U.S. patent application number 13/596999 was filed with the patent office on 2012-12-20 for mutagenized tobacco plant as seed culture for production of oil for energetic, industrial and alimentary uses.
This patent application is currently assigned to AEP-ADVANCED ECOPOWER PATENTS S.A.. Invention is credited to Corrado FOGHER.
Application Number | 20120323027 13/596999 |
Document ID | / |
Family ID | 39106288 |
Filed Date | 2012-12-20 |
United States Patent
Application |
20120323027 |
Kind Code |
A1 |
FOGHER; Corrado |
December 20, 2012 |
MUTAGENIZED TOBACCO PLANT AS SEED CULTURE FOR PRODUCTION OF OIL FOR
ENERGETIC, INDUSTRIAL AND ALIMENTARY USES
Abstract
The present invention relates to the development of tobacco
plants, modified through mutagenesis techniques, interspecific
hybridisation followed by poliploidisation and recombinant DNA
technologies, characterised by the fact of being capable of
producing a very high amount of seeds and their use for the
production of oil for energetic and industrial scopes, such as
combustion oil, biodiesel and lubricating oil, and for animal and
human alimentation.
Inventors: |
FOGHER; Corrado;
(Casalmaggiore, IT) |
Assignee: |
AEP-ADVANCED ECOPOWER PATENTS
S.A.
Locarno
CH
|
Family ID: |
39106288 |
Appl. No.: |
13/596999 |
Filed: |
August 28, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12531202 |
Nov 12, 2009 |
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PCT/IB2007/053412 |
Aug 27, 2007 |
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13596999 |
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Current U.S.
Class: |
554/12 ;
554/9 |
Current CPC
Class: |
Y02E 50/10 20130101;
A01H 1/06 20130101; Y02E 50/30 20130101 |
Class at
Publication: |
554/12 ;
554/9 |
International
Class: |
C11B 1/06 20060101
C11B001/06; C11B 1/10 20060101 C11B001/10 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 14, 2007 |
IT |
RM2007A000129 |
Claims
1-13. (canceled)
14. A method for the extraction of oil from tobacco seeds wherein
the oil yield is between the 70 and the 95% of the oil contained in
said seeds comprising the following steps: a) mechanically
extracting said oil by pressing with helical press and obtaining
oil and a residual oilcake; b) filtering said oil extracted in step
a) with paper or cloth filters.
15. The method according to claim 14 further comprising step c)
chemically extracting by the use of solvents the residual oil
contained in the oilcake obtained in step a).
16. The method according to claim 14 wherein said oil is extracted
from seeds.
17. The method according to claim 15 further comprising step d)
refining the oil obtained in steps a) or b), and c).
18-23. (canceled)
Description
BACKGROUND ART
[0001] Tobacco has been cultured, in the beginnings, as an
ornamental and as a medical plant, imposing itself subsequently as
an essentially luxury good getting into human culture and modifying
human customs and habits.
[0002] Tobacco has, amongst the agricultural plants, a position
that is not comparable with other plant crops and presents certain
peculiarities such as:
[0003] 1. it is one of the few plants marketed only for its
leaves;
[0004] 2. it is the major non-alimentary plant in the world with a
production extension higher than four million hectares in the whole
world;
[0005] 3. in many countries it is a very important instrument for
economical and financial politic;
[0006] 4. its consumption is based on the transformation of the
leaves into smoking products, inhaling powders and chewable
products;
[0007] 5. considering its narcotic substance characteristics and
its dangerousness for human health, there have always been attempts
aimed to forbid its use and hence its production.
[0008] The evolution of the Nicotiana genus into different
habitats, initially through natural selection and poliploidisation
and, later on, through human-driven selection, has brought to the
appearance of a vast range of kinds, all selected on the basis of
the leaf properties being the leaf considered as the only valuable
part of the plant.
[0009] Recently, alternative uses of tobacco have been indicated in
addition to the above-listed ones:
[0010] 1. the production of alimentary proteins through
purification thereof from leaves (Long R. C. 1979. Tobacco
production for protein. Project n. 03245. North Carolina State
university, Raleigh N.C.);
[0011] 2. the extraction of pharmacologically useful active
ingredients normally present in the leaves (Baraldi M. et al. 2004.
Presenza di sostanze Benzodiazepino-simili in estratti di foglie di
tabacco (Nicotiana tabacum). Atti 1st. Sper. Tab., 23 Aprile, Roma,
pp. 45-52);
[0012] 3. the production of recombinant proteins expressed in the
leaves or in the seeds of genetically modified plants (Twyman et
al. 2003. Molecular farming in plants: host systems and expression
technology. Trends Biotechnol. 21:570-578).
[0013] The tobacco plant presents a very large leaf area, a small
inflorescence and a ratio aerial part:roots that is the highest
observed among agricultural plants (Went, 1957. The experimental
control of plant growth. pp. 343. Chronica Botanica, Waltham,
Mass.).
[0014] Taking into account the economic relevance exerted by
tobacco's cultivation, notwithstanding the alarming increase of
tabagism amongst the youngest, Europe provides grants for its
cultivation giving rise to perplexity both of economical and
ethical nature.
[0015] The European Commission on its internet site
(www.ec.europa.eu/agriculture/publi/fact/tobacco) affirms: "there
are no economically valid alternatives to this culture that does
not use good soils. The incentives to tobacco's culture permits the
survival of the rural tissue and produces an industrial activity
that contributes to the survival of regions menaced by
desertification".
[0016] The negative consequences, in environmental terms, of the
use of fossil combustibles and the limited availability of
petroleum, require the search of new energetic sources. Amongst
these, biofuels are the best choice in a future perspective due to
their renewability.
[0017] Considering biofuels of agricultural origin, up to date, the
attention has focused on the production of bioethanol starting form
simple (i.e. saccharose) or complex (i.e. cellulose) sugar
producing plants. Model plants for such production has been
identified in sugar cane, corn, wheat, potato, tapioca, sugar beet,
barley, sorghum etc. The development of cultures aiming to the
maximisation of the production of biomass to be transformed in
ethanol through fermentation processes or for the production of bio
fuels or gas through gasification may have the same scope.
Alternatively, the state of the art aims to the production of fuel
oil and biodiesel starting form oleaginous or non oleaginous
species but rich in oil such as soybean, sunflower, rape, peanut,
flax, corn, sesame, palm, palm-kernel, coconut, ricinus etc.
[0018] The choice of the ideal species for the production of
biofuels shall relate on the fulfillment of requirements such
as:
[0019] 1. determining a net energetic gain in the difference
between culture's input and output, comprising in the said
calculation the energetic costs for the production of the
agricultural machinery and for the processing for the extraction
and transformation/purification of the oil;
[0020] 2. determining environmental benefits deriving from the
supportability of the agricultural production, decrease of the CO2
and particulate matter (e.g. PM-10) emission after combustion and
limited use of agrochemicals such as pesticides herbicides and
fertilizers;
[0021] 3. being economically competitive and, possibly, determining
social benefits that may increase the system's economy, e.g. by
lowering indirect costs on the sanitary system, considering also
that the fossil energy used at presents imposes environmental costs
that are usually not in the cost determination; a bio fuel shall
envisage benefits both on the cost competitiveness side and on the
environmental side;
[0022] 4. being available in large quantities without decreasing
the alimentary availability; the use of agricultural plants
traditionally used for food production does not reasonably allow
their use for the production of biofuels without determining a
reduction of the food sources deriving from said plants hence
increasing the costs of the raw materials;
[0023] 5. the plant culture from which it derives shall possibly
concern marginal lands that are not likely to be used for
alternative cultures.
