Abstract:

In Uganda, vitamin A deficiency (VAD) and iron deficiency anaemia (IDA) are major
public health problems with between 15-32% of children under 5 years of age
showing VAD and 73% being anaemic. This is largely due to the fact that the staple
food crop of the country, banana, is low in pro-vitamin A and iron, therefore leading
to dietary deficiencies. Although worldwide progress has been made to control VAD
and IDA through supplementation, food fortification and diet diversification, their
long term sustainability and impact in developing countries such as Uganda is
limited. The approach taken by researchers at Queensland University of Technology (QUT), Australia, in collaboration with the National Agricultural Research Organization (NARO), Uganda, to address this problem, is to generate consumer
acceptable banana varieties with significantly increased levels of pro-vitamin A and
iron in the fruit using genetic engineering techniques. Such an approach requires the use of suitable, well characterised genes and promoters for targeted transgene
expression. Recently, a new banana phytoene synthase gene (APsy2a) involved in
the synthesis of pro-vitamin A (pVA) carotenoids was isolated from a high 0-
carotene banana (F'ei cv Asupina). In addition, sequences of banana ferritin, an iron
storage protein, have been isolated from Cavendish banana. The aim of the
research described in this thesis was to evaluate the function of these genes to
assess their suitability for the biofortification of banana fruit. In addition, a range of
banana-derived promoters were characterised to determine their suitability for
controlling the expression of transgenes in banana fruit. Due to the time constraints involved with generating transgenic banana fruit, rice
was used as the model crop to investigate the functionality of the banana-derived
APsyZa and ferritin genes. Using Agrobacterium-mediated transformation, rice
callus was transformed with APsyZa +/- the bacterial-derived carotene desaturase
Organization (NARO), Uganda, to address this problem, is to generate consumer
acceptable banana varieties with significantly increased levels of pro-vitamin A and
iron in the fruit using genetic engineering techniques. Such an approach requires
gene (Crt/) each under the control of the constitutive maize poly-ubiquitin promoter
(ZmUbi) or seed-specific rice glutelinl (Gtl) promoter. The maize phytoene
synthase (ZmPsyl) gene was included as a control. On selective media, with the
exception of ZmUbi-Crt/-transgenic callus, all antibiotic resistant callus displayed a yellow-orange colour from which the presence of 0-carotene was demonstrated
were
To investigate the potential of the banana-derived ferritin gene (BanFerl) to
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enhance iron content, rice callus was transformed with constitutively expressed
BanFerl using the soybean ferritin gene (SoyFer) as a control. A total of 12 and 11
callus lines independently transformed with BanFerl and SoyFer, respectively, were
multiplied and transgene expression was verified by RT-PCR. Pearl's Prussian blue
staining for in-situ detection of ferric iron showed a stronger blue colour in rice
callus transformed with BanFerl compared to SoyFer. Using flame atomic
absorption spectrometry, the highest mean amount of iron quantified in callus
transformed with BanFerl was 30-fold while that obtained using the SoyFer was 14-
fold higher than the controls. In addition, ~78% of BonFerl-transgenic callus lines
and ~27% of SoyFer-transgenic callus lines had significantly higher iron content than
the non-transformed controls.
Since the genes used for enhancing micronutrient content need to be expressed in
banana fruit, the activity of a range of banana-derived, potentially fruit-active
promoters in banana was investigated. Using u/dA (GUS) as a reporter gene, the
function of the Expansinl (MaExpl), Expansinl containing the rice actin intron
(MaExpla), Expansin4 (MaExp4), Extensin (MaExt), ACS (MaACS), ACO (MaACO),
using Raman spectroscopy. Although the regeneration of plants from yellow-orange
callus was difficult, 16 transgenic plants were obtained and characterised from
callus transformed with ZmUbi-APys2a alone. At least 50% of the T1 seeds
developed a yellow-orange coloured callus which was found to contain levels of Pcarotene
ranging from 4.6-fold to 72-fold higher than that in non-transgenic rice
callus. Using the seed-specific Gtl promoter, 38 transgenic rice plants
generated from APsy2a-Crt/-transformed callus while 32 plants were regenerated
from ZmPsyl-Crt7-transformed callus. However, when analysed for presence of
transgene by PCR, all transgenic plants contained the APsy2a, ZmPsyl or Crtl
transgene, with none of the plants found to be co-transformed. Using Raman
spectroscopy, no P-carotene was detected in-situ in representative T1 seeds.