REVIEW

Genetic enhancement of Brassica napus seed quality

Abdelali Hannoufa • Bhinu V. S. Pillai •

Sreekala Chellamma

Received: 30 April 2013 / Accepted: 17 August 2013 / Published online: 27 August 2013

� Her Majesty the Queen in Right of Canada 2013

Abstract The ultimate value of the Brassica napus

(canola) seed is derived from the oil fraction, which

has long been recognized for its premium dietary

attributes, including its low level of saturated fatty

acids, high content of monounsaturated fatty acids,

and favorable omega-3 fatty acid profile. However, the

protein (meal) portion of the seed has also received

favorable attention for its essential amino acids,

including abundance of sulfur-containing amino acids,

such that B. napus protein is being contemplated for

large scale use in livestock and fish feed formulations.

Efforts to optimize the composition of B. napus oil and

protein fractions are well documented; therefore, this

article will review research concerned with optimizing

secondary metabolites that affect the quality of seed

oil and meal, from undesirable anti-nutritional factors

to highl value beneficial products. The biological,

agronomic, and economic values attributed to second-

ary metabolites have brought much needed attention to

those in Brassica oilseeds and other crops. This review

focuses on increasing levels of beneficial endogenous

secondary metabolites (such as carotenoids, choline

and tochopherols) and decreasing undesirable

antinutritional factors (glucosinolates, sinapine and

phytate). Molecular genetic approaches are given

emphasis relative to classical breeding.

Keywords Brassica napus � Seed quality �Genetic manipulation � Anti-nutritional factors �Secondary metabolites

Introduction

Research and development efforts to improve seed

quality in Brassica napus are well-established and

based on nutritional concerns driven by consumer and

food industry priorities. Brassica oilseeds generate a

large proportion of the world’s edible oil production,

with canola oil itself contributing 13–16 % between

1999 and 2009 (USDA-ERS 2012). Total USA supply

of canola oil in 2011–2012 was estimated at 4.71

billion pounds and consumption at 4.52 billion pounds

(USDA-Economic Research Service, United States

Department of Agriculture 2012). Desirable oil and

meal quality traits such as reduced contents of erucic

acid and glucosinolates were introduced into B. napus

using conventional breeding approaches during the

1970s and 1980s (Kimber and McGregor 1995).

However, conventional breeding is time consuming,

less straightforward and less predictable than modern

and directed genetic engineering.

Transgenic technologies to over-express or sup-

press single or multiple genes allow for rapid and

A. Hannoufa (&)

Agriculture and Agri-Food Canada, 1391 Sandford Street,

London, ON N5V 4T3, Canada

e-mail: Abdelali.Hannoufa@agr.gc.ca

B. V. S. Pillai � S. Chellamma

Dow AgroSciences, LLC, 9330 Zionsville Road,

Indianapolis, IN 46268, USA

123

Transgenic Res (2014) 23:39–52

DOI 10.1007/s11248-013-9742-3

directed engineering of plant metabolism to achieve

specific plant traits. Genetic engineering of B. napus

can benefit from the wealth of genomics resources that

have been developed in recent years in the Brassica

family, including a large collection of expressed

sequence tags (www.brassicagenomics.ca/ests),

genetic mapping populations (www.brassica.ca) and

genome sequences (www.jic.ac.uk/staff/ian-bancroft/

research_page4.htm; www.bayercropscience.ca/our-

company/news/Bayer-CropScience-first-to-sequence-

the-entire-genome-of-rapeseed-canola). In addition,

B. napus has a unique advantage over other major

crops in that it shares high genomic colinearity with

Arabidopsis thaliana (Parkin et al. 2005; Cheung et al.

2009); the model plant with a fully sequenced genome,

large sets of mutants, and massive amounts of genomic

information (Robinson et al. 2009; The Arabidopsis

Genome Initiative 2000; http://arabidopsis.org/).

These resources combined with the ease of Agrobac-

terium-mediated transformation have allowed for

major advances in metabolic engineering of B. napus

for seed quality improvement in recent years (Bhinu

et al. 2009a, b; Fujisawa et al. 2009; Husken et al.

2005).

Modifying factors affecting oil quality

It has long been known that genotype and environment

(G 9 E) affect seed oil content and fatty acid profiles

in B. napus. This awareness had led to the pursuit of an

improved understanding of the right combination of

genotype and favorable environment and has

remained a subject of high interest for breeding and

production of higher quality oilseeds, but with little

progress. Recently, Faraji (2012) reported that oil

content in B. napus is a function of environmental

conditions during the seed filling period, which takes

our understanding a little further. For example, high

temperature (over 17 �C) stimulates oleic acid bio-

synthesis leading to a 60 % increase in content

(Tremolieres et al. 1978), while temperature and light

variations alter the levels of both saturated and mono-

unsaturated (C18:1) fatty acids and linolenic acid

(C18:3) (Deng and Scarth 1998; Si and Walton 2004;