[0024] In the state of the art the plants taken into account for
oil production are: soybean (Glycine max), sunflower (Helianthus
annuus), rape (Brassica napus), peanut (Arachis hypogaea), ricinus
(Ricinus communis), flax (Linum usitatissimum), corn (Zea mais),
sesamus (Sesamum indicum), palm (fruit, Aracaceae), palm-kernel
(seed, Aracaceae), copra (coconut, Cocos nucifera), safflower
(Carthamus tinctorius), olive (Olea europea), cotton (Gossypium
sp.), acajou (Anacardium occidentale), hemp (Cannabis sativa),
poppy (Papavers sp.), mustard (Brassica sp.), grape (Vitis sp.),
apricot (Prunus armeniaca), pine (Pinus sp.), argan (Argania
spinosa), avocado (Persea americana), almond (Prunus amygdalus),
hazelnut (Corylus avellana), nut (Juglans regia), neem (Azadirachfa
indica), niger (Guizotia abyssinica), jojoba (Simmondsia
chinensis), rice (Oryza sativa), pumpkin (Cucurbita sp.), crambe
(Crambe abyssinica).
[0025] On the contrary, in the prior art, tobacco has always been
considered as an agricultural plant apt for the production of
leaves.
[0026] The only three publications in literature, listed below,
suggesting further uses for tobacco, take into account the present
tobacco varieties, that have been selected for the production of
leaves, as a source of the seed by-product for oil extraction.
[0027] In particular Giannelos et al. (Tobacco seed oil as an
alternative diesel fuel: physical and chemical properties.
Industrial Crops and Products, 2002, 16:1-9) declaring that "the
seed is a by-product of the leaf production in Greece" suggest the
possibility of using said seeds for the production of fuels
describing methods for the extraction of oil form tobacco seeds
that uses solvents, indicating, however, that the oil extracted
from tobacco may not be used as such as biodiesel due the high
iodine value in it.
[0028] Usta N. (Use of tobacco seed oil methyl ester in a
turbocharged indirect injection diesel engine. Biomass and
Bio-energy, 2005, 28:77-86) declares that tobacco seed oil is a
by-product of the world production of leaves, estimates the
worldwide production of seed deriving from tobacco's cultivation
for leaves and describes the oil extraction from seed through the
use of solvents.
[0029] Finally, Patel et al. (Production potential and quality
aspects of tobacco seed oil. Tobacco Research, 1998, 24:44-49)
estimate the production of tobacco seed as a by-product of leaves
in India equal to 1,171 kg/ha with a content of oil of the 38% by
weight and describe its extraction by the use of solvents.
[0030] The technological processes for oil extraction comprise
mechanical (pressure) and chemical (solvents) techniques. In
practice, the two systems are often combined. In general the
mechanical extraction is carried out on seeds containing more than
20% of fat material (e.g. rape and sunflower) wherein the seeds
dimensions are favourable for the pressing technique. Tobacco seed,
by way of example, due to its very tiny dimensions, is subject to
oil extraction by chemical treatments.
[0031] Generally, the possibility of extracting oil mechanically,
facilitates the direct extraction in the seed production sites,
hence also at the farm's level, with small plants.
[0032] For lower quantities of fat material chemical extraction is
used, and can be applied also to the oilcake, leftover of the
mechanical extraction, in order to recover the remaining 6-12% of
oil left after the mechanical treatment. The oils extracted by the
use of solvents (e.g. hexane) prior to commercialisation for
alimentary uses, require a refinement step. The main product of the
extraction process is crude oil; the mechanical extraction further
produces the protein oilcake whereas the chemical one produces
flour. The latter, used in animal feeding, weights in a critical
way upon the production and processing of oily seeds economy.
[0033] In certain cases the production is bound to the protein
flour request (e.g. soybean). The crude oil may subsequently be
rectified with a series of physicochemical treatments (e.g. pH
adjustment, filtration, degumming, discolouration, etc.) depending
on the intended use.
[0034] The mass balance of the entire process varies from species
to species, by way of example considering a content in oil of 42%
for the sunflower seeds, for a ton of seeds (that are the main
product) 2.6 by-product (biomass) tons are considered, with a
production of 420 kgs of crude oil, 580 kgs of oilcake, obtaining a
final production of 390 kgs of refined oil and 30 kgs of process
residuals. Taking into account that the average yield of sunflower
seeds is about 2.6 t/ha (+/-15%) it can be calculated that the
yield/hectare of oil is equal to about one ton. This relation is
valid also for other species, in particular for rape, depending on
the percentage in oil. Vegetable oils may be used directly as fuel
oils for heat production (ovens or boilers) or mechanic energy
production (engines), utilizing their gross calorific value that is
about 8,500 kcal/kg or, after transesterification, transformed as
biodiesel.
[0035] The use of vegetable oils in boilers may be carried out with
conventional burners by substituting the industrial or the heating
diesel oil with vegetable oil. This kind of solution appears quite
interesting due to the fact that: (i) the price of substituted
fossil fuel is often quite similar to the one of the automotive
diesel oil and is subject to high excise duties; (ii) the use of
oil in boilers requires the organisation of a very simple
agroenergetic thread that can end directly in the rural
environment, where the fuel producers and the fuel users can be
located very near to each other or can even correspond. The higher
or lower easiness of the oil extraction process is another
important aspect to take into account when an local use of the bio
fuel is envisaged. The production economy and the more or less
favourable energetic balance will depend mainly on the production
per hectare of fuel oil.
[0036] The use of vegetable oils in diesel engines requires, on the
other hand, a chemical process of transesterification with methanol
and a certain fatty acid composition, which may be summarised in a
iodine value that has to be equal or lower than 120. Vegetable oils
are also often used for alimentary scopes. Depending on the plant,
the productions can be mainly directed to alimentary or energetic
scopes, or both.
[0037] On the light of the above mentioned problems, it would be
highly desirable to recycle tobacco's industry for ecological
scopes and harmless for human beings.
[0038] The identification of an alternative and economically valid
use of tobacco does hence constitute a clear worldwide economically
interesting topic.
SUMMARY OF THE INVENTION
[0039] The present invention discloses the realisation of tobacco
plants suitable for a very high production of a particular
component of the plant: the seed. Said plants have been realized
through somatic in vitro, chemical or physical mutagenesis
techniques and/or by interspecific crossbreeding and subsequent
chromosomal duplication. Said plants are optionally further
modified by genetic engineering techniques. The tobacco's plant
selection in the state of the art has always been directed towards
the leaf as final product, the focusing of the attention and,
hence, the plants selection for the maximisation of the seeds
production has never been suggested before and allows the use of
tobacco in order to maximise the production of seed to the
detriment of the leaf production.
[0040] It has been surprisingly found that the selection of tobacco
plants, carried out using non biological techniques of chemical,
physical and somatic in vitro mutagenesis techniques, of crossing
also between different species, followed by the induction of
amphidiploids and, optionally by recombinant DNA techniques, has
allowed to obtain tobacco plants presenting the following
characteristics: [0041] they present the ideal characteristics for
the transformation of the tobacco agricultural plant from a plant
of leaf production to a plant for seed production; [0042] they have
the capability of producing seeds up to values of from 20
quintals/hectare, to 50 quintals/hectare, or to 70
quintals/hectare, or even to 90 quintals/hectare with the
possibility of further improvements in the yield of seed/hectare;
[0043] they present a seed oil content up to 38% of the seed
weight, or up to 40%, or up to 48%, or up to 52% or up to 58% or
even up to 60%; [0044] they have a low necessity of agronomical
inputs for the defense against parasites and weeds.
[0045] Furthermore, completely unexpectedly, due to the small
dimensions of the tobacco seed that is, amongst the cultivated
plants, one of the smallest seeds, the present invention shows that
it is possible to extract oil from the seed by pressing reaching
extraction yields that are about the 80% of the total oil present
in the seed, about the 90% of the total oil present in the seed or
even about 95% of the total oil present in the seed, thus granting
the possibility of carrying out the extraction also in small plants
for farms use.
[0046] Hence, object of the invention are tobacco plants that are
mutagenised and/or obtained by interspecific crossing followed by
diploidisation and selection, characterised in that they produce a
seed quantity equal to at least 20 quintals/hectare, said plants
optionally further modified by genetic engineering, the use of said
plants for the production of seeds, for the manufacturing of oil
and derivatives thereof, the use of said plants for the production
of biomass for the biochemical or thermo chemical conversion, the
method of producing and selecting said plants, the seeds of said
plants and their use for the manufacturing of oil and derivatives
thereof, the oil deriving from said seeds, the biodiesel obtained
from said seeds, food supplement derived from tobacco, solid fuels
comprising the oilcake resulting from pressing tobacco seeds, the
method for oil extraction from tobacco seeds by pressing.
DETAILED DESCRIPTION OF THE FIGURES
[0047] FIG. 1. General characteristics of the tobacco plant
realised through chemical mutagenesis, intra and interspecific
crossing, poliploidisation, selection, induction of somaclonal
variability, genetic transformation, aimed to maximise the seed
production per surface unit, with a high oil content and having
characteristics suitable for the use as energetic source, for
industrial uses and for human and animal nutrition. Principal
induced and selected characteristics; deep and wide radical
apparatus; thin and erected lanceolate leaves; robust stalk at the
basis with long internodes; cob-like inflorescence compact, wide or
columned; short pedicellate, non-dehiscent capsules, di- or
multi-valve, straight apex, with at least 5000 seeds/capsule; oval
or elliptical seeds of a length>than 1 mm; plant height between
50 and 120 cm, insect resistance, herbicides resistance, fungi
resistance, drought resistance, variable ratio among the fatty
acids components.
[0048] FIG. 2. Example of the product oil and oilcake obtained from
tobacco seed by pressing with a screw-press mod. Komet (IBG). After
pressing the oil has been filtered in paper and presents high
limpidity characteristics.
DETAILED DESCRIPTION OF THE INVENTION
[0049] The present invention, hence, relates to the realization of
plants of the Nicotiana genus, as ideal plants for the production
of seed from which fuel oil, biodiesel, proteins, oil for
zootechnical use, for industrial use, for human alimentary use is
obtained.
[0050] The varieties of the Nicotiana genus that may be used as
parental plants for the carrying out of the plants of the invention
may be, by way of example, comprised among the following species:
N. tabacum, N. rustica, N. glauca, N. paniculata, N. knightiana, N.
solanifblia, N. benavidesii, N. cordifolia, N. raimondii, N.
thyrsiflora, N. tomentosa, N. tomentosiformis, N. otophora, N.
setchellii, N. glutinosa, N. ondulata, N. arentsii, N.
wigandioides, N. trigonophylla, N. palmeri, N. sylvestris, N.
langsdorffii, N. alata, N, forgetiana, N. bonariensis, N.
longiflora, N. plumbaginifolia, N. repanda, N. stocktonii, N.
nesophila, N. moctiflora, N. tomentosiformis, N. otophora, N.
setchellii, N. glutinosa, N. petunioides, N. acaulis, N. ameghinoi,
N. acuminata, N. pauciflora, N, attenuata, N. miersii, N.
corymbosa, N. linearis, N. spegazinii, N. bigelovii, N.
clevelandii, N. nudicaulis, N. maritima, N. velutina, N. gossei, N.
excelsior, N. megalosiphon, N. exigua, N. goodspeedii, N. ingulba,
N. stenocarpa, N. occidentalis, N. rotundifolia, N. debneyi, N.
benthamiana, N. fragrans, N. suaveolens, N. obtustfolia.
[0051] According to the present invention, the plant will be
achieved by means of mutagenesis techniques that will allow to
develop plants capable of producing a quantity of seeds higher than
the average with respect to the starting individuals. Mutagenesis
may be induced by standard chemical and/or physical treatment
techniques of tobacco seeds, or also by in vitro culture for the
induction of somaclonal mutants. The higher production of seed may
be obtained also by generating hybrid plants produced by
interspecific crossing followed by diploidisation of the chromosome
set (in order to obviate to the sterility events displayed by
interspecific hybrids) by chemical treatment with colchicine. The
plants produced by interspecific crossing may be further
mutagenised by chemical and/or physical standard techniques.
[0052] Once the mutants are obtained, they will be selected for the
seed production character, so to isolate and select plants
producing at least 20 quintals of seed per culture hectare.
[0053] According to the present invention, the plant will be hence
realised starting from seeds produced by:
[0054] 1. crossing between individuals of the same species or
[0055] 2. crossing between individuals of different species having
the same chromosome number (e.g. N. tahacum.times.N. clevelandii)
as such or wherein said crossing is followed by embryo culture
methods and poliploids induction by treating with the mutagen
colchicine, using techniques well known to the skilled in the art,
in order to obtain amphidiploids or,
[0056] 3. crossing between individuals of different species having
a different chromosome number (e.g. N. tabacum.times.N.
trigonophylla) followed by embryo culture methods and poliploids
induction by treating with the mutagen colchicine, using techniques
well known to the skilled in the art, in order to obtain
amphidiploids.
[0057] As indicated above, when there is no induction of
diploidisation, as in the cases 1 and 2, the seeds will be
mutagenised by chemical and/or physical techniques, when mutation
of the chromosome number is induced by diploidisation, as in cases
2 and 3, the selection may be carried out on said mutated seeds so
produced or on said seeds further mutagenised by chemical and/or
physical techniques. Standard mutation techniques known to the
skilled person may be used, such as, by way of example, the
treatment of the seed with Ethyl Methane Sulfonate (EMS) (e.g. in
aqueous solution at a 0.5% concentration) and living EMS in contact
with the seed for variable times e.g. as indicated in example 1,
or, as already said with colchicine, so to induce poliploidisation,
by X or Gamma radiations performed in suitable fields or, anyhow,
following any protocol available in literature used for plant
mutagenesis and for performing large scale screenings. The so
treated seeds will be allowed to germinate and the plants of the
M.sub.2 generation will be selected on the basis of the following
characteristics: inflorescence shape, number of capsules, number of
seeds per capsule, seed dimensions, leaf shape, dimensions of the
root apparatus, bearing of the leaf, etc.
[0058] According to the present invention, plants having the
following characteristics will be selected: plant height 80-120 cm,
leaf with thin lamina and straight bearing, compact inflorescence,
number of flowers higher than 100, number of capsules higher than
100, number of seeds per capsule higher than 5000, lignified and
strong stalk, deep roots.
[0059] The quantity of seeds produced by the selected plants will
be than verified in open field and only the plants producing at
least 20 quintals of seed/hectare at a seeding density of about
from 125,000 to 250,000 plants/hectare will be selected.
[0060] By chemical and physical mutagenesis and by somatic
mutagenesis, mutants of different classes are obtained, hence
increasing the probability of finding the desired variants. As an
example, a mutant with a lanceolate leaf having an erect bearing
allows the increase of the seeding density without compromising the
light reception that is important for the photosynthetic activity
and hence to increase the seed production per ha. By way of
example, a mutant having a deeper root apparatus allows a better
anchorage and nourishment of the plant. As an example, a mutant
with a compact inflorescence and a higher number of capsules allows
to increase the quantity of seed produced by every single
plant.
