Research Project Report

On

PLANT BREEDING
Submi ed To :

Mall road, Kanpur

(Affiliated By: C.S.J.M.U., Kanpur)

For the partial fulfillment of the requirment

for the award of the
Degree of Bachelor of Science
(2025 - 26)

Under the Guidence of

Submi ed To: Submi ed By:
Dr. PRITI SINGH LAXMI
Botany Department [Link]. - 6th Sem.
INTRODUCTION
Organic farming looks at the breeding of new varieties in a holistic way. Thus, not only the
variety itself but also the process of varietal development must comply with the guiding
principles of organic agriculture. Important criteria are taken into account such as
preserving the integrity of the plant, increasing genetic diversity, respect for crossing
barriers, and interactions of the plant with the living soil and climate. Breeding techniques
that are used to produce genetic variation for selection and propagation must be
evaluated for compatibility with organic farming. Genetically modified organisms (GMOs)
are excluded from organic farming, as isolated DNA is exchanged within or across
species, which would not occur under natural conditions. Other methods that do not
currently need to be declared, such as protoplast fusion, in vitro propagation, induced
mutations and hybrid breeding are critically discussed in the sector.

The discussion about the compatibility of modern breeding techniques with organic
farming has been central to the organic sector for several years as part of national and
international workshops and conferences (Wyss et al., 2001; Arncken, 2002; Wilbois,
2002; Arncken and Thommen,2002; Arncken and Dierauer, 2005; Lammerts van Bueren
and Wilbois, 2003; Lammerts et al., 2007; Bill-mann et al., 2008; Oehen and Thommen,
2009). Until now, a comprehensive assessment of modern breeding techniques has not
been realised due to the complexity of the issue which is emotionally charged by the GMO
debate. New techniques (i.e. smart breeding) are constantly emerging out of research and
development activities that are rapidly implemented in conventional breeding
programmes raising new questions. The complexity of these new technologies can cause
great scepticism by consumers and stakeholders of organic farming. However, if modern
breeding techniques are rejected a priori, the organic farming sector risks to be left out of
the breeding progress unable to compete with growing challenges of productive and
sustainable food production (BMVEL 2002). Therefore, it is important to gain an overview
of current breeding techniques, along with those still under development, and to define
criteria that ensure an objective and transparent assessment of each technique in light of
the principles of organic farming. An adapted organic plant breeding approach must
include the development of improved varieties for organic farming without violating its
ethical and ecological principles. The use of genetically modified varieties is prohibited in
organic farming according to EC Regulation No 834/2007. The same regulation requires
to use only organically produced seeds and propagating material. For this, the mother
plant (in the case of seeds) or the parent plant (in the case of vegetative propagating
material) must have been produced under the provisions of this regulation for at least one
generation (for annual crops) or for a period of two growing seasons (for perennial crops).
TECHNIQUES FOR THE BREEDING AND
PROPAGATION OF PLANTS
How are plants bred and propagated?
Plant breeding describes all activities that aim to improve the genetic characteristics of a
crop. The art of the process is to find new positive traits that are heritable, and to combine
these with other desirable traits. In each case, the best compromise should be sought
from the large number of possible target traits.
For this purpose, parental plants with the desired traits must be collected, often from other
varieties, from gene banks or related wild species. These are then crossed to obtain the
desired combinations of traits in the progeny. The result of such crosses is a large number
of seeds with different genetic constitutions (i. e. a segregating population). Within the
next few generations, those plants with the best combination of desired traits must be
selected. To assist with this, a number of techniques are available from which the breeder
chooses the most appropriate depending on the crop, mode of reproduction (e. g. self-
pollinators, cross-pollinators or those which require vegetative propagation) and the
desired traits. It must be ensured that the desired traits are stably transmitted to the
progeny, which may require 6 to 10 generations.
In official variety testing, new varieties are grown in parallel with standard varieties and
traits are determined and compared. If the variety is new, different from all existing
varieties, the traits are homogeneous and stable over time, and it has a name, it can be
awarded plant variety protection. To be accepted as a variety under the Seed Act, most
arable varieties must also have a value for cultivation, i.e. have an advantage in one or
more traits over existing approved varieties.
In general, plant breeding techniques can be categorised according to the
following three areas:
› Generation of genetic variation through collection of genetic resources, mutation and
recombination of genes
› Selection and narrowing of genetic variation for genotypes which best exhibit the
desired combination of traits.
› Maintenance and propagation of the best varieties In each of the three steps, various
techniques can be applied to different anatomical levels of a plant:
› Whole plant level, i.e. the single plant, its progeny or a population
› Tissue level, i.e. the plant parts, organs or cell cultures
› Cell level, i.e. an isolated single cell, protoplasts, pollen or egg cell
› DNA level, i.e. the nuclear DNA or extra-chromosomal DNA
The techniques can also be differentiated by the environment in which they are
applied:
› Field experiments in interaction with soil and climate (e. g. under organic growing
conditions)
› Pot experiments using an artificial substrate under standardised conditions (e. g.
cultivation in the greenhouse)
› In vitro experiments using artificial nutrient medium under sterile conditions (e. g.
meristem cultures, leaf cell cultures)
› Cell suspensions (e. g. protoplast cultures)
POLYPLOIDISATION

