Biotechnology and Genetic Engineering Reviews

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Green biotechnology, nanotechnology and bio-

fortification: perspectives on novel environment-
friendly crop improvement strategies

Shikha Yashveer, Vikram Singh, Vineet Kaswan, Amit Kaushik & Jayanti
Tokas

To cite this article: Shikha Yashveer, Vikram Singh, Vineet Kaswan, Amit Kaushik & Jayanti
Tokas (2014) Green biotechnology, nanotechnology and bio-fortification: perspectives on novel
environment-friendly crop improvement strategies, Biotechnology and Genetic Engineering
Reviews, 30:2, 113-126, DOI: 10.1080/02648725.2014.992622

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Published online: 19 Jan 2015.

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Biotechnology and Genetic Engineering Reviews, 2015
Vol. 30, No. 2, 113–126, http://dx.doi.org/10.1080/02648725.2014.992622

Green biotechnology, nanotechnology and bio-fortification:

perspectives on novel environment-friendly crop improvement
strategies
Shikha Yashveera, Vikram Singhb, Vineet Kaswanc*, Amit Kaushikd and Jayanti Tokase
a
Department of Molecular Biology & Biotechnology, College of Basic Sciences & Humanities,
CCS HAU, Hisar, Haryana, India; bDepartment of Genetics & Plant Breeding, Wheat Section,
College of Agriculture, CCS HAU, Hisar, Haryana, India; cDepartment of Biotechnology, College
of Basic Science & Humanities, SDAU, Sardarkrushinagar 385506, Gujarat, India; dAmity
Institute of Biotechnology, Amity University Uttar Pradesh, Noida, India; eDepartment of
Biochemistry, College of Basic Sciences & Humanities, CCS HAU, Hisar, Haryana, India
(Received 13 September 2014; accepted 24 November 2014)

Food insecurity and malnutrition are prominent issues for this century. As the world’s
population continues to increase, ensuring that the earth has enough food that is
nutritious too will be a difficult task. Today one billion people of the world are
undernourished and more than a third are malnourished. Moreover, the looming
threat of climate change is exasperating the situation even further. At the same time,
the total acreage of arable land that could support agricultural use is already near its
limits, and may even decrease over the next few years due to salination and desertifi-
cation patterns resulting from climate change. Clearly, changing the way we think
about crop production must take place on multiple levels. New varieties of crops
must be developed which can produce higher crop yields with less water and fewer
agricultural inputs. Besides this, the crops themselves must have improved nutritional
qualities or become biofortified in order to reduce the chances of ‘hidden hunger’
resulting from malnourishment. It is difficult to envision the optimum way to
increase crop production using a single uniform strategy. Instead, a variety of
approaches must be employed and tailored for any particular agricultural setting.
New high-impact technologies such as green biotechnology, biofortification, and
nanotechnology offer opportunities for boosting agricultural productivity and enhanc-
ing food quality and nutritional value with eco-friendly manner. These agricultural
technologies currently under development will renovate our world to one that can
comfortably address the new directions, our planet will take as a result of climate
change.
Keywords: malnutrition; transgenic; vitamin deficiency; green innovations; precision
farming

1. Introduction
Bringing to an end, food insecurity, hunger, and malnutrition is a pressing global prior-
ity. The current global architecture for governing food, nutrition, and agriculture has not
been able to adequately address the challenges the system now faces and ensure pro-
gress toward food security. New high-impact technologies such as Green Biotechnology,
Biofortification, and Nanotechnology now offer opportunities for boosting agricultural

*Corresponding author: Email: vineetkaswan@gmail.com

© 2015 Taylor & Francis

114 S. Yashveer et al.

productivity and enhancing food quality and nutritional value. Plant varieties are
required which are capable of surviving and even thriving in a variety of rapidly chang-
ing and extreme environmental conditions.
Much attention has been placed on generating crops which are tolerant to heat,
drought, and other environmental stresses. The methods by which scientists are address-
ing this challenge are creative and green to say the least. Plant architecture, for example,
can be modified to enable plants to resist adverse environmental conditions. The shape,
distribution, and consistency of plant roots and leaves can be designed to better catch
and retain water in times of extreme drought. Roots can be altered for shallow growth
so that they remain close to the surface, the better to collect dew and runoff from
precipitation. Similarly, leaves can be modified to trap moisture from escaping by
strictly controlling their stomata (pores) (Bhatnagar-Mathur, Vadez, & Sharma, 2008;
Somvanshi, 2009; Tester & Langridge, 2010).
Plants with modified photosynthetic machinery can be tailored to be more receptive
to changing weather patterns. This is a considerable challenge, resting on the hope that
‘greener’ innovations – mostly based on molecular biology and genetic manipulations
of plants – will be environmentally safer, although this is not a straightforward path in
many cases. At the same time, efforts are being made on the breeding of new varieties
of staple crops that are rich in micronutrients (biofortification). New high-impact tech-
nologies, such as nanotechnology and its applications, might allow people to eat foods
without absorbing harmful allergens and cholesterol, and modifies food taste and nutri-
tional value. For such technologies, however, research efforts should be devoted to care-
fully studying both benefits and hazards early on in the application process.
Nanotechnologies, genomics, and electronics can also be useful for improving disease
diagnostics, the delivery of pesticides, fertilizers, and water, or for monitoring and man-
aging soil quality.