[0061] The present invention encompasses the thus obtained and
selected plants producing a seed quantity higher or equal to 20
quintals/hectare. The plants of the present invention are modified
in order to produce a seed quantity up to 90 quintals per
cultivated hectare, hence 20, 30, 40, 50, 60, 70, 80, 90 quintals
per cultivated hectare against the 10-12 normally produced by the
tobacco plants selected for the leaf production.
[0062] The plants of the invention may be obtained also by
induction of somatoclonal variants, wherein the above indicated
seeds may be treated with sodium hypochlorite and then with 70%
ethanol, from the plants germinated from said seeds leaf parts are
taken and the formation of calluses is induce and reproduced for
variable times in vitro is hence induced from said parts. From said
calluses plants having variable characteristics such as:
inflorescence shape, number of capsules, number of seeds per
capsule, seeds dimensions, leaf shape, dimensions of the root
apparatus, leaf bearing, seed production, oil content of the seed,
fatty acid composition of the oil, protein content of the seed etc.
regenerate, said plant being selectable on the basis of the above
mentioned parameters.
[0063] The plants of the invention may further be selected for the
presence of characteristics such as: percentage of oil content of
the seed, fatty acid composition of the oil, protein content of the
seed, etc.
[0064] The above described plants may further be modified by
recombinant DNA techniques in order to obtain further advantageous
characteristics, in case said characteristics were not already
present in the selected mutants, such as: increase in the
percentage of the oil content of the seed, variable fatty acid
composition depending on the uses envisaged for the oil, insects
resistance, herbicides resistance, fungi resistance, etc.
[0065] For the genetic transformation of the plants of the
invention vectors suitable for plant cells transformation may be
used as well as expression cassettes allowing the in plant
expression of the genes of interest. Depending whether the genes of
interest are to be expressed in the green part of the plant (i.e.
genes for parasites or herbicides resistance) or in the seeds (e.g.
genes involved in the fatty acids metabolism) vectors known in the
art, ensuring the expression of said genes in the organs of
interest may be selected. Hence, vectors with constitutive
promoters known in the state of the art, or with inducible
promoters, e.g. by the parasites attack or in the forming capsule
may be used. In particular, as the plants of the invention are
selected for the high seed production and, being the product of
said seed of particular interest, vectors comprising seed specific
expression cassettes that will guarantee the expression of the
heterologous inserted genes in the seeds of the plant of the
invention will be particularly suitable.
[0066] For the genetic transformation the Agrobacterium tumefaciens
or physical DNA transfer systems may be used.
[0067] In an embodiment of the invention, it will be particularly
advantageous to realise plants presenting not only a high seed
productivity but also insect resistance, herbicides resistance,
fungi resistance, drought resistance, this will allow to reduce
significantly the cultivations inputs, thus increasing the culture
productivity and reducing the environmental impact.
[0068] In this case vectors comprising the kanamycine resistance
gene as marker, regulating regions allowing the constitutive
expression (e.g. 35S or the ubiquitin promoter) of the genes of
interest such as the cry gene of Bacillus thuringiensis, the aroA
gene of Salmonella typhimurium, the Rpt1 gene of N. obtusifolia,
may be used. Said genes allow, in the same order, the production of
plants resistant respectively, to: insects, herbicides, fungal
diseases and may be introduced according to standard gene transfer
techniques known to the skilled person.
[0069] Resistances can be introduced following one or more
transformation events with several vectors or, alternatively, due
to the fact that plants may be very easily crossed, the resistances
can be introduced singularly in different individuals of the same
selected variety and subsequently assembled together in the same
individual by crossing.
[0070] In this case it will be easy to obtain homozygosis for all
the characters by duplication of haploids obtained from in vitro
anthers cultures.
[0071] The genetic transformation can be carried out in an
analogous way for metabolic engineering purposes aimed to the
increase of the amount of oil accumulated in the seeds and to the
fatty acids metabolic pathway modification. In this case, it is
possible to use regulating regions with seed specific activity such
as the globulins promoter, and directing the enzymatic proteins to
the endoplasmic reticulum where they can be stabilise inserting a
specific signal, such as KDEL, or where from there can be
translocated to plastids, inserting amino acid specific signals,
e.g. the leader sequence of the small RuBisCO subunit.
[0072] By way of example, the oil quantity and the fatty acids
composition of the same, may be modified by modifying the
expression of genes coding for enzymes such as, merely by way of
example, acetyl-CoA carboxylase (ACCase), diacyl-glycerol
acyltransferase (DGAT), lysophosphatidate acyl transferase (LPAT),
Phosphatidate phosphohydrolase (PAPase) acyl protein carrier (ACP),
malonyl-CoA:ACP transacylase, ketoacyl-ACP synthase (CAS),
ketoacyl-ACP reductase, 3-hydroxyacyl-ACP dehydrase, enoyl-ACP
reductase, stearoyl-ACP desaturase, acyl-ACP thioesterase,
glycerol-3-phosphateacyltransferase, 1-acyl-sn-glycerol-3-phosphate
acyltransferase, cytidine-5-diphosphate-diacylglycerol synthase,
phosphatidyl glycerophosphate synthase, phosphatidyl
glycerol-3-phosphate phosphatase, FAD1-8 desaturase, phosphatidic
acid phosphatase, monogalactosyl diacyl glycerol synthase,
digalactosyl diacyl glycerol synthase, sulpholipid biosynthesis
protein, long chain acyl-coA synthase, glycerol-3-phosphate
acyltransferase (GPAT), diacylglycerol cho linephospho transferase,
phosphatidylinositol synthase, acil-CoA diacylglycerol
acyltransferase, acyl-ACP desaturase, lineoyl desaturase,
sphingolipid desaturase, oleate 12-desaturase, fatty acid
acetylenase, fatty acid epoxygenase, diacylglycerol kinase,
cholinephosphate cytidyl transferase, choline kinase,
phospholipase, phosphatidylserine decarboxylase,
phosphatidylinositol kinase, ketoacyl-CoA synthase, CER
transcription factor, oleosin, 3-ketoacyl-CoA thiolase, acyl-CoA
dehydrogenase, enoyl-CoA hydratase, acyl-CoA oxidase.
[0073] According to the invention, an increase in the total seed
oil content can be obtained determining an over expression of the
tobacco Acetyl CoA carboxylase or the same enzyme of another
species (e.g. rape). As an example, a variation in the fatty acid
profile and hence in the iodine number, can be obtained by
silencing through the expression of antisense constructs, the gene
coding for oleate desaturase of the plastid and of the endoplasmic
reticulum. The expression or the silencing of one or more of said
genes in the seeds of the plants of the invention, results in the
fact that the oil produced by said seeds can be directly used for
the production of biodiesel, as it has a iodine number inferior or
equal to 120.
[0074] The expression of said genes can also affect the percentage
of oil in the seed and the plants of the invention can be further
selected for the seed's oil content that can be equal to about 38%
of the seed weight, the 40%, the 48%, the 52%, the 58% and even the
60%.
[0075] The above mentioned genes can be introduced in vectors for
the seed specific expression such as the ones described in patent
application WO03073839 following the teachings disclosed in said
application. The expression vector used for the said embodiment
will hence be a vector comprising: a. a promoter of a plant gene
specific for the expression in the seed storage organs; b. a DNA
sequence coding for the signal sequence of a plant protein capable
of direct the product of the gene of interest into the seed storage
organs via the endoplasmic reticulum; c. a DNA sequence coding for
said gene of interest deprived of the native signal sequence; d. a
stop signal. The promoters and the leader sequences may belong,
e.g., to the 7S soybean globulin or to the beta conglycinin soybean
gene, or to genes coding for tobacco seed storage proteins.
[0076] The above mentioned genes can be introduced following one or
more transformation event, or by transformation with several
vectors, or, alternatively, as plants may be easily crossed, said
genes may be introduced singularly in different individuals of the
same selected variety and subsequently grouped together in the same
individual through crossing.
[0077] In this case it will be easy to obtain the homozygosis for
all the characters by duplication of haploids obtained by in vitro
anther cultures.
TABLE-US-00001 TABLE 1 Content in fatty acids of some tobacco
varieties selected only after mutagenesis, or engineered and
selected in order to change the fatty acids metabolic pathway, and
selected for the stability of the character. The table points out
the result obtained with mutagenesis and the genetic intervention
carried out by introducing some of the listed genes in order to
change the acidic composition; the oil of the three last columns
has a iodine title suitable for the transformation of said oil into
biodiesel. Component PLT 103 PLT 256 PLT 318 PLT 335 Palmitic Acid
6.31% 8.26% 7.15% 17.20% Palmitoleic Acid 0.11% 0.18% 0.18% 1.25%
Stearic Acid 2.58% 5.20% 8.50% 12.50% Oleic Acid 12.62% 22.58%
25.56% 53.27% Linoleic Acid 77.48 58.78% 52.00% 6.45% Linolenic
Acid 0.65% 4.15% 5.25% 7.80% Arachidic Acid 0.13% 0.85% 0.80% 0.85%
Eicosanoic Acid 0.13% 0.58% 0.56% 0.68%
[0078] Object of the invention are also the seeds of the plants as
described above, that, being mutant plants' seeds, will be as well
mutant and will thus contain DNA modifications that will make them
differ form wild type seeds. Moreover, as indicated above, said
seeds can have a different chromosome number compared to the
parental plants (e.g. poliploids) and in most cases will also be
transformed with the above mentioned vectors.
[0079] When the transformation is made with vectors expressing
genes related to the fatty acids metabolism listed above, said seed
will also be characterised in that it contains an oil having a
iodine title lower or equal to 120 and an oil percentage comprised
between the 38% and the 60% of the seed's total weight.
[0080] Object of the present application is also the method for the
production of the plant of the invention comprising the following
steps:
[0081] a) seed produced by starting crossings between individuals
of the same species belonging to wild type or selected varieties
are subject to mutagenesis;
[0082] b) said seeds are allowed to germinate and the plants of the
M2-M4 generations are selected according to the following
parameters:
[0083] i) presence of characteristics that manifest at the
phenotypic level selected in the group comprising height of the
plant of 80-120 cm, leaves with thin lamina and straight bearing,
compact inflorescence, number of flowers higher than 100, number of
capsules higher than 100, number of seed per capsule higher than
5,000, strong and lignified stalk, deep roots;
[0084] ii) stability of the selected character in generations after
the M2 generation;
[0085] iii) testing of the hereditability of the selected
character;
[0086] c) the seeds selected at point b) are allowed to germinate
and plants are regenerated starting from the callus obtained from
the in vitro induced leaf mesophyll in the presence of
phytohormones, the plants maintaining the characters selected at
point b) in R0-R2 generations are selected;
[0087] d) the plants selected at point c) are seeded in open field
and plants producing at least 20 quintals per hectare are
selected.
[0088] In the method described, the plants obtained at point a) can
also be obtained by
[0089] a') carrying out interspecific crossings in the Nicotiana
genus, followed by the backcrossing of the F1 or by the induction
of amphidiploids by treatment of the vegetative apex with
colchicine.
[0090] The plants obtained with the above described methods may be
submitted to further steps e) and/or f) and to a passage g) as
indicated below:
[0091] e) genetic transformation of the plants obtained at point
a-d or a'-d with vectors comprising expression cassettes expressing
in plant genes for insects, herbicides and/or fungal diseases
resistance selected in the group comprising the cry gene of
Bacillus thuringiensis, the aroA gene of Salmonella typhimurium,
the Rpt1 gene of N. obtusifolia and selecting the thus transformed
plants in the T0-T4 generations for the for insects, herbicides
and/or fungal diseases resistance;
[0092] f) genetic transformation of the plants obtained at point
a-d or a'-d with one or more vector comprising expression cassettes
expressing in seed genes of the fatty acids metabolism selected in
the group comprising, acetyl-CoA carboxylase (ACCase),
diacyl-glycerol acyltransferase (DGAT), lysophosphatidate acyl
transferase (LPAT), phosphatidate phosphohydrolase (PAPase) acyl
protein carrier (ACP), malonyl-CoA:ACP transacylase, ketoacyl-ACP
synthase (KAS), ketoacyl-ACP reductase, 3-hydroxyacyl-ACP
dehydrase, enoyl-ACP reductase, stearoyl-ACP desaturase, acyl-ACP
thioesterase, glycerol-3-phosphateacyltransferase,
1-acyl-sn-glycerol-3-phosphate acyltransferase,
cytidine-5-diphosphate-diacylglycerol synthase, phosphatidyl
glycerophosphate synthase, phosphatidyl glycerol-3-phosphate
phosphatase, FAD1-8 desaturase, phosphatidic acid phosphatase,
monogalactosyl diacyl glycerol synthase, digalactosyl diacyl
glycerol synthase, sulfolipid biosynthesis protein, long chain
acyl-coA synthase, glycerol-3-phosphate acyltransferase (GPAT),
diacylglycerol cholinephospho transferase, phosphatidylinositol
synthase, acil-CoA diacylglycerol acyltransferase, acyl-ACP
desaturase, lineoyl desaturase, sphingolipid desaturase, oleate
12-desaturase, fatty acid acetylenase, fatty acid epoxygenase,
diacylglycerol kinase, cholinephosphate cytidyl transferase,
choline kinase, phospholipase, phosphatidylserine decarboxylase,
phosphatidylinositol kinase, ketoacyl-CoA synthase, CER
transcription factor, oleosin, 3-ketoacyl-CoA thiolase, acyl-CoA
dehydrogenase, enoyl-CoA hydratase, acyl-CoA oxidase, followed by
selection of the plants in the T0-T4 generations for
characteristics such as total oil content of the seed and fatty
acid composition of the same;
[0093] g) crossing of the materials obtained at points a-f or a'-f
and selecting the resulting progenies for characteristics such as:
high seed productivity, high oil content in the seed, variable
fatty acids oil composition depending on the intended use, insects
resistance, herbicides resistance, fungal resistance.
[0094] The plants at points comprised between b. and f. may, by way
of example, be selected for the presence of characteristics that
can be pointed out by chemical analysis such as the total oil
content of the seed and/or the content in single acidic components
of the seed and/or the protein content of the seed.
[0095] A further object of the invention is a method for extracting
oil from tobacco seeds wherein the oil yield is equal to values of
70 to 95% of the oil contained in said seeds comprising the
following steps:
[0096] a) mechanically extracting of said oil by pressing producing
oil and a residual oilcake;
[0097] b) filtering said oil produced in step a) with paper or
cloth filters.
[0098] The above indicated method has, surprisingly, a yield higher
than 70%, which is a totally unexpected yield after using said
pressing methods on seeds as small as the tobacco ones. In the
present invention, wherein plants having a high seed production are
selected with the aim of increasing the tobacco oil production per
plant, the discovery that the pressing method applied onto tobacco
seeds has a yield comparable to the yield obtainable on large sized
seeds, has extremely advantageous applications.
[0099] Besides the lower production costs and the totally
unexpected yields given the state of the art, said method allows to
extract tobacco oil directly in the sites where it can be used for
energetic scopes.
[0100] In one embodiment, the seed of the invention can be subject
to cold pressing using a screw-press or another kind of press,
loaded with the seeds. The press may reach temperatures up to about
60.degree. C. when it is steady operating and the oil pressed from
the seed is collected and filtered on paper or with cloth
filter-press. Other pressing systems suitable for seeds may be used
for pressing tobacco seeds.
[0101] In order to further improve the yield of the method of the
invention, where the oil productivity from the seed is to be
exploited at its maximum, it is possible to perform a further step
of
[0102] c) chemically extracting with solvents the residual oil
present into the oilcake obtained at point a).
TABLE-US-00002 TABLE 2 Characteristics of the tobacco oil obtained
by pressing the seed and filtering. The table points out the higher
gross calorific value, the low sulphur content, the low viscosity
when compared to other vegetable oils. Determination Result Mis.
Un. Methods Flash point 236.0 .degree. C. UNI EN ISO 27 19 2005
Sulphur <0.01 % m/m ISO 8754 1992 Ashes 0.005 % m/m EN ISO 6245
2002 Viscosity at 50.degree. C. 21.630 mm2/s UNI EN ISO 3104 200
Melting point -18 .degree. C. ISO 3016 1994 Calorific value 9,670
Kcal/Kg ASTM D240-97 Volumic mass at 925.0 Kg/m3 UNI EN ISO 3675
2002 15.degree. C. Saponification value 193.6 mg KOH/g ASTM
D94-02
[0103] It is also object of the invention the use of the plants of
the invention and/or of their seeds for the production of liquid or
solid fuels, biodiesel, industrial lubricants, plastic materials
such as linoleum, dietary supplements for animal feeds, dietary
supplements for human use.
[0104] The plants of the invention, in fact, do show
characteristics that are extremely advantageous for the production
of said products as: [0105] they produce an oil obtained by
pressing that is ideal, also without refinement as a simple
filtering is sufficient, to be used as combustible oil as it has a
clear aspect, a cinematic viscosity at 40.degree. C. of 29.11 mm2/s
and at 50.degree. C. of 21.63 mm2/s and a sulphur content lower
than the 0.01%, entering in a class with very good physical and
thermodynamic characteristics; [0106] they produce an oil, obtained
from pressing the seed that, even in the simplest embodiment of the
invention, hence without the transformation for the expression of
genes of the fatty acids metabolism indicated above, may be used
for the production of biodiesel once mixed, e.g., with 25% of palm
oil, or with other vegetable oil percentages having a lower iodine
title, in order to lower the iodine value under the value 120 and
that, in the embodiment comprising the expression of one or more of
said genes, with the aim to change the fatty acids metabolism, can
be directly used for transforming it in biodiesel, having a iodine
number equal to 120, or better equal to 100, or even better, equal
to 80; [0107] they produce an oilcake, resultant from the pressing
of the seed, having an oil content variable form about 6 to 12% and
a protein content of about 35%, which is ideal for the dietary
supply of animal feeds given its high content of omega 6 fatty
acids (linoleic acid); [0108] they produce an oilcake resultant
from the pressing of the seed that, as an alternative to its use as
animal feed, may be used as solid combustible in coal or biomass'
pellet working plants due to its calorific value higher than 4,950
KCal/kg; [0109] they produce an oil with a flashpoint of
236.degree. C. and a melting point of -18.degree. C. thus being
suitable for use as non polluting lubricant, e.g. for chainsaw
chains, or as lubricant in general; [0110] they produce an oil
that, taking into account its composition abounding in
polyunsaturated fatty acids (C18:2, PUFA) that are essential for
humans and are hence requested for healthiness scopes (omega 6),
may be used as food or as dietary supply for humans; [0111] they
produce a residual biomass (leaves, stalks, inflorescences,
capsules' coating) after the seed harvest, that may reach values of
100 quintals per hectare or, better, of 200 quintals per hectare
or, even better, of 300 quintals per hectare with the possibility
of further improving the yield per hectare; [0112] they produce a
residual biomass that may be used for the uses normally ascribed to
it, such as, e.g., gasification, combustion, pyrolisis, anaerobic
digestion, fermentation or steam explosion, thus contributing to
ameliorate the economical yield of the culture.
[0113] Consequently, object of the invention is the use of the
plants of the invention in general for the large-scale production
of tobacco seeds.
[0114] Object of the invention is the use of the said plants and/or
seeds for the production of tobacco oil.
[0115] Also object of the invention is the use of the plants and/or
the seeds for the preparation of tobacco-oil-based or essentially
tobacco-oil-consisting fuels for boilers or diesel engines.
[0116] In a particularly advantageous embodiment of the invention,
said tobacco oil is obtained with the pressing method of the
invention and not by solvent extraction.
[0117] The extraction method of the invention, in fact, allows the
production of an oil that is directly usable as fuel due to the
fact that said oil has a kinematic viscosity of 29.11 mm2/s at
40.degree. C. and of 21.63 mm2/s at 50.degree. C. which allows to
nebulise it into burners without the need of a fluidifying
pre-heating treatment.
[0118] A further object of the invention is the use of the plants
and/or the seeds of the invention for the preparation of biodiesel
by admixing tobacco's seeds oil with, e.g., 25% of palm oil or with
other percentages of plant oils reducing the iodine final title.
The production of biodiesel according to the invention may be
carried out admixing the tobacco oil extracted with the above
described method (pressing) with plant oils capable of lowering its
iodine title to a value equal or lower than 120.
[0119] Also object of the invention is the use of the plants and/or
seeds of the invention for the preparation of tobacco's seeds oil
having a iodine title equal or lower than 120 without carrying out
any procedure aimed to reduce the iodine title on the oil extracted
according to the method of the invention. In this embodiment of the
invention, transgenic plants and/or transgenic seeds expressing one
or more genes, among the ones listed above, of the fatty acids
metabolism according to the invention producing a tobacco oil
having a iodine title equal or lower than 120 will be used.
[0120] In this case, hence, no refining or treating process of the
oil will be carried out after the pressing according to the
invention, in order to lower the iodine title of the oil thus
obtained. The biodiesel according to the invention may hence have a
composition of 100% tobacco oil. The biodiesel will be obtained
from the tobacco oil according to the normal transesterification
procedures with methanol known to the person skilled in the
art.
[0121] In an embodiment of the invention the biodiesel will consist
entirely of tobacco oil having a iodine title lower than 120, or
even equal or lower than 100, or even equal or lower than 80, said
oil being transesterificated with methanol according to the methods
known to the skilled person.
[0122] Object of the invention is also the use of the plants and/or
the seeds of the invention for the preparation of solid fuels for
coal or biomass pellets functioning plants. In this case the
biomass resulting from the cultivation may be used, said biomass
having a calorific value higher than 4,200 KCal/kg.
[0123] Object of the invention are also the fuels obtainable as
indicated above, i.e. fuels comprising the oilcake obtained from
tobacco's seeds pressing.
[0124] Alternatively, the plants and/or the seeds of the invention,
may be used for the preparation of dietary supplements for animal
feeds. The oilcake resulting from the pressing of tobacco's seeds
according to the invention, has an oil content from about the 6% to
about the 12% and a protein content of about the 35% of the oilcake
weight and a high omega 6 fatty acids content (linoleic acid) that
renders it ideal for said scope. In trials carried out on rearing
piglets, the substitution of soybean protein flour with the tobacco
oilcake in percentages variable from 3% to 7% in isoproteic diets,
did not show significant differences in the animals
development.
[0125] In a further embodiment of the invention, the plants and/or
the seeds of the invention may be used for the preparation of
non-polluting lubricants. By way of example, the oil obtained with
the method of the invention, from plants producing the seed of the
invention having no other modifications besides the mutations for
the appearance of the basic character (i.e. the high seed
production) already show a flash point of 236.degree. C. and a
melting point of -18.degree. C., characteristics that renders it
suitable for use even as such as non-polluting lubricant e.g. for
chainsaw chains or for engines in general.