Method:
In polyploidisation, the chromosome number of a plant species is multiplied. Whereas,
most species are diploid and have two copies of each chromosome, the doubling of
chromosomes, for example, gives rise to tetraploid plants with four copies each. The
doubling of chromosomes affects all genes of a genome and is also referred to as genome
mutation. In nature, there are various types of polyploidy. A distinction is made between
autopolyploids, which have two or more sets of homologous chromosomes derived from a
single species (e. g. in the tetraploid potato AAAA), and allopolyploids, which are
polyploids produced from several different genomes (e. g. hexaploid wheat, which has
three different genomes, AABBDD). Polyploidy may occur spontaneously or can be
induced by chemicals (e. g. colchicine). The resulting autopolyploid plants are usually
more vigorous and robust, and have larger fruits than the original diploid plants.
Application:
Polyploidy is used to grow more robust and higher yielding crops (e. g. autopolyploid rye
and red clover), to restore the fertility of inter-specific crosses (e. g. allopolyploid triticale,
canola and cotton) and to develop double haploid plants (see chapter ‘Double haploid
(DH) plants‘, page 20). Furthermore, tetraploid cross-breeding plants are required to
produce seedless fruit.
Critical issues from the perspective of organic farming:
› If tetraploid and diploid plants cross, the resultant triploid progeny is sterile.
› The most commonly used anti-mitotic drugs, e. g. colchicine and the herbicide oryzalin,
are not permitted in organic farming.
CYTOPLASMIC MALE STERILITY (CMS)

Method:
Cytoplasmic male sterility (CMS) occurs when the male flowering organs (anthers, pollen
or male gametes) develop incompletely due to a malfunction of the mitochondria. This
malfunction occurs when the interaction between the nuclear genome and mitochondrial
DNA is disturbed.
CMS is initiated in nature by spontaneous mutations in mitochondrial DNA. Depending on
the mutation, it may be that no pollen or only malformed pollen is produced. It may also be
that pollen is produced, but it is incapable of germinating and thus sterile. Since the
mitochondria are passed to the progeny almost exclusively via the egg cell, this is referred
to as maternal inheritance. In most cases, it is possible to restore male fertility by crossing
with chromosomal ‘restorer genes’. To produce a hybrid, line A is crossed with a male
sterile plant that possesses the mutated mitochondria. The seeds harvested from the
sterile mother plant will also develop into male sterile plants. In this way, through repeated
backcrossing, line A can be produced in a male sterile version (CMS line). The sterile line A
is then grown together with the fertile line B and both flower together. The seed harvested
from the mother plant is hybrid seed (AxB).To ensure that these hybrid seeds produce
fertile progeny and can produce yield via generative organs (seeds), line B must have a
chromosomally inherited restorer gene that can induce male fertility again. With
vegetables like cauliflower, however, this is not strictly necessary as it is the flower heads
that are harvested and not the seeds
SYNTHETIC BIOLOGY

Method:
With the help of synthetic biology, new DNA blueprints can be created. Individual
components, such as nucleotides or amino acids, are reassembled or synthesised
without a natural precursor. Current research mainly deals with the synthesis of bacteria to
produce new enzymes in large quantity and high purity. In this case, the original DNA of
the bacterial cell is replaced by a synthetic genome. In fundamental plant research, it is
already possible to create artificial mini-chromosomes (see previous chapter) and
synthesise novel chloroplasts.
Application:
Early applications of synthetic biology are tested on simple bacteria. In comparison to
standard methods of genetic engineering, rather than constructing new individual gene
segments (e. g. integrating a Bt gene from a bacterium into the maize genome), synthetic
biology can produce new genes and genomes from chemicals based on computerised
DNA layouts. In this way, DNA sequences can be synthesised without a natural template,
e. g. to create new proteins. It is being attempted, for example, to obtain novel substances
in pure form via synthetic bacterial DNA templates.
Critical issues from the perspective of organic farming:
› Genetically modified organisms are not allowed in organic farming.
› The method raises ethical concerns about illusions of omnipotence and the reduction of
organisms to the sum of their individual components. Synthetic biology represents a
strong intervention in creation.
REVERSE BREEDING

Method:
In reverse breeding, the process of hybrid creation is reversed. The technique attempts to
reproduce genetically identical progeny from a selected, heterozygous plant with all
desired positive features. This would usually not be possible because genes are
recombined during meiosis when selfing heterozygous plants. Thus, a hybrid can be
selfed, but the traits of the progeny segregate widely due to novel [Link]
breeding attempts to prevent recombination of genes by suppressing crossover events
and, thus, divide hybrids in reproducible hereditary components. Recombination is
suppressed using RNA interference (see previous chapter) and double haploid
technology is used to create homozygous inbred lines in a single step. These can be
propagated as often as required. The homozygous inbred lines are subsequently crossed
with each other to reproduce the original heterozygous genotype.
Application:
Reverse breeding can be used in heterozygous allogamous plants for generative
propagation. Until recently, this was only possible for vegetatively propagated genotypes.
Critical issues from the perspective of organic farming:
› Isolated DNA or RNA sequences are brought into the nucleus via technical
interventions, thus, violating the integrity of the cell as a functional unit.
› Reverse breeding interferes in the overall control of gene expression, and the self-
organisation of the cell is disturbed.
› The variety must be recreated from each of the hereditary components. Seed saving is
not possible without a decline in performance.
TRANSFORMATION VIA MINI-CHROMOSOMES

Method:
To incorporate a large number of new genes into the plant genome, a whole group of gene
constructs can be transferred by means of artificial mini-chromosomes. These mini-
chromosomes can be prepared in two different ways: by shortening the natural
chromosomes, or by de-novo synthesis of all the functional elements of a chromosome.
Subsequently, the desired gene is inserted into the mini-chromosomes and introduced
into the cell nucleus. Mostly, recombination sites are transferred at the same time to
integrate further gene constructs at a later stage.
Application:
This method is useful when many existing gene constructs are to be transferred into a
variety / species, or if entirely new metabolic processes are to be induced for industrial or
pharmaceutical purposes. The mini-chromosomes are not incorporated into the existing
plant chromosomes avoiding undesired position effects. The localised foreign genes on
the mini-chromosomes show stable expression and heredity.
So far, the method has mainly been used to increase biomass production.
Critical issues from the perspective of organic farming:
› Genetically modified organisms are not allowed in organic farming.
› The integrity of the plant genome is disrupted.
› The plant is degraded to purely function as a metabolic producer.
› There is a ready transition of this technique to synthetic biology.
PLASTID TRANSFORMATION

Method:
In plastid transformation, the gene construct is not integrated into the nuclear genome
(nuclear DNA), but instead into the extra-chromosomal DNA of the plastids (chloroplastal
or mitochondrial DNA). An advantage of transferring foreign DNA into plastids is the high
level of gene expression due to the large number of gene copies. In addition, plastids are
controlled differently to chromosomal genes and, as a consequence, there is no risk of
gene silencing by RNA interference (see chapter ‘Gene silencing – RNA interference’,
page 26). Multiple genes can be transmitted simultaneously and the plastids are almost
exclusively maternal in origin, i.e. inherited via the egg cell. Therefore, the risk of
outcrossing via pollen is substantially lower compared with plants that have foreign DNA in
the nucleus.
Application:
To date, this method has only been successfully applied in tobacco to produce bioplastics.
Critical issues from the perspective of organic farming:
› Genetically modified organisms are not allowed in organic farming.
› The integrity of the nuclear DNA is retained, but the extra-chromosomal DNA is altered
which violates cell integrity
SITE-DIRECTED MUTAGENESIS TRIGGERED BY
PLASTID TRANSFORMATION ZINC-FINGER NUCLEASES
Method:
Zinc-finger nucleases are
synthetic proteins containing
zinc-finger domains which bind to
a specific DNA triplet and
nucleases that can cleave the
DNA double helix. By coupling
several zinc-finger domains, the
DNA double-strand can be cut at
a specific point. Via the plant’s
own subsequent repair
mechanisms, base substitution
or base shift (frameshift) may
occur which can create a
mutation or loss of gene function.
In this method, no recombinant
DNA is introduced into the cell,
only the synthetic zinc-finger
nucleases.
This is usually achieved by transfection, electroporation or via Agrobacterium. Zinc-finger
nucleases may, in addition, be coupled with short, isolated DNA sequences
(oligonucleotides) that are used as a template after the double-strand break and can thus
be used for targeted alteration of the gene sequence (also known as targeted
mutagenesis via oligonucleotides). If zinc-finger nucleases are coupled with larger
functional gene constructs (foreign genes), the foreign gene is inserted at the exact
location in the genome where the zinc-finger domain attaches to the DNA. This increases
the efficiency of gene transfer and simultaneously prevents undesired position effects
(also known as gene transfer with targeted integration).
Application:
There have already been some applications in plant breeding. Apart from mere mutation
using the plant’s own repair mechanisms, zinc-finger nucleases combined with isolated
gene constructs are said to have great potential for use in targeted gene transfer. Due to
the high sequence specificity of the zinc-finger, it can be assumed that all copies of the
genes have mutations. This is particularly advantageous in polyploid species.
Critical issues from the perspective of organic farming:
› Zinc-finger nucleases are synthetic proteins, which do not occur in nature.
› The technological use to transfer zinc-finger nucleases into the nucleus of the plant cell
compromises the integrity of the cell.
› When zinc-finger nucleases are transferred into the nucleus of the plant cell, the
integrity of the cell is compromised.