2. Green biotechnology
Green biotechnology is defined as the application of biological techniques to plants with
the aim of improving the nutritional quality, quantity, and production economics. Green
Biotechnology can help farmers produce food sustainably through:

2.1. Less fuel consumption on farms

Genetically modified (GM) herbicide tolerant (HT) crops help farmers by reducing the
need to plough fields in preparation for planting crops saving fuel. This resultant reduc-
tion in tractor use also helps to protect the structure of the soil which reduces erosion.
The agricultural practice of ploughing is also known as ‘tillage’. The adoption of reduced
tillage or no-tillage systems in respect of fuel use results in reductions of carbon dioxide
emissions. In addition, GM insect resistant crops have been developed to require fewer
insecticide treatments. This in turn means a reduction in fuel use and lower CO2 emis-
sions since farmers need to spray pesticides less frequently on their fields.

2.2. Carbon sequestration

As previously mentioned, crops developed with agricultural biotechnology reduce the
need for tillage or ploughing, allowing farmers to adopt conservation or ‘no-till’ farming
practices. As a result, over time soil quality is enhanced and becomes carbon-enriched
 Biotechnology and Genetic Engineering Reviews 115

since more crop residue can be left on the fields. In addition, since the soil is not
inverted by ploughing, less carbon in the soil becomes oxidised through exposure to the
air and therefore less CO2 is released into the atmosphere. In 2007, the no-till area
nearly doubled in the US and a 5-fold increase was recorded in Argentina, with GM
HT soybeans. Besides soil preservation, no-tillage agriculture saves fossil fuel use in
tractors, and decreases the economic costs and environmental impact of productive
farming. According to Barfoot and Brookes (2009), the additional amount of soil carbon
sequestered since 1996 has been equivalent to 83,179 million tonnes of carbon dioxide
which would otherwise have been released into the global atmosphere. GM HT technol-
ogy has been a key contributor to this increase in soil carbon sequestration, though it is
not the only influential factor.

2.3. Reduced fertilizer use

Today around 120 teragrams (Tg) of nitrogen are chemically fixed every year, around
80% of which is used as agricultural fertilizer (Galloway et al., 2003, 2004). Typical
nitrogen-use efficiencies for wheat, rice, and maize indicate that around 66% of this
nitrogen is lost to the environment either in the form of nitrous oxides, which are potent
greenhouse gases, or as soluble nitrates that find their way into aquatic systems.
Around 100 million tonnes is deposited globally in terrestrial, freshwater, and mar-
ine environments every year, a threefold increase over preindustrial levels (Galloway
et al., 2008; Rockström et al., 2009). Nutrient excesses are especially large in China,
northern India, USA, and western Europe, leading to widespread nutrient pollution
(Foley et al., 2011). In addition, it is predicted that the energy-intensive production of
nitrogen fertilizers will consume around 2% of global energy by 2050. For all these
reasons, it is desirable to reduce agricultural reliance on nitrogen fertilizers. However,
reducing reliance on agricultural fertilizers must be balanced with the predicted
requirement of doubling agricultural productivity to meet the demands of a growing
global population with changing consumption patterns (Beddington, 2010). Different
biotechnological approaches can be used to reduce fertilizer use:

2.3.1. Nitrogen use efficiency technology

Crops which are efficient in nitrogen usage and/or have lower nitrogen requirements are
much needed. GM rice and canola has been developed that uses nitrogen more effi-
ciently, so the plants need less fertilizer. This so-called ‘nitrogen use efficiency’ technol-
ogy produces plants with yields equivalent to conventional varieties but which require
significantly less nitrogen fertilizer because they use it more efficiently. This technology
has the potential to reduce the amount of nitrogen fertilizer lost by farmers every year
due to leaching into the air, soil, and water ways.

2.3.2. Engineering cereal crops to fix their own nitrogen

A biotechnological approach where cereal crops are engineered to fix nitrogen has the
potential to reduce fertilizer use and greatly reduce the environmental impact of increas-
ing yields. However, replacing nitrogen fertilizer would require levels of nitrogen fixa-
tion in cereals equivalent to those that occur in legumes, and would therefore be
extremely challenging.
There are multiple biotechnological approaches currently being explored that could
deliver fixed nitrogen to cereal crops (Beatty & Good, 2011; Oldroyd & Dixon, 2014).
116 S. Yashveer et al.

