Genetic engineering can be used to develop drought tolerant crops. Approaches include engineering genes for organic osmolytes like glycine betaine, trehalose, proline, and mannitol. Genes encoding regulatory enzymes and LEA proteins can also be engineered for drought tolerance. Transgenic crops overexpressing antioxidant enzymes or transcription factors involved in the plant stress response have shown enhanced drought tolerance. Commercially available drought tolerant crops include Monsanto's DroughtGard maize which expresses a cold shock protein from bacteria.

1. GENETIC ENGINEERING FOR
DROUGHT
Presented by-
B. RACHANA
RAD/18-18
Dept. of Genetics and
Plant Breeding
Submitted to-
Dr. S. Vanisri
Professor
Institute of Biotechnology (IBT)

2. Contents
• Introduction
• Critical stages for drought stress
• Methods of crop improvement
• Genetic engineering
• Approaches for engineering drought
tolerance
• GM crops for Drought tolerance
• Marker Assisted selection (MAS)
• Conclusion

3. INTRODUCTION
• Drought is “ the inadequacy of water availability, including precipitation and
soil moisture storage capacity, in quantity and distribution during the life cycle of
a crop to restrict expression of its full genetic yield potential” (Sinha, 1986).
• Drought is governed by various factors, the most prominent being extremes in
temperature, photon irradiance and scarcity of water.
• It has marked effect on cellular processes, plant growth, development and
economic yield.

4. CRITICAL GROWTH STAGES OF CROP FOR DROUGHT STRESS

5. Stress adaptive response to Drought in Plants

6. Conventional breeding:
It involves the crossing between
two individuals from a same
species, produces a improved
variety.
Genetic engineering:
It involves the DNA
manipulation to alter the
plant/organism by introducing
genes from another
plant/organism.
Methods of crop improvement for drought tolerance

7. Why Genetic engineering for Drought Tolerance?
• The conventional breeding has contributed significantly in the
development of drought tolerant high yielding crop varieties
since centuries.
• However the pace to develop new crop cultivars has been
relatively slow, laborious, undesirable linkage and limitation of
fertility barriers for hybridization.
• The advent of genetic engineering approaches enabled the
researchers to overcome such problem.

8. Genetic engineering

9. APPROACHES
1. Engineering of genes for organic osmolytes
2. Engineering of genes encoding enzymes for regulatory genes
3. Engineering of genes encoding LEA Proteins
4. Genetic engineering of detoxifying genes
5. Engineering of genes encoding transcription factors

10. • Osmotic stress induced by high drought is one of the major causes for cellular
and metabolic dysfunction which ultimately affects plant growth and
productivity.
• To cope up with such harsh environmental conditions, plants alter
physiological, molecular and metabolic function to produce low molecular
weight, electrochemically neutral small molecules, maintaining cellular
osmotic homeostasis by lowering osmotic potential of a cell under stress.
• Such compounds are known as compatible solutes or osmolytes, which are
nontoxic and do not interfere with cellular metabolism when accumulated in
cytosol.
• Many genes play an important role in the synthesis of osmoprotectants in
stress tolerant plants like proline, glycine betaine and polyamines, mannitol,
trehalose and galactinol which accumulate during osmotic adjustment, these
organic solutes play an important role in induction of drought tolerance.
1. Engineering of genes for Organic osmolytes

11. Genes for Glycine betaine Biosynthesis-
- Effective osmolyte accumulated during water stress by Bacteria,
Cyanobacteria and members of Chenopodiacae.
- Several crop like potato, tomato, rice, tobacco do not accumulate it
but can be made to accumulate by developing transgenics.
- GB is synthesized from choline in two-step oxidation processes
- It is obtained in two step-
Choline Betaine aldehyde Glycinebetaine
CDH BADH
1. Choline DeHydrogenase (CDH) in E. coli and
Choline monoxygenase in Spinach.
2. Betaine Aldehyde DeHydrogenase (BADH).
E. coli betA gene encoding CDH has been cloned and used in
transgenesis.

