Basics and recent developments in developing Genetically engineered male sterile plants

1. Genetic engineering for male sterility
in plants
Navaneetha Krishnan J
L-2016-A-18-D

2. What is male sterility?
• Male sterility is a situation where the male reproductive parts of a plant are either
absent, aborted, or nonfunctional, and hence they fail to participate in the process of
natural sexual reproduction.
• This situation can arise due to any developmental defect at any stage of
microsporogenesis or release of pollen grains.
• First observed by Koelrouter in 1763-anther abortion within intraspecific hybrids of
tobacco.
• Darwin (1877) recognized the importance of this phenomenon and hypothesized that the
loss of reproducing ability of plant helps evolutionary processes in enhancing adaptation
through gene transfer from various related and unrelated individuals through cross-
pollination.
Sterile Fertile

3. What about female sterility?
• Occurs rarely in nature.
• Not useful in Plant Breeding.
• Very difficult to detect when compared to male sterility (large
no: of pollens produced).
• Does not have the ability of self propagation(seed set).
• Cannot be stained for preparing assays as that of male
sterility.

4. Need for male sterility ???

5. Classification of male sterility
1) Based on the type of malfunctioning of the androecium,
(i) Structural (absence or deformity of anthers)
(ii) Sporogenous (defective microsporogenesis) and
(iii) Functional (failure of mature pollen to germinate) male sterility
2) On the basis of genetic control mechanisms,
(i) Genetic Male Sterility (GMS)
(a) Environment insensitive GMS
(b) Environment sensitive GMS
(ii) Cytoplasmic Male Sterility (CMS) and
(iii) Cytoplasmic Genetic Male Sterility (CGMS)
3) Artificiallly induced male sterility
(i) Chemically induced male sterility and
(ii) Genetically engineered male sterility

6. Generalized hereditary constitution of the nucleus and cytoplasm of the
three male sterility systems (B. Bahadur et al. 2015)

7. Environment sensitive Genetic male Sterility(EGMS)
• Induction of male sterility in response to fluctuations in
environmental conditions.
• Particularly
Photoperiod(PGMS) and
Temperature(TGMS).
• Eg:Rice
• Controlled by nuclear genes.
• No need to use maintainer line.
• Two-line hybrid system-Only male sterile line(female parent) and
Pollinator(male parent) present.

8. • PGMS lines(Rice) : Nong-Ken 58 S,Zennong s,X-88.
• TGMS lines(Rice):Annong S,Hennong S,Novin PL 12,IR 68945.
• Any genotype can be used as a Pollinator parent.
INHERITANCE OF EGMS
Contd…

9. Origin of male sterility systems
• Selection from natural variation
• Integration of cultivated genome in to alien cytoplasm (using wild sp.
as female parent)
• Intergeneric and interspecific hybridisation
• Selection from recombinant populations
• Induced mutations (physical and chemical mutagens)

10. Chemical induced Male Sterility
• The chemical which induces male sterility artificially called as
male gametocides are used.
• These chemicals are also known as Chemical hybridizing agents.
• It is rapid method but the sterility is non-heritable.
• In this system A, B and R lines are not maintained.
• Some of the male gametocides used are Gibberellins (Rice,
Maize), Sodium Methyl Arsenate (Rice) and Maleic hydrazide
(Wheat, Onion).
• Could be used in the large scale commercial production of
hybrid seed (Hybrid wheat in UK, Germany).