One scenario focuses on engineering is a nitrogen fixing symbiosis in cereal roots, either
through transferring the legume-rhizobial interaction to cereals or through improving
pre-existing associations in cereal roots. Alternatively, the nitrogenase enzyme itself
could be introduced into organelles of plant cells to create a new nitrogen-fixing capa-
bility. This is an attractive solution and is currently underway in at least two major pro-
jects but has two notable challenges. Firstly, nitrogenase is a highly complex enzyme
and would require the coordinated expression of at least 16 nif genes (Temme, Zhao, &
Voigt, 2012). Secondly, nitrogenase activity has high energetic demands but while aero-
bic respiration is therefore essential, nitrogenase is irreversibly denatured by oxygen.
Unlike symbiotic nitrogen fixation, no eukaryotes have evolved a nitrogen-fixation capa-
bility, despite plastids being derived from endophytic cyanobacteria. This might indicate
that there are fundamental barriers to nitrogenase activity in plant plastids. Engineering
a nitrogen-fixing symbiosis can provide solutions to these problems found in nature, by
adapting existing signalling and developmental mechanisms to provide a suitable envi-
ronment for nitrogenase activity in the plant nodule. Both of these approaches are
highly challenging and it is unlikely that in the short term any plant will deliver the lev-
els of fixed nitrogen equivalent to fertilizer application rates in the developed world.
However, even low levels of nitrogen fixation could be transformative for crop yields in
the developing world. It is hoped that these biotechnological approaches may gradually
reduce the requirement in agriculture for inorganic fertilizer.

2.4. Crop adaptation to water use efficiency

Agriculture accounts for 70% of all water use; if current trends continue, predicted
water shortages in agriculture have been identified as the single most significant con-
straint on crop production over the next 50 years. Agricultural biotechnology can play a
significant role in enabling farmers to improve yield by using water more sustainably
and helping to cope with water scarcity. This works in two main ways:

2.4.1. Minimising water loss from agriculture

Agricultural biotech practices have been developed to reduce the amount of ploughing
required before planting their crops. This means the soil surface is not inverted which
helps to trap soil moisture. Under drought conditions this can mean the difference
between having a crop to harvest and crop failure.

2.4.2. Improving drought tolerance

Plants react to stresses such as drought by consuming large quantities of stored energy
normally used for growth and seed production. Drought conditions can, therefore, drain
the plant’s energy reserves, resulting in irreversible damage to the plant or even death.
Agricultural biotechnology practices which improve drought tolerance have an immedi-
ate positive impact on the plant’s resilience and the energy available to it for growth to
maturity and seed production. Drought-tolerant maize has now entered the regulatory
phase of development in the US, demonstrating that a GM solution to this important
issue is well beyond the theoretical stage.
The Water Efficient Maize for Africa partnership, led by the African Agriculture
Technology Foundation, is a five-year public–private partnership aiming to develop new
African drought-tolerant maize varieties incorporating the best technology available
 Biotechnology and Genetic Engineering Reviews 117

internationally. The national agricultural research systems, farmer’s groups, and seed
companies participating in the project will contribute their expertise in field testing, seed
multiplication, and distribution. The current timing for the availability of the crop is
2017. Kenya has recently announced its intention to commence field trials with this type
of maize. Hybrid crops have been developed to tolerate drought and periodic water defi-
cits. Over the next decade, several companies plan to introduce GM crops that will fur-
ther improve drought tolerance.

2.5. Increased photosynthetic activity

Corn and sugarcane grow like weeds. They typically get yields that are about 70%
higher, need less water, less nitrogen, and are less vulnerable to the droughts than wheat
and rice.
Corn and sugarcane have the advantage of a form of photosynthesis called C4
instead of C3. Plants that use C3 photosynthesis have to open their pores to absorb CO2
during the heat of the day, while the sun is shining on them. As a result, they lose mois-
ture to the warm air. Plants that use C4 photosynthesis inhale carbon dioxide at night,
when it’s cool, and save it for combining with sunlight during the day. By doing that,
they hang on to more water and capture almost twice as much of the sun’s energy as
calories than C3 plants. And the system they have to turn CO2, water, and sunlight into
sugars needs 30% less nitrogen than the system in C3 plants.
Now an international team of scientists funded in part by the Bill and Melinda Gates
Foundation is working to move the genes for C4 photosynthesis from corn and sugar-
cane into rice – ‘C4 Rice’ (http://www.irri.org/c4 rice). If the projects succeed, there
will be rice and wheat that produce one and a half times the yield per acre, that require
less water per calorie, that need less fertilizer per calorie, and that are more resistant to
drought.
The C4 genes are totally natural. What the C4 rice project is doing, in a sense, is
cross-breeding rice with the plants that have evolved to use C4. The resulting crop will
be more than 99.9% rice, but with the energy capturing genes that evolved in thousands
of other plants, and have been present in nature for 30 million years.
Another example where there is enhanced utilization of solar energy is of winter-
beet. At present, sugarbeet is sown in the spring and harvested in the fall. The vegeta-
tion period can be prolonged when the beets are already sown in the fall, making it
possible to increase the sugar yield by 20 to 30 percent due to better photosynthetic effi-
ciency. However, the development of a cold-resistant beet is no easy task. Not just
because a beet of this kind has to withstand frost and cold, it can also not be allowed to
produce any undesired inflorescence (‘bolters’). In order to prevent bolting and flower-
ing triggered by cold stimulus, certain genes involved with these processes have to be
activated or shut off. Many years of research and development will be needed before
cold-resistant sugarbeet varieties can make their way to the market.