12. Genes for Trehalose Biosynthesis -
• Trehalose is a non-reducing sugar.
UDP-Glucose-6-phosphate Trehalose-6-phoshate
TPS
TPP
(TPS-Trehalose-6-phosphate synthase )
(TPP- Trehalose Phosphatase) Trehalose
• TPS1 Gene from budding yeast have been cloned and used for
engineering drought and salinity resistance in crop plants.
( Dan Tau et al.,2008)

13. SPONTANEOUS
CYCLIZATION
Genes for Proline Biosynthesis -
• In plant it is produced from ornithine under normal
condition but under stress it is made directly from
glutamate.
• P5CS – PYRROLINE-5-CARBOXYLATE
SYNTHATASE
• P5CR- PYRROLINE-5-CARBOXYLATE
REDUCTASE
• Gene was obtained from
Soybean and Mothbean
(Baocheng Zhu et al.1998. ,Moss J.P.Etal.1992.)
P5CS
P5CS
GLUTAMATE
ᵞ-GLUTAMYL PHOSPHATE
GLUTAMIC -ᵞ-SEMIALDEHYDE
∆-PYRROLINE-5-CARBOXYLATE
P5CR
PROLINE

14. Genes for mannitol biosynthesis
HPI hexose phosphate isomerase;
MTLD mannitol-1-phosphate dehydrogenase;
M1PP mannitol-1-phosphate phosphatase.
mtlD1 gene was isolated from E.coli and it imparts drought tolerance

15. Examples of organic osmolytes in Transgenic plants
ORGANIC
OSMOLYTES
CROP GENES
INTRODUCED
REFERENCE
Glycine betaine Tobacco CMO Shen et al, 2002;
Zhang et al, 2008
Maize betA Quan et al, 2004
Potato COX Ahmad et al, 2008
Rice codA Kathuria et al, 2009
Proline Soybean - Ronde et al, 2004
Tobacco P5CR Gubis et al, 2007
Popisilova et al, 2011
Trehalose Tobacco TPS1 Romero et al, 1997;
Karim et al, 2007
Rice otsA and otsB Garg et al, 2002
Mannitol Wheat mtlD Abebe et al, 2003
Baksh et al. (2015), Emir. J. Food Agric.

16. Overexpression of Bacterial mtlD Gene in Peanut
Bhauso et al. (2014)
Bacterial mtlD gene coding for
mannitol 1-phosphate dehydrogenase
under the control of constitutive
promoter CaMV35S was introduced
and overexpressed in the peanut.

17. Stress-induced expression of choline oxidase in potato
Drought stress analysis of non-transgenic (NT) and
transgenic (SC) plants
• This study demonstrated
that engineering for GB
synthesis is an effective
way to impart stress
tolerance to non
accumulators, such as the
potato plant.
• Was conducted using the
popular and economically
important cultivar,
‘‘Superior’’.
• The transgenic lines
harboring the codA gene
enhanced tolerance, was
driven by a stress-inducible
promoter.
Ahmad et al. (2008)

18. Trehalose accumulation in rice plants confers high
tolerance levels to drought
NTC – non transformed
R80, A05 – transgenic; A-Control B-drought
Garg et al. (2000)

19. • The various stress-related genes, metabolites such as abscisic acid (ABA)
or osmotically active compounds and some other specific proteins are
trigged under stress condition.
• Several groups of kinases like Mitogen-activated protein kinase (MAPK),
Calcium-Dependent Protein Kinase (CDPK) and Sucrose Non-fermenting-1
(Snf1)-Related Protein Kinase (SnRK) are also involved in perceiving the
stress stimuli, along with ABA, and transduce the signal downstream to the
transcription factor and their target genes.
• ABA is a significant player that is involved in plant growth and
development when plants are subjected to stressed condition.
• This group also includes protein phosphatases such as PP2C, PI turn over
related proteins such as PLC, PLD, PIP5K, DGK, and PAP, and
calmodulin-binding protein and Ca 2+ -binding proteins.
2. Engineering of genes encoding enzymes for
regulatory genes

20. Kinase families overexpressed to generate drought-tolerant
transgenic plants
Paul and Roy Choudhury (2018), Springer.

