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Golden bananas in the field: elevated fruit pro-vitamin A from the expression of a single banana transgene Jean-Yves Paul1, Harjeet Khanna1,†, Jennifer Kleidon1, Phuong Hoang1, Jason Geijskes1,‡, Jeff Daniells2, Ella Zaplin1,§, Yvonne Rosenberg3, Anthony James1, Bulukani Mlalazi1, Pradeep Deo1, Geofrey Arinaitwe4, Priver Namanya1,4, Douglas Becker1, James Tindamanyire1, Wilberforce Tushemereirwe4, Robert Harding1 and James Dale1,*

1Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Qld, Australia 2Agri-Science Queensland, Department of Agriculture and Fisheries, South Johnstone, Qld, Australia 3PlantVax Inc, Rockville, MD, USA 4National Agricultural Research Laboratories, National Agricultural Research Organization, Kampala, Uganda

Received 15 August 2016;

revised 6 October 2016;

accepted 7 October 2016.

*Correspondence (Tel +61 7 3138 2819; fax

+61 7 3138 4132; email [email protected])

Present addresses: †Sugar Research Australia,

Brisbane, Qld, Australia. ‡Syngenta Asia Pacific, Singapore,

Singapore. §Charles Sturt University, Wagga Wagga,

NSW, Australia.

Keywords: Vitamin A deficiency,

Uganda, pro-vitamin A, staple food

crop, banana, biofortification, genetic

modification.

Summary Vitamin A deficiency remains one of the world’s major public health problems despite food

fortification and supplements strategies. Biofortification of staple crops with enhanced levels of

pro-vitamin A (PVA) offers a sustainable alternative strategy to both food fortification and

supplementation. As a proof of concept, PVA-biofortified transgenic Cavendish bananas were

generated and field trialed in Australia with the aim of achieving a target level of 20 lg/g of dry weight (dw) b-carotene equivalent (b-CE) in the fruit. Expression of a Fe’i banana-derived phytoene synthase 2a (MtPsy2a) gene resulted in the generation of lines with PVA levels

exceeding the target level with one line reaching 55 lg/g dw b-CE. Expression of the maize phytoene synthase 1 (ZmPsy1) gene, used to develop ‘Golden Rice 2’, also resulted in increased

fruit PVA levels although many lines displayed undesirable phenotypes. Constitutive expression

of either transgene with the maize polyubiquitin promoter increased PVA accumulation from the

earliest stage of fruit development. In contrast, PVA accumulation was restricted to the late

stages of fruit development when either the banana 1-aminocyclopropane-1-carboxylate oxidase

or the expansin 1 promoters were used to drive the same transgenes. Wild-type plants with the

longest fruit development time had also the highest fruit PVA concentrations. The results from

this study suggest that early activation of the rate-limiting enzyme in the carotenoid biosynthetic

pathway and extended fruit maturation time are essential factors to achieve optimal PVA

concentrations in banana fruit.

Introduction

Micronutrient deficiency, often referred to as hidden hunger,

occurs when intake and absorption of vitamins and minerals are

too low to sustain good health and development. The World

Health Organization (WHO) estimates that 190 million pre-school

children are deficient in one of the major micronutrients, vitamin

A. Vitamin A deficiency (VAD) alone is responsible for almost 6%

of child deaths under the age of 60 months in Africa and 8% in

South-East Asia (WHO, 2011). For the vast majority of these

children, VAD is almost exclusively the result of inadequate intake

of dietary vitamin A or pro-vitamin A (PVA) although exacerbated

by other health conditions. Similar levels of VAD are also evident

in women of childbearing age in these same regions (WHO,

2011). These levels of VAD continue despite the implementation

over many years of extensive alleviating strategies such as

supplements and food fortification. These strategies have been

demonstrably successful, but there remain persistently high and

unacceptable levels of VAD particularly in sub-Saharan Africa and

south Asia (Stevens et al., 2015).

In an effort to significantly reduce VAD in these regions,

strategies aimed at increasing the dietary intake of particularly a- and b-carotene together as PVA are being developed or imple- mented. These include programmes to encourage growing and

consuming staple foods with high levels of PVA. In some

instances, such foods or crops with the desired agronomic and

consumer traits are already available and can therefore be easily

deployed (HarvestPlus, 2012). However, the majority of accepted

cultivars and landraces of staple crops are low in micronutrients

such as PVA and iron, and therefore, it is necessary to develop

new varieties with enhanced levels of these micronutrients. This

can be achieved either through conventional breeding or by

genetic modification where the traits are not available within the

accessible germplasm or cannot be easily introgressed into

acceptable cultivars. These two approaches are known as

biofortification.

The best-known example of biofortification by genetic mod-

ification, and the most advanced in terms of development, is

‘Golden Rice’. In Ye et al., 2000 and colleagues reported the

generation of transgenic rice expressing the daffodil phytoene

synthase (Psy) gene under the control of an endosperm-specific

rice glutelin promoter together with the bacterial (Pantoea

ananatis formerly known as Erwinia uredovora) phytoene desat-

urase (CrtI) gene under the control of the constitutive CaMV 35S

promoter. The endosperm of selected lines was yellow, and one

heterozygous line contained 1.6 lg/g dry weight (dw) total carotenoids. Paine et al. (2005) subsequently reported the

development of the second generation of ‘Golden Rice’, which

520 ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use,

distribution and reproduction in any medium, provided the original work is properly cited.

Plant Biotechnology Journal (2017) 15, pp. 520–532 doi: 10.1111/pbi.12650

was engineered with a maize (Zea mays) Psy gene and the CrtI

gene under the control of the glutelin promoter. One Golden Rice

2 elite event had a 23-fold increase in total carotenoids over the

original Golden Rice with a total carotenoid level of up to 37 lg/g dw in the endosperm of which 31 lg/g was b-carotene. A number of other important food crops have been or are being

developed to enhance the level of PVA through genetic modi-

fication.

Bananas are the world’s most important fruit crop and one of

the top 10 crops by production. They are widely grown in the wet

tropics and subtropics forming an important dietary component

both raw as a dessert fruit and cooked often as the major source

of carbohydrate. In a number of countries, bananas are the

principal staple food including Uganda where consumption levels

average 0.5 kg per person per day rising to around 1 kg per

person per day in some regions (Komarek, 2010; Smale and

Tushemereirwe, 2007). In East Africa, the staple cultivar is East

African highland banana (EAHB) (M. acuminata AAA-EA) pre-

pared primarily by steaming or boiling whereas, in West Africa,

plantains are dominant and are usually fried or roasted (Fungo

and Pillay, 2011). In both regions, the level of VAD is high. In

Uganda, it varies from 15% to 33% in children under 60 months

with similar levels in women of childbearing age (UDHS - Uganda

Demographic and Health Survey, 2006). Unlike rice endosperm,

banana fruit contains PVA and, in some instances, very high levels

particularly Fe’i bananas of Micronesia and Papua New Guinea

(Englberger et al., 2003). Bananas with b-carotene equivalent (b- CE) levels of 340 lg/g dw have been reported whereas the dominant dessert banana cv ‘Cavendish’ has between 1 and

4 lg/g dw b-CE and the EAHB clone, ‘Nakitembe’, has approx- imately 10 lg/g b-CE dw (Englberger et al., 2006a; Mbabazi, 2015). Unfortunately, domesticated bananas have very low male

and female fertility rendering conventional breeding extremely

difficult. Thus, the introgression of the high PVA traits of Fe’i

bananas for instance into farmer preferred EAHB selections would

be practically impossible. However, genetic modification of

bananas is well established.