3. Biofortification
Unfortunately, agricultural systems have never been explicitly designed to promote
human health and, instead, mostly focus on increased profitability for farmers and agri-
cultural industries. Agriculture met the challenge of feeding the world’s poor during the
‘Green Revolution,’ focusing primarily on three staple crops rice (Oryza sativa L.),
wheat (Triticum aestivum L.), and maize (Zea mays L.). These crops provided enough
118 S. Yashveer et al.

energy to prevent widespread famines in many developing nations. An unforeseen con-

sequence of that agricultural revolution was the rapid rise in micronutrient malnutrition
in many nations that adopted the cropping systems that prevented large-scale starvation.
Humans require at least 44 known nutrients in adequate amounts and consistently to
live healthy and productive lives. Many agricultural tools (e.g. diversification, crop
selection, fertilizers, cropping systems, soil amendments, small livestock production,
aquaculture, etc.) could be used to increase the nutrient output of farming systems. Bio-
fortification (developing food crops that fortify themselves) is the first agricultural tool
now being employed to address micronutrient malnutrition worldwide.
Biofortification is a process for increasing the bioavailable concentrations of essen-
tial elements in the edible portions of crops. Biofortification capitalizes on the consistent
daily intake of food staples, thus indirectly targeting low-income households who cannot
afford a more diverse diet. After the initial investment of developing fortified crops, no
extra costs are met, making this strategy very sustainable. Furthermore, the improved
varieties can be shared internationally. Biofortified seeds are also likely to have an indi-
rect impact in agriculture, as a higher trace mineral content in seeds confer better protec-
tion against pests, diseases, and environmental stresses, thereby increasing yield (Welch
& Graham, 2004). Biofortification is not a panacea in itself but a very important com-
plement to dietary variety and to supplementation.

3.1. Crops for biofortification

A significant portion of the developing world’s population relies largely on one or more
of the staple crops such as rice, maize, wheat, and non-cereal food crops such as potato
and cassava for their nutrition, and these are the subject of biofortification projects, both
by conventional breeding and by modern biotechnology methods.

3.2. Methods to achieve biofortification

Biofortification can be achieved either by conventional breeding or genetic modification,
both approaches have unique capabilities and constraints. A plant breeding strategy is a
long-term process requiring substantial effort and resources, but is a sustainable and
cost-effective approach useful in improving micronutrient concentrations. Substantial
genetic variation is the basis for crop improvement through plant breeding strategies
(Ortiz-Monasterio et al., 2007). Adequate genetic variation in concentrations of
β-carotene, other functional carotenoids, iron, zinc, and other minerals exists among cul-
tivars, making selection of nutritionally appropriate breeding materials possible. Also,
micronutrient-density traits are stable across environments. In all crops studied, it is pos-
sible to combine the high-micronutrient-density trait with high yield, unlike protein con-
tent and yield, which are negatively correlated; the genetic control is simple enough to
make breeding economic. Therefore, it is possible to improve the content of several
limiting micronutrients together, thus pushing populations toward nutritional balance.
Conventional breeding has been employed in different parts of the world for enhanc-
ing the levels of provitamin A carotenoids in cassava, sweet potato, iron in cassava,
banana and beans, and zinc in rice, pearl millet, and wheat. Many of the biofortified
products are now available in the market and are well accepted in rural population.
In spite of the gains that have been made in using conventional breeding for biofor-
tification, there are limitations to the use of the technique including the very limited
 Biotechnology and Genetic Engineering Reviews 119

number of traits that can be improved at the same time, available genetic variation for
the trait, and the long period required.
Transgenic approaches are advantageous when the nutrient does not naturally exist
in a crop (e.g. provitamin A in rice) or when sufficient amounts of bioavailable micro-
nutrients cannot be effectively bred into the crop. However, once a transgenic line is
obtained, several years of conventional breeding are needed to ensure that the transgenes
are stably inherited and to incorporate the transgenic line into varieties that farmers pre-
fer. Genetic modification allows more traits to be improved in much shorter periods of
time and attains much higher levels of nutrient enhancement relative to conventional
breeding. While transgenic breeding can sometimes offer micronutrient gains beyond
those available to conventional breeders, many countries lack legal frameworks to allow
release and commercialization of these varieties.

