This document summarizes information about cytoplasmic genomes and their applications in plant breeding. It discusses how cytoplasmic DNA located in plastids and mitochondria can influence agronomic traits such as male sterility and disease resistance. Common techniques used in chloroplast transformation are also outlined, including vector design and selection markers. The advantages of chloroplast transformation over nuclear transformation are highlighted, such as high levels of transgene expression and gene containment due to maternal inheritance of plastids. Potential applications of chloroplast transformation include developing herbicide and insect/pathogen resistance in crops.

2. Speaker
SANJAY KUMAR SANADYA

3. Content
 Introduction
 Cytoplasmic genome
 Agronomic traits influenced
 Breeding using cytoplasmic factors
 Genetic transformation of chloroplast
 Application of chloroplast transformation

4. Introduction
 Nucleus is the main organelle where genetic
information is stored in the form of DNA.
 DNA is found in the cytoplasm as plasmone.

5.  Plasmone DNA is located as plastome in plastids
(pt-DNA) and as chondriome in mitochondria (mt-
DNA).
 Plasmone DNA size is small compared to the
genomic DNA (About 200kb/plastid, up to 2500kb/
mitochondrion).
 Despite smaller size of organelle DNA compared to
that of genomic DNA, its importance in inheritance
of few specific traits cannot be undermined.

6. Cytoplasmic Genome
 Synonyms : Plasmone, Organellar DNA, Extra
nuclear DNA, non-nuclear DNA.
 Show cytoplasmic inheritance

7. THE MITOCHONDRIA

8. Characteristics of Cytoplasmic Inheritance
First time reported by Correns in Mirabilis jalapa
and by Baur in Pelargonium zonale in 1908.
 Maternal & Uniparental inheritance
 Reciprocal differences present
 Lack segregation
 Show somatic segregation during mitosis
 Biparental inheritance show irregular segregation
ratio

9. Mitochondrial genome
 Size: vary from 200kb in Brassica species to 2500kb
in musk melon
 Contain protein encoding genes, rRNA, and tRNA
genes, non-functional genes etc.
 Play pivotal role in cytoplasmic male sterility,
susceptibility to systematic insecticides and
sensitivity to fungal toxins

10. Plant Genome size (kb)
Brassica compestris 218
Brassica oleracea 219
Triticum aestivum 440
Zea mays 570
Raphanus sativa 242
Helianthus annus 300
G. barbadense 677
Cucumis melo 2500
human 16
Sizes of plant mitochondrial genomes

11. Gossypium barbadense
Tang, M. et al., 2015

12. Gene Enzyme/ subunit of functional complex
CoxI, coxII, coxIII Cytochrome Oxidase
cob Apocytochrome b of cc1 complex
nad1, nad5 NADH-Ubiquinone oxidoreductase
atp6, atp8, atp9, atpA F0-F1 ATPase complex
rps4, rpsl3, rpsl4 Ribosomal proteins
Mitochondrial genes

13.  The lower plant liverwort (Marchantia polymorpha)
is the first plant from which the mitochondrial
genome has been sequenced.
 Arabidopsis - 57 mitochondrial genes
Mt-DNA of higher plants has a large coding capacity.

14. Chloroplast DNA (cp-DNA) is also known as
plastid DNA (pt-DNA).
Circular double stranded DNA molecule
Chloroplast genome size range between 120-
217kb with majority of plants fall into 120-160kb.
(Pelargonium has a chloroplast genome size
217kb)
Chloroplast Genome

15. Crop Inverted
sequence (bp)
SSCs (bp) LSCs (bp)
Arabidopsis 26284 17780 84170
Spinach 25073 17860 82719
Maize 22748 12536 82355
Tobacco 25339 18482 86684
• Tobacco
•Contain about 100 genes to synthesize proteins
• CpDNA regions includes Large Single-Copy (LSC) & Small Single-
Copy (SSC) regions, and Inverted Repeats (IRA & IRB).
•Conifers and a group of legumes lack Inverted Repeats

