IAEA-TECDOC-342

MUTATION BREEDING
FOR DISEASE RESISTANCE
USING IN-VITRO
CULTURE TECHNIQUES
REPORT OF AN ADVISORY GROUP MEETING
ON MUTATION BREEDING FOR DISEASE RESISTANCE
USING IN-VITRO CULTURE TECHNIQUES
ORGANIZED BY THE
JOINT FAO/IAEA DIVISION OF ISOTOPE AND RADIATION APPLICATIONS
OF ATOMIC ENERGY FOR FOOD AND AGRICULTURAL DEVELOPMENT
HELD IN VIENNA, 8-12 OCTOBER 1984

-O) A TECHNICAL DOCUMENT ISSUED BY THE

age INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1985
MUTATION BREEDING FOR DISEASE RESISTANCE
USING IN-VITRO CULTURE TECHNIQUES
IAEA, VIENNA, 1985
IAEA-TECDOC-342

Printed by the IAEA in Austria

July 1985
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 CONTENTS

Introduction ............... ............. ............................ 5

Conclusions and recommendations ..................................... 7

A. General considerations on the use of in-vitro culture in

breeding for disease resistance ............................... 9

B. In-vitro mutagenesis ........................................... 13

C. In-vitro selection of disease resistant plants ................. 15

D. Successes with tissue culture efforts for improved disease

resistant plants ........................................ ....... 19

E. Crop pathogen systems where in-vitro techniques may be

appropriate to use ............................................. 19

F. Specific examples as to where and when to use in-vitro

techniques for improving disease resistance .................... 21

G. The use of in-vitro techniques for studies of host-parasite

interactions ................................................... 25

References .......................................................... 29

List of participants and observers .................................. 41

 EDITORIAL NOTE
In preparing this material for the press, staff of the InternationalAtomic Energy Agency
have mounted and paginated the original manuscripts and given some attention to presentation.
The views expressed do not necessarily reflect those of the governments of the Member States
or organizations under whose auspices the manuscripts were produced.
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 INTI'RODUCT'ION

A. Micke
Joint FAO/IAEA Division

In 1982, the Joint FAO/IAEA Division arranged an expert consul.ation to

discuss the problems arising in mutation breeding by the need to find rel.ativel.y
rare mutants with desired traits or characteristics in large populations.
(Selection in Mutation Breeding, IAEA 1984). It was pointed out that choosing a
high standard variety and applying an effective mutagen treatment is not enough:
Clear definition of breeding objectives and the establishment of appropriate
selection criteria are crucial prerequisites for the success of a mutation
breeding programme. Since mutation induction cannot be directed in any way
towards particular traits (not even to think about changing that trait into a
desired direction), mutant selection plays a crucial role.

Mutant selection generally is phenotypic selection, which means that the

mutated gene has to have a fairly strong expression with little disturbance by
environmental interaction for a reasonable chance of being detected.
Unfortunately, many genes do not have such a strong phenotypic expression because
they share responsibility for a trait or characteristic with several or many
other genes. Nevertheless also these genes have a gene product and thus a signal
that could be identified, if one would know what to look for. Selection gets of
course more complicated by the fact that many genes are "silent" through long
periods of the plant's life and are only activated at certain stages of plant
development, in certain organs, at certain times of the daily metaboli.c cycle etc.

The phenotype looked at is always the result of genotype/environment

interactions. The plant breeder is used to deal with environmental problems by
using established experimental designs, which by standardization, replication,
randomization etc. silence the environmental noise and thus let the relevant
gene/environment interaction become more clearly recognizable. Going one step
further, the breeder' may even provoke a particular genotype/envi.ronment
interaction, e.g. by varying planting dates, by different fertilizer levels or by
inoculation with pathogens. Such practices are common, but considering the
persisting difficulties in plant selection, one wonders whether there are not
more possibilities to manipulate the environment so that gene expression can be
more distincly recognized.

Earlier selection, e.g. using seedlings, could save breeder's time and allow
the screening of larger populations, which would increase the probability of
success. Unfortunately, many useful plant characters are not expressed in the
seedling stage. When adult plants are required for selection, this poses
limitations on the number of individuals that can be handled and considerably
extends the time required for decision making. The technology of in-vitro
culture stimulated hopes for new dimensions in plant breeding as far as
population sizes are concerned, and certainly if one considers single cell
cultures, there is virtually no limit as to the number of individuals that can be
handled. On the other hand, the problem with gene expression, pointed out for
seedlings, is worse for single cells or protoplasts.
Of course, in-vitro cultured plant cells are amenable to various kinds of
genetic manipul].ation, thus producing not only genetic variability, but eventually
genotypes hitherto not existing in nature. However, the phenotype is subject of
selection and if we want to make use in plant breeding of the immense population
sizes manageable through in-vitro culture, we will have to use single cell
selection, For that we might have to learn the manipulation of gene expression
at the cell level. There is a wide field open for' research. Once we know how to

5
turn on and off particular genes, once we have learrned how to make genes
expressing more distinctly, once we are able to recognize genes through signals
rather than through the phenotype of' differentliated plants in the field, we can
design ef:i.c:ient .n--vitro selection procedures comparable to those applied in
microbiology.

The Adslvisory Groul: Meeting was rot convened to deal with such broad issues.
It was focussed on disease' resistance for two reasons: First, diseases are among
the biggest probl.ems in crop produc-tion. Although resistant varieties appear to
be the most elegant and acceptable way of solving these problems, breeders and
patholog.ists see(m to tottter between frustration arnd desperation, ornly consoled by
the assurance of permanent employment. Secondly, there is optimism that in-vitro
culture technology could be particularly useful for solv:ing problems of
r.si.stance to pathogens.

The prospects and limitations of using irn-..vi.tro culture for mutation

breeding have been discussed already once in October 1983 at a meeting here in
Vienna and the recommendations are now followed by a group of 20 research
institutes in 17 countries as well as by the FAO/IAEA Agricultural Biotechnology
Unit of the Seibersdorf Laboratory. Experiences and future trends will be
reviewed during an international symposium to be held in Vienna, 19 - 23 August
1.985.

The subject matter of disease resistance is likewise not new to the

FAO/IAEA programme. Following a Panel of experts in 1970, the Joint FAO/IAEA
Division organized a co--ordinated research programme aimed 'at the development of
improved techniques for induction, selection and utiliz'ation of mutations for'
better disease resi.stance. This work, financially supported by the Swedish
Government through SIDA involved more than 30 institutions in 21 countries. In
its first phase from 1971-1979, it focused primarily upon cereals and their
diseases, but from 1978 on more and more emphasis has been given to grain
legumes. Results of this work have been very much up to expectations with regard
to cereal diseases caused by a number of fungi and bacteria, but not so much
regarding virus diseases which are particularly important in leguminous plant
species. It is anticipated that in-vitro culture techniques will be particularly
useful in selecting for resistance to facultative parasites, many of which seem
to be necrotrophs and enter the host plant via toxins. Whether in-vitro culture
can help in making advances in breeder for virus resistance remains to be seen.
So far we recognize the possibilities for virus-free clonal propagation.

Conclusions and recommendations from this group will be useful for FAO,
IAEA, their Member States and numerous scientists in making a decision .lbout
priorities in research efforts and funding.

6
 COI\CLUSIIONS ARID RE.COMMIM\DA) IIOMS

Bre:ed:i.nig for disease resistance :i.s a major aspect of plant br.ee:dingl, whiich
may take at least 20% of a plant breeder's time, effort and budlgt..
Nevertthe lesss, numerous resistance problems remain unsolved and present major
constraints to the production of food, f'eed, fiber' arnd industrial commodi ties.

The application of novel bi.otechnology and( genet;ic enri.cnee(ring w:i.ll. extend

the possibilities of conventional plant breeding. Th.erefore a meet'ingc of exper"ts
in plalt protect ion, plat reeant ad in-.vitro culture technolotgy was convened
by the Joint F1::/IAEA Division in Vienna. The experts were asked to discuss and
give advise on prospects of biotechnology, espe.ciall.y plant in.-vitro culture s, to
contribute towards impr'oved chances of success in mutati.on br'eedling for d'isease
resistance.