3.3. Examples of biofortification projects

3.3.1. Biofortification for increased protein content
Human cells can produce only 10 out of the 20 amino acids, the building blocks of pro-
teins, and so the missing essential amino acids must be supplied in the food. As the
body cannot store excess amino acids, their intake must be daily. In many poor develop-
ing countries, the daily intake of essential amino acids is often not sufficient due to the
scarcity of high-protein sources such as meat, fish, or soybean. Rice, cassava, and potato
are important sources of carbohydrates, but they are low in protein content. Suitable
protein candidates for biofortification include the storage protein Sporamin A from
sweet potato, the seed albumin AmA1 protein from Prince’s Feather (Amaranthus hypo-
chondriacus), and ASP1, an artificial storage protein rich in essential amino acids.
ASP1 has been introduced and expressed successfully in rice and cassava, and efforts
are under way to optimize expression and increase the level of protein accumulation in
transgenic plants.

3.3.2. Combating vitamin A deficiency

Vitamin A deficiency, particularly prevalent among children in Africa and southeast
Asia, causes irreversible blindness, and increased susceptibility to disease and mortality.
Orange sweet potato (OSP), rich in vitamin A, is the first biofortified crop to be
released. OSP varieties that are suited to African tastes and environments have been
developed and distributed in parts of Africa where prevalence of vitamin A deficiency
is high and where white or yellow varieties – which provide little or no vitamin A – are
traditionally consumed.
Rice plants produce β-carotene (provitamin A) in green tissues, but not in the seeds.
A public–private partnership to produce rice varieties rich in provitamin A culminated
in the development of golden rice, in which two genes were introduced by genetic engi-
neering. These encode the enzymes phytoene synthase (PSY) and phytoene desaturase
(CRTI). Golden rice 1 contains the PSY gene from daffodil (Narcissus pseudonarcissus)
and the CRTI gene from the bacterium Erwinia uredovora, both expressed only in the
rice seed (Ye et al., 2000). Replacing PSY with genes from maize and rice increased the
level of β-carotene by 23 times in golden rice 2 (Paine et al., 2005). Half the daily rec-
ommended allowance of vitamin A for a 1–3 year old child would therefore be provided
for in by 72 g of golden rice 2.
120 S. Yashveer et al.

In addition to rice, other crops engineered for higher β-carotene content include
potato, canola, tomato, carrot, and cauliflower (Diretto, Al-Babili, & Tavazza, 2007;
Sautter, Poletti, Zhang, & Gruissem, 2006). The first generation of provitamin A-rich
orange open-pollinated maize varieties developed using conventional breeding was
released by the Institute for Agricultural Research in Nigeria in June 2012. A human
bioavailability study using transgenic provitamin A banana began in late 2013 (http://
www.banana21.org/index.html). Queensland University of Technology and the National
Agricultural Research Organization of Uganda are developing transgenic provitamin A
bananas for Uganda. Bananas with up to 20 ppm provitamin A have been developed
and trials have commenced in Uganda. Provitamin A bananas are expected to be
released in 2019. The first field trials for a genetically engineered provitamin A bioforti-
fied cassava began in 2009. Delivery of the biofortified crops is expected in 2017.

3.3.3. Iron and zinc-rich crops

Iron deficiency anemia affects more than 2 billion people in virtually all countries,
which makes iron deficiency by far the most common micronutrient deficiency world-
wide. Iron is found in vegetables, grains, and red meat. However, the bioavailability of
iron in plants is low.
In terms of biofortification, improvement of common bean is advantageous precisely
because the baseline grain iron content is high at 55 ppm (mg/kg) and variability for the
trait is great, ranging up to 110 ppm, allowing initial breeding attempts to be much more
successful than in the cereals (Beebe, Gonzalez, & Rengifo, 2000). In addition, unlike
many cereals that are polished before eating, resulting in significant loss of nutrients, com-
mon beans are consumed as a whole, thus conserving all their nutritional content. The tar-
get areas for biofortified beans are in iron deficiency anemia prone areas of Latin America
and eastern and southern Africa where the crop is important and consumption is high, such
as the Central America, northeast Brazil, and the Great Lakes region of Africa.
The bioavailability of iron in rice is very low; the problem is aggravated by the
presence of phytate, a potent inhibitor of iron resorption, and by the lack of iron resorp-
tion-enhancing factors. Therefore, scientists have had to increase the iron content in
grains, reduce the level of phytate, and add resorption-enhancing factors. Expression of
the iron storage protein ferritin from French bean and soybean in the endosperm of rice
results in a 3-fold increase of iron in seeds. In order to decrease the level of phytate, an
enzyme that degrades it (known as phytase) has also been transformed into rice, and
efforts are currently under way to optimize the construct. Finally, over-expression of a
cysteine-rich protein that transports metals in rice can improve the rate of iron resorption
during digestion. A transgenic high-iron rice variety has been developed by the Univer-
sity of Melbourne and IRRI that contains 14 ppm iron in the white rice grain and trans-
locates iron to accumulate in the endosperm, where it is unlikely to be bound by phytic
acid and therefore likely to be bioavailable (Johnson et al., 2011).
Teams at IRRI have produced several thousand transformants of IR64 and IR69428
that carry the soybean or rice ferritin and rice nicotianamine synthase (NAS2) over
expression genetic constructs and, in the field, demonstrate the target level for iron and
surpass that for zinc. Achieving the iron and now higher zinc levels in the field requires
both a ferritin and NAS gene to be expressed correctly. The project at IRRI is now
moving beyond proof of concept to product development for high-iron and high-zinc,
highly adapted rice genotypes. Bioavailability trials are expected to begin next year, and
release is projected for about 2022 in Bangladesh.
 Biotechnology and Genetic Engineering Reviews 121

