Plant biotechnology for crop improvement

Pergamon Biotechnology Advances, Vol. 13, No. 4, pp. 673-693, 1995

Copyright © 1995 Elsevier Science Inc. Printed in Great Britain. All fights reserved

0734-9750/95 $29.00 + .00

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PLANT BIOTECHNOLOGY FOR CROP IMPROVEMENT

K.P. PAULS

Department of Crop Science, University of Guelph, Guelph, Ontario, Canada, N1G 2JV1

ABSTRACT

The typical crop improvement cycle takes 10-15 years to complete and includes germplasm

manipulations, genotype selection and stabilization, variety testing, variety increase, proprietary

protection and crop production stages. Plant tissue culture and genetic engineering procedures

that form the basis of plant biotechnology can contribute to most of these crop improvement

stages. This review provides an overview of the opportunities presented by the integration of

plant biotechnology into plant improvement efforts and raises some of the societal issues that

need to be considered in their application.

Key Words: Plant bioteehnology, crop improvement, genetic engineering, plant tissue culture.

INTRODUCTION

Humans began to modify the characteristics of plants used for food and fibre

approximately ten to twenty thousand years ago. Even primitive seeding, cultivating, harvesting

and storing practices would have exerted selection pressures on those plant species which

became domesticated that were different from the pressures their progenitors encountered in the

wild. Over time, but particularly in the last 150 years, plant breeding has developed into a

complex discipline that now incorporates information from many branches of science and

mathematics. The most recent development is the utilization of biotechnology for plant

improvement (Ratner, 1989).

Plant biotechnology can be defined as the application of tissue culture and molecular

genetics to develop or produce a commodity from plants. Tissue culture refers to the 673

674 K.P. PAULS

maintenance and propagation of plant parts (as small as a single cell) in biologically pure

(axenic) and controlled environments (Fig. 1; Evans et al., 1983; Vasil, 1984). Molecular

~ . . ~ . ~ ~ plant explant mature plant hUl~ujedewith plant

a,,us

"germination" ~ P s r ~ l l ~ P ~ ~ ~ "

embryos ~ embryogenic cells collected on mesh ~ ~) hormone t ~ ~ L ~v( [ treatment and ~ somatic / ~'~ ~ / /~] / desiccation embryos ¢

suspension culture

large cell clumps removed by sieving

embryogenic callus

Figure 1. Plant tissue culture. Plant cells can be induced to proliferate in a variety of forms including nondifferentiated callus and suspension cultures in vitro. Single ceils like protoplasts express totipotency, i.e. the ability of a single cell to regenerate into a whole plant. Regeneration may occur via various processes including somatic embryogenesis. In this process structures that resemble zygotic embryos are formed in the tissue culture.

PLANT BIOTECHNOLOGY FOR CROP IMPROVEMENT 675

genetics includes techniques for isolating, characterizing, recombining and multiplying and

transferring discrete fragments of DNA that contain genes coding for specific traits (Fig. 2;

foreign gene and

, right hand

~actefium ~aciens

transgemc ~lant containing the selectable marker gene and the foreign gene of interest

callus forming on co-cultivated leaf disks from cells that have taken up the gene that confers resistance to the selective agent

shoots developing from resistant callus

Figure 2. Plant molecular genetics. A foreign gene, coupled to a selectable marker gene, is cloned into a disarmed Ti plasmid of Agrobacterium tumefaciens. The foreign gend and the selectable marker gene axe transferred to the plant by cocultivating the bacteria with the explant (leaf disk) and regenerating plants from the cells that express resistance to the selective agent in tissue culture. The foreign gene becomes stably integrated into the DNA of the plant and is inherited by future generations of plants derived from the original transgenic plant.

676 K, P. PAULS

Maniatis et al., 1982; Gelvin et al., 1988; Watson et al., 1987). The fact that a whole plant

can be regenerated from a single cell makes tissue culture a valuable procedure for proliferating

genetically identical material and selecting interesting variants. Totipotency also allows a

genetic change, made at the cellular level, to become an established trait of a whole plant. The

newly introduced or selected trait can, subsequently, be passed on to future generations of the

species by conventional crossing methods.

To be effective, plant biotechnology must be well integrated into established plant

breeding and crop production practices. For many field crop species the average amount of

time that is required to develop, test and release a new variety is 10-15 years. The procedure

has many stages including: germplasm manipulation, parent selection, genotype selection,

genetic stabilization, testing, variety increase, proprietary protection, crop production and crop

quality control. Fig. 3 illustrates that biotechnology can contribute to most stages of crop

development and production.

GERMPLASM MANIPULATIONS

Germplasm Preservation

The variety of forms that occur in a plant species reflect its genetic diversity. Since

this germplasm represents the raw material with which plant breeders work there is considerable

interest in preserving representatives of old varieties, land races and related wild species

(Plucknet et al., 1987). These lines may be a valuable source of agronomic genes in the future.

Biotechnology makes diverse germplasm sources more valuable than ever before because

genetic material from different species, genera and even kingdoms can now be stably

incorporated and expressed in plants.

Techniques such as minimal growth in tissue culture and cryopreservation (in liquid

N2) have been used to store plant materials from a wide variety of species (Kartha, 1985;

Withers, 1987). These techniques are particularly important for plant species that are

vegetatively propagated since they reduce the labour associated with maintaining a line as well

as the risk of losing the material to disease. International exchange of germplasm is also

facilitated because of the disease-free nature of the plant materials in culture.

Molecular techniques can be used to make libraries of the genetic material of plants

in bacteria or viruses (Sambrook et al., 1989). Germplasm can be stored in these forms for an

indefinite period in low temperature freezers (-70°C) and the libraries take up little space.


Minimal growth Invi tm

C r y o ~ o n

Gene libraries ' ~ GERMPLASM

Related wild species

Protoplast fusion Unimproved somadon~ , germplasm

variation i Land races

In vitro selection p 9r GeneticallYmodified

Agrobacfe#um genotypes transformation

Direct D ~ uptake

Registered varieties

Genetic distance test

T PARENT I F

1 SELECTION

RECOMBINATION

F 2 i N

/ F : 3 t ° o° °°o"

,,ng,= ng SEED F "" . Pollination control INCREASE larker

Agrobacterium assisted V transformation selection

Pest monitoring • CROP Direct DNA Uptake To,in =says r PRODUCTION

Figure 3. Inputs from biotechnology into the plant improvement cycle. In a typical plant breeding program parents for new varieties are selected from a germplasm pool that contains currently registered varieties, land races, unimproved germplasm collected from the wild and related plant species. Today genetically-modified plants created by biotechnology are also used for plant breeding. Parents for crosses on which new varieties are based are selected on the basis of their proven agronomic characteristics (like high yield) or because they have useful traits like disease resistance. The best characteristics of the parents are combined into one population by making crosses among the parents. The progeny are evaluated on the basis of a variety of agronomic criteria and the best few percent of the plants are used for further rounds of crossing and testing. The genetic makeup of the plants in the breeding population is stabilized (fixed) by the crossing program and can be accelerated by crossing the plants to themselves or by crossing closely related plants. The filial (F 1 to Fs) generations that result are increasingly uniform. This process ensures that the variety that is produced breeds true and gives uniform crop stands in the field. Today haploidy can be used to rapidly produce homozygous lines of breeding material. High performing plant lines are introduced into registration trials to test their performance against current standard varieties. After demonstrating superior performance in these trials a new line may be licensed for sale as a variety in Canada. Several years of seed increase are required to obtain sufficient seed to establish a new variety. Bioteclmological procedures can enhance this crop improvement cycle in the various ways described in the text.

678 K.P. PAULS

Thus, gene libraries and individual genes (Newman et al., 1994) are rapidly becoming important

components of germplasm exchange among plant breeders.

Germ~lasm D/versification

A common first step in plant breeding is to create a genetically diverse population by

crossing unrelated parents or by inducing mutations in the breeding germplasm. Several

biotechnological procedures have greatly expanded the possibilities for increasing the genetic

diversity of crop species including: molecular assisted selection of potential parents, tissue

culture-induced variability, protoplast fusion and the production of transgenic plants.