-lThe plant blee(der, in searchi.ng for resis talnce to a particul'ar pathogen,

like for any other desirable character, needs genetic variation to bIegin with.
In addition he nee.ds an appropriate screennin.l method to detectl the desireel
character. Science has developed so fast that it is now time to apply the
existin.g knowledge of biotechnology to practical problems in ag ricultu.re , al.so
in deve(loping countriies. In the near future this may be Lrue also for novel
te(chniques of genet ic engineerig . Thie usefu.lness ando feasibii.ty of the
appl..icaatLion of .in.-vitr!o techniques for' these purposes vari.es with cr'ops and
pathogens, but also depends on the strength of plant bree(ddinig and plant pathology
arid the facilities available in a particular country. The members of the
Advi.sory Group attempted to discuss the various aspects and to reach sound
conclus :i.OnS .

7
A. GENERAL COISIDERA:IIOINS ON 'THEUSE: OF :LI\I-.VIT:"RO CULIJITUR :[IV 13REIEDI:G FOR Dl:SEASE
I
RES]:STAIUt:E:

F. 1 Problem definition
Before starting breedin.g for resistance and considering the inclusion of
in-vitro culture techniques, the disease or pest problems have to be defined
precisely, talkin i.nto account
the crop, (its cultivation and use, genetics, type of propagation)
the pathogen (its life cycle, evolutionary capacity etc.)
-the losses caused by the pathogen or pest
the cost of alternative ways of control, considering also the ecological
impact
- the type and level of resistance required
-the extent of anld experiences with conventional breeding for resistance
the ava:i.lability of established i- i._.litro culture methods and of suitab:le
in.--vitr'o screening procedures
suppl:ies
the avai.lab:i.l.ity of the necessary laboratory fac:i.lit:i.s andrl
(including reliable water and power supply)
thellavailability of sufficient aln qualified personnel.
-the costs of in-vitro technique application in relation to avai:l.ble
resources
the possi.b:i..i.ty of using material resu lt:i.ng from the application of i.n!:-vi.tro
techniques in regular plant breeding programmes for cultivar development.

"he potential. economic impact of a successful in....-vitro disease res:istance

breedin.g programme depends not only upon the magnitude of the crop losses to
be avoided, but also on the rapidity with whichl su.ccess can be obtain.ed.
In--vitro techniques properly applied could be a means to speed up conventional
plant breeding as exemplified by the use of anther culture-derived haploids in
breeding programmes of rice and barley.

Regarding the cost/benefit ratio of the application of in.--vitro culture

techniques, the possible multiple use of in--vitro facilities (e.g. clonal
multiplication) must be kept in mind, especially in vegetatively propagated
crops, since it can make the investment into establishment of in vitro culture
facilities more economical.

Genetic vulnerability has been identified in several crops due to widespread

use of a certain germplasm. The risk of crop losses, however, is particularly
great (and the expenses for fungicides particularly high) in vegetatively
propagated crops, which generally consist of uniform clones and in addition often
have to fulfil particularly high consumer requirements. Here, in--vitro culture
offers a chance additional to or in combination with classical mutation breeding
to create genetic diversity without disrupting the basic genotype, and it
facilitates selection of mutant clones possessing specific improvements. The
situation of bananas in the Caribbean area provides an instructive example for
the approach needed (ref. chapter F,1.2.).

A good plant breeding infrastructure would be a very positive asset, but it

is not necessarily a prerequisite for starting in-vitro culture work. Some
in--vitro techniques applications are so simple that they can be recommended,
without hesitation, even to less-developed countries. In-vitro selection for
disease resistance must in any case be followed--up by good field experiments
along conventional lines of plant breeding.

A.2 Genetic variation in the host species

Without genetic variation there is no chance for developing improved
varieties. In many cases sufficient genetic variation for the desired character
is available in cultivar or other germ plasm collections. In other cases

9
scouting in centres of high genetic diversity would be needed to eventually find
the desired character. Not infrequently, the desired variation is missing in the
cultivated species, but present in more or less related species. To create
interspecific hybrids, sterility barriers often have to be overcome and in this
context i.n-.vitro techniques were found to be useful or even indispensable (e.g.
for embryo rescue).

In several cases intensive germ plasm studies have led to the conclusion
that suitable genetic variants do not exist and therefore variation must be
induced (e.g. for resistance against bean golden mosaic virus of Phaseolus beans
or against swollen shoot virus of cocoa). The common way td create genetic
variation is mutation induction by radiation or chemical mutagens (ref. chapter
B), but apparently also in-vitro culture as such leads, under certain
circumstances, to considerable genetic variation ("somaclonal variation"). Novel.
biotechnology has extended the means of inducing genetic variation by using
tissue, cell and protoplast cultures, and is also providing new ways of mutant
selection, including screening for disease resistance. Therefore, it is worth
examining which advantages are offered by in-vitro cultures to solve disease
resistance problems.

The majortypes of plant in-vitro culture are

- meristem culture
- anther culture
-embryo culture
- callus culture
- cell culture, and
- protoplast culture.

Each of these cultural types has specific advantages and disadvantages for
induction of variation, for sel.ection and for subsec(uent regeeleration arnd
propagation. The use of a particular type of culture in breeding for di.sease
resistance wil. also depend upon the pathogen and the host/pathogen interaction.

For resistance selection purposes, the in.-vitro techni.ques present an

attractive advantage in that large populations can :be screened: easily. The
population sizes required For selection depend upon the expected success, which
may be in the order of 1 in 104 to 106 individuals. Such populations would
require, for example, in cereal breeding approxi.mately 1 ha nursery. Using
in-vitro culture, only a few square meters of laboratory space would be
required. inr_-vitro cultured material can also be subjected to various
I'lhe
stresses, such as high temperature or salinity, so that simil.taneous or
consecutive selection for multiple objectives is possible using the same
established cultures. After passing through the selection screens, however, the
in--vitro material. first has to be regenerated to normal plants, then propagated,
and finally subjected to usual procedures of repeated testing and evaluation in
the field.

It has been found that plant genotypes vary in their suitability for culture
and regeneration, and this "culturab:i.lity" seems to be :inheited. Therefore, in
passing plant populations through i.n--vitro culture there is a risk of reducing
variation of germ plasm by inadvertent selection for culturability. It would, of
course, also be a disadvantage to rely in biotechnology only on plant varieties
that comply with the rather unusual conditions of in-vitro culture. In training
and technology transfer attention should be called to this problem.

A.3 Tlpys _or.oppla.. nts

Considering different types of crop plants, with regard to the use of
in-vitro culture in breeding for resistance, there are first the perennial tree
crops. A characteristic of these crops is that their life time is a mu].tiple of

10
their generation Lime and therefore genetic improvement by classical means is
rather cumbersome. Many of them are clonally propagated in order to maintain
their desired heterozygous genotypes: rubber, cocoa, citrus, apple, banana aind
black pepper, to mention a few. As p]antation crops they are characterized by
genetic uniformity over large areas. The cultivation of many thousands of
hectares of genetically uniform clones, however, presents, a serious risk of
vulnerability, if an epidemic occurred. .n--v.itro culture mult:i.pr>lic:<.ion as is
used commercially in oil.. palms acids to the problem. Durable resistance is
therefore a necessity if one wants to get away from the costs and hazards oF
chemical protecti.on. Resistance screens during the in-vitro propagation phase
are indisplensable, but genetic diversification is also mandatory.

Among the vegetatively propagated crops are also annual or biennial. ones
such as potatoes, sugar cane and cassava, which are likewise highly heterozygotus
and for which classical cross breeding has the disadvantage of breaking up many
useful gene combinations. In-vitro irnduction of genetic variation, followed by
n- vitro screening and i!-.-vitro disease-.free multiplication will be an attractive
methodological package. A special plea is made here for cassava and two of its
diseases that are very important in Africa, namely cassava mosai.c virus and
cassava bacterial. blight.

