Tobacco / Molecular Farming

Question 1

Tobacco Nicotiana tabacum L.

Answer 1

Solanaceae

Annual plant (some other species perennial)

origin: south and central america; arrived in Europe via Kolumbus

Alloploid genome (2n = 4x = 48)

Harvest organs: leaves

Question 2

contents of tobacco

Answer 2

harvest 1-3 t/ha dry matter

4000 - 6000 different substances

proteins ca. 1.5 %

1 to 10% nicotine in dry matter

Question 3

How can tobacco be used and how can it be classified?

Answer 3

Tobacco can be smoked, sniffed or chewed

classification accordin gto its use in:
Cigars and pipes tobacco
orient tobacco (added to strengthen taste)
cigarettes tobacco
chewing tobacco

Question 4

Biggest tobacco producers

Answer 4

China
USA
India
Brazil
EU

Question 5

Types of tobacco in CH

Answer 5

Variety Burley
type of drying: natural
drying device: shelter
color after drying: brown

variety virgin
type of drying: Artificial
drying device: Oven
color after drying: yellow

Question 6

biology of tobacco

Answer 6

shoot: erect, up to 2m

short day plant

large, egg shaped leaves with gland hairs containing nicotine

Nicotine is produced in roots and is delivered to leaves via xylem

seeds in capsules (1500 - 4000 seeds per capsule)

one plant develops up to 150 capsules

oil content of seeds: 35%

Question 7

Production of plantlets

Answer 7

Mostly production of plantlets on site (in greenhouses or plastic tunnels)

Sowing: 1. – 15. March

Seed quantity for 1 ha: 5 g seeds ~ 24’000 – 30’000 seedlings

Sowing method:
- mixed with sand
- in water with the watering can

Picking at 4-leaf-stage (approx. 4 – 6 weeks after sowing)

Greenhouse area requirements for one ha: 50 – 75 m2

Hardening before planting: reduction of water supply (better root formation)

Sometimes floating nursery cultivation

Question 8

Planting in field

Answer 8

Optimal: 15. – 30. May

Planting density: 25’000 – 30’000 plants/ha

Planting depth: see page 17

Question 9

Environment & Soil

Answer 9

Light soils: promote the development of lateral roots
Heavy soils:
- only main root develops, lateral roots develop weakly
- limited capacity to acquire water and nutrients
- bad air circulation: risk of root diseases
The heavier the soil, the worse is tobacco quality.

Virgin tobacco is very sensitive to N. High wind sensitivity (leaf breakage)

Question 10

Temperature requirements

Answer 10

Minimal germination temperature 10 – 12 °C

Optimal growth temperature > 20 °C to 30 °C

Temperatures > 38 °C: burning symptoms in leaves - Temperatures < 3 °C: chilling and frost damages

Optimal night temperature: approximately 20 °C

Question 11

Water requirements

Answer 11

High during main growth period (approx. 4 weeks after planting) → 100 mm/month; irrigation is often required

Water requirements are even higher for Virginia tobacco

Water supply must be optimal:
- optimal supply: thin, soft leaves
- water deficit: thick leaves, difficult to dry

No rainfall during maturation → Maturation (import of nicotine)
requires senescence processes; leaves must start to turn yellow

Question 12

Harvest of different leaf positions

Answer 12

Harvest starts at the bottom leaves and proceeds to the top leaves (following maturation)

Total harvest period: 30 – 40 days

Question 13

Harvest timepoint

Answer 13

Virgin and Burley are usually decapitated to facilitate prolonged leaf growth (break apical dominance)

Technical maturity: leaves reach maximum weight → best for drying

Leaf tips turn yellow, most parts of the lamina are green, veins are white

Leaves detach easily from the stem

Question 14

Harvesting process

Answer 14

Only dry leaves are harvested

First selection in the field (too small, damaged or bad leaves)

Harvested leaves should not be exposed to direct sunlight

Leaves should be bundled 12-24 h after harvest

Question 15

Storage and processing in CH

Answer 15

Air drying (Burley)
Hot air drying (Virgin)
Air drying takes place in a dry shelter

Question 16

First phase of air drying

Answer 16

Leaves turn yellowish: degradation of proteins, sugars and starch. Takes 5-10 days, water loss 20-25 %, dry weight loss 10-15 %

Question 17

Second phase of air drying

Answer 17

Leaves turn brownish: water loss 25-30 %, dry weight loss 8 -12 %.