Si et al. 2003). Although more extensive investiga-

tions are required to clarify the effect of temperature

on oil production, compounding these environmental

effects with genotypic differences complicates the

process of conventional breeding for oil quality. The

perceived opportunity is that a high level of genetic

diversity naturally exists in the Brassica C (oleracea)

genome,which makes up half of the B. napus genome,

and this diversity has already been used to alter the oil

composition of B. napus through pathway engineering

(Barker et al. 2007). Despite these important advances,

the quest to understand the overall effect of genotype

and environment on oil quality in B. napus is ongoing

and continuously evolving. Hence, it may be worth

pursuing research opportunities in other areas of seed

biochemistry and the modification of biochemical

components that are more easily defined and con-

trolled than seed oil levels or composition. For

instance, oil-soluble metabolites derived primarily

from the isoprenoid pathway, including sterols,

carotenoids, chlorophylls, and tocopherols may be

more ideal candidates for investigation.

Carotenoids

Carotenoids are antioxidant pigments that are recog-

nized for their overwhelming health benefits; how-

ever, the ability of carotenoids to impart a yellow color

to seed oil is considered undesirable for some

conventional food applications. Carotenoids find

applications in the food and cosmetics industries as

colorants, supplements in livestock and fish feed

formulations, and nutraceuticals with potential thera-

peutic properties (Fraser and Bramley 2004; Taylor

and Ramsay 2005; Botella-Pavıa and Rodrıguez-

Concepcion 2006). Additionally by virtue of their

antioxidant properties, carotenoids provide oxidative

stability to B. napus oil (Frankel 2005), and so an

obvious priority is to increase carotenoid accumula-

tion in B. napus seeds. Carotenoid accumulation in B.

napus seed varies throughout development with the

highest levels detected at approximately 35–45 days

post anthesis (Yu et al. 2008). Levels of carotenoids

drop substantially during the later stages of seed

development, with b-carotene and lutein accounting

for more than 90 % of the mature seed carotenoid

content (Yu et al. 2008). In addition, carotenoids are

heat labile and hence a significant portion of seed

carotenoids are degraded during oil processing.

The desirable attributes of carotenoids have inten-

sified interest in enhancing their levels in several

crops, including B. napus. Among numerous potential

metabolic engineering strategies to alter seed oil

40 Transgenic Res (2014) 23:39–52

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quality, altering the upper carotenoid biosynthesis

pathway at the initial step catalyzed by phytoene

synthase (PSY; CRTB; Fig. 1) seemed promising. A

straight-forward approach involving over-expression

of a bacterial CRTB in B. napus seeds resulted in

visibly orange embryos and a 50-fold increase in seed

carotenoids (Shewmaker et al. 1999). This dramatic

increase in carotenoids was accompanied by an

increase in the proportion of desirable oleic acid

(18:1) and a concomitant decrease in the proportions

of linoleic acid (18:2) and linolenic acid (18:3), which

are undesirable in food-grade oil. These studies

pointed to the significant metabolic flux (and poten-

tial) that exists in the carotenoid biosynthesis pathway

in plants. Consequently, to increase flux through this

pathway, Ravanello et al. (2003) used the crtB-

expressing B. napus transgenic lines as a platform

for additional transformation with bacterial genes

encoding geranyl geranyl diphosphate synthase

(GGPS), phytoene desaturase (PDS), and lycopene

b-cyclase (b-LYC). However, total carotenoid levels

in the enhanced transgenic seeds (expressing four

transgenes) were similar to those of the crtB-overex-

pression line, which suggests that substrate availabil-

ity may be a limiting factor in carotenoid biosynthesis.

It was suggested proteins coded by the three bacterial

genes (CRTB, PDS, b-LYC) form a multi-enzyme

complex required for proper substrate channeling in

Fig. 1 General carotenoid pathway showing the main carotenoids in B. napus seed and two novel carotenoids, astaxanthin and

canthaxanthin. Enzymatic steps that were targeted for metabolic engineering are indicated by asterisk

Transgenic Res (2014) 23:39–52 41

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plants. However, geranyl geranyl pyrophosphate

(GGPP) in the upstream mevalonate pathway, is a

multi-use substrate involved in the synthesis of

tocopherols, gibberellins and chlorophylls as well as

carotenoids (Naik et al. 2003). Competition between

these four pathways for phytoene may not always

allow for a net direction of GGPP into carotenoids

even if PSY is enhanced. Similarly, carotenoid

enzymes downstream of PSY would also be limited

by the amount of GGPP synthesized and to which

pathway it is directed. Another important aspect to be

considered is that the activity of phytoene synthase is

influenced by light (Cazzonelli et al. 2009). Given

these constraints, the step directed by PSY is often

described as a bottleneck in the carotenoid biosynthe-

sis pathway (Cazzonelli et al. 2009).

