Technical Paper January 2014

Establishing intracellular symbiotic nitrogen

fixation in crop plants for reduced inputs of
synthetic nitrogen fertilizers




Prof Edward Cocking FRS
University of Nottingham, UK


The oil crisis of the early 1970s highlighted the ever increasing cost of synthetic
nitrogen fertilizers for crop production, and their pollution of the atmosphere and
water systems has become a major environmental concern throughout the world.
Since then in the Centre for Crop Nitrogen Fixation at the University of Nottingham
we have sought ways to find a biological symbiotic substitute for synthetic nitrogen
fertilizers in agriculture. Our aim was to match Habers ingenuity, in producing NH
3

from N
2
and H
2
at high temperature and pressure, by the use of naturally occurring
nitrogen-fixing bacteria interacting symbiotically with crop plants. This would utilise
their nitrogenase enzyme to reduce N
2
to NH
3
at ambient temperature and pressure
and thereby grow and sustain agriculture without sacrificing the environment.

Legume crops such as peas and beans which already fix nitrogen from the air
symbiotically, thereby eliminating the need for synthetic nitrogen fertilizers, are
able to interact with nitrogen fixing rhizobia bacteria which become established
intracellularly within nodules on their roots. Our approach for more than two
decades was to attempt to imitate this nodulation interaction of legumes by
inoculating cereals and other non-legume crops with a wide range of species of
rhizobia in national and international collaborations with support from BBSRC, the
Rockefeller Foundation, The Royal Society and DFID. We clearly established that in
response to inoculation nodulation, which is genetically controlled by the plant,
could occur but that there was no intracellular colonization of the cells of the
nodules by rhizobia and therefore no resultant symbiotic nitrogen fixation.

Establishing intracellular symbiotic nitrogen fixation in crop plants for
reduced inputs of synthetic nitrogen fertilizers

The challenge for global agriculture is to grow more food on not much
more land, using less water, fertilizer and pesticides than we have
historically done
Sir John Beddington FRS
UK Government Chief Scientific Adviser

The oil crisis of the early 1970s highlighted the ever increasing cost of
synthetic nitrogen fertilizers for crop production, and their pollution of
the atmosphere and water systems has become a major environmental
concern throughout the world. Since then in the Centre for Crop
Nitrogen Fixation at the University of Nottingham we have sought ways
to find a biological symbiotic substitute for synthetic nitrogen fertilizers
in agriculture. Our aim was to match Habers ingenuity, in producing
NH3 from N2 and H2 at high temperature and pressure, by the use of
naturally occurring nitrogen-fixing bacteria interacting symbiotically
with crop plants. This would utilise their nitrogenase enzyme to reduce
N2 to NH3 at ambient temperature and pressure and thereby grow and
sustain agriculture without sacrificing the environment.

Legume crops such as peas and beans which already fix nitrogen from
the air symbiotically, thereby eliminating the need for synthetic nitrogen
fertilizers, are able to interact with nitrogen fixing rhizobia bacteria
which become established intracellularly within nodules on their roots.
Our approach for more than two decades was to attempt to imitate this
nodulation interaction of legumes by inoculating cereals and other non-
legume crops with a wide range of species of rhizobia in national and
international collaborations with support from BBSRC, the Rockefeller
Foundation, The Royal Society and DFID. We clearly established that in
response to inoculation nodulation, which is genetically controlled by
the plant, could occur but that there was no intracellular colonization of
the cells of the nodules by rhizobia and therefore no resultant symbiotic
nitrogen fixation.
THE PATH TO SYMBIOTIC NITROGEN FIXATION IN NON-LEGUME CROPS

A. Our Earlier Attempts

Plant cell walls act as a major barrier to the invasion of plant cells by
bacteria and other microorganisms.

My development in 1960 (1) of an enzymatic method to degrade
plant cell walls using cellulases resulted in the availability of plant
protoplasts plant cells lacking their cellulosic walls which could be
used to investigate the extent they could be intracellularly colonized
by bacteria, other microorganisms, viruses and also inert latex
particles of various sizes.

Interaction directly with the plasma membrane of protoplasts was
readily possible, and uptake by endocytosis was extensively
investigated by light and electron microscopy. This was in parallel
with extensive studies on the fusion of protoplasts to produce novel
plant somatic hybrids not possible by sexual hybridisation (2).

What became clear from these investigations of endocytosis in
protoplasts was that intracellular colonization of bacteria and viruses
into membrane bound vesicles was taking place (3).

My desire to focus on further investigations using nitrogen-fixing
rhizobia, isolated from legume nodules, was enabled by a generous
unsolicited project grant from Rank Hovis McDougall. This desire
arose from my PhD on nitrogen metabolism in barley plants and
being fascinated as to why legume crops, but not cereals such as
barley, could establish symbiotic nitrogen fixation with rhizobia.