Efforts to increase iron concentrations in wheat by conventional breeding have not

been successful, and there are currently no iron-biofortified wheat varieties available for
farmers. Whole wheat grain contains approximately 30 ppm iron, of which only 5% is
estimated to be bioavailable. It is estimated that wheat requires an additional 22 ppm
iron in the whole wheat grain, for a total concentration of 52 ppm iron, to adequately
biofortify a wheat-based diet with iron.
The University of Melbourne, Australia, has been employing an approach that has
proven highly effective in rice, using NAS to increase iron concentrations in wheat and
produce biofortified wheat varieties with 52 ppm iron in whole grain. The project places
strong emphasis on multi-location field trials of wheat plants transformed with a rice
nicotianamine synthase gene (OsNAS2) under regulatory control of the maize ubiquitin
promoter, Ubi1, to provide proof of concept of the transgenics approach in wheat. Addi-
tionally, selectable marker-free transgenic populations will be developed and evaluated
in commercially important wheat backgrounds.
The John Innes Centre, Norwich UK, has been investigating several independent
strategies to increase iron concentration and bioavailability in wheat grains through
transgenic means. The approaches will target distinct stages and tissues including uptake
from the soil, remobilization from vegetative tissue during grain filling, and accumula-
tion within grains to enhance total iron in the grain (Borrill et al., 2014).
A number of zinc rice advanced breeding lines for both Boro (irrigated) and
T. Aman (rainfed) seasons are under development through a breeding program at the
Bangladesh Rice Research Institute. The first zinc rice aman variety, ‘BRRI dhan 62,’
that contains 19 ppm of zinc and 9% protein and yields 4.2 tons per hectare (yield is
similar to that of other popular, conventional varieties like BRRI dhan33 and BINA
dhan7) was released in 2013. Crop duration from seed to seed is 112–115 days for
BINA dhan7 and BRRI dhan33 while BRRI dhan62 can be harvested within 100 days,
which is the shortest duration T. Aman variety in the country. BRRI dhan62 can also
escape terminal drought because of its short duration. At least one zinc rice boro variety
with 22–24 ppm is expected to be released in 2014. In India, the first varieties are
expected to be commercialized in 2015.

3.4. Increased folic acid in tomato

Folic acid deficiency is a global health problem that affects mainly, though not exclu-
sively, women over the age of 30, and it is the main cause of anemia in at least 10 mil-
lion pregnant women in developing countries. In food, most of the folic acid occurs as
folate. In order to engineer tomatoes with higher level of folate, scientists have over-
expressed in the fruit the genes encoding the enzymes catalyzing the synthesis of two
folate precursors (Díaz de la Garza, Gregory, & Hanson, 2007). In plants both traits
were combined by crossing, vine-ripened transgenic fruit accumulated up to 25 times
more folate than controls.

4. Nanotechnology
Nanotechnology could provide possible solutions to many of the major risks in agricul-
ture. It could improve our understanding of the biology of different crops, thus poten-
tially enhance yields and nutritional values with greater control over various plant
diseases and pest incidences. It could also help in developing improved systems for
monitoring environmental conditions and delivering nutrients and/or plant protecting
122 S. Yashveer et al.

chemicals in the needed concentrations, thus controlling various plant diseases in the
right manner at the right time (Sharon, Choudhary, & Kumar, 2010). These applications
of nanobiotechnology in agriculture are gradually moving from the theoretical possibili-
ties into the applicable area and play an important role in improving the existing crop
management techniques as follows:

