Development of New Biotech Food Products:

Fruits

Presented by

Nafisa Nawal IslamClass: M.S., Roll No.: 14, Session: 2014-15

Dept. of Genetic Engineering and Biotechnology University of Dhaka

Course No.: 504

- employs the tools of modern genetics to achieve/ enhance desired traits of plants, animals & microorganisms for food production by adding / extracting selected genes

- benefit environment, society & consumer health.

Food Biotechnology

Nafisa Nawal Islam

• Fruits with vaccines

• Transgenic bananas- constitutively expressing the hypersensitive response-

assisting protein (Hrap) or plant ferredoxin-like protein (Pflp) gene from sweet pepper

- to combat Banana Xanthomonas wiltcaused by Xanthomonas campestris pv. musacearum.

• Melons, strawberries, kiwi, pineapple, mango, other fruits

Biotechnology is needed because nature does not contain all the genetic variation men desire

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Genetically engineered fruitsPapaya Resist papaya ringspot virus (PRSV) by over-expression of

the viral coat protein

Squash Resist watermelon mosaic 2, zucchini yellow mosaic, cucumber mosaic viruses

Tomato - Altered ripening to enhance fresh market value- Thicker skin & altered pectin to enhance processing value

Sugarbeet Resist glyphosate (herbicide)

Non-browning apple

- does not go brown after fresh cut; - retains all its natural flavor & taste

RSVresistant papaya

Susceptible plants

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Trait/Gene ExamplesTrait Gene

Insect Resistance Bt-toxin protein

Glyphosate resistance Bacterial EPSP synthase enzyme

Plant Virus Resistance Viral Coat Protein

Plant Bacterial Resistance P35

Male Sterility Barnase

Salt tolerance Glyoxylase I, AtNHX1

Cold Tolerance CBF transcription factors

Increased Vitamin-A content Vitamin A biosynthetic pathway enzymes

Mercury Resistance Mercuric ion reductase

Modern Plant Biotechnology & Transgenic

Plant technologies are reliant on Tissue Culture

• Propagating plants “in vitro”• Tissue culture is required to generate transgenic

plants

Reasons to use tissue culture:• Virus free reproduction

– Bananas

• Make many identical clones

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Plant Biotechnology Techniques Fall Into 2 Classes

• Identify a gene from another species which controls a trait of interest

• Or, modify an existing gene (create a new allele)

1) Gene Manipulation

Gene of Interest

IS IS

T-DNABorder

T-DNABorder

SelectableMarker

• Transferring gene into an organism to develop transgenic organisms• Technique called transformation

2) Gene Introduction

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Field test the plants- Multiple sites

multiple years

Culture plant tissue- Develop shoots

- Root the shoots

Introduce Gene to the target Plant- Agrobacterium- Gene gun

Prepare tissue for transformation

Transformation Steps

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Field Testing

Non-transgenicsTransgenics

Herbicide Resistance

Final TestConsumer Acceptance

RoundUp Ready Corn

Before After

Lab Testing The Transgenics

Regenerate Transgenic Plants on Selection Medium

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* Induced Mutation Assisted Breeding (IMAB) Many plant varieties were subjected to mutagenic agents (like

radiation) to induce mutations & then selected for the desired “new”traits that appeared.

IMAB has resulted in the introduction of new varieties of apples,citrus fruits, sugar cane & banana.

* Micro-propagation involves taking small sections of plant tissue, or entire structures such

as buds, & culturing them under artificial conditions to regenerate complete plants.

In Kenya, banana shoot tips have been successfully tissue-cultured to regenerate disease-free banana plantlets from healthy tissue.

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Food Biotechnology Applications1. High yield / agricultural productivity 2. Insect-resistant plants

- Bt toxin, Cowpea trypsin inhibitor etc.3. Disease-resistant plant4. Herbicide-resistant plant

Genetic Modificaton of fruit crops include

- Gene transformation: Apple, Cherry, Papaya

- Somaclonal variation: Orange5. Edible vaccines6. Improve value of feed crops

- Transfer a fungal enzyme (phytase) to crops to remove phytic acid

from feed & improve phosphate availability

7. Development of tools for breeding for

- improved food processing & fruit quality (texture, taste, storage ability)

- phytopathogen resistance (both in the field & post-harvest)

7) Virus-resistant plants

Figure: Procedure for putting CuMVCoat protein into plants

• Overexpression of the virus coat protein, e.g.

- cucumber mosaic virus (CuMV) in

cucumber

- PRSV in papaya

- tobacco mosaic virus in tomato.