Another category are the annual or biennial. seed-propagated crops. These

can be differentiated into self-pollinators and cross-pollinators, and the latter
may be monoecious or. dioecious. 'The distinction between these groups hIas
implications for the strategy of genetic manipulation. The production of
haploids from anther cultures has already been used successfully for a few
self-pollinating species (namely cereals), but the method offers great
opportunities for the future also for cross pollinators. Diploidization of
haploids leads immediately to homozygous forms with genetically fixed traits
including resistances. This would be true for "qualitative" and "quantitative"
resistance, for immunity, hypersensitivity or partial resistance, no matter
whether controlled by one or several genes.

The development of technology for in-vitro disease resistance breeding is

still in an infant stage for most seed propagated species. Efforts seem
particularly urgent for leguminous crops, like Phaseolus bean, cowpea (Vigna
urnuiculata), and groundnut (Arachis hypogaea), because of their importance as
food crops, the difficulty encountered in conventional disease resistance
breeding, and the great number of diseases, especially virus diseases, several of
which are still ill described.

A.4 Type.s of pathogens

In principle, any kind of plant pathogen can also be used for in-vitro
screening, whether fungi, bacteria, mycoplasms, viruses, nematodes and possibly
even insects. However, there are practical differences: with fungi there are
already a number of positive experiences, bacteria cause some technical problems,
mycoplasms have hardly been studied, viruses and nematodes seem to offer good
possibilities, but information on insect in-vitro screens is scanty.

Among fungi, the toxigenic ones, usually of the necrotrophic type, provide
particularly good prospects (e.g. Helmint_!osorium in sugar cane) (ref. chapter
E.2). Among biotrophic fungi, opportunities are indicated by results obtained
with Peronospora spp., Phytophthora spp. and Plasmodiophora brassicae. Care must
be taken to use the proper part of the life cycle of the fungus in a resistance
screen. Two groups of fungi demand special attention:
(1) Soil-borne fungi, especially in clonally propagated plantation crops with
high genetic vulnerability.
(2) Leaf-flecking toxigenic fungi that are particularly relevant to crops grown
in low-input agriculture.

11
 Bacteria as a group have been neglected because of specific technical
difficulties. Since bacteria are potential toxin producers, their toxigenic
properties should be exploited as in Pseudomonas s.ringae pathovar phaseolicola
on Phaseolus beans.

Viruses as a group need to be studied urgently with a view to develop

appropriate screens, with special emphasis on tree viruses and viruses of
leguminous plants (ref. G.2).

Nematodes and insects producing physiological damage need further

investigation with respect to in--vitro screens. Some plants produce undesirable
insect attractants, and it might be possible to screen them out. If insect
repellents are identified, it may be possible to select for them.

Many harmful biological agents (including fungi, bacteria, viruses,

nematodes and insects) are able to undergo genetic differentiation and, as a
consequence, pathogenic specialization for particular host genotypes through
physiologic races (in a particular context also called biotypes, pathotypes,
pathovars, isolates or strains). In plant breeding for disease resistance the
appearance and spread of new physiologic races is causing a rapid turnover of
cultivars but nevertheless is accepted as a "fact of life". The effective
countermeasure is the avoidance of genetic uniformity within and among crop
cultivars. Fundamental research on resistance phenomena may profitably use
in--vitro cultures to find means for resistance which would exclude the pathogen's
possibility for response in terms of physiologic specialization.

.5 Typ.es of resistance
The type of resistance to be recognized determines the type of in-vitro
culture. Resistances which depend on cellular physiology, such as restriction
of virus replication, can be identified in protoplast and cell cultures. This
type of resistance against viruses is likely to be expressed also in regenerated
p].ants. However, no information is available as to the durability of this
cellular type of resistance, nor of its compatibility with plant vigour as
required in farmers' fields.

In another type of resistance, internal plant structures are involved (e.g.

restriction of movement of virus from cell to cell). Such types of resistance
probably can only be observed in tissues (callus) or differentiated organisms
(embryo, plantlet).

Other types of resistance certainly require normally developed and

differentiated plants or plant parts. For example, to judge resistance against
powdery mildew an intact epidermis is needed. To this category also belong
mechanical barriers such as hairiness in resistance to insects. Such resistances
may be tested using detached plant parts in-vitro, as is done routinely for
cereal powdery mildew.

Detection of major differences between susceptible and resistant is

relatively easy. More fundamental research is needed, however, to detect smaller
differences. For this purpose, refined testing techniques (perhaps the use of
chemical markers) combined with appropriate statistical analyses are required.
In-vitro culture offers the possibility of quickly multiplying any selected plant
by meristem culture. In certain cases anther cultures could be used to advance
development of homozygous lines.

Epidemiological analysis should usefully be undertaken prior to the breeding

programme to determine the required level and the most effective type of
resistance. Different mechanisms of resistance for one and the same
host/pathogen system may possibly be detected with the help of in-vitro

12
techniques. These could then be combined to provide more complex, and therefore
likely more durable resistance.

A.6 nvailable resources

For the establishment of in-vitro laboratories, particularly in developing
countries, several aspects have to be taken into consideration.

In-vitro laboratories can serve multiple purposes:

increase of genetic variation and in-vitro screening in support of an
ongoing breeding programme.
disease-free in-vitro mu]tiplication of cultivars for commercial use or
newly selected genotypes conserving their desirable heterozygosity.
convenient and more economical maintenance of germ plasm collections,
especially of trees, eventually combined with cryopreservation
- disease-free international exchange of germ plasm (as already practiced
by the International Potato Centre (CIP) in Peru).
In view oF the limited resources and scarcity of qualified personnel. in most
develop:ing countries it is suggested that first national or regional iJi-v..it.ro
laboratories are established. Such centres should then offer training and
services for plant breeders, but also for associated phytopatlholoj:i.sts,
virologists, nematologists and entomologists. Desirable would also be training
of scientists having respolnsible positions in devel.oping countries to pave the
way for appropriate use of new technologies that can be oF benefit for these
countries.

An important question is also whether facil.ities are available for'

confirming in-v.it.! se].lected resistance in whole plants, for testing th.:h selected
lines, clones and populations using colimmon greenhouse or field procedures and for
selecting the same original populations under natural conditions. This is a
serious matter since the expression of resistance in culture is often abnormal..
It is vital that any resistance breeding programme involving in-vitro cultures is
. ....... . .... .. . ......
... .........
..........
................ ......... .......... ............................ .............
. ...........................................
............
supplemented
1.J... ~ em.e...
s u.p..p.. ...... by
. 1~.d..... b.. ... intact
. .......... ..plant . testing
....... .. ... ..using
......
...
u.... - established
.....-...
t..„.....1.n „ ..... ...... ....... . procedures.
.......
C......... ..................

B. IN--V:LIRO MUTFACEIMESIS

Resistance of agricultural crops can be improved by selection,

hybridization, induced mutations or in-uvitro manipul.ation and various combined
approaches.

The first and second approach can only be successful when the resistance to
a certain disease is at hand in the parental genotypes. On the other hand, the
selected type or the recombinant may not be resistant enough or resistant only
for a short time or may possess some other characters that limit the practical
utilization and therefore requires additional crosses or mutation induction.

Certain types of in--vitro culture create genetic variation which may contain
new resistance traits useful for the creation of new cultivars. The genetic
changes observed, however, are often very complex. In most cases, such somatic
mutants require genetic reconstruction by hybridization from which segregants
with desired resistance and agronomically acceptable performance have to be
se]ected. Unfortunately such recombination is not feasibl].e in most vegetatively
propagated plants and this limits the usefulness of in-vitro culture derived
variation. Experimental mutagenesis, on the other hand, offers the possibility
(using appropriate doses) to induce changes for desired traits while limiting the
disruption of the genetic constitution of the valuable original. material.
Experimental mutagenesis combined with in-vitro culture offers certain
advantages. For example, dominant mutations are a relatively rare event, but

13
in.-vitro mutagenesis in large populations will give an increased probability of
obtaining such mutants. T'his would be particularly relevant for improving
disease resistance of cross pollinating crop plants.

