This study establishs a basis for growing plantlets without sucrose and investigating other factors like carbon dioxide and light regime to improve the in vitro growth performance.

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In vitro growth and hardening performance of
yam plantlets from Temporary Immersion
Bioreactor system at different sucrose
regimes
Akeem. O. NOFIU, Morufat. O. BALOGUN, Norbet
MAROYA and Adeniyi. O. TOGUN
21st Annual symposium of IARSAF,
IITA conference center, Ibadan Nigeria.
18th April, 2018.

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Introduction
▪ In West Africa, yam (Dioscorea spp.) plays a very
important role as a
• source of income,
• food security,
• and livelihood systems for at least 60 million
people(Maroya et al., 2015)
▪ Six species are economically important out of 600
species identified
▪ The variety D. rotundata is the principal commercial yam
(FAOSTAT 2014,)
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Yam Productivity constraints
✓ Ultimate constraints
• Limited supply of affordable improved quality seed yam
(Maroya et al., 2015, Asumugha and Ogbona, 2013)
✓ Other constraints
• Seasonality
• on-farm losses of tubers during harvesting and storage
• incidences of pests and diseases,
• high labour cost
▪ More so, the seed yam system in West Africa is mainly
informal and entirely market driven (Maroya et al., 2015).
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High propagation ratio technology
▪ Tissue culture is a technology employed by YIIFSWA to
rapidly propagate disease-free plantlets of yam. (Balogun
et al.,2014)
▪ Conventional plant tissue culture (CTC) is the growth of
plant tissues in a closed, static sterile and laboratory
environment.
▪ Its efficiency is limited by;
• Increased labour cost for frequent sub-culturing
• Small size of the culture container
• limits plantlets access to nutrient and air.
• Fragile plantlets
▪ As a result, plant propagation ratios in CTC are
suboptimal. 4

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High propagation ratio technology
▪ CTC limitations led to the development of TIBS(Paek et
al., 2005)
▪ TIBS is an advanced tissue culture technology, where
plants are intermittently ventilated and immersed in
nutrient solution.
▪ Advantages of TIBS over CTC
• Increased productivity (more vigorous plantlets)
• Reduced labour cost
• Eliminates cost of agar
• Less time to label and seal vessels
• Possibly automated
• Ability to combine research with production
• Internet compatibility
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Justification
▪ However, Photoautotrophic micropropagation (PAM),
unlike the CTC techniques is assumed to reduce;
• in vitro biological contamination
• expensive sucrose containing medium
• high rate of acclimatization loss and
• its efficient use in TIBs because of the aeration system.
▪ PAM is carried out using the natural ability of plant to
prepare its own food in controlled environment.
• under high CO2 and high PPF (photosynthetic photon
flux) conditions in sugarless nutrients medium.
▪ Sugar in the medium is considered an essential cause
for the high production costs of plantlets in CTC (Kozai
1991). 6

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Objective
▪ In order to establish a baseline;
• a preliminary study to investigate the effects of
sucrose regime on in vitro and post-flask growth
performance of yam plantlets (TDr 95/19177) in SETISTM
type of temporary immersion bioreactor system (TIBs)
and hardening performance was carried out.
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Materials and methods
Study Location:
The Cell biology unit of the Bioscience centre, IITA Ibadan.
Source of yam plantlets(TDr 95/19177):
The YIIFSWA project of IITA.
Materials:
Murashige and Skoog (MS) medium, Sucrose, vented
vessels,beakers, measuring cylinder, electric magnetic
stirrer, magnetic rod/retriever, paper towel, pH meter,
weighing balance, laminar flowhood, distiled water,
autoclave e.t.c.
Experimental Design:
Completely Randomized Design (CRD) with 2 treatments
and 3 replicates 8

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Materials and Methods
In vitro growth performance experiment
▪ 50 clean, 3 weeks old CTC plantlets of Dioscorea
rotundata (TDr 95/19177) were transferred into TIBs
containing modified MS medium (with or without 30 g/l
sucrose)
▪ Growth performances observed for 8weeks and
transferred to the glass house for hardening.
▪ A set of plantlets were hardened through gradual
acclimatization
▪ Another sets were hardened directly for each of the
treatments.
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Materials and Methods
Plate 1. a. in vitro yam plantlets grown in 0g/l sucrose media.
b. in vitro yam plantlets grown in 30g/l sucrose media.
a b
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Plate 2. a. Height of in vitro yam plantlets grown in 30g/l sucrose media
b. Height in vitro yam plantlets grown in 0g/l sucrose media
Materials and Methods