21. Paul and Roy Choudhury (2018), Springer.

22. Overexpression of SlMAPK3 Positively Regulates Tomato
Tolerance to Drought Stress
Muhammad et al. (2019), Molecules
Overexpression of SlMAPK3
increased tolerance to Cd2+
and drought as reflected by an
increased germination rate
and improved seedling
growth.

23. Overexpression of the AtLOS5 gene increased abscisic acid level
and drought tolerance in transgenic cotton
• Six-week-old seedlings of non-transgenic
Z35 and the transgenic cotton lines L5 and
L8 subjected to drought stress for 5d.
• LOS5 -overexpressing cotton showed less
wilting compared with Z35 plants
• Overexpressed Arabidopsis
LOS5 gene in cotton variety.
• The developed lines of
transgenic cotton had increased
endogenous ABA (25%) and
proline (20%) when compared
with the control plants.
• In addition to it, increased
activity antioxidants
(superoxide dismutase,
peroxidase, and ascorbate
peroxidase) was also recorded;
leading ultimately to better
drought tolerance response.
Yue et al. (2012), J. Exp Bot.

24. • Late Embryogenesis Abundant (LEA) proteins, are highly
expressed during later phase of embryo development.
• This group of proteins are induced under water-limiting
condition and also accumulated during seed or pollen
development or some stages of shoot and root development
when water is limiting.
• They help to maintain structure of cellular membranes, ionic
balance, water binding and acts as molecular chaperons under
drought stress conditions.
• LEA proteins were classified into at least seven groups (nine
groups in Arabidopsis thaliana based on amino acid sequence
homology and specific motifs).
3. Engineering of genes encoding LEA Proteins

25. Examples of LEA proteins in transgenic crop plants
Gene Source Crop Reference
HVA1 Barley Rice Xu et al., 1996
HVA1 Barley Wheat Siwamani et al.,
2000
PMA1959 and
PMA80 LEA
Wheat Rice Cheng et al.,
2002
ME-lea N4 Brassica napus Lactuca sativa L. Park et al., 2005
ME-lea N4 Brassica napus Brassica
campestris L.
Park et al., 2005
Baksh et al. (2015), Emir. J. Food Agric.

26. ZmLEA14tv gene from Maize Landrace Enhance the Drought
Tolerance of Transgenic Maize and Tobacco
Minh et al. (2019), Agronomy.
• The coding region of ZmLEA14tv was
isolated from Z. mays cv. Tevang 1 and
then cloned.
• Tobacco significantly improved the
recovery ability, while the enhanced
ZmLEA14tv transgenic maize showed
better germination and growth in drought
simulation conditions.

27. • Oxidative stress caused by high drought leads to the generation of
ROS which is detrimental for plant growth and survival.
• Excess ROS accumulation causes protein oxidation, membrane
lipid peroxidation, DNA and RNA damage and may even lead to
cell death.
• In order to maintain cellular homeostasis, plants have developed a
complex system of ROS scavenging enzymes.
• The common antioxidant enzymes are superoxide dismutase
(SOD), ascorbate peroxidase (APX), catalase (CAT) and
glutathione reductase (GR).
• Peroxidases and catalases operate with SOD for antioxidant
mechanism.
4. Genetic engineering of detoxifying genes
O2 2 H2O2 H2O + O2
SOD CATALASE

28. Examples of antioxidants for inducing drought
tolerance in transgenic plants
Gene Source Crop Reference
Cu/Zn SOD Oryza sativa Tobacco Badawi et al., 2004
Cu/Zn SOD Avicennia
marina
Rice Prashanth et al., 2008
Mn-SOD Nicotiana
tabacum
Alfalfa McKersie et al. 1993, 1996
Capx Pisum sativum Tomato Wang et al., 2005, 2006
APX3 Arabidopsis
thaliana
Tobacco Yan et al., 2003
Cu/Zn SOD Saussurea
involucrata
Tobacco Zhang et al., 2017
Hussain et al. (2018), Frontiers in plant science.