Here, we report the ‘proof-of-concept’ technology required

towards the generation of PVA-biofortified EAHB varieties in

Uganda. The ‘Cavendish’ dessert banana was genetically modi-

fied, and greatly enhanced PVA levels were demonstrated in the

fruit of plants grown in the field in Australia.

Results

The target

At the outset, it was important to identify a target fruit level of b- carotene equivalents necessary to help alleviate VAD in Uganda.

The target was set at delivering 50% of the estimated average

requirement (EAR) of vitamin A in vulnerable populations which

for children under 60 months is 120 lg/day and for females ranging from 235 lg/day up to 445 lg/day for lactating mothers. An estimated bioconversion 6:1 ratio of b-carotene equivalents (b-CE) to vitamin A from cooked banana pulp was used (Bresnahan et al., 2012) with an estimated consumption of

cooked bananas of 300 g/day for children and 500 g/day for

women. The a- and b-carotene retention after steaming or boiling was also estimated at 70% (Mbabazi, 2015). Using these

parameters, banana fruit needed to contain b-CE levels of at least 20 lg/g dw to achieve 50%of the EAR.

There were three major technical constraints at the com-

mencement of this project that influenced the research strategy:

(i) very little information was available regarding the expression of

transgenes in bananas generally and more specifically in banana

fruit, (ii) the time from transformation to harvestable fruit ranges

from 2 to 2½ years, and (iii) it was clearly impractical to take large numbers of transgenic bananas through to fruit in the green-

house. Therefore, a large number of independent transgenic lines

were generated to enable the testing of a wide range of

promoter and transgene combinations. Initially, a single plant per

transgenic line was planted in the field with, in most instances,

between 10 and 30 transgenic lines per construct. For more

information, refer to the ‘History of the project’ section of the

Supplementary information document.

Promoter characterization

Three promoters were selected as possible candidates for

expressing PVA-related transgenes in banana fruit, and these

were characterized for levels and patterns of expression in

transgenic bananas. The promoters included the constitutive

maize polyubiquitin promoter (Ubi) and two promoters isolated

from banana, the expansin 1 promoter (Exp1) and the ACC

oxidase promoter (ACO) which were predicted to be fruit

specific. These promoters were fused to the b-glucuronidase reporter gene (uidA), the cassettes transformed into bananas

and the transgenic plants established in the field. b-glucur- onidase (GUS) protein levels were measured in the fruit pulp

using ELISA; however, this approach could not be used for leaf

or peel material because of very high background levels.

Therefore, for leaf and peel samples, MUG fluorometric assays

were used to estimate enzyme activity rather than protein

levels.

GUS activity was measured in the leaves of six independent

transgenic lines for each promoter. As expected, there were high

but variable levels of GUS activity in the leaves of all six plants

where uidA was under the control of the Ubi promoter

(Figure 1a). In the leaves of the wild-type control plant and

plants where uidA was under the control of either the Exp1 or

ACO promoter, there was undetectable to negligible GUS activity

(Figure 1b and c).

Pulp samples from the fruit of the same lines described

above were collected at 3, 6, 9 and 12 weeks post-bunch

emergence (S3, S6, S9 and S12) and also at ‘full green’ (FG),

when the bunches were harvested, and ‘full ripe’ (FR). The FG

stage is equivalent to the stage when cooking bananas are

harvested in Uganda. GUS protein was not detected in the

wild-type at any fruit development stage. In contrast, appre-

ciable but varying levels of GUS protein were detected in the

pulp of banana fruit from S3 through to FR in the six Ubi-uidA

lines (Figure 1d) confirming the constitutive nature of the Ubi

promoter. No reproducible trend in GUS protein levels across

the six lines was observed as fruit matured from S3 to FR

except that average protein accumulation was lowest at the

earliest stage, S3. In the six Exp1-uidA lines examined, no

appreciable GUS expression was detected in fruit pulp from S3

through to FG with the exception of FR fruit from lines FT258

and FT263 (Figure 1e). This indicated that the Exp1 promoter is

activated very late during fruit development. Very low levels of

GUS expression were detected in the fruit pulp of the ACO-

uidA lines from S3 to S9 (Figure 1f). With the exception of line

FT736 which peaked fourfold higher than any other line, the

overall trend was that GUS expression slowly increased from S9

through to FG and plateaued at FR. These results indicated that

the ACO promoter was activated earlier than Exp1 during fruit

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 520–532

Pro-vitamin A biofortified Cavendish bananas in the field 521

development and more consistently at the FG and FR stages

(Figure 1e and f).

The pattern of Ubi driven GUS expression in fruit peel was

unexpectedly different from fruit pulp with low expression from

S3 to FG followed by a substantial increase at FR (Figure 1g). In

the peel of Exp1-uidA lines, the pattern of GUS expression was

similar to that observed in the fruit pulp (Figure 1h). ACO-uidA

lines had very little GUS expression in the peel up to FG; however,

there was a dramatic increase to levels higher than either the Ubi

or Exp1 lines at FR (Figure 1i).

PVA analysis: plant and ratoon crops

Domesticated bananas grow as a perennial crop. The initial plant,

the plant crop, develops a corm and a pseudostem from which

the original bunch is produced. After the bunch is harvested, the

pseudostem dies and is replaced by a second pseudostem

producing the first ratoon crop which develops from a sucker

originating from the corm. Similarly, a second ratoon crop is

produced and so on.

The same three promoters used to assess GUS expression were

used to drive the expression of three carotenoid biosynthesis

transgenes. These transgenes were the phytoene synthase 1 gene

from maize (ZmPsy1) used in Golden Rice 2, a phytoene synthase

2a gene isolated from the Fe’i banana cultivar Asupina (MtPsy2a)

(Mlalazi et al., 2012) and the bacterial phytoene desaturase gene

PaCrtI also used in Golden Rice 2. ZmPsy1 and MtPsy2a were

transformed into Cavendish banana singly and in combination

with PaCrtI.