B
B.1*i*Objects of .mutagen
9 J..bj..c..s.......... treatment
u.........~....n...
...
The target of an in-ltvitro mutagenic treatment may differ in ploidy level,
being haploid, diploi.d or polyploid (eu- or aneuploid).

B.1.1 lThe..hap.lo.id syst.em (anthers containing microspores or pollen grains,

protoplasts, meristem tissue, etc.) allows phenotypic expression of all. mutations
directly in the M1 generation. Selection could be applied to haploid cells or
haploid plants regenerated from them. But there would still be1a big advantage
From haploid mutagenesis, even if selection for mutants is carried out later,
following genome duplication of the mutagen treated hapl.o:id i.nd i.iduals. The
reason is that the M 2 would only contain homozygous mutants and original types,
thus the selection of mutants (e.g. by chemical analysis) would be free from the
problems created by the presence of heterozygotes. On the other hand, a.l
mutants would be fixeds. without any further segregation and recombination of
multiple mutations. This system is applicable for species, where haploids can
easily be obtained (at present e.g. rice, barley, tomato, pepper).

B.1.2 The di.ploid or polyploidsystems (usually buds, embryos, shoot tips, but
potentially also protoplasts, cells, tissues, calli etc.) require, in the case of
homozygous material of seed propagated species, for selection of recessive
mutations a sexual. cycle and screening in subsequent generations. Therefore,
in-vitro mutagenesis offers little advantage. Screening for recessive mutations,
however, may be performed already in the M 1 generation if the original, material
is heterozygous. TIhis is particularly the case in most vegetatively propagated
crops like banana, cassava, yam, potato, sweet potato, as well as many fruit
crops and ornamentals. Here irn-vitro mutagenesis offers many advantages ranging
from mutagen treatment of large cell numbers, possible test tube selection,
non--chimeric regeneration for in-vivo selection to quick clonal mass propagation
of promising mutants for field evaluation and subsequent distribution to
farmers. The target of an in-vitro mutagenic treatment may be a single cell or a
multicellular entity. In the latter case one must be aware of possible chimerism
and may have to dissolve the chimeric structure of regenerant M 1 plants.
Somatic embryogenesis generally has single cell origin so that M1 plants
regenerated in this way are supposed to be homohistonts.

B.1,3 The cytoplasm

In-viitro culture methods may be necessity when the trait needed is
cytoplasmically inherited. Some resistances to pathotoxins were shown to be
based upon mitochondrial characteristics. Mitochondrial DRNIA will likely respond
to the same mutagenic treatments as chromosomal DNA, but the selection of mutated
mitochondria by application of toxins will require in-vitro techniques. Research
should be encouraged on the role of mitochondrial plasmids, their interaction
with nuclear DNA and on practical aspects of cytoplasmic mutagenesis. (Ref.
Report of FAO/IAEA Consultants Meeting on the Induction of Mutation in
Extranuclear Hereditary Cell Elements, STI/PUB/591, IAEA, Vienna 1981).

B.2 Choice of_mutagens

Basically, both physical or chemical mutagens can be used, the choice
depending usually upon availability. The spectrum of mutation may differ, but
nothing specifically is known in this respect concerning disease resistance
mutations. Using several different mutagens will ascertain a wide spectrum of
mutations. Both categories of mutagens offer advantages and disadvantages.

B.2.1 Physical mutagens

Ionizing radiations like X-rays, gamma rays, fast neutrons, but also UV.

14
.Advantages:
Good dosimetry and reasonable reproducibil.ity. High and uniform penetration of
multicellular systems particularly by gamma rays.

Disadvantaes.. :
Radiation source required.
Unlike seeds, culture]. flasks cannot easily be shipped long distances to the site
of the source.
Simultaneous induction of chromosomal and gene mutations.
UV can only be used for single cell layers.

B.2.2 Chemical mutagens

Most commonly used are ethyl-nitroso.urea (EIUH), methyl-nitroso--urea (MNH),
ethyl-methane--sulphonate (EMS), di-ethylsulphate (DES), ethyl.eneiLmine (El),
sodium azide (SA), ethidium bromide (EB).

Advan
_t.,ages:
Point mutations may predominate for some of the chemical mutagens. High mutation
rates can be obtained from certain mutagens.

Disadv ant.ges~:
Difficulties in effective dose assessment and reproducibility.
Non uniform penetration of multicellular systems.
Problems with breakdwon compounds of the mutagens applied, as they cannot easily
be removed.
Several mutagenic chemicals are cancerogenic.

Compared to seed or dormant bud mutagenesis, the cells of in-vitro

cultivated targets generally lack synchronization as to the cell cycle stage,
which limits the reproducibility of experiments. Means for synchronization may
be found. Many relevant aspects of mutation breeding are dealt with in the
"Manual on Mutation Breeding" and other books published by IAEA (see
bibliography). However, little is known so far as to the optimal doses, time of
treatment, physiological conditions of objects for in-vitro mutagenesis.
Research in this respect is needed and dissemination of results would seem an
appropriate task for the Joint FAO/IAEA Division.

C. I:I--V'ITRO SELEC'"'TON OF' DISEASE RESI:S'TANT PLANTS

n-vit.ro selection may be applied irrespective of the source of genetic

variation. (e.g. "somaclonal variation" or induced mutations). Also the
distinction between monogenic or polygenic inheritance is rather irrelevant,
since at the selection stage only the phenotype difference between susceptible
and resistant is important. (The genetic classification is a matter of later
concern, more critical is the verification of in--vitro selected resistance under
field conditions). However, in-vitro culture conditions must be suitable to
screen for resistance to the various plant pathogens. It would seem most
efficient if such screening could be performed immediately after the generation
of genetic variation, e.g. protoplasts subjected to mutagens could subsequently
be plated on toxin-containing media. Survivors resistant to the toxin should be
regenerated and tested for resistance to the pathogen as such.

It is one of the main attractions of in-vitro techniques that they can

provide a scale for selection that would be difficult, if not impossible, to
achieve in other ways. For example, a potato breeder may screen in the
greenhouse or the field perhaps 50,000 to 100,000 seedlings per year for disease
resistance. On the other hand, 20 million protoplasts obtained from only 1 g of

15
leaf issue, could easily be cultured in a laboratory. If a given beneficial
mutation such as disease resistance had a probability of 10--O,then perhaps one
such mutant would occur among the 100,000 seedlings. However, 200 mutant plants
might be obtained from protoplast culture screening.

Thus, a serious consideration of the potential of in-.vitro selection, both

with respect to various pathogen types and to various host plant materials is
clearly warranted.

C.1 The use of various tissue culture systems for selectirng disease resistant
p.l. .Ants.
Several systems of different complexity can be subjected to screening.
Single cells without walls (protoplasts) or with walls (cells), multicellular
suspension cultures, callus cultures, cultured anthers or microspores,
regenerated plantlets and even intact plants may be useable in such selection
procedures.

C.l.l Protoplast. cultures

Protoplasts can be adequate for screening for toxinr resistance. If
resistance is not the result of cell wall impermeability but really of
insensitivity to the toxin (inability for certain reactions or detoxification by
certain reactions), then such screens can be quite effective. Much information
is needed, however, on the biochemical and physiological effects of toxins, on
the required purification of the toxic materials and particularly on the
importance of such toxins in host--pathogenl interactions.

Interact:i.on of protoplasts with intact pathogens may differ from that of

intact plants. Whereas there are indications that protoplasts resistant to virus
infection yield plants that are resistant too, particularly 'viruses often infect
protoplasts which are derived from resistance plants.