29. Cu/Zn superoxide dismutase gene (SiCSD) from Saussurea
involucrata, enhances drought stress in tobacco
Zhang et al. (2017), Canadian Journal of
Plant Science.
Comparision of survival rate between
SiCSD-overexpressing transgenic and
WT plants under drought stress. Seeds
of WT and transgenic lines (3 and 5)
were germinated and grew on soil and
then water was withheld for 11 d.
SiCSD transgenic tobacco plants
have higher relative survival rate,
relative water content, antioxidant
enzyme activity, and higher
photosynthesis efficiency
compared with WT plants.

30. • Transcription factors are the proteins that bind DNA specifically, transcribe
and regulate genes.
• They are terminal transducer in a signaling cascade and are able to regulate the
expression of downstream genes involved in stress responses by recognizing
and binding to conserved cis-acting sequences in their promoter
• Identification and characterization of several groups of TFs and numerous
studies have been made to improve stress tolerance by genetic manipulation of
these TFs.
• The most important TF families involved in abiotic stress tolerance are
AP2/EREBP (Apetala2/Ethylene-Responsive Element- Binding Protein),
MYB, WRKY, NAC (NAM, ATAF, and CUC) and bZIP (basic leucine zipper
domain) (Golldack et al. 2014).
5. Engineering of genes encoding transcription factors

31. TF’s families overexpressed to generate drought-
tolerant transgenic plants
Paul and Roy Choudhury (2018), Springer.

35. Maize TF ZmWRKY40 Confers Drought Resistance in Transgenic
Arabidopsis
Wang et al. (2018), Int. J. Mol. Sci.
Overexpression of
ZmWRKY40 could
improve drought
tolerance in transgenic
plants possibly by
regulating ROS
scavenging and
enhancing the expression
levels of stress-
responsive genes.

36. Monsanto’s Transgenic Drought Tolerant Maize
31-03-2015
• The first drought-tolerant GM crop is
MON 87460, a maize (Zea mays L.)
product developed by Monsanto
Company in 2009 and first planted in the
United States in 2013.
• Drought Gard™ increased 5.5-fold from
50,000 ha in 2013 to 2,75,000 ha in 2014.
• MON 87460 expresses cold shock
protein B (CSPB) from Bacillus subtilis
to impart drought tolerance.
• CSPB protein in MON 87460 maintains
normal cellular functions under drought-
stress conditions by preserving RNA
stability and translation
Wang et al. (2015), Regulatory Toxicology and Pharmacology.

37. • Reduces crop losses only modestly during moderate droughts and
will not reduce the crop’s water requirements.
• Despite many years of research and millions of dollars in
development costs, Drought Gard doesn’t outperform the non-
engineered alternatives.
• The fact that drought is not predicable also makes it difficult for
farmers to decide whether it is worthwhile to buy Drought Gard seed
prior to the growing season.
• The report estimates that cspB corn would increase the overall
productivity of the U.S. corn crop by only about one percent.
• It does not improve water use efficiency.
Monsanto’s “DroughtGard”
Corn Barely a Drop in the Bucket

38. GolS2 - Transgenic Drought Tolerant Rice
• Collaboration with researchers from CIAT in
Colombia and the Japanese International
Research Center for Agricultural Sciences
(JIRCAS) in Japan and was developed in 2017.
• Galactinol synthase (GolS) is an enzyme
needed to produce important sugars called
galactinol.
• The transgenic rice modified with a gene
(AtGolS2) from the Arabidopsis plant yield
more than unmodified rice when subjected to
stress brought by natural drought.
• The greatest barrier to commercial availability
is that they used genetically modified (GM)
technology to generate the GolS2 transgenic
rice.
Selvaraj et al. (2017), Plant Biotechnology.