4.1. Detection of plant diseases

One of the major problems associated with plant disease management is the detection of
correct stage of disease. Most of the times, plant protection chemicals such as fungicides
and pesticides are applied only after the appearance of symptoms thus causing some signif-
icant crop losses. Hence, it is essential to detect and diagnose plant diseases at their early
stage itself, so that plant protecting chemicals could be applied at correct dose at the right
time thus avoiding residual toxicity and environmental hazards. Many of the molecular
systems for the detection of micro-organisms are primarily based on specific nucleotide
probe detection or on specific antibodies and such systems are highly sensitive and selec-
tive and hence mostly suited for laboratory uses only. Proper sensing systems that could
detect and quantify pathogens in defined positions of the field would help the growers in
site-targeted and optimized application of agrochemicals with minimal environmental haz-
ards. In this scenario, nanobiosensors, once installed in the field, could detect pathogens
with high sensitivity and specificity. Such nanosensors are highly potable systems with
‘real-time’ monitoring of results. They do not need extensive sample preparations and
detection is label-free also (Sadanandom & Napier, 2010). Cell biologists of Cornell Uni-
versity are investigating on nanofabrication technologies to understand how the fungi and
bacteria feel their way on plant surface, to induce infection (Mccandless, 2011).

4.1.1. Smart delivery systems for fertilizers, pesticides, and for the control of plant
diseases
Agrochemicals are conventionally applied to crops by spraying and/or broadcasting. In
order to avoid the problems such as leaching of chemicals, degradation by photolysis,
hydrolysis, and microbial degradation, a concentration of chemicals lower than mini-
mum effective concentration to reach the target site of crops is required. The nanocapsu-
lated agrochemicals are designed in such a manner that they hold all essential properties
such as effective concentration, time-controlled release in response to certain stimuli,
enhanced targeted activity, and less eco-toxicity with safe and easy mode of delivery,
thus avoiding repeated application. The best example is the reduction of phytotoxicity
of herbicides on crops by controlling the parasitic weeds with nanocapsulated herbicides
(Pérez-de-Luque & Rubiales, 2009).

4.2. Improved germination

Cañas and co-workers (2008) reported the effects of functionalized single-walled carbon
nanotubes (SWCNTs) and non-functionalized SWCNTs on root elongation of six differ-
ent crop species, such as cabbage (Brassica oleracea), cucumber (Cucumis sativus), car-
rot (Daucus carota), onion (Allium cepa), lettuce (Lactuca sativa), and tomato
(Solanumly copersicum). They showed that the root elongation in onion and cucumber
was enhanced by non-functionalized SWCNTs, and the interaction of both functional-
ized SWCNTs and non-functionalized SWCNTs with root surface, resulted in the
 Biotechnology and Genetic Engineering Reviews 123

formation of nanotube sheets on cucumber root surface, without entering into the roots.
However, cabbage and carrot remained unaffected by either form of nanotubes. Further-
more, functionalized SWCNTs inhibited the root elongation of lettuce, while tomato
was found to be most sensitive to non-functionalized SWCNTs with significant root
length reduction, whereas a positive response has been shown on the seed germination
and growth of tomato plants upon interaction with multi-walled carbon nanotubes
(MWCNTs) (Khodakovskaya et al., 2011). They showed that the presence of MWCNTs
increased water uptake by seeds which in turn enhanced the germination process. Simi-
lar positive effects of MWCNTs on seed germination and root growth of six different
crop species – radish (Raphanus sativus), rye grass (Lolium perenne), rape (Brassica na-
pus), lettuce (Lactuca sativa), corn (Zea mays), and cucumber (Cucumis sativus) – was
also reported (Lin & Xing, 2007). Remya et al. (2010) also reported the positive effects
of both SWCNTs and MWCNTs on the germination of rice seeds and observed an
enhanced germination for seeds germinated in the presence of nanotubes.
Nonetheless, the interaction of different nanomaterials with plants and their mecha-
nism for genetic and molecular modification of plants are still unpredictable.
The interaction of nanomaterials with plants differs with type and time of exposure
to nanomaterials, so these facts should be kept in mind while performing nanotoxicity
studies. Additionally, the orientation of nanotubes with respect to the plant cell wall
might be important for their penetration, but the mode of entry of nanotubes through
the cell wall remains mysterious which still needs more studies.

4.3. Agricultural diagnostics and drug delivery with nanotubes

Agriculture is seasonal in nature and depends on many variables such as soil, crops,
weather, etc. Sensing, acquisition, manipulation, storage, and transfer of reliable and
accurate data about the plant and production handling environment is, therefore, crucial
in managing this variability to optimize both inputs and outputs and reduce impacts on
the environment to meet the demand for high and good quality products. Based on
advances in nanotechnology research, a new scanning probe-based data storage concept
called ‘millipede’ was developed that combines ultrahigh density, terabit capacity, small
form factor, and high data rate. Other developments include nanosensors to monitor the
health of crops and magnetic nanoparticles to remove soil contaminants. Dispersed
throughout fields, a network of nanosensors would relay detailed data about crops and
soils. The sensors will be able to monitor plant conditions, such as the presence of plant
viruses or the level of soil nutrient. Nanofabricated devices offer the scope for their
injection into plants to detect tissue parts affected by rare phenomena such as diseases,
nutrient deficiency, and developmental abnormalities (Opara Linus, 2004).