On the other hand, protoplasts may not be affected substantially by viruses,

and detection of infection may only be possible by destructive procedures such as
staining with flourescent antibodies not allowing any selection with subsequent
regneration. Bacterial and fungal infections, where pathotdxins (either
host-specific or host non-specific) are not involved, may not be usable as
protoplast screens. Often protoplasts from resistant and susceptible plants are
killed at equally rapid rates. For selection of resistance against bacterial
diseases, perhaps in some cases changes in bacterial attachment to cells could be
observed and used. Resistance against certain bacteria, where the bacterium
doesn't cause rapid death of protoplasts, may be screened for by direct mixing of
viable plant and bacterial cells.

Recently it has been suggested that protoplasts may redpond to

fungus-derived elicitors by producing phytoalexins and that these responses are
under genetic control. Thus screening for resistance at the protoplast level in
the near future may involve the use of pathogen components (which could more
easily be standardized). When molecular aspects of host--pathogen interactions
become clearer, more effective screens with protoplasts can probably be devised.

C.1.2 Cell suspension cultures

Cell]. suspension cultures have been used for studies of metabolic responses
to pathogens and as systems to give cell lines that are resistant to various
toxin analogs of amino acids. However, few rigorous studies have been made using
these as means to screen for disease resistant plants. Considerable research is
needed e.g. on the means that host cells use to recognize pathogens, and vice
versa. Such information could provide a basis for devising screening procedures.

16
C.1.3 Callus cultures
Callus cul.tures have been used already to select for resistant plants.
Toxic crude filtrates of fungal broth killed many cells but some survivors showed
enhanced resistance when intact plants derived from the cultures were inocul.ated
with the fungus. Reaction of callus tissues to inoculation with living fungi has
also been exemplified. When tobacco leaves were inoculated with virus and callus
derived from the leaves were selected for resistance, the resistant mater:i.al
appeared green, the susceptible yellow. More studies wi.th viruses producing good
visible responses such as these are desirable.

In:..vitro screening for resistance to bacteria can be difficult if no toxin

production is involved in pathogenesis. Often bacteria colonize the tissues and
media very quickly; structural requirements for recogniz:ing differences in
pathogenesis may not be fulfilled in callus cultures.

Finally, expression of resistance in callus tissue may be quantitative and

in that case difficult to identify by assessing a slightly more or less vigorous
growth of pathogens. More research would be needed as to thie expression of
levels of resistance against various pathogens in callus cultures.

C.1. 4 Embryo cultures

Embryos in culture might be useful in screens by virtue of their organized
structure. For any selected embryo, there is also a greater likelihood of
regenerating intact whole plants. By somatic embryogenesis, large numbers of
embryos for screening could be generated. It seems feasible for example, to have
embryos of Brassica or Hyoscyamus plated on toxin containing med:i.a or be
challenged by the fungus itself. Susceptibility to loose smut could be detected
using in-vitro inoculation of young embryos.

C.1.5 Anther cultures

Anther and microspore cultures seem particularly attractive for induced
mutant screening. Dominant as well as recessive traits for resistance might be
shown immediately and doubl.ing genomes wi.l then give homozygous breedi.ng lines.
One still needs, however, reliable methods for obtaining embryogenic microspores
of many crop plants.

C.1.6 Whole.plant in-vitro cultures

In-vitro screening methods can also be applied to intact in-vitro cultured
plants using the opportunity of standardization, of total environmental control
and of environmental manipulation for accentuating disease reactions. In
particularly difficult cases, like long-generation-cycle host plants and long
latent-infection-periods, intact plant in-vitro culture might open ways to early
selection and thus represent a real great advantage. By examining changes in
properties of diseased versus non-diseased susceptible plants, "early screening"
could possibly be further improved. Perhaps changes in conductivity could
indicate the state of plant infection, and thus differences in susceptibility.

C.2 General recommendations on in-vitro selection for disease

C.2.1 Fungal toxins at present are the easiest applicable screens for
resistance. Therefore, where possible, determine if a toxin is involved in
pathogenesis and then use it in screening, Whether toxin selection leads to
plants with satisfactory resistance remains to be seen.

C.2.2 Careful study of host, pathogen and environmental parameters in

pathogenesis should be made prior to attempts of designing a screen. Resistance
may be needed at particular stages of host's life cycle. It may or may not be
expressed at other stages. Biotrophs and necrotrophs will behave quite
differently in culture.

17
00

CASES OF:' SUCCESSFUL SELECTION OF RESISTANCE PLAN"S OR CE.L.. LINES USING IN--VI'"RO CUL.TURES

PATHOGEN HOST PLANT RESISTANCE TO LEVEL OF' AUTHOR

ANALYSIS

Helminthosporium maydis Zea mays toxins c Gengenbach et al. 1977

Brettel et al. 1979

Phoma lingam Brassica napus toxin, germinating conidia c Sacristan 1982

Phytophthora infestans Solanum tuberosum culture filtrate, infection a Behnke 1979
Alternaria solani Solanum tuberosum toxin, infection a Matern et al. 1978
Pseudomonas tabaci Nicotiana tabacum toxin c Carlson 1973
Sclerospora sacchari Saccharum officinarum infection a Heinz et al. 1977
Helminthosporium sacchari Saccharum officinarum infection a Heinz et al. 1977
Fusarium oxysporum Lycopersicon esculentum culture filtrate b Scala et al. 1984
Fusarium oxysporum Solanum tuberosum culture filtrate b Behnke 1980
Pseudomonas syringae Nicotiana tabacum toxin a Thanutong et al. 1983
Alternaria alternata Nicotiana tabacum toxin a Thanutong et al. 1983
Fusarium oxysporum Medicago sativa culture filtrate a Hartman et al. 1984
Helminthosporium sativum Triticum aestivum culture filtrate, toxins a Dutrecq 1981.

__..__ ._ ___
_ _ _ . ______.....................................................
. .. __ ........................................................................................

a = Cell lines
b = Regenerated plants
c = Progeny of regenerated plants
C.2.3 Attempts to determine correlations between properties of the tissue culture
system and resistance under field conditions should be made. Studies on means to
enhance disease resistance expression in tissue culture should be conducted. It
would be useful to carefully examine many tissue culture/pathogen systems to
obtain the best criteria for disease resistance differences.

C.2.4 Too little information is currently available on in-vitro culture screening

for nematode, viroid or mycoplasma resistances. Most work with nematodes appears
to have been done using intact plants, because there exist specific structural.
requirements for infection, but also root tip cultures have been used.

C.2.5 It would appear most useful to choose a selection system that would lead in
the shortest and most direct way to intact plants. Regeneration from rl.-vi.tr_
o
cultures still presents problems for many important plant species and therefore
is a major handicap of in-vitro selection for disease resistance.

C.2.6 There is much need for information on the genetics of host-pathogen

interactions and on the components of the interactions. With this information,
screens for the presence or absence of specific molecules involved and for the
operation of particular genes will facilitate screening at the single cell level,
the level that will allow to handle a maximum number of individuals with the
minimum of effort.

D. SUCCESSES WITHI TISSUE CULTURE EFFORTS FOR IMPROVED DISEASE RESISTANT PLANTS.

First, in the early seventies Carlson suggested that tissue culture methods
could give disease resistance and a few years later this was proven by obtaining
maize lines resistant to Helminthosporiu_ maydis. Screening for resistance was
done at the callus level, then plants were regenerated and subjected to genetic
analysis and evaluation. Good examples of the potential of in-vitro disease
resistance breeding are the recessive resistance against barley yellow mosaic
virus found in self--pollinating barley and the dominant resistance to Pho!ma
liqngam in cross-pollinating Brassica najus.In the meantime, a number of other
examples have been published. Some are presented in the table on page 18.

E. CROP--PATHOGEN SYSTEMS W-IERE INI--VITRO TECHNIQUES MAY BE APPROPRIATE TO USE

The choice of the host/parasite systems where in-vitro techniques could be

used as an aid to disease resistance breeding has to be made taking into
consideration both the crop and the pathogen as mentioned before. The
investigator should have a thorough knowledge of the interaction between the
host, the pathogen and the environment. Some more general guidelines given here
are supplemented by specific examples in Chapter F.1.

