Mechanis of increaseing the production of garlic

1. Jimma University College of Agriculture and Veterinary
Medicine
1
M.Sc. Open thesis defense
Protocol Optimization for in vitro Propagation of Garlic (Allium sativum L.)
Variety Tseday 92 Using Basal Plate Culture
BY:
Kasahun Amare
Major Advisor: Techale Birhan(Assistant Professor)
Co-advisor: Milkiyas Ahmed(MSc.)
December 2017
Jimma, Ethiopia

2. Presentation out lines
2
1.Introduction
2.Materials and methods
3.Result and discussion
4.Summary and conclusion

3. 1. Introduction
3
 Garlic (Allium sativum L.) is a monocotyledonous aromatic
belongs to Alliaceae family and genus Allium (Osman et al.,
2007).
 It is the second most widely distributed species of Allium genus
throughout the world (Robledo and Tovar, 2012).
 Garlic has a wide area of adaptation and refinement all over the
world.
 Its center of origin believed today is central Asia (Espino et al.,
2012).

4. Cont…
4
 On the global scale, top 5 producing countries are:
 China producing, 20.06 million tons
 India 1.25 million tons.
Bangladesh, 0.31
 Egypt,0.26 and
Russia, 0.26 million tons per year (FAOSTAT,
2014).

5. Cont…
5
91.7%
Out of world total production,
24,939,965 ton

6. Cont…
6
 Ethiopia shares 0.39% of the world total production and it
occupies 12th in the series of top producing countries (FAOSTAT,
2014).
 According to CSA (2015/16) report, its production covers
6,789.11 hectare with a total production of 5,781.767 tons and 8.5
ton/ha in average.
 Garlic is predominantly used for flavoring and serving dishes and
as a medicinal plant all over the world (Wiczkowski, 2011; Nagy
et al., 2015).

7. Cont…
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 In Ethiopia, it is `produced as a spice and income generating for
famers as well as it adds to the national economy as export goods
(DARC, 2006; Gebreyohannes and Gebreyohannes,2013).
 Conventional crossing in garlic is actually low, or negligent.
 Thus, clonal selection, induced mutations, somaclonal variation or
genetic engineering are the only options to develop cultivars for
breeding (Robledo & Tovar 2012).

8. Cont…
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 Traditional propagation method has low:
 Multiplication rate,
 Transmission of pathogens (Walkey, 1990; Nagakubo et
al., 1993).
 Because of its vegetative propagation, it could be easily infected by
pathogens.
 Which results in yield drop from 20% to 60% depending on the
stage of infection and cultivar type (Lot et al., 1998; Pérez et al.,
2014).

9. Cont…
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The present production of garlic cannot meet up the increasing
demand; the area and production of garlic has not been improved
at desired level (Haque et al., 2013).
Plant tissue culture techniques have provided many solutions to
basic questions and practical difficulties in plant biology.
However, tissue culture protocols are influenced by genotype and
explant used.
so it is necessary to optimize culturing conditions for different
species (Barandiaran et al., 1999).

10. Cont…
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 To date, copious of reports have been documented for direct
organogenesis from several explants such as
 stem disc (Ayabe and Sumi, 1998)
 shoot apex (Roksana et al., 2002),
 Shoot meristem (Gull et al., 2014)
 root tip (Hassan et al., 2014).
 Kamenetsky et al. (2015) reported the distribution of garlic virus in six
organs:
cloves (32–41%),
roots (32–50%),
inflorescence (8–20%),
basal plate (5–7%),
flowers (1–7%) ideal to reduce viral content
leaves (1–4%).

11. Cont…
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 Beside explant source plant growth regulators along with additive
compounds accelerate regeneration of full plants in vitro
(Gantaint et al., 2010).
 Therefore, present study intended to develop in vitro mass
propagation protocol for garlic with 6-Benzylaminopurine (BAP)
and 1-Naphthaleneacetic acid (NAA), with the following
objectives:

12. Cont…
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 General objective
 To develop a suitable protocol for in vitro mass propagation of
garlic variety Tseday92 using basal plate culture.
Specific objectives
To determine the optimum concentration of BAP for shoot
initiation
To determine the optimum combinations of BAP and NAA for
shoot multiplication.
To determine the optimum concentration of NAA for in vitro
rooting.

