
vuestro seso que dolor en vuestro
pesar; la fortuna os quitó lo que
pudo, pero no la virtud que en vos
queda que es más.
Señora, dixo Vasquiran, plega á
Dios que tanta parte os dé la
tierra quanta en vuestra
hermosura nos ha dado de lo del
cielo, pues que está en vos mejor
aparejado el merecer para ello
que en mí el consuelo para ser
alegre. Bien sé yo que si posible
fuera que en mí pudiera hauer
remedio para mi tristeça, el
esperança de vos sola la
esperara.
Al qual respondio la señora
Persiana: Vasquiran, por la
compasion que tengo de ver
vuestra tristeça, quiero consentir
que me siruays e sin perjuizio mio
yo hare que perdays mucha parte
de vuestra passion con mis
fauores.
Assi tornado a la señora duquesa
se despidio con todos aquellos
caualleros que con él hauian
venido, e quedose alli el marques
Carlerin. De alli se fueron a visitar
al señor visorey con el que
hallaron al cardenal de Brujas y el
cardenal de Felernisa, los quales
todos con mucho amor le
recibieron. El restante de lo que
alli passó, por abreuiar aqui se
acorta. Assi se tornaron á su
posada. Otro dia fue a besar las
manos a la reina Noplesana e a
su madre, e despues a otras
muchas señoras que a la sazon
en Noplesano se hallaron.

LO QUE DESPUES DE LAS

VISITACIONES E HAUER
REPOSADO ALGUNOS DIAS,
ENTRE FLAMIANO Y
VASQUIRAN PASSÓ SOBRE
SU QUESTION
Estando vn dia acabado de comer
Vasquiran e Flamiano en vna
huerta de su posada acostados
de costado sobre vna alfombra
debaxo vnos naranjos, comemço
Vasquiran en esta manera de
dezir. No quiero, Flamiano, qu'el
plazer de nuestra visita con su
plazer ponga silencio en nuestra
question a sus pesares, porque
tanto por dalle fin a nuestra
question soy venido, quanto por
verte; a tu postrera carta no
respondi por hazerlo agora.
Muchas variedades he visto en
tus respuestas assi de lo que en
mi contradizes como de lo que en
ti manifiestas, en especial agora
que a Belisena he visto, e digo
que todo el fin de tu mal seria
perder la vida por sus ameres;
digote vna cosa, que si tal
perdiesses el más de los bien
auenturados te podrias llamar,
¿pues si tu muerte seria
venturosa, tu pena no es
gloriosa? claro está. Todas las
cosas que me has escripto en
cuenta de tus quexas, agora que
lo he visto juzgo en cuenta de tus
glorias; quando nunca más bien
tuuiesses de verte su servidor es
mucho para hacerte ufano,
quanto más que tus ojos la
pueden ver muchas veces, que
más bien no le hay. Quantas
cosas me podrias encarecer de
los males que pregonas no son
nada, por que Quiral en tu egloga
te ha respondido lo que yo podria;
digote vna cosa, que te juzgo por
mas dichoso penando en seruicio
suye que no si alegre te viese sin
seruilla. Si assi supiesses tú suffrir
contento tu pena como supiste
escoger la causa della, ni comigo
competerias como hazes, ni yo te
reuocaria como hago. No plega a
Dios que mi mal sepas a qué
sabe, ni de tu pena sanes porque
viuas bien auenturado. Mirado el
lugar do tu desseo e voluntad
possiste, de todo lo possible
gozas; visto lo que quexas, todo
lo impossible desseas. Visto lo
que yo perdi no hay mas bien que
perder; visto lo que yo desseo no
hay mas mal que dessear, pues
que al fin con la vida se acaba
todo.
A todas las cosas que me has
escripto te he respondido; a lo
que agora me querras dezir
tambien lo verás, oyrte quiero.
 RESPUESTA DE FLAMIANO
Vasquiran, todo quanto hasta
agora en mis cartas y de palabra
te he escripto y enbiado a dezir,
en dos cosas me parece que
consiste. La vna, ha sido
parecerme que quexas mas de lo
que deues e que no perdiste sino
que se acabó tu plazer, e que
demasiado estremo dello
muestras. La otra ha sido que mi
mal es mayor qu'el tuyo. Agora
quiero que despacio juntos lo
determinemos, e quiero començar
por mí. Dizesme que las virtudes
e merecimientos de Belisena con
quantas excelencias en ella has
visto, me deuen hazer ufano y
contento, e que si por ella
perdiesse la vida seria bien
auenturado, e que no puedo mas
perder, e que cada hora la veo,
que no hay más bien que perder e
que desseo lo impossible y gozo
lo possible. ¿Cómo se podra
hazer que las perficiones de
Belisena si estas mismas
encienden el fuego do m'abraso
hagan mi pena gloriosa? quanto
más de su valer contento, más de
mi remedio desconfio, e si como
dizes por ella la vida perdiesse,
bien dizes que seria bien
auenturado, mas no la pierdo y
muero mill vezes cada hora sin
que agradecido me sea; el bien
que me cuentas que por su vida
gano, es todo el mal que cada
hora renueua mis males, pues
que para más no la veo de para
mis pesares. Pues mi desseo es
impossible, ¿qué bien puedo
hauer que sea lo posible como tú
dizes? A mi me pareze que el fin
de todas las glorias está en
alcançarse e no en dessearse,
porque el desseo es un acidente
que trae congoxa, e quanto mayor
es la cosa deseada mayor es la
congoxa que da su desseo;
¿pues cómo me cuentas tu a mi
el desseo por gloria siendo él
mismo la pena? Visto estar claro
que de todas las cosas e desseos
se espera algun fin, de todos los
trabajos se espera algun
descanso. Todos los desseos se
fundan sobre alguna esperança,
porque si cada cosa destas esta
causa no la caussase, no ternia
en si ninguna razon, pues que no
tuuiesse principio donde naciesse
no ternia termino do acabase,
pues no teniendo principio ni cabo
consiguiente caduca seria. Pues
luego si mi desseo es impossible
y es grande y grande la pasion
que me da, ¿qué cuenta haura en
mi mal? no otra sino que no hay
remedio para él? Pues si el
remedio le falta, el mio es grande,
que el tuyo no.