The selection of parents is one of the most critical decisions a plant breeder makes

because the amount of genetic diversity that is incorporated into the first cross determines the

range of characteristics that can be expressed in the varieties that develop from it as well as the

time it takes to achieve the goals of the breeding program. DNA analyses results can be used

to maximize genetic distances among parents in a conventional cross, thus assuring that a broad

base of genetic diversity exists in the population from which selections are made. Genetic

distances, based on DNA analyses, have also been shown to be good predictors of hybrid vigour

(heterosis) in single cross hybrid combinations of corn inbreds (Smith et al., 1990).

Although the purpose of tissue culture can be to preserve the genetic fidelity of the

stocks, long-term tissue culture can also be used to increase useful genetic variation. Genetic

variability in tissue culture-derived material, called somaclonal variation (Larkin & Scowcroft,

1981) is especially prevalent if the material is kept in a rapidly dividing, non-differentiated

state, (callus or cell suspension) for an extended period. In fact, the frequency of somaclonal

variation can be 10,000 times higher than spontaneous mutation rates in whole plants (Larkin

& Scowcroft, 1981). Variants such as lettuce with improved seedling vigour, a scented

geranium, rice with improved protein quality, flax tolerant to salt, and disease-resistant tobacco

have been isolated from mutant cell lines that arose spontaneously in tissue culture and some

of these variants have been used to develop plant varieties (Evans, 1989).

Protoplast fusion is a method for making large changes in the genetic composition of

plants. Protoplasts are released from plant tissues after incubation in cell wall degrading

enzymes (Cocking, 1960). Several procedures, including incubation in PEG (Kao &

Michayluk, 1974) or treatment with electrical pulses (Zimmerman, 1982), can induce protoplasts

from different plants to fuse. Somatic hybrid plants can be regenerated from cultures of these

fusion products. Protoplast fusion has been used to:


I. make very wide combinations between distantly related species that are not possible

by conventional crossing procedures [like tomato and potato (Melchers et al., 1978)

or mustard (B. juncea) and rapeseed (B. napus) (Sjodin & Glimelius, 1989)],

2. bring about limited gene transfer, [for example, disease resistance from a wild relative

of potato (Solanum brevidens) to potato (S. tuberosura) (Gibson et al., 1988)], and

3. create new nuclear cytoplasmic combinations [for example, to obtain new cytoplasmic

male sterility systems for hybrid variety production oferops like canola and rice

(Pelletier et al., 1983; Pelletier and Chupeau, 1984; Kyozaka and Shimamoto, 1989;

Schell & Vasil, 1989; Pauls, 1991)].

Recombinant DNA procedures can be used to make discrete changes in the genetic

makeup of plants. The most successful systems to date harness the natural ability of a

tumorigenic plant pathogen called Agrobacterium tumefaciens to transfer genes into plants

(Schell & Vasil, 1989). Agrobacteria are usually altered by recombinant DNA technology to

remove tumour-inducing genes and introduce a foreign gene(s) before they are used for plant

transformation. The modified Agrobacteria no longer induce the disease state but retain the

ability to transfer foreign genes into plants. Applications of the Agrobacterium-mediated gene

transfer techniques have been limited by a host range that is restricted primarily to dicots but

successes with direct DNA uptake methods, like protoplast electroporation (Potrykus, 1990) and

biolistic introduction (Klein et al., 1987), indicate that plant transformation will be achievable

with virtually all crop species. The most significant development in the past few years in plant

transformation has been the demonstration that transgenic monocots like rice (Datta et al.,

1990), corn (Gordon-Kamm et al., 1990) and wheat (Vasil et al., 1992) can be created.

Transgenic plants of over 45 species that contain genes from other plant species,

bacteria, viruses and animals, are currently available (Oxtoby & Hughes, 1990; Fraley, 1992).