13. 2. MATERIALAND METHODS
13
The experiment was conducted at Jimma University College of
Agriculture and Veterinary Medicine from December, 2016 to August,
2017.
 Experimental material
 The garlic variety Tseday92 (G-493) was obtained from Debrezeit
Agricultural Research Center.
 The variety has high yield potential (8.13 ton/ha) and early maturity
time(135days)
 Good marketability and high consumer preference.
(Tadesse and Kassa, 2004; Bezu et al., 2014).

14. Explant surface sterilization
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 Garlic cloves were separated from healthy and mature bulbs and
carefully peeled the outer protective leaves
 Afterward it was washed thoroughly in running tap water with
powder soap (omo) for several times.
 Next it was taken to laminar airflow cabinet and
 First washed with autoclaved distilled water for three minutes.
 Then immersed in 70% ethanol for one minute, followed by
repeat rinsing with autoclaved distilled water for three times.

15. Cont…
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 0.2 %( W/V) HgCl2 was
used for two minutes under
constant hand agitation .
 After that it was thoroughly
washed with autoclaved
distilled water four times.
Finally, basal plates were sliced to
2-3 cm in diameter and 0.5 cm in
length

16. Preparation of stock solution
16
 Stock solutions of macro and micronutrients, and vitamins are
prepared in distilled water.
 Calcium chloride was dissolved separately and then added to the
rest solution to avoid precipitation.
 MS Iron EDTA stock was prepared first warming the water near
boiling point in 500 ml beaker and then added Na2 EDTA.2H2O .

17. Cont…
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 After Na2EDTA.2H2O was dissolved FeSO4.7H2O was added
gradually under mild magnetic stirring.
 Immediately after adding FeSO4.7H2O; the bottle was closed
and kept on stirring for an hour.
 Finally, it was kept in a refrigerator for further use.

18. Growth regulators preparation
18
 The growth regulators were prepared by weighing the
powder in 1mg/1ml (W/V) ratio.
 It was dissolved by 0.5-1ml of 1NNaOH for BAP and
70% ethanol for NAA.
 Then the total volume was adjusted to 100ml by distilled
water.
 Afterward stored in a refrigerator at 4°C for further use.

19. Preparation of culture Media
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 A liter of MS Basal medium was prepared taking
 10ml of macronutrient,
 10ml of iron EDTA,
 1ml of micronutrients, and
 1ml of vitamins from stock solutions and added
sequentially in Erlenmeyer flask.
 3%(W/V) sucrose was weighed and added while dissolving by
magnetic stirring.

20. Cont…
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 The medium pH was adjusted to 5.8 using 1N NaOH and 1N HCl.
 Afterward the media was solidified by addition of 0.8% (W/V)
agar.
 Finally, the final volume was adjusted to 1000ml with distilled
water and dispersed 50ml per jar and autoclaved at 121ºC for 20
min.

21. Culture Establishment
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 The explants were placed in a glass jar containing 50 ml of MS
media.
 The glass jars with cultured explants were properly sealed with
parafilm.
 Afterward, it was maintained on shelves at:
• Temperature of 25±2oC
• Photo period of 16:8 hours light and dark
• With photo flux density of 200lux

22. Experiments
22
 Experiment 1 effect of BAP on shoot initiation
 The sliced cloves with the basal portion cultured on MS
medium provided with eight different BAP concentrations(0.0,
0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0mg/l).
 There were three explants per jar and three replications for
each treatment was used in Completely Randomized Design.