16. Wakasugi, et al., 1998

17. Genes Encoded by Chloroplast DNA
Function Number of genes
Genes for the genetic apparatus
rRNAs (23S, 16S, 5S, 4.5S) 4
tRNAs 30
Ribosomal proteins 21
RNA polymerase subunits 4
Genes for photosynthesis
Photosystem I 5
Photosystem II 12
Cytochrome bf complex 4
ATP synthase 6
Ribulose bisphosphate
carboxylase (RUBISCO)
1

18. Agronomic traits influenced
 Cytoplasmic Male Sterility. Eg: Cotton, Maize etc.
 Yield and quality parameters.
 Disease resistance. Eg: Yellow berry in wheat
 Combining ability. Eg: Pearl millet
 Adaptability
 Fertility
Frei, U., et al, 2003

19. Cytoplasmic male sterility (CMS)
 Cytoplasmic factors are responsible for male sterility
 Maternally inherited trait
 Chimeric mt-genes believed to disturb mitochondrial
function at critical stages of tapetum development and
thus cause MS.
 Eg: Maize- a specific S-plasmid of mitochondria can
integrate into mtDNA of wheat.
Frei, U., et al, 2003

20. A line:-
• It is male sterile line which is used as female parent in hybrid seed production.

21. B line
•It is a male fertile line which is used to maintain male sterility in A line called
maintainer line.
•Both A and B line are isogenic type.
R line
Which is used to restored male sterility in male sterile plant, used as male parent in
hybrid seed production.
Produced F1 fertile progeny which called hybrid.
A and B-lines:
Production of A and B-lines is done by growing the A-line in four rows alternating
with the corresponding B-line in two rows.
Three criteria are used in the selection of parents for this purpose: genetic diversity,
the per se performance of the lines and the average performance of a line in crosses
with other lines [called general combining ability (GCA)].
Identification of B and R line in sorghum
Improved breeding lines, named/released varieties and landraces from the pollinator
collection are the sources that can be used as pollen parents or pollinators.
more reliable method is the bagging test, i.e., covering 4-6 panicles with a paper bag
before anthesis, and observing the seed-set after 2-3 weeks.
Ashok Kumar, et al.

22. MAINTENANCE OF CMS LINES & HSP

23. Molecular mechanism of CMS
 1. CMS is associated with
i. Altered mt-genome organization,
ii. Altered mt-gene expression at transcriptional or post-
transcriptional level.
• Mostly novel transcripts with new ORFs (open reading frames)
associated with male sterility.
• Eg: In Brassica napus, in nap and pol CMS lines atp6 transcript
contain additional orf222 and orf224, respectively.
Xiu-Qing Li, et al., 1998

24.  In alloplasmic plants with nuclear genome of Brassica
napus and mitochondria of Arabidopsis thaliana, products
of CMS-determining genes alter the expression of class-B
floral identity gene.
 Male-sterile plants show premature degeneration of anther
tissues that cause mitochondria-signaled programmed cell
death (PCD):- cytochrome-c release from mitochondria,
nuclear DNA cleavage and cellular condensation occur.
 In Zea mays, plants with the Texas type of CMS, the tapetal
cells show features of necrotic cell death.

25. Christine D. Chase, 2006
 2. TISSUE-SPECIFICITY: in Phaseolus vulgaris, the
CMS-associated protein ORF239 is degraded in
vegetative tissues but accumulates in anther tissue.
 Negative consequences of CMS: In vegetative tissues of
T-CMS maize, the product of the mitochondrial gene
that determines CMS functions as a pore-forming
receptor for a toxin produced by the fungus Bipolaris
maydis, thereby inducing susceptibility to this
pathogen.