A key feature of symbiotic nitrogen fixation in legume crops is the
establishment of rhizobia in membrane bound vesicles in the
cytoplasm of their nodule cells. My thinking was that this might now
be imitated in non-legume crop protoplasts, but without the need for
nodule formation, since we had developed the technology for
protoplasts to re-form a cell wall, divide and regenerate into plants
that would be systemically colonized by intracellular rhizobia in
membrane bound vesicles fixing nitrogen symbiotically.

In extensive investigations we demonstrated the uptake of rhizobia
by non-legume protoplasts (4), and also the uptake by endocytosis of
other nitrogen-fixing microorganisms (5). But regenerating plants
from these protoplasts was not possible, probably due to negative
effects of endocytosis on cell division and the difficulty of
regenerating plants from cultured protoplasts.

Because of these difficulties our approach shifted to attempts to
stimulate nodulation of cereals and other major non-legume crops by
inoculation with a range of rhizobial strains, coupled with various
chemical and enzymatic treatments. We successfully nodulated rice,
wheat, maize and oilseed rape and collaborated in an international
programme supported by the Rockefeller Foundation, but we could
not get the rhizobia to intracellularly colonize nodule cells and
establish symbiotic nitrogen fixation (6, 7). We abandoned any
approach to further obtain nitrogen fixing nodulation when other
researches showed that all genes for nodulation were plant genes,
and that nodulation could occur in the absence of rhizobia. It was
clear that the basic key requirement was the intracellular
colonization of plant cells by nitrogen-fixing bacteria to achieve
symbiotic nitrogen fixation, and that nodulation was not an essential
requirement.

The findings in Brazil that in sugarcane colonization of xylem and
intercellular spaces by nitrogen-fixing bacteria provided fixed
nitrogen suggested to us another approach to establishing symbiotic
nitrogen-fixing interaction with rhizobia. This resulted in our
undertaking an extensive investigation of the internal colonization of
rice, wheat, maize, oilseed rape and the model non-legume
Arabidopsis by a wide range of labelled rhizobia (8, 9) in the
presence of compounds likely to stimulate entry and internal
colonization. We observed rhizobia growing in xylem and in
intercellular spaces and some suggestions of nitrogen fixation but it
was clear that intracellular colonization of living cells by rhizobia was
not taking place. All the indications were that rhizobia were not
suitable for the establishment of the intracellar colonization of living
cells of the roots and shoots of these non-legumes and that a totally
fresh approach was required using nitrogen-fixing bacterial species
other than rhizobia.

(1) Cocking, E.C. (1960) A method for the isolation of plant protoplasts
and vacuoles. Nature 187, 927-929.

(2) Evans, P.K., Berry, S.F., and Cocking, E.C. (1976) Somatic
hybridisation of Petunia hybrida and Petunia parodii. Nature 263,
500-502.

(3) Cocking, E.C. (1977) Uptake of foreign genetic material by plant
protoplasts. International Review of Cytology 48, 323-343.

(4) Davey, M.R. and Cocking, E.C. (1972) Uptake of bacteria by isolated
higher plant protoplasts. Nature 239, 455-456.

(5) Davey, M.R. and Cocking, E.C. (1980) Tissue and cell cultures and
bacterial nitrogen fixation. In: Subba Rao, N.S., ed. Recent advances
in nitrogen fixation. London: Edward Arnold, 281-343.

(6) Al-Mallah, M.K., Davey, M.R. and Cocking, E.C. (1989) Formation of
nodular structures on rice seedlings by rhizobia. J. Exp. Bot. 40, 473-
478.

(7) Al-Mallah, M.K., Davey, M.R., and Cocking, E.C. (1990) Nodulation of
oilseed rape (Brassica napus) by rhizobia. J. Exp. Bot. 41, 1567-1572.

(8) Webster, G. and Cocking, E.C. (1997) Interactions of rhizobia with
rice and wheat. Plant and Soil 194, 115-122.
(9) Stone, P.J., OCallaghan, K.J., Davey, M.R. and Cocking, E.C. (2001)
Azorhizobium caulinodans ORS571 colonises the xylem of Arabidopsis
thaliana. Mol Plant Microbe Interact 14, 93-97.