The foreign genes expressed in transgenic plants confer on them a variety of important

agronomic traits including:

1)

2)

3)

4)

5)

6)

herbicide resistance (Oxtoby, 1990; Quinn, 1990; Schulz et al., 1990),

insect resistance (Htfte & Whiteley, 1989; Ryan, 1990; Koziel et al., 1993),

viral resistance (Baulcombe, 1989; Beachy et al., 1990; Hull & Davies, 1992),

microbial resistance (Lamb et al., 1992; Staskawicz, 1995),

altered macromolecular composition (Hiatt et al., 1989; Altenbach et al., 1990;

Krebbers & Vandekerckhove, 1990; Knutzon et al., 1992; Voelker et al., 1992; Visser

& Jacobsen, 1993; Ttpfer et al., 1995),

modified reproductive capacity (Mariani et al., 1990; Mariani et al., 1992; Lee et al.,

680 K.P. PAULS

1994) and

7) delayed senescence (Sheehy et al., 1988; Smith et al., 1988 Oeller et al., 1991; Gray

& Grierson, 1993).

Completely new roles for crop plant as factories for recombinant DNA products such as

antibodies have been suggested (Conrad and Fielder, 1994; Ma et al., 1995). Proponents of this

approach cite high quality products, easy scale-up and cost effective development and

production economics as some of the attributes of using plants to produce speciality protein

products.

The transgenic plants created by Agrobacterium transformation or direct DNA uptake

procedures have also been very useful for isolating genes when used in conjunction with

transposon tagging (Springer et al., 1995) or T-DNA tagging procedures (Gierl & Saedler,

1992). Furthermore, transgenic plants have been used for testing the functions of genes and

their promoters (Schell, 1987). These studies have resulted in a more profound understanding

of plant biology and have led to the evolution of strategies for tissue developmental- and

environmental-specific expression of inserted genes (Katagiri, 1992; Quail, 1995).

GENOTYPE SELECTION AND GENETIC STABILIZATION

In most plant breeding programs line selection and genetic stabilization occurs

simultaneously and consists of a procedure whereby the breeder selects a few of the best lines

to advance to the next round of selfing or backcrossing. The process serves to fashion the raw

germplasm into varieties that are superior to those that already exist and to stabilize their

genetic makeup by making them homozygous. Biotechnological techniques that can contribute

to this stage of variety development are: in vitro selection, haploidy, and marker-assisted

selection.

Selection

An efficient method for obtaining plants with desired characteristics is to add a

selective agent that will kill the majority of the cells (except the resistant ones) to a tissue

culture. This procedure is called in vitro selection (Chaleff, 1983). Since the in vitro unit of

selection can be a single cell the selection pressure can be uniformly and reproducibly applied.

Also, in vitro selection is potentially more efficient than whole plant selection. For example,

a single flask of cells can have the same number of selection units as 30,000 plants in a three


hectare nursery. This method has been particularly effective for selecting herbicide- and

disease-resistant plants (Shaner & Anderson, 1985; Swanson et aI., 1989; Dix, 1990; Frame et

al., 1991; ller et aI., 1993).

The difficulties that are associated with selecting plants that express recessive alleles,

genes that have only minor effects on plant phenotypes or genes whose expression is strongly

modified by the growth environment are major limiting factors in plant improvement. The

utilization of molecular marker-assisted selection may allow plant breeders to overcome some

of these limitations. Differences (polymorphisms) in isoenzyme, DNA restriction fragment and

polymerase chain fxagment patterns are useful as molecular markers and have been shown to

be genetically linked to both simple and complex traits such as disease resistance and fruit

composition in crop plants (Bernatzlcy & Tanksley, 1986; Lander et al, 1987; Landry et al.,

1987; Tanskley et al., 1989; Landry et al., 1992).

In a breeding program which uses the molecular markers, progeny from each mating

cycle are selected on the basis of the presence of a particular electrophoretic band that has been

shown to be tightly linked to the desired characteristic (Ralfalski & Tingey, 1993). This

approach circumvents masking effects by dominant alleles, eliminates variability due to

environmental effects and can greatly simplify the patterns of inheritance for complex traits.

Marker-assisted selection also allows early stage selection to be carried out since the genetic

pattern does not change during plant development. Furthermore, after a linkage map of

molecular markers has been constructed a whole genome selection procedure can be utilized,

thus, decreasing the time required to fix a gene in an agronomically useful background

(Paterson et al., 1988; Lander & Botstein, 1989).