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Materials and Methods
Data collection:
• Numbers of nodes at 8weeks after transfer (WAT)
• Numbers of shoots at 8WAT
• Numbers of roots at 8WAT
• Fresh weight per plantlet at 8WAT
• Height per plantlet at 8WAT and
• survival of plantlets in hardening 2weeks after hardening
(2WAH).
STATISTICAL ANALYSIS:
• The data were subjected to analysis of variance
(ANOVA) using Microsoft excel package.
• Differences in means were considered significant at
P ≤ 0.05.
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Result
In vitro growth performance traits
▪ There were no significant differences between treatments in all
growth traits
Table 1: Effects of sucrose regime on in vitro growth performance of
yam plantlets (TDr 95/19177) in SETISTM type of temporary immersion
bioreactor system (TIBs).
Sucrose regime
growth performance traits
Numbers
of Nodes
Numbers
of Shoots
Numbers
of Roots
Fresh weight
per Plantlets
(g)
Height per
plantlets (cm)
30 g/l sucrose media 6.40±3.50 2.20±2.41 3.30±0.63 944.04±766.49 7.82±2.41
0 g/l sucrose media 5.8±2.47 1.8±0.78 3.9±1.96 725.08±572.41 7.46±2.46
LSD value(P ≤ 0.05) 1.25 0.30 0.90 280.39 1.00
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Results
Figure 1: Percentage survival of gradual hardened in vitro
yam plantlets grown in 30g/l sucrose media and 0g/l
sucrose media at 2WAH
0
10
20
30
40
50
60
70
80
90
30g/l sucrose media 0g/l sucrose media
%survival
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Results
0
60
0
10
20
30
40
50
60
70
30g/l sucrose media 0g/l sucrose media
Figure 2: Percentage survival of direct hardened in vitro
yam plantlets grown in 30g/l sucrose media and 0g/l
sucrose media at 2WAH
▪ Post-flask growth performance
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Discussion
▪ The results indicated that the growth performance traits
were not significantly different between the 30g/l sucrose
media and 0g/l sucrose media.
▪ This result is in agreement with Kozai et al. (2002)
▪ Low photosynthetic ability of in vitro plantlets is
considered to be the cause of the low survival and
growth rates in the acclimatization stage (Grout, 1978;
Grout and Aston, 1978; Preece and Sutter, 1991)
▪ Deng and Donnelly, (1993) reported that sucrose in the
medium promoted plantlet growth but depressed
photosynthesis and reduced in vitro hardening.
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Conclusion
▪ In vitro Yam plantlets grown in 30g/l sucrose media
are less adapted to the stress of hardening and
require gradual acclimatization.
▪ Hence, sucrose free media in TIBS can produce
yam plantlets with better adaptive performance to
the natural environment than sucrose containing
media.
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Recommendation
❖ This study has established a basis for growing
plantlets without sucrose and investigating other
factors like carbon dioxide and light regime to
improve the in vitro growth performance which is
the actual study and still in progress.
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Acknowledgements
▪ The most Merciful God
▪ IITA
▪ Yam Improvement for Income and Food security
in West Africa (YIIFSWA) Project, IITA
▪ ALL YIIFSWA team members
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References
Asumugha, G.N. and M.C. Ogbonna. 2013. Promotion of improved yam
minisett technology to improve productivity and reduce excessive
use of food yam for planting in Ghana, Nigeria, Togo and Benin:
Nigeria Report. Ref.: 2010/0022/SN/EA.PS/SE: Consultancy
baseline study on CORAF/WECARD commissioned projects on
staple crops. Submitted to CORAF/WECARD secretariat, Dakar,
Senegal. 27 pp.
Maroya et al., 2015. A new paradigm for improving yam systems. IITA R4D
Review.http://r4dreview.iita.org/index.php/2015/03/20/a-new-
paradigm-for-improving-yam-systemsparadigm-for-improving-yam-
systems.
Deng, R., Donnelly, D. (1993). In vitro hardening of new raspberry by CO2
enrichment and reduced medium sucrose concentration. Hortic. Sci.,
281:1048-1051.
Kozai T, Koyama Y and Watanabe I (2002). Multiplication of potato plantlets
in vitro with sugar-free medium under high photosynthetic photon
flux. Acta. Hort. pp.230.
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