Good ventilation is essential to avoid humidity accumulation.

Question 18

Hot air drying of Virginia tobacco

Answer 18

Drying through supply of warm air

First at relatively low temperatures (28-35 °C), then at higher temperatures (65-75 °C)

Whole duration of drying process: 4-7 d; Air drying: 4-10 weeks !

Question 19

Taxation

Answer 19

weight and humidity of each bale are measured

taxation commission (two representants of grower and two representants of factory) determines the quality

Question 20

Fermentation and final processing

Answer 20

Tobacco leaves are piled, temperature increases to 55-60 °C.

Enzymatic degradation of various proteins (weeks to 6 months)

During fermentation, typical tastes of tobacco develop

After fermentation, cigars are rolled with special leaf sequences

Question 21

Why tobacco as a production platform?

Answer 21

Genetically well studied and can easily be manipulated (transgenic plants since 1980s)

One of the best-studied platforms today for expressing recombinant biopharmaceuticals

Classified as a non-food, non-feed crop

High biomass production in field & greenhouse

Question 22

Which products might be interesting (produced in tobacco)

Answer 22

Therapeutic proteins (antibody)
Industrial enzymes
Biopolymers

Question 23

What are antibodies

Answer 23

Bacteria and viruses (‘antigens’) are recognized by human immune system via proteins at their surface

‘Antibodies’ bind at these sites and activate our immune response

They are composed of a heavy and a light chain which need to be glycosylated and combined via sulfide-bonds

Important: cancer therapy, diagnostics

Question 24

Advantages of plant-based systems vs. hybridoma systems to produce Mabs (Monoclonal antibodies)

Answer 24

Lower production costs

Higher flexibility (rapid establishment of platform; easier to generate platform at large scales)

No contamination risk with mammalian pathogens

Thereby: Potential establishment of personalized medicine

Question 25

Three forms of production systems

Answer 25

Stable expression (mostly leaf-based): Transgenic plants
Nuclear or plastidic expression, depending on post-translational requirements of the target protein: Glycosylated antibody proteins need to be coded in nuclear genome and need to have peptide signal sequences to be transported to the endoplasmatic reticulum for assembly of light and heavy chain

Transient expression system: Infiltrated plants
Foreign genes are introduced via vacuum-infiltration of leaves of intact, non-transgenic plants with Agrobacterium solution; T-DNA with or without additional, virus-derived components for extra- chromosomal gene-expression. Production for several days; mostly in N. benthamiana (Fig. to the right); also in potato, pea

In vitro culture systems: Plant biomass in sterile conditions
Large-scale production in bioreactors of plant suspension cells, mosses, hairy roots (they are elongating forever and are transformed with Agrobacterium
rhizogenes containing Ri-plasmid; similar to A. tumefaciens)

Question 26

Success with plant-made monoclonal antibodies

Answer 26

1.) Recent success stories were achieved by the use of N. benthamiana for diseases such as…
* Non-hodgkin lymphoma (blood cancer)
* Anthrax (infectious disease)
* Ebola (infectious disease)
* West Nile Encephalitis (infectious disease) *…

2.) Still hampering the success: Recombinant proteins produced e.g. in N. benthamiana are degraded to a high extent by proteases within the leaf once the proteins are produced. Researchers are now on their way to find specific protease inhibitors to act against this (Grosse-Holz et al. 2018, Plant Biotechnology Journal 16, 1797-1810).

3.) Outlook also into other processes…

Question 27

Slides 47 - 51

Answer 27

47 - 51