12. Induced GMS (Transgenic male sterility)
Promoter which
induces transcription
in male reproductive
specifically
Gene which disrupts
normal function of cell
Agrobacterium-
mediated
transformation
regeneration
male-sterile
plant

13. First successful experiment on GE male sterility…

14. • The first transgene designed to confer GMS was reported and were
used to transform Tobacco and oilseed rape plants.
• Tapetal-specific transcriptional activity of the tobacco TA29 gene.
• Upstream regulatory elements of TA29 gene used to drive the
expression of transgenes (extracellular RNAses from bacteria). Two
genes were used:
 barnase from Bacillus amyloliquefaciens
 RNAse-T1 from Aspergillus oryzae
• RNase genes selectively destroyed the tapetal cells during anther
development and prevented pollen formation
• Herbicide (bialophos) resistant gene (bar) used as selectable marker
Abstract

15. Robert B. Goldberg et.al. 1993

16. Pollen grain differentiation and release

17. Gene expression is temporally and spatially regulated
17
Koltunow et al. (1990)

18. Fig 1. RNA gel blot analysis of organ-specific gene expression
Results…

19. Fig 2. Floral morphology of male sterile (transgenic) and untransformed plants

20. Fig 3. Tissue abnormalities in male sterile tobacco
and oilseed rape anthers (Bright field microscopy)
Fig 4. SEM micrographs of pollen grains produced by
male sterile tobacco and oilseed rape anthers
E- epidermis; C- connective tissue; F- filament;
PS- pollen sac and T- tapetum

21. Another Breakthrough

22. • Barstar gene codes for an intracellular bacterial protein which is an
inhibitor of barnase.
• Oilseed rape plants were transformed with a binary vector containing
barstar gene along with bar gene as selectable marker using
agrobacterium mediated transformation.
• When the transgenic plants containing barstar were crossed with the
male sterile transgenic plants (barnase), the F1 progenies segregated
in the ratio of 2:1 (male fertile : sterile) (viable progenies) after
selection using herbicide spray at the seedling stage.
• Around 25% male sterile plants produced in the F1 will be lacking bar
gene and they will die.
• The plant transformed with the barstar gene serves as the restorer of
fertility.
Abstract

23. Fig 1. Restoration of male fertility by crossing oilseed rape plants
containing the TA29-barstar and TA29-barnase genes
Note: ms, rf and hr refer to the
hemizygous chromosomal loci that
lack the TA29- barnase, TA29-barstar
and bar genes respectively

24. Fig 2. Oilseed rape flowers and anther cross sections
Male fertile plants containing the TA29-barstar gene
Male sterile plants containing the TA29-barnase gene
Male fertile plants restored to fertility containing
both the TA29-barstar and TA29-barnase gene
E-epidermis; En-endothecium; PG- pollen grain; PS- pollen sac and T-tapetum
Results…

25. Fig 3. Scanning electron micrographs of oilseed rape pollen grains and dehiscing
anthers
Dehiscing anther from
an untransformed plant
Dehiscing male sterile
anther from a plant
containing TA29-barnase
gene
Dehiscing anther from a
plant restored to male
fertility containing both
the TA29-barstar and
TA29-barnase gene
Pollen grains –
untransformed anthers
Pollen grains – anthers
containing both the TA29-
barstar and TA-29 barnase
genes – restored to fertility

26. Fig 4. Presence of barnase and barstar mRNAs in anthers of oilseed rape
plants restored to male fertility

27. Fig 5. Presence of barstar and barnase proteins in oilseed rape anthers
restored to male sterility
MS plants (TA29-
barnase gene)
Anther proteins from
wild type plants
MS/RF plants (TA29-
barnase+TA29-barstar)
Barstar and barnase
proteins circled in the
immunoblot
Purified barstar-barnase
complexes (denatured,
fractionated by 2D gel
electrophoresis)

28. in a
nutshell
…

29. Whats going on now??

30. • L-ornithinase (argE) gene of E.coli was fused to the OSIPA promoter
sequence which is known to function specifically in the pollen grains.
• OSIPA – Oryza sativa indica pollen allergen
• argE gene – involved in arginine biosynthesis in E.coli and also can
deacetylate N-acetyl Phosphinothricin (N-ac-PPT) to yield
phosphinothricin (PPT).
• N-ac-PPT, a non toxic compound, but when converted in to PPT
becomes cytotoxic.
• Phosphinothricin is the active ingredient of the herbicides Basta
(bialophos) and Glufosinate.
• Homozygous transgenic rice (variety BPT 5204) plants (T2) were
obtained with argE gene by selection with hygromycin (hyg) following
Agrobacterium mediated transformation.
Abstract