E.1 The crops

Crops which are normally propagated vegetatively, or which have a long
generation time, are obvious prime candidates for the use of in.-vi.tro culture
techniques. There are several reasons for this: commercial cultivars of these
crops generally are expected to have very homogeneous phenotypes. On the other
hand, their level of heterozygosity is generally very high. It is therefore
difficult to introduce alien resistance gene or even to uncover a recessive
resistance gene possibly present in a heterozygous clone, without disrupting the
complex valuable type of an existing cultivar.
Moreover, in several instances, crosses both within and between species are
difficult due to a non-flowering habit, incompatibility barriers, and different

19
ploidy levels. Reconstruction of a cultivar after a cross is extremely
difficult. TIhe time required for reselection using traditional tools may be
quite long and the development of a resistant variety may not be fast enough to
cope with the urgency of a disease problem and the evolutionary capacity of a
pathogen. For these reasons, disease resistance breeding in this group of plants
is well advised to look for spontaneous or induced mutations. But even for the
latter, their Frequency requires large scale screening by effective techniques.
Iln-vitro cultures promise such possibilities.

The obstacles to traditional selection for disease resistance increase with

the length of the plant generation time and are almost unsurnountable in cases
where vegetative propagation is obligate. It is rather unfortunate that in-vitro
culture techniques for these groups of plants, with a few notable exceptions (as
we shall see later), are far from being established. In case of urgency,
however, the methodology may only be a matter of time, provided that general
feasibility criteria have been fulfilled. To increase genetic variability may be
a crucial point in cases where vegetative propagation has restricted or
eliminated previously existent genetic variability. Where cross breeding is
possible, the introduction of al.ien resistance genes via intra- and interspecific
crosses may be facilitated by in-vitro fertilization, embryo rescue, the use of
irradiated protoplasts as donors in fusion or by donating DI:A from in-vitro
germinating pollen.

In the case of long-generation-time seed propagated plants the breeder may

also be lacking genetic variability, which could be augmented by mutagenesis and
selected usi in-gvitro culture. But the breeder will face segregation, and
therefore has to screen large populations for the most desired recombinant.
Anther cultures, as far as available, could provide a real help for quickly
developing improved cultivars or populations once the desired recombinants have
been identified. In the case of short-generation-time seed propagated crops, the
use of in_-vitro cultures may be of only subsidiary value, at least when suitabl.e
sources of disease resistance genes are available. Direct in-.vitro selection for,
disease resistance does not seem generally recommendable in this case, even if
better selection techniques are becoming available. But in-_vitro systems could
of course be helpful if used For the increase of genetic variability in
self-pollinated crops, for overcoming incompatibility barriers through one of the
already mentioned techniques and as an aid to establish homozygosity via haploids.

Two general. considerations may be added which should be taken into account
in deciding whether a disease resistance breeding programme involving in-v.itro
techniques should be started. Priority should be given to plants for which
reliable in-vitro techniques are already available. When this is not yet the
case, preference should be given to species which belong to groups where at least
good tissue culture model systems have been developed. For example, dicots at
the present state of the art are easier to handle than monocots and gymnosperms,
Secondly, it should be obvious that progress in breeding for disease resistance
using in-vitro cultures will be more likely if sound breeding experiences as well
as a good knowledge of the etiology of the disease in question exist.

E.2 Thepathogen
To judge whether it would be appropriate to embark on the use of in-vitro
procedures in breeding for resistance against a particular pathogen, the
following questions should be considered:
(a) Is the life cycle of the pathogen, its means of infection and the cause of
disease sufficiently understood? Which is the most infectious spore stage?
What is the survival mechanism? Are there alternative hosts? Is a
particular environment required for infection? Does the pathogen produce a
toxin? Is toxin production a means of infection or the result of the
established infection? In which organs and stage of host plant development

20
 is the disease expressed? Are there several organs or stages where the
disease is detectable?

It will be crucial to have this information for devising in-vitro selection

procedures and therefore it is very important to have a plant pathologist's
co-operation.

(b) Is it likely possible to devise an in-vitro screening procedure7 The

prediction will be difficult because the existing evidence and precedents
are limited. The following guidelines, however, may be useful:
If the pathogen is a necrotrophic fungus or bacterium, it is possible that a
toxin (or toxins) is involved in pathogenesis. There is reasonably good
evidence that screening in-vitro using crude toxin preparations or culture
filtrates is feasible. If the pathogen is not producing a toxin, it will
probably be necessary to screen by using whole organisms. A major practical
difficulty in this procedure would be excessive growth of the pathogen on
the culture medium as such.

If the pathogen is a biotrophic fungus or a bacterium, or a nematode, it

will probably be necessary to screen by using the whole organisms. There
are strong indications from published work that the expression of resistance
to all these groups of pathogens in culture is very variable and depends
upon the nature of the culture environment. Dual cultures would be valuable
for maintaining pathogen collections and as sources of aseptic inoculum.

- The type of resistance searched for will have a strong bearing on the
probability of success. For example, structural barriers are less likely to
be expressed in cell or tissue culture than physiological mechanisms, and
partial resistance is likely to be more difficult to identify in-vitro than
complete resistance or hypersensitivity.

If the pathogen is a virus or mycoplasma it will most likely be necessary to

use the disease causing agent as such. Means for detection of
resistance/susceptibility must be developed by strong research efforts.

F. SPECIFIC EXAMPLES AS TO WHERE AND WHEN TO USE IN-VITRO TECHNIQUES FOR

IMPROVING DISEASE RESISTANCE

F.1. Crops that are usually vegetatively propagated

F.1.1 Sugarcane
The sugarcane breeding system is commonly based upon hybridization followed
by vegetative propagation through stem cuttings and subsequent selection of
clones meeting agronomic and industrial requirements. However, in some breeding
stations mutation induction as well as in-vitro culture have already been used
successfully for new variety development.

Disease problems are many and losses are high due to smut, rust, Fusarium
sp., Helminthosporium sp., ratoon stunt (virus), Fiji diseases (virus) etc.
Breeding for resistance is recognized as the most desirable means of controlling
crop losses, but traditional breeding methods (incl. distant hybridization and
clonal selection) are hampered by space, time, labour and other requirements.

Present role of in-vitro techniques

Plant regeneration (via organogenesis and somatic embryogenesis) is well
established from different explants including cell and protoplast cultures.
However, dedifferentiated cell cultures usually possess high genetic instability

21
(especially chromosomal irregularities). Somatic variation in regenerated plants
appears to be a useful source of genetic variability. Induced mutations may
supplement this genetic variability.

In breeding for disease resistance, in--vitro cultures currently could serve

(a) as a quick tool for selection against fungal pathogens producing
toxins, e.g. HeJ.minthospo.rium_
(b) as a source of genetic variability
(c) for rapid propagation of selected types by using meristem culture.

Among limitations of the application of in-vitro techniques for disease

resistance breeding of sugarcane are the non-controllable genetic variability and
abnormal metabolism of cultured cells which might bias in-vitro selection
procedures.

Prospects for the future

In--vitro technology has been used since 1960 in sugarcane and must be
considered as a regular component of sugarcane breeding. Research on somatic and
induced genetic variability however is still needed. Protoplast technology
should be developed for use in solving important breeding problems. In-vitro
technology will certainly be useful for germ plasm preservation
(cryopreservation) and for international germ plasm distribution,

F.1.2 Banana
Commercial bananas and plantains are vegetatively propagated
parthenocarpic triploids. Genetic variability is very narrow in cultivated
types. In commercial banana plantations mainly two groups of clones are found
i.e. Gros Michel and Cavendish.