B. More Recent Investigations
In 1988 a new species of a non-nodulating, non-rhizobial nitrogen-fixing
bacterium (Gluconacetobacter diazotrophicus, G.d) was isolated in
Brazil from the intercellular juice of sugarcane (non-legume) stems, and
it was shown that the bacterium grew in the sucrose-rich intercellular
environment and could supply 50% of the plants overall fixed nitrogen.
Somewhat similar results were obtained with Mexican sugarcane
varieties. We thought that in other non-legumes, with much lower
levels of intercellular sucrose, that inoculation with G.d might enable
even more effective nitrogen fixation if G.d could become intracellular
in roots and stems rather than just growing in the sucrose-rich
intercellular spaces as in sugarcane. This would imitate the
intracellular colonization of legumes resulting in symbiotic nitrogen
fixation, but without the need for actual nodule formation. Our
discovery that this is indeed possible using a Mexican strain of G.d is
now enabling symbiotic nitrogen fixation to be established in a range of
non-legumes, and legumes.
We have shown in patents granted in the US and EU, and in subsequent
primary publications, that interaction of G.d with maize, rice, wheat,
oilseed rape, tomato and the model non-legume Arabidopsis
results in extensive intracellular colonization of roots and shoots
and into seed progeny. We demonstrated the generality of intracellular
colonization in this wide range of plant species (both non- legumes and
legumes), its stimulation by sucrose and the need for interaction with
very few G.d bacteria. To visualise in sections the bacteria
unambiguously within membrane bound compartments in the
cytoplasm, G.d labelled with a blue histochemical marker gene was
used to inoculate seedlings (Fig 1). In maize for instance we
demonstrated that 7 days post inoculation 91% of inoculated plants
had intracellularly colonized root tips as in Fig 2. 21 days post
inoculation G.d had spread into the shoot system and blue-stained G.d
could be detected in chloroplast-containing leaf cells (Fig 3). In
divisional US and EU patents we showed microscopically and by
molecular characterisation of seed progeny from inoculated seedlings
that G.d in membrane bound cytoplasmic compartments could be
transmitted to seed progeny.
It has been shown that G.d is able to excrete the nitrogen it fixes from
the air in a form (NH3) that is potentially available to plants. In all
the non-legume crops investigated, using inoculation with G.d
carrying a nitrogenase gene promoter linked to the blue
histochemical gene marker, expression of this construct to turn the
G.d bacteria blue demonstrated that intracellular conditions in the
cytoplasmic compartments containing G.d were suitable for nitrogenase
gene expression and nitrogen fixation. This was confirmed by direct
measurement of the nitrogenase enzyme activity of G.d inoculated wheat
seedlings using the acetylene reduction gas chromatography assay
during 24 hours, and an activity level per wheat seedling of 50% of that
of clover inoculated with rhizobia was recorded. Additionally this was
also confirmed in maize intracellularly colonized by G.d, using the heavy
isotope of nitrogen (
15
N) to show that up to 30% of total nitrogen per
plant was derived from atmospheric nitrogen by symbiotic nitrogen
fixation in young plants.
Further proof of concept has been obtained in oilseed rape seedlings
inoculated under controlled growth room conditions and systemically
intracellularly colonized by G.d. Inoculated seedlings were deprived
of any inputs of fixed nitrogen synthetic fertilizer but nevertheless
they were able to grow for many months forming green shoots and
leaves (Fig 4), proving that oilseed rape plants, initially colonized in
their root meristem cells (Fig 5) can obtain all their required nitrogen
from atmospheric nitrogen as a result of systemic intracellular
colonization and resultant symbiotic nitrogen fixation.

Fig 1
Section of region of tomato root tip showing blue-stained G.
diazotrophicus bacteria (arrows) within cells (scale bar = 10 m).

Fig 2
Section of edge of maize root tip showing blue-stained G.
diazotrophicus bacteria within cells (scale bar = 10 m).

Fig 3
Section of region of maize leaf showing blue-stained G. diazotrophicus
bacteria within cells and closely associated with chloroplasts.

Fig 4
Oilseed rape, inoculated with G. diazotrophicus and systemically
intracellularly colonized, growing for many months without any inputs of
fixed nitrogen synthetic fertilizer, and forming green shoots and leaves.

Fig 5
Schematic representation of the interaction of G. diazotrophicus with
roots: (a) meristematic zone of the elongating root, (b) G.
diazotrophicus penetrates the epidermal cell wall by secretion of
cellulase enzymes, (c) the plasma membrane pinched off via
endocytosis forms a membrane surrounding vesicles containing G.
diazotrophicus, (d) vesicles with G. diazotrophicus are surrounded by a
membrane analogous to the symbiosome membrane of rhizobia.

References
Cocking EC, Stone PJ, Davey MR (2005) Symbiosome-like intracellular
colonization of cereals and other crop plants by nitrogen-fixing bacteria
for reduced inputs of synthetic nitrogen fertilizers. Sci China C Life Sci.
48, Special Issue 888-896.
Cocking EC (2005) OBPC Symposium: Maize 2004 & beyond-
intracellular colonization of cereals and other crop plants by nitrogen-
fixing bacteria for reduced inputs of synthetic nitrogen fertilizers. In
Vitro Cell Dev Biol Plant 41, 369-373.
Cocking EC, Stone PJ, Davey MR (2006) Intracellular colonization of
roots of Arabidopsis and crop plants by Gluconacetobacter
diazotrophicus. In Vitro Cell Dev Biol Plant 42, 74-82.
Cocking EC (2009) The challenge of establishing symbiotic nitrogen
fixation in cereals. In: Nitrogen Fixation in Crop Production. Agronomy
Monograph 52, 35-63 (American Societies of Agronomy, Crop Science
and Soil Sience).
Prof Edward Cocking FRS
April 2013




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Azotic Technologies Ltd was established in January 2012 to develop and commercialise a coating technology,
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