callus induction and tillering capability of 4 sugarcane cultivars (saccharum officinarum l.) under...
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Callus Induction and Tillering Capability of 4 Sugarcane Cultivars (Saccharum officinarum L.) under In Vitro CultureTRANSCRIPT
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SPECIAL PROBLEM
TITLE
Callus Induction and Tillering Capability of 4 Sugarcane Cultivars
(Saccharum officinarum L.) under In Vitro Culture
By:
Arghya Narendra Dianastya
DEPARTEMENT OF AGRONOMY
FACULTY OF AGRICULTURE AT KHAMPHAENGSAEN
KASETSART UNIVERSITY
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SPECIAL PROBLEM
TITLE
Callus Induction and Tillering Capability of 4 Sugarcane Cultivars
(Saccharum officinarum L.) under In Vitro Culture
By:
Arghya Narendra Dianastya
DEPARTEMENT OF AGRONOMY
FACULTY OF AGRICULTURE AT KHAMPHAENGSAEN
KASETSART UNIVERSITY
2014
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Special Issue Certificate
Department of Agronomy, Faculty of Agriculture, Kasetsart
University, Kamphaeng Saen Campus
Tittle Callus Induction and Tillering Capability of 4 Sugar
Cultivars (Saccharum officinarum L.) under In Vitro
Culture
Author Arghya Narendra Dianastya
Advisor................................................................................................................................
(Assoc. Prof. Dr. Sontichai Chanprame)
Date___Month______Year_______
Approved by Departement of Agronomy
....................................................................................
(Assist. Prof. Dr. Chanate Malumpong)
Head of Departement
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ACKNOWLEDGEMENTS
First and foremost, praise be upon The Lord Almighty, The Creator of the
universe which embodies complex and orderly system of nature as well as extends
contentment throughout lifetime.
I would like to express my deep gratitude and sincere appreciation to Assoc.
Prof. Dr. Sontichai Chanprame as my extraordinary kind advisor for his patience,
valuable advice, encouragement and guidance throughout this special problem research.
Thanks are also addressed to Departement of Agronomy, Faculty of Agriculture
at Kamphang Saen, Kasetsart University Kamphaeng Saen Campus as my current
department. Thanks for all useful knowledge and memorable atmosphere.
My sincere thanks are also given to Plant Cell Tissue Culture and
Transformation Laboratory provided by Center for Agricultural Biotechnology (CAB)
for permission and offering place and resources to conduct daily operation for my
special problem research.
Thanks to all my Thailand friends and my Indonesian counterpart students for
their companionship, joyfulness and blissful memories in Thailand. Last but not least,
special thanks are also dedicated to Soerodjos Family, my beloved one, and my two
awesome young brothers for the strength given.
This exchange program would not have been possible without the support and
genuine cooperation between Faculty of Agriculture, Jember University and Faculty of
Agriculture at Kamphang Saen, Kasetsart University Kamphaeng Saen Campus. This
program expansively augments my point of view about education, culture and life
ultimate goal.
Arghya Narendra Dianastya
July, 2014
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ABSTRACT
Callus Induction and Tillering Capability of 4 Sugarcane Cultivars (Saccharum
officinarum L.) under In Vitro Culture; Arghya Narendra Dianastya; 5620000087;
Departement of Agronomy; Faculty of Agriculture; Kasetsart University Kamphaeng
Saen Campus.
Sugarcane is (Saccahrum spp. hybrid) is high polyploid parennial grass belong
to the family Poaceae and tribe Andropogoneae. Biotechnology approach such as in
vitro culture is needed to fulfill the growing demand of sugarcane, since it offers
advantages for rapid multiplication of cultivars and produces a healthy and disease-free
plants. The objectives of this study was to investigate the capability of callus induction,
shoot regeneration and tiller growth of 4 Thai local sugarcane cultivars (K92-80, KK3,
LK95-127, and K93-219). The callus induction medium was MS + 3.0 mg/L of 2,4-D +
2% of sucrose + 10% (V/V) of CW + 0.7 % of agar. Medium for shoot regeneration and
tiller production was MS + 10% (V/V) of CW + 2% of sucrose + 0.7% agar. In callus
induction stage, K92-80, KK3 and K93-219 had the highest callus induction percentage
(100%). The best cultivar in shoot regeneration stage was LK95-127 which had 72.72%
in shoot regeneration percentage and 5.27 on the average number of shoots produced.
The best cultivar in tillering capability stage was KK3 which had 4.54 on the average
number of tillers produced. Gene factor is highly responsible in the callus induction,
shoot regeneration and tillering capability of 4 sugarcane cultivars under in vitro
culture. The result in in vitro culture is argued to have slightly different result in ex vitro
culture due to environmental factors which might affect plant growth.
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TABLE OF CONTENTS
Page
COVER ........................................................................................................................... i
SPECIAL ISSUE CERTIFICATE ................................................................................ iii
ACKNOWLEDGEMENT .............................................................................................. iv
ABSTRACT ................................................................................................................... v
TABLE OF CONTENTS .............................................................................................. vi
LIST OF TABLES ......................................................................................................... x
LIST OF FIGURES ....................................................................................................... xi
LIST OF APPENDIX TABLES ................................................................................... .xiii
I. INTRODUCTION .................................................................................................... 1
1.1 Background ................................................................................................... 1
1.2 Objective ......................................................................................................... 4
II. LITERATURE REVIEW ...................................................................................... 5
2.1 General aspects of Saccharum officinarum L. ................................................ 5
2.2 Classification of Saccharum officinarum L.. ................................................. 6
2.3 Morphology of Saccharum officinarum L. .................................................... 6
2.3.1 The root ............................................................................................... 6
2.3.2 The stem.............................................................................................. 6
2.3.3 The leaf ............................................................................................... 7
2.3.4 The infloresence.................................................................................. 7
2.4 Tillering ........................................................................................................... 7
2.5 Sugarcane in Thailand ..................................................................................... 8
2.5.1 sugarcane cultivar ............................................................................... 8
2.5 Tissue culture .................................................................................................. 9
2.6. Totipotency .................................................................................................... 10
2.8 Micropopagation stages of tissue culture ........................................................ 11
2.9 Organogenesis ................................................................................................. 12
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TABLE OF CONTENTS (contd)
Page
2.10 Callus culture ................................................................................................ 12
2.11 Component of in vitro culture media ............................................................ 13
2.11.1 Inorganic nutrient.............................................................................. 13
2.11.2 Carbohydrate..................................................................................... 14
2.11.3 Plant growth regulator ...................................................................... 14
a. auxin ............................................................................................... 15
b. cytokinin ......................................................................................... 15
c. coconut water ................................................................................. 16
d. agar ................................................................................................. 16
2.12 Culture condition ........................................................................................... 17
2.13 Sugarcane micropropagation ......................................................................... 17
2.13.1 Organogenesis in sugarcane micropropagation ................................ 18
2.13.2 Shoot regeneration in sugarcane in vitro culture .............................. 18
III. MATERIALS AND METHODS ......................................................................... 20
3.1 Time and date .................................................................................................. 20
3.2 Materials .. ....... 20
3.3 Methods ...... 20
3.3.1 Explant collection .. 20
3.3.2 Surface sterilization . .. 20
3.3.3 Callus induction ..... 21
3.3.4 Shoot regeneration .............................................................................. 21
3.3.5 Tillering capability.............................................................................. 22
3.3.6 Data collection and statistical analysis ............................................... 22
IV. RESULTS
4.1 Callus induction .............................................................................................. 23
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TABLE OF CONTENTS (contd)
Page
4.1.1 Physical properties of callus ............................................................... 23
4.1.2 Percantage of explant inducing callus ................................................ 26
4.2 Shoot regeneration .......................................................................................... 29
4.2.1 Physical properties of shoot ................................................................ 29
4.2.2 Percentage of callus producing shoot ................................................. 33
4.2.3 Average number of shoot ................................................................... 36
4.3 Tillering capability .......................................................................................... 38
4.3.1 Physical properties of tiller ................................................................. 38
4.3.2 Percentage of explant producing tiller ................................................ 41
4.3.3 Average number of tiller ..................................................................... 44
V. DISCUSSION
5.1 Callus induction .............................................................................................. 47
5.1.1 Effects of leaf properties in callus growth .......................................... 47
5.1.2 Effects of 2,4-D in callus growth ........................................................ 47
5.1.3 The roles of sucrose as a source of carbohydrate in callus growth .... 48
5.1.4 Effect of light in callus growth ........................................................... 48
5.1.5 Effects of phenolic compound on callus properties ............................ 49
5.1.6 Effects of genotype in callus growth .................................................. 49
5.2 Shoot regeneration .......................................................................................... 51
5.2.1 The roles of coconut water as plant growth regulator
in shoot regeneration ......................................................................... 51
5.2.2 Effects of callus properties used in shoot regeneration ...................... 52
5.2.3 Effects of cytokinin and genotype in shoot regeneration ................... 53
5.3. Tillering capability ......................................................................................... 54
5.3.1 Determinants of variation in in vitro tillering capability .................... 54
5.3.2 Effects of genotype in tillering capability ......................................... 55
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TABLE OF CONTENTS (contd)
Page
5.3.3 Compatibility of tillering capability result from in vitro culture
for understanding tillering properties in ex vitro culture ................... 56
VI. CONCLUSION AND RECOMENDATION
6.1 Conclusion .......................................................................................................... 59
6.2 Recommendation ................................................................................................ 61
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LIST OF TABLES
Page
Table 1 Murashige and Skoog medium composition ................................................. 13
Table 2 Callus physical properties of 6 sugarcane cultivars in callus induction
stage for 2 months .......................................................................................... 24
Table 3 Number and percentage of explant inducing callus of 6 sugarcane
cultivars cultured in callus induction stage for 2 months .............................. 26
Table 4 Shoot physical properties of 4 sugarcane cultivars in shoot
regeneration stage for 2 months ..................................................................... 30
Table 5 Number of callus producing shoot of 4 sugarcane cultivars in shoot
regeneration stage taken every 1 week .......................................................... 34
Table 6 Percentage of callus producing shoot of 4 sugarcane cultivars in shoot
regeneration stage taken every 1 week .......................................................... 34
Table 7 Average number of shoot of 4 sugarcane cultivars in shoot
regeneration stage taken every 1 week .......................................................... 37
Table 8 Tiller physical properties of 4 sugarcane cultivars in tillering
capability stage taken every 1 week for 2 months ......................................... 39
Table 9 Number of explant producing tiller of 4 sugarcane cultivars in tillering
capability stage taken 2 weeks ....................................................................... 42
Table 10 Percentage of explant producing tiller of 4 sugarcane cultivars in
tillering capability stage taken 2 weeks ......................................................... 42
Table 11 Average number of tiller of 4 sugarcane cultivars in tillering capability
stage taken every 2 weeks.............................................................................. 45
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LIST OF FIGURES
Page
Figure 1 Regeneration pathway in plant tissue culture .............................................. 10
Figure 2 Callus induction of 6 sugarcane cultivars from innermost spindle leaf
explant after 2 months inoculation. ............................................................. 25
Figure 3 Number of explants inducing callus of 6 sugarcane cultivars after 2
months inoculation....................................................................................... 28
Figure 4 Percentage of explant inducing callus of 6 sugarcane cultivars after 2
months inoculation....................................................................................... 28
Figure 5 Initiation of shoot growth from callus explant of 4 sugarcane cultivars
in shoot regeneration stage after 3 weeks culturing..................................... 31
Figure 6 Initiation of multiple shoot growth of 4 sugarcane cultivars in shoot
regeneration stage after 5 weeks culturing. ................................................. 32
Figure 7 Expanding and differentiated shoot of 4 sugarcane cultivars in shoot
regeneration stage after 7 weeks culturing. ................................................. 32
Figure 8 Long and expanding green shoot of 4 sugarcane cultivars in shoot
regeneration stage after 9 weeks culturing. ................................................. 33
Figure 9 Number of callus producing shoot of 4 sugarcane cultivars in shoot
regeneration stage after 2 months culturing................................................. 35
Figure 10 Percentage of callus producing shoot of 4 sugarcane cultivars in shoot
regeneration stage after 2 months culturing................................................. 35
Figure 11 Weekly graphic of average number of shoot produced of 4 sugarcane
cultivars in shoot regeneration stage............................................................ 37
Figure 12 Average number of shoot produced of 4 sugarcane cultivars in shoot
regeneration stage taken every 1 week. ....................................................... 38
Figure 13 Explant used for tiller production from cut shoot. ....................................... 40
Figure 14 Initial growth and elongation of shoot in the 1st week................................. 40
Figure 15 Tiller growth in the last week observation. ................................................. 41
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LIST OF FIGURES (contd)
Page
Figure 16 Number of explant producing tiller of 4 sugarcane cultivars in tillering
capability stage taken every 2 weeks. .......................................................... 43
Figure 17 Percentage of explant producing tiller of 4 sugarcane cultivars in
tillering capability stage taken every 2 weeks. ........................................... 43
Figure 18 Weekly graphic of average number of tiller produced of 4 sugarcane
cultivars in tillering capability stage. ........................................................... 45
Figure 19 Number of tiller produced in each explant of 4 sugarcane cultivars in
tillering capability stage taken 9 weeks after explanting. ............................ 46
Figure 20 Average number of tiller produced of 4 sugarcane cultivars in tillering
capability stage taken every 2 weeks. .......................................................... 46
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LIST OF APPENDIX TABLES
Page
Table 1 Number of shoot of 4 sugarcane cultivars in shoot regeneration stage
in the first week of observation (February 19th
2014). ................................ 73
Table 2 Number of shoot of 4 sugarcane cultivars in shoot regeneration stage
in the second week of observation (February 26th
2014) ............................. 73
Table 3 Number of shoot of 4 sugarcane cultivars in shoot regeneration stage
in the third week of observation (March 5th
2014) ...................................... 74
Table 4 Number of shoot of 4 sugarcane cultivars in shoot regeneration stage
in the forth week of observation (March 12th
2014) .................................... 74
Table 5 Number of tiller of 4 sugarcane cultivars in tillering capability stage
in the third week of observation (April 3rd
2014). ....................................... 75
Table 6 Number of tiller of 4 sugarcane cultivars in tillering capability stage
in the fifth week of observation (April 17th
2014) ....................................... 75
Table 7 Number of tiller of 4 sugarcane cultivars in tillering capability stage
in the seventh week of observation (Mei 1st 2014) ...................................... 76
Table 8 Number of tiller of 4 sugarcane cultivars in tillering capability stage
in the ninth week of observation (Mei 1st 2014) .......................................... 76
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I. INTRODUCTION
1.1 Background
Sugarcane (Saccharum spp. hybrids, family Poaceae, tribe Andropogoneae) is a
high polyploid (2n = 36-170) perennial grass (Gallo-Meagher et al., 2000). It has ability
to store high concentration of sucrose in the stalk and grows relatively rapid and
produces high yields (Singh, 2010). It is commonly known that sugarcane is one of the
most efficient photosynthesizer, C- 4 plant in plant kingdom (Yadav and Ahmad, 2013).
Commercial sugarcane today is mainly derived from the hybridization of the noble cane
(Saccharum officinarum) with the cultivated species such as S. sinese and S. barberi or
sometimes is the result of hybridization process of S. officinarum with the other two
wild species which are S. spontaneum and S. robustum (Peng,1984).
Sugarcane (Saccharum officinarum L.) is one of the most important cash and
industrial crop and is widely cultivated for white refined sugar (Khamrit et al., 2012).
Nowadays, sugarcane is also used for ethanol due to its inexpensiveness, abundant and
can be planted in vast region of the world. In 2013, approximately 104 million m of
ethanol produced worldwide, and approximately 50% of production was from sugarcane
crops (Singh, 2010; CropEnergies, 2014). Besides that, sugarcane also produces
valuable products such as biofibres, waxes, and bioplastic (Singh et al., 2013).
Sugarcane is cultivated in 127 countries in both the tropics and subtropics and
covering an area up to 25.4 million hectares worldwide with a production of 1.79 billion
tons in 2011, providing approximately 70% of the worlds sugar supply. (Singh, 2010;
Joshi et al., 2013). The top 5 largest exporters are Brazil, Thailand, European Union,
Australia, and Cuba. As number one exporter since 1985, Brazil has exported ten-fold,
to over 10 million tonnes in 2003 and control the world sugarcane price (Kole, 2007).
Thailand as one of the largest producer of sugarcane also increases the number of
sugarcane production up to 99.5 million metric tons in 2012 (Prasertsri, 2013).
According to the Departement of Agriculture (2001), Thailand become the biggest
sugarcane exporter in 1998/99 when the sugarcane production was about 50 milion
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tonnes with an average yield of 55 tons/ha. The exported quantity at that time was
approximately 2.6-3.9 million tonnes.
Sugarcane breeding programmes have focused on the production of cultivars
with high yield, higher sucrose content, pest and disease resistance, tolerance to abiotic
stress and improved rooting ability (Yadav and Ahmad, 2013). However, improvement
of sugarcane cultivar via conventional breeding is relatively slow due to the large and
variable in genome size, complex ploidy levels, narrow genetic base, limited gene pool,
and meiotic instability (Joshi et al., 2013). The other problems of conventional breeding
of sugarcane are lack of rapid multiplication due to multiplication rate through sett by
conventional propagation is 1:8 (Abbas et al., 2013) and continuous contaminations by
systemic diseases (Visessuwan et al., 1999). Consequently, breeding for superior traits
is a difficult and taking 10-14 years to realease (Snyman et. al., 2010).
It has been realized that the growing demand of newly released sugarcane
cultivars could not be fulfilled by only use the conventional methods of plant
multiplication (Sengar et al., 2011; Yadav and Ahmad, 2013). Using new technology
such as biotechnology offers excellent opportunities to improve sugarcane crop for
specific targeted objectives such as high productivity and disease resistance in the short
period of time (Sengar et al., 2011). There are several areas of biotechnology research
in sugarcane improvement today including: (1) cell and tissue culture techniques for
molecular breeding and propagation; (2) engineering novel genes into commercial
cultivars; (3) molecular diagnostics for sugarcane pathogens to improve exchange of
Saccharum germplasm (Lakshaman et al, 2005).
Understanding tissue culture technique becomes the basic tools to conduct plant
propagation via biotechnology (Neumann et al., 2009). According to Hartmann et. al.
(1990), tissue culture can be defined as an aseptic culture of a wide range of excised
plant parts. Plant tissue culture offers advantages over conventional methods of
propagation for a large and rapid multiplication of cultivars with desirable traits and
production of healthy and disease-free plants in any season with conservation of space
and time (Ahmadian et al., 2013; Kataria et al., 2013). Propagation by tissue culture
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also becomes an attractive and powerful tool in the research field throughout the world,
especially in the area of large scale clonal propagation, crop improvement through
genetic manipulation, conservation of plant genetic resources and valuable germplasm
(Tarique et. al., 2010).
As mentioned by Snyman et al. (2006), plant tissue culture of sugarcane offers
the best methodology for quality and phytosanitary planting material at a faster rate in a
shorter period of time as well as increases the propagation potential by 20-35 times.
This is because regerenation through tissue culture can produce rapid production of
sugarcane clones (Jabbott and Atkin, 1987). Sugarcane tissue culture also gives an
advantage which effectively reduces the time period between selection and commercial
release of new sugarcane cultivars (Abbas et al., 2013) as well as provides an
alternatives control practice to overcome various kind of viruses and diseases
(Visessuwan et al., 1999).
Numerous studies on sugarcane plant regeneration have been reported.
Successful culture and regeneration of plants from protoplasts, cells, callus and various
tissue and organs have been achieved in sugarcane crops (Yadav and Ahmad, 2013).
Attempt to measure callus growth and tillering capability using in vitro culture is
particularly important. Understanding callus growth capability can be used as futher
development of biotechnology in sugarcane, while understanding number of tiller under
in vitro culture can be used as a guidance to understand the production yield. As it is
mentioned by Yadaf (1991) that optimal number of millable canes dirrectly effect to the
sugar and yield production. However, every sugarcane cultivar has different responses
and variations from the treatment given. Attempt to conduct tissue culture experiment in
different sugarcane cultivars is needed to find the disirable trait using short period of
time. This research is conducuted to observe callus induction and tillering capability of
4 sugarcane cultivars using tissue culture technique. Sugarcane cultivars used are
Thailand local cultivars : K92-80, KK3, LK95-127, and K93-219.
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1.2 Objective
a. To investigate the callus induction capability of 4 Thai local sugarcane cultivars
under in vitro culture.
b. To investigate the shoot regeneration via callus of 4 Thai local sugarcane
cultivars under in vitro culture.
c. To investigate the tillering capability of 4 Thai local sugarcane cultivars under in
vitro culture.
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II. LITERATURE REVIEW
2.1 General aspects of Saccharum officinarum L.
Sugarcane belongs to the genus Saccharum, that firstly established by Linnaeus
on Species Plantarum in 1753 with two species: S. officinarum and S. spicalum. The
genus belongs to the tribe Andropogenae in the grass familiy, Poaceae. The tribe
includes other tropical grass such as Sorghum and Zea (maize) (Kole, 2007).
The generic name Saccharum could be traced back to the Sanskrit word Karkara
or Carkara, meaning gravel (Bakker, 1999). It symbolizes prosperity for it adorns the
goddness of wealth, Sri Laxmi (Hunsigi, 1993). Records of sugarcane in history have
been in existence since 510 BC where reeds which produce honey without bees were
first indicated by soldiers of the emperor Darius near the Indus river, India. However,
The conquest of Alexander The Great of India in 327 BC made the sugar start to spread
in the western world (Kole, 2007).
Modern sugarcane as we know it today evolved in 1893 with the successful
crossing program between S. officinarum Black Chirebon (2n=80) and the wild S.
spontaneum Kassoer (2n=40-128) (Kole, 2007; Joshi et al., 2013). According to Sengar
et al. (2011), a series of backcrosses to S. officinarum resulted in cultivars with higher
yields, improved ratooning ability and disease resistance in which Java breeder called
this process as nobelization (Babu,1990). The process of nobelization of sugarcane
as we know has resulted in a highly complex interspecific aneupolyploid genomic
organization in sugarcane crops (2n=99130). (Joshi et al., 2013). Nowadays, over 400
clones of S.officinarum have been recorded. S.officinarum is generally characterized by
having chromosome number of 2n=80, with basic chromosome number of x=10 (Kole,
2007). Most modern sugarcane breeding programs rely on extensive intercrossing of
elite cultivars derived from these early hybrids (Lakhsaman et al., 2005).
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2.2 Classification of Saccharum L.
Sugarcane belongs to the genus Saccharum L., traditionally placed in the tribe
Andropogoneae of the grass family (Poaceae). This tribe includes tropical and
subtropical grasses and the cereal genera Sorghum and Zea (known as maize or corn).
The taxonomy and phylogeny of sugarcane is complicated and comes from five genera
which share common characteristics and form a closely related interbreeding group
known as the Saccharum complex. The Saccharum complex comprises Saccharum,
Erianthus section Ripidium, Miscanthus section Diandra, Narenga and Sclerostachya.
These genera are characterised by high levels of polyploidy (polyploids have more than
two sets of chromosomes) and frequently unbalanced numbers of chromosomes
(aneuploidy) (Kole, 2007).
2.3 Morphology of Saccharum officinarum.
2.3.1 The root
The sugarcane root system is fibrous and shallow. There are two kinds of root of
sugarcane. The first root is from primordial of the cutting, which are thin and branched,
and the second root is from the primordial of the tillers that are thick, fleshy and much
less branched. In the sugarcane, the top 25 cm of soil contains 50% of the plant roots,
with the next 35 cm containing a further 40% of the roots. However, the effective root
zone varies depending on the soil type (Peng, 1984).
2.3.2 The stem
Sugarcane has multiple stems or culms which height of mature sugarcane stem
varies in the range of 3-5 meters and the diameter of stem varies in the range of 2-4 cm,
depending on cultivars, internal and external growth factors. In every stem consists of a
series of nodes separated by internodes. Each node consists of a growth ring or
intercalary meristem. The node is the place where a leaf scar remain after the leaf has
dropped (Peng, 1984). Internode length varies from each cultivar (Bakker, 1999). The
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basal region of internode, just above the leaf scar, is the root band (root ring) where the
root primodia (root initials) are located. Below the root band is the wax band, a zone
covered with a layer of wax in varying density (Peng, 1984).
2.3.3 The leaf
The leaf of sugarcane consists of two parts, the blade and the sheath which
separated by a leaf joint. The sheath which clasps the stem may be smooth or hairy. The
leaves are attached alternately to the nodes. The basal leaves are very small but up to the
stem, blades develop, gradually growing longer till they reach a maximum size. The leaf
joint is located at the juction of the blade and the sheath. The number of green leaves
increase as the plant grows older. During the boom phase of growth, the stalk of a
healthy plant may contain as many as 17 20 leaves (Bakker, 1999).
2.3.4 The infloresence
The sugarcane inflorescence is an open branched panicle which also known as
an arrow whose shape, degree of branching and size are highly cultivar specific. The
arrow can bear thousands of flowers, and is estimated to average 24,600 florets. The
arrow consists of a main axis and first, second and third order branches. Attached to the
branches are spikelets arranged in pairs, one of which is sessile and one pedicellate, that
bear individual flowers. At the base of each spikelet is a row of silky white hairs.
Sugarcane flowers consist of three stamens as a male organ and a single carpel with a
feathery stigma as a female organ. Sugarcane flower is a wind pollinated flowers. The
male stamens may be abortive and reduced the pollen production (Australian
Goverment, 2004).
2.4 Tillering
Tillering is characteristic of the grass family. In field propagation, tillering is
defined as underground branching of sugarcane. Tillering is a phenomenon when the
buds of a cutting start developing into shoots called mother shoots or primaries. The
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little stem of these primaries consists of many shoots which in turn may produce tertiary
shoots. Tillering phase prevailds only during the early growth stage. After an appropiate
number of tillers are formed, each begins to undergo the elongation phase until
maturity. Only a certain number of tillers will successfully become millable stalks, due
to competition for nutrients (Peng, 1984).
2.5 Sugarcane in Thailand
Sugar was known to the Thai people as sugar cake in the Sukhothai Dynasty
(1219-1438 A.D.). The sugar producers during that time were cottage industries from
Sukhothai, Phitsanulok and Kamphaeng Phet Province. In modern times, the sugar mill
industry began in 1937 by the government. Lampang Sugar Mill was the first state
enterprise sugar mill, followed by a second mill in Uttaradit in 1942 (Departement of
Agriculture, 2001).
Sugarcane in Thailand grows best in deep, well drained loamy to loamy sand
soil textures that have pH range between 6.1- 7.7 and an organic matter content not less
than 1.5 %. In Thailand, clay textured soils are unfavorable to sugarcane growth.
Optimal temperatures for growth are between 20 and 35o Celcius. The water
requirment is 1,200-1,600 mm/year (Departement of Agriculture, 2001).
There are many cultivars of sugarcane in Thailand. Cultivars should be chosen
that are specifically adapted for that region. Cultivars such as K 88-92, U Thong 3 and
U Thong 1 are favorable because can be grown in almost every place in Thailand
(Departement of Agriculture,2001).
2.5.1 Sugarcane cultivars
K 92-80 is a non flowering cultivar as a result of hybrid cross between K84-200
and K 76-4. K 92-80 cultivar has a yield potential up to 118.8 ton/ha. K 92-80 has fast
growing capability with moderate tillering. In the case of ratooning, this cultivar has a
very good ratooning and moderate drought stress tolerance.
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KK3 is non flowering cultivar that very popular in Northeast region of Thailand
which has sandy loam soil characteristic. KK3 is a progeny of 85-2-352 and K 84-200.
This cultivar has desirable traits such as fast growing, good ratooning, and high yield
with average 113.1 ton/ha. One plant of KK3 can have tiller up to 5 tiller per plant
which is considered to be moderate tillering capability.
K 93-219 is characterized by fast germination and growing with potential yield
up to 125 ton/ha. This cultivar is non flowering cultivar as a result of hybrid cross
between U-thong 1 and K 84-200. Tillering capability of this variety is considered to be
moderate with good ratooning capability. K 93-219 also known as drought tolerant
cultivar.
LK 95-127 is a non flowering cultivar and also known as a cultivar that good for
fresh juice cane. It is high yield cultivar with average of yield up to 112.5 ton/ha. This
cultivar is good in ratooning with moderate tillering capability (4-5 stalks/plant).
2.6 Tissue culture
The concept of plant tissue and cell culture was mentioned in 1902 by the
German botanist Gottlieb Harberlandt. Gottlieb Harberlandt published a paper entitled
Experiments on the culture of isolated cells. Haberlandt had attempted to culture
chlorophyll-containing cells and demonstrated the totipotency of cells. That experiment
initiated a new method of plant propagation, which has known as 'Plant Tissue Culture'
(Singh, 2003).
Tissue culture is a term used to indicate the aseptic culture (in vitro) of a wide
range of excised plant parts. In many practice, propagators use the term
micropropagation, in vitro culture and tissue culture interchangeably to mean any plant
propagation using aseptic culture (Hartmann et al., 1990). This definition also extends
to the culture of excised embryos and protoplast culture. There are other terms have
been used in micropropagation and tissue culture based on explant selection in relation
to life cycle. These terms are meristem-tip culture, axilary shoot proliferation,
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10
adventitious shoot induction, organogenesis and somatic embryogenesis (Hartmann et
al., 1990).
According to Hartmann et al. (1990), there are several important pathways of
development of explant from tissue culture. The pathways are as follow:
1. Organogenesis may occur within the callus mass, to produce new plantlets.
2. Specific treatments may cause the cells to disassociate and develop a cell
suspension culture.
3. Cells may be treated to produce a protoplast culture.
4. The regenerative potential may be shift toward somatic embryogenesis.
Figure 1: Regeneration pathways in plant tissue culture.
Source: Hartmann et al., 1990
2.7 Totipotency
The basic concept of tissue culture is totipotency. Totipotency means an ability
in individual plant cells to be regenerated to a whole plant by controlling culture
conditions (Lee and Huang, 2013). In nature, totipotency can happen in the response of
fast restoration of the lost or stress-damaged parts of shoots and roots. In in vitro
conditions, practically any living cell with a nucleus can experience the process of
dedifferentiation under the influence of nutrient medium components (Ezhova, 2003).
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11
2.8 Micropropagation stages of tissue culture
Generally, according to Beyl and Trigiano (2008), there are 5 stages to establish
micropropagation in plant, start to stage 0 to 4. Those stages are: (stage 0) donor plant
selection and preparation, (I) establishment of aseptic culture, (II) proliferation of
axillary shoot, (III) pretransplant or rooting, (IV) tranfer to natural environtment.
Stage 0 refers to selection and maintenance of the stock plants that used as the
source of explants. Stock plants are maintained in clean and controlled environtment to
avoid specific pathogens and unfavorable environtments. General objectives of Stage I
are to place an explant into aseptic culture by avoiding contamination and provide an in
vitro environment that promotes stable production. Contamination avoidance is
conducted by sterilization. Sterilization is usually accomplished through surface
disinfecting by alcohol or sodium hypochlorite to eradicate any kinds of bacteria, fungal
and virus from plant. Most of Stage I media consist of mineral salts, sucrose, and
vitamins, supplemented with plant growth regulator (PGR) (Beyl and Trigiano, 2008).
Stage II is also known as Multiplication Stage has a purpose to mantain the explant in a
stabilized state and multiply the microshoots to the number that suitable for rooting.
Media used are slightly similiar with Stage I and commonly cytokinin is mainly used to
shoot initiation process. Stage III has a function to produce root in explants and to
prepare them for transplanting out of the aseptic protected environtment to the outdoor
condition. Subculture is needed in this process and required an auxin hormone to induce
root. The last stage is Stage IV in which the explant rooted are transplanted out side the
culture vessel. In this stage, the microplants are transplanted into standard pasteurized
rooting or soil mix in a small pots or cells in more or less conventional manner. Once
the microplants are established in the rooting medium, the microplants should be
gradually exposed to a lower relative humidity and higher light intensity (Hartmann et
al., 1997).
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12
2.9 Organogenesis
Organogenesis involves the formation of organized structure like shoot and root
from pre-existing structures such as unorganized mass of cells known as callus. Plant
cultured through organogenesis can be achieved by two ways. The first is organogenesis
through continuous development of callus formation with de novo origin also known as
indirect pathway and second is from emergence of adventious organs such as lateral or
axillary buds directly from the explant which also known as direct pathway (Chawla,
2003). Indirect regeneration often results in somaclonal variation making the strategy
less desirable for large scale clonal multiplication. Therefore, direct regeneration
without a callus phase is a reliable method for clone production (Kataria et al., 2013).
2.10 Callus culture
Callus is an actively dividing non-organized mass of undifferentiated and
differentiated cells often developing either from injury or wounding. In in vitro culture,
callus is produced on explants from peripheral layers as a result of wounding and in
response of growth regulators either endogenous or supplied in the medium. Callus
provides an important tissue culture system because it can be subculted and mantained
more or less for an unlimited or unspecified period of time (Hartmann et al., 1990).
Explants from both mature and immature organs can be induced to form callus.
However, explants with an active cells such as young and juvenil cells are generally
good for callus initiation. Callus tissue form different plant species may be different in
structure and growth habit. The callus growth differs among plant species. It depends on
various factors such as the origin, position of the explant and the growth conditions
(Chawla, 2003).
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13
2.11 Component of in vitro culture media
The main components of most in vitro culture media are mineral salts and sugar
as carbon source and water. Other components may include organic supplements,
growth regulators and gelling agent.
2.11.1 Inorganic nutrients
The inorganic nutrients of a plant cell culture are those required by the normal
plant. The inorganic nutrients consist of macronutrients and also micronutrients.
Macronutrients are reqired in millimmole quantities and micronunutrients are required
in micromolar concentration (Thorpe, 1981). For most purposes a nutrient medium
should contain at least 25 and up to 60 mM inorganic nitrogen. There are various
ingredients of inorganic nutrient in in vitro culture for different stages of culture and
plant species, but the basic MS (Murashige & Skoog) (Table 1) and LS (Linsmaier &
Skoog) are most widely used (Kataria et al., 2013). The Murashige and Skoog medium
has been used widely for a range of culture types and species, particularly herbaceous
plants and tissue culture. This medium is rich in marcoelements, particulary nitrogen,
including nitrate (NO3) and ammonium ions (NH4) and vitamins (Hartmann and Kester,
1983).
Table 1 Murashige and Skoog medium composition.
Medium composition mg l-1
NH4NO3 1650.00
KNO3 1900.00
CaCl2H2O 440.00
MgSO4.7H2O 370.00
KH2PO4 170.00
KI 0.83
H3BO3 6.20
MnSO4.4H2O 22.30
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14
Table 1 (continue)
ZnSO4.7H2O 0.86
Na2MoO4.2H2O 0.25
CuSO4.5H2O 0.025
CoCl2.6H2O 0.025
FeSO4.5H2O 27.85
Na2EDTA.2H2O 37.25
Myo-inositol 100.00
Nicotinic acid 0.50
Pyridoxine-HCl 0.50
Thiamine-HCl 0.10
Glycine 2.00
2.11.2 Carbohydrate
During in vitro culture, carbohydrate plays an important role and act as an
energy source required for growth, maintenance and differentiation of xylem and
phloem element (Kataria et al., 2013). Carbohydrate is also needed for inducing primary
root and acts as an osmoticum and regulates the in vitro shoot proliferation. The most
commonly used carbohydrate source is sucrose, but other sugar like glucose, fructose,
dextrose, mannitol and sorbitol are also used. According to Lee and Huang (2013),
explants uptake sucrose from the medium and hydrolyze it into glucose. Cell wall-
bound invertase (CIN) and sucrose transporter (SUT) are the main routes for sucrose
absorption and transportation in higher plants.
2.11.3 Plant growth regulator
Plant growth regulators (PGRs) have an important role in cell growth and
differentiation. Both exogenous and endogenous levels of PGRs are highly related to
shoot organogenesis (Lee and Huang, 2013). Among various growth regulators, auxins
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15
(NAA, IAA, IBA and 2, 4-D), cytokinins (BAP, Kinetin, Zeatin), ABA, gibberellins
and ethylene are very important. In in vitro culture, the nature of organogenic
differentiation is determined by the relative concentration of auxins and cytokinins.
Higher cytokinins to auxins ratio promotes shoot formation, while higher auxins to
cytokinins ratio favours root differentiation (Kataria et al., 2013).
a. Auxin
Auxin is synthesised by plant and it owes its name due to its effect on elongation
of cells (auxesis). In in vitro culture, auxin plays an important role to induce cell
multiplication and rhizogenic activity (Auge et al., 1995). Indole-3-acetic acid (IAA) is
the primary auxin in plants. IAA is a weak acid (pKa = 4.75) that is synthesized in the
meristematic regions at the shoot apex and transported to the root tip in plants (Yong et
al., 2009). The strongest form of auxin is 2,4-D. According to Nikolaeva et. al. (2008),
2,4-D promotes active proliferation of the cells and steady growth of callus and
suspension cultures with the rate of callus formation depending on 2,4-D concentration
and cultivar characteristics.
b. Cytokinin
Cytokinin is one of the plant hormones that crucial for plant growth and
development and it is known to promote cell division and differentiation. Cytokinin can
also stimulate lateral bud growth and cause multiple shoot formation by breaking shoot
apical dominance (Jana et al., 2013). Different concentration of cytokinin used affects
the percentage of shoot regeneration, shoot numbers and shoot lenght (Bohidar et al.,
2008).
The compounds of cytokinin include N6-benzyladenine (BA), kinetin, N
6-
isopentenyl-adenine (2iP) and zeatin (Hartmann et al., 1997). According to Rui and
Vujovi (2008), cytokinins are classified into two major groups by their chemical
structures which are synthetic phenylurea derivates and adenine derivates which may
occur naturally. Zeatin and 2-isopentyladenine (2iP) are naturally occurring cytokinins,
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16
whereas, N6 benzyladenine (BA), 6-Benzylaminopurine (BAP), 6-furfuryl-aminopurine
(kinetin, Kin), and [1-Phenyl-3-(1,2,3,-thiadiazol-5-yl)] urea (thidiazuron, TDZ) are
synthetic cytokinins (Jana et al., 2013).
c. Coconut water
Coconut water is traditionally used as a growth supplement in plant in vitro
culture. This is because there are many phytohomones in coconut water such as auxin,
cytokinin and gibberelline. The effect of coconut water on micropropagation was first
discovered by Van Overbeek in 1942. The study focused the stimulatory effect of
coconut water on the embryo development and callus formation in Datura and
concluded that there are some complex substances in coconut water which are
sometimes required in addition to growth hormones for callus induction and
regeneration (Yong et al., 2009).
Some of the most significant and useful components in coconut water in
micropropagation are cytokinins, which are a class of phytohormones. Cytokinins can
be found in young green coconut fruit. Coconut water contains various cytokinins such
as kinetin and trans-zeatin. Kinetin is the first form of cytokinin discovered by human.
It is a degradation product of herring sperm DNA and it is found to be able to promote
cell division in plants. Kinetin is one of the cytokinins that has the effects on plant
developmental processes that could be influenced by cytokinins, such as leaf expansion
and seed germination. The other form of cytokinin found in coconut water is trans-
zeatin. Trans-zeatin is the first naturally-occurring cytokinin identified from a plant
source (Zea mays). Trans-zeatin is normally used to induce plantlet regeneration from
callus in plant tissue culture (Yong et al., 2009).
2.11.4 Agar
Agar is a powdered product obtained from certain species of red algae. Agar is
used as a solidifying agent and assumed to be an neutral support for callus growth and
multiplication (Kataria et al., 2013). There are two factors that affect agar usage. Those
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two factors are concentration and pH (Hartmann and Kester, 1983). Normally, 0.8
percent agar is used for culture medium. A higher concentration of solidifying agent in
the medium reduced vitrification, but in certain cases, an increase in amount of agar
causes adverse effect (Kataria et al., 2013). A pH of 5.0 to 6.0 is usually used. Acid or
very low pH can cause deteroriation of agar and unproper solidify of agar (Hartmann
and Kester, 1983).
2.12 Culture condition
Light is an important factor for the success of an in vitro experiment. The
intensity, quality and extent of daily exposure of light are the determining factors in the
in vitro culture. Cultures are usually maintained in a photoperiod of 16 hours of light
photon flux density of 60 mol m2 s1 and 8 hours of darkness. Temperature for in
vitro culture is about 26 C (Joshi et al., 2013). The pH of the medium is also an
important factor for tissue culture. The pH of the medium is usually adjusted to between
5 and 5.8 before autoclaving and extremes of pH are avoided. Light and temperature
will give effect in humidity of the culture vessel and pH of the medium plays a role in
osmotic potential of the medium. Mantaining humidity and osmotic potential is very
important due to its capability to affect the growth and development of plantlets in vitro
in different ways (Kataria et al., 2013).
2.13 Sugarcane micropropagation
Sugarcane is a perennial grass that normally reproduces vegetatively through
nodal buds and rhizomes but seed propagation also occurs. Commercial sugarcane is
propagated vegetatively by nodal cuttings and for this reason, micropropagation offers a
practical and fast method for mass production of clonal material (Bakker, 1999). In
vitro techniques for the mass propagation of healthy sugarcane plantlets can be achived
via organogenic and/or somatic embryogenic (direct and indirect) pathways (Synman et.
al., 2010).
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2.13.1 Organogenesis in sugarcane micropropagation
Organogenesis begins with dedifferentiation of parenchyma cells to produce
centers of meristematic activity called meristemoids. Dedifferentiation of parenchyma
cells form a clumps of cell which also known as a callus (Hartmann et. al., 1997).
Callus can be initiated from any sugarcane tissue such as root apical meristems, young
root, leaves, node tissue, immature florescence, pith and parenchyma (Patil et al., 2010).
But present investigation demonstrates that inner fresh leaves and shoot apical meristem
of sugarcane are highly amenable to in vitro callus culture (Ali et al. 2008). According
to Tiwari (2013) callus volume is found to be larger for the young leaf rather than the
apical meristem explants.
In sugarcane, callus is induced in the presence of auxin, either 2,4-D (2,4-
dichlorophenoxy-acetic acid) or picloram (Ali et al., 2012). But among the auxins
presented, 2, 4-D at 3.0 mg/l is more potent for callus induction than other auxin
hormones (Ali et al., 2008). Yellow callus is typically produced from 2,4-D containing
culture media. Beside its amenability, the in vitro sugarcane regenerated from callus is
susceptible to somaclonal variation for different traits like high yield, more sugar
recovery, disesase resistance, early maturity and drough tolerant (Ali et al., 2012).
2.13.2 Shoot regeneration in sugarcane in vitro culture
Shoot regeneration of sugarcane can be achived by both organogenesis and
somatic embryogenesis (Khan and Khatri, 2006). In most cases, shoot regeneration of
sugarcane are come from callus culture also known as organogenesis (Yadav and
Ahmad, 2013). According to Tarique et al. (2010), shoot regeneration from sugarcane
callus was first demonstrated by Heinz and Mee in 1969. High level of cytokinin and
low level of auxin is essential for regeneration of shoots in sugarcane leaf sheath callus
(Smiullah et al., 2013). Combination between BAP, kinetin and NAA mostly give the
best response in shoot regeneration of sugarcane (Yadav and Ahmad, 2013). Callus can
also be transferred to 9.3 mM kinetin and 22.3 mM -naphthaleneacetic acid (NAA) to
obtain rapid regeneration of shoot (Chengalrayan and Gallo-Meagher, 2001). However,
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19
thidiazuron aslo known as TDZ recently becomes superior plant growth regulator to
other cytokinins tested for shoot regeneration of sugarcane from callus. TDZ treatments
give faster shoot regeneration than the kinetin/NAA treatment (Gallo-Meagher et al.,
2000).
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III. MATERIALS AND METHODS
3.1 Date and place
The special problem reseach entitled Callus Induction and Tillering Capability
of 4 Sugarcane Cultivars (Saccharum officinarum L.) under In Vitro Culture was
conducted on November 12th
2013 at Tissue Culture Laboratory, Center for Agricultural
Biotechnology, Kasetsart University Kamphaeng Saen Campus.
3.2 Materials
Healthy leaves (innermost spindle leaf) of 4 field-grown sugarcane cultivars,
K92-80, KK3, LK 95-127 and K 93-219 were used as special problem material. MS
(Murashige and Skoog) medium was used with additional of 2,4-D and coconut water
as plant growth regulator. Sucrose was given as carbohydrate source. All of the
ingradients were solidified using agar powder.
3.3 Methods
3.3.1 Explant collection
The cane top containing young leaves of 4 field-grown sugarcane cultivars,
K92-80, KK3, LK 95-127 and K 93-219 were cut approximately 2030 cm below the
uppermost internode of sugarcane.
3.3.2 Surface sterilization
The outer whorls of cane tops were removed and remaining 1-2 centimeters in
diamater of immature leaf segments. The explants were surface sterilized with 20% and
15% of comercial bleach for 10 minutes each and subsequently rinsed with steriled
water 3 times for 5 minutes each.
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3.3.3 Callus induction
Surface sterilized immature leaf segments were used for callus induction. The
outer two or three whorls of leaves were aseptically cut and removed remaining
innermost whorls containing more or less 2 mm in diameter of immature leaf. Immature
leaves segments were cut into 0.5 cm-long in aseptic condition. Each cultivar has 10
replications which was used in callus induction stage. The callus induction medium was
MS (Murashige and Skoog) medium supplemented with 3.0 mg/L of 2,4-D, 2% of
sucrose, 10% (V/V) of coconut water and 0.7 % of agar. The pH of the medium was
adjusted to 5.7 and autoclaved at 121o C for 15 minutes. Callus induction was initiated
under complete darkness at 25o C 1 for 60 days. The calli were subcultured to the
fresh medium every 30 days. The data collection were callus physical properties and
callus induction percentage. They were done every 3 weeks for 2 months from
November 12th
2013 to January 10th
2014.
3.3.4 Shoot regeneration
The healthy and uncontamined calli were transferred onto plant regeneration
medium. There were 11 replications in each cultivar used in this stage. Shoot
regeneration medium was MS (Murashige and Skoog) containing 10% (V/V) of coconut
water for plant growth regulator. The MS medium also suplemented with 2% of sucrose
as carbon source and 0.7% agar as solidifying agent. Explants were cultured under
white florescent light with intensity of 55 M.m-2.s-1 and 16 hours photoperiod at 25C
1. The explants were subcultured to the fresh medium every 30 days. The data
collected in shoot regeneration medium were shoot physical properties, number of
callus producing shoot ( 2 cm) and average number of shoots produced ( 1 cm). All
the data were collected every 1 week for 2 months from January 11th
to March 12th
2014.
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3.3.5 Tillering capability
The healthy and uncontamined shoots that had 2-4 cm in height were seperated
into a single shoot. Each shoot was transferred to tillering induction medium. There
were 11 replications in each cultivar used in this stage. The medium used was MS
(Murashige and Skoog) containing 10% of coconut water for plant growth regulator.
The MS medium also suplemented with 2% of sucrose as carbon source and 0.7% agar
as solidifying agent. Explants were cultured under white florescent light with intensity
of 55 M.m-2.s-1 and 16 hours photoperiod at 25C 1. The explants were subcultured
to the fresh medium every 30 days. In tillering capability stage, data collected were
tiller physical properties, number explant producing tiller ( 1cm) and average number
of tillers produced ( 1 cm). They were recorded every 2 weeks for 2 months from
March 12th
to Mei 12th
2014.
3.3.6 Statistical analysis
A completely randomized design (CRD) was used with 4 different sugarcane
cultivars. The data of callus induction, shoot regeneration and tillering capability were
collected and analyzed using ANOVA statistical analysis to find out the significant
effects of the source variables. Duncans multiple range test (DMRT) was futher applied
to the data to test the significant differences between the treatment means (p 0.05).
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IV. RESULT
4.1 Callus induction
4.1.1 Physical properties of callus
In callus induction stage, 6 cultivars were used which young inner spindle leaves
were used as a source of explant. Those cultivars were LK92-11, K88-92, K92-80,
LK95-127, K93-219 and KK3. Each cultivar was done with 10 replications. During the
callus induction process which was conducted from November 12th
2013 to January 10th
2014, there were several data were recorded (Table 2). Callus was initiated 20 days after
culturing on MS medium containing 3.0 mg/L of 2,4-D and 10% (V/V) of coconut
water.
The first emerged callus was noticed in the 3rd
week of culture. It was also
showed that after 2 months of callus induction process, from 6 cultivars used LK95-127
and K93-219 had the best physical properties showed by vigorous growth with light
yellow in color (Figure 2) as well as relatively 2 cm in length. KK3 and K92-80 showed
vigorous callus properties with less compact callus properties (Figure 2), while LK92-
11 and K88-92 showed the worst result with black viable callus appearence (Figure 2)
as well as approximately 1.5 cm and 2 cm in length, respectively.
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Table 2 Callus physical properties of 6 sugarcane cultivars in callus induction stage for
2 months.
Date Cultivar Callus
colour
Size of
callus
(The best
sample)
Note
12/11/2013 LK92-11 - - Intial callus induction
K88-92 - - Intial callus induction
K92-80 - - Intial callus induction
KK3 - - Intial callus induction
LK95-127 - - Intial callus induction
K93-219 - - Intial callus induction
3/12/2013 LK92-11 Brown-green 0.5 cm Small callus
K88-92 Black-green 0.0 cm Small callus
K92-80 Light yellow 0.5 cm Small callus
KK3 Light yellow 1.0 cm Small callus
LK95-127 Light yellow 1.0 cm Small callus
K93-219 Light yellow 1.0 cm Small callus
24/12/2013 LK92-11 Brown-green 1.0 cm Callus sub-culture
K88-92 Black-green 0.5 cm Callus sub-culture
K92-80 Light yellow 1.0 cm Callus sub-culture
KK3 Light yellow 1.5 cm Callus sub-culture
LK95-127 Light yellow 1.5 cm Callus sub-culture
K93-219 Light yellow 1.5 cm Callus sub-culture
10/01/2014 LK92-11 Brown-green 1.5 cm Dark and viable callus
K88-92 Black-green 1.0 cm Dark and viable callus
K92-80 Light yellow 1.5 cm Less compact callus
KK3 Light yellow 2.0 cm Less compact callus
LK95-127 Light yellow 2.0 cm Compact callus
K93-219 Light yellow 2.0 cm Compact callus
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Figure 2 Callus induction of 6 sugarcane cultivars from innermost spindle leaf explant
after 2 months inoculation.
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4.1.2 Percentage of explant producing callus
During 2 months of observation in callus induction stage, the number of explants
producing callus and the percentage of explant producing callus were recorded (Table 3,
Figure 3 and 4). After 2 months of inoculation, cultivar K92-80, KK3 and K93-219 had
the best callus induction percentage in which 100% of explants produced callus,
followed by cultivar LK95-127 which showed 95% explants produced callus. Cultivar
K88-92 and LK92-11 on the other hand had the smallest of callus induction percentage
which were 30% and 10% respectively. Those two last cultivars started producing callus
in the 7th
weeks after inoculation.
Table 3 Number and percentage of explant inducing callus of 6 sugarcane cultivars in
callus induction stage in 2 months period.
Date Cultivar No. of
explants
No. of explants
producing callus
% of callus
induction
12/11/2013 LK92-11 10 - -
K88-92 10 - -
K92-80 10 - -
KK3 10 - -
LK95-127 10 - -
K93-219 10 - -
3/12/2013 LK92-11 10 0 0
K88-92 10 0 0
K92-80 10 10 100
KK3 10 10 100
LK95-127 10 9 95
K93-219 10 10 100
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Table 3 (continue)
Date Cultivar No. of
explants
No. of explants
producing callus
% of callus
induction
24/12/2013 LK92-11 10 0 0
K88-92 10 2 20
K92-80 10 10 100
KK3 10 10 100
LK95-127 10 9 90
K93-219 10 10 100
10/01/2014 LK92-11 10 1 10
K88-92 10 3 30
K92-80 10 10 100
KK3 10 10 100
LK95-127 10 9 90
K93-219 10 10 100
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Figure 3 Number of explants inducing callus of 6 sugarcane cultivars after 2 months
inoculation.
Figure 4 Percentage of explant inducing callus of 6 sugarcane cultivars after 2 months
inoculation.
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29
4.2 Shoot regeneration
4.2.1 Physical properties of shoot
The calli were tranferred to shoot regeneration medium. The medium consisted
of MS inorganic and organic salts + 10% (V/V) CW + 20 g/L sucrose + 7 g/L agar. The
cultures were kept in 16 hr photoperiod. Cultivar used for shoot regeneration were K92-
80, KK3, LK95-127 and K93-129 which had 11 replications each. Shoot regeneration
was performed on January 11th
to March 12th
2014. The data of physical properties
were recorded (Table 4).
There was no distinct difference of physical properties of shoot among 4
cultivars used except for the number of shoot produced. Multiple green spots initiated
after 1 week of transferring to regeneration medium. The callus differentiated into
multiple small shoots 2 weeks after transferring (Figure 5). Countable multiple shoots
appeared at the 7th
week (Figure 7). After 8 weeks of shoot regeneration, the shoots
developed up to 7 cm in height relatively. Cultivar LK95-127 and K93-129 had the
most vigorous growth with many healthy shoots produced and average of 7 cm in height
compared with 2 other cultivars K92-80 and KK3 which only had average of 6 cm in
height (Figure 8).
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Table 4 Shoot physical properties of 4 sugarcane cultivars in shoot regeneration stage
in 2 months period.
Date Cultivar Average hight
of shoot (cm)
Note
11/01/2014 K92-80 - First time shoot regeneration
KK3 - First time shoot regeneration
LK95-127 - First time shoot regeneration
K93-219 - First time shoot regeneration
28/01/2014 K92-80 Undetermined Green spot appeared
KK3 Undetermined Green spot appeared
LK95-127 Undetermined Green spot appeared
K93-219 Undetermined Green spot appeared
03/01/2014 K92-80 0.5 cm Multiple small shoot
KK3 0.5 cm Multiple small shoot
LK95-127 1.0 cm Multiple small shoot
K93-219 1.0 cm Multiple small shoot
12/02/2014 K92-80 1.0 cm Subculture
KK3 1.0 cm Subculture
LK95-127 2.0 cm Subculture
K93-219 2.0 cm Subculture
19/022014 K92-80 2.0 cm Microshoot appeared
KK3 2.0 cm Microshoot appeared
LK95-127 4.0 cm Microshoot appeared
K93-219 3.0 cm Microshoot appeared
26/02/2014 K92-80 3.0 cm Shoot appeared
KK3 3.0 cm Shoot appeared
LK95-127 4.0 cm Shoot appeared
K93-219 3.0 cm Shoot appeared
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Table 4 (continue)
Date Cultivar Average hight
of shoot (cm)
Note
05/03/2014 K92-80 5.0 cm All the explants grew the big shoot
KK3 6.0 cm All the explants grew the big shoot
LK 95-127 6.0 cm All the explants grew the big shoot
K93-219 6.0 cm All the explants grew the big shoot
12/03/2014 K92-80 6.0 cm Last day shoot regeneration
KK3 6.0 cm Last day shoot regeneration
LK95-127 7.0 cm Last day shoot regeneration
K93-219 7.0 cm Last day shoot regeneration
Figure 5 Initiation of shoot growth from callus explant of 4 sugarcane cultivars in
shoot regeneration stage after 3 weeks of culturing.
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32
Figure 6 Initiation of multiple shoot growth of 4 sugarcane cultivars in shoot
regeneration stage after 5 weeks of culturing
Figure 7 Expanding and differentiated shoot of 4 sugarcane cultivars in shoot
regeneration stage after 7 weeks of culturing.
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33
Figure 8 Long and expanding green shoot of 4 sugarcane cultivars in shoot
regeneration stage after 9 weeks of culturing.
4.2.2 Percentage of callus producing shoot
After 2 months of incubation from February 19th
to March 12th
2014, the data
showed number of explants produced shoot (Table 5 and Figure 9) and shoot
regeneration percentage (Table 6 and Figure 10). Totally 11 calli from each cultivar
were used in shoot regeneration process. When the shoot height of > 2 cm was
accounted, among 4 cultivar used, LK95-127 showed the best shoot regeneration
percentage of 72.72% or 8 out of 11 calli were successfully regenerated, following by
KK3 and K93-219 which 63.63% or 7 calli regenerated shoots. The lowest shoot
regeneration percentage was found in K92-80, in which only 54.54% or 6 out of 11
calli were able to regenerate shoots.
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Table 5 Number of callus producing shoot of 4 sugarcane cultivars in shoot
regeneration stage taken every 1 week.
Cultivar Number of callus produced shoot ( 2 cm)*
Week 1 Week 2 Week 3 Week 4
K92-80 3 5 6 6
KK3 1 4 7 7
LK 95-127 4 8 8 8
K 93-219 2 6 7 7
* 11 calli from each cultivar were transferred to regeneration medium.
Table 6 Percentage of callus producing shoot of 4 sugarcane cultivars in shoot
regeneration stage taken every 1 week.
Cultivar Percentage of callus regenerated shoot
Week 1 Week 2 Week 3 Week 4
K92-80 27.27 % 45.45 % 54.54 % 54.54 %
KK3 9.09 % 36.36 % 63.63% 63.63 %
LK 95-127 36.36 % 72.72 % 72.72 % 72.72 %
K 93-219 18.18 % 54.54 % 63.63 % 63.63 %
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Figure 9 Number of callus producing shoot of 4 sugarcane cultivars in shoot
regeneration stage after 2 months of culturing.
Figure 10 Percentage of callus producing shoot of 4 sugarcane cultivars in shoot
regeneration stage after 2 month of culturing.
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4.2.3 Average number of shoots
The average number of shoots in each cultivar were taken every one week from
February 19th
to March 12th
2014. Shoot which had height of > 1 cm was accounted.
The data were collected and analyzed using ANOVA statistical analysis. It displayed
that since the first week to the forth week of observation, LK95-127 had the highest
average number of shoots among other cultivars, showing significantly different to
K92-80 and KK3, but was not significantly different to K93-129.
In the first week of observation (Figure 12 and Table 7), it observed that the
average number of shoots in cultivar LK95-127 was not significantly different to
cultivar K93-219, but was significantly different to K92-80 and KK3. The average
number of shoots produced by LK95-127 and K93-219 in the first week were 4 and
3.09, respectively. On the other hand, the average shoot numbers of KK3 and K92-80
showed no significant difference between them, producing 2.45 and 2.18 shoots in
average, respectively.
There was no changed in the result during the second and third week of
observation (Figure 12 and Table 7). In the second week, between cultivar LK 95-127
and K93-219 still had not significantly different which producing 5.00 and 4.30 on the
average number of shoots, respectively although K93-219 did show significantly
different compared to K92-80 (3.09 shoots) and KK3 (2.90 shoots). In the third week,
LK95-127 still had the highest average shoot number of 5.09, followed by K93-219,
K92-80, and KK3 which had the average number of tillers 4.45, 3.18 and 3.00,
respectively.
In the forth week (Figure 12 and Table 7), LK95-127 still showed significant
different among other cultivars. LK95-127 gave the highest average number of shoots
produced of 5.27 and followed by K93-219 which was 4.63. On the other hand, KK3
and K92-80 had 3.27 and 3.18 on average number of shoots produced respectively with
no significantly different between them. Based on the graphic given (Figure 11), it was
also noticed that cultivar K92-80 was able to surpass the average number of shoots
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produced by cultivar KK3. In the first week of observation, cultivar K92-80 showed
2.18 on the average number of shoots produced, lower than KK3 which showed 2.45.
But the conditon was slightly changed in the forth week, where the avarage shoot
number of K92-80 was 3.27, higher than cultivar KK3 which was only 3.18.
Table 7 Average number of shoots of 4 sugarcane cultivars in shoot regeneration stage
taken every 1 week.
Cultivar Average number of shoot ( 1 cm)
Week 1 Week 2 Week 3 Week 4
K92-80 2.18 b 3.09 b 3.18 b 3.27 b
KK3 2.45 b 2.90 b 3.00 b 3.18 b
LK95-127 4.00 a 5.00 a 5.09 a 5.27 a
K93-219 3.09 ab 4.30 ab 4.45 ab 4.63 ab
P. Value 0.0092 0.0340 0.0267 0.0148
Means in the same column followed by the same letter are not significantly different (p
0.05) by DMRT.
Figure 11 Weekly graphic of average number of shoots produced of 4 sugarcane
cultivars in shoot regeneration stage.
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Figure 12 Average number of shoots produced of 4 sugarcane cultivars in shoot
regeneration stage taken every 1 week.
4.3 Tillering capability
4.3.1 Physical properties of tiller
Four cultivars were used in the tillering capability stage which were K92-80,
KK3, LK95-127 and K93-219. The multiple shoots that higher than 2 cm of each
cultivar were used. The multiple shoots were separated into a single shoot and than cut
into 1 cm length (Figure: 13) to conducted tillering capability experiment. The cut
shoots than were tranfered into a new MS medium containing 10% (V/V) CW + 20 g/L
sucrose + 7 g/L agar. There were 11 replications in each treatment (cultivar). The data
collected for 2 months observation, from March 12th
to May 12th
2014 (Table 8).
The data showed that in the first week of observation, the explants performed
shoot elongation had not produced tiller yet (Figure 14). The shoot started producing
tiller in the third week after transferring which then was continued with subculture in
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the forth week. In general physical appearance, it noticed that KK3 had the highest
tillering capability compared to the other cultivars (Figure 15).
Table 8 Tiller physical properties of 4 sugarcane cultivars in tillering capability stage
taken every 1 week for 2 months.
Date Cultivar Note
12/03/2014 K92-80 Used 1 cm shoot
KK3 Used 1 cm shoot
LK95-127 Used 1 cm shoot
K93-219 Used 1 cm shoot
15/03/2014 K 92-80 Shoot elongated but no tiller
KK3 Shoot elongated but no tiller
LK95-127 Shoot elongated but no tiller
K93-219 Shoot elongated but no tiller
3/04/2014 K 92-80 Initial tillering
KK3 Initial tillering
LK95-127 Initial tillering
K93-219 Initial tillering
10/04/2014 K 92-80 Subculture
KK3 Subculture
LK95-127 Subculture
K93-219 Subculture
17/04/2014 K92-80 Normal growth
KK3 The best tillering capability
LK95-127 Normal growth
K93-219 Normal growth
01/05/2014 K92-80 Normal Growth
KK3 The best tillering capability
LK95-127 Slowest growth
K93-219 Normal growth
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Table 8 (continue)
Date Cultivar Note
12/05/2014 K92-80 Normal Growth
KK3 the best tillering capability
LK95-127 Slowest growth
K93-219 Normal growth
Figure 13 Explant (cut shoot) used for tiller production.
Figure 14 Initial growth and elongation of shoot in the 1st week.
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Figure 15 Tiller growth in the last week of observation.
4.3.2 Percentage of explants producing tiller
In tillering stage, the number of explants produced tiller (Table 9 and Figure 16)
as well as the percentage of explants produced tillers (Table 10 and Figure 17) were
recorded. Shoot which had height of > 1 cm was accounted. Four different cultivars
were used for comparing tillering capability. Those cultivars were K92-80, KK3, LK95-
127 and K93-219. The data of number of explants produced tillers were taken in the 3rd
week after subculturing and recorded every two weeks from April 3rd
to May 12th
, 2014.
In the 3rd
week, the result displayed that there were 8 out of 11 explants or
72.72% produced tillers from KK3 and K93-219 cultivars, respectively, followed by
K92-80 and LK95-127 which showed 5 (45.45%) and 4 (36.36%) of the explants
produced tiller, respectively.
The increment of number of explants produced tillers was noticed in the 5th
week of observation. Cultivar KK3 and K93-219 were considered to be the cultivars
that yielded the highest number of explants produced tillers. Both of them showed 10
out of 11 explants (90.90%) produced tillers at the 5th
week. On the other hand, K92-80
showed 8 explants (72.72%) produced tillers, followed by LK95-127 which showed 6
explants (54.54%) produced tillers.
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KK3 and K93-219 showed no different in number of explants produced tillers in
the 7th
week. Both of them had 10 explants (90.90%) successfully produced tiller. On
the other hand, cultivar K92-80 and LK95-127 had the same number of tillers produced
as in the 5th
week of observation. Both of those cultivars showed 8 explants (72.72%)
and 6 explants (54.54%) produced tillers, respectively. However, in the last week of
observasion, ten of the explants (90.90%) from all cultivars were successfully produced
tillers.
Table 9 Number of explant producing tillers of 4 sugarcane cultivars in tillering
capability stage taken every 2 week.
Cultivar Number of explant produced tiller ( 1 cm)
Week 3 Week 5 Week 7 Week 9
K92-80 5 8 8 10
KK3 8 10 10 10
LK95-127 4 6 6 10
K93-219 8 10 10 10
Table 10 Percentage of explants producing tillers of 4 sugarcane cultivars in tillering
capability stage taken every 2 week.
Cultivar Percantage of explant produced tiller
Week 3 Week 5 Week 7 Week 9
K92-80 45.45% 72.72% 72.72% 90.90%
KK3 72.72% 90.90% 90.90% 90.90%
LK95-127 36.36% 54.54% 54.54% 90.90%
K93-219 72.72% 90.90% 90.90% 90.90%
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Figure 16 Number of explants producing tillers of 4 sugarcane cultivars in tillering
capability stage taken every 2 weeks.
Figure 17 Percentage of explants producing tillers of 4 sugarcane cultivars in tillering
capability stage taken every 2 weeks.
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4.3.3 Average number of tillers
The average number of tillers in each cultivar was also recorded in this study.
The data of average number of tillers in each cultivar were taken every two weeks from
April 3rd
to May 12th
, 2014. Shoot which had height of > 1 cm was accounted. Cultivar
used were K92-80, KK3, LK95-127, and K93-219. The data were analyzed using
ANOVA statistical analysis (Table 11 and Figure 20). It observed that cultivar KK3 has
the most abundance average number of tillers regenerated among other cultivars as well
as the most progressive growth since the first week of observation (Figure 18 and 19).
Observation in the 3rd
week demonstrated that there was not significantly
different between cultivar KK3 and LK93-219. Among 4 cultivars used, cultivar KK3
had the best average number of tillers, producing 2.54 tillers on average, followed by
K93-219 which had 2.18 on the average number of tillers produced. In case of K92-80
and K95-127, the number of average tillers produced were lower, each of them only
produced 1.54 and 1.36 respectively.
There was not significantly different on the average number of tillers between 2
cultivars, KK3 and K92-80 in the 5th
week of observation, although KK3 showed
significantly different compared to LK 95-127 and K93-219. The average number of
tillers produced by KK3 was 3.45, followed by K93-219 which had 2.72. The other
cultivars, K92-80 and LK95-127 displayed the lower average number of tillers, in
which each of them produced 2.27 and 1.90, respectively.
There was significantly different on the average number of tillers in the 7th
week
of observation. It was found that the average number of tillers of KK3 was higest (4.09)
and significantly different among the others, while the other cultivars did not show any
significantly different result. The average number of tillers of K93-219, K92-80 and
LK-95-127 were 2.81, 2.54 and 2.18 tillers, repectively.
At the 9th
week of observation, The highest average number of tillers produced
was obtained in cultivar KK3 with significantly different among the other cultivars
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tasted. The average number of tillers produced by KK3 cultivar were 4.54, while the
other cultivars, K93-219, K92-80 and LK95-127 had the average number of tillers 3.00,
2.90 and 2.72, respectively.
Table 11 Average number of tillers of 4 sugarcane cultivars in tillering capabilty stage
taken every 2 week.
Cultivar Average number of tillers ( 1 cm)
Week 3 Week 5 Week 7 Week 9
K92-80 1.54 bc 2.27 ab 2.54 b 2.90 b
KK3 2.54 a 3.45 a 4.09 a 4.54 a
LK95-127 1.36 c 1.90 b 2.18 b 2.72 b
K93-219 2.18 ab 2.72 b 2.81 b 3.00 b
P. Value 0.0082 0.0108 0.0184 0.317
Means in the same column followed by the same letter are not significantly different (p
0.05) by DMRT.
Figure 18 Weekly graphic of average number of tillers produced of 4 sugarcane
cultivars in tillering capability stage.
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Figure 19 Number of tillers produced in each explant of 4 sugarcane cultivars in
tillering capability stage taken 9 weeks after explanting.
Figure 20 Average number of tillers produced of 4 sugarcane cultivars in tillering
capability stage.
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V. DISCUSSION
5.1 Callus induction
5.1.1 Effects of leaf properties in callus growth
Inner spindle of young leaf was used as a source of explant in callus induction
stage. Young leaf segment becomes the best source of explant used in vitro culture,
specially for monocotyledonous plant. Various sugarcane genotypes have been cultured
in vitro using immature leaves establishing a good callus induction (Gallo-Meagher et
al., 2000). According to Lakshmanan et al. (2006) in monocotyledonous plant,
especially poaceae the young leaf is the only organogenically responsive explant. Ali et
al. (2008) also mentioned that young and newly formed leaf has the highest potential for
callus formation and proliferation as well as resulting better callus formation. Newly
formed whorl of young leaf exhibites maximum morphogenic potential due to their
greater meristematic nature and oftenly contains high level of cytokinins to support cell
proliferation. Young meristematic tissue also has an advantage compare to old tissue,
such as from pith. Smiullah et al. (2013) mentioned that leaf explant performs better
and statistically different on the average callus growth compare to the pith explant. This
is because pith or old tissue excretes phenolic compounds, which turns the whole pith
brown and hinders proliferation.
5.1.2 Effects of 2,4-D in callus growth
Medium used in callus induction stage was MS medium containing 3.0 mg/L of
2,4-D, 2% of sucrose, and 10% of coconut water. 2,4-D is one of the artificial auxin that
promotes active proliferation of the cells (Nikolaeva et al., 2008). Among different
concentration proