PART I
ESSENTIAL BASIC INFORMATIONMutation Breeding Manual

1. AN OVERVIEW OF THE METHODS OF CROP IMPROVEMENTOBJECTIVES- To increase yield- To develop plants that are resistant to pests and diseases- To improve quality- To develop plants that have resistance to adverse conditions

Selection

Introduction

Hybridization

Mutation Breeding

PIONEERS- 700 BC, Assyrians & Babylonians werealready artificially pollinating Date Palm
- 1694, Camerarius - reported sex in plants- 1717, Thomas Fairchild - Demonstrated
artificial hybrid in plants- 1758-1835, Thomas Andrew Knight - Firstman to use hybridization for practical plant
improvement- 1866, Gregor Mendel - First auto-ritative
establishment of the principles of inheritance- 1868, Meischer - discovered DNA- 1900, Hugo De Vries, Carl Correns & Erich
Tschermak - rediscover the work of Mendel
independently- 1953, Watson & Crick - discovered DNA
materials that carry the hereditary charactersof organisms- 1960-66, Joseph Kolreuter - did the systematic
study of artificial hybridization

Biotechnology

PIONEERS- 1896, Becquerel - discovered
radiosensitivity- 1896, De Vries - proposed the useof radioactivity for inducing
mutation- 1927, Muller - showed that x-ray
induce genetic deviants in
Drosophila- 1928, Stadler-reported highmutation rates in x-rayed maizeand barley- 1934-35, Herman Nilsson-Ehle &Ake Gustaffson-started mutation
experiment in plants & reported
erectoids mutants in barley withcompact head type & stiffcr straw

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Mutation Breeding Manual

PIONEERS
I. 1854 - 1945, Gottlieb Haberlandt - father of plant tissue culture. He was the first person to culture
isolated fully differentiated cells and ovary.
II. 1901, Winkler - observed that pollen tubes stimulate growth in ovules and ovary.
III. 1904, Manning - successfully grew excised nearly mature embryo.
IV. 1922, Robbins and Kotte - reported some success with growing isolated root tips.
V. 1925-29, Laiback - demonstrated the practical application of embryo culture in plant breeding by
isolation the embryo from a non-viable seeds of the cross Linum perenne x L. austriacum and rearedthem to maturity on a nutrient medium.
VI. 1934, White - reported successful growing for the first time continuous culture of tomato root tips.
VII. 1939-50, Street and his Students - conducted an extensive work on root culture to understand therole of vitamins in plant growth and shoot - root relationship
VIII. 1939, Gautheret - first to continuously grew tissue culture from carrot roots.
IX. 1939, Gautheret, White and Nobercourt - were credited for laying the foundation for further workin plant tissue culture.
X. 1941, Van Overbeek and Co-Workers demonstrated for the first time the stimulatory effect of
coconut milk on the embryo development and callus formation in Datura
XI. 1963, Raghavan and Torry; 1965, Norstog and others - successfully developed synthetic media
for culture of younger embryo.
XII. 1944, Scoog; 1951, Scoog and Tsui - demonstrated that the addition of adenine and high levelsof phosphate increased callus and bud formation in tobacco pith tissue culture.
XIII. 1953, Muir - demonstrated that transferring callus of Tageta erecta and Nicotiana tabacum toliquid medium and agitating the culture on a shaking machine broke the tissue into single cells andsmall cell aggregate.
XIV. 1954, Muir et al, - succeeded in mechanically picking single cell from the shaked culture aswell as callus tissues and making them individually on separate filter resting on the top of the wellestablished callus-culture.
XV. 1960, Jones - designed a micro-culture method for growing single cells in hanging drops in aconditioned medium.
XVI. 1960, Cocking - demonstrated the feasibility of enzymatic degradation of cell walls to obtainlarge quantities of viable naked cells called protoplast.
XVII. 1965, Vasil and I lildebrandt - raised whole plants starting from single cells in tobacco.
* Prepared by Mr. A. B. Asencion, PNRI

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Mutation Breeding Manual

Electromagnetic Radiation- X-rays from X-ray generator- Gamma rays froma. Co-60 (half-life –5.27 yrs.)
b. Cs-137 (half-life – 30 yrs.)
Corpuscular Radiation- -Thermal or Slow Neutrons fromreactors- -Fast Neutrons from reactors- -Beta Particles
a.
a. Phosphorus-32 (half-life-14.3 days)b.
b. Sulfur-35 (half-life-87 days)

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Mutation Breeding Manual

2. MOLECULAR BASIS OF MUTATION: THE NUCLEUS, CHROMOSOMES AND DNA
2.1 THE NUCLEUS
Cells whether of animal or plant (except for very few exceptions) are composed of two most importantprincipal recognizable parts, i.e. nucleus and cytoplasm. The nucleus is bounded by a membrane within
the cytoplasm. It is separated form the cytoplasm by a membrane. Generally, these two major parts of acell are function-wise separate. The nucleus coordinates and directs cellular activities whereas thecytoplasm carries out the directions made by the nucleus. The cytoplasm is the site of metabolism in thecell, i.e. building up of protein and other essentials and the tearing of worn out structures with theconsequent production of waste materials. But the nucleus orders times and directs this metabolism. The
separation of the nucleus and cytoplasm does not imply independent, but are dependent upon each other.
Neither could survive without the other. Events happening in either the nucleus or cytoplasm will haveimportant effects in the other major parts of the cell. Changes occurring in the nucleus can alter its ability
to direct metabolism in the cytoplasm. Likewise, alterations in the cytoplasm or even in its membranethat surrounds it, can exert an effect within the nucleus since it is the metabolic activity of the cytoplasm
that supplies the materials the nucleus needs for its own existence, i.e. its repair and reproduction and its
function of coordination and direction of cellular activity. Hence, due to its intimate interdependentchanges detected in the nucleus in structure and function may have been due to events taking place ineither itself or in the cytoplasm and vice-versa. Changes in the cytoplasm that bring about anomalies innuclear material will almost unavoidably have further consequences in the cytoplasm. An injured nucleus,
no matter the cause of the anomaly or change will not be capable of properly directing cytoplasmic
activity. Changes in cytoplasmic function then may be the result of impairment of the nuclear function
which may have resulted due to an injury in the cytoplasm.
2.2 THE CHROMOSOMESThe apparatus within the nucleus that directs nuclear activity are the chromosomes. They are present inmost cells in specific and fixed numbers. The number of chromosomes in a cell is characteristic of thespecies. All members of a species carry the same number of chromosomes in every cell. The germ cells(male and female gametes) and the somatic cells (the cells composing the body) differ in chromosome
number. The gametes have half the number of chromosomes (n) found in the somatic cells. The somatic
cells have twice (2n) as the gametes. The chromosomes which constitute the n number is said to be a setof chromosomes. The n number is made up of a particular number of chromosomes each of which isdifferent from the other. The chromosomes will often differ among themselves in size or shape. More
importantly, they may differ in content. All of them taken together form a set, a complete ofchromosomes, the n set. It is very necessary for in most organisms, it is a minimum requirement for life.
Cells that in some way lose chromosomes or even only parts of it, so that the basic set number isincomplete, cannot live. Even if the cell has more than the n of chromosomes, it will not live if the set isincomplete. The somatic cells have twice the number of chromosomes as are found in germ cells. They
actually have two n sets of chromosomes, i.e. each chromosome in the n set of the germ cells in present insomatic cells in duplicate. The symbol used is 2n and the somatic cells are said to have the 2n number ofchromosomes with the knowledge that they really represent n + n sets. In man, 2n = 46, n = 23; in onions
2n = 16, n = 8; Tradescantia 2n = 12, n = 6, etc). The chromosomes are the structures which carries thegenes. They are the vehicles of the molecules that direct the metabolism of each cell and hence of thetotal organism. In order to have a complete set of genes, and to have the proper balance between genes
for metabolism to be carried out properly, a 2n set of chromosomes is a fundamental requirement. Lossof a chromosome or loss of even a small part of a chromosome will almost inevitably result in the loss of
one or more genes, which may produce metabolic anomalies, for unless another is present to take overthe function of the lost one, the function itself will, in most likelihood disappear. Or worse, if more are
lost,life is impossible. The chromosomes are generally characterized morphologically by the position of
the centromeres (the primary constriction ) and according to its size (Fig.I-1a, b)

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Mutation Breeding ManualFig. I-1a
C-Centromere or primary constriction with four centromeric granules orspindle sphericle; Ch-chromonemata; NO-nucleolus organiser, or
secondary constriction; S-satellite; T-Telomere; SC-secondary
constriction; M-matrix

Fig. I-1b Mitotic metaphase chromosome
A- Median position-Metacentric; B- Both arms nearly but not quite equalSubmetacentric; C- If short arm is approximately spherical, has the diameter of thelong arm subaciocentric; D- short arm can be seen, has a diameter clearly less than the
diameter of the long arm-acro- centric; E- Telocentric, no observable short arm
whatsoever.

2.3 THE GENES
The structures in the nucleus of the cell of which the chromosomes are the vehicle which control
inheritance and metabolism are called the genes. The genes are composed of very large molecules called7

Mutation Breeding Manual

deoxyribonucleic acid (DNA). The type of molecules of which DNA is a representative is called amacromolecule. The macromolecules are made up of subunits which are stable molecules withcharacteristics peculiar to themselves. It is a large molecule, having certain characteristics, which is madeup of smaller yet discrete molecular units. DNA as in most other macromolecules, the subunits of which
the whole is composed of, are joined together end-to-end to form a long chain, in what is calledpolymerization. Such molecules are said to have been polymerized. DNA is usually described as a longchain polymer, a olymer composed of many subunits connected end-to-end as links in a chain. Eachmolecule of DNA is composed of two unbranched threads. The two chains or threads are joined in adderfashiion. The DNA is twisted or spiral structure reseming a spiral staircase. The lnkage fo the subunits inthe chains and the chains to each other is very important, as its is the basis of the mechanism by which
genetic information is coded and stored in the cells, as well as the basis where this coded information istransmitted to the cytoplasm. It is also important in the proper carrying of genetic information from one
cell generation to the next.
2.4 THE DNA
The subunits of which DNA is made up of are themselves composed of phosphoric acid, the five carbon
sugar, deoxyribose, and an organic, nitrogenous base. These components taken together are called
nucleotides, specifically deoxyribonucleotides. It is these nucleotides that are arranged in sequence ineach chain making up DNA. In the macromolecule (the double chain) the side pieces or backbones of theladder are sugar-phosphates, while the rungs are nitrogenous bases. Only four nitrogenous bases arecommonly associated with DNA. They are categorized as purines and pyrimidines. Close examination of
the DNA will show that each is made up of nucleotides which differ only in their nitrogenous bases. Thisdifference is specific and constant. Whenever adenine occurs in one chain, thymine will be in the opposite
chain. Whenever cytosine occurs in one chain, guanine will occur in the opposite chain. Hence DNA
chains have polarity or direction. In each DNA macromolecule chain, a complement appears in the
opposite chain. In most molecules of DNA in nature, there will be equal amounts of adenine and thymine
and equal amounts of guanine and cytosine, as they occur in pairs. The two chains of the DNA
macromolecule are held together by a very weak form of chemical bond called a hydrogen bond, where
two molecules share one hydrogen atom and are held together by the atom they share. In DNA, each basepair shares two or three hydrogen atoms so that each is bonded at two or three points, where the chainsare joined, the rungs of the ladder are complete (Fig. I-2a, b).

Fig. I-2a Hydrogen-bonding between the bases as it occurs in DNA

8Mutation Breeding ManualFig. I-2b The double helix model of DNA
Cellular metabolism as stated earlier is under the control of the gene. The genes and consequently, theDNA which makes them up, direct all the activities of any given cell, hence the DNA must contain all
information necessary for the activities of any given cell to direct those activities. A cell’s activities
include, not only the chemical reactions going on within it but will also include whatever intercellularinteractions in which the cell might participate. Such information is indeed present and is stored in the
DNA molecule. The sequence of the nitrogenous bases, the purines and pyrimidines along both sides of
the DNA macromolecule is the genetic information. The sequences of the purines and pyrimidines along
the strands of the DNA polymer is, in a highly distilled or modified mannier, i.e. containing all of theinformation required by the cell for all its metabolic activity. The sequence or linear arrangement of thesebases contains the genetic information and not the bases themselves. It is the linear arrangement orsequence that holds the genetic information. Hence any force which changes this sequence will produce achange in the information stored in the molecule. Such changes will almost always result in an importantchange in the metabolism of the cell in which they happen. The changes are called mutations and theindividual that bear them are mutants.
9Mutation Breeding Manual

Mutations have been known for many years as a naturally occurring phenomenon. Almost every
species of plant studied, including the very simples, is known to have members arise in its midst which
differ from the rest. These are mutants which occurred after a change in the genetic material. Thechanges and the mutant individuals appeare apparently at random in any population of living things. No
cause is now known that explains their appearance. As a result, they have been called spontaneous
mutations.
Spontaneous mutations are permanent, heritable changes in individual genes. When genes mutate, thechange will, for any given change in any given gene, a rate event, sudden and in discrete fashion, i.e., thegene will pass from one state to another, both of which are stable indicating the genes undergo a definite
transformation when they mutate, and that, between mutations they are fixed in composition. The
appearance of spontaneous mutations then is an indication that the genes have a distinctive chemical
structure, which, like any chemical change, is subject to the all-or-nothing rule.
All somatic cells within an individual contain the 2n number of chromosomes. The genes (or the DNA)
are actually part of the chromosomes, and a typical somatic cell contains the 2n number of genes and the
2n amount of DNA. When cells have reached the point where they need to divide it will be necessary forthe daughter cells of this division to be provided with the 2n number of chromosomes and with the 2nnumber of DNA. If this provision were not made, the daughter cells resulting from the division of amature somatic cell might have different numbers of chromosomes and genes and a different balance
between genes. Daughter cells would differ from their parent cell and other cells in the body of which
they are part of. To preserve the 2n quantities and balance relationship between genes, the number ofchromosomes and the quantity of DNA in the nucleus, must double just before cell division. The DNA
and the chromosomes duplicate themselves. Before the cell division, the cell about to divide will have the
4n number of chromosome and the 4n quantity of DNA. Half of this 4n quantity (two copies of each set)
will go to each daughter at division, paving the way for each daughter cell to receive the proper 2namount of genetic material and have genetic balance similar to its parent.
When the DNA duplicates itself, the hydrogen bonds which link the chains of the DNA molecule break,and the chains separate. Each then directs the synthesis of the complementary chain, the chain similar tothe one from which it just separated. The synthesis of the new DNA (the complementary chain) begins at
one end of an existing chain and continues to its terminus closing zipper-like. In the nucleus, all of thefour nucleotides will be available for the existing DNA to incorporate into the newly synthesizing
complementary chain. As a new chain is synthesized, only the proper nucleotide, the right member of thenucleotide pair will occupy each place in the new chain. Only the particular bases, i.e. the proper memberof each pair can form a hydrogen bond with the other member of the base pair while at the same timepresenting the phosphate group of the new nucleotide in the proper position to be bonded in the growing
new chain. DNA synthesis under ordinary circumstances is really DNA duplication, for the new DNA is
identical to the old, i.e. no change comes about. Hence, at cell division, each daughter cell which
provided no error in the DNA transmission will get exactly the same genetic information as its parent had.
Another sequence of events, however, may take place in rare situations where the electrons and theprotons of the molecules of nitrogenous bases can undergo rearrangement. The distribution changes
within these molecules so that they actually have more than one possible structural configuration. The
various forms have the same number of protons and electrons, they are however, arranged somewhat
differently in each molecule, resulting into the rearrangement of the parts of the molecule called atautomeric shift. The rearranged structural configurations, where molecules may exist are calledtautomers. One tautomeric form will be, by far, the most commonly occurring one. Most of themolecules of these bases will exist in this form. But sometimes on rare occasions, an internalarrangement (a tautomeric shift) will take place in another molecule in a common form to produce
another uncommon form, a rare one. These shifts are not necessarily permanent. These rare forms canundergo rearrangement either to other rare forms or back to the common one. When a tautomeric shift on
one of the bases in a nucleotide occurs during DNA synthesis, the resultant new DNA may not be a
duplicate of the old. It may differ from the old in some very significant detail. Many consider thisphenomenon of tautomerism of the nitrogenous bases as the molecular basis of the mechanism of
mutation.

10Mutation Breeding Manual

The base when it is in its common form during DNA synthesis can form a hydrogen bond with only oneof the other bases, that is adenine-thymine, guanine-cytosine and yet presents each phosphate group in theproper position for bonding in the molecule. But if one of the bases should be in a rare tautomeric state
during DNA synthesis, it may not be able to form a hydrogen bond with its partner, i.e. adenine will not
be able to pair with thymine and neither will guanine in its rare form pair with cytosine in their tautomericstates. They will find paring in the DNA with adenine and guanine difficult. The rare tautomers will beable to pair though with the base of which pairing is forbidden, i.e. rare adenine pairs with cytosine andrare thymine with guanine resulting in directing the incorporation of the wrong partner, the forbidden
partner, into a new DNA chain at the level of rare tautomer. The sequence of bases of such rare
tautomers will be different from the sequence of the old chain so that the genetic information may be
drastically changed. Although at this stage of events a full blown mutation does not as yet exist, it is atransition stage between the old and the new, and the inception of a possible mutation. The rare tautomer
in the old chain will probably return to its common state and is not permanent. Change will have ensued
in that part of the molecule. In the new DNA chain, the sequence of the bases is permanent. The genetic
information it represents is inheritable. Such a sequence of events take place in a cell giving rise to
gametes, the mutation will be handed on to the future generations of the individuals through the gametes.
When the cell that inherits this changed DNA chain prepares to divide the chain will, of course, direct thesynthesis of a chain complementary to itself. It will do so according to the permitted (altered) pairing ofbases, so that the complementary chain synthesized will be different from the one that made them before.
Thus, this change too is inheritable. Both chains of DNA have undergone the change and the mutation issaid to be accomplished and completed.

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Mutation Breeding Manual

3. MUTAGENESIS
In nature, sudden, rare, discrete changes in the genetic material which result in a permanent change inthe expression of the genes take place spontaneously may be due to an interplay of the combined effect ofbackground radioactivity, extremely high or low temperatures, presence of chemicals and age of seeds
and plant nutrition. These sudden heritable changes in plants or animals are called mutation and may beclassified as spontaneous or induced, somatic or genetic, chromosomal or extra-chromosomal. Althoughmutations occur spontaneously with fixed frequencies, it does not mean that the frequency of eachoccurrence is not changing. Certain agents, known by its generic name mutagens can change the rate ofits appearance. Mutagens as the name implies, (mutation-change; genesis – to give rise to or generatechange) as an agent of mutation can increase its frequency and rate. There are a number offundamentally different types of mutagens completely unrelated in structure and mechanism of action. A
large general type of rich diversified agents can be found in chemical mutagens, can change the genetic
material of incorporated cells. Ionizing and those of lesser energy (ultraviolet) radiations are mutagens
that are quite efficient for producing changes in genetic material. But it must be emphasized that
mutation is basically a property of the genetic material and not a property of the mutagens themselves.
Since mutagens are so different from each other both in what they are and how they act, mutation isprobably a response of the genetic material common to any agent that produces ultramicroscopic, pointlike discrete changes in it.
3-1 RADIATION MUTAGENESIS
It has been suggested that ionizing radiation acts by ionizing nitrogenous bases in the DNA chains inparticular during the DNA synthesis in the production of mutations. If ionization of the base occurs
during DNA synthesis, forbidden base pairs can come into existence in the new macromolecule just as
they are formed following tautomeric shifts in the structure of the bases. As a result, the sequence ofbases in the newly synthesized chain would be wrong, and a permanent, heritable, discrete change in the
DNA would be produced. This change(s) or mutation would be similar to the one that occurs
spontaneously, but the frequency of its appearance would have been increased. Aside from ionization of
the bases of DNA, base change or base deletions, effects that can change critical base sequences of themolecule, may occur. If sufficient numbers of ionization take place within one or more bases or ifinteraction with free radicals so alter one or more bases they are deleted from the DNA molecule. Base
deletion occurs.
There are available a number of radiation types and radiation sources to mutation breeders. Aside fromultraviolet, several types of ionizing radiation, i.e. X- and gamma rays, alpha and beta particles, protons
and neutrons, which has in common the ability to form discrete releases of energy called ionization or ion
pairs, as they pass through matter. X-rays, like gamma rays and the UV light are electromagnetic
radiations emitted as quanta (100.001nm for X- and gamma rays in contrast to 2,000 to 3,000 nm for UV
light). In an X-ray machine electrons are electrically accelerated in a high vacuum and then stopped
abruptly by striking a target of tungsten or molybdenum, the deceleration of the moving electric charge isaccompanied by the emission of radiation. For mutation induction it is desirable to use X-ray machines
producing short wavelength radiations (hard X-rays) as the penetration is greater than with longer
wavelengths. The short wavelengths emitted is related to the peak operating voltage (kVp) of the tube.
The higher the kVp, the shorter the wavelength of the X-ray that is produced. When X-ray irradiation isused, voltage (kVp), current (mA), thickness and type of filter, distance of the tube to target, dose anddose rate should be noted as well as the half value layer (expressed in millimeters of aluminum or copper)
i.e. the thickness of an absorber required to attenuate a beam of radiation to one half. This value is themeasure of the effective energy of the X-ray machine or the quality of the beam.
3.1.1 GAMMA RAYS
Gamma rays have generally a shorter wavelength and hence possess more energy per photon than Xrays. Gamma radiation in usually obtained from radioisotopes in contrast to X-rays. A gamma radiation
facility can be used essentially in the same manner as an X-ray machine for acute or semi-acute exposures.
The most distinct advantage of gamma radiation source for prolonged treatments is that it can be placed ina greenhouse or field so that plants can be exposed as they develop over long periods of time. Cobalt-60

12Mutation Breeding Manualand Cesium -137 are the main sources of gamma rays used in mutation breeding. They are stored in leadcontainers when not in use and operated by remote control mechanisms to irradiate plant material.
3.1.2 UV RAYS
Ultraviolet light has limited penetrating ability, therefore its use is limited to treating spores, pollen
grains cells and cultured tissue. Wavelengths in the range of 2,500 to 2,800 nm are biologically mosteffective because this is the region of maximal light absorption by nucleic acids.
3.1.3 BETA PARTICLES
Beta particles such as those from Phosphorus-32 and Sulfur- 35 produce effects in tissues similar to
those of X- or gamma rays. The penetrating ability of beta particles is lower than that of X- and gamma
rays. The lower penetrating ability of beta particles can be overcome by putting the radioisotope in asolution and administering them to the plant material. P-32 and S-35 may then be incorporated directly
into cell nucleus giving a somewhat greater localization of the site of action. But, because of thevariability from tissue to tissue and cell to cell, it is difficult to determine the exact dose given by aninternal emitter in plant material.
3.1.4 NEUTRONS
Neutrons have been shown to be highly effective for the induction of mutation in plants but, a certain
degree of confusion exists concerning the results of early experiments and due to lack of adequate
dosimetric techniques.
3.1.5 ION BEAMS
Ion beams can give a large amount of energy with high LET (Linear Energy Transfer) to the localized
position in tissues. Therefore, we can expect different biological effects will be given to plant materials
compared with low LET radiations, such as, gamma- and x-rays. Also ion beams can produce large
structural changes in chromosomes and DNA. So we can expect to induce different kinds of mutations inplants with high frequency than gamma- and x- rays. But further basic and practical researches should bemade to use ion beams efficiently in the future.
3-2 CHEMICAL MUTAGENSThe number of chemical mutagens is numerous and continuously increasing. But for mutation inductinin cultivated plants only a few are readily very useful and most of them belong to the special calss ofalkylating agents such as, ethyl methane sulhonate (EMS), diethyl sulfate (dES), ethyleimine (EI), ethyl
nitroso urethane (ENU), ethyl nitroso urea (ENH), and methyl nitroso urea (MNH). A number of workers
also found azides as effective mutagens.
3.2.1 BASE ANALOGUES AND RELATED COMPOUNDS
True base analogues are closely related to DNA bases, adenine, guanine, cytosine or thymidine and canbe incorporated into DNA without affecting its replication. But analogue differs from the normal base incertain substitutes hence its electronic structure is modified and it can be expected that occasional pairing
errors will occur at the time of DNA replication after the analogue has been incorporated. The mostfrequently used analogues are 5-bromo-uracil (BU) and 5-bromo-deoxyuridine (BUDR), which areanalogues of thymine. Apart from true analogues, it has been found that N-methylated oxypurines have achromosome effect. The most efficient compounds are 8-ethoxy caffeine (EOC) and 1,3,7,9 tetramethyluric acid (TMU), but their mode of action is still unknown.
Maleic hydrazide (MH), an isomer of uracil, induces chromosome breaks in cell and aberrations are
localized in heterochromatic regions near the centromere of the chromosomes.
3.2.2 ANTIBIOTICS
Antibiotics such as azaserine, mitomycin C, streptonigrin and actinomycin D have been found to havechromosome breaking properties, but their usefulness are limited.
3.2.3 ALKYLATING AGENTS
This is the most important group of mutagenic chemicals for mutation induction in cultivated plants.
They have one or more reactive alkyl groups which can be transferred to other molecules. They react
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Mutation Breeding Manualwith DNA by alkylating the phosphate groups as well as the purine and pyrimidine bases. One should be
extremely careful in using them because most are potential carcinogens, such as EI, EMS, MNH andshould be used in small units.
3.2.4 AZIDE
Azide is an effective mutagen under certain treatment conditions. It is possible to obtain high mutation
frequencies with azide. Most mutations are gene mutations with some minor frequencies of chromosome
aberrations. It is relatively safe, non-persistent and inexpensive but are also potential carcinogens.

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4. OTHER MECHANISMS OF MUTAGENESIS
Mutations are found to occur without any apparent cause and are usually referred to as spontaneous
mutations. A closer analysis of the origin of spontaneous mutations showed that factors both intrinsic andextrinsic to the organism may be responsible both for spontaneous mutations and for increased
frequencies over the spontaneous mutation rate. The important intrinsic factors are genetic constitution
and physiological conditions and the important extrinsic factors are nutrition, temperature, naturalradiations and chemicals and very high oxygen pressure.
4.1 GENETIC CONSTITUTION
Spontaneous chromosome structural changes with high intensity and frequency has been alluded toeither hybridity, polyploidy or either both during meiosis or mitosis.
4.2 PHYSIOLOGICAL CONDITIONS
It has been found that sex and age influence on spontaneous mutation rate.
NUTRITION
Nutrition is known to influence spontaneous mutation rate in many plants. Spontaneous mutability is
increased by sulphur, phosphorus and nitrogen deficiencies. In plants grown in a magnesium or calcium
deficiency, a very high incidence of chromosome breakage occurs in the order of up to seventeen times
the control values in the case of calcium deficiency. An extensive analysis on the effects of deficienciesof essential elements has shown that radiosensitivity is increased by zinc deficiency and to a lesser extent
by copper, sulphur and molybdenum deficiencies.
TEMPERATURE
In higher plants, dry seeds subjected to heat treatment induced chromosomal aberrtions and mutations.
The mutagenic effect in the heat treatments is so low to be useful in mutation experiments.
NATURALLY OCCURING RADIATIONS
It was thought that natural radiation may be responsible for spontaneous mutations in plants. However,it can not be excluded that mutations may occur at increased rate in organisms living in areas with amarkedly increased natural radiation such as exists in India, Brazil, Southern France and other countries
unless hormesis exists.
NATURALLY OCCURRING MUTAGENSNaturally occurring metabolites of unknown composition, sulphur-containing substances, amines,amino acids, amides, nitrogen-free acids, aldehydes, alkaloids, phenols, quinines, degradation products ofnucleic acids and the widespread applications of chemicals in present-day civilization and the industrial
pollutions from factories, motor vehicles, energy production, etc. the genetic implications of chemicalmutagens may tend to increase.

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5. CELL CYCLE
THE NON-DIVIDING NUCLEUS
The non-dividing cells, whether interphase or postmitotic stage is not characterized as much byobservable structures as by physiological activities especially the nuclei part. At the end of mitotic
telophase chromosomes practically disappear into the non-dividing, the energic nucleus. The term
energic emphasizes its biochemical activity and at the same time does not imply that it is neither
interphase or postmitotic. Non-dividing nuclei and cells are of two sorts, i.e. those in the interphase,
which are preparing for another division and those at postmitotic which will normally not divide again.
The postmitotic do not replicate their DNA during the synthetic phase but differentiate in a permanent
Gap 1 (G1) condition.
In eukaryotic cells one or more nucleoli forms in each telophase nucleus. Nucleoli are formed by
specific regions of specific chromosomes called nucleolus organizers while which are usually located in
short arms of certain chromosomes. If they lie near the ends of chromosome arms, the tip of the arm
beyond the nucleolus organizer constriction is called a satellite. Nucleolus organizers are usuallydetectable only at metaphase in higher plants. In a cell of a diploid plant species there are at least two,
one in each chromosome set. Nucleoli are large nuclear organelles that are not bounded by memberane,
usually lie in the nucleus and are rather spherical. The nucleoli represents the fist phases of ribosomal
formation. The DNA genes (cistrons) of the nucleus organizer region (rDNA) aided by RNA polymerase
transcribe RNA (rRNA). Protein appears to be synthesized immediately in the nucleolus. The chromatin
within the non-dividing cell nucleus is mostly invisible by the optical microscope. The biologicalsignificance of the nondividing cell nucleolus is its physiological activity which has been worked out inconsiderable detail during the past 50 years or so.
THE CELL CYCLE
It is common knowledge that during the interphase between two mitotic divisions the DNA and histones
replicate during the synthetic (S) period. The time between the preceding telophase and the beginning of
the S period is called the Gap 1 (G1) period and the time between the end of the S phase period and the
beginning of the prophase is the Gap 2 (G2) period. The mitosis itself is called the M-period. In diploidorganisms the relative amount of DNA in a diploid telophase and during the G1 period is 2C and from G2
to telophase a diploid cell has the 4C amount of DNA (Fig. I-3).

Fig. I-3 The cell cycle

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5.1 MITOSISNext to DNA replication, mitosis is the fundamental form of reproduction in all organisms. Life canexist at no level lower than the complete cell with its hereditary and informational DNA, its organelles,
enzymes, and other material needed for all the numerous processes necessary for life. Mitosis generally
divides this mass into more or less equal and self-sufficient daughter cells after the DNA and otheressentials have been exactly or approximately doubled. During the interphase following mitosis, theDNA is exactly doubled quantitatively and qualitatively by the semi-conservative process, followed rightaway or even later by structural doubling into chromosomes consisting of two cytogenetically identicalchromatids. Followed by events that divide the chromatids into two identical groups and the cytoplasm
is divided also into two very similar portions. Hence, the daughter cells almost always are genetically
identical to each other. And each is identical genetically to the cell where it came from. Cells may enter
mitosis from interphase rather inevitably or cells in the post mitotic condition may be stimulated naturally
or by treatment to do so. In the case of post mitotic cells some sort of inhibition must be removed tostimulate DNA replication by the application of auxin. (Fig. I-4)

Fig. I-4 Diagrammatic representation of different stages of mitosis

PROPHASE
The onset of nuclear division is characterized cytologically as the gradual disappearance of thechomatinic network coincidental with the emergence of the long thready chromosomes. Throughout
17Mutation Breeding Manualprophase, the chromosomes become progressively shorter and thicker as a result of coiling as if eachchromosome is a coiled spring enclosed within matrix. At mid-prophase, each prophase, eachchromosome is seen to have two chromatids. Each chromosome has poorly staining portion (thecentromere) which will be the site for the production of the spindle apparatus at metaphase.
PROMETAPHASE
By the end of prophase in typical plant cell mitosis, the nucleolus and the nuclear membrane disappears.
This relatively very short stage of mitosis has been termed prometaphase where the axis of division isevident and the spindle is forming and the chromosomes are nearly as short as they ever will be.
Prometaphase is the transition period from prophase to metaphase called metakinesis. It is a dynamic
period when the centromeres of the chromosomes (or chromatids) acquire spindle fibers to opposite poles
and during which the chromosomes are moved to their metaphase position, i.e. equidistant from the twopoles of the spindle. The movement is the orientation of the chromatids in the region of the centromere
such that one of them lies toward each of the spindle poles.
METAPHASE
The alignment of the chromosomes in a plane formed by their centromeres midway between two polar
bodies of the spindle apparatus characterizes metaphase. The spindle apparatus is basically a complex offibers extending from pole to pole and from poles to centromeres. At metaphase, the chromosomes of the
set are not always arranged at random, the centromoeres lie toward the center of the plate with its arms
directed outward. Those at one pole will be included in one nucleus hence at metaphase the spindle
continue to develop.
ANAPHASE
The separation of the sister chomatids of each chromosome at the centromere signals the beginning ofanaphase. The centromeres of the anaphase chromosomes lead toward the poles. The arms seem tofollow passively or to be dragged. Often the ends of the arms of the sister chromatids seem to stick
together so that they must be eventually be pulled apart. There is one anaphase error that is of
cytogenetic and biological importance, the nondisjunction. This happens when both sister chromatids ofa metaphase chromosome end up in same telophase nucleus. It may be a regular gene-determined process
or it may be an error which may later produce cells that are aneuploid if one or a few of the chromosomesof the set undergo nondisjunction. Anaphase is usually very regular but when it is not, there is any
chromosomal breaks and reunions so that an anaphase chromatid is formed with two centromeres andanother without centromeres. And if the two centromeres go to the opposite poles, a bridge is formed
along with an acentric fragment. Pieces of unicentric chromosome without centromeres are produced,
because of the absence of centromeric spherules. These and their contained genes are not included inother daughter cells and may give rise to micronucleus outside the main nucleus.
TELOPHASE
It is characterized by the conversion of anaphase into interphase chromosomes, the reformation of
nuclear envelope, formation of a second centriole, formation of one new nucleolus or more,disappearance of spindle and astral fibers and the division of the cell into two complete and distinct cells,
the two daughter cells. The nucleoli become enlarged. Within each nucleus, the chromosomes whichstarted elongation at anaphase become progressively longer and thready until the telophase nuclei merge
into resting nuclei.
5.2 MEIOSIS I
Meisos evolved in part as a chromosomal number reduction process and in part to produce
recombination. It evolved out of mitotic divisions one or more times but has remained very sensitive toenvironmental conditions as well as to mutations that act various times and to other anomalies such as
hybridity may create. Many variations of it have evolved as normal for certain species, such as various
forms of apomeiosis, non-disjuntion, directed chromosomal eliminations, multiple or unipolar spindles
etc. Sexual reproduction in eukaryotes involves the production of nuclei, with the haploid chromosome
number and the means for delivering the diploid chromosome number in the zygotic nucleus. The
production of haploid nuclei from diploid cells involved a process called meiosis. The immediate
products of meiosis in plants are the spores. In plants microgenesis yields microspores andmegasporogenesis yields megaspores. In terms of sexual differences, microspores furnish the male18

Mutation Breeding Manual

gametic nucleus whereas megaspores supply the females gametic nucleus. Meiosis or reduction division
gives rise to daughter cells that are not identical with the parent cell. It consists of two successive
divisions (called I and II) of a nucleus accompanied by one division of its chromosomes. Each of the fourdaughter cells produced by one meiotic division contains only half of the number of chromosomes of theparental cells (the diploid, 2n generation). Meiosis is a compensatory reduction mechanism for thechromosome doubling that occurs through a cell fusion at fertilization in sexually reproducing organism.
Without meiosis, the chromosome number would double at each fertilization, giving rise to bigger cellswith greater chromosome numbers in each succeeding generation indefinitely (Fig.I-5).

Fig. I-5 Diagrammatic representation of different stages of meiosis

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PROPHASE I
It has a longer duration than the prophase of mitosis and has five separate stages. Meiocytes continue
DNA synthesis beyond interphase into prophase and the RNA continues to be synthesized after
completion of DNA duplication.
Leptotene - is the first generally accepted stage, the chromosomes are very thin and the homologue are
randomly distributed and unpaired. There are many tiny swellings called chromomeres along the threads.
Leptotene ends when the single threads pair or synapse with their homologues.

Zygotene - is marked by the lateral associations of the segments of two chromosomes. This pairing
called synapsis, is the expression of the homology of the associated segments. Pairing continues
throughout zygotene in a manner resembling a zipper.
Pachytene - at this substage, the synapsed chromosomes become shorter and thicker, and a highlyorganized structure of filaments called the synaptonemal complex between the paired chromosomes
appears. The complex appears as parallel dense bands in a single plane curving and twisting along theaxis between chromosomes.
Dipotene - The synapsed homologous chromosomes separate from each other except at the point where
they are held together by the mutual switching of non-sister chromosomes at one or more sites along thechromosomes. This visual consequence of crossing over is called the chiasmata.
Diakinesis - is the end of diplotene where the associations of bivalent chromosomes are scatteredthroughout the nucleus. Terminalization occurs and the chiasmata moves away from the centromere andeventually slip off.
Prometaphase - the nucleolus disappears, the nuclear envelope breaks down, the spindle fibers forms
and chromosomes are moved on the equatorial plane.
Metaphase I - the alignment of bivalents midway between the poles of the spindle apparatus
characterizes the metaphase I. The centromere of each chromosome in the bivalents is associated withfibers from only one pole.
Anaphase I – the loss of all chiasmata for one or more bivalents marks the onset of anaphase I. Eachgroup with the haploid number proceeds to its respective pole within the confines of the spindle
occupying increasingly smaller volumes. The arms of each chromatid separate and each chromosome isobviously a dyad.
Telophase – each group of chromosome is enclosed within a nuclear membrane before the chromosomes
reach the pole. The spindle apparatus disappears and nucleolus reappears and the chromosomes become
longer and threadier.
Interkinesis – interval between the first and the second meiotic division where the primary meiocyte has
produced either one secondary meiocyte with two haploid nuclei or two secondary meiocytes with
haploid nucleus.
5.3 MEIOSIS II
The second meiotic division is essentially a mitotic division.
Prophase II – each nucleus enters prophase II at the same time and synchronously proceeds through thesubsequent stages.
Metaphase II – the nuclear membrane disappears and the chromosomes are aligned in a plane midway
betweent poles of the spindle apparatus. The centromeres of chromosomes at metaphase II are associated20Mutation Breeding Manualwith fibers from opposite poles.
Anaphase II - the separation of the chromatids at the centromere signals the onset of anaphase II. Eachchomatid has a centromere and therefore is a chromosome at this time. Each group with the haploid
chromosome number proceeds toward each respective pole.
Telophase II – each group of chromosomes is now enclosed within a nuclear membrane, the spindle
apparatus disappears, the chromosomes become larger and threadier, and finally the chromatinic networkof the interphase nucleus appears.

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6. SPOROGENESIS AND FERTILIZATION
The four haploid nuclear products of microsporogenesis for the microsporocyte or pollen mother are
packaged to give four viable uninucleate microspores. After the microspores separate, the microspore
nucleus divides mitotically producing a generative nucleus and a tube nucleus. In some species, thesecells are shed as pollen grains and after landing on the stigma germinate to produce a pollen tube which
grows in the direction of the female gametophytes in the ovary. The generative nucleus proceeds down
the pollen tube and divides mitotically to produce the two haploid sperm nuclei. In most higher plants,
each haploid product of megasporogenesis is enclosed within a megaspore. The three of the four
megaposres forming a linear tetrad degenerate and the basal megaspore develops into the female
gametophyte, or embryo sac. The pollen tube of the male gametophyte delivers the two sperm nuclei tothe embryo sac where one nucleus fertilizes the egg and produces the diploid nucleus (Fig. I-6a, 6b).

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Mutation Breeding ManualFig. I-6a Development of the male gametophyte(pollen grain) in higher plants

Fig. I-6b The basic pattern of the development of the female
gametophyte (embryo sac) in higher plants

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Mutation Breeding Manual

7 EFFECTS OF IONIZING RADIATION
Radiations both ionizing and those of lesser energy (ultra violet light) are physical mutagens that arequite efficient at producing changes in genetic material. Its production if mutations is its most importantbiologic effect. Nonif its other effects is so far reaching in consequences both for the individuals in which
mutation occurs (whether single cell or multicellular organism) and for the population of which a mutant
bearing individual is a member. Radiation as a mutagen is important because: 1) it is the most effective
one known for producing mutations in quantity; 2) it is almost an indispensable tool for geneticists as its
affords an opportunity to study the very nature of the gene; 3) mutational frequency is increased so thatchanges are produced in genes at will; 4) changes that are made give an important clue as to what has
been changed; 5) by producing mutations in abundance radiation facilitates the study of consequences ofgenetic mutation in individuals (cells or organisms) and in population in which these mutations arise.
7-1 RADIATION EFFECTS ON DNA
In the production of mutations, radiation acts by ionizing nitrogenous bases in the DNA chainsspecifically at DNA synthesis. Base-change or base-deletion gives change in critical base sequence of themolecules. Ionization of one or more of the bases with free radicals that radiation produces may alterbase structure that they no longer possess the characteristics which they had before exposure to radiation.
7-2 RADIATION EFFECTS ON CHROMOSOME STRUCTURE
Ionizing radiation can, either by direct or indirect action, break chromosomes. Structural
rearrangements of chromosome and even loss a part of chromosome from cells as well as the wholechromosomes may follow a radiation induced break. Loss of chromosomes or chromosome fragments
produces distinct deviations from the cellular status quo. Even the loss of very small pieces ofchromosomes gives damage to the cells in which it happens, and the loss of large chromosomes is serious
enough to bring about cell death within a few cell generations.
The type of chromosome structural change induced by ionizing radiation have been well documented
and detailed descriptions and illustrations of those seen to occur in irradiated samples may be found inmany publications.
There are three basic categories of “break”, a) the chromosome type where lesions involved are seen at
metaphase to affect both chromatids of a chromosome at the same locus (balanced types) corresponding
to chromosomes unsplit at the time of irradiation; b) the chromatid type of aberration where the lesions
involved affect only one chromatid at any particular locus (unbalanced types) corresponding tochromosomes which had split longitudinally before irradiation and sub chromatid type of aberration
where the lesions involved affect only one chromatid at a very minute locus, corresponding tochromosomes exposed at prophase and may affect only one chromatid at sub-chromatid locus.
A. CHROMOSOME-TYPE ABERRATIONS
1. Exchange aberrations involve the interaction of two or more lesions.
2. Interchange-when lesions occur in the arms of different (homologous or non-homologous)
chromosomes
3. Interchange-when lesions are small and all within the same chromosome.
a. Inter-arm interchanges when the lesions are in opposite arms with respect to the centromere.
b. Intra-arm interchanges when both lesions are in the same arm.
B.
1.
2.
3.

CHROMATID TYPE ABERRATIONS
Exchange-similar to description above
Interchange
Intra-changea. Interchromatid- when the interacting lesions are in different (sister) chromatids
b. Intra-chromatid-when both lesions are in only one of the sister chromatids.

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7.3 EFFECTS OF RADIATION ON MITOTIC APPARATUS
Ionizing radiation interferes with the normal orderly process of cell division. This interference with cell
division is the most immediate conspicuous effect of ionizing radiation on the transmission of the genetic
material. All ionizing radiations as well as ultra-violet radiations and other mutagens inhibits mitosis.
All mitoses are not stopped, cells in mitosis as far along as mid or late prophase when radiation is given,
may complete their divisions. But those in interphase and those about to enter prophase, will be
prevented from doing so and their divisions will be inhibited. Those in the early stages of prophase may
not complete division but instead regress, returning to the interphase condition. When inhibition is over,normal cells would begin to enter the mitotic cycle to divide, hence a profusion of mitoses following aperiod of inhibition, i.e. synchronization of mitoses in irradiated group of cells or tissue. The inhibitory
effect is dependent upon dose but is transitory. Radiation also brings about non-disjunction ofcentromeres at metaphase and the unequal distribution of chromosomes to daughter cells at anaphase.
Ionizing radiation in addition causes lagging of chromosomes on the spindle at anaphase, an effectascribed to changes brought about in the centromere. Ionizing radiation promotes cross-over in the regionof the centromere due to an effect of radiation on the centromere.

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8. PLANT BIOTECHNOLOGY (IN VITRO CULTURE)
A wide variety of technologies in tissue culture, genetic manipulation and molecular biology have beendeveloped in a number of plant species. These laboratory-based technologies are now jointly referred toas plant biotechnology. Plant biotechnology had direct application in mutation breeding, genetic
improvement and production of plants. The fundamental basis for the application of plant biotechnology
is the capacity of plant cells and organs to develop into complete plants which can be grown to maturity.
1. Axillary buds. One of the most exciting and important aspects of in vitro cell and tissue culture is thecapability to regenerate and propagate plants from cultured cells and tissues. The simplest type of in-vitro
plant propagation is the stimulation of axillary bud development. The technique exploits the normal
ontogenetic route for branch development by lateral (axillary) meristems. The buds are treated withhormones to break dormancy and produce shoot branches. The shoots are then separated and rooted to
produce plants. The shoots are also used as propagules for further propagation. Many ornamental plants
and woody species are propagated commercially by axillary bud proliferation. Axillary bud proliferation
results in an average tenfold increase in shoot number per monthly culture passage, that in 6 months it ispossible to obtain as many as 1,000,000 propagules or plants from a single explant.
2. Adventitious shoot and organogenesis. The technique for adventitious shoot organogenesis from leaf
sections covers several defined steps in a typical micropropagation system. The first step is the initiationof a sterile culture of the explant (stage I). The second step is the multiplication of shoots or otherpropagules from the explant (stage II). Adventitious shoot proliferation is the most frequently used
multiplication technique in micropropagation systems. The culture media and growth conditions used instage II systems are optimized for maximum rates of multiplication. The third step is the development ofroots on the shoots to produce plantlets (stage III). Specialized media may or may not be required toinduce roots depending on the species. The final step is to produce self-sufficient plants (stage IV) whichusually involves a hardening-off process and acclimatization to growing in soil mixes under greenhouseconditions for later transplanting to the field.
3. De novo organogenesis. The first step in plant regeneration by organogenesis for callus and cellsuspension or de novo regeneration is to establish callus or cell suspension cultures. Explant tissues
generally show distinct planes of cell division, various specializations of cells, and organization into
specialized structures such as the vascular system. Callus formation from explant tissues involves thedevelopment of more random planes of cell division. There are five phases in callus growth:a. A lag phase, where cells prepare to divide.
b. An exponential phase, where cell division is highest
c. A linear phase, where cell division slows but the rate of cell expansion increases
d. A deceleration phase, when rates of cell division and elongation decrease, ande. A stationery phase, where the number and size of cells remain constant.
Cell suspensions are initiated by transfer of callus pieces into flask with a liquid medium placed on agyratory shaker to provide aeration to the cells. As new cells are formed they are dispersed into liquidmedium and become clusters and aggregates. Cell suspension can be maintained by subculturing in flask
called batch cultures. Cells from suspension cultures can be harvested by filtration or transferred to agar
medium and grown as callus.
4. Somatic embryogenesis from de novo. In somatic embryogenesis, somatic cells develop by division toform complete embryos similar to zygotic embryos. The bipolar structure of the somatic embryo contains
both shoots and root meristems. As the embryos develop, they progress through distinct structural steps
i.e. the globular, heart torpedo, cotyledonary and mature stages.
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5. Direct somatic embryogenesis. This is the formation of somatic embryos or embryogenic tissue
directly from explant without the formation of an intermediate callus phase. To induce direct
embryogenesis, explant most often used is the immature zygotic embryo. This tissue is alreadyembryogenic in nature and apparently requires less nurturing.

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9. DOSE DETERMINATION OF RADIATIONS
Several methods are used to determine the dose of radiations. Among the commonly used are:
1. Ionization Chamber
These are chambers whose walls and gas consist of materials that have been elemental
composition similar to the body tissue. These are called tissue ionization chambers. In mixed
radiation field, two chambers are used, one sensitive to both fast neutrons and gamma radiation andthe other insensitive to neutrons.
2. Fricke Dosimeter
This method is useful for gamma dose measurement with dose ranging from 400 – 40,000 rads.
The standard Fricke solution is composed of the following:
Fe(NH4) 2.6 H2O ------------------ 10 mol/ lSulfuric chloride, NaCl ----------- 10 mol/l
Sulphuric Acid, H2SO4 ----------- 0.4 mol/l
The absorbed dose D is obtained from the formula9.64 x 10 (d-d)
D=
G (Fe +3 ) . p.l.e.
Where : d
= optical absorption of irradiated solution at 304 nmd= absorption of unirradiated solution (control)
G (Fe +3 ) = G-value 100 eV -1= 15.5 for 60Co radiationp= density of the solutionl= thickness of solution (normally 1 cm quartz cell are used)e= extinction coefficient of Fe +3 (1/mol cm)= 2190 at 304 nm and 25 oC(e increases with temperature by 0.7% per 1 oC
( *Prepared by: Mr. A.B.Asencion, PNRI )3. FeCu Dosimeter
This method is used when gamma and fast neutron doses from mixed field are to be determined
separately. Usually two chemical systems with different yields are used.
4. Fast Neutron Dose Estimation by Sulfur Activation
This method make use of the 32 S (n,p) 32 P reaction to estimate the fast neutron fluence and fastneutron dose. Since the actual neutron spectrum is not known, results of measurement are given interms of equivalent tissue fluence:
Of
Where :As · T (n/cm2 )=(6 f ) · nAs= specific saturation activity (dps/g )(6 f) = average cross-section of the reaction for a fission neutron
spectrum= 65 x 10 – 27 (cm 2 )T= irradiation time (sec)N= number of 32 S atoms/ g sulphur= 1.791 x 10 23

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10. TREATMENT METHODS
10.1 RADIATION TREATMENT
There are several methods in treating materials for irradiation. Among them are acute, chronic,
recurrent and combined.
Acute treatment is done in a few minutes or a few hours. Usually the dose is at the optimum level andapplied only once.
Chronic treatments are applied over long periods of time. The dose is small, fractionated and deliveredin minutes or few hours.
Recurrent treatment on the other hand is carried out on materials that had been irradiated already in theprevious generation
Based on the results obtained from several experiments, the beneficial effects come mainly from acuteand chronic treatments. The benefits derived from the recurrent treatment appear not to compensate forthe extra labor and time in handling materials.
Any plant part can be used for mutagenic treatment and the most commonly used ones are seeds, pollen,
whole plant, cuttings, tuber, corm, bulbs, stolons and cultured cells, tissue and organs. Seeds can beeasily irradiated using X-ray machines or gamma sources in a greenhouse or shielded room or gamma
cells. The advantage of using seeds is that they can be irradiated in many physical environments. They
can be dessicated, soaked, heated or frozen. They can be maintained for a long period of time. This isadvantageous because under dry conditions, no significant damage can affect the seeds. Also under thiscondition, it is favorable since the factors that modify the radiation damage are controlled. One very
important factor however is the control of the moisture content. It should be maintained between the 12 –14 %. This is accomplished by putting the seeds in mesh bags and storing them in a dessicator with 60 to
70% (by volume) glycerol solution for 4 days. This is important to control damage to the seeds. At highwater moisture content the molecule is changed when exposed to radiation. The following results can beseen when free→ radicals recombine after irradiation.
E – aq + e-aq
H + H
OH + OH--------Æ--------Æ--------Æ

H2 + 2 H2O
H2
2 H2O2All the radicals are highly reactive thus resulting in chemical changes that are similar to the effect after
direct ionization of the molecule.
After the moisture is stabilized the seeds are pasted in appropriate cardboard with stand so that the seeds
are perpendicular to the radiation source during irradiation. This is to make the treatment evenparticularly when it is for radiosensitivity studies. Irradiation should be done as soon as the set up iscompleted. For mutation breeding purposes, the seeds can be placed inside a small coin envelope or
plastic bag and attached to a card board so that during irradiation it is perpendicular also to the source. As
soon as the seeds are irradiated, they should be packed in a plastic bag and stored in a refrigerator ifsowing cannot be done immediately. The doses found appropriate for cereals and other seed crops ranges
from 100 – 600 Gy.
For cuttings and other asexually propagated crops, care should be made not to expose the rootsystem. Placing lead blocks in front of the base during irradiation is sufficient to protect the rootsystem from being irradiated. The dose range found to be appropriate including cultured cells
between 5 – 6 Gy.
WHOLE PLANT
For the whole plant, they can be irradiated in gamma field, gamma room or “Greenhouse” with lighting
and water provisions to maintain the plants especially when the method of treatment is chronic and lasting
for a number of weeks or more. Seedlings and small plants can be irradiated by same way.

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POLLEN GRAINS
Pollen grain is not as convenient as in seed irradiation. In the first place, collecting pollen is a problem
and sometimes not enough can be collected and viability is not long in some cases. Besides special
provision is needed for chronic irradiation. So one should work fast when it comes to irradiating pollen.
One advantage however, is that pollen rarely produce chimera.
Pollen can be irradiated using U.V. machine, X-ray machine, gamma cell, or ion beams. In cases ofpotted plants that are flowering, gamma garden or gamma field is ideal. In the case of cereals like rice,panicles can be collected in the early hours, irradiated, wrapped in moist cloth and then used inpollination at 10 – 12 a.m. where the stigma is receptive.
CULTURED CELLS AND TISSUESWith the popularity of biotechnology, irradiation of cultured cells and tissues will become very
common in biological studies (Fig. I-7). The advantage of treating single somatic cells is that large
numbers of homogenous individuals are available in small area. Another is that mutant characteristics are
manifested almost immediately because of the limited metabolic product in the cell. Still anotheradvantage is that a single mutated character is not masked by a dominant allele. Selection can also bemade efficient and specific since the cells are cultured according to the biological need to isolate mutantsfrom a large mix cells. Furthermore, selected single haploid can be regenerated and diplodized forbreeding purposes or genetic studies. This makes the operation less costly.
MERISTEM
Whether one deals with seeds or any plant part in mutagenic treatment, one deals with meristem. In thecase of seeds, the resting embryo is the target and it is composed of a few primordial leaves, a number ofaxillary buds and the apical meristem. In the case of cuttings, one is dealing with the shoots thatdeveloped from a single epidermal cell of the petiole. Thus treating with mutagens will in almost
homohistant or solid mutants instead of chimeric plants. Chimera, however, can be observed especiallyin merstem. Three types of chimera are mericlinal, periclinal and sectoral.
DEVELOPMENT OF CHIMERA (Fig. I-8, 9a, 9b)30

Mutation Breeding Manual31Mutation Breeding ManualFig. I-8

Development of Chimeras in Shoot tips (apical meristems)

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Mutation Breeding ManualFig. I-9a Types of chimeras and vegetative propagation of mutated sector
Source: Research Highlights on: The Use of Induced Mutations for Plant
Improvementin Malaysia. National Committee on the Use of Induced Mutations in Plant breeding
Malaysian Institute for Nuclear Technology Research (MINT) Bangi, 1998

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Mutation Breeding ManualFig. I-9b Schematic presentation of mode of origin and chimaericalstructure of somatic mutations

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10.2 CHEMICAL TREATMENT
The use of chemicals to induce mutation vary widely but most of the treatments involves seeds rather
than other parts of the plants. The reasons for this is that chemicals are not readily absorbed to planttissues as by seeds. Techniques however, to overcome this problem were developed and one of these isby placing a cotton on the part to be mutated and the mutagen is put on the cotton cot it can be retained
for sometime.
Only two examples of treatment procedures will be highlighted here for chemical treatment since themethodology is more or less representative of the technique.
ETHYL METHANE SULPHONATE (EMS) TREATMENT FOR SEEDS
Prepare seeds with more or less 95% germination and place them in mesh bags. About 200 seeds per
bag would be enough so that the seeds will be in one layer. Stabilize the moisture content of the seeds tobetween 12-14% by putting them in dessicator with 60% glycerol solution for 4 days.
Prepare water bath to stabilize the temperature during treatment with mutagen. Pre-soak the seeds indistilled water for up to 16-20 hours at room temperature (20-22ºC).
One half hour or a few minutes before the end of presoaking time, prepare the mutagen. At least 1 ml
per seed should be made so that prepare 1 liter if the number of seeds to be treated is 1000. Use distilledwater. Usually it is recommended to use mole in preparing the solution although the percentage issometimes used. For EMS, the molecular weight is 124.2, so to prepare for 1 mole simply weigh 124.2 g.
of EMS and dissolve it in distilled water to make a final volume of 1000 ml. Based on this, one canadjust his/her requirement by ratio and proportion.
Soak the seeds for two hours at constant temperature of 30-35ºC. When temperature rises, add ice until
the desired temperature is maintained.
Remove the seeds after soaking and wash them in running tap water for at least 4 hours.
The seeds can be planted immediately after a short drying under an electric fan. If the seeds cannot be
planted right away, they should be dried back to 13% moisture content and stored in a refrigerator toretain their viability.
For rice seeds, it is advisable to de-hull them before any chemical mutagenic treatment. A word ofcaution when doing the treatment. Do it always inside a fumehood.
SODIUM AZIDE (NAM3) TREATMENT FOR SEEDS
Basically the preparation is the same as that in EMS except that in sodium azide, pH has to be adjusted
to 3 during treatment because it has been found that this mutagen is effective only under acidic condition.
After stabilizing the moisture content of the seeds, the seeds are pre-soaked in free flowing water for 2-16
hours at 20-23ºC with continuous bubbling of the water.
Stock solution of 1 M should be prepared ahead using distilled water. The stock solution can be stored
in a refrigerator for long period of time without losing its potency. Sodium azide has a molecular weightof 65 so that to prepare 1 M stock solution, simply weight 65 g of the NaM3 crystals and dissolve indistilled water to make a final volume of 1000 ml using a volumetric flask.
Since sodium azide requires low pH, a buffer of 0.1 M is needed and should be prepared one half hour
before the treatment using mono basic potassium phosphate, the molecular weight of which is 136.09.
Thus, to prepare 1 M, weigh 136.09 g of the crystals and dissolve in distilled water in a volumetric flask
to make a final volume of 1000 ml. The pH can be adjusted to 3 by adding drop by drop concentrated
phosphoric acid using pipette, reading the pH of the solution with an appropriate pH meter.
To prepare the 10-3 M sodium azide solution, add 1 ml of the 1M sodium azide to 999 ml phosphatebuffer (pH 3) to make a final volume of 1000 ml. One ml of this solution must be used for each seed sothat 5,000 ml should be prepared for 5,000 seeds.

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Put the pre-soaked seeds in the solution for 2-4 hours with the temperature of the solution maintained at
20ºC and continuously bubbled.
After the treatment, the seeds are washed in cold flowing water for two hours. The seeds can be planted
immediately after short drying period. Alternatively, the seeds can be dried back to 13% and stored in arefrigerator if they cannot be planted immediately to maintain their viability.

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11. HANDLING OF THE TREATED MATERIALS AND THEIR SUCCEEDING
GENERATIONS
Schematic mutation breeding for seed crop plants is shown in Fig. 10.
When seeds are exposed to ionizing radiation or chemical mutagen, several effects can be observed.
Measure and analyze the physical damage and chromosomal aberrations. The physical damage can be
determined by cytological analysis and by measuring plant injury in the M1 generation. Plant injury canbe measured by taking :Seedling height (determined 10-14 days after)
Root length (determined soon after germination in the laboratory)
Emergence under field or laboratory condition
Number of spikes per plant
Number of florets per spike
Number of seeds per spike
Fruits and/or seeds per plant
Determination of M1 injury using seedling height and survival should be a routine procedure in
mutation breeding, because it has been established that these characters are correlated with M1 mutation
frequency.
There are three methods in raising seedlings to determine seedling height, namely, Flat Method, PetriDish Method and Growing-Rack Method.
In the Flat Method, the seeds are sown in boxes filled with a sterile mixture of sand and soil (50:50) or
any convenient medium. The cheapest way of sterilization can be done by firing the media in a suitablecontainer for at least one hour with complete stirring from time to time or it can be accomplished usingchemicals. The sterilized mixture is placed in seed boxes and covered with plastic film and can be used
for planting the following day. The treatments are arranged in a Randomized Complete Block Design(RCBD) and replicated at least 4 times. Randomization can be accomplished by using random table or by
drawing lots. The seedling height is measured on the first leaf when the growth stop already(10-14 days
after).
The second is the Petri-Dish Method. This is done by putting wet blotting paper in the petri-dish and theseeds are sown on them. The seedling height and root length are determined 7 days after.
The third is the Growing-Rack Method. In this method, the seeds are placed between two wet blottersand supported vertically by wire or PVC rack. The racks are placed in aluminum, plastic or wooden trays
lined with plastic and water is added to the tray making sure that the lower edge of the filter paper isimmersed. The racks are placed in a controlled uniform environment either in a "Greenhouse" or achamber.
Measurement of the seedlings height and root length can be made 7 days.
For the other data, gathering can be done in the field or "Greenhouse". Planting for this purpose shouldneed more plants (1000-5000) and spacing should be closer to each other to minimize tillering especially37

Mutation Breeding Manualfor cereals. The seeds gathered from this (M1) planting can be used also for scoring chlorophyll andmorphological mutations in the M2. Sometimes for critical experiment, it is necessary to bag the plant
individually to prevent out-crossing. This will make the result more reliable.

38

Mutation Breeding Manual

Fig.I-10 Schematic mutation breeding for seeded crop plants
Source: Research Highlights on: The Use of Induced Mutations for Plant Improvement in Malaysia,
National Committee on the Use of Induced Mutations in Plant Breeding. Malaysian Institute for Nuclear
Technology Research (MINT) Bangi 1998

39

Mutation Breeding Manual

11.1 CHLOROPHYLL AND MORPHOLOGICAL MUTATION IN THE M2 GENERATION
Two methods can be used in scoring chlorophyll mutation in the M2. One method is based on 1000 M2
plants or seedlings and the other is based on the M1 plant and M2 seedlings. The M1 plant method makeuse of the number of M1 plants sown and the number of plants that have mutations. The M2seedling
method is done by counting the total number of mutants and also the total number of seedlings. The typesof chlorophyll and morphological mutations should be separately counted and computed based on the
total number of seedlings. The data can be tabulated and entered in the appropriate Tables below. (Table
I-1-5)
Table I-1.
Treatment

Table I-2.
Treatment

Frequency of Chlorophyll Mutation
No of M2
No. Studied
No. of M1%Chlorophyll

M1 Plt. M2

Plant

Mutant

M1

M2

Seedling

Segregating

Seedling

PlantsSeedling

Frequency of Chlorophyll Mutation Types in M2 SeedlingTypes of Seedling Mutation
Total M2
Seedling

No.

%

Table II-3.
Treatment

No.

%

Virescent

Chlorina

No.

No.

%

XanthaAlbina%Mutation Spectrum in M2
Chlorophyll Mutation
Total
MutantViable Type
Lethal Type
Seedlings
No

%

40

No

%

Mutation Breeding Manual

Table I-4.
Treatment

Table I-5.

Treatment

Frequency of Morphological Mutations
No. of M1 Plant
No. of M2
No. StudiedSegregating
Mature Plts.
M2
M1 Plantsmature% Mutation
M1Plts.

M2Plts.

M2 Morphological Mutation

No. of Mutant Plants

Characteristics of Mutant

Handling of M2 and Succeeding segregating Population

Several methods are employed to handle the M2 and succeeding segregating population (Fig. I-10
Among them are the bulk method and pedigree methods. Sometimes the two are combined.)41

Mutation Breeding Manual

11.2 BULK METHOD
Bulk of M1 population - the bulk M1 seeds are planted and observed for variability. This method isefficient if the mutated character is easily identifiable. This method is also efficient in the case of
mechanize screening for size of seeds, weight and shape.
a.
b.

M1 Population bulk ------ M2 single seed bulk --------- M3 ear to row progenies
M1 Population bulk ------ M2 ear to row progenies (this is a combination of Bulk and Pedigree)

11.3 PEDIGREE METHODS
a. M1 ear to row bulk - The M1 ear in this method is randomly selected and small number ofprogenies are needed.
b.

M1 single-seed or multiple seed bulk - In this method a single seed is selected from each plantand constituted as M2 population. This is followed by a single plant progeny test in M3 orsingle M2 ear for progeny selection in the M3. This is the most efficient, low cost and spacewise method but requires the competency of the Plant Breeder in identifying the mutants.

c.

M1 plant to row - This method utilizes all the seeds from a single plant. However, about 50 or
so seeds is enough to plant in each pedigree row. The advantage of this method is that one cannote the specific origin of the mutant-lines. The disadvantage is that one will need a bigger
space to plant the segregating population and therefore a little costly.

d.

M1 ear branch, pod, fruit (within plant) to row- This method is the most precise when it comesto identifying the origin of the mutant but is also the most expensive.

11.4 HOW THE MUTANTS ARE SELECTED
For the trained eye of the breeders, visual selection of the mutants phenotypic variation is the mosteffective. Depending on the breeder's objectives, mutants that meet them are selected disregarding theseemingly worthless mutants. Since the value of the other mutants cannot be ascertained immediately, it
is worthwhile to select and preserve them for future studies and possible utilization. For example in dwarf
rice mutants, their worth in breeding cannot be fully appreciated unless the genes controlling them arestudied and compared to already established genes like sd1, sd4, sd6 etc. This is to preclude uniformity ofsource of dwarfing genes in the released varieties which may prove disastrous if the varieties succumb toa particular disease or pest.
Visual selection is effective in identifying earliness, plant height, color changes, non-shattering,
adaptation to soil, climate, day length, resistance to pests and diseases etc.
Another method of selection is mechanical or physical method. This method is very effective inhandling large quantities of M2 seeds particularly for sieving size, shape, weight, density etc.
Chemical, biochemical, physiological, physio-chemical and other screening procedures may also beneeded for selecting certain kind of mutants but almost all employ visual observation including the muchnewer biotechnology techniques like PCR and related technologies.
Selection usually start from the M2 and confirm in the M3. Since it is not sure for the mutant to behomogenous already in all characters in the M2, selection is necessary even in the M3 generation. This isso because of the epistatic interaction among genes. In some cases, it may be necessary to reselect even inan M3 line and grow the M4 particularly in polyploids.
11.5 HOW TO DETERMINE WHETHER A VARIANT IS A MUTANT OR CONTAMINANT
A variant can be considered a mutant if the frequency of appearance is very low even in a bulk M2. It isalso a mutant if it appears as one to a few individuals in branch, spike or plant progenies and neverappearing in a 3:1 ratio (but lower) in more than one or few of several branches or spike or progenies. In
addition, the variant can be considered a mutant if similar variants have been isolated from several
different progenies in a treated population at a much higher frequency than that obtained in the control.
Aside from the above criteria, selected variants should breed true already in M3 if not in the M2 and thevariation should be limited to one or few traits.
When the variants back-crossed to the parent variety and to some other strains should show a simple
genetic segregation.
Phenotypically, the mutants should show similarity to the parent variety except for the modification
42

Mutation Breeding Manual

involved. Even in complex changes in phenotype, similarity in many features shall still remain.
Aside from this similarity of the mutants to the parent phenotypically , the variant should also be similarin traits governed by several different gene complexes like quality characteristics, disease and pest
resistance and biological characteristics as in several methods of protein identification.
11.6 ASEXUALLY PROPAGATED CROPS
Induced mutation is very useful to improve asexually propagated crops particularly for those that do not
flower and those that take long period to flower.
Chimera formation however, becomes a problem because the nature of mutation which is a one-cell
event. Thus when multi-cellular meristem is irradiated, the mutated cell has to compete with the unmutated ones and this is called diplontic selection. Usually solid mutants are not expressed readily.
The problem of diplontic selection can be overcome by cutting back the top portion of the plant toforce out the development of the side branches. When solid mutants are observed, they can be isolated
through cuttings or tissue culturing to develop them into new plants.
Another approach is the use of adventitious bud techniques. This is based on the knowledge that theapex of adventitious buds originates from only one epidermal cell, thus solid mutants or homo-histantmutants can be recovered.
Chimera formation, however, is not a problem in ornamentals because any variation in color form areconsidered beneficial.
11.7 Yield Testing of Mutants
For seed crops, once the mutants are stabilized (M2 to M4), performance test should be conducted tofind out the yield potential of the mutant(s). This is carried out in several locations and climaticconditions to find out the adaptability of the mutants. The entries should include the original
variety/cultivar and a standard variety for comparison. A randomized complete block design with at least
four replications will be sufficient. For testing two or more factors, split-plot design would be useful.

43Part IIAPPLIED MUTATION BREEDING

Mutation Breeding Manual

The categories now known as Basic and Applied Research did not exist prior to the 21st century. A
number of researchers/scientists practice both basic and applied research. Basic and applied research
while distinct in many ways, tend to merge in the process of scientific discovery. Today, basic research
involves theoretical or experimental investigation to advance scientific knowledge, without immediate
practical application as a direct objective, whereas the goal of applied science is essentially to create asituation that will eventually lead to a product or service being developed. But applied research uses
knowledge gained through theoretical or experimental investigation to make things or create situations
that will serve a practical purpose.
The scientific community comprises of researchers in both basic and applied research. Their research
projects overlap and feed one another. It is difficult to make clear distinctions between basic and appliedresearch just by looking at the results. Whether researchers call their work “basic” or “applied” is more areflection of how they picture themselves and how much they aspire to interest with innovation system.
The difference between basic and applied science is narrowing rapidly, people who are working inapplied research need to know the basics, because the demand for new, improved and better products thatwill feed the hungry world and increase the dwindling biodiversity which mutation breeding may help
alleviate.
This practical section will follow the chronological discussions of agricultural commodities as presented
by the previous Mutation Breeding Workshops/Seminars in an effort to combine all the methodologies
observed to be useful for mutation breeding by the researchers of this aggrupation.

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Mutation Breeding Manual

1. COMMODITY I: CEREALS
Cereals or grains are members of the grass family that are cultivated primarily for their starchy seeds or
dried fruits (the grain) which is used for human food, feed for livestock and as a source of industrial raw
materials. They are the most important food crops in the world. Rice, wheat, maize, barley, sorghum, oats,
rye and millets are some of the most common cereals.
Cereals were among the first plants to be domesticated having been grown before the beginning ofrecorded history.
Rice, wheat and rye are grown primarily for consumption as human food while much of maize, barley,oats and sorghum and millet is mostly fed to livestock.
Rice is believed to have originated in the orient more than 5,000 years ago. It is the principal cerealgrown for food in the tropics and warmer, humid parts of the temperate zone.
Wheat has been an important cultivated crop since prehistoric times. Many scientists believe it
originated in Southwestern Asia.
Rye is botanically related to wheat and barley, all belonging to the tribe Hordeae. It was probably
domesticated first in Turkestan having grown as a weed for many years in barley and wheat fields
Barley is a crop of ancient origin having been cultivated by Swiss lake dwellers in the Stone Age 11 andused for livestock feed and malt production and to some amount for human food.
Maize originated in the western hemisphere and was introduced into Europe and Asia by early explorers
It is a highly domesticated crop that has been cultivated for 6,000 years or longer. It is the highest
yielding grain crop.
Sorghum an important grain crop probably originated in tropical Africa. Its growth requirements anduses are similar to those for maize but is more heat and drought tolerant.
Oats are believed to have been cultivated first in Western Europe during the Iron and Bronze Ages.
They are now grown mostly in temperate regions especially in corn belt of the US, Canada and in
Northern Europe and in Australia.
1.1 RICE MUTATION BREEDINGINTRODUCTION
Rice (Oryza sativa, L.) is one of the two leading cereals in the world. It is feeding over half of theworld’s population. Rice is the staple food for the people of East, Southeast Asia, where 90% of theworld’s rice is grown and consumed.
Rice is grown in over 110 countries of the world, making 126 ha and 579 million tons of grain in 1990.
Among the major producing countries, India leads in hectareage (29% of the world’s total) while China
follows in planted (23%) but leads in production (36%). Other Asian countries and India contribute about
56% of the production , while Latin America and African countries provide 3% and nearly 2%
respectively. The United States contributes 1.35% to the world’s crop. Thailand and the U.S.A. areleading exporters of rice and lately the Peoples Democratic Republic of Vietnam is exporting some of its
harvest.
Rice provides more food energy yield per hectare than other cereals and the food protein yield per
hectare is also high for rice because of its high mean yield. The superior quality of rice proteins provides
a higher net utilization value of 73.8%.
In the rice-growing countries of the world, 83% of the rice land is irrigated; about 23% is rained-wet
land fields followed by rain fed-dry land, 13%.
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Mutation Breeding Manual

The genus Oryza belongs to the the tribe Oryzeae under the sub family Poo idea in the grass family
gramine (Poaceae). The two cultivated species (cultigens) in the Genus are O. sativa L. (common rice)and O. glaberrima Steud (African rice).
The cultivated rice plants is characterized as an annual grass, with round, hollow, jointed culms, flat
sessile leaf blades and a terminal panicle. The roots are fibrous, possessing rootlets and root hairs. It is asemi-aquatic plant, capable of both aerobic and anaerobic seed germination. It has aerenchyna tissues in
the leaf sheaths and transport air to the submerged roots in the rhizosphere (by bacteria) and in the water(by blue green algae associated with azolla). The plant can supply a part of the nitrogen needed for plant
growth from this non-symbiotic existence.
Some workers confused by certain terms in rice plants and its parts “paddy” refers to the grain or roughrice (in boiled form) and it also denotes the crop or harvested grains. “Paddies” refer to we land fields ofrice. The inflorescence is a panicle. Rice seeds refers to the grain, the mature fruit (caryopsis) with its
enveloping glumes (lemma, palea, awn, sterile lemmas) and the rachilla. The bulging sheath pulvinus isoften mistakenly called the “node”. In a germinating seedling, only the second leaf is a complete withsheath, blade, liqule and auricles. An adult plant height should include both culm length and panicle
length. A grain is a filled spikelet.
Current advances in planting methods from direct seeding to transplanting and multiple cropping in ayear from a single crop to two (main and off, kharif and rabbi) or three (aus, aman, and boro) have further
enriched the genetic diversity in the rice of tropical and subtropical areas (Fig. II-1). Threeecogeographic races if rice are generally recognized, Indica, Sinica/Japonica and Javanica (Table II-1).

47

Mutation Breeding Manual

Fig.I-1 Complex array of varietal designations by topohydrologic, edaphic,
cultural, and seasonal criteria (Chang, 1985a)

48

Mutation Breeding Manual

Objectives
Introduction to mutatioan for improved variety of rice with respect to any of the following traits:
I.

Morphologic Traits Improvement (Fig. II-2)
A.
B.
C.
D.
E.
F.

Pigmentation of plant parts
Pubescence
Dwarfs and semi-dwarfs
AwningPanicle morphology
Grain

49

Mutation Breeding Manual

II.

III.
``

Quantitative Traits Improvement (Fig. II-2)
A.
B.
C.

Plant stature
Lodging resistance
Plant type
1. Culm length
2. Tiller number
3. Culm angle
4. Culm stiffness
5. Leaf dimension
6. Leaf angle
7. Panicle characters

D.

Grain characteristics
1. Grain width
2. Grain length
3. Grain shape
4. Grain thickness
5. Heritability estimates and character asscociationa) Number of days from seeding to headingb) Spikelet lengthc) Plant heightd) Panicle lengthe) Spikelet widthf) Grain weightg) Number of grains per panicleh) Panicle weighti) Panicles per plantj) Tillers per plantk) Grain yield per plant

Growth Characters and Ecological Adaptation
A.

B.
C.
D.
E.
F.

Growth duration
1. The basic vegetative phase
2. The photoperiod-sensitive phase
3. The optimum photoperiod where the shortest growth duration is obtained
Response to temperatures
Trait complexes contributing to wide-adaptability and high yielding ability
Growth habit
Grain shattering and dormancy
Tolerance to adverse environment
1. Adaptation to deep water or floating ability
2. Acidity
3. Drought
4. Temperature50Mutation Breeding ManualIV.

Hybridization
A.

Grain yield and crop productivity
1. Yield potential under most favorable condition
2. Yield stability across seasoned locations
3. Crop productivity on a daily field-use basis

B.

Grain quality
1. Physical appearance of grain (market quality)a) Size and shapeb) Colour of brown rice or milled ricec) Test weight of raind) Percentage of head rice (whole kernels) and brokense) Transluscencyf) Whiteness and chalkinessg) Cooking characteristicsh) Purity and trueness to type or namei) Percentage of discolored or red kernels
2. Cooking and eating qualitya) Water absorption (kernel elongation and volume expansion duringcookingb) Cohesiveness, texture and softness of cooked ricec) Hardness of cooked rice after cooking and upon cooling and standingd) Absence or presence of aromae) Absence or presence of red ricef) Nutrition quality

C.
D.
E.
F.

Resistance to diseases and insects
Resistance and tolerance to hydrologic stress
Tolerance to extreme temperatures
Tolerance to adverse soil factors

51

Mutation Breeding ManualFig. II-2 A part of the panicle of rice bearing single-flowered spikelets (left).
A single spikelet (above, right) and a flower (below, right)

52

Mutation Breeding Manual

MATERIALS
Rice Seed (caryopsis), 5000 per envelope/dose
Manila (brown) coin envelopes at least 3” x 5” (7.5 x 12.8 cm)
Seed boxes with fine sandSeed bed with garden soil-sand mixture (50 : 50)
Experimental paddies
Irradiation facility (Gamma cell 220, AECL), X-ray machine, etc.
METHODOLOGY
Select the base or parent variety in which mutations will be induced following criteria in Section I.
Essential basic information and placed in envelopes at 5,000 seeds per dose and stabilized moisture
between 13 – 14%. Expose each envelope to desired dose (20% higher and about 20% longer from thatfound in literatures or historical data of your laboratory for example 0.1 to 0.5 kGy. Set aside to 150
exposed seeds from each dose range for germination prior to replanting. For germination, seedling andsurvival tests, 50 seeds per treatment will be sown in basin filled fine sand. Sow the seeds closely within
the rows and space the rows at 6 cm apart. Set up the experiment in a randomized complete block design
with a number of germinations. Take percentage of germination after and seedling height measurementtake survival data on the 21st day seeding.
For field planting, the seeds sown in seedbeds should be transplanted in the field on the 21st day aftersowing. The seedlings (M1) should be planted singly with spacings of 10 cm within the rows and 30 cm
between rows. Record date of maturity. Harvest one panicle from the primary tiller of each plant in eachdose at maturity, combine all panicles from each dose and plant again as M2. Score chlorophyll mutation
rates per 1000 seedlings/dose. Transfer M2 seedlings singly in the field 20 x 25 cm apart between hills
and rows. Score for normal morphologic mutations for preliminary yield trial seed of M2, i.e.., M3 should
be sown in seedbed and transplanted 21 days after in a well-prepared field. Prior to final harrowing, 8020-20 Kg, NPK per ha should be applied and incorporated during the final land preparation. Three tofour seedlings should be planted per hill with spacing of 20 x 20 cm between hills and rows. At panicle
initiation, 20 Kg N per ha must be applied. Keep the experimental field cleared by constant weeding andapply insecticide to control insect infestation. Design the experiment in a randomized complete block
design with replications.
I.

Early radiation effects should be observed at M1
1.Percentage germination
2.Survival rate
3.Growth (height) 7 days after sowing
4.Growth (height) 14 days after sowing

II.

Observation on the frequency of chlorophyll and morphological mutations at M2
1. Chlorophyll mutations per 1000 seedlingsa. Striata
b. Virescemt
c. Albina
d. Others
2. Morphological mutation frequency (see list objective)
a. Morphology
b. Quantitative traits
1)Stature2)Lodging resistance3)Plant type4)Grain characteristics

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Mutation Breeding Manual

III. Yield performance of the mutants based on agronomic traits M3
1.
Days to heading
2.
Nature plant height3.
Productive tillers
4.
Panicle length5.
Number of grains per panicle
6.
Percentage sterility
7.
Weight of 100 grains
8.
Yield (T/ha)
HANDLING OF M1 GENERATION
a. TREATMENT CLASS
Always prepare small amount of control (zero – dose). This assures you a good handling during
treatment and planting. If the growth is inferior to the usual operation, some mishandling might be there.
If it is routine mutagenesis work, numbers of plant may be some 100 or less. It often helps you topreserve the original variety.
As a general rule, the smaller the treatment dose, the better. Although it requires larger population size,
the unfavorable accompanied mutation can be suppressed to low level. Growth reduction by 20% (height,
fresh weight, etc) or 80% growth, would be a recommended level. To be sure of the appropriate dose,some sample should be treated to find the 50% growth reduction dose, making at least 4 classes, e.g.
control, 10%, 20%, and 50% or more reduction, the size of the population being e.g. 1, 10, 20 and 1. (Fig.
II-3)
b. HARVESTING SCHEME
This depends on the crop species, or how the seed are produced. Numbers of seeds in one unit (panicle,
spike, ear, capsule, fruit, etc) and M2 population plan must be considered.
Planting of M2 plants
M1 plant basis

Pooled within M1 plant. Many chimeric sector gives high Frequency ofmutants
M1 spike basis
Pooled within spike etc, Generally easiest to handle
Pooled within M1 population. Similar mutants might be from the sameM2 seed basis
mutation

You may use the last method by dividing the M1 population into small subgroups of about 300 plants orso, so that similar mutants in different subgroup can be assured to be derived from independent mutation.
Application
Size of 1 unit (beware of chimera within the unit)
Rice, millet
100 seeds or more
Barley, wheat
20-50 seeds
Beans, peas5-20 seeds/capsule, 1-3 capsule per bunch
Soybean
1-3 seeds per capsule, 2-4 capsule per node.
Harvest scheme used for rice (E. Amano) (Fig. II-4)
Plant M1 as ordinary planting pattern
Harvest 3 rounds, 1 panicle at a time from each M1 plant
1st harvest is for endosperm character screening
2nd harvest is planted to the field as M2 generation (panicle basis)
3rd harvest is stored in cold storage for future use
In case of japonica race which is non-shattering, panicles can be tied together until use, but for othershattering crops, paper bags or other storage containers will be necessary in large numbers.

54

Mutation Breeding ManualFig. II-3 Dose-survival responce
LD50 is good to define the sensitivity, but 80% survival may be recommended forbetter surbival and less accompanied unfavorable mutation.

Fig. II-4 Example of harvest method (rice)55

Mutation Breeding Manualc. SCREENING/SELECTION METHODSCONCEPT OF RESISTANT/TOLERANT SCREENING (SURVIVAL SCREENING)

Factors to be considered :- uniformity of the screening agent (including temperature, etc.)- Resasonable selection level- Environmental condition
Light, water, humidit, air circulation
Field, greenhouse or laboratory
Isolation of a few generations before the mutagenesis work is recommended to ensure the purity of thematerial.
SCREENING BY MEASUREMENT
Protein quality and other biochemical component must be measured one by one. The method used inselection of rice protein mutant by electrophoresis assay from 1600
M1 panicles was as follows :-One panicle (one from each M1 plant) was considered as the unit of mutation.
One grain from each panicle was assayed in each assay cycle.
If variant in electrophoreogramme was found, the panicle was separated for further
analysis
Grain analyses continues for 8 cycles(Prepared by E. Amano)56

Mutation Breeding Manual

FURTHER EXAMPLES OF MUTANT EVALUATIONGRAIN QUALITY EVALUATION
The development of rice varieties provide more choices to farmers, traders, millers, processors as well
as household consumers who in turn become more selective. The grain quality plays a key role in theassessment of selections according to standards that are beneficial to the various sectors concerned.
The project specifically provides information on grain quality characteristics of rice selections. These
characteristics include : 1) milling potential (% brown rice, % total milled rice, and % head rice), 2)
physical attributes (% chalkiness, % immature grains, grain length and grain shape); 3) physico-chemical
properties (%amylose, gel consistency, gelatinization temperature and % crude protein); 4) cooking
parameters (optimum cooking water, % height increase and cooking time), and 5) sensory qualities (%acceptability, preference score and descriptive characteristics as aroma, flavor, tenderness, color, gloss,
cohesiveness, translucency and brittleness of grains).
METHODOLOGY
Rice samples for grain quality tests come from the breeders usually starting from the preliminary yieldtrial. Standard yield check varieties for each group and a sensory quality check are grown along with theentries. Rice samples are aged for three months before actual analysis. Breeding materials in the earlier
generation are usually screened for kernel quality along with the agronomic and field reactions to insect
pests and diseases.
The flow chart of grain quality evaluation is shown in Fig. II-5.

57

Mutation Breeding ManualFig. II-5 Flowchart of Grain Quality Evaluation of Rice Entries

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Mutation Breeding Manual

a. MILLING POTENTIALS
Milling yield of rough rice is the estimate of the quantity of total milled rice consisting of head rice andbroken grains that can be produced from a unit of rough rice. It is generally expressed in percentage.
The percentages obtained are classified based on a scheme developed from research experiences andreadings. For each of the parameter, a recommended value/classification is used as reference for varietal
recommendation as shown in Table 2.
Table II-2. Classification and recommended value for parameters of milling potentials
Milling Potentials
Classification
Recommended Value% Brown RiceGood (G)80.0% and above
75.0% and above
Fair (F)75.0 – 79.9%(Fair to Good)
Poor (P)below 75.0%% Total Milled Rice
Premium (Pr) 70.1% and above
65.1% and above
Grade 1
65.1% – 70.0%(Grade 1 to Premium)(G1)
Grade 2
60.1% – 65.0%(G2)
Grade 355.1 – 60.0%(G3)% Head Rice
Premium (Pr) 57.0% and above
48.0% and above
Grade 1
48.0% – 56.9%(Grade 1 to Premium)(G1)
Grade 2
39.0% – 47.90%(G2)
Grade 3
30.0% - 38.9%(G3)
b. PHYSICAL ATTRIBUTESThe physical attributes consists of four (4) parameters namely: % chalky grains, % immature grains,
grain length and grain shape. Chalky grains are whole or broken grains, one half or more of which iswhite like the color of a chalk and is brittle. Immature grains are light green and chalky with soft texture.
Grain length, on the other hand, is the length in millimeters of the rice grain, while grain shape is theratio of length and width of the grains.
Grain Shape (length/width in mm)

Grain Length (in mm)
Extra Long (EL)Long (L)Medium (M)Short (S)

7.5 and above
6.6 – 7.4
5.5 – 6.5
5.4 and below

Slender (S)Intermediate (I)Bold (B)more than 3.0
2.0 – 3.0
less than 2.0

Results of the consumer preference studies for more than 20 years revealed that Filipinos prefer ricegrains which are long and slender with grain size of 6.6 mm to 7.4 mm and grain shape of more than 3.0.

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Table II-3. Classification and recommended value for % chalky and immature grains in rice

% Chalky Grains% Immature
GrainsClassification
Premium (Pr)Grade 1 (G1)
Grade 2 (G2)
Grade 3 (G2)

< 2.0 %
2.0% - 5.0%
5.1% – 10.0%
10.1% – 15.0%

Premium (Pr)Grade 1 (G1)
Grade 2 (G2)
Grade 3 (G3)

< 2.0 %
2.0% - 5.0%
5.1% – 10.0%
10.1% – 15.0%

Recommended Valueless than 5.0 %(Grade 1 to Premium)less than 2.0 %( Premium)c. PHYSICO-CHEMICAL CHARACTERISTICS
The physico-chemical characteristics of the rice grains are important indicators of grain quality. They
are referred to as indices of cooking qualities and cooked rice texture. These characteristics aregelatinization temperature, gel consistency, amylase content, and crude protein.
1. Gelatinazation Temperature. Gelatinization temperature is estimated by the extent of alkali
spreading of raw milled rice soaked in 1.70% (anhydrous) potassium chloride for 23 hours at 30o
C or at room temperature. Thus, rices with low gelatinization temperature disintegratecompletely, whereas rices with high gelatinization temperature remain largely unaffected in thealkali solution.
2. Crude Protein Determination. Rice grain contains 8 – 10% protein. It is an important factor fromthe viewpoint of nutrition and taste. There is a tendency of decreased palatability as the content
of protein increase (Ishima et al, 1974). Very common among Philippine varieties register 6- 7 %protein.
3. Amylose determination. Amylose and amylopectin are major ingredients of starch of nonglutinous and glutinous rice, respectively. It is also closely related to the palatability of rice.
Varieties with high amylose content are less sticky and fluffy while low amylose rices are more
sticky.
Classification of rice according to amylase content is listed below
Waxy/glutinous (w)Very low
(vl)
Low
(l)
Intermediate (i)
High
(h)

- 1.0% - 2.0%
- 2.1% - 10.0%-10.1% - 20.0%-2 0.1% - 25.0%
more than 25.0%

For Filipinos, intermediate amylose content is most preferred.
Gel c o nsi s ten c y i s a me a s u r e o f t h e f lo w
4. Gel Consistency Determination.
characteristics of milled rice gel. The test differentiates in terms of tenderness of
cooked rice. Within high amylose group, varieties with soft/medium gel consistency
are more tender when cooked than rices with hard gel consistency. The test separates rice into
the following classification :RatingLength in mm

Hard
MediumSoft

25 - 40
41 – 60
61 – 10060

Mutation Breeding Manual

d. COOKING PARAMETERS AND SENSORY EVALUATION OF MILLED RICE
The milling and cooking procedures affect the quality of the milled raw and cooked rice grains, hence a
standardized method to evaluate the cooking parameters and sensory attributes is very necessary.
Components for cooking parameters include optimum cooking water, height increase and exact cooking
time.
Sensory evaluation is used to interpret sensations perceived by the human senses of sight, smell, touchand hearing. It is conducted using either or both trained laboratory and consumer panels.

1.

Laboratory Panel
Consumer Panel
Measures sensory differences. Describes or 1. Measures preferences and acceptability
characterizes a food product in terms of(likes or dislikes) preference where choice iscooked rice attributes as aroma, color, flavor
involved. General characteristics aretenderness, gloss, cohesiveness and texture
considered.
as well as raw rice attributes like aroma,color, gloss, translucency of grains,
2. The number of panel members is large
wholeness of grains and brittleness ofUsually 30 to several hundred of persons as
grains.
the case warrants.

2.

The number of panel members is small.
Consist of five (5) to ten (10) panelists

3.

Trained. The members are selected from alarge group for their acuity and consistency
in recognizing differences.

Untrained. The ability of its members todiscriminate according to degree for specificattributes is not considered as long as they are
representative of the consuming public and arewilling to participate.

Based on the above considerations, preference is determined by ranking and is expressed inscores of ranked data following the procedure of Lamond (1985). Acceptability is indicated by a “yes” ora “no” response.
1.2 MUTATION BREEDING IN WHEATINTRODUCTION
Wheat (Triticum aestivum) is one of the two leading cereals in the world. The total wheat area
harvested worldwide in 1992 was 220 million ha. Representing 31.4% of the global area planted tocereals. Wheat area harvested in developing countries has 100 million ha or 23.5% of the cereal
hectareage for developing countries. Increases both in yield and area in the last two decades in thedeveloping countries are due to the spread of high yielding semi dwarf wheat varieties, the expansion ofirrigation and the increase in use of chemical fertilizers. The Third World’s share of global bread wheat
production has increased from 27 to 41% in the past 40 years. In the developing countries, the researchon yield is a high priority where mutation breeding can perhaps contribute to augment existing
programmes (Fig. II-6).

61

Mutation Breeding ManualFig. II-6 Spikelet of wheat (center) in which the primary floret on the left isopen showing the three anthers and a part of the feathery stigma. Anotherprimary floret and the secondary floret are closed. On the left is a floret
showing anthers excerted after blooming and on the right is the pistil with
two feathery stigmas.
OBJECTIVES FOR MUTATION BREEDING
1. Breeding for high genetic yield potential, stability, and wide adaptability
2. Breeding for pests and diseases resistance/tolerance
3. Breeding for drought tolerance
4. Breeding for mineral stress conditions
5. Breeding for salt tolerance and other abiotic stresses
MATERIALS AND METHODOLOGY
MATERIALS
1. Wheat seeds 5,000/dose
2. Manila (brown) coin envelopes to contain seeds for irradiation
3. Seed boxes
4. Seedbed
5. Experimental field
6. Irradiation facility
a. Co-60 or Cs-137, etc.
b. X-ray machines, etc.
METHODOLOGIES
1. Determine radiosensitivity of seeds of selected variety to establish lethal dose
2. Starting from lethal dose, expose seeds at incremental decrease of radiation doses
3. Germinate in seedbox for response studies
4. Plant in seedbed for germination prior to field transplant
5. Transfer to experimental field

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2. COMMODITY II: LEGUMESDATA GATHERING AND SOURCES OF DATA
M1 - Materials are space planted. Outstanding plants are identified and selected on the basis similar torice breeding (i.e. percentage germination, survival rate, growth after sowing,etc). Transfer seedbed
planted to experimental plots. Selection are advanced to M2.
M2 - Two to three thousand plants are grown with space in M2 in well watered, high fertility conditions.
Individual plants are characterized for disease resistance and agronomic suitability (i.e. tillering, lodging,maturity, etc).
M3 - Progeny of selected M2 are grown solid in 2 m three-row plots. Each plant is treated as a separate
population. Selection is done by plot first and individual heads in the selected plot are bulked. The
environment of the M3 field depends on the conditions for which materials are being selected. Irrigated or
rainfed environments, acid soils, hot climate, etc.
M4 - Bulk of M4 is planted solid and selection criteria and choice of environment are the same as M3.
M5 - Approximately 100 plants/plot selected in M4 are space planted in a favorable, high yielding
environment. Individual plants are selected on the basis of disease resistance, acceptance, agronomic
characters and spike fertility.
M 6 - Each plant selected in M 5 is grown individually as M 6 in plots of three rows, 2 m long.
Agronomically outstanding lines acceptable in resistance (diseases, pests, environmental) are bulked inadvance to yield evaluations on the basis of choice in rainfed, acid soil and hot humid climate
environments according to breeding objectives. The lines developed in purely irrigated condition of highfertility are also checked in rainfed condition of low fertility and vice-versa.
BREEDING MUNGBEAN
The mungbean breeding program of the PNRI dates back to the 60s when Dr. I.S. Santos, the former
Head of the Agricultural Research Section, isolated a multifoliata mutant after seed treatment of the small
seeded, shiny yellow mungbean variety with EMS (ethyl methyl sulfonate), at 0.04 M. At first, themutant appeared to have no economic value being small seeded, had poor seedling vigor, very leafy andlate maturing. Being leafy however, the mutant appeared to be useful only as green manure crop to
improve soil fertility. While studying its later progenies, it was observed that this mutant possessed somedegree of male sterility or reduced cleistogamy - allowing natural outcrossing with other varieties. To
mungbean breeders, the trait appeared very useful since conventional breeding by emasculation was
difficult. This unique trait allows transfer of the mutated gene into different genetic backgrounds, thus
allowing introduction of desirable genes into other varieties. The discovery urged plant breeders to work
further for its improvement to produce the variety with the needed genotypes.
Thus, starting in 1972, a cross breeding program was initiated to improve the mutant by natural
outcrossing using mungbean varieties recommended by the Philippine Seedboard. Varieties developedby the Bureau of Plant Industry and the University of the Philippines at Los Banos were used as parentsof the crosses made. In 1975, after several plantings, observation and selection, a large-seeded variety
was isolated. It was early-maturing, non-lodging, less leafy and had shiny yellow seedcoat color. Further
selection resulted in the development of the many mungbean varieties developed at the Institute. A
schematic diagram of the breeding procedure is illustrated below.

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Mutation Breeding Manual

Original Parent(small-seeded, native variety)------------------------Æ Original Multifoliata mutant
EMS (0.04M)

Crosses Made :Orig. Mutant

X CES 14
Selection
CES 28 -------Æ for desirable plant types
CES 55(U.P. Los Banos Varieties)

Orig. Mutant

X MD 15-2 -------Æ Selection for desirable plant types
MGS-10 A(Bureau of Plant Industry varieties)

F7 of the cross : MG50-10A X Orig. Mutant -------Æ Large-seeded Multifoliata was isolated
Further selection and purification resulted in the development of the following varieties :PAEC 1 - Medium-size seed : shiny yellow seedcoat; matures in 70 to 75 days; yields about 10% lessthan the high yielding Seedboard recommended green-seeded CES 55; should not be grown in May to
July, because it grows too tall, and sets very few pods; recommended for planting in November to January
when it grows taller than most mungbean varieties and therefore competes well against weeds.
PAEC 2 - Multifoliata leaf; some seeds have shriveled or wrinkled seedcoat; seeds as small as those of
the yellow native mungbean; matures later than PAEC 1 by about five days, grows about 10 cm shorterand yields about 5 to 10% less; recommended only for green-manuring; might have superior eating
quality and/or nutritional value because of the wrinkled seedcoat.
PAEC 3 - Large-seeded; seedcoat very shiny yellow; matures in 60 to 65 days; in the dry season planting
for which it is recommended, grows to a desirable height of 40 to 60 cm and yields as much as CES 55
PAEC Mungbean No. 5 - Seeds as small as those of native yellow mungbean; yelds as much as CES 55 in
the dry season; Recommended to replace the still-popular small-seeded native yellow mungbean which
has the undesirable characteristic of shattering pods; grows to a very desirable height of 40 to 60 cm.
PAEC Mungbean No. 7 - Very large-seeded; matures in 60 to 65 days; grows to a very desirable height of
40 to 60 cm and yields a little less than CES 55. Recommended for growing in the dry season forsotanghon production.
PAEC Mungbean No. 9 - Dull yellow seedcoat; medium-size seed; matures in 60 to 65 days; with wider
and longer pods than PAEC Mungbean No. 7; grows to a desirable height of 40 to 60 cm and yields atleast as much as CES 55. Recommended for use by farmers who would want to produce their own pure
seeds
PAEC Mungbean No. 10 - Multfoliata leaf; otherwise it has the same characteristics as PAEC Mungbean
No. 9. Unlike PAEC Mungbean No. 2 (multifoliata), has vigorous, fast growing seedlings and does nothave the yield depression associated with the multifoliata leaf. Recommended for growing in shady areas
and for green-manuring. Even easier to maintain pure than PAEC Mungbean No. 9.

64Mutation Breeding Manual

3. COMMODITY III: ASEXUALLY PROPAGATED ORNAMENTALSINTRODUCTION
Ornamentals have become an important commodity, a promising dollar earning crop in the country. It
has a big market potential both here and abroad. Development of improved varieties of these ornamentals
through mutation breeding will enhance export capacity, which will greatly help in the country’s
economic recovery.
Furthermore, this will serve as encouragement for enthusiasts to grow more of these ornamental cropsthat would create livelihood opportunities. This will help also in realizing the goals of becoming anexport winner and sunshine industry.
OBJECTIVESGeneral: To develop improved varieties of ornamental crops
Specific:

1. To develop a wide array of cutflower (orchids and chrysanthemum) with improved
characteristics (color, texture, substance) earliness to flower (Fig. II-7)
2. To induce variation in form or color of the leaves, size and growth habit in Foliage
and plants that will cater to changing consumer demands. (Fig. II-8)

65

Mutation Breeding ManualFig. II-7 Schematic diagram for mutation breeding in cut flower ornamentals

66

Mutation Breeding ManualFig. II-8 Schematic diagram of mutation breeding of foliage ornamentals

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Mutation Breeding Manual

3.1 CUTFLOWER ORNAMENTALSStudies on the tissue culture of orchids and chrysanthemum are presently being done to induce variationand to provide a rapid means of propagation.
Murashige and Skoog’s (MS) medium is used for the in-vitro micropropagation of chrysanthemum.
Irradiated and non-irradiated stem explants were planted in MS medium for the regeneration of plantlets.
Sub-culturing of the plantlets were done until roots are well-developed. Whorling and changes in leaf
color were observed at 10 Gy and doubling of the leaf growth at the node in 20 Gy of the 3rd vegetative
generation. Explants (shoot tips and nodal sections) were obtained from this generation and planted in
MS medium as M1V4 generation. At present, there 783 seedlings micropropagated. Morphologicalchanges observed in the fourth generation were multiple branching per node in the 10 Gy dose as well as
in the 20 Gy dose.
For orchid tissue culture (Fig. II-9a-d), the Knudson C (KC) medium with organic additives is used forembryo culture. Irradiated immature embryo were inoculated in KC medium and allowed to develop into
callus and protocorms. There was proliferation of growth of callus and protocorm tissues with repeatedsubculture in Dendrobium pattaya Beauty. Protocorms are sub-cultured for plantlet regeneration.
In Vanda sanderiana, reflasking of the plantlets was done until roots are well-developed and ready forcompotting. Seedlings were compotted in orchid pots containing osmunda fiber and charcoal and grownunder greenhouse conditions. A decrease in seedling height, number of roots and rootlength wasobserved with increasing dose of gamma radiation of 10 and 20 Gy.
MURRAYA EXOTICA (Local name: Kamuning)
Seeds of Murraya exotica (local name, Kamuning) was obtained from sexually precocious plantselection of Dr. Ibarra S. Santos were exposed to 10, 20, and 30 Gy dose of gamma rays. These weresown in seedbed and grown under greenhouse conditions. Seeds were germinated 45 days from day ofsowing and bore flowers from 30 to 60 days. Seeds that developed from M1 generation were harvested
and grown as M2 generations. These were isolated and grown as M3 generation. Selection andpurification of mutants were done in the M4 to M7 generations. In the M7 generation, a dwarf mutant ( 1to 2 feet) was obtained from a 30 Gy dose of gamma rays. This mutant is characterized with branchingand spreading habit of growth in multi-flowered chymes that are prolific. Unirradiated Kamuning usually
grows to a height of 7 feet or 210 cm.
The dwarf Murraya mutant was approved for registration by the Technical Working Group in
Ornamentals.(Fig. II-10)
DRACAENA SANDERIANA (Local name: Corn plant)
Stem cuttings of Dracaena sanderiana (local name, corn plant) were exposed to 10 and 20 Gy of
gamma rays and planted in seedbeds and grown under greenhouse conditions. M1V1 generation cuttings
were obtained in the M1V1 plants and planted as M1V2 generations. Chlorophyll mutants were induced in
the M1V2 generation. The mutants were selected and grown in the M1V3 and succeeding generations. In
the M1V7 generation, a Dracaena sanderiana mutant was induced. (Fig. II-11a.b) It is a stiff and robust
plant with modified shape and stripes of the leaves. The center is silver-green with white stripes andbroad margins in deep green.

68

Mutation Breeding Manual69

Mutation Breeding Manual70Mutation Breeding Manual71

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3.2 ORNAMENTAL PINEAPPLEINTRODUCTION
Pineapple (Ananas comosus (L.) Merr) is one of the most important crop of the Philippines. The
pineapple industry provide employment and income to thousands of Filippinos.
The pineapple Queen variety is the most desirable for fresh consuption because of its sweetness of fiber
content and crispiness of the flesh. However one objection to this variety is the presence of troublesome
hard spines which interfers with cultivation, weeding and harvesting.
Mutation breeding through the use of ionizing radiation (gamma rays) coupled with in-vitro culture
techniques was undetaken for the developement of improved pineapple Queen variety with the following
objectives:(1) To eliminate or reduce spines/thorns along the margin of the leaves. (Fig. II-12, 13a,b)(2) Progation of the chlorophyll mutation which can be the bases of ornamental types(3) To establish protocl for the tissue culture techniques for the Queen variety pineapple.
(4) To make use of the in vitro culture techniques as a tool for induced mutation breeding.
Induction of mutations, using ionizing radiations in vegetively propagated crops, one of the problems
encountered is diplontic selection and chimera formation which lower the efficiency of mutation
breeding. This problem can be avoided by developing a single-cell approach which can be made possibleby the development of cloning techniques through tissue culture and the use of axillary buds. Buds
consist of few cells provide larger mutated sectors in the growing shoot. Hence, the use of the in-vitro
approach (through irradiation of axillary buds) and the in-vitro technology (through tissue culture
techniques) were the methods used to attain the induction above-mentioned objectives.
MATERIALS AND METHOD
a. MATERIALS
Pineapple (Ananas comosus (L.) Merr.) variety Queen obtained from Imelda, Camarines Sur, Bicol
Region was used as tthe test material for mutation studies. Crowns of uniform size were selected, and thebud at the base of each leaf was separated carefully by a scalpel, taking into consideration not to separate
the bud from leaf.
b. IN-VITRO CULTURE APPROACH
Establishment of the protocom for the tissue culture of pineaple Queen variety
Protocol:1. Crowns of pineapple variety Queen were used as sources of explants.
2. Crowns were washed in tap water with detergent to get rid of soil particles.
3. Leaves were removed from the crown and surface-sterilized with 70% ethanol for 1 minute andin 5.25% sodium hypochlorite for 20 minutes and rinsed several times with sterile distilledwater.
4. Crown sections without buds of about 0.5 cm3 were inoculated in soil Murrashige and Skoog(MS) medium with 10% coconut water (cw), sucrose and varying levels of benzyl adenine(BA) in combination with naphthalene acetic acic (NAA), autoclaved prior to inoculation at 15
psi for 20 minutes at 120°C
5. Callus from the crown section were induced with the best treatment combination of BA and
NAA that would enhance plantlet morphogenesis
6. Series of sub-culturing in fresh medium were done until plantlets were ready to be transferredto the soil.

73

Mutation Breeding ManualFig. II-12 Stages of Development

74

Mutation Breeding ManualFig. II-13 Mutants induced in pineapple(a) chlorophyll mutant(b) plant with reduced spines75

Mutation Breeding Manualc. IRRADIATION TREATMENT
Eighteen lots were divided into 6 groups and irradaited with dose levels of 10, 20, 30, 40, and 50 rad of
gamma rays from a Cobalt-60 facility of the Philippine Nuclear Research Institute, Diliman, Quezon City.
The remaining non-irradiated axillary buds were used as control (0 rad) Immediately after irradiation,these were rehydrated for 30 minutes in a water with a constant temperature of 32℃℃.
d. GROWING OF MV1 GENERATION
The axillary buds in each dose were planted in wooden boxes contsining sterilized soil and grown undergreenhouse conditions. The distance of the planting were 7.0 cm betweem rows and 3.0 cm between eachleaf containing the bud.
The experiment consist of 50 samples per dose level with 3 replications and arranged in a randomized
complete (RGB) design. In the MV1 generation, data on the percentage of buds emerged were taken 65
days after irradiation. Seedling height, rootlenght, number of plantlets developed from bud and othermorphylogical changes were recorded 155 days after irradiation. Percentage survival was reckoned 95
days after transplanting.
e. GROWING OF MV2 GENERATION
All MV2 plants were harvested 250 days after irradiation on individual plant basis. The bud at the baseof each leaf was separated and planted in a plant-to-row plan in boxes. The distance of planting used in
the MV2 was the same with that of the MV1. The frequency of plantlet with chlorophyll mutations andother morphological changes were obtained in the MV2 generation. Chlorophyll mutants were selected
and grown in the M1V3 and succeding generations. The scheme of mutation breeding in pineapple isshown as follows.
Fig. 1 Scheme of mutation breeding in pineapple
Breeding a new variety of crop takes anywhere from 12 to 15 years of intensive effort. The steps include:
Generation
M1V1
M1V2
M1V3-M1V8
Next 2-3 generations
Next 2-3 generations

Characterization axillary buds or tissue cultures treated by gamma
rays.
Plants grown from axillary buds
Populatiion of plants grown from vegetative parts harvested from
M1V1 Selection of desired mutants may start in this generation or later.
Continuing selection, genetic confirmation, multiplication andstabilization of field performance of mutant lines.
Comparative analyses of mutant lines during different years and indifferent locations.
Official testing before release as a new variety.

3.3 CHRYSANTHEMUM
Chrysanthemum is one of the four leading cut flowers in the world with Netherlands, South Africa,
Spain and Israel being the major world producers. The current demand in Thailand for cut
chrysanthemum outstrips national production. The market trend in the country suggests that in the future,
new chrysanthemum varieties will be required for exotic flower sscolour, size and shape. This project
aims at improving the chrysanthemum variety 'Reagan White' by in vitro culture in combination withinduced mutation using gamma rays. Ray florets of chrysanthemum variety 'Reagan White' were cultured
on MS medium supplemented with 3 concentrations of BA i.e. 1, 5 and 10 mg/l. Shoot formation wasobserved only on the medium supplemented with 5 and 10 mg/l BA. The shoot formation percentage was
21 and 67% for the cultures supplemented with 5 and 10 mg/l BA, respectively. By culturing ray floretson the medium supplemented with 5 mg/l BA, it took 50-60 days to produce shoots whereas it took only
21-30 days to produce shoots on the medium supplemented with 10 mg/l BA (Table II-4)

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Table II-4. Percentage of shoot formation from ray florets and duration for shoot formation ofchrysanthemum variety 'Reagan White' on MS medium supplemented with different concentrations of BA
Concentration of BA (mg/1)
Percentage of shoots
Duration for shoots
formation from ray florets
formation(days)
(%)
0
1
5
10

0
0
21
67

－
－
50-60
21-30

From these results, we then used the MS medium supplemented with 10 mg/l BA to initiate the in vitro
culture of chrysanthemum from ray florets in subsequent experiment. Multiple shoots produced were
irradiated with gamma rays of 10, 20 and 30 Gy. The irradiated shoots were then transferred to fresh
medium and multiplied two times from single-node cuttings. M1V3 shoots were rooted on half-strength
MS medium and were irradiated with 10 Gy acute gamma rays. After irradiation treatment, rooted shoots
were transferred to soil in the greenhouse at Mae Sa Mai Experimental Station, Chiang Mai. They were
multiplied two times by conventional cutting and then transplanted in the field for observation andselection of desirable variants at flowering time. Changes in flower color, form and shape were observedin plants treated with gamma rays of 20, 30 and 40 Gy. Mutation frequency of flower form and colour
mutation frequency among the irradiated plants with 20, 30 and 40 Gy was 2.9, 4.2 and 17.8%,
respectively. (Table II-5).
Table II-5. Phenotype of chrysanthemum variety 'Reagan White' derived from in vitro irradiation withgamma rays.
No. of plants
Flower form mutation
Colour mutation
Radiation dose (Gy)
investigatedfrequency (%)
frequency(%)
10+10
68
14.7
2.9
20+10
48
22.9
4.2
30+10
28
39.3
17.8
Five promising mutants were selected - three from plants irradiated with 30 Gy and two from the plants
irradiated with 40 Gy. Phenotypic characteristics of selected variants are shown in Table II-6.

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Table II-6. Colour and size of selected M1V3 variants derived from in vitro acute irradiation ofchrysanthemum variety 'Reagan White'.
Selected
variants

Radiationdose(GY)

Diameter offlower(cm)Diameter of
discflorets(cm)Length ofrayflorets(cm)No. offloretwhorlsFlowercolourWW2S7 1a20+10

6.43±0.06

1.63±0.15

3.17±0.29

3

White

WW2S9

20+10

8.27±0.31

1.80±0.10

3.97±0.25

2

WW2S1020+10

7.63±0.15

1.77±0.06

3.67±0.21

2

Yellow
2D
White

WW3S1

30+10

6.40±0.26

1.13±0.15

3.53±0.06

3

WW3S1330+10

6.70±0.26

1.20±0.26

3.53±0.06

3

Yellow
2C
White

From the original white variety, mutants with attractive colour, form and size were generated. They
were multiplied by in vitro culture (Fig. II-14) as well as by conventional cutting. They will be evaluatedfor their performance stability and uniformity in the field during the next planting season.

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Mutation Breeding ManualFig. II-14 Micropropagation in Chrysanthemum

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3.4 CANNA
The garden canna is a wide spread ornamental in several tropical countries especially in Thailand. In
general, canna varieties arise from several hybridizations, resulting in wide variation in flower colour,
shape, size and plant morphology. The scientific name of garden canna is Canna general/s. The
chromosome of canna is 2n = 18 (diploid) or 3n = 27 (triploid). The former produces good seeds withhigh pollen fertility while the latter is virtually seedless with very low fertility. Wide diversity in canna
could be achieved not only from hybridization but more rapidly from induced mutation. Gamma
radiation is a good tool for improving seedless canna where the conventional crossing is ineffective. This
study aims to produce new novel canna varieties from canna plants grown in the Kasetsart University
Campus, by using gamma irradiation technique and selection of various characteristics such as plantmorphology, flower colour, shape and size.
The initial step in the experiment was irradiation with acute gamma rays (15-25 Gy) of rhizomes andyoung shoots of canna. The irradiation samples were planted in pots to produce new shoots. In order toisolate mutants, progenies were observed for variation in flower colour, shape and size. When variants
appeared, they were separated and grown for observation of stability and uniformity of the above
characteristics. As stable mutants were obtained, they were given names and registered with the Kasetsart
University' Board of Plant and Animal Varieties Registration.
Two types of variants were observed, i.e., stable and unstable. Only the stable variants could be
multiplied and could give rise to new varieties. Whereas, the unstable ones tend to revert to the original
parent. Ten stable mutants were obtained and were multiplied to produce new varieties. The Royal
Horticulture Society colour chart was used to classify the flower colours of mutants and their controls.
Plant characteristics of mutant varieties and their controls are shown in Table II-7.
Table II-7 Some characteristics of the controls and new mutant varieties of canna from gamma rays
induced mutations
Control plants
LineNo.1

LineNo.6

Line No. 12

Line No. 24

Characteristics
Flower deeppink(Red Group #48A-B), medium
bloom, large cluster.
Flower lightpink(Red Group #49 C), medium
bloom, large cluster.
Flower-red (RedGroup #45A-B),
small bloom, large
cluster; broad leaf.
Flower-darkorange(Orange
Group 28# A-B),
medium bloom,
large cluster; tall
plant.

Radiationdose (Gy)
20

15

20

Mutants

Characteristics(1) Pink
Peeranuch

Flower-pale pink (Orange
Group #28 C) with yellow
splash, medium bloom,
large cluster
Flower-creamy white(Yellow Group # 2 D),
medium bloom, large
cluster.
Flower-yellow (Yellow
Group # 12 A), small
bloom, large clusters;
broad leaf.
Flower-yellow-orange(Yellow-Orange Group#21 A-B), medium bloom,
compact and large cluster;
thick leaf and short plant.

(2) Cream
Prapunpong(3) Yellow
Arunee(4) Orange
Siranut

25

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Mutation Breeding ManualControl
plantsLine No.10

Line No.11

Characteristics
Flower-red (RedGroup # 54 A)withyellow spots(YellowOrangeGroup # 18A),medium bloom,
large cluster.

Flower-orange-red(Orange-Red Group# 32 A), small
bloom, large cluster,
red-green leaf.

Radiationdose (Gy)

Mutants

Characteristics

20(5) Napavarn

Flower-yellow- orange(Yellow- Orange Group #18 A) with red spots (RedGroup # 37 A), medium
bloom, large cluster.

20(6) Pink
PanneeFlower-light red(RedGroup # 37A), withyellow- orange spots(Yellow-Orange Group #18B), medium bloom,
large cluster.

20(7) Orange

Flower-orange (Orange
Group # 28 B), small
bloom/ large cluster,
green leaf,

20(8) Pim Ngern

Flower-orange-red(Orange-Red Group # 32A), small bloom, large
cluster; variegated leaf.

Line No.24

Flower-darkorange(Orange
Group #28 B),
medium bloom, large
cluster.

20(9) Wanwisa

Flower-yellow-orange(Yellow-Orange Group#14 A) with red spots,
medium bloom, large
cluster.

LineNo.2

Flower-red(RedGroup # 44 B)
with sparse yellow
streaks on petal edge,
large bloom, large
cluster.

20(10) Red
Ridthee

Flower-red (RedGroup #44 B)with large yellow
splash(Yellow-Orange
Group #17 B), large
bloom, large cluster.

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PART III:
CELL AND MOLECULAR BIOTECHNOLOGYTECHNIQUES IN MUTATION BREEDING

Mutation Breeding Manual

DNA ANALYSIS ON ORCHID MUTANTSMETHOD
1) Grind plant material to a fine powder using liquid nitrogen, mortar and pestle.
2) Transfer the powder into 15ml of prewarmed (60°C) isolation buffer in a capped polypropylene tube.
3) Incubate for 30-90 min at 60○C. Mix every 10 min.
4) Add 1 vol. of chloroform-isoamyl alcohol, cap the tube and extract for 10 min on rotary shaker/by
hand.
5) Centrifuge for 10 min (5000 x g, room temperature). Rcextract the aqueous phase once with fresh
chloroform-isoamyl alcohol and centrifuge again.
6) Transfer final aqueous phase into a new tube. Add Rnase A to a final concentration of 100µg/ml. Mix& incubate at room temperature for 30 min.
7) Add 0.6 vol. of ice cold isopropanol, and mix gently to precipitate DNA.
8) Centrifuge for 10 min (5000 x g, 4○C)
9) Wash pellet with 20 ml of washing solution for a few minutes and collect by centrifugation (10min,5000 x g, 4○C)
10) Invert tubes and drain on paper towel for about one hour.
11) Add appropriate TE buffer and allow the pellets to dissolve overnight (4○C) without agitation.
12) Add o.5 vol. of 7.5 M ammonium acetate solution, mix and chill on ice for 15 min.
13) Centrifuge for 30 min (10,000 x g, 4○C). Transfer supernatant to a new tube, add 2 vol. of 96%
ethanol, mix by inversion, and store for 1 hour at -20○C.
14) Centrifuge for 10 min (5000 x g, 4○C), wash pellet in 70% ethanol and centrifuge again.
15) Drain final pellet, and dissolve in an appropriate volume of TE buffer.
DNA ISOLATION
A modified CTAB procedure based on the protocol on Doyle and Doyle (1987) was used to obtained
good quality DNA (Weising et al. 1991).
MATERIALS AND REAGENTS
3g fresh leaf/flower
Isolation buffer:
2% CTAB(cetyltrimethylammonium bromide), 1.4 M NaCl, 20mM EDTA,
100mM Tris-HCL(pH 8.0), 0.2% β-mercaptoethanol (added just before use).
Chloroform-isoamyl alcohol (24:1)
Rnase A solution:
10mg/ml Rnase A in 10mM Tris-Hcl, 15mM Nacl (pH7.5)
100%isopropanol
Washing solution:
76% ethanol, 10mM ammonium acetate
TE Buffer:
10 mM Tris-HCL, 1 mM EDTA (PH 8.0)
7.5 M ammonium acetate
RAPD ANALYSISOLIGONUCLEOTIDE 10BP PRIMERS
Primer used for RAPD are: Operon Kit A & Kit B. Primers that generated bands are:
OPB-04,OPA-10
OPA-13, OPA-03

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REACTION(PCR KIT)
Genomic DNA
PrimerMgCl2
DNTPs
Reaction buffer(10x)
Taq polymerase
Deionised water
Total volume:
PCR PROGRAMME
Denaturation:
1min
Cycles 1-3:30s
30s
1 min 30s
Cycles 4-38:
15s
30s
1 min 30s

Volume(ul)
2.0
2.0
3.5
1.0
5.0
0.5
36.0
50.0

Final concentration
10ng/µl
20 pmol
1.75mM
0.2 mM1x
2.5 unit

94○C
94○C(Denaturation)
35○C (Annealing)
72○C (Extension)
94○C
35○C
72○C

GEL ELECTROPHORESIS
Amplified fragments were separated by electrophoresis on 2% agarose gels with a TAB running bufferand 100bp DNA ladder (Promega,USA) was used to determine the size of the fragments. Gels were
stained with ethidium bromide and visualized on UV transilluminator.
METHODOLOGY FOR IN VITRO MUTAGENESIS OF ORCHIDS
Meanwhile, studies on the radiosensitivity of orchid protocorms showed that the results vary with timeof irradiation and source of explants and also species [8]. The radiosensitivity is affected by the age of the
protocorms and differences in ploidy level of different species or hybrids. Furthermore, problems indetermining the optimum dose were also caused by the difficulties determining the age of the protocorms
correctly and the sensitiveness of protocorms to manhandling during culturing and irradiation procedures.
Factors affecting radiosensitivity for in vivo plant parts must also be considered in vitro. In addition,evidence also indicates that concentration of auxin and/or cytokinin in the culture medium can influence
the radiosensitivity of the cultured cell [9]. The medium itself when exposed to irradiation has beenshown to have both stimulatory and deleterious effects on different cultured cells and tissues. High doseof irradiation may produce radiolysis products from sucrose in the medium that have effects on cell
growth and differentiation [10]. To circumvent these associated problems, fresh explants and callus
material can be irradiated on moist and sterile filter paper. Treated materials are then immediatelytransferred onto fresh non-irradiated medium.
1. Matured self-pollinated seeds from Demlrobium 'Sonia Kai' are collected.
2. Working in the laminar flow cabinet, seeds were dipped in 90% alcohol and flamed. The seeds werethen left in a sterile Petri plate for a few minute.
3. Using a pair of sterile forceps and scalpel, the seed pods arc cut open longitudinally.
4. Using a new set of sterile forceps, the seeds are scooped out of the seed pod and placed on half
strength macro and microelements of Murashige and Skoog (1962) medium, which is supplemented
with 3% sucrose, solidified with 0.24% (wt/vol) Gelrite.
5. Cultures were maintained at 26○C with 12h photoperiod until protocorm-like bodies (PLBs) wereformed.
6. Regenerated PLBs are then selected according to size and placed on wetted sterile filter paper in a
Petri plate.
7. PLBs are then irradiated.
8. Irradiated PLBs are immediately transferred onto fresh 1/2 MS medium and incubated at 26○C with
12h photoperiod.
9. Irradiated PLBs were multiplied by subculturing every three weeks until four subculture cycles.
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10. At the end of the fourth subculture cycle, the PLB are allowed to regenerate into whole plantlets.
11. Once the plantlets are about 1-2 cm in height and have several roots, they are transferred into a thumb
pot and acclimatised in the greenhouse/shadehouse.
12. The plants are transferred into bigger pot when they reached a suitable size, and remain in this pot
until mature and flower.
13. Selection for flower characteristics are done by harvesting sprays with 70% blooming flowers. Data
are taken using the attached data sheet.

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Mutation Breeding Manual

Data taken include:
Morphology of Orchid Flower
Mutant Line No.:Species/Hybrid Name:
Locality/Origin:
Altitude:Habitat:Purpose:Study of Orchid Flower
Measurement and colour description
1. Horizontal measurement (spread):
2. Vertical measurement (spread):
3. Length of spray/inflorescence:
4. No. of flower and buds:
5. Length of scape (length between the base of stalk to the point where the 1st flower starts:
6. Length of pedicel:
7. Internode length:
8. Date of first flower to bloom:
9. Date of first flower to wilt:
10. Length of flower life on plant:
11. Texture of flowers
Sepal description:
Petal description:
12. Sepals
length:Width:
Thickness:
Colour:13. Vertical sepal length:
Width:
Thickness:
Colour:14. Petals
length:Width:
Thickness:
Colour:15. Column
height:Width:
Colour and description:
16. Lip
lengthWidth:
Colour and description:
17. Bracts
lengthWidth:
Colour and description:
18. The position/no, of flower the first to wilt:
19. No. of flowers and buds at time of wilts:
20. Shelflifc (harvesting at 70%) flowering
Date of 1st flower
Date of 70% flowering
Date when the 1st flower wilts
Shelflife
21. In florescence/flower stalk - erect/pendulous
22. Brief description of the flowers:

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BREEDING TECHNIQUES OF MUTATION INDUCTION COMBINED WITH TISSUE
CULTURE FOR CHRYSANTHEMUM

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PRODUCER OF RAPID PROPAGATION WITH TISSUE CULTURE FOR OVARY OF
GLADIOLUS GANDAVENSIS

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MICROPROJECTILE BOMBARDMENT FOR MAIZE TRANSFORMATIONa:Particle accelerates DNA-coated tungsten microprojectiles at high speed into plant cells such asembryogenic callus. Inset shows the supercoiled DNA in the tungsten particle.
b:
DNA-coated microprojectiles are accelerated into a large number of plant cells where DNA migrates off
the particles, and for a small proportion of cells the vector DNA integrates into plant chromosomal DNA.
c:
Transgenic cells with integrated vector DNA divide and can regenerate plants in the presence of a
selective agent such as Bialaphos, while nontransgenic cells do not grow or regenerate plants.
d:Regenerated transgenic plants produce roots in the presence of Bialaphos in preparation for transfer togreenhouse growth, while nontransgenic plants will not produce roots.
e:Transgenic plants are grown to maturity in the greenhouse and are wither selfed or crossed to obtain seed.

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ANOTHER CULTURE OF RICE

Steps involved in another culture of rice. (Graphic by Deren Hansen)

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