Disease problems
Panama disease caused by Fus.arium oxysporum f.sp, cubense is a serious
problem in Gross Michel clones. The major limitations of the Cavendish clones on
the other hand are their susceptibility to the Yellow Sigatokka or banana leaf
spot disease caused by My.co.sehaerella musicola and to the black leaf streak or
Black Sigatoka disease caused by Mycoshaerella fij.iensis var. diformis. Other
serious problems are bunchy top virus, Moko disease (Pseudonionas solanacearum)
and nematodes (Radopgholus similis).

Breeding for disease resistance

Germ plasm for disease resistance is available among diploid wild species
but their very inferior bunch characteristics have handicapped the synthesis of
resistant clones with good commercial characteristic, The contemporary breeding
strategy is based on the development of acceptable banana diploids from crosses
with disease resistant wild species which then could be crossed with tetraploids
for obtaining synthetic triploids. Mutation breeding is considered a tool to
improve currently cultivated clones without disrupting their commercially
valuable genetic structure.

Role of in-vitro techniques

The established steps of banana in-vitro culture are:
(1) banana shoot tip culture
(2) stimulation of multi-shoot formation from adventitious buds
(3) shoot rooting and plantlet development.

Prospects for the future

In-vitro cultures must be considered as a future regular component of banana
breeding. This technology will certainly become useful also for germ plasm
storage and for international germ plasm distribution. In-viitro cultures as a

22
means of clonal propagation will have wide application for both bananas and
plantains, The production of regenerating callus and morphogenetically competent
cell suspensions will be the major requirement for successful application of
in-vitro technology, for obtaining new genetic variability and for making use of
in-vitro selection procedures.

F.2. Crops that are usually vegetatively propagated but produce also seeds

F.2.1 Cassava
This is one of the most important tropical tuber crops used as a staple
food in many developing countries but also as an export commodity for animal
feed. It is vegetativel.y propagated by stem cuttings. The main sources of
genetic variability are cultivars used in crosses. Genetic variability appears
to be fairly broad for' various agronomically useful characteristics, but limited
for disease resistance. The main disease problems are caused by virus (Cassava
mosaic virus) and bacteria (bacterial blight).

.Breeding
. for disease resistance
Sources of virus resistance are limited. Selection of virus symptom free
plants and the use of thermotherapy are practical ways of providing healthy
planting material, but this is only a short term solution. Shoot tip culture can
be used for production of virus free material. Screening of germ plasm for
resistance or tolerance to bacterial and virus diseases is urgently recommended.

Present role of in-vitro techniques

Shoot tip culture and in-.vitro multiplication of cuttings are established
procedures in certain clones. High genetic stability is associated with these
in--vitro systems. However, no established method for plant regeneration from
cell and callus culture is available.

Prospects for the future

Virologica]. research should be strengthened, particularly the development of
indexing techniques to identify virus free plants in the field and to check
re-infection of clean stocks. In-vitro technology will certainly be useful for
germ plasm preservation and for international germ plasm distribution. Research
on using shoot tip culture technology for induction of mutations and for in-vitro
selection is advisable. Development of procedures of plant regeneration from
single cells is urgent. This will offer the scope for in--vitro selection against
bacterial, fungal and virus diseases.

F.3 Seed propatedpp crops with a_very longeneration time

F.3.1 Cocoa
Cocoa is among the most important export crops of West Africa and, to a
lesser extent, of many South American, Caribbean and South East Asian countries.
Swollen shoot virus is in West Africa perhaps the most important disase of cocoa
and the crop losses caused by this disease are dramatic, Other important
diseases include black pod (Ph^y.to_.~hthora spp.) and witches' broom (Crinipellis
perniciosa).

Breeding systems
Manual pollination for the production of seed is a common practice.
Seedlings of evaluated hybrids are supplied to farmers from seed gardens.
However, the period between planting and fruiting ranges from 3 years for
improved hybrids to about 10 years for other stocks. Vegetative propagation has
been developed, but so far has been used mainly for experimental purposes. The
genetic base of the cultivated cocoas unfortunately is narrow, and progress in

23
the breeding programme has relied on the introduction of germ plasm from the
forests of South America.

Breedn.in. ...fo.r disease .resistance

Breeding for swollen shoot and black pod resistance has been going on for
many years. For swollen shoot resistance, a screening technique using seedlings
has been developed which is based on the inoculation of seeds using purified
virus or virus infected vectors. Appropriate methods for initial screening have
also been developed for fungal and nematode diseases. These screening methods
are, however, time consuming. Recently the early detection of swollen shoot
virus by ELISA has been developed, and its inclusion in the breeding programmes
will be a useful asset.

Present role of in-vitro techno9lo9

In--vitro technology has, so far, not been extensively used in practice but
some tissue culture methods are available, especially shoot tip culture.
In--vitro culture has, so far, not been used for disease resistance breeding.
Attempts are now being made to induce mutations by irradiation of vegetative
materials and pollen,-so as to produce genetic variability for disease reaction,
and there are plans to combine mutation induction with in-viLtro culture.

Pr.ospects for the Future

Shoot tip culture to produce virus free plants should be an essential
supplement to traditional breeding, to be able to retain precious germplasm in
the field. For breeding purposes, tissue culture technology may also help in
speeding up traditional breeding programmes, eventually permitting early and
quick screening for disease resistance.

When an in-vitro system for clonal progation is established it could be used

for mutation breeding by irradiating the initial explants (shoot tips) and
subsequently isolating non-chimeric material to be screened for disease resistant
mutants.

A recently initiatedprogramme of inducing mutations in cocoa to introduce

genetic variability into breeding materials should be vigorously pursued. Such
variability is desired not only for disease resistance but also for other useful
characters such as drought resistance, dwarfing of plants, and for overcoming
genetic incompatibility.

F.4 Seedpraa_ ropsaa

w__ith a short_
eneration time

F.4.1 Soybean
Soybean is considered here as an example of the large group of important
crop plants belonging to the family of Leuminose. It is one of the major
protein food and feed crops in the world. Its importance as vegetable oil and
industrial crop is also well recognized. It is an autogamous species. Many
developing countries such as Indonesia, India and Thailand use soybean as a major
protein food source, but production is usually unsatisfactory.

Diseas.e problems
Soybean rust caused by Phakopsori
a pachyrhizi is a serious disease limiting
soybean production in the tropics. This disease may become a potential threat
for temperate climate soybean growing regions as well. A number of virus
diseases such as soybean mosaic, yellow mosaic and black eye cowpea mosaic are
also serious problems.

24
B reediln. for disease. resistance
The entire world soybean germ p]asm collection has been screened for soybean
rust and virus resistances. However, for soybean rust only a limited level of
resistance is available. Wild perennial Glycine species seem to include types
possessing better soybean rust resistance, but most are polyploids and cannot
easily be crossed due to hybrid breakdown. Resistances to at least some of the
virus diseases have been identified and are used in conventional breeding.

.. .. . . .... ..
......
................. . ......................
So far a wild perelnnial, Glycine canescens, and one particular variety of G.
max (cultivated soybean) have been successfully regenerated through. organogenesis
and embryogenesis using unique media and specialized procedures. In G,
canescer!s, the regeneration occurred after two years in culture. Add i :i.onral
research is needed to understand the finer detail.s involved in problems of
in-vitro culture of soybean and other leguminous crop plants.

Embryo culture has been successfully used to rescue hybrids between G. max
and G. canescens. This method as well as other ini-vitro culture techniques will
hopefully pave the way for interspecific hybridization to transfer soybean rust
resistance from other species.

Future prospects
Inr!-..vitro culture methods so far are not well established for grain legumes.
It can be expected that current large efforts will soon lead to advances in view
of the unique role legumes play in symbiotic fixation of atmospheric nitrogen.
Without such advances in in.-vitr.o technology, there are little prospects for
using in-vitro cultures in breeding of grain legumes for disease resistance.

F.4.2 Rice
Rice is considered as an example of a cereal crop in which good
conventional breeding procedures are well established, and rice breeding has led
to major advances in productivity as well as disease resistance. Nevertheless,
rice breeders are eager to make use of in-vit!ro culture procedures, especially
anther cultures, for the purpose of rapidly fixing recombinants and obtainig
homozygosity. However, such methods are not yet applicable to all rice types and
to other important cereals. Efforts should therefore be exerted to develop
efficient haploid production in recalcitrant genotypes of rice (indica) and other
cereals.

Futu.re _.Prospects
The use of in--vitro cultures in rice breeding for improving disease
resistance may not be limited to the support of classical cross breeding via
anther culture. There are many disease and insect pest problems in rice and the
possibility for screening huge populations under in-vitro conditions may provide
a chance for new genetic resources. The same would apply for other cereals.

G. THE USE OF IN-VITRO TECHNIQUES FOR STUDIES OF HOST - PARASITE INMERACTI:ONS

In-vitro culture techniques may have an important role to play in

elucidating host - parasite interactions. The results of such studies could be
of direct value in the development of practical procedures for the use of
in-vitro techniques in disease resistance breeding, but in many instances would
lead to fundamental knowledge useful in advancing current concepts of plant
protection.

G.1 Research with pathoge nic fungi, bacteria and nematodes

Several topics of research may be identified in which in-vitro cultures, by
providing a controlled experimental systems, could be an relevant aid:

25
 - Mutual recognition of host and parasite.
- Resistance reactions (phytoalexiris, hypersensitivity) vs. tolerance.
- Tumour physiology.
- The mode of action (interaction) of pathotoxins and enzymes.
- Systemic infections.
- utrient and signal exchange between host and parasite.
- Cross protection.
-Sexual and asexual sporulation.
Evolutionary capacity of pathogens.

Three of these topics have particular relevance to the development of

methods for in-vitro screening: studies about recognition, about resistance
reactions and about the mode of action of pathotoxins. Most useful. would also be
progress in the in-vitro culture of obligate parasites. This could be
advantageous with regard to providing identified and stanldardized inoculum and
would allow maintenance as well as transport of collections of such pathogens.

Despite the potential importance, studies of the interaction between

cultured plant material and pathogenic fungi, bacteria and nematodes have so far
been rather limited. In the case of bacteria, most attention has been focussed
on the crown gall syndrome (Aqrobactterium
. tumefac:ie.ns). In the case of fungi and
nematodes, the dual culture of obligate or semiobligate parasites as well as
their hosts and the mode of action of pathotoxins have received most attention.
lhe well defined model. system of tobacco and Phytophthora pArasitica nicotianae
is available for resistance studies, but only few researchers have taken
advantage of the possibilities. Too little attention has been devoted to
studying the effects of cell free components of pathogens on the physiology of
host cells and tissues, which should allow to dissect complex interactions.

There are three probable reasons for this lack of efforts. Firstly, plant
pathologists seem to have been unwilling to explore the possibilities offered by
in-vitro techniques, perhaps partly through prejudice and partly through
ignorance of what is available. Second, by an irony, the lack of understanding
of the genetical and biochemical basis of many aspects of the interaction between
plants and their pathogens may have prevented recognition of both the strengths
and the weaknesses of in-vitro systems and, therefore, the correct identification
of those circumstances under which they could be of value. Finally, in-v_itro
culture technology itself, despite its massive research effort, still has many
unsolved problems. Those most relevant to studies of host - parasite interaction
(and, incidentally, to the use of in-vitro culture in plant breeding) are: (1)
genetic instability; (2) abnormal and stress metabolism in culture; and (3) the
control of differentiation and regeneration.

G.2. Research with viruses

Plant virology provides a marked contrast to the above, The development of
meristem culture techniques during the fifties led to the perfection of methods
for the production of virus free clones of a wide range of vegetatively
propagated crop and ornamental species. The techniques are still of great
importance. Later, in the sixties, the development of methods for culturing and
infecting plant protoplasts revolutionised many aspects of plant virology, by
opening up new avenues of research not possible with intact plants. The
following topics are currently under active investigation using protoplast
systems:
- The infection processes.
-Replication and the expression of the viral genome
- Genetic variation
- Cross protection and other virus interactions.
- Resistance mechanisms
- DNA-viruses as tools for genetic modification of plants

26
 The first five topics have relevance for the development of in-vitro
screening procedures but also for classical disease resistance breeding. The
last topic may become relevant for the development of new in-vitro plant breeding
procedures.

The momentum of the research on viruses is considerable, and there is every

reason to suppose that it will continue. It should be strongly encouraged,
Important challenges exist for the future, especially the solution of the problem
of metabolic stress imposed by the artificial environment of protoplast culture,
the understanding of how the events in isolated protoplasts are related to those
in intact tissues, and the uncovering of the interrelationship between cell
growth and virus multiplication.

G.3 Recommendation
Studies of the interaction between plant pathogens and their hosts using
in-vitro techniques should be strongly encouraged and supported because they will
form the basis for the development of new, reliable and efficient in-vitro
screening procedures. But such research will also produce fundamental knowledge
about plant diseases, contribute to the understanding of resistance mechanisms
and thus help in designing more effective and durable measures for controlling
crop losses.

27
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..-.-.- . oo..
........._......

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33
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36
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DUTRECQ, A., Studies of the effect of toxic preparations of Helminthosporium

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37
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Virus

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39
 LIST OF PARTICIPANTS AND OBSERVERS

Bieberach, C. Institute de Investigacion Agropecuaria

de Panama
Panama 6A
Panama

Buiatti, M. Istituti di Anatomia Comparata

Biologia Generale e Genetica
Via Romana 17
Firenze
Italy

Dutrecq, A. Faculte des Sciences Agronomiques

Lab. de Pathologie Vegetale
13 Av. Marechal Juin
B-5800 Gembloux
Belgium

Guanren, Xu Institute for Application of Atomic

Energy
Chinese Academy of Agricultural Sciences
Beijing
People's Republic of China

Helgeson, J.P. Dept. of Plant Pathology and

Horticulture
University of Wisconsin
Madison WI 53706
USA

Ingram, D.S. Botany School

Downing Street
Cambridge CB2 3EA
United Kingdom

Kiraly, Z. Research Institute for Plant

Protection
P.O. Box 102
H-1525 Budapest
Hungary

Owusu, G.K. Cocoa Research Institute

P.O. Box 8
Tafo, Akim
Ghana

Menten, O.M. Universidade de Sao Paulo

Centro de Energia Nuclear na Agricultura (CENA)
Cx/P 96
13400 Piracicaba SP
Brazil

Peacock, J. CSIRO
Division of Plant Industry
P.O. Box 1600
Canberra City, A.C.T. 2601
Australia

41
Sacristan, M.D. Institut fur Angewandte
Genetik der Universitat
1000 Berlin 33 (West)
Federal Republic of Germany

Scala, A. Istituto di Anatomia Comparata

Biologia Generale e Genetica
Via Romana 17
Firence
Italy

Shanmugasundaram, S. The Asian Vegetable Research and

Development Centre (AVRDC)
P.O. Box 42, Shanhua, Tainan 741
China

Takebe, I. Department of Biology

School of Science
Nagoya University
Furo-cho, Chikusaku, Nagoya
464 Japan

Wenzel, G. Biol. Bundesanstalt

Institut f. Resistenzgenetik
D-8059 Grinbach b. Munchen
Federal Republic of Germany

Zadoks, J.C. Institute of Phytopathology

Binnenhaven 9
Wageningen
The Netherlands

Joint FAO/IAEA Division P.O. Box 100, A-1400 Vienna (Austria)

SigurbjiJrnsson, B. Director

Atkins, C. Soil Fertility, Irrigation and

Crop Production Section

Donini, B. Plant Breeding and Genetics Section

Maluszynski, M.

Division of Research IAEA, P.O. Box 100

and Laboratories A-1400 Vienna (Austria)
Seibersdorf Laboratory

Brunner, H.
Daskalov, S.
Hermelin, T.
Novak, F.

Scientific Secretary

Micke, A. Joint FAO/IAEA Division

Plant Breeding and Genetics Section

42
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