Improvement of drought tolerance through Genetic Transformation

Improvement of drought tolerance through Genetic Transformation

Drought tolerance

Drought tolerance refers to the degree to which a plant is adopted to arid or drought conditions.

Desiccation tolerance is extreme degree of drought tolerance.

Drought tolerant plants typically make use of either C4 carbon fixation or crassulacean acid metabolism (CAM) to fix carbon during photosynthesis.

Many adaptations for dry conditions are structural, including the following:

Adaptations of the stomata to reduce water loss, such as reduced numbers or waxy surfaces.

Water storage in succulent above-ground parts or water-filled tubers.

Adaptations in the root system to increase water absorption.

Trichomes (small hairs) on the leaves to absorb atmospheric water.

Drought Tolerance – is it a Complex Trait?

Drought tolerance is a very complex trait.

However, both conceptually and functionally, drought tolerance is not a complex trait if one consider the following axioms:

1. Most of the crucial plant traits that control plant water status and plant production under drought are constitutive and not stress adaptive.

2. Plant water status, more than plant function, controls crop performance under drought.

Response of Plants to drought Response of Plants to drought

Constitutive Plant Traits Controlling Plant Water Status and Productivity under Drought

Phenology

Root traits

Plant and organ size

Leaf surface properties

Non -senescence

Stress Adaptive Plant Traits Controlling PlantProduction under Drought

Cellular compatible solutes

Anti oxidant agents

Heat shock proteins

Molecular chaperone proteins

Methods of crop Improvement for drought Methods of crop Improvement for drought

• Traditional Breeding Crossing two individuals from the same species; produces a new, improved variety; not a biotechnology procedure

• Genetic Transformation Adding a gene from another species; the essential biotechnology procedure to produce transgenics

Source: USDA

Source: USDA

Umezawa et al 2006, 17:113-122

Strategies for the genetic engineering

of drought tolerance.

Success of getting drought tolerant plants

Through…. Conventional breeding : Undesirable

linkage, time consuming and laborious. Molecular breeding : Improvement of

drought tolerance by this method overcomes the bottlenecks of traditional breeding.

Further molecular breeding may be of 2 Types they are :

Marker assisted selection :

Marker-assisted selection, are costly,

slow, require massive screening

labours to identify specific

quantitative traits, while linkage of

Agronomically important QTL´s to

undesirable traits can sometimes

occur.

Best OptionBest Option

Transgenics : A transgenic crop plant contains a

gene or genes which have been artificially inserted instead of the plant acquiring them through pollination.

The inserted gene sequence (known as the transgene) may come from another unrelated plant, or from a completely different species

What is a transgenic?What is a transgenic?

Transgene – A gene introduced in to an organism by means of genetic engineering. This gene may be from the same or a different organism or even a synthetic sequence

Ex. – Daffodil gene in golden rice

Transgenic – an organism containing a transgene introduced through genetic engineering and has become integrated in its genome.

Ex. – BT-COTTON

We can develop crops that express a “novel” trait not normally found in the existing plant

Why are Transgenics important?

Extended shelf-life tomato (Flavr-Savr)

Herbicide resistant soybean (Roundup Ready)

Steps in transgenic development

Nucleic acid extraction

Gene cloning

Gene design and packing

Transformation

Back cross breeding

Introducing the Geneor

Developing Transgenics

Steps

1. Create transformation cassette

2. Introduce and select for transformants

Transformation Cassettes

Contains

1. Gene of interest

• The coding region and its controlling elements

2. Selectable marker

• Distinguishes transformed/untransformed plants

3. Insertion sequences• Aids Agrobacterium insertion

Transformation Steps

Prepare tissue for transformation

Introduce DNA

Culture plant tissue

• Develop shoots• Root the shoots

Field test the plants

• Leaf, germinating seed, immature embryos• Tissue must be capable of developing into normal plants

• Agrobacterium or gene gun

• Multiple sites, multiple years

Plant Transformation Plant Transformation

• Plants are the easiest of higher organisms to transform

• Both physical and biological methods exist for transformation

• Until recently, only transgenic organisms in wide public release were plants

Plant Transformation Methods

Physical Chemical Biological

MicroinjectionBiolistics - gene gun/particle bombardment Electroporation Liposome mediated transformationSilica/carbon fibersmediated Transformation

PEGCalcium phosphate co-precipitationPolycation DMSO technique

A. Tum e fa c ie nsA. Rhiz o g e ne s

Virus-mediated

In planta

Microinjection This Electroporator is for low current applications such as those using small electrodes

Biolistic / Gene Gun

Biological Transformation

Agrobacterium tumefaciens Agrobacterium tumefaciens & & AgrobacteriumrhizogenesAgrobacteriumrhizogenes

Selection of transgenic plantsSelection of transgenic plants

May be of two type Negative selection : The transgenic cells acquire the ability to

survive on selective media while the non-transgenic cells are killed .

positive selection : Actively favours regeneration and growth of the transgenic cells while the non-transgenic cells are starved but not killed.