23. Experiment 2 effect of BAP and NAA on shoot
multiplication
23
 Explants from the initiation stage about 2cm length was cultured on
MS medium containing BAP (0.0, 0.5, 1.0, 1.5, 2mg/l) and NAA
(0.0, 0.25, 0.5, 0.75mg/l) concentrations .
 The experiment was designed as 5x4 factorial arrangement in CRD
with three replicates.
 There was two shoots per culture.
 Sub-culturing was done twice at 15 days interval.

24. Experiment 3 effect of NAA on root formation
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 shoots from the multiplication stage was excised in 2cm length
and cultured in MS medium supplemented with different NAA
concentrations (0.0,0.25, 0.5, 0.75, 1.0,1.25, 1.5,1.75, 2.0, 2.25,
2.5mg/l).
 There were 11 treatments replicated thrice in Completely
Randomized Design.
 Two shoots were cultured per each replication.

25. Cont…
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 Acclimatization
 The well-rooted shoots washed thoroughly to remove any
medium residual.
 The plantlets having a length of 10-15 cm were transplanted in
small pots containing sterile soil, compost, and sand in the ratio
of 2:1:1.
 Then covered with plastic bags (in order to keep RH) for one
week in culture room.

26. Cont…
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 Data collected
 Shoot initiation percentage
 Days took to shoot initiation
 Number of shoots per explant
 Shoot length (cm)
 Leaf number per main shoot
 Root number per explant
 Root length (cm)
 Rooting percentage (%)
 Acclimatization percentage (%)

27. Cont…
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 Statistical Analysis
 The analysis of variance was performed using SAS version 9.3.
 The significant differences between mean values were
compared using Least Significant Difference (LSD) at P≤ 0.05.

28. 3. RESULTS AND DISCUSSION
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 Experiment 1: Effect of BAP on Shoot Initiation
BAP(mg/l) IP (%)
0 54.44c
0.25 77.77b
0.50 86.66a
1.00 77.77b
1.50 86.66a
2.00 77.77b
2.50 77.77b
3.00 77.77b
CV (%) 5.46
LSD(0.05) 7.28
Roksana et al. (2002) recoded the
earliest (three - four days) shoot
initiation form supplementation of
0.5mg/l 2ip+0.25 mg/l NAA and
1.5mg/l BAP+0.5mg/l NAA.
Del Pozo and Gonzalez (2005)
reported early growth of garlic is suited
by exposure of cloves to low
temperature.
Haque and Haque (2017) recorded
75% of shoot initiation from root
segments. However, in the current
work, 86.66 shoot initiation percentage
was recorded.

29. Experiment 2: Effect of BAP and NAA on Shoot
Multiplication
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BAP(mg/l) NAA(mg/l) NSPP NLPP SL (cm)
0.0
0.0
0.0
0.0
0.0 0.67±0.16k 1.83±0.16c 3.67±0.08i
0.25 4.00±0.28fg 2.83±0.16b 4.00±0.08i
0.50 5.00±0.28de 2.23±0.14c 3.67±0.16i
0.75 8.33±0.16ab 2.83±0.16b 5.00±0.28h
0.5
0.5
0.5
0.5
0.0 3.67±0.16gh 2.00±0.0c 7.00±0.28efg
0.25 3.67±0.16gh 2.17±0.16c 6.67±0.44fg
0.50 3.17±0.28hij 2.17±0.16c 5.00±0.28h
0.75 8.00±0.28b 3.83±0.16a 6.63±0.73fg
1.0
1.0
1.0
1.0
0.0 5.00±0.28de 2.83±0.16b 10.83±0.16a
0.25 5.67±0.33cd 3.83±0.16a 7.20±0.15ef
0.50 9.00±0.28a 2.00±0c 8.16±0.16cd
0.75 8.67±0.33ab 2.00±0c 7.27±0.14ef
1.5
1.5
1.5
1.5
0.0 3.67±0.33gh 2.67±0.16b 9.57±0.21b
0.25 5.00±0.28de 2.67±0.16b 8.63±0.38c
0.50 3.50±0.28ghi 2.00±0.16c 8.73±0.14c
0.75 6.00±0.28c 2.17±0.16c 7.50±0.25de
2.0
2.0
2.0
0.5
0.0 4.67±0.33ef 2.83±0b 6.27±0.14g
0.25 2.67±0.33j 2.00±0.16c 6.4±0.14g
0.50 2.67±0.33j 1.83±0.16c 5.07±0.17h
0.75 2.83±0.16ij 1.83±0.16c 5.17±0.08h
CV (%) 9.89 10.21 7.30
LSD(0.05) 0.78 0.40 0.79