39. Other GM crops for Drought – in Pipeline
Sugarcane
• Gene from a grass species called Erianthus
arundinaceus, which is related to the Saccharum or
sugarcane sub-family have been inserted for drought
tolerance and developed 18 transgenics at SBI,
Coimbatore.
• The Institute has applied for permission from the
Review Committee on Genetic Manipulation (RCGM)
for Biosafety Research Level 1 trials in confined field
conditions.
Wheat
• A gene (HB4) that confers sunflower seed with drought
tolerance was identified in 1990’s.
• In 2007, HB4 was transferred to wheat and yields 25%
more than conventional wheat under drought condition.
• Syngenta plans to launch hybrid wheat in the U.S. by
2020 and new durum wheat will be a breakthrough in
drought-tolerant technology.

40. Marker Assisted Selection (MAS)
• The high-yielding wheat cultivar ‘HD2733’ sensitive to drought and is used as the
recurrent parent.
• ‘HI1500’ released for water-limiting conditions and carrying drought-tolerant QTLs
(Xbarc68-101+ Xgdm93+ Xgwm165) was used as donor parent.
• This study developed five wheat lines with inbuilt tolerance to drought stress using
MABC approach employing three QTLs in an initial population of 516 BC1F1 plants.

42. Grain yield in five improved lines of ‘HD2733’
• The three QTLs combined through MABC led to the development of improved lines that
are tolerant to drought stress.
• The improved lines have desirable morpho-physiological characters, in tandem with high
chlorophyll content, low canopy temperature, high normalized difference vegetation index
and at par grain yield compared to the original parent ‘HD2733’.
• These drought stress-tolerant lines could be the future products for release as new
improved wheat cultivars.

43. • FUNAABOR-2 is a popular rice variety grown across western states of Nigeria.
• A combination of phenotypic and MAS to improve grain yield of FUNAABOR-2
under drought stress (DS) at the reproductive stage via introgression of two drought
quantitative trait loci (QTLs), qDTY12.1 and qDTY2.3.

44. • Foreground selection was carried out using peak markers RM511 and
RM250, associated with qDTY12.1 and qDTY2.3, respectively, followed
by recombinant selection with RM28099 and RM1261 distally flanking
qDTY12.1.

45. BC1F2-derived introgressed lines and their parents were evaluated under DS and non-
stress (NS) conditions during the 2015–2016 dry season.
• Overall reduction of grain yield under DS compared to NS was recorded.
• Introgressed lines with qDTY12.1 and qDTY2.3 combinations showed higher yield
potential compared to lines with single or no QTL under DS, indicating significant
positive interactions between the two QTLs under the FUNAABOR-2 genetic
background.
• Pyramiding of qDTY12.1 and qDTY2.3 in the FUNAABOR-2 genetic background led to
higher grain yield production under DS and NS.

46. Conclusion
• Although few transgenic plants have been developed so far, progress has been
rather limited regarding producing such crops for field condition or
commercialization.
• There is a need to address various regulatory obstacles for commercial release of
various transgenic crops so that the benefit of this wonderful technology may reach
the consumers, the end users.
• Application of techniques which are efficient, precise and reliable, like the
CRISPR/Cas system will result in the development of non-genetically modified
(Non-GMO) crops with the desired trait that can contribute to increased yield
potential under stress conditions.
• Also alternatives to GE—classical breeding, improved farming practices, or crops
naturally more drought-tolerant such as sorghum and millet—can produce better
results, often at lower cost.