[0126] In a further embodiment, the plants and/or seeds of the
present invention, can be used for the preparation of foods or food
supplements for human use.
[0127] In this case, the oil obtained by the process of the
invention, will be further refined in order to eliminate waxes,
gums, complex carbohydrates, phospholipids and de-acidified. Once
refined it can be used as such or as dietary supplement for human
use. The advantage of said use is given by the richness of said oil
in polyunsaturated fatty acids (C18:2, PUFA) that are essential for
humans and that are required for health reasons (omega 6).
[0128] It is obvious that all the embodiments envisaging the use of
tobacco's seeds oil, preferably obtained by pressing, or of oilcake
resulting from the seed pressing, can be carried out also on
non-mutagenised tobacco plants. The unquestionable advantage of the
plants of the invention is obviously given by the high seed
production and the resulting higher yield of product obtainable by
the plants of the invention and by their seeds compared to the
yield obtainable from wild type plants and tobacco seeds planted in
a comparable cultivating area and with comparable cultivating
methods.
[0129] The use of tobacco oil for the preparation of lubricants, of
human or animal dietary supplements have never been disclosed in
the art. Obviously, also all the embodiments in which an oil having
a iodine title equal or lower than 120 extracted by pressing and no
further treated have never been described.
[0130] The plants of the invention, also show a residual biomass
(leaves, stalks, capsules and inflorescences coating) that, after
the seed harvest, can reach values between 100 and 300 quintals or
more per hectare, the use of the plants of the invention as a
biomass source for gasification, combustion, pyrolisis, anaerobic
digestion, aerobic digestion, feimentation or steam explosion
processes as described in the art in order to further increase the
economic yield of the culture, is hence an object of the
invention.
[0131] Object of the invention are also: a tobacco's seeds oil
obtainable from the seeds of the plants of the invention with the
extraction method of the invention, a biodiesel obtained by said
oil by transesterification, fuels comprising said oil, food
supplements for humans deriving from said oil further refined,
lubricants comprising said oil, tobacco's seeds oil obtainable from
the seeds of the plants of the invention by the extraction method
of the invention characterised in that it has a iodine title equal
or lower than 120 without the need of admixing it with other oils
for lowering said title, biodiesel comprising the said oil having a
iodine title equal or lower than 120.
EXAMPLES
Example 1
Chemical Mutagenesis
[0132] The seed of the variety selected for the mutants induction
by chemical mutagenesis, has been placed in a 100 ml flask in the
amount of 20 gr per experiment, corresponding to about 200,000
seeds. 50 ml of deionised water have been added to the flask and
the seeds have been rehydrated for 14 hours at 25.degree. C. The
water has been than substituted with an 0.5% EMS aqueous solution.
The seeds have been shacked using a magnet for a time variable from
0.5 to 5 hours, depending on the variety, that in preliminary
trials showed a higher mutation frequency for morphological
characters such as height of the plant, seeds dimensions, leaves
shape, capsules numbers, seed production per plant etc.
[0133] Once the treatment period was ended, the mutagenic solution
has been discarded by pouring the seeds in a thin sieve and rinsing
them for several minutes under running water. Afterwards, the seeds
have been rinsed for 6 times in a flask, by adding 50 ml of water
and shaking them for 10 minutes at each rinsing.
[0134] The seeds have than been dried on filter paper and have been
sent to a specialised firm for pelleting.
[0135] The pelleted seeds had a final diameter of 1.2 mm and have
been used for direct in field seeding (M.sub.1) at a 100,000 plants
per hectare density. The M.sub.1 generation has been allowed to
flower, and has been examined for the possible presence of dominant
mutations and, once the seeds were ripe, a capsule per plant has
been hand-harvested in order to obtain the mass seed that has been
used for the open field seeding of the following year and for the
phenotypic screening of the M.sub.2 generation. Each mutant of
interest has been singularly harvested and controlled in the
following generations.
Example 2
Somaclonal Variants Induction
[0136] The seed of the variety selected for inducing somatoclonal
variants has been sterilised by immersion for 5 minutes in a 20%
sodium hypochlorite solution followed by an immersion for 1 minute
in 70% ethanol followed by 5 washes in sterile water. The seed has
been allowed to germinate in rectangular plastic containers of 10
cm per side and of 12 cm of height containing agarised MS medium.
From the fully developed plants, parts of 0.5 cm per side of leaf
have been cut and placed in Petri discs with MS1 (MS+1 mg/lt 2,4-D)
medium in order to induce the formation of the callus. Also
individuals F.sub.1 obtained from interspecific crossings have been
used. The callus collected from leaf discs has been placed in
liquid MS1 medium in 250 ml flasks containing 50 ml of medium and
shacked at 80 rpm on a rotating plate maintained at 28.degree. C.
with a 16 hours photoperiod.
[0137] The callus has been maintained in liquid culture for several
generations renewing the culture every 20 days and inoculating a
new 50 ml liquid media flask with 2.5 ml of the preceding culture.
At each generation a part of the callus was distributed on Petri
dishes containing agarised MS2 (MS+1 mg/l NAA+1.5 mg/l Kinetin)
medium in order to induce the formation of sprouts that, once
reached 2 cm of length were transferred on MS3 (MS+1 mg/l IBA)
medium to root and for the subsequent transfer in pot in a
greenhouse. The obtained variants were screened in the R.sub.1 and
R.sub.2 generations and concerned the leaves dimensions, the leaves
shape, the capsules dimensions, the number of seeds per capsule,
the oil content of the seeds, etc.
Example 3
Crossing and Chromosomal Duplication
[0138] Many Nicotiana species are sexually compatible with
Nicotiana tabacum and, even if the crossing product is sterile, it
is possible to backcross using the parental plants as pollinators
or induce the formation of amphidiploids. In this species, the work
is facilitated by the fact that it is possible to obtain
intergeneric hybrids F.sub.1 populations of many thousands of
individuals, allowing to perform the improvement program based on
amphidiploids. The crossings have been carried out both using
parental Nicotiana spp. having the same chromosome number (e.g. N.
paniculata.times.N. solanifolia; N. tabacum.times.N. rustica) or a
different chromosome number (eg. N. tabacum.times.N. paniculata; N.
tabacum.times.N. longiflora). The F.sub.1 individuals obtained by
some cross combination have been cultivated in greenhouse, in order
to verify their phenotypic characteristics and to cross them with
both the parental plants, as well as allowed to germinate in vitro
and micro propagated. The micro propagated material has been used
to carry out chromosome duplication experiments by treatment with
colchicine in the sprouts reproduction phase or later after the
transplantation in pot and before flowering. The sprouts of the
germinated seeds have been cut at the basis and transferred on MS
media containing 2 mg/litre of benzylaminopurine (BAP). After about
4-5-weeks the lateral sprouts formed were excised and maintained on
the same medium. In order to obtain entire plants the transfer was
performed on MS medium without hormones in order to induce roots
formation. After few days from the excided sprouts transfer on the
rooting medium, a drop of a 0.5% colchicine solution was laid onto
the same. Once the plants rooted, they were transferred into pots
in a greenhouse and allowed to flower in order to verify their
fertility and their capability of forming vital seeds. In some
cases, in order to allow the hybridisation between different
species, it has been necessary to duplicate the aploids and to
perform the hybridisation on autotetraploids.
[0139] The materials obtained by the backcrossing generations or by
the in vitro propagation followed by the chromosome duplication
were screened for their phenotypic characteristics and optionally
used in the genetic improvement programs.
[0140] The chromosome number control in the stabilised
amphidiploids lines has been carried out using the root apex.