31. • Transgenic rice plants expressing argE gene became completely male
sterile after application of N-ac-PPT (inducer) due to the pollen
specific expression of argE.
• The argE transgenic plants produced fertile seeds in the absence of N-
ac-PPT treatment.
• Normal fertile seeds were obtained when male sterile argE
transgenics were cross pollinated with untransformed control plants.
• Female fertility male of sterile argE transgenics is not affected by the
N-ac-PPT treatment.
• First report of induction of complete male sterility in Rice.
• This system does not require the use of restorer line.
Contd…

32. Results…

33. Table 2. Effect of N-ac-PPT on pollen fertility of argE rice transformants after treatment
with irrigation (0.2 mg/ml @ 80-90 days, 50ml/plant)
Table I. Effect of N-ac-PPT on pollen fertility of argE rice transformants after topical
application (0.075 mg/ml @ 100-110 days, 25 ml/plant)

34. Fig 2. Induction of pollen sterility in argE transgenic rice plants by N-acetyl-PPT treatment.
(Alexander staining was carried out before and after N-ac-PPT treatment)
Transformed
Untreated anther and gynoecium Treatment with N-ac-PPT
through irrigation
Treatment with N-ac-PPT by
spraying
Control

35. Fig 3. Pollen grains germination ability of UC and argE-transgenic rice plants before
and after N-ac-PPT treatment
Treated with N-ac-
PPT by spraying
Treated with N-ac-
PPT by irrigation
Untreated pollen
grains
Transformed
Control

36. (B) Plants treated with N-acetyl-
PPT through irrigation
Fig 4. Effect of N-acetyl-PPT on male fertility of argE transgenic rice plants
(A) Plants treated with N-acetyl-PPT
by spraying topically

37. (A) Treated with N-ac-PPT by
spraying
Later, they were allowed to self pollinate. UC: Panicle of untransformed control showing normal
seed setting. 9-6 & 25-3: Panicles of two argE transformants showing the failure of seed setting.
Fig 5. Seed setting ability of argE transgenic rice plants after treating with N-acetyl- PPT
(B) Treated with N-ac-PPT through
irrigation

38. Fig 6. Seed setting ability of argE male-sterile rice lines after pollination with UC plants.
(A) Treated with N-ac-PPT by
spraying
(B) Treated with N-ac-PPT
through irrigation

39. The list continues…

43. Commercial exploitation of GE male sterility
GM Canola – barnase/barstar system
• Aventis has successfully introduced a GE canola hybrid, using the barnase/barstar gene
system in 1996.
• Regulatory agencies in such countries as Canada , USA , Mexico , Europe, Australia and
Japan have approved consumption of this GE canola

44. Issues in developing Genetically engineered hybrid crops – the Indian case
• One of the DMH-11 genes, called the bar gene, made the plant resistant to a herbicide (or weed killer)
brand-named Basta, a product sold by multinational company Bayer Cropscience.
• Two other genes — that weren’t patented in India — called barnase and barstar — were used to make
mustard varieties more amenable to becoming hybrids.
• DMH-11 developed by crossing Indian and East European mustard varieties. 25-30% more yield.

45. • Hybrid varieties have had the greatest impact on increasing the world’s
feed or food resources.
• Usable male sterility systems have not yet been generated through
conventional breeding methodologies for several important crop plants.
• In such a scenario, genetic engineering becomes an efficient and rapid
approach for developing male sterile lines in these crops.
• The creation of new and efficient means of pollination control to
produce better hybrids will soon be the plant biotechnologist’s
contribution to this effort.

46. Thank You…
Queries?