STANDARDIZATION OF PROTOCOL FOR GENETIC TRANSFORMATION OF ANNEXIN GENE IN ...

98 Pages·2014·1.34 MB·English

STANDARDIZATION OF PROTOCOL FOR
GENETIC TRANSFORMATION OF ANNEXIN
GENE IN Musa acuminata cv. Patakpura
FOR DROUGHT RESISTANCE

A

THESIS SUBMITTED TO

THE ORISSA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY
BHUBANESWAR

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF

MASTER OF SCIENCE IN AGRICULTURE
(AGRICULTURAL BIOTECHNOLOGY)

BY

DRAMADRI GERALD AFAYO

DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY
COLLEGE OF AGRICULTURE
ORISSA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY
BHUBANESWAR-751003, ODISHA
2014

THESIS ADVISOR: Dr. A.B.DAS
CONTENT
____________________________________________________________
CHAPTER PARTICULARS PAGE
______________________________________________________________________
I INTRODUCTION 1-17

II REVIEW OF LITERATURE 18-32

III MATERIALS AND METHODS 33-40

IV RESULTS 41-55

V DISCUSSION 56-62

VI SUMMARY AND CONCLUSION 63-64

VII REFERENCES i-xxv

VIII APPENDICES xxvi-xxviii
LIST OF TABLES

TABLE PARTICULARS PAGE NO.
NO.
1.1 Production of top 5 world producers of banana in 2012 4
1.2 Banana exports of top 5 world exporters in 2011 4
1.3 Production of top 5 world producers of plantains in 5
2012
1.4 Exports of top 5 world exporters of plantains in 2011 5
2.1 In vitro regeneration studies of banana 29
2.2 Genetic transformation of banana via Agrobacterium 30
4.1 Effects of surface sterilization on aseptic culture and 41
survival of explants
4.2.1 Effect of growth regulators on callus induction from 43
corm slices of banana cv. patakpura
4.2.2 Effect of various concentrations og growth regulators 44
BAP and IAA along with Adenine sulphate on multiple
shoot induction from corm slices of Musa acuminate
cv. patakpura
4.3 Effect of various concentrations of cytokinin BAP and 45
auxins IAA and NAA on in vitro shoot multiplication
4.4 Effect of various concentrations of IAA on root 46
formation from multiple shoots of Musa acuminate cv.
patakpura
4.6 Kanamycin based selection 47
4.7 Effect of cefotaxime on shoot induction in the medium 48
containing different concentrations of cefotaxime
4.8 In vitro transformation studies using Annexine BJ2 48
gene
LIST OF FIGURES

FIG. PARTICULARS PAGE
1.1 Production of top 5 producers of banana in 2012 7
1.2 Banana export of top 5 exporters in 2011 7
1.3 Plantain production by top 5 producers of the world in 8
2012
1.4 Plaintain export of top 5 exporters of the world in 2011 8
2.1 An illustration of how Agrobacterium can be used to 28
transform plant cells in order to regenerate transgenic
plants
2.2 Mechanism of T-DNA transfer 28
3.1 Gene construct of AnnBj2 and AnnBj3 gene 35
3.2 Gene construct of GUS marker 35
3.3 Schematic map of binary vector pCAMBIA2301 35
4.1 Effect of growth regulators on callus induction 49
4.2 Effect of IAA concentration on root formation 49
4.3 Kanamycin based selection 50
4.4 Effect of cefotaxime concentration on shoot survival 50
LIST OF PLATES

PLATE PARTICULARS PAGE
Plate 1 A banana plant with a bunch of female flowers 6
Plate 2 1. Shoot initiation on MS +8mg/l BAP+1mg/l IAA after 3 weeks. 51
2. Shoot buds after 4 weeks.
3. Shoots after 4.5 weeks.
4. Elongated shoots after 5 weeks of culture.
5. Elongated shoots after 6 weeks.
6. Shoot multiplication on MS+4mg/l BAP+0.5mg/l IAA and
NAA.
7. Shoots after 1 month of culture on multiplication medium.

Plate 3 8. Root initiation and development on MS+1mg/l IAA+0.5mg/l 52
activated charcoal.
9. Prehardening of rooted plants on 1:1:1(sand: soil:
vermicompost) in culture room after 7days.
9a. Prehardened plants after 12 days.
10. Final hardening in poly house On 1:1:1 (sand: soil:
vermicompost) after7 days of transfer.