Example 4
Genetic Transformation Mediated by Agrobacterium tumefaciens
[0141] Day 1: a small quantity of Agrobacterium tumefaciens of the
EHA 105 strain containing the plasmid of interest, collected from a
culture on Petri dish with a sterile handle, has been inoculated in
2 ml of sterile LB medium. Subsequently, a leaf of a healthy plant
presenting no alterations whatsoever and showing, on the other
hand, ideal turgidity conditions, has been collected. The leaf has
been briefly rinsed in bi-distilled water in order to eliminate the
superficial impurities and dipped in a 20% sodium hypochlorite 0.1%
SDS solution for 8 minutes and allowed to dry in a sterile cabinet
flux. and all the successive operations have been carried out under
sterile conditions. In particular the leaf has been dipped into 95%
ethanol and shacked in order to fully wet both sides for about
30-40 sec. The leaf has been than allowed to dry completely.
[0142] With an ethanol sterilised punch discs from all the leaf
surface have been obtained and have been dropped onto plates
containing antibiotics-free MS10; in detail, not more than 30 disks
per plate have been placed. Subsequently, 2 ml of LB plus
agrobacterium (freshly inoculated) have been poured on the plate
and the bacterial suspension has been uniformly spread with a
gentle rotating movement in order to obtain a homogeneous bacterial
distribution onto the discs. The LB in excess has been carefully
aspirated with a pipette. A negative control where nothing or mere
LB has been added has been carried out.
[0143] The plates have hence been incubated for 24-48 hours at
28.degree. C., with constant illumination and the bacterial growth
has been pointed out by the appearance of a thin opaque halo
diffused onto all the plate.
[0144] Day 2: the leaf discs have been carefully transferred on a
plate containing MS10+cephotaxime 500 mg/l, and incubated for 6
days at 28.degree. C., in constant lighting. This step determines
the agrobacterium inactivation.
[0145] Day 8: the leaf discs have than been carefully transferred
onto MS10+cephotaxime 500 mg/l and Kanamicyne 200 mg/l, and
incubated for 14 days at 28.degree. C., in constant lighting. This
step determined the selection of the transformed plants since the
kanamycine resistance gene is carried by the plasmid inserted in
Agrobacterium.
[0146] Day 22: the leaf discs that have grown in the meantime, thus
forming a callus, have been carefully transferred onto
MS10+cephotaxime 500 mg/l, kanamycine 200 mg/l and carbenicillin
500 mg/l, and incubated for 6 days. This step determines the
elimination of the agrobacteria possibly survived to the preceding
antibiotic treatments.
[0147] Day 28: the leaf discs have been once more transferred onto
MS10+cephotaxime 500 mg/l and kanamycine 200 mg/l, and incubated up
to the appearance of sprouts. Once the sprouts presented at least
two leaves, they have been separated from the callus mass and
transferred onto rooting medium MSO+cephotaxime 500 mg/l and
kanamycine 200 mg/l.
[0148] Once the roots appeared, the small plants have been
extracted from the plate, freed from the agar residues, gently
rinsed in running water and planted in small plastic pots in soil
and sand (2:1). The soil has been previously saturated with water,
subsequently, the pots have been covered with plastic transparent
lids in order to maintain high humidity conditions and have been
placed in a growing chamber at 25.degree. C. with a daily lighting
period of 16 hours. The transgene presence was screened on all
plants collecting a leaf portion (250 mg), extracting the DNA and
carrying out a first PCR analysis and than, on positive plants, a
Southern analysis in order to verify the number of copies of the
transgene.
Example 5
Oil Extraction from the Seed
[0149] The tobacco seed produced by certain selected varieties has
been used for oil production. In one case the analysed seed has a
humidity content of 7.01% and a fatty substances content
(extraction with hexane) of 39.4%. The oil extraction has been
carried out as a cold extraction, using a screw press mod. Kornet
(IBG, Germany), manually loaded with tobacco seeds. At steady state
the press has reached and maintained a temperature of 60.degree. C.
After pressing the oil has been filtered on paper and shows high
clearness characteristics. The yield of oil extracted has proven to
be of the 81.1% of the total oil contained in the seed. The
residual oilcake had a oil content of 0.74% and a protein content
of 34.5%.
Example 6
Combustion Tests
[0150] The oil coldly extracted and filtered with paper filters as
in example 5, has been transesterified with methyl alcohol in
presence of NaOH. The oil has been heated up to.sub.--55.degree. C.
and the methanol-NaOH solution added and mixed for 90 minutes. At
the end of this step, after cooling down, the mixture has been
allowed to stratify thus dividing the lower glycerol layer from the
ester. The ester has been washed two times with water and in the
first washing phosphoric acid (2.5 ml/litre) has been added to the
water. At the end of the process the oil has been heated under
vacuum at 90.degree. C. in order to remove all water residuals and
the ester obtained is named with the abbreviation TOE (Tobacco Oil
Ester). The trials have been carried out with an indirect injection
diesel engine (details: 4 cylinders, rotary injection pump,
compression ratio 21.5:1, maximum power 55 kW at 4500 rpm). Prior
to the combustion trials, analytic parameters such as viscosity at
50.degree. C. (21.63 mm2/s), melting point (-18.degree. C.),
superior calorific value (9,670 KCal/kg), volumic mass at
15.degree. C. (925.0 kg/m3), sulphur (<0.01% m/m), ashes (0.005%
m/m) have been verified. During the trial a gas analyzer and an
hydraulic dynamometer have been used. The oil ester has been used
admixed at 20% with diesel having the following main
characteristics in comparison with TEO (indicated in brackets):
density 840.8 (886.6), viscosity at 40.degree. C. 2.9 (3.3),
sulphur content (mg/kg) 6,750 (6). The test results have not
pointed out detectable variations in the engine performance using
the two products and showed a more complete combustion that
resulted in a higher thermal efficiency when the diesel was added
up with TEO. The maximum power increase has been observed at 2,200
rpm with a 3.5% increase in comparison with pure diesel (29.86 kW
against 28.85 kW). The produced CO is lower in the mixture in
comparison to the sole diesel and also the sulphur content is
lower, which determines a decrease in the SO.sub.2 emission (up to
the 40%).
Example 7
Animal Feeding Trials
[0151] The oilcake obtained from the tobacco's seeds pressing has
been analysed in order to evaluate its suitability for using as a
protein dietary supplement in animal feeds. The analytical data of
the oilcake resulting from the pressing point out a fatty
substances content of the 10.74%, a protein content of 34.5% and a
humidity and volatile substances of 5.97%. The total aminoacids
after the sample hydrolysis are: aspartic acid 2.40%, threonine
1.06%, serine 1.17%, glutamic acid 5.53%, proline 0.83%, glycine
1.29%, alanine 1.18%, valine 1.27%, methionine 0.45%, isoleucine
1.13%, leucine 1.84%, tyrosine 0.97%, phenylalanine 1.43%,
histidine 0.72%, lysine 0.72%, arginine 3.36%. Of the total content
of fatty substances, the 76.59% consists of linoleic acid that
ascribes to the Omega 6 class, that are of particular relevance for
animal diet.
[0152] The feeding trial has been carried out on piglets having a
starting weight of about 8 kg, divided in two groups, each of 20
individuals, a control group fed with a feed having a soybean
protein supplement (C), the second one having the same protein
amount with respect to the first, wherein soybean was partially
substituted with 3% of the tobacco oilcake (T). The animals treated
with the oilcake did not show any problems in beginning and
continuing for all the test's duration the intake of said feed. The
test was constantly monitored and the piglets sowed no
gastroenteric trouble. The animals showed a different daily weigh
growth (237 gr/head/day with tobacco oilcake, 170 gr/head/day with
soybean oilcake), and a final, after 27 days, weigh of 15.2 kg for
T and 12.8 for C.
* * * * *