30. Experiment:3 Effect of NAA on root formation
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NAA (mg/l) NR (Mean±SE) RL (cm)
(Mean±SE)
RP (%)
0.0 0h 0f 0f
0.25 15.83±0.44c 1.88±0.04a 83.33b
0.50 10.67±0.16d 1.9±0.04a 100.00a
0.75 9.67±0.44ed 1.87±0a 66.67c
1.00 9.27±0.37ef 1.73±0.06b 50.00d
1.25 18.50±0.28b 1.3±0.05cd 83.33b
1.50 22.00±0.28a 1.29±0.04d 83.33b
1.75 6.50±0.28g 1.07±0.06ed 50.00d
2.00 10.17±0.44ed 1.42±0.01c 66.67c
2.25 8.43±0.34f 0.97±0.03e 33.33e
2.50 6.67±0.16g 1.2±0.05d 33.33e
CV% 5.22 5.9 6.16
LSD 0.95 0.13 6.17

31. 5. SUMMARY AND CONCLUSION
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 Low crop yield on this crop is due to the contamination of different
pathogen attack and its vegetative propagation nature.
 The main goal of this study was to optimize the culturing condition
for in vitro mass propagation of garlic variety Tseday92.
 Specifically to determine the optimum concentrations of BAP and
NAA for shoot initiation, shoot multiplication and root formation.

32. Cont…
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 MS medium supplemented with BAP and explants from basal plate
0.5cm in length and 2-3cm in diameter were used for shoot
initiation.
 All concentrations of BAP used for shoot initiation induced the
only single shoot within four days.
 The supplementation of 1mg/l BAP+0.5mg/l NAA resulted in the
highest (9±0.28) number of shoots.

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 The highest leaf number (3.83±0.28) was recorded at 0.5mg/l
BAP+0.75mg/l NAA, 1mg/l BAP+0.25mg/l NAA.
 The lowest number of leaves (1.83±0.28) were obtained at
control, 2mg/lBAP+0.25mg/l NAA and 2mg/lBAP+0.5mg/l
NAA respectively.
 The longest shoot (10.83±0.28 cm) was measured at
1mg/lBAP+0mg/l NAA followed by (9.57±0.37cm) at 1.5mg/l
BAP+0mg/l NAA .

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 The shortest shoot length was obtained from control, 0mg/l
BAP+0.25mg/lNAA,0mg/l BAP+0.5mg/l NAA
correspondingly
 The highest root number (22±0.5) was recorded at 1.5mg/l
NAA with 83.33% of root formation.
 The highest root length was observed at 0.5mg/l (1.9±0.07cm)
followed by, 0.25mg/l (1.88±0.07cm) respectively. And the
lowest root length (0.97±0.05cm) was recorded at 2.25mg/l.

35. Conclusion
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 The result obtained from this work proved that micro propagation
protocol for garlic variety Tseday92 at :
 1mg/l BAP +0.5mg/l NAA concentration was adequate for shoot
multiplication.
 1.5mg/l NAA for in vitro root formation.
 In vitro rooted plantlets were transferred to acclimatization and
survived up to 55.55%.

36. Cont…
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Basal plate derived explants are proficient of for in vitro
propagation.
Hence, this finding will be utilized for propagation of
elite genotype at commercial-scale.
Future line of the work
 Based on the present result transferring of in vitro
propagated plantlets to soil needs further investigation

37. The end
37