E. coli -glucuronidase gene has become the most frequently used reporter gene for the analysis of plant gene expression. Major advantage of this reporter is that it does not require DNA extraction, electrophoresis or autoradiography.

Drought Stress Genes Identified

Low temperature responsive genes/proteins in plants

Plant species Genes /ProteinsArabidopsis Rab 18, Kin 1,2

Brassica Oleracea 7K .Da proteins

Cucumis sativus Proteins of 25,50,70,80 K Da

Soyabean HSP 70

Oat 45,75 K Da

Tomato Proteins 27,35 K

Rice Proteins of 95,75,50,25, K Da

Wheat Wes 19, 120

Maize Adh-1

Water stress responsive genes/proteins in plants

Plant species Genes /Proteins

Sorghum BADH 1,15 clone

Cotton LEA Proteins

Sunflower Hsp 17

Soya bean P 5 CR Clone

Oat LEA Proteins

Rice Rab 16 A, Rab 16 B, Rab 16 C

Peas PPs B 12 Clone

Wheat Emla, lb WSP-23

Maize Rab 17,28,LEA Proteins

Expression of the Nicotiana protein kinase (NPK1) enhanced drought tolerance in transgenic maizeExpression of the Nicotiana protein kinase (NPK1) enhanced drought tolerance in transgenic maize

Huixia Shou, Patricia Bordallo and Kan Wang (2004)Huixia Shou, Patricia Bordallo and Kan Wang (2004)

Effect of drought on Apparent Photosynthesis (AP) rate of transgenic and non-transgenic maize plants

Events Apparent photosynthetic rate decrease (%)

Transgenic event

A4-1 19

A4-2 13.8

A4-9 5.3

P84 24.4

Non-transgenic control

Hi II 47Huixia e t a l (2004)

Effect of drought on final leaf number of transgenic and non-transgenic maize

Events Leaf number increases (%)

Transgenic event

A4-1 3.3

A4-2 9.6

A4-9 3.3

P84 3.1

Non-transgenic check

Hi II -2.2Huixia e t a l (2004)

Kernel appearance of transgenic (A4-1, A4-2, A4-9, P84-12) and non-transgenic (Hi II) plants under well-watered (WW)

and drought-stressed (DS) conditions.

Kernel appearance of transgenic (A4-1, A4-2, A4-9, P84-12) and non-transgenic (Hi II) plants under well-watered (WW)

and drought-stressed (DS) conditions.

Modulation of the polyamine biosynthetic pathway in transgenic rice confers tolerance to drought stress

Teresa Capell, Ludovic Bassie, and Paul Christou (2004)

Response of rice plants to drought stress

(A) Phenotype of 2-montholdwild-type (WT) and transgenic plants with adc gene of Datura stramonium (84-2 and 84-9 lineages) growing in soil after drought stress (6 days).

(B) Close-up of rice leaves (wild type on the left and 84-2 on the right)

Response of rice plants to drought stress

(A) Phenotype of 2-montholdwild-type (WT) and transgenic plants with adc gene of Datura stramonium (84-2 and 84-9 lineages) growing in soil after drought stress (6 days).

(B) Close-up of rice leaves (wild type on the left and 84-2 on the right)

Tolerance of Mannitol-Accumulating Transgenic Wheat to Water Stress and Salinity

Tilahun Abebe, Arron C. Guenzi, Bjorn Martin, and John C. Cushman(2003)

Effect of water stress and salinity on the growth of -mtlD and +mtlD plants.

A)The mannitol-accumulating transgenic wheat line P2-19-1 (-mtlD) and the non accumulating P1-13-1 (+mtlD) were stressed by withholding water. B) supplementing the nutrient solution with 150 mM NaCl (B) for 30 days

Effect of water stress and salinity on the growth of -mtlD and +mtlD plants.