Genetic stabilization

Homozygosity is required for many crop varieties to ensure their uniformity in the

field as well as their stability from year to year. Generally, three to four years are required to

achieve homozygosity in a field crop by conventional techniques. However, haploidy can

replace several generations of inbreeding (usually 7 or 8) normally required to achieve

homozygosity. Haploid plants are obtained by culturing gamete cells and they are,

subsequently, doubled by a treatment with colchicine to produce homozygous lines (Kasha,

1974; Hu & Yang, 1986). The use of doubled haploid populations also makes breeding for

recessive traits much simpler because the homozygous recessive individuals occur at a greater

frequency in these populations than in conventional breeding populations (Siebel & Pauls, 1989;

682 K.P. PAULS

Henderson & Pauls, 1992). Haploids have been obtained from over 200 species, and varieties

based on double haploid lines have been produced in a variety of field crops including: barley,

wheat, tobacco, rice and rapeseed (Morrison & Evans, 1988).

VARIETY TESTING

After a variety is developed it is tested in several locales over several years. In some

countries the results of these trials are used to decide whether the variety can be licensed for

sale. The movement of genetically altered material from the laboratory or greenhouse to the

field is highly regulated. In many countries the regulations state that field tests of transgenic

material can only be done under a permit that is granted on a case by case basis (Anonymous,

1988; Anonymous, 1995 a,b).

Some of the concerns that have been raised about the production and use of transgenic

plants include:

1) escape of genes from the transgenics to related wild species to produce super weeds

(Wrubel et al., 1992; Dale, 1992),

2) intergeneric or interkingdom transfer of genes like antibiotic resistance genes

(Am/Lbile-Cuevas, 1993; Dixon, 1993),

3) questions of the safety of foods derived from plants possessing crop protection genes

like the insecticidal Bacillus thuringiensis (Erickson, 1992; Beck & Ulrich, 1993) and

4) issues related to ownership of genetic resources (Witt, 1985; Berland & Lewontin,

1986; Stone, 1995).

The results from initial studies indicate that the use of biotechnology does not impose

extraordinary risks on society (Flavell & Fraley, 1992; Huttner et al., 1992). However,

considerable knowledge gaps exist in these areas of concern and a cautious approach to the

introduction of transgenic plants into the environment or the utilization of transgenic foods has

been advocated (Bryant & Leather, 1992)

VARIETY INCREASE

The plant propagation step cain be a major cost in a variety development program. For

many field crops this is simply done by producing seed from pure stands of one variety.

However, the interest in using pollination control systems such as cytoplasmic male sterility,

self incompatibility and artificial male sterility to produce hybrid seed in a wide variety of


crops has grown as the techniques to manipulate these traits have developed. This interest can

be attributed to the facts that hybrid varieties generally have higher yields than conventional

varieties and that the investment associated with their development is protected because they

do not breed true.

Molecular biology has been used to identify some of the genes (Cornish et al., 1987;

Dzelzkalns et al., 1993) that determine self incompatibility and there is some hope that this

information can be used to develop hybrid seed production systems for crops that are now sold

as inbreds or homozygous varieties. Protoplast fusion has been used to transfer organelles (in

particular mitochondria) from one species to another. This approach has been used to introduce

cytoplasmic male sterility into crop plants for use in pollination control and hybrid seed

production (Pelletier & Chupeau, 1984; Kyozuka et al., 1989; Scbell & Vasil, 1989; Pauls,

1991).

An artificial genetic male sterile system has been created by transforming plants with

a RNAse gene from Bacillus amyloliquefaciens under the control of a promoter that is specific

for cells (called tapetal cells) that surround the pollen sacs (Mariani et al., 1990). In these

transgenic plants the tapetom is destroyed and they do not produce pollen. They represent the

female parent in a hybrid seed production system. The male parents are transgenic for a RNAse

inhibitor gene which is also under the control of the tapetum promoter (Mariani et al., 1992).

Both the male parents and F I hybrids obtained by crossing the female and male transgenics

produce pollen.