26. CMS Utilization
Significance
Hybrid seed production
Eg. Bajra, Sorghum, rice
Pollen control
Reduces labour cost
Time saving

27. Pearl millet :- (Tift 23A1) CMS lines 97111B, 97444B, J 2450, HBL 11, 21A/S 10, MS
93333B and 85A/S-10 were also found drought tolerant.
•Three hybrids, (Kafir 60 derivatives) 1111A x CB43, 2714A x CB11 and 4114A x
CB11 recorded 20% increase in grain yield over the check, CSH 25.
•The sweet sorghum hybrids ICSA 38 x [(RSCN 2103 x SSV84)-2-1]-1 and IMS9A
x [(RSCN2103 x SSV84)-2-1]-1 recorded the highest fresh biomass of 80 t/ha and
78 t/ha respectively.
Rice
Sorghum

28. Sorghum hybrid Year Release
CSV 24SS 2011 ICAR- IIMR
CSV 26R 2012 ICAR-IIMR
CSV 27 2012 ICAR-IIMR
CSV 29R 2012 Rahuri
CSV 30F 2014 Bijapur
CSV 31 2014 Palem
CSV 32F 2015 ICAR-IIMR
CSV 33MF 2016 TNAU
Rice hybrid (Rajasthan) Cross
BK 79 (TN 1 x NP 130) x Basmati 370
BK 770 BK 79 x Basmati 370
Mahi Sugandha BK 79 x Basmati 370
Pusa Sugandha - 4 P 614-12 x Pusa 614-35
Pusa Sugandha - 5 P 3A x Haryana Basmati
IET 13549 (Malva Basmati) -

29. Maize hybrid
MPUAT, Udaipur released hybrids-Pratap Sankar-3, Pratap hybrid Maize -1 and Pratap
QPM Hybrid-1 which are performing well in the rainfed kharif and rabi seasons in region.
Vivek Maize Hybrid 51 released from ICAR-VPKAS, Almora which is suitable for
rajasthan Gujarat & Madhya Pradesh. Moderately resistant to Post-flowering Stalk Rot,
(PFSR) Rajasthan Downy Mildew (RDM) and shoot borer (Chilo partellus).

31. Yield and quality parameters
 Ekiz et al.(1998) reported small but significant
differences between reciprocal crosses of
alloplasmic lines for kernel weight and protein
percentage in bread wheat.
 Alloplasmic lines have showed significant
cytoplasmic effects on grain weight and Spikelet
fertility in indica rice (Tao et al., 2011).

32.  Alloplasmic lines with cytoplasm Aegilops
squarrosa have shown rather stable resistance to
a rust Puccinia recondita.
 Maize - Texas male sterile cytoplasm, highly
susceptible to leaf blight.
 In barley resistance against Fusarium culmorum
decreased when Hordeum vulgare cytoplasm was
replaced by H. bulbosum cytoplasm.
Reaction to disease

33. Disease resistance

34. Combining ability

35. Fertility

37. Breeding using cytoplasmic factors
 Cytoplasmic factors are important in qualitative
traits such as CMS and disease resistance.
 The contribution of cytoplasmic factors to
quantitative agronomic traits is probably of minor
significance.
 To enhance the performance of cultivars breeders
should exploit such minor effects.

38. Characterization has been done in several crops
Potato :
 Origin of tetraploid potato traced via plasmone,
predominantly pt-DNA in addition to genetic relationships
among cultivars.
 Five different classes (ε,δ,γ,β,α) of mtDNA identified in
potato.
Maize
 Peiretti (2003) evaluated the plasmone of 30 dent and 32
flint lines using 23 mt-probes and found clear
polymorphisms.
 A genetic distance analysis revealed three clusters viz. dent,
flint and both lines.

39. Frie, U., et al. 2003
Rice
 Cytoplasmic variability of cultivated species Oryza sativa
and Oryza glaberrima indicates high conservation of cp
and mt genomes.
 Lin performed pedigree analysis to quantify ancestral
contributions to 27 rice cultivars released at IARI

40. Cytoplasmic DNA can also have other roles
 Contribute in photosynthesis and respiration
 Evolution of plant species
 Phylogenetic studies
 Production and use of alloplasmic lines by
combination breeding
 Direct transfer of cytoplasmic DNA genes into
organelles

42. An overview of tools and techniques
 To develop homoplasmic transplastomic clones:
i. transformations vectors that harbour a
selectable marker gene, a reporter gene and the
passenger gene(s) flanked by homologous
targeting sequences.
ii. A totipotent explant,
iii. an efficient DNA delivery method,
iv. selection agents (antibiotics) and a reproducible
regeneration protocol.
 Transgenes integrated in plastome through
homologous recombination.

44. 1. Directly Transformation method
 PEG-mediated transformation method also applied
 Generally achieved by the biolostic process due to
high efficiency & simple operation.

45. 2. Vector mediated transformation
Selectable marker
The first selection marker gene used in chloroplast
transformation was plastid 16S rRNA (rrn16) gene.
Others are:
 aadA gene- aminoglycoside 3′-adenylyltransferase
 bar gene- phosphinothricin acetyltransferase
(PAT), etc

46. • Plastid expression vectors possessed left and right flanking
sequences –which are used for foreign gene insertion into
plastid DNA via homologous recombination.
• Insertion of foreign DNA occur in intergenic regions of the
plastid genome.
Regulation sequences
• Expression level in plastids is predominately determined by
promoter and 5′-UTR elements.
• Most laboratories used the strong plastid rRNA operon (rrn)
promoter (Prrn).

47. Comparison of Chloroplast and Nuclear Genetic
Engineering
Transgene Chloroplast Genome Nuclear Genome
Copy number 10-100 plastid/cell with 100
identical genome/plastid
Two copies of each
chromosome per cell
Level of gene
expression
High accumulation of
transgenes
Less accumulation of
transgenes
Gene
arrangement and
transcription
Genes are often arranged in
operons and transcribed into
polycistronic RNA
Independently inserted
into chromosome and
transcribed into
monocistronic mRNA
Position effect Absent Present
Gene silencing Not reported Present

48. Cont..
Transgene Chloroplast Genome Nuclear Genome
Integration Site-specific integration: eliminates
the harmful effects of vector
sequences and transgene silencing
Both are serious
concerns in nuclear
transformation
Gene
containment
Lack of plastid DNA in pollen
minimizes the possibility of leaking
transgenes to related weeds or crops
and potential toxicity of transgenic
pollen to non-target insects
Chances of paternal
inheritance results in
outcrossing among
crops and weeds
expression Uniform gene expression Highly variable gene
expression
Grevich & Daniell (2005)

49. Advantages of chloroplast
engineering
Multigene
engineering
Hyper
expression
No Vector
Sequences
NoGene
Silencing
No
positional
Effect
No
pleiotropic
Effect
Gene
containment
Maternal
Inheritance

50. Agronomic Traits Expressed Via The Plastid
Genome
1. Herbicide Resistance:
Example:-
Glyphosate resistance gene
engineered to overexpress 5-
enolpyruvyl shikimate-3-
phosphate (EPSP) synthase.
Because the target of
glyphosate residues within the
chloroplast, such transgenic is
an ideal strategy for
developing glyphosate
resistance in plants.
Grevich & Daniell, (2005)

51. 2. Insect And Pathogen Resistance:
 Tobacco chloroplasts were shown to correctly process a
bacterial operon and express the Cry2Aa2 proteins at
levels up to 46% tsp (total soluble proteins), the highest
levels ever recorded.
 Chloroplast transgenic leaves for MSI-99, an analog of
maganin-2, is a helical antimicrobial peptide inoculated
with the phytopathogen Pseudomonas syringae pv. tabaci
showed no sign of necrosis even 100-times more cells were
used for inoculation.

52. 3. Drought Resistance:
Example:-
Trehalose phosphate synthase gene, chloroplast
transgenic plants grew normally and accumulated
25-fold higher trehalose.

53. 4. Phytoremediation:
Chloroplast is the primary
target of Hg damage in
plants, so is an ideal place
to engineer resistance and
detoxification of mercuric
compounds.
Eg: bacterial enzymes,
mercuric ion reductase
(merA) and
organomercurial lyase
(merB), in tobacco.

54. Case study

55. Other uses of transgenic chloroplast genome beside crop
improvement are:
 Pharmaceutical production:
Eg: Human serum albumin, somatotropin,
interferon-alpha, interferon-gamma and
antimicrobial peptide.
 Vaccines:
Eg: cholera toxin beta subunit (CTB), Anthrax
vaccine, Plague vaccine etc.
 Enzymes:
Eg:Anthranilate Synthase, xylanase

56. Plastid transformation in flowering plants: methods of
transformation and gene targeting sites in the plastomes
Molecular Plant Breeding , 2012

57. Milestone of chloroplast transformation
New agronomic trait: B.
thruingiensis
Marker gene elimination: co-
transformation
targeting
Year Milestone DNA
delivery
Approach Selection Reference
1988 Chlamydomonas reinhardtii Biolistic Homologous Photosynthetic Boynton & Gillham
1st stable plastid transformation targeting competence (Science, 240)
1990 Nicotiana tabacum Biolistic Homologous Spectinomycin Svab et al (PNAS,
1st stable plastid transformation targeting (rrn16) 87)
1993 Nicotiana tabacum PEG Homologous Spectinomycin Golds et al
1st high level foreign protein
(2.5% GUS)
targeting Kanamycin (Biotech. 11)
O’Neill et al (Plant
1995 Nicotiana tabacum Biolistic Homologous Spectinomycin
J. 3)
McBride et al
(Biotech. 13)
Carrer and Maliga
(Biotech. 13)
1st stable plastid transformation
Oryza sativa (rice)
1st stable plastid transformation
targeting
1998 Arabidopsis thaliana Biolistic Homologous Spectinomycin Sikdar et al (Plant
1999
1st stable plastid transformation
Solanum tuberosum (potato) Biolistic
targeting
Homologous Spectinomycin
Cell Rep. 18)
Sidorov et al
(Plant J. 19)
Khan and Maliga
(Nat. B iotech. 17)

58. Marker gene elimination: CRE-lox
New agronomic traits: glyphosate
tolerance and PPT resistance
Year Milestone DNA
delivery
Approach Selection Reference
2000 Nicotiano tabacum Biolistic Homologous Spectinomycin Staub et al (Nat.
1s t human protein expression targeting Biotech. 18)
2001 Lycopersicon esculentum
(tomato)
Biolistic Homologous
targeting
Spectinomycin Ruf et al(Nat.
Biotech. 19)
1s t foreign protein in fruit Corneille et al (Plant
J. 19)
Ye et al (Plant J.
25)
Lutz et al (Plant
Physiol. 125)
Biolistic Spectinomycin
Biolistic
Homologous
targeting
Homologous
targeting
Spectinomycin
2002 Porphyridium sp.
1s t stable plastid transformation
2003 Chlamydomonas reinhardtii :
Foot-and-mouth disease virus
VP1 protein expression
Brassicacea (oil seeds)
1s t stable plastid transformation
Phytoremediation: Mercury
2004 Gossypium hirsutum (cotton)
1s t stable plastidtransformation
Glycin max (soybean)
1s t stable plastid transformation
Linum usitatissimum L. (flax):
PHB polymer expression
Biolistic Homologous
targeting
aph A-6
npt II
Spectinomycin
Lapidot et al (Plant
Physiol. 129)
Sun et al
(Biotechnol Lett. 25)
Skarjinskaia et al
(Transgenic Res.
12)
Ruiz et al (Plant
Physiol. 132)
Kumar et al (PMB.
56)
Dufourmantel et al
(PMB. 55)
Wrobel et al (J.
Biotech. 107)

59. Foreign gene expression in chloroplasts of higher plants

60. Limitations
1. Crop plants, including cereals, has been their
regeneration from non-green embryonic cells
(containing proplastids) rather than leaf cells
(containing chloroplasts). Identification of
promoters and UTRs active in non green tissues
should help to overcome this limitation
2. Lack of information on genome sequences for
several important crop species to locate
intergenic sequences for integration of
transgenes.
3. Delivering foreign DNA through the double plastid
membrane.