The branch point in carotenoid biosynthesis at e,b-

lycopene and b,b-lycopene cyclization (giving rise to

a-carotene or b-carotene, respectively) has also been

exploited for its potential to benefit B. napus. In

potato, tuber-specific antisense silencing of lycopene

e–cyclase (e-LYC) resulted in a substantial increase in

b,b-carotenoid and a relatively smaller increase in

total carotenoid levels with no effect on lutein content

(Diretto et al. 2006). In Arabidopsis, a mutant

defective in e-LYC was almost devoid of lutein, but

had high levels b-carotene from the competing step

(Pogson et al. 1996). In contrast, RNAi silencing of e-

LYC in B. napus resulted in a general increase in total

seed carotenoids (Fig. 2), including b-carotene,

zeaxanthin, violaxanthin, and (unexpectedly) lutein

(Yu et al. 2008). However, transgenic RNAi e-LYC

seeds also showed reduced levels of fatty acids,

although changes in the ratios of different fatty acids

were minimal (Yu et al. 2008).

Recently, strategies to modify carotenoids have

also included impacts on other desirable plant qual-

ities, and several new carotenoid regulatory genes

appear to accomplish this with a single transgene

insertion. For example, the DE-ETIOLATED 1

(DET1) gene is a negative regulator of light-mediated

responses in Arabidopsis (Schroeder et al. 2002), and

suppression of DET1 in tomato fruits led to an increase

in levels of both carotenoids and flavonoids (Davuluri

et al. 2005). In B. napus, RNAi silencing of DET1 led

to an increase in seed carotenoids and a decrease in the

level of the anti-nutritional factor 1,2-di-O-sinapoyl-

glucose, while flavonoid levels remained unaltered

(Wei et al. 2009). Nevertheless, these combined

changes resulted in an overall improvement of the

nutritional quality of the B. napus seed. Over-expres-

sion of microRNA156 in B. napus and Arabidopsis led

to enhanced carotenoid levels in seeds, while also

increasing vegetative shoot branching, although leaf

carotenoids were not significantly affected (Wei et al.

2010, 2012). When Arabidopsis cpSRP54 encoding

the 54 kDa subunit of the chloroplast signal recogni-

tion particle was expressed in B. napus, seed caroten-

oid content was enhanced with minimal impact on the

oil content and the fatty acid profile (Yu et al. 2012).

Efforts have also been made at engineering novel

carotenoids in B. napus seeds when Fujisawa et al.

(2009) simultaneously expressed seven Agrobacte-

rium aurantiacum genes involved in ketocarotenoid

biosynthesis. This comprehensive expression strategy

led to a 30-fold increase in total carotenoids, including

a-carotene, echinenone, phytoene, lutein, and cantha-

xanthin; the latter compound is not naturally synthe-

sized in B. napus.

Chlorophylls

The presence of functional chloroplasts in developing

green B. napus seeds makes them photosynthetically

active until the onset of desiccation (Eastmond et al.

1996). Photosynthesis in the chloroplasts of develop-

ing seeds is driven by the approximately 20–30 % of

light transmitted through the walls of the seed pod

(Eastmond et al. 1996; King et al. 1998; Ruuska et al.

Fig. 2 Elevated carotenoid pigmentation of seed carotenoid

extracts of B. napus with silenced lycopene epsilon cyclase.

Control, wild type B. napus DH12075; and three transgenic lines

BY223, BY365 and BY54 (Hannoufa, unpublished data)

42 Transgenic Res (2014) 23:39–52

123

2004). However, a requirement for photosynthesis and

chlorophyll synthesis in developing seeds is not well

established, since seeds are largely dependent on ATP

generated from cytosolic processes (Eastmond and

Rawsthorne 1998). Chlorophyll is one of the major

factors that negatively impact oil quality. In addition

to increasing oil rancidity (Dahlen 1973), chlorophyll

imparts a green appearance to oil that is unaccepted by

the oil industry and consumers. Most chlorophyll is

degraded during seed maturation, but if present, it is

difficult to remove when co-extracted with oil. The so-

called ‘green seed’ problem resulting from premature

harvest or unfavorable environmental factors, such as

frost, can significantly diminish the quality of B. napus

seed crops. Enzymatic degradation of chlorophyll is

regulated by pheophorbide a oxygenase and activity of

this enzyme during the early stages of seed develop-

ment is reduced by freezing temperatures (Chung et al.

2006). Even though chemical and physical processes

have been developed to reduce chlorophyll in B. napus

oil (Singh and Chuaqui 1991), only limited efforts

have exploited molecular approaches to reduce chlo-

rophyll in B. napus seed. An example is the use of

antisense RNA driven by a napin promoter to achieve

seed-specific silencing of glutamate l-semialdehyde

amino-transferase, an enzyme involved in chlorophyll

biosynthesis (Tsang et al. 2003). The resulting trans-

genic seeds with depressed enzyme activity had

reduced levels of chlorophyll, with no noticeable

effect on other seed characteristics.

Tocopherols

The importance of tocopherols, collectively known as

vitamin E (Kamal-Eldin and Appelqvist 1996), stems

from their strong antioxidative properties that scav-

enge oxygen radicals and slow down the oxidation of

unsaturated fatty acids. As antioxidants, tocopherols

quench singlet oxygen and are desired by consumers

for ingestion (Ajjawi and Shintani 2004). B. napus

seed contains small amounts of tocopherols, but like

carotenoids, much is degraded during oil extraction

processes. Approximately 30–40 % of tocopherols are

also lost during oil processing in soybean (Jung et al.

1989), however, considerable concentrations of toc-

opherols can be recovered regardless. For example, Ito

et al. (2007) reported that the molecular distillation

process reduces the degradation of soybean tocophe-

rols during oil processing. This suggests that

increasing tocopherol levels or modification of pro-

cessing methods are potential avenues for retention of

these antioxidants in B. napus oil.

The beneficial properties of tocopherols have

encouraged research efforts to profile these com-

pounds as a function of development and to find ways

to enhance their levels in B. napus seed. Zhang et al.

(2007) observed that the overall level of tocopherols in

B. napus sprouts remained unchanged during seed

germination (1–10 days), while the proportion of a-

tocopherol (the most active form) increased and c-

tocopherol decreased. As development progressed,

tocopherol levels in 20-day-old B. napus seedlings

increased to sixfold higher than levels in seeds. The

high concentration of these valuable phytochemicals

in seedling oil fractions may result from the depletion

of seed oil reserves during seedling establishment in

addition to new light-induced synthesis of a-tocoph-

erol (Zhang et al. 2007). Transgenic methods to

improve tocopherol levels initially focused on tocoph-

erol cyclases from Arabidopsis and Zea mays. When

expressed in B. napus seeds, these genes stimulated a

28 % increase in total tocopherols (Kumar et al. 2005).

Expression of the Arabidopsis c-tocopherol methyl

transferase (c-TMT) in Brassica juncea resulted in a

sixfold increase in a-tocopherol content (Yusuf and

Sarin 2007) and improved tolerance to abiotic stress

(Yusuf et al. 2010). Due to the genetic closeness of

these two species, it is likely that expression of (c-

TMT) in B. napus would have a similar effect, but this

awaits experimental confirmation. Using a classical

breeding approach, Fritsche et al. (2012) reported that

seed tocopherol content and composition were highly

heritable traits in an evaluation of 229 accessions of B.

napus. High polymorphism was also revealed in the

tocopherol genes BnaX.VTE3.a and BnaA.PDS1.c,

and pointed to the value of these two genes as

polymorphic markers for selecting germplasm with

the high tocopherol trait.

Phytosterols

Plant sterols (phytosterols) are known for their health

benefits, especially for their contribution to lowering

the levels of serum and low-density lipoprotein

cholesterol in animals and humans by inhibiting the

absorption of dietary and biliary cholesterol (Akihisa

et al. 1992; Ling and Jones 1995). B. napus seed

contains small quantities of oil-soluble phytosterols,

Transgenic Res (2014) 23:39–52 43

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and their total content is slightly influenced by

genotypes and growing locations (Hamama et al.

2003). Although the levels of phytosterols vary little

during seed germination, this process eventually

depletes seed oil reserves and sterol concentrations

increase in seedling oil by up to fivefold (Zhang et al.

2007). Stanols are hydrogenated sterols which occur

naturally in soybean oil, wood pulp, and tall oil (a by-

product of paper manufacturing), and can also be

manufactured from plant sterols (Woodgate et al.

2006). Hydrogenation of phytosterols results in phy-

tostanols that are more potent than phytosterols in

lowering cholesterol (Heinemann et al. 1991). Meta-

bolic engineering has provided avenues for modifying

the phytosterol pathway to benefit human health. For

instance, expression of 3-hydroxysteroid oxidase from

Streptomyces hygroscopicus in B. napus resulted in

the production of phytotanols that corresponded to

their phytosterol counterparts, in addition to novel

phytostanols not normally produced by chemical

hydrogenation (Venkatramesh et al. 2003).

Altering factors that affect meal quality

Studies on the chemical composition of B. napus meal

highlight the relatively high protein content compared

to the meals of other major oilseed crops, except

soybean (Enami 2011). However, large-scale inclu-

sion of crude B. napus protein in livestock and fish

feed is hindered by the presence of high levels of anti-

nutritional factors, such as glucosinolates, sinapine,

fibre and phytate. On the other hand, levels of

desirable (valuable) compounds such as choline are

relatively low. To address this problem, several groups

have embarked on research to improve the composi-

tion of B. napus meal by reducing levels of anti-

nutritional factors and enhancing levels of favourable

metabolites using a range of strategies.

Glucosinolates

Glucosinolates (GS) are a class of amino acid-derived

secondary metabolites that predominantly occur in the

Cruciferaceae family and particularly in the Brassica

genus. To date, more than 100 different aliphatic and

aromatic glucosinolates have been identified in plants

belonging to the family Brassicaceae. Many endoge-

nous myrosinase enzymes also are known and are

compartmentalized separately from GS until released

as a defense response to plant injury, whereupon they

convert glucosinolates into highly reactive isothiocy-

anates (ITCs), thiocyanates, epithiocyanates, nitriles,

and other unstable breakdown products. Although

their roles are debated and are isomer-specific, gluc-

osinolates or their breakdown-products have been

variously reported as natural host-specific feeding

attractants for Brassica-specialized insects or repel-

lants with insecticidal, bactericidal or fungicidal

properties (Sang and Salisbury 1988; Gruber et al.

2009; Brader et al. 2001; Tierens et al. 2001). Some

glucosinolate breakdown products (some nitriles and

some ITCs) are associated with toxic effects to insect

pests such as housefly, lesser grain borer, and Amer-

ican cockroach (Tsao et al. 2002), while ITCs impart a

pungent bitter flavor characteristic of mustard. There-

fore, utilization of B. napus meal (which contains the

GS fraction) has been limited in animal feed in spite of

the fact that *40 % of the seed is composed of

proteins with a relatively well-balanced amino acid

content (Underhill 1980; Fenwick et al. 1983).

The aliphatic glucosinolates in B. napus seeds

include sinigrin, progoitrin and gluconapin (Gland

et al. 1981) derived from methionine and the indolyl

glucosinolates derived from tryptophan (Underhill

1980). Since the discovery that the Polish cultivar

‘Bronowski’ contains considerably reduced glucosino-

late levels, low glucosinolate cultivars (known as canola

quality types) containing less than 30 lmol/g glucosin-

olates in the meal have been the standard in the canola

industry (Uppstrom 1995). The majority of these

cultivars have been derived from this single genetic

resource for low glucosinolate alleles. Recently, rape-

seed cultivars with as low as 10 lmol/g glucosinolates

were developed (Khajali and Slominski 2012). How-

ever, ultra-low glucosinolate cultivars only have a

reduced level of aliphatic glucosinolates. Consequently,

seeds of low glucosinolate B. napus cultivars contain

relatively high concentrations of indole glucosinolates

(4-hydroxyglucobrassin, glucobrassicin, neogluco-

brassicin), and other phenolic glucosinolates. The

oligomeric compounds generated from indole-3-carbi-

nol breakdown products of ingested glucobrassicin in

the stomach can have toxic effects on mammalian health

and resemble the carcinogenic dioxins (Bjeldanes et al.

1991). For these reasons, genotypes with reduced indole

glucosinolate (indole-GS) contents have received par-

ticular attention from B. napus breeders.

44 Transgenic Res (2014) 23:39–52

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Modification (raising) of indole-GS profiles was

achieved via metabolic engineering in Arabidopsis by

raising levels of CYP79 gene expression, but a

knockout failed to lower indole-GS levels (likely due

to gene duplication) (Mikkelsen et al. 2002). CYP79

transcript levels and glucosinolate levels also

increased in response to the application of defense

signaling compounds (Mikkelsen et al. 2003). In

contrast, Chavadej et al. (1994) reported transforma-

tion of B. napus with tryptophan decarboxylase that

blocked the conversion of tryptophan to indole-GS,

reducing indole-GS and re-directing tryptophan into

tryptamine. These different results illustrate the

potential that metabolic engineering holds for modu-

lating glucosinolates in Brassica species, but also

points to stumbling blocks that will impact on success.

Sinapine

Sinapine (sinapate choline ester) is unique to crucif-

erous oilseeds and is the predominant phenolic

compound in B. napus seed (Clauss et al. 2011), with

levels ranging from 0.7 to 4 % of the seed (Blair and

Reichert 1984) and *90 % located in the embryo

(non-hull) fraction (Velasco and Mollers 1998; Wang

et al. 1998). Sinapine is considered anti-nutritional

despite its beneficial role in the plant, in which it

supplies an important component to the biosynthesis

of plasma membrane phosphatidylcholine (Strack

1981) and contributes to UV protection (Sheahan

1996; Booij-James et al. 2000). The anti-nutritional

nature of sinapine in B. napus meal is linked to an

unpleasant flavor that accumulates in the meat and

milk of animals fed on B. napus rich in sinapine

(Pearson et al. 1980), as well as its bitter flavor in feed

resulting in poor palatability for livestock (Ismail et al.

1981). Consumption of B. napus meal rich in sinapine

(a precursor of trimethylamine, TMA) by hens is

known to impart a fishy odour to brown-shelled eggs

due the buildup of TMA in layers which are deficient

in trimethylamine oxidase (Pearson et al. 1979).

Consumption of large amounts of sinapine may also

cause serious growth and reproductive problems in

chicks (Pearson et al. 1980).

Development of B. napus germplasm with reduced

sinapine content is economically more sustainable

than reducing meal sinapine by chemical treatment

due to higher cost of meal processing (Husken et al.

2005). Approximately 80–85 % reduction in seed

sinapine content is considered sufficient for use in

animal feed. Given the substantial reduction of

sinapine required in B. napus seed, molecular engi-

neering approaches are more promising than tradi-

tional breeding or molecular marker-assisted selection

methods, although both metabolic engineering and

molecular breeding techniques have been used (Ve-

lasco and Mollers 1998; Nair et al. 2000; Bhinu et al.

2009a). Attempts to identify Brassica varieties with

naturally low sinapine content to incorporate into

breeding programs were met with limited success

(Velasco and Mollers 1998). A combinatorial

approach achieved up to 40 % reduction in sinapine

content in seeds of B. napus by silencing the mid-

stream enzyme ferulic acid 5-hydroxylase (Nair et al.

2000). Up to 76 % reduction was achieved by

suppression of the down-stream terminal enzyme

UDP-glucose: sinapate glucosyltransferase (SGT)

(Husken et al. 2005). Suppression of SCT was

effective in reducing sinapine, but with little impact

on the total content of upstream sinapoyl moieties

(Weier et al. 2007). In contrast, Bhinu et al. (2009a)

developed a more refined strategy that first relied on

the model plant Arabidopsis to identify rate-limiting

steps in sinapine biosynthesis using knockout mutants

affecting each step in sinapine biosynthesis. Arabid-

opsis mutants deficient in ferulic acid 5-hydroxylase

(FAH) and sinapoylglucose:choline sinapoyltransfer-

ase (SCT) gene activity had the lowest levels of

sinapine (Bhinu et al. 2009a; Huang et al. 2008). They

then applied this knowledge derived from Arabidopsis

to ensure maximal reduction of sinapine by simulta-

neously down-regulating FAH and SCT in B. napus

seed using antisense technology (Bhinu et al. 2009a).

As a result, sinapine reductions of up to 90 % were

achieved in seeds harbouring the FAH–SCT fusion

antisense construct, and these reductions were main-

tained without visible pleiotrophic or growth problems

even when transgenic B. napus lines were grown in

field trials, indicating the stability of the low sinapine

trait under cropping conditions.

Because sinapine is synthesized from the trans-

esterification of sinapoylglucose and choline, inhibi-

tion of its biosynthesis would be expected to result in

an enhanced level of free choline. Choline is a

valuable feed additive in livestock and fish feed

(Chung et al. 2009; Shiau and Lo 2000). It is also a

nutraceutical with health claims associated with the

prevention of a range of neurodegenerative diseases in

Transgenic Res (2014) 23:39–52 45

123

humans (Burgess et al. 2009). Bhinu et al. (2009a) and

Huang et al. (2008) showed that reducing sinapine in

B. napus and Arabidopsis led to a substantial increase

in seed choline content, resulting in two improvements

to seed composition in a single transgenic event.

Threfore, the accumulation of choline in low sinapine

Arabidopsis seeds was exploited to produce betaine,

an effective stress-alleviating metabolite in plants.

Expression of the choline oxidase (COX) gene of

Arthrobacter pascens in the Arabidopsis sct mutant

resulted in nearly twofold greater levels of betaine

relative to that of wild type Arabidopsis seeds

expressing COX (Huang et al. 2008). However,

Arabidopsis sct and fah mutants are more susceptible

to fungal infection, and expression of several flower-

ing genes are also altered in these mutants under salt

stress (Huang et al. 2009). Moreover, a range of other

changes discovered in gene expression and secondary

metabolite accumulation were not directly related to

sinapine In contrast, pleiotrophic effects on growth

and development were not found with the down-

regulated FAH–SCT antisense B. napus plants, These

findings point to the importance of testing for pleio-

tropic effects resulting from silencing SCT, FAH,and

other genes; especially the antisense plants should be

tested for a broader range of metabolic and stress-

related changes.

Various other molecular approaches, including

suppression or over-expression of various target genes,

have also been employed to reduce sinapine in B. napus

seeds. A recent approach involved over-expressing the

B. napus sinapine esterase (BnSCE3) gene under the

seed-specific napin promoter (Clauss et al. 2011).

BnSCE3 is involved in the conversion of sinapine to

sinapate and choline, and is normally expressed at the

early stages of seedling development. Over-expression

of BnSCE3 during seed development resulted in 95 %

reduction in the sinapine content of mature seed

(Clauss et al. 2011). Milkowski and Strack (2004,

2010) had suggested earlier that a feedback loop might

control sinapate levels. However, sinapate accumu-

lated after knockdown of sinapine by Clauss and co-

workers was not reduced; instead it was conjugated to

several small molecules. Based on these findings, it

was hypothesized that accumulated sinapate might

serve as a feedback regulation for further sinapate

biosynthesis (Milkowski and Strack 2004, 2010). The

REF1 gene (reduced epidermal fluorescence1) encod-

ing a bi-functional cinnamaldehyde dehydrogenase

involved in sinapaldehyde metabolism (Milkowski and

Strack 2010) was also the target of genetic engineering

to reduce sinapine levels. RNAi silencing of REF1 in B.

napus resulted in reduced levels of sinapate esters,

altered accumulation patterns of kaempferol glyco-

sides, changes in minor conjugates of caffeate ferulate

and 5-hydroxyferulate, and the appearance of conju-

gated mono-, di- and tri-lignols (Mittasch et al. 2013).

However, reduction was more pronounced for minor

sinapate conjugates than for the major component

sinapine. Harloff et al. (2012) used the TILLING

(Targeting-induced local lesions in genomes) technol-

ogy to identify a total of 579 mutations in two genes,

BnaX.SGT and BnaX.REF1, involved in sinapine

biosynthesis. Unfortunately, a significant reduction in

sinapine was not detected in these single mutants,

likely due to the multi-copy nature of the two affected

genes. Nevertheless, if these mutants were to be

crossed to generate combinatory mutant lines, it might

be possible to achieve substantial reductions in sina-

pine content using the TILLING approach.

Phytate

Phytate (myoinositol-1,2,3,4,5,6-hexakisphosphate) is

the main storage form of phosphorus in B. napus

seeds, with levels ranging from 2 to 4 % on a whole

seed basis. Phytate impacts both animal nutrition and

the environment. It lowers the bioavailability of

mineral nutrients as a result of chelation and also

reduces phosphorus digestibility, thereby increasing

livestock and fish phosphate waste (which can pollute

water systems). The most common strategy for

reducing phytate in feed is through treatment with

the phytase enzyme (Teskeredzic et al. 1995; Maenz

et al. 2003). This is an expensive process and is

economically unsustainable especially in areas of the

world that lack strict regulations for the disposal of

manure with high phosphorus content. The most

effective strategy, therefore, is to reduce the actual

levels of phytate in seed, and a number of approaches

have been followed in B. napus for this purpose (www.

genomeprairie.ca/project/previous/designing-oilseeds-

tomorrows-markets). However, successes in obtaining

low phytate mutants in some plant species (Badone

et al. 2010 and references therein) have not resulted in a

breakthrough in lowering phytate in B. napus. Over-

expression of the B. napus phosphatidylinositol–

phospholipase C2 enhanced the level of phytate in B.

46 Transgenic Res (2014) 23:39–52

123

napus seed (Georges et al. 2009), and thus one would

expect that silencing this gene would result in lower

phytate levels. However, so far there have been no

reports in the literature on such an experiment. Muta-

tion of the MRP5/ABCC5 phytate transporter gene

reduced seed phytate levels in the closely related plant,

Arabidopsis, (Nagy et al. 2009). Again, this experiment

has yet to be conducted on B. napus.

Fiber

Dietary fibre represents approximately one-third of the

B. napus meal remaining after oil extraction, with a

significant portion of fiber being in the form of

indigestible lignin (Bell 1995). High fibre reduces seed

oil and protein yields and can have adverse effects on

livestock diet digestibility and feed efficiency (Jarrige

1980; Jovanovic and Cuperlovic 1977). Compared to

other crops, B. napus contains high amounts of lignin

(8 % of the oil-free meal), while in soybean lignin

represents only 1 % of the meal. Lignin is one of the

most important compositional factors affecting feed

utilization by ruminants. Therefore, efforts have been

made to reduce lignin content in B. napus seeds.

Breeding research at Agriculture and Agri-Food

Canada has resulted in the production of the yellow-

seed B. napus YN01-429 line, which contains signif-

icantly lower levels of lignin due to a thinner seed hull

and reduced content of proanthocyanidin flavonoids

(Akhov et al. 2009; Relf-Eckstein et al. 2007). Recent

research by Bhinu et al. (2009b) also developed a

strategy for producing B. napus seeds with reduced

lignin content using metabolic engineering

approaches. Four genes were targeted for silencing

by RNAi independently or in combination; caffeic

acid O-methyltransferase (COMT), cinnamate 4-

hydroxylase (C4H); coumarate 3-hydroxylase (C3H),

and ferulic acid hydroxylase (FAH). The best lignin

reduction was observed in lines carrying a crucifer-

inp::COMT RNAi construct with a decrease of up to

40 % relative to the wild type control plant. Although

no visible phenotypic effects on plant growth were

observed in the transgenic lines under greenhouse

conditions, anatomical variations were detected when

stem sections were examined under the microscope

(Fig. 3). These included deformation of vessel ele-

ments, and minor changes in the syringyl units of the

lignin monomers. Taken together, these results show

that genetic engineering approaches are effective in

altering lignin content and composition in a manner

non-detrimental to B. napus plants (Bhinu et al.

2009b).