A)The mannitol-accumulating transgenic wheat line P2-19-1 (-mtlD) and the non accumulating P1-13-1 (+mtlD) were stressed by withholding water. B) supplementing the nutrient solution with 150 mM NaCl (B) for 30 days

Phenotypes observed in transgenic wheat plants. LinesP2-16-1 and P2-19-1 were transformed with plasmid pTA2 for accumulation of mannitol in the cytosol (mtlD). Line P1-13-1 was transformed with pAHC20 (mtlD) and did not accumulate mannitol. In the fertile mtlD plants, mannitol content ranged from 0.4 to 0.7 micro mol/g fresh weight.

Phenotypes observed in transgenic wheat plants. LinesP2-16-1 and P2-19-1 were transformed with plasmid pTA2 for accumulation of mannitol in the cytosol (mtlD). Line P1-13-1 was transformed with pAHC20 (mtlD) and did not accumulate mannitol. In the fertile mtlD plants, mannitol content ranged from 0.4 to 0.7 micro mol/g fresh weight.

A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances cold tolerance in transgenic plants

Jong Cheol Kim, Sang Hyoung Lee, Yong Hwa Cheon, Cheol-Min Yo, Soo In Le, Hyun Jin Chun, Dae-Jin Yu. (2001)

Freezing tolerance of SCOF-1transgenic Arabidopsis plants . The transcription of SCOF-1 is specifically induced by low

temperature and absicsic acid.

WT-wild type

Cold tolerance of SCOF-1 transgenic tobacco plants

Wide range of substrate specificity

Highly conserved structure (NADH or NADPH binding region, catalytic tetrad)

Occurrence: from bacteria to Homo sapiens

polyol pathway

detoxification of reactive aldehydes

About the aldose reductase superfamily in general:

Development of first shoots on medium

Transgenic plantletsin soil

Role of ALR For Regeneration of the Transgenic wheat for drought tolerance

Fertile ALR spikes

ALR EXPRESSING TRANSGENIC WHEAT LINES WITH IMPROVED DROUGHT TOLERANCE

CTR

AL

R A

CT

. (A

. U

.)

10

50

ALR ACTIVITY IN LEAF EXTRACTS

20

30

40

TR304 TR288

CTR

(% D

RO

UG

HT

ST

R./

UN

ST

R.)

90

110

HARVEST INDEX(% DROUGHT STR./UNSTR.)

95

100

105

TR304 TR288

CTR

85

125

95

105

115

TR304 TR288

THOUSAND KERNEL WEIGHT(% DROUGHT STR./UNSTR.)

(% D

RO

UG

HT

ST

R./

UN

ST

R.)

Is there any commercial success for drought tolerance through Transgenics ?

Though research on developing transgenic crops has gained momentum across the world and producing commercial results, but they were restricted to biotic stresses only.

As most abiotic stresses are quantitative traits and commercial success for these traits specially for drought is still far from reality.

Agriculture Transgenics On the Market

Source: USDA

Insect resistant cotton – Bt toxin kills the cotton boll worm• Transgene = Bt protein

Insect resistant corn – Bt toxin kills the European corn borer• Transgene = Bt protein

Normal Transgenic

Virus resistance - Papaya resistant to papaya Ring spot virus• Transgene = virus coat protein

Source: Monsanto

Herbicide resistant crops Now: soybean, corn, canola Coming: sugar beet, lettuce, strawberry alfalfa, potato, wheat • Transgene = modified EPSP Synthase or Phosphinothricin-N-acetyltransferase

Next Generation of Ag Biotech Products

Source: Minnesota Microscopy Society

Golden Rice – increased Vitamin A content (but not without controversy)Transgene = three pathway enzymes

Sunflower – white mold resistanceTransgene = oxalate oxidase from wheat

Transgenics in world scenario

Top transgenic crop producers 2010

Ranking by million hectares in GM crops Ranking by % of farmland in GM crops1. USA 64 Paraguay 66% 12. Brazil 21.4 Argentina 65% 23. Argentina 21.3 Uruguay 35% 34. India 8.4 USA 32% 45. Canada 8.2 Brazil 23% 56. China 3.7 Canada 13% 67. Paraguay 2.2 South Africa 11% 78. South Africa 2.1 India 4% 89.= Uruguay 0.8 China 2% 99.= Bolivia 0.8

1. Source: ISAAA Brief 41-2010, www.isaaa.org/resources/publications/briefs/41/default.asp2. Source: Heinemann, Jack A. (2010) Hope Not Hype, Penang: Third World Network

ConclusionConclusion

The conventional breeding strategies have been useful in some cases for the improvement of the drought tolerance. But the success rate is few and far.

Hence Gene manipulations are being looked hopefully for raising the drought tolerant genotypes

Thank youThank you