A total of 244 transgenic lines were confirmed to contain the

respective PVA transgene(s) by PCR. Southern blot analyses

showed that the transgene copy number varied from one to more

than 10 copies (Figure S1 and S2). For each line, a single plant

was established in the field together with 50 non-transgenic

control plants. This initial randomized trial was designated Field

Trial 1 (FT-1), and the plant crop was assessed over a period of

16 months. Forty-eight transgenic lines either died or were

stunted and did not produce fruit. Therefore, fruit was harvested

from a total of 196 transgenic lines of which 153 samples were

selected for the initial plant crop fruit analysis (Table 1). Fruit was

harvested at FG, ripened to FR and sampled at both stages. The

sample used for the initial PVA level screen consisted of a single

fruit taken from the middle of the bunch from each of 153

transgenic lines together with the equivalent sample from the

fruit of 15 non-transgenic control plants. Following lyophilization

and total carotenoids extraction, each sample was analyzed by

HPLC and b-CE levels calculated. The most important outcomes

Figure 1 Analysis of promoter activity in wild-type and transgenic Cavendish banana lines. GUS activity was measured in leaf (a, b, c), and peel (g, h, i),

while GUS protein concentration was measured in pulp tissue (d, e, f). Ubi promoter (a, d, g); Exp1 promoter (b, e, h); and ACO promoter (c, f, i). WT, wild-

type FT432. S3, S6 and S9 represent 3, 6 and 9 weeks post-bunch emergence, respectively. FG, full green and FR, full ripe. Error bars: �SD.

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 520–532

Jean-Yves Paul et al.522

of this initial screening included: (i) there was obvious variation

between individual control plants and also variation within lines

with the same promoter-transgene combination, (ii) the highest

expressing transgenic line for each of the different transgenes or

combinations of transgenes contained higher levels of b-CE than the highest control plants except those lines containing Exp1-

PaCrtI or Ubi-PaCrtI alone or where Ubi-PaCrtI was combined with

Exp1-MtPsy2a or Exp1-ZmPsy1, (iii) in all transgenic lines and

controls, the fruit contained higher levels of a-carotene than b- carotene (Figure S3), and (iv) PaCrtI alone had little effect on fruit

PVA levels when driven by either the Exp1 promoter or the Ubi

promoter; as such, none of the single transgene PaCrtI lines were

progressed through for further analysis.

From this initial plant crop screen, 63 transgenic lines were

selected for more comprehensive analysis. The selection included

lines representing high, average and low PVA accumulation. As

preliminary data revealed considerable variation in PVA accumu-

lation across the bunch (data not shown), carotenoids were

extracted from a composite sample including equal amounts of

fruit taken from the top, middle and bottom of the bunch.

Further, although fruit samples from the selected lines were to be

analyzed across three generations (plant crop followed by first

and second ratoon crops), the trial was hit by a severe cyclone in

February 2011. As a consequence, all lines were blown over

resulting in fruit from some lines in either the first or the second

ratoon crops not being available for analysis.

For this project, the PVA levels at FG were considered more

important as cooking bananas are harvested at this stage in

Uganda. However, FR data were also collected as cooking

bananas are usually consumed over a number of days post-

harvest. The b-CE levels in FG and FR fruit from each of the selected 63 transgenic lines are presented in Table 2. Where

expression of MtPsy2a or ZmPsy1 was controlled by the Exp1

promoter, the level of b-CE in the plant crop and first ratoon increased from FG to FR in 43 of 53 samples analyzed (81%)

(Table 2). A similar increase was seen in 82% (14 of 17 samples)

of the analyzed samples where the Ubi promoter was used to

drive the expression of the same transgenes. However, when

ACO was used as a promoter, this number reduced to 50% (13

of 26 samples).

In the plant crop, there were no lines that met the target level

of 20 lg/g dw b-CE where the composite sample of three fruit per bunch was analyzed. The highest b-CE level at FG was 18.7 lg/g dw in Ubi-MtPsy2a line FT328 that subsequently died. In addition, only eight lines had PVA levels greater than 10.0 lg/g dw b-CE at FG. These were two Ubi-ZmPsy1 lines (FT287 and FT 309 with 13.4 and 11.9 lg/g dw b-CE, respectively), two Ubi- MtPsy2a lines (FT328 and FT324 with 18.7 and 11.7 lg/g dw b- CE, respectively), two ACO-MtPsy2a lines (FT504 and FT518 with

16.6 and 15.9 lg/g dw b-CE, respectively) and two ACO- ZmPsy1 + Exp1-PaCrtI lines (FT584 and FT587 with 17.1 and 11.5 lg/g dw b-CE, respectively). To investigate whether a correlation existed between transgene expression levels and

accumulation of carotenoids, the expression of the ZmPsy1 and

MtPsy2a transgenes was determined in the FG fruit of a selection

of lines using RT-PCR (Figure S4 and S5) and qRT-PCR (Figure 2).

MtPsy2a line FT246 had the highest relative expression of the

transgene followed by line FT324, FT518 and FT295 (Figure 2a).

Expression was considerably lower in the other three lines tested.

Expression of ZmPsy1 was highest in line FT584 followed by

FT309 while similar, but lower expression was seen in lines FT287,

FT467, FT475, FT479 and FT585 (Figure 2b). Lines FT187 and

FT192 had low expression.

When FG fruit from the next generation (first ratoon crop)

were analyzed, 68% (23 of 34) of samples showed an increase in

b-CE from the plant crop (Table 2). Interestingly, every line

Table 1 Data summary from the transgenic banana field trial

Promoter-transgene

Number of plants

in the field

Number of plants

harvested

Number of plants

analyzed (first cut)*

b-CE in FG fruit

(lg/g dw)

b-CE in FR fruit

(lg/g dw)

Range Average Range Average

Wild-type 50 50 15 0.6–3.8 1.5 0.8–5.8 3.1

Exp1-MtPsy2a 33 28 28 1.2–8.6 3.3 0.8–10.0 2.8

Exp1-MtPsy2a + Ubi-PaCrtI 13 8 5 0.2–2.3 1.0 1.3–3.4 2.1

Exp1-ZmPsy1 32 26 26 1.2–4.6 2.6 1.3–15.2 4.5

Exp1-ZmPsy1+ Ubi-PaCrtI 18 11 8 0.2–1.8 1.0 1.5–3.6 2.4

Exp1-ZmPsy1+ Exp1-PaCrtI 7 7 2 – – 2.1–11.0 6.5

Exp1-PaCrtI 29 26 3 – – 1.7–2.6 2

ACO-MtPsy2a 30 29 28 11.2–15.3 13.3 3.4–13.4 8.3

ACO-ZmPsy1 30 18 18 5.4–10.9 7.6 2.5–24.6 9.0

ACO-ZmPsy1+ Exp1-PaCrtI 6 4 3 10.6–25.7 17.1 7.8–16.7 13.2

Ubi-MtPsy2a 9 7 7 1.4–19.1 6.8 3.5–16.1 7.4

Ubi-ZmPsy1 10 5 5 0.3–13.6 6.1 1.0–16.1 7.9

Ubi-PaCrtI 27 27 20 – – 1.0–3.7 1.8

Total 294 246 168 – – – –

⁄ First cut relates to an initial screening of the fruit of transgenic banana lines done by HPLC and using only a single fruit collected from the middle position of the bunch. FG, full

green and FR, full ripe.

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 520–532

Pro-vitamin A biofortified Cavendish bananas in the field 523

Table 2 PVA carotenoid concentration in the fruit pulp of selected wild-type and transgenic Cavendish banana lines across four generations

Promoter-transgene Line #

Plant crop 1st ratoon crop 2nd ratoon crop Sucker crop

FG FR FG FR FG FR FG FR

b-carotene equivalents (lg/g dw)

Wild-type Average (n≥ = 6) 2.6 3.1 2.2 2.2 1.7 2.3 6.0 7.4

Exp1-MtPsy2a FT246 7.3 9.3 8.3 8.5 NA NA 18.2 19.6

FT544 3.4 4.6 NA NA NA NA NA NA

FT545 3.4 4.7 NA NA NA NA NA NA

FT342 2.8 4.2 7.4 4.6 NA NA 10.6 9.0

FT335 2.5 4.0 2.4 3.3 NA NA NA NA

FT343 2.3 3.8 2.9 3.4 NA NA NA NA

FT242 2.2 3.7 2.0 2.0 NA NA 8.6 8.0

FT233 2.0 3.3 1.9 2.6 NA NA NA NA

FT341 1.4 3.1 1.8 2.0 NA NA 9.3 8.1

Exp1-MtPsy2a + Ubi-PaCrtI FT244 1.5 2.8 2.6 1.9 NA NA NA NA

FT245 0.9 1.8 1.8 4.6 NA NA NA NA

FT220 0.7 1.4 1.7 1.9 NA NA NA NA

FT232 0.2 1.9 1.8 1.5 NA NA NA NA

Exp1-ZmPsy1 FT534 9.3 11.9 NA NA NA NA NA NA

FT536 9.1 8.9 NA NA NA NA NA NA

FT317 6.6 7.9 NA NA NA NA 11.4 15.6

FT192 4.3 6.4 NA NA NA NA 20.3 31.2

FT538 3.8 9.4 NA NA NA NA 10.9 14.8

FT187 3.5 5.3 1.8 1.8 9.5 9.9 7.0 11.0

FT311 2.9 6.1 3.1 2.5 NA NA NA NA

FT201 2.3 3.2 1.9 3.2 NA NA 9.1 15.0

FT319 1.9 3.2 3.5 2.6 NA NA NA NA

FT318 1.5 2.7 1.9 2.4 NA NA NA NA

FT210 1.3 2.8 1.2 2.5 NA NA NA NA

Exp1-ZmPsy1 + Ubi-PaCrtI FT217 1.8 2.4 1.5 2.4 NA NA NA NA

FT196 1.6 4.2 2.9 2.5 NA NA NA NA

FT207 1.5 3.5 2.4 2.7 NA NA NA NA

FT195 1.0 2.6 NA NA NA NA NA NA

FT208 0.8 2.7 2.1 2.1 NA NA NA NA

FT205 0.7 2.6 1.0 7.4 NA NA NA NA

FT213 0.4 1.8 NA NA NA NA NA NA

ACO-MtPsy2a FT504 16.6 12.0 12.1 11.7 NA NA 20.0 24.7

FT518 15.9 10.7 NA NA NA NA 23.1 35.9

FT511 9.4 7.3 5.3 6.0 NA NA 14.4 13.6

FT508 9.2 7.1 4.2 3.8 NA NA 15.6 19.0

FT498 8.9 9.9 8.6 8.4 NA NA NA NA

FT506 6.0 5.9 NA NA NA NA NA NA

FT516 4.2 7.4 NA NA NA NA NA NA

FT497 4.1 10.5 NA NA NA NA 13.4 12.9

ACO-ZmPsy1 FT492 9.4 8.5 NA NA NA NA NA NA

FT467 7.3 10.4 NA NA NA NA 10.5 13.4

FT468 NA NA 7.3 6.6 NA NA NA NA

FT475 4.3 10.5 NA NA NA NA 20.7 22.0

FT493 4.3 18.7 7.0 6.0 NA NA NA NA

FT476 2.9 5.4 4.5 4.7 NA NA NA NA

FT487 2.7 15.1 NA NA NA NA NA NA

FT479 2.7 13.6 NA NA NA NA 20.5 16.2

FT483 1.7 9.1 NA NA NA NA 18.8 16.9

ACO-ZmPsy1 + Exp1-PaCrtI FT584 17.1 11.2 NA NA NA NA 27.0 32.8

FT587 11.5 NA NA NA NA NA 21.5 22.2

FT588 7.7 10.1 NA NA NA NA NA NA

FT585 7.3 5.6 NA NA NA NA 12.2 11.5

Ubi-MtPsy2a FT328 18.7 18.9 NA NA NA NA NA NA

FT324 11.7 16.1 NA NA 26.6 33.4 55.0 50.1

FT294 6.6 9.7 13.5 9.4 13.5 12.0 29.0 25.2

FT295 5.4 6.1 6.6 6.6 4.8 4.5 10.5 12.0

FT296 4.2 6.0 5.6 4.1 12.5 13.1 NA NA

FT327 3.0 4.9 NA NA NA NA NA NA

FT330 2.5 4.9 3.6 4.0 NA NA 11.9 15.1

Ubi-ZmPsy1 FT287 13.4 15.8 NA NA 24.3 21.8 39.7 60.9

FT309 11.9 14.7 NA NA 40.4 39.3 46.9 18.5

FT298 0.7 2.5 1.1 1.3 NA NA NA NA

FT302 0.6 1.5 1.4 2.0 NA NA NA NA

FG, full green and FR, full ripe.

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 520–532

Jean-Yves Paul et al.524

analyzed that contained either Ubi-ZmPsy1 or Ubi-MtPsy2a

showed an increased accumulation in b-CE from the plant crop. However, again, no line accumulated over the 20 lg/g dw b-CE target level. FG fruit from two lines had PVA levels above 10 lg/g dw b-CE in the ratoon crop: ACO-MtPsy2a line FT504 with 12.1 lg/g dw b-CE down from 16.6 lg/g dw b-CE in the plant crop and Ubi-MtPsy2a line FT294 with 13.5 lg/g dw b-CE up from 6.6 lg/g dw b-CE in the plant crop. Due to the impact of the 2011 cyclone, only seven lines could be assessed in the

second ratoon. FG fruit from three of these lines accumulated

above target levels of PVA. Ubi-ZmPsy1 line FT309 had FG fruit

with 40.4 lg/g dw b-CE, more than double the target, while Ubi- MtPsy2a line FT324 and Ubi-ZmPsy1 FT287 accumulated 26.6

and 24.3 lg/g dw b-CE, respectively (Table 2). Carotenoid accumulation throughout fruit development was

also monitored in selected lines from each of the single promoter-

Psy combinations in the second ratoon crop at 3, 6 and 9 weeks

post-bunch emergence as well as FG and FR (Figure 3). For the

four lines where the transgene was under the control of the Ubi

promoter, PVA levels were elevated (above 15 lg/g dw b-CE) from the earliest fruit collection time point (S3) irrespective of

whether the transgene was ZmPsy1 or MtPsy2a (Figure 3a and d).

For the two lines with the highest PVA levels, the general trend

was increasing PVA during fruit development to a maximum at

FG for Ubi-ZmPsy1 line FT309 or at FR for Ubi-MtPsy2a line

FT324. In contrast, accumulated PVA levels in the fruit of Exp1-

ZmPsy1 and Exp1-MtPsy2a lines remained below 5 lg/g dw b-CE from the emergence of the bunch all the way through to S9 (with

the exception of line FT342 which accumulated 6.6 lg/g dw b-CE at S9) followed by an increase towards maturity with a maximum

of 9.9 lg/g dw b-CE at FR in Exp1-ZmPsy1 line FT187 (Figure 3b and e). During fruit development of lines containing ACO-

ZmPsy1, PVA levels were lowest at S3 and S6 for two of the lines

but moderately higher than the Exp1-ZmPsy1 lines at those stages

(Figure 3c). PVA levels peaked for those same two lines at either

S9 or FG. In contrast, the highest PVA level in line FT476 was at

S3. For the ACO-MtPsy2a lines, again one line, FT511, had

maximum PVA accumulation at S3, while the other two lines had

maximums at S9 or FG (Figure 3f). Overall, the PVA accumulation

pattern during fruit development reflected the expression profiles

previously observed in transgenic lines where the same three

promoters were used to drive the expression of uidA (Figure 1).

The constitutive Ubi promoter provided consistent stronger

expression throughout fruit development followed by the ACO

promoter and finally Exp1.

During the plant and ratoon crops, the phenotype of each

plant was recorded at regular intervals from planting to bunch

harvest. None of the 50 wild-type control plants showed altered

phenotypes and fruit developed normally (Figure 4a–c). The presence of the PaCrtI transgene did not appear to affect

phenotype. However, three categories of altered phenotypes

were observed in the transgenic lines: stunting, ‘golden leaf’ and

‘golden bunch’. For the ‘golden leaf’ phenotype, the youngest

leaf would consistently unfurl with a bright yellow colour

(‘golden’) and progressively turn to green as it matured (Fig-

ure 4g). Fruit on the ‘golden bunch’ emerged bright orange

instead of green (Figure 4d). As the fruit matured and filled, it

progressively turned greener to a mixture of green and orange at

harvest (Figure 4e and f). Fruits with increase PVA levels displayed

a pulp ranging from deep yellow to bright orange (Figure 4h and

i). Of the original 244 transgenic lines planted in the trial, 65 had

the ‘golden leaf’ phenotype of which 29 were also stunted; 29

had the ‘golden bunch’ phenotype. The ‘golden leaf’ and ‘golden

bunch’ phenotypes were highly transgene dependent where lines

with those phenotypes invariably contained the ZmPsy1 trans-

gene in contrast to lines containing the MtPsy2a transgene. The

‘golden leaf’ phenotype was observed in 27 of 57 (50%) lines

observed carrying Exp1-ZmPsy1 alone or together with either

Exp1-PaCrtI or Ubi-PaCrtI. More importantly, the ‘golden bunch’

phenotype which only occurred in ACO-ZmPsy1 or ACO-

ZmPsy1 + Exp1-PaCrtI lines was recorded in 94% (29 of the 31) of the lines assessed. In contrast, across all 85 lines containing

Figure 2 Analysis of mRNA expression levels in

the FG fruit pulp of selected transgenic Cavendish

banana lines by qRT-PCR. (a) MtPsy2a lines and (b)

ZmPsy1 lines. FG, full green. WT, wild-type with

WT1 = FT167 and WT2 = FT430. Values are

normalized expression levels � SEM.

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 520–532

Pro-vitamin A biofortified Cavendish bananas in the field 525

MtPsy2a, only 7 (8%) had the ‘golden leaf’ phenotype and none

displayed a ‘golden bunch’.

The b-CE levels in the pulp and peel of FG and FR fruit from lines with the ‘golden bunch’ phenotype were analyzed and

compared with the levels in fruit from phenotypically normal

lines. In the FG fruit from four lines of ACO-MtPsy2a, the levels of

b-CE in the peel were similar to those in the three non-transgenic controls (Table 3). However, the peel b-CE levels in four ACO- ZmPsy1 containing lines were more than ninefold higher than in

fruit peel from the control lines (Table 3). Importantly, the peel

b-CE levels in the ACO-ZmPsy1 lines did not influence fruit pulp b-CE levels as the four ACO-ZmPsy1 lines had a similar range of b-CE levels in their fruit pulp at FG to fruit from the four ACO-MtPsy2a lines (Table 3).

PVA analysis: sucker crop

Following the initial plant and ratoon crop assessment with single

plants per line, 30 lines selected from seven promoter/transgene

combinations and five wild-type lines were multiplied through

suckering to a maximum of 10 replicates per line. A total of 239

transgenic and 48 wild-type plants derived from suckers were

planted in a second field trial (FT-2). Each plant was harvested,

and PVA levels were measured in the fruit at FG and FR by HPLC.

In all transgenic and wild-type lines, the averaged fruit PVA

level was higher in the sucker crop than in either the plant or

ratoon crops. The highest average PVA level was 55.0 lg/g dw b- CE found in the FG fruit of Ubi-MtPsy2a line FT324 (Table 2) with

one individual plant of this line reaching 73.8 lg/g dw b-CE (data not shown). Fruit from this plant had bright orange pulp

compared with the fruit of non-transgenic control plants

(Figure 4h). Of the 27 transgenic lines shown in Table 2, 11

had fruit PVA levels equal to or greater than the 20 lg/g dw b-CE target. However, the fruit PVA levels in the wild-type controls

were also higher than in the plant and ratoon crops with an

average of 6.0 lg/g dw b-CE. Interestingly, the top four sucker crop lines all contained an Ubi-Psy promoter/transgene combina-

tion. Furthermore, six of 12 ACO promoter lines had PVA levels

equal to or greater than target level, compared to only one Exp1

promoter line of 9 (Table 2). Analysis of the carotenoid compo-

sition of fruit pulp from wild-type as well as transgenic fruit in the

Figure 3 PVA carotenoid accumulation in the pulp of wild-type and selected transgenic Cavendish banana lines during fruit development. S3, S6 and S9

represent 3, 6 and 9 weeks post-bunch emergence, respectively. FG, full green and FR, full ripe. Error bars = �SD.

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 520–532

Jean-Yves Paul et al.526

sucker crop revealed that, like the plant crop fruit, samples

contained higher levels of a-carotene than b-carotene and in similar proportions (Figure 5).

Variation in fruit PVA levels in wild-type banana

The average PVA levels in the FG fruit of non-transgenic control

plants varied considerably throughout the two field trials from a

low of 1.0 lg/g dw b-CE in March-harvested fruit in the plant crop of FT-1 to 8.1 lg/g dw b-CE in September-harvested fruit of FT-2 (Figure 6). When analyzed together, a strong correlation was

observed between the level of accumulated PVA in the fruit and

the number of days from bunch emergence to harvest (bunch

filling time). Indeed, fruit harvested in March had the shortest

bunch filling time (94–97 days) and the lowest accumulated PVA level in the fruit compared with 142 days for September-

harvested fruit which had the highest fruit PVA levels (Figure 6).

Longer bunch filling time and associated increased levels of PVA

also appeared temperature dependent where fruit maturing

during the cooler months had higher levels of accumulated PVA.

Discussion

Micronutrient deficiencies remain a substantial burden on the

public health of populations particularly in developing countries.

The ‘poorest of the poor’ are particularly impacted by micronu-

trient deficiencies because of their increasing dependence on

nutrient poor single staple crops for the majority of their calorific

intake (Muthayya et al., 2013). There have been significant

inroads into reducing VAD in children aged 6 to 59 months

where worldwide prevalence has fallen from 39% in 1991 to

(a)

(b) (e)

(c) (f)

(d)

(h)

(i)

(g)

Figure 4 Characteristic phenotypes observed in wild-type and transgenic Cavendish banana lines. (a) Bunch from wild-type line FT448; (b) fruit from wild-

type line FT448; (c) longitudinal section of fruit from wild-type line FT448; (d) immature bunch from ACO-ZmPsy1 line FT477; (e) fruit from ACO-ZmPsy1

line FT477; (f) longitudinal section of fruit from ACO-ZmPsy1 line FT477; (g) Exp1-ZmPsy1 line FT192; (h) Ubi-MtApsy2a line FT324; and (i) Ubi-MtApsy2a

line FT294. WT, wild-type.

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Pro-vitamin A biofortified Cavendish bananas in the field 527

29% in 2013 in low- and middle-income countries (Stevens et al.,

2015). However, VAD prevalence in children age 6–59 months in Uganda has increased from 20% in 2006 to 38% in 2011 and a

similar increase was seen from 19% to 36% in women age 15– 49 years (UDHS - Uganda Demographic and Health Survey, 2006,

2011). In Uganda, the staple crop is cooking bananas, more

specifically East African highland bananas (EAHB). These bananas

are a group of very similar triploid clones of Musa acuminata with

low male and female fertility levels.

Therefore, a metabolic engineering strategy that could provide

the basis for elevating fruit PVA levels in EAHB was developed.

Previously, the most common strategy used to achieve transgenic

elevated PVA has been the Golden Rice 2 strategy where seed

expression of both ZmPsy1 and PaCrtI has been reported to

significantly increase seed PVA in rice (Paine et al., 2005), maize

(Naqvi et al., 2009; Zhu et al., 2008), wheat (Cong et al., 2009)

and sorghum (Lipkie et al., 2013) with levels up to 31 lg/g dw in rice and 59.3 lg/g dw in maize. A range of other transgenes have also been tested either alone or in combinations including PaCrtB

in potato (Ducreux et al., 2005), tomato (Fraser et al., 2002),

canola (Shewmaker et al., 1999), maize (Aluru et al., 2008), soya

bean (Schmidt et al., 2015) and cassava (Sayre et al., 2011),

AtDXS in cassava and sorghum (Lipkie et al., 2013; Sayre et al.,

2011) and the brassica Or gene, BaOr, in potato (Li et al., 2012).

In the present study, the dominant dessert banana, Cavendish,

was used as a model for EAHB as it is also a triploid M. acuminata.

Two phytoene synthase transgenes were tested with and without

co-expression of PaCrtI. Interestingly, over-expression of phytoene

synthase as a strategy to increase PVA had previously only been

reported in cereal crops for which the transgene used was always

ZmPsy1 except in Golden Rice 1. In this work, ZmPsy1 was tested as

well as MtPsy2a, a banana phytoene synthase gene previously

cloned from the fruit of a naturally high PVA Fe’i banana called

Asupina (Mlalazi et al., 2012). This approach allowed the gener-

ation of eleven transgenic lines which produced fruit that contained

greater than the target level of 20 lg/g dw b-CE. The highest level of PVA in the fruit of a single banana plant was from line FT324

(73.8 lg/g dw b-CE) which averaged at 55.0 lg/g dw b-CE. This line contained the banana phytoene synthase gene (MtPsy2a)

under the control of the constitutive ubiquitin promoter. The

highest PVA levels with the phytoene synthase transgene under the

control of an apparently fruit-specific promoter with or without co-

expression of PaCrtI were also above target and included one ACO-

ZmPsy1+ Exp1-PaCrtI line with 27.0 lg/g dw b-CE, one ACO- ZmPsy1 line with 20.7 lg/g dw b-CE and one ACO-MtPsy2a line with 23.1 lg/g dw b-CE. The levels of PVA in Line 324 are equivalent to the highest levels obtained in any other crop where a

plant phytoene synthase gene has been over-expressed. In maize

Figure 5 Percentage accumulation of individual carotenoids in the fruit pulp of wild-type and transgenic bananas. Percentage (%) carotenoid content

calculated based on total carotenoid content measured in the pulp of full green fruit collected from the sucker crop. Biological replicates: wild-type (n = 5),

ACO-MtPsy2a (n = 5), ACO-ZmPsy1 (n = 4), ACO-ZmPsy1 + Exp1-PaCrtI (n = 3), Exp1-MtPsy2a (n = 4), Exp1-ZmPsy1 (n = 5), Ubi-MtPsy2a (n = 4) and

Ubi-ZmPsy1 (n = 2). All samples were analyzed in three technical replicates.

Table 3 PVA carotenoid concentration in the fruit pulp and peel of

wild-type and selected transgenic Cavendish banana lines

Promoter-transgene Line

b-CE in pulp

(lg/g dw)

b-CE in peel

(lg/g dw)

FG FR FG FR

Wild-type FT166 6.6 7.3 74.2 32.1

Wild-type FT430 6.5 8.3 126.5 45.4

Wild-type FT448 8.0 9.4 99.5 38.8

ACO-MtPsy2a FT504 20.0 24.7 108.3 72.7

ACO-MtPsy2a FT508 15.6 19.0 68.3 72.6

ACO-MtPsy2a FT511 14.4 13.6 105.7 75.0

ACO-MtPsy2a FT518 23.1 35.9 129.2 131.2

ACO-ZmPsy1 FT467 10.5 13.4 742.1 703.2

ACO-ZmPsy1 FT475 20.7 22.0 1112.2 1132.6

ACO-ZmPsy1 FT479 20.5 16.2 740.9 833.0

ACO-ZmPsy1 FT483 18.8 16.9 1155.1 837.3

FG, full green and FR, full ripe. All samples were collected from the sucker crop.

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Jean-Yves Paul et al.528

transformed with the seed-expressed ZmPsy1 and PaCrtI transge-

nes,Zhuet al. (2008)andNaqvi et al. (2009) bothreported levels of

57.4 and 59.3 lg/g dw b-CE, respectively. However, Schmidt et al. (2015) recently reported that soya beans transformed with PaCrtB

alone under the control of the seed-specific Le1 promoter

accumulated up to 845 lg/g dw b-carotene in the seed. Although two banana lines containing ACO-ZmPsy1 had above

target levels of PVA, nearly all lines transformed with this

construct had a ‘golden bunch’ phenotype which was never

observed in ACO-MtPsy2a transformed plants. That ACO-ZmPsy1

expression should result in a ‘golden bunch’ phenotype while

Exp1-ZmPsy1 expression resulted invariably in a ‘golden leaf’

phenotype suggests that these two promoters express in tissue

other than fruit pulp and most likely in either leaf primordia for

Exp1 or ‘bunch’ primordia for ACO. The molecular basis for both

the phenotypes has not yet been determined and is under

investigation.

An important outcome from this study was that the levels of

PVA in most banana lines increased through the ‘generations’

from the plant crop through the first ratoon, second ratoon to

finally the sucker crop. Line FT324 had only 11.7 lg/g dw b-CE in the FG plant crop to 55.0 lg/g dw b-CE in the sucker crop. Bananas are vegetatively propagated and do not go through a

seed phase, and thus, all generations are T0. The explanation for

the phenomenon of increasing PVA levels with successive

vegetative generations is not entirely understood but it did

demonstrate that, rather than there being a reduction of

expression with successive vegetative generations as a result of

transgene silencing, the trait was stable. Further, it is possible that

this phenomenon might also occur in other vegetatively propa-

gated crops demonstrating the importance of monitoring trans-

genic traits in vegetative propagated crops in the field through

multiple ‘generations’.

Two outcomes from this study indicated that the final level of

PVA was a result of accumulation through the development of

the banana fruit. Firstly, the levels of PVA in non-transgenic

Cavendish fruit were found to vary considerably. Our observa-

tions indicated that these variations correlate with time to fruit

maturity. Indeed, the highest PVA levels were obtained in fruit

with the longest maturity time. This probably accounts for the

varying reported levels of fruit PVA levels in cultivars such as

Cavendish (Davey et al., 2007; Englberger et al., 2006b; Fungo

and Pillay, 2011) as well as much of the variation observed

within transgenic lines in different seasons. This variable was

controlled for in the ‘sucker generation’ where all suckers were

planted on the same day at the same location. Secondly, the two

lines that accumulated the highest PVA concentration contained

the phytoene synthase genes under the control of the Ubi

promoter. This promoter was previously demonstrated to be

active from the earliest stages of banana fruit development in

contrast with the late fruit expression from the ACO and Exp1

promoters.

Figure 6 Influence of temperature and time to fruit maturity on the concentration of PVA carotenoids in the FG pulp of wild-type Cavendish banana. For

each month, b-CE levels and time from bunch emergence to harvest were averaged from all samples collected. FG, full green.

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 520–532

Pro-vitamin A biofortified Cavendish bananas in the field 529

Two constructs containing Ubi-MtPsy2a and ACO-MtPsy2a

have been transferred to the National Agricultural Research

Organization (NARO) in Uganda and have been transformed into

two Ugandan cultivars including EAHB.

Experimental procedures

Vector construction

The coding regions of Musa troglodytarum x acuminata culti-

var Asupina phytoene synthase 2a (MtPsy2a; GenBank #:

JX195659), Zea mays cultivar B73 phytoene synthase 1 (ZmPsy1;

GenBank #: U32636) and Pantoea ananatis phytoene desaturase

(PaCrtI; Genbank #: D90087) genes were used to facilitate PVA

enhancement in banana. The uidA gene from Escherichia coli,

encoding the enzyme b-glucuronidase (GUS), containing a catalase intron (Khanna et al., 2004), was used to assess

promoter activity. Each of these genes was fused to the

Agrobacterium tumefaciens nopaline synthase (Nos) 30 transcrip- tion termination regulatory sequence (Depicker et al., 1982).

The banana expansin (Exp1; GenBank #: JN172931) and 1-

aminocyclopropane-1-carboxylate oxidase (ACO; GenBank #:

AF221107) promoters, and the maize polyubiquitin1 promoter

(Ubi, Dugdale et al., 2000) where characterized in banana using

each of them to drive uidA expression, and the resulting

expression cassettes: Exp1-uidA-nos, ACO-uidA-nos and Ubi-

uidA-nos were assembled in the pBIN-19 (GenBank #: U09365)

binary vector backbone. These three promoters were also used

to regulate the expression of the MtPsy2a and ZmPsy1 genes,

resulting in the production of six expression cassettes in the

pCAMBIA-2300 (GenBank #: AF234315) binary vector back-

bone. Expression of the PaCrtI gene was driven by the Ubi or

Exp1 promoter, and the resulting Ubi-PaCrtI-nos and Exp1-

PaCrtI-nos expression cassettes were assembled in pBIN-19.

Selection of transgenic plants was mediated by the neomycin

phosphotransferase II gene (nptII; Beck et al., 1982) in both

pCAMBIA-2300 and pBIN-19.

Plant transformation and regeneration

Transgenic Musa acuminata (AAA Group) ‘Dwarf Cavendish’ lines

were generated via Agrobacterium-mediated transformation of

embryogenic cell suspensions (ECS) using A. tumefaciens strain

AGL1 (Khanna et al., 2004). Binary vectors containing MtPsy2a or

ZmPsy1 were used for transformation of banana ECS either alone

or in combination with one of the vectors containing PaCrtI, while

vectors containing uidA were all used individually for transfor-

mation.

Plant material and field trials

Transgenic and wild-type banana lines established in tissue

culture were transported to the field trial site at the Department

of Agriculture and Fisheries (DAF) South Johnstone Research

Facility (Queensland, Australia) according to conditions on the

Office of the Gene Technology Regulator (OGTR) licence number

DIR109. Plants were acclimatized in soil and grown in a

glasshouse for 12 weeks before planting and maintenance in

the field according to standard agronomic procedures. The initial

field trial (FT-1) was conducted between 2009 and 2012.

Promising transgenic lines and selected wild-type control lines

(up to 10 sucker plants per line) were established for 12 weeks

in the glasshouse before being transferred to a second field trial

(FT-2) at the same facility, which commenced in September

2012.

Field sample collection and processing

Mature green (full green—FG) fruit was harvested from each plant and sent to the Centre for Tropical Crops and Biocom-

modities (CTCB) laboratory in Brisbane, Australia, within 48 h of

harvest. When analysis of developing fruit was required, fruit

was taken at 3, 6 and 9 weeks post-bunch emergence (S3, S6

and S9) prior to harvesting the entire bunch at FG. Fruit was

handled under low-light conditions and was processed either

immediately (for FG analysis) or at 7 days post-exposure (24 h)

to ethylene for full ripe (FR) analysis. Representative fruit from

the top, middle and bottom of the bunch was received at all

stages of fruit development from plants transformed with Psy or

CrtI genes, while fruit from the top of the bunch only was

obtained from lines transformed with the uidA gene. Leaf

samples from the field were collected from the first fully

expanded leaf prior to bunch emergence. Prior to further

analysis, all samples were freeze-dried in a Benchtop 4K Freeze

Dryer (VirTis�) and homogenized in a Mini-Beadbeater-8TM

(Biospec Products) tissue disruptor.

Nucleic acid isolation

Genomic DNA (gDNA) for PCR and Southern blot analysis was

isolated from 50 mg of homogenized freeze-dried leaf tissue

using a modified CTAB method (Stewart and Via, 1993). Isolation

of plasmid DNA (pDNA) was done using the Wizard� Plus SV

Minipreps DNA Purification System (Promega) according to the

manufacturer’s instructions. Total RNA was extracted from

50 mg of homogenized freeze-dried banana fruit tissue essen-

tially as described by Valderrama-Ch�airez et al. (2002).

DNase treatment and complementary DNA (cDNA) synthesis

For cDNA synthesis, 3 lg of total RNA was DNase treated using an RQ1 RNase-free DNase Kit (Promega). DNA-free RNA samples

(1.8 lg) were reverse-transcribed to cDNA using an oligo(dT20) primer and the GoScriptTM Reverse Transcription System (Pro-

mega) in 25 lL reactions according to the manufacturer’s instructions.

Polymerase chain reaction (PCR) and reverse- transcription PCR (RT-PCR)

Putatively transgenic tissue culture plants were tested under

standard PCR conditions using oligonucleotide primers designed

to amplify gene fragments spanning the MtPsy2a, ZmPsy1 and

PaCrtI genes and their respective promoter region and using

GoTaq� Green master mix (Promega). Transgene-positive plants

were further PCR tested for the presence of contaminating

Agrobacterium using VirC gene primers (Haas et al., 1995).

Complementary DNA (cDNA) from PCR-positive plants was then

used in RT-PCR to verify gene expression.

Quantitative real-time PCR (qRT-PCR)

Quantitative RT-PCRs were done in a CFX384 TouchTM Real-Time

PCR Detection System (Bio-Rad) using GoTaq qPCR Master Mix

(Promega). Each sample was analyzed in three technical replicates

in addition to the inclusion of ‘no template’ and ‘RT-negative’

controls. The following amplification parameters were used: Hot-

Start polymerase activation at 95 °C for 2 min, followed by 45 cycles of 10 s denaturation at 95 °C and 30 s annealing/extension at 60 °C. At the end of the reaction, a dissociation curve was

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 520–532

Jean-Yves Paul et al.530

produced from 65 to 95 °C to confirm the specificity of the amplicon from each primer set. Fluorescence was recorded in real

time and detected at 470 nm. Relative expression levels were

calculated using the CFX Manager 3.1 (Bio-Rad) software and the

DCT method (Schmittgen and Livak, 2008). Ct data obtained from target gene of interest (GOI) were normalized using Ct

values from the two stable reference genes cyclophilin (CYP) and

ribosomal protein S2 (RPS2). All primers were designed using the

Primer3Plus freeware (http://www.bioinformatics.nl/cgi-bin/pri-

mer3plus/primer3plus.cgi) (Table S1).

Southern hybridization

For determination of transgene copy number integration by

Southern analysis (Southern, 1975), genomic DNA (10 lg) as well as positive control plasmid DNA (20 ng) were digested for 16 h

and 1 h, respectively, by incubation at 37 °C with 20 U of restriction enzyme. Restriction enzymes were selected to only cut

once within the binary vector T-DNA region without cutting the

region to which the probe would hybridize. PCR based DIG-

labelled probes (Roche) were designed targeting the coding

regions of MtPsy2a and ZmPsy1. Digested DNA was elec-

trophoresed, blotted and detected under standard Southern

blotting conditions (Sambrook and Russell, 2001).

Carotenoid content quantification

Carotenoids were extracted from banana pulp (200 mg) or peel

(25 mg) tissue and analyzed by HPLC as previously described

(Buah et al., 2016). Total carotenoids and b-carotene equivalents (b-CE) were expressed in lg/g dry weight (dw).

Assessment of promoter activity

b-glucuronidase (GUS) content was measured in banana pulp tissue (40 mg) by ELISA as per Dugdale et al. (2013), while

fluorometric quantification of GUS activity was determined in leaf

and peel tissue as described by Jefferson et al. (1987).

Acknowledgements

The Banana21 project is supported by the Bill & Melinda Gates

Foundation and the Department for International Development

(United Kingdom).

Conflict of interest

Authors declare no conflict of interest.

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Supporting information

Additional Supporting Information may be found online in the

supporting information tab for this article:

Figure S1. Determination of transgene copy number in trans-

genic Cavendish banana lines by Southern blot analysis.

Figure S2. Determination of transgene copy number in trans-

genic Cavendish banana lines by Southern blot analysis.

Figure S3. Representative HPLC chromatogram of the main

carotenoids in wild-type and transgenic Cavendish banana.

Figure S4. Transgene expression analysis in selected MtPsy2a

transgenic Cavendish banana lines by reverse transcriptase-PCR

(RT-PCR).

Figure S5. Transgene expression analysis in selected ZmPsy1

transgenic Cavendish banana lines by reverse transcriptase-PCR

(RT-PCR).

Table S1. List of oligonucleotide primer sequences used for RT-

PCR and qRT-PCR.

ª 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 520–532

Jean-Yves Paul et al.532