• Expression of antiviral proteins (pokeweed)

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8) Fungus- & bacterium-resistant plants

Addition of two bacterial genes in plants

Overproduction of salicylic acid Over-expression of NPR1 gene

- encodes the master regulatory protein

for

pathogenesis-related (PR) proteinProduction of PR proteins

(b1,3-glucanases, chitinases, thaumatin-

like proteins, & protease inhibitors)

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• Over-expression of the gene encoding a Na+/H+

antiport protein in vacuole membranewhich transports Na+

into the plant cell vacuole

• Done in tomato allowing them to survive on 200 mM salt (NaCl)

• If transporters known, engineer plants to phyto-remediate toxic soils Figure: Schematic representation of ion transport in the

plant A. thaliana showing the Na+ ions being sequestered in the large vacuole

Control tomatoes at 200 mM NaCl

Transformed tomatoes at 200 mM NaCl

Tolerance to Aluminium (a growth limiting factor in acid soils; is in the early phase of R&D for several crops, including papaya.)

9) Development of stress (i.e. salt)-tolerant plants

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10) Development of senescence-tolerant plants:

Flavorful Tomatoes

• Fruit ripening is a natural aging process that involves 2independent pathways: flavor development & fruit softening.

• Tomatoes are picked when not very ripe (hard & green) toallow for safe fruit-shipping, shipped to market & treatedwith ethylene before sale at wholesale level

• Flavr Savr tomato blocked polygalactonurase (a plantenzyme that degrades plant cell wall pectins & contributes tofruit softening) synthesis by antisense.

Flavor development pathway

Fruit softening pathway

Green Red

Hard Softpolygalacturonase

antisense polygalacturonase

• Future Prospective: Could be used on any climacteric fruitsNafisa Nawal Islam

11) Modification of plant nutritional content

• Use metabolic engineering

to insert new pathways into plants or

improve expression of enzymes in existing ones

• Amino acids (corn is deficient in lysine, methionine &tryptophan; legumes are deficient in methionine &cysteine)

• Lipids (altering the chain length and degree of unsaturation is now possible since the genes for such enzymes are known)

• Low fat content

• Increased levels of antioxidants

- vitamin C, phenolics, etc.

• Improving vitamin content of plants

- Vitamin E (a-tocopherol) & vitamin ANafisa Nawal Islam

11) Modification of plant nutritional content:

Increasing the vitamin E (a-tocopherol) content

• Plants make g-tocopherol but very little a-tocopherol; they do not produce enough methyltransferase (MT)

• The Arabidopsis MT gene was cloned, expressed under the control of a seed-specific carrot promoter,and found to produce 80 times more vitamin-E in the seeds.

• Similarly, GE ‘super banana’ can be produced.Nafisa Nawal Islam

Strawberry Production in Bangladesh

• 3 varieties (RB-1, RB-2 & RB-3) have been innovated & found adaptive to the region’s soil & environmental conditions.

• 2 out of the 3 were found very impressive in size, taste & flavor.

Future prospects: Sweetness increase

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Development of cost-effective high throughput sequencing technologies

Significant increase in availability of genomic information for fruit species

Improved understanding of many important crop traits (timing of flowering, control of juvenility, ripening, shelf life etc.)

Recently developed genome-editing tools for fruit improvement

Novel biotechnological tools:

• Zinc finger nucleases (ZFNs)• Transcription activator-like effector nucleases (TALENs) • Clustered regulatory interspaced short palindromic repeat

(CRISPR) - the latest development in the genome-editing toolbox- based on bacterial form of adaptive immune system- simple, most efficient, cheap, easy to design & user-friendly

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Opportunities:

• The advent of new genome-editing tools makes it possible to manipulate the plant genome with unprecedented control.

• Engineered nucleases (i.e., ZFNs & TALENs) generate dsDNA breaks at almost any specific genomic location, allowing precise genome editing.

Absence of foreign DNA, notably

selectable markersin the final product

Introduction of genes derived from same

plant species throughsite-specific insertion,

deletion, mutation

Development of novel, consumer-acceptable GM products developed with

these technologies

Recently developed genome-editing tools for fruit improvement

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Concluding Remarks & Future Perspectives

• Fruit crops with superior phenotypes developed with the recent genome-editing tools - might be considered as non-transgenic genetically altered plants.

- can be commercializeded even in countries where GM crops are poorly accepted.

Food biotechnology has the potential to:

• Reduce levels of natural toxins in plants

• Provide simpler & faster ways to locate pathogens, toxins & contaminants

• Keep products fresher longer

• Identify ways to eliminate allergens from foods

• Ensure food security

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Thank You

Nafisa Nawal Islam