Prospects for future genetic improvement of B.

napus

Genetic improvements in B. napus seed quality can be

broadly classified into two focus areas, namely oil

quality and meal quality. Secondary metabolites

impact both oil and seed quality parameters. Even

though the seed protein in B. napus is superior to many

other plant proteins (reviewed by Bhinu et al. 2009a),

the presence of anti-nutritional factors prevents the use

of this protein in human and animal diets. Develop-

ment of B. napus varieties with high seed protein, high

levels of beneficial metabolites, and significantly

reduced levels of anti-nutritional factors would repre-

sent a major contribution towards improving the B.

napus meal not only for livestock feed, but also for

potentially direct human consumption.

Conventional approaches for genetic enhancement

are limited due to difficulties in transferring desirable

traits from distantly related species and the long

duration time required to develop new germplasm, as

well as disadvantages associated with genetic link-

ages. Recent advances in molecular, biochemical, bio-

informatics and genomic techniques have allowed for

a deeper understanding of various processes and

mechanisms in plant metabolism. The availability of

whole genome sequence information has resulted in

identification of many genes involved in primary and

secondary metabolism in B. napus and other related

Brassica crops. Advances in understanding of com-

plex physiological and metabolic processes have also

revealed the need to understand broader metabolic

cascades and interdependent processes. Efforts are

ongoing to understand regulatory mechanisms and the

significance of engineering metabolic networks in a

precise and predictable manner.

Genetic engineering approaches have proven effec-

tive at introducing changes to the secondary metab-

olite profile of B. napus. With these technological

advancements there is ample scope for genetic mod-

ifications to improve seed quality. However, negative

public perception of genetically modified (GM) crops

has adversely impacted the development and adoption

of GM crops, including GM B. napus. Concerns about

Transgenic Res (2014) 23:39–52 47

123

GM crops may be partially addressed by advances in

TILLING technology (Till et al. 2003). TILLING

allows for the exploitation of some of the knowledge

derived from genetic engineering and molecular

genetics to develop non-GM plant varieties with

desired genetic modifications. The availability of a

B. napus TILLING facility (www.botany.ubc.ca/can-

till) will likely accelerate the development of non-GM

varieties with enhanced seed characteristics and other

desired traits.

Another tool that molecular biologists could exploit

to improve B. napus seed composition is the combi-

natorial potential of metabolic pathways. This is

possible by employing directed molecular evolution

accomplished through multi-gene shuffling, which

may involve an iterative gene shuffling process. This

technique has been used to modify protein character-

istics, including improvement in specific activity or

enzyme kinetics (Castle et al. 2004; Zhang et al. 2012),

formation of novel substrate specificities (Rao 2008),

and optimization of performance during various stages

of seed development. Enzymes with useful yet insuf-

ficient activities can be improved by applying directed

evolution until the desired activity is gained. It is

possible to capitalize on interspecies variation in

substrate and product selectivity to harness the com-

binatorial potential of several biosynthetic pathways.

This approach has been successfully employed in the

generation of novel carotenoids (Umeno et al. 2005;

Nishizaki et al. 2007). Molecular shuffling technology

has been practised for over two decades to develop

superior proteins, including development of a Rubisco

activase shuffled for enhanced thermostability (Kurek

et al. 2007). Likewise, fully annotated genome infor-

mation available from various model species and oil

seed crops (including the Brassica family) are valuable

resources that should lead to a favorable outcome with

molecular shuffling, enabling the re-working of an

extensive foundation of biosynthetic pathways and

offering new avenues to produce novel products in B.

napus. While improving the metabolic profile is one

challenge, numerous other challenges persist; for

instance achieving consistent oil and meal quality in

B. napus or transferability of traits across different

geographic conditions given that GxE is a complex

attribute. While the lack of molecular switches and

tools (including novel seed specific promoters) that

function consistently across changing physiological

phases of Brassica seed development is not helpful,

the growing repertoire of metabolic engineering tools

and a combination of methodologies are most likely to

lead to more diverse libraries of novel compounds and

thus encourage new pursuits and new improvements to

B. napus seed.

Fig. 3 Histochemical analyses of lignin in stem sections of

transgenic and control plants. a Maule staining of transformant

DE178 (912), b Maule staining of transformant DE178 (9125),

c Maule staining of control (912), d Maule staining of control

(9125), e Weisner staining of transformant DE178 (912),

f Weisner staining of transformant DE178 (9125), g Weisner

staining of control (912), h Weisner staining of control (9125).

Transgenic primary xylem (PX) appears slightly distorted or

misshapen. The secondary xylem (SX) and primary phloem

fibers (PP) appear normal (adapted from Bhinu et al. 2009b with

kind permission of the Agricultural Institute of Canada)

48 Transgenic Res (2014) 23:39–52

123

Acknowledgments We would like to thank Dr. Margie

Gruber, Dr. Carol Powers and Ms. Teresa Fruits for language

editing of the manuscript.

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