Tobacco / Molecular Farming Flashcards
Tobacco Nicotiana tabacum L.
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
contents of tobacco
harvest 1-3 t/ha dry matter
4000 - 6000 different substances
proteins ca. 1.5 %
1 to 10% nicotine in dry matter
How can tobacco be used and how can it be classified?
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
Biggest tobacco producers
China
USA
India
Brazil
EU
Types of tobacco in CH
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
biology of tobacco
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%
Production of plantlets
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
Planting in field
Optimal: 15. – 30. May
Planting density: 25’000 – 30’000 plants/ha
Planting depth: see page 17
Environment & Soil
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)
Temperature requirements
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
Water requirements
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
Harvest of different leaf positions
Harvest starts at the bottom leaves and proceeds to the top leaves (following maturation)
Total harvest period: 30 – 40 days
Harvest timepoint
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
Harvesting process
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
Storage and processing in CH
Air drying (Burley)
Hot air drying (Virgin)
Air drying takes place in a dry shelter
First phase of air drying
Leaves turn yellowish: degradation of proteins, sugars and starch. Takes 5-10 days, water loss 20-25 %, dry weight loss 10-15 %
Second phase of air drying
Leaves turn brownish: water loss 25-30 %, dry weight loss 8 -12 %.
Good ventilation is essential to avoid humidity accumulation.
Hot air drying of Virginia tobacco
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 !
Taxation
weight and humidity of each bale are measured
taxation commission (two representants of grower and two representants of factory) determines the quality
Fermentation and final processing
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
Why tobacco as a production platform?
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
Which products might be interesting (produced in tobacco)
Therapeutic proteins (antibody)
Industrial enzymes
Biopolymers
What are antibodies
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
Advantages of plant-based systems vs. hybridoma systems to produce Mabs (Monoclonal antibodies)
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
Three forms of production systems
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)
Success with plant-made monoclonal antibodies
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…
Slides 47 - 51
47 - 51