4.4. Precision agriculture

Precision agriculture refers to new farming methods based on optimizing resources and
minimizing inputs, including water and fertilizer. Precision agriculture can include sophisti-
cated devices such as global positioning system to identify factors ranging from moisture
and nutrient content of soils to pest infestation of a given crop. Using this approach, optimal
inputs can be applied to a specific region of a given crop when required, rather than uni-
formly and at predetermined times across the entire field, whether the crop requires inputs
or not. The great advantage of this technique is the avoidance of overuse of pesticides,
herbicides, fertilizer, and water (earthobservatory.nasa.gov, www.ghcc.msfc.nasa.gov). The
124 S. Yashveer et al.

same principles of precision farming can also be applied to developing countries, without
the requirement of advanced technologies. For example, the concept of drip irrigation, a
practice by which small amounts of water are applied to plant root systems by a network of
irrigation pipes, has been demonstrated to work successfully for drought-prone areas.
Similarly, some resource-poor countries utilize a farming technique whereby tiny amounts
of fertilizer are applied to the roots of crops at specific times in the growing season. These
low-tech farming practices have enabled farmers who have poor access to water or artificial
fertilizers to make the most of their crop yield.

4.5. Developing transgenic plants with novel properties

Nanotechnology has shown its ability in modifying the genetic constitution of plants by
introducing novel genes, thus resulting in crop improvement. The application of fluores-
cent labeled starch-nanoparticles as plant transgenic vehicle, was reported in which the
nanoparticle biomaterial was designed in such a way that it binded the gene and trans-
ported it across the cell wall of plant cells by inducing instantaneous pore channels in
the cell wall, cell membrane, and nuclear membrane with the help of ultrasound (Jun
et al., 2008). It is possible to integrate different genes on the nanoparticle at the same
time and the imaging of fluorescent nanoparticle is possible with fluorescence micro-
scope, thus understanding the movement of exterior genes along with the expression of
transferred genes. It was reported that polyamidoamine (PAMAM) dendrimer acts as a
nanocarrier for delivering genes into plant cells with intact cell wall. Supramolecular
complexes of PAMAM dendrimer-DNA are formed through electrostatic interactions
and these complexes are penetrated through the cell walls of turf grass calli, expressing
foreign genes within the cells (Pasupathy, Lin, Hu, Luo, & Ke, 2008).
Surface functionalized mesoporous silica nanoparticles (MSNs) provide new ways to
precisely manipulate gene expression at single cell level by delivering DNA and its acti-
vators in a controlled fashion (Torney, Trewyn, Lin, & Wang, 2007) by penetrating
through plant cell wall. MSNs are loaded with gene and its chemical inducer and the
ends are capped with gold nanoparticles to protect the molecules from leaching out.
Uncapping of capping agents results in stimuli responsive release of chemicals, thus
triggering gene expression in plants. It is found that surface modification of MSNs with
triethylene glycol promotes their easy penetration into cells and also allows plasmid
DNA to absorb on MSN surface. In this method, the minimum amount of DNA
required to detect marker expression is 1000-fold less than that required for the conven-
tional delivery method and such delivery method has significant applications in various
gene expression studies. Such nanoparticle-mediated plant transformation allows site-
targeted simultaneous delivery of both DNA and effector molecules. Future possibilities
include enlargement of pore size and multifunction-alization of MSNs, which support
site-targeted delivery of proteins, nucleotides and chemicals in plant biotechnology. The
ability of carbon nanotubes to penetrate intact plant cell wall and cell membrane has
already been reported (Liu et al., 2010). Thus, the nanomaterial-mediated transformation
methods will provide great possibilities in developing transgenic plants.

5. Conclusions
Climate change brings with it some daunting challenges. More food must be produced
on less arable land than is available today. New agricultural technologies and farming
practices must be developed and implemented. This chapter has attempted to address
 Biotechnology and Genetic Engineering Reviews 125

some of the strategies currently under development in the agricultural sciences. One
way to achieve global food security requires the utilization of novel plant breeding strat-
egies which will quickly find helpful traits that enable plants to thrive under adverse
environmental conditions. Green technology will bring an extensive transformation of
agriculture to increase production and improve quality in an equitable and sustainable
manner without compromising the environment (Godfray et al., 2010). Biotechnology
and nanotechnology will play a paramount role in these approaches. Revolutionary
farming techniques, led by precision agriculture, will keep crop yields high while main-
taining water, pesticide, and nitrogen inputs to a minimum. Key food crops have already
been biofortified with micronutrients such as iron and vitamin A. With these and other
strategies in place, the world will be better prepared to address the future challenges that
will result from climate change and increasing population.

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