This document provides an overview of reverse breeding, a novel plant breeding technique to directly produce homozygous parental lines from any heterozygous plant. It discusses how reverse breeding uses RNA interference to suppress meiotic recombination and produce doubled haploids from gametes, generating homozygous parental lines. The document summarizes a case study applying this to Arabidopsis thaliana and discusses applications like reconstructing hybrids, breeding at the chromosome level, and implications for food safety. Limitations and future research directions are also outlined.

2. Credit Seminar
on
Reverse Breeding: A novel approach for
development of homozygous plants
Department of Genetics & Plant Breeding
College of Agriculture
S.K. Rajasthan Agriculture University, Bikaner
Major Advisor
Dr. Vijay Prakash
Seminar Incharge
Dr. A.K. Sharma
By
Sanjay Kumar
Ph.D. (Ag.) GPB

3. Contents
 Introduction
 Concept of Reverse Breeding
 Why Reverse Breeding ?
 Steps of Reverse Breeding
 Applications of Reverse Breeding
 Case study
 Consequences for food and environmental safety
 Limitations
 Conclusion
 Future Thrust

4. Reverse Breeding - Introduction
 A novel plant breeding technique designed to directly produce
homozygous parental lines from any heterozygous plant.
 It was proposed by Dirks et al. (2009) in Arabidopsis
thaliana.
 Homozygous parental lines are produced from selected plants
by suppressing meiotic recombination.
 Gametes are directly converted into adult plants, which after
chromosome doubling are used as homozygous parental
lines.
 The main objective of reverse breeding is to generate
homozygous parental lines (complementing parents) that can
be mated to recreate a desired heterozygous genotype (i.e. the
initial hybrid; Wijnker et al. 2012)
 It has not been commercialized yet and very limited work has
been done.

7. “Forward
breeding”
“Reverse
breeding”
Production of
hybrid plants
Selection of
heterozygous
plants
Selection of “end product” at the start of
breeding cycle
Production of lines
Selection of lines

8.  To enhance the hybrid performance, first the parental
lines have to be improved.
 Difficulty in maintaining hybrid stability.
 Breeders cannot produce parents of a hybrid.
 Clonal propagation (Apomixis) preserves the
parental genotypes but prevents its further
improvement. Moreover these techniques are
restricted to limited crops.
To solve all these problems,
Reverse Breeding is the answer.
Why Reverse Breeding ?

9. Reverse breeding includes the following Steps
 Reverse breeding comprises two essential
steps:
1) The suppression of crossover
recombination in a selected plant.
2) The regeneration of double haploids
(DHs) from spores containing non-
recombinant chromosomes.

10. Step 1: Suppression of crossing over

11. RNA interference
 RNA interference (RNAi) is a
biological process in which RNA
molecules are involved in sequence-
specific suppression of gene
expression by double-stranded
RNA, through translation or
transcriptional repression.
 It was first discovered by Andrew
Fire and Craig Mello (1998) in the
nematode worm Caenorhabditis
elegans.

12. The Gene Silencing Mechanism
Two-step model to explain RNAi
I. ds-RNA is diced by an ATP-
dependent ribonuclease
(Dicer) into short interfering
RNAs (siRNAs).
II. siRNAs are transferred to a
second enzyme complex,
designated as RISC (RNA
induced silencing complex).
 The siRNA guides RISC to
the target mRNA, leading to
its destruction.
 The anti-sense strand of the
siRNA is perfectly
complementary.

13. Step 2. Production of Doubled Haploids
Using tissue culture technique referred to as “Anther
culture” and “Isolated microspore culture”,
immature pollen grains grow to produce colonies of
cells which are then transferred to different media to
induce growth of shoots and then roots.

14. Step 3: Selection of complimentary parents through
marker assisted selection
F1
Step 4: Crossing appropriate DH lines on the basis of matching
molecular markers.

15. 1. Reconstruction of heterozygote:
 For crops where an extensive collection of
breeding lines is still lacking, RB can
accelerate the development of varieties.
 In these crops, superior heterozygous
plants can be propagated without prior
knowledge of their genetic constitution.
 Examples – Arabidopsis thaliana, Maize
etc.
Applications
(Dirks et al. 2009)

16. F1
Segregating F2
Starting hybrid
Gametes
Population
double haploid
Reconstituted hybrid

17. 2. Breeding at single chromosome level:
 Reverse Breeding explains how chromosome
substitution lines can be obtained when RB is
applied to a F1 hybrid of known parents.
 These homozygous chromosome substitution
lines provide novel tools for the study of gene
interactions.
 Offspring of plants in which just one
chromosome is heterozygous, will segregate for
traits present on that chromosome only.
 Development of improved breeding lines
carrying introgressed traits.
(Dirks et al. 2009)

19. 3. Reverse breeding and Marker assisted breeding:
 High throughput genotyping speeds up the process
of identification of complementing parents in
populations of DHs.
 Helps in the study of populations that segregate for
traits on single chromosome allow the quick
identification of QTLs, when genotyping is
combined.
 Aids in generation of chromosome specific linkage
maps.
 Fine mapping of genes and alleles.

20. Case study

21. Major objective: To produce homozygous parental lines from the heterozygous
plant of Arabidopsis thaliana.
(Wijnker et al. 2012)

22. Materials and Methods
Plant materials:
1. Arabidopsis thaliana plants were grown in glass house in
standard conditions.
2. Plant transformation
Method:
RNAi knock downs the function of RecA, DMC1 (a
meiosis-specific recombinase essential for the formation of
crossovers).
RNAi used – Brassica carinata DMC1 gene.
Recombinase silenced- A. thaliana DMC1 gene.
PCR amplified cDNA of Brassica carinata DMC1 gene
was cloned to pKANNIBAL Hairpin RNAi vector.
The vector was subsequently cloned into pART27 binary
vector and transformed into Col-0

23. 3. Quantitative RT PCR:
4. Microscopy and FISH.
5. Genetic Analysis: SNP markers.
6. Marker segregation in WT and RB
haploids.
7. Development of Homozygous
diploids, each having half the genome of
the original hybrid.

24. Fig: Meiosis in wild-type (WT; above) and RNAi:DMC1 transformants (below) in
Arabidopsis thaliana.

25.  Reverse bred crops are similar to those of parental
lines and F1-hybrids obtained by conventional
breeding.
 The parental lines produced by reverse breeding and
the subsequent produced F1-hybrids do not contain
any genetic modification-related DNA sequence and
the RNA silencing signal itself will not be transmitted
through seeds, so it is not covered under GMO
regulations.
 So Reverse bred crops said to be safe.
Consequences for food and environment
safety

26.  Development of RB is limited to those crops
where DH technology is common practice e.g.
cucumber, onion, broccoli, sugarbeet, maize,
pea, sorghum.
 There are some exceptions such as soybean,
cotton, lettuce and tomato where doubled
haploid plants are rarely formed or not
available at all.
 The technique is limited to crops with a haploid
chromosome number of 12 or less and in which
spores can be regenerated into DHs.
Limitations

27.  One important application is the
production of complementary
homozygous lines that can be used to
generate specific F1 hybrids.
 Additionally, when RB is applied to F1
heterozygotes, it is possible to generate
chromosome substitution lines that allow
targeted breeding on the single
chromosome scale.
Conclusion

28.  RNAi mediated Reverse Breeding is a young
work, requires extensive study to overcome
technical problems.
 Additional research is required to improve the
efficiency of the DH production.
 Emphasis should be given for the production of
hybrids in crops like cucumber, onion, broccoli,
cauliflower where seed production is problematic.
Future Thrust

29.  Rob Dirks, Kees van Dun, C. Bastiaan de Snoo, Mark van den Berg, Cilia L. C. Lelivelt,
William Voermans, Leo Woudenberg, Jack P. C. de Wit, Kees Reinink, Johan W. Schut,
Eveline van der Zeeuw, Aat Vogelaar, Gerald Freymark, Evert W. Gutteling, Marina N.
Keppel, Paul van Drongelen, Matthieu Kieny, Philippe Ellul, Alisher Touraev, Hong Ma,
Hans de Jong and Erik Wijnker (2009). Reverse breeding: a novel breeding approach based
on engineered meiosis. Plant Biotechnology Journal, 7: 837–845.
 Erik Wijnker, Kees van Dun, C Bastiaan de Snoo, Cilia L C Lelivelt, Joost J B Keurentjes,
Nazatul Shima Naharudin, Maruthachalam Ravi, Simon W L Chan, Hans de Jong & Rob
Dirks (2012). Reverse breeding in Arabidopsis thaliana generates homozygous parental lines
from a heterozygous plant. Nature Genetics, 1-5.
 Aude Dupré , Louise Boyer-Chatenet, Rose M Sattler, Ami P Modi, Ji-Hoon Lee, Matthew L
Nicolette, Levy Kopelovich, Maria Jasin, Richard Baer, Tanya T Paull, Jean Gautier (2008).
Nature Chemical Biology, 4(2): 119-125.
Referances