Plate 4 11. Final hardening after 12 days of transfer to poly house. 53
12. Hardened plants after 2 months in polyhouse.
13. Hardened plants after 3 months in polyhouse.
Plate 5 14. Callus initiation after 45 days of culture on MS +2mg/l BAP + 54
4mg/l 2, 4-D
15. Organogenesis from callus on MS + 4mg/l BAP + 1mg/l 2, 4-
D
16. Somatic embryos scanned with S-3400N scanning Electron
Microscope
16a. Somatic embryos scanned with S-3400N scanning Electron
Microscope
Plate 6 17. Bacterial culture on selection medium containing 55
kanamycin and rifampicin

18. Co-cultivation of embryogenic cell mass with bacteria
after infection

19. GUS stained embryogenic mass with distinctly visible
somatic embryos.

20. Shoot bud initiation on selection medium

21. Shoot regeneration on selection medium.
LIST OF ABBREVIATIONS USED

2, 4-D 2, 4-Dichlorophenoxy acetic acid
Ads Adenine sulphate
BAP Benzyl aminopurine
CaMV Cauliflower Mosaic Virus
cm centimeter
d day
DNA Deoxyribonucleic acid
ECS Embryogenic cell suspension
EDTA Ethylene diamine tetra acetic acid
FAO Food and Agriculture Organization
FAOSTA Online FAO Statistical Database containing statistics on agriculture,
nutrition, fisheries, forestry, food aid, land use and population
Fig. Figure
GUS beta-glucuronidase
HCl Hydrochloric acid
Hrs Hours
IAA Indole-3- acetic acid
IBA Indole-3- butyric acid
LB Left T-DNA border sequence
LB Luria Bertani
mg/l milligram per litre
Min. Minute
ml mililitre
mm millimeter
MS Murashige and Skoog
NAA Napthalene acetic acid
NaOH Sodium hydroxide
nm Nanometer
OD Optical density
PCR polymerase chain reaction
pH Hydrogen ion concentration
RB Right T-DNA border sequence
rpm Revolutions per minute
TAE Tris acetic acid EDTA
TBE Tris boric acid EDTA
T-DNA Transferred DNA
TDZ Thidiazuron
v/v volume/volume
w/v weight/volume
X-Gluc 5-bromo-4-chloro-3-indolyl-β-D-glucuronide
µg Microgram
µl Microlitre
µM Micromolar
I. INTRODUCTION
1.1 Back ground
Bananas and plantains are monocotyledonous plants in the genus Musa
(Musaceae, Zingiberales). They are giant herbs, commonly up to 3 m in height, with
no lignifications or secondary thickening of stems that is characteristic of trees
(Tomlinson, 1969). The centre of origin of the group is in South-East Asia, where
they occur from India to Polynesia (Simmonds, 1962). The centre of diversity has
been placed in Malaysia or Indonesia (Daniells et al., 2001), although considerable
diversity is known throughout the range. The plants are distributed mainly on margins
of tropical rainforests (Wong et al., 2002).
Banana is one of the most important staple food crops in the tropics and a
source of income to millions of poor subsistence farmers. It is the fourth most
important food crop in developing world's after rice, wheat and maize (Bioversity
International, 2006). Banana is grown in over 120 countries worldwide (Thangavelu
and Mustaffa, 2012) covering about 10 million hectares, with an annual world
production estimated at 107 million tones. India is the largest producer in the world
(24.8 million tonnes) followed by China (10.8 million tonnes). Production share by
region indicated that Asia produced 56%, Americas 26.6%, Africa 15.6%,
Oceania1.5% and Europe 0.4%. In Africa, the total production of banana was
estimated at 15.8 million tonnes of which Uganda produced an estimated 5.7 thousand
tonnes (FAOSTAT, 2012). In Uganda, it is one of the most important staple crops
contributing about 30% of the total food consumption and 14% total crop value
(Kalyebara et al., 2005). About 24% of the agricultural households are engaged in
banana production. Banana being a year round fruiting crop ensures food security at
household level, providing food to more than 70% of Uganda’s population on a
regular basis. Banana is primarily grown for subsistence needs and any surplus for
sale to local markets.
Worldwide, well over a thousand banana cultivars or landraces are recognized.
The vast majority of the cultivated bananas (Pollefeys et al., 2004) are derived from
inter- and intraspecific crosses between two diploid (2n = 2x = 22) wild species, Musa
acuminata and Musa balbisiana (Simmonds and Shepherd, 1955). In terms of the
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chromosome sets, these are designated as having the genome constitution AA (M.
acuminata) or BB (M. balbisiana). These diploid Musa species have seeded fruit with
little starch and only a small amount of fleshy pith, and are of not much value as a
crop. The cultivated bananas and plantains differ from their wild relatives by being
seedless and parthenocarpic – the fruit develops without seed development or
pollination and fertilization. The genetic basis of the mutation (or mutations) in the A
genome that gives rise to parthenocarpy has not been characterized, and no
parthenocarpy has been identified in B genome diploids, although hybrids of A and B
show the character. Most of the cultivars are wild collections made by farmers of
spontaneously occurring mutants with parthenocarpic fruit production, which were
brought into cultivation and then multiplied and distributed by vegetative propagation.
There is no straightforward botanical distinction between bananas and plantains but,
in general, bananas refer to the sweeter forms that are eaten uncooked, while starchy
fruits that are peeled with a knife when unripe and then cooked are referred to as
plantains and cooking bananas, while some cultivars are ‘beer bananas’ for
fermentation of the juice, or used for deep frying as banana chips.
Many of the domesticated bananas have proved to be triploid, 2n = 3x = 33,
with genome constitutions of AAA (mainly the sweet dessert bananas), AAB or ABB
(mainly but not exclusively starchy plantains eaten after cooking). There are also
seedless cultivated AA and AB diploids, and tetraploids (2n = 4x = 44) with genome
constitutions of AAAA, AAAB, AABB and ABBB. These various plants have been
collected from multiple, independent sources in the wild, so the hybridization events
and mutations giving rise to the seedless and parthenocarpic characters have occurred
many hundreds of times. Where fertile plants occur together, hybridization continues
to produce new diversity (Pollefeys et al., 2004) and parental combinations.
Simmonds (1962) considered five plant characteristics that lead to farmers picking
plants for cultivation: plant vigour, yield, seedlessness, hardiness and fruit quality, the
first four of which are related to polyploidy (triploidy).
1.2 Description of banana plant
Banana is a monoecious plant having male flowers at the tip of inflorescence and
female flowers behind (Fig. 1). The fruit of banana or plantain is a product of
parthenocarpy and characterised as berry with a leathery outer peel that contains much
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collenchyma (Daniells et al., 2001). The fruits are formed in layers called combs or
hands, consisting of 10–20 bananas, and there are 6–15 combs per stalk. The latter
equals 40–50 kg per stalk or ten or more tons per acre. If commercially grown, the
large terminal bud and bracts are removed to redirect sugars to the developing fruits.
An unripened banana and the plantain have high starch and low sugar levels plus
copious amounts of bitter-tasting latex. Starch is converted to sugar as the fruit ripens,
so that bananas can eventually contain about 25% of total sugars. As the banana
ripens, the latex is also decomposed. Plantain has the stinging, bitter latex, so the peel
is removed with a knife and the pulp is soaked in salt water for 5–10 min prior to
cooking (http://www.crfg.org). Bananas are harvested unripe and green, because they
can ripen and spoil very rapidly. The fruits are cleaned of old floral parts, combs and
divided into smaller bunches. Poorly formed fruits are removed, and bunches are
thrown into a water bath, where latex is washed away. Then fruits are dried and
usually placed in a ripening room for several days before their transfer to market, or
exported after storing and packing with cushion (usually paper). Presence of naturally
formed ethylene gas, produced by ripe fruits, hastens considerably the ripening of
surrounding, greener fruits (http://www.botgard.ucla.edu)
1.3 Importance of Banana and Plantains
Banana is having great importance in the world due to their commercial and
high nutritional value. Bananas are multipurpose plants because most of their parts
can be used in various ways, depending on the species. The most important part is the
edible fruit, which can be eaten either ripe as a dessert, or unripe as boiled, fried or
roasted food (Smith et al., 2005). Nutritionally, the fruit is rich in carbohydrates,
vitamins A, B, and C, and potassium (Aurore et al., 2009). The unripe fruit can be
brewed to form beer and wine, or processed into sauce, flour, chips, crisps, smoked
products, and confectionary. Unripe fruit is also a source of amylase and starch (van
den Houwe et al., 2000). Male floral buds can be eaten as a boiled vegetable, whereas
pseudostems are a source of fiber for the manufacture of rope, paper, and textiles.
Banana leaves are used for thatching, in the production of fabric and cordage, and as
mulch and animal forage (Smith et al., 2005). Species such as M. ornata and M.
veluntina are popular ornamental plants (Heslop-Harrison and Schwarzacher 2007).
Banana has also been found effective against colorectal cancer (Deneo-Pellegrini et
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al., 1996) breast cancer, (Zhang et al., 2009) and renal cell carcinoma (Rashidkhani et
al., 2005).
Bananas are popular as fresh fruit in temperate countries. In 2011, the world
export of bananas, consisting mainly of Cavendish-type dessert bananas, was
estimated to be 18 million tons (20% of world production), (FAOSTAT 2011). The
most important attributes that make the Cavendish subgroup the main bananas for
export are related to their reliability during transport and their shelf life, rather than
taste. In economic value, banana fruit ranked fifth in the world trade for agricultural
crops (Aurore et al., 2009).

Table 1.1: Production of top 5 world producers of banana in 2012
Country Production (× 1,000,000 tonnes) Percentage
India 24.8 24.38
China 10.8 10.63
China mainland 10.5 10.34
Philippines 9.0 9.04
Ecuador 7.0 6.87
Source: FAOSTAT 2012

Table 1.2: Banana exports of top 5 world exporters in 2011
Country Export (×1,000,000 tonnes) Percentage
Ecuador 5.7 30.86
Philippines 2.0 10.93
Costa Rica 1.9 10.22
Colombia 1.8 9.76
Guatemala 1.4 7.61
Source: FAOSTAT 2011
Uganda is the leading producer and consumer of plantains in the world
(FAOSTAT, 2012). East African Highland Bananas (EAHB) serves as the principle
staple food (‘matooke’) in Uganda with an average daily consumption of 0.6 kg/capita
(FAOSTAT, 2004). This is due to the continuous fruiting habit of EAHB varieties, an
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