Plant cloning is a method that has been used with success by horticulturaiists and

ornamentalists for many years to rapidly propagate desired genotypes. The multiplication of

fruit varieties by bud grafting is an example of vegetative propagation. Vegetative propagation

can be greatly accelerated by utilizing tissue culture procedures because a small piece of tissue

can be used to produce hundreds of plants. This procedure has been called micropropagation

and has been applied to a wide variety of plant species (Klausner, 1986). In addition, the

material that is produced in vitro is disease free which facilitates international plant exchange

Giles & Morgan, 1987).

For many field crops vegetative propagation has not been used because the cost of

production greatly exceeds the value of an individual plant (Sluis & Walker, 1985). However,

from recent work on somatic embryogenesis (Gray & Purohit, 1991) and automated plant tissue

culture (Levin et aL, 1988; Vasil, 1991) it appears that it may be possible, in some instances,

to produce large numbers of cheap synthetic seed that are vegetative clones of one plant. Such

techniques may be particularly important for cross pollinating species like alfalfa where it is

684 K.P. PAULS

difficult to produce seed from self pollinations and, therefore, difficult to maintain and increase

superior plants (McKersie & Bowie)', 1993).

PROPRIETARY PROTECTION

Because of the long time it takes to develop a variety there is a need to prevent its

unauthorized multiplication and sale. The ability to distinguish one variety from another is

important in establishing ownership and protecting investments related to variety development.

Isoenzyme, restriction enzyme and PCR fragment polymorphisms have been used to describe

the uniqueness of plant varieties. In particular, the latter two techniques, can be used to obtain

fingerprints of a plant that are based on the sequences in the DNA itself (SoUer & Beckman,

1983; Beckman & Soller, 1986). A DNA branding procedure has been described where a

unique sequence of DNA is inserted into nontranslated regions of the DNA used for

transformation (Beckman & Bar-Joseph, 1986). This positively identifies plant material derived

from a molecular transformation experiment.

Over 100 US patents have been issued since the 1980's for transgenic plants or genetic

engineering approaches to altering plants (Stone, 1995). However, some very broad patents

issued in the United States and Europe for rights to all genetically altered forms of a particular

crop or all crops that utilize a particular molecular engineering procedure have raised concerns

that this practice will severely restrict the utilization of plant biotechnology.

CROP PRODUCTION AND QUALITY CONTROL

Once the crop is standing, biotechnology can play a role in its management and in the

quality control of products derived from it. For example, some DNA hybridization-based

disease assays, and antibody-based toxin assays can be used to monitor disease levels in crops

and products derived from them (Klauaner, 1986). Early detection of diseases in the field is

valuable where economical control measures exist and they can be applied before major

outbreaks occur (Miller et al., 1988). Also, rapid immunoassays for toxins produced by plant

pathogens such as Fusarium toxins in corn or agrochemical residuals such as herbicides and

insecticides (Vanderlaan et al., 1988), can be used to test crops before they are used for animal

or human consumption.


SUMMARY

Biotechnology is applicable to many aspects of plant improvement and crop

production. Seed, agrochemical and food processing companies have made substantial financial

commitments to implement these technologies in plant improvement programs and research

carried out at universities and in government research institutes have been important in

developing and evaluating these techniques (Moses et al., 1988; Kalton et al., 1989; Hodgson,

1992). A measure of the level of interest in plant biotechnology can be obtained by examining

the number of field trials of transgenic plants conducted throughout the world in the last five

years (Beck and Ulrich, 1993). For example, in Canada this value has grown from less than 10

in 1985 to almost 500 in 1993 (Tomlin, 1993). Plant varieties based on transgenic plants are

just beginning to be introduced into the market. However, it is obvious from the level of

activity in this area that the pipeline is full with a large variety of potential products to follow

the recent introduction of the Flaw Saw tomato by Calgene.

Given the range of expertise and level of integration that is required in a modem plant

breeding program it is important that mutually beneficial partnerships among industry,

government and university institutions are developed in the application of plant biotechnology

to plant improvement. Not only is this partnership approach likely to be necessary to collect

a sufficient critical mass around a particular topic, but it is probably the only way in which the

societal issues related to the application of biotechnology will be properly addressed (Wrubel

et al., 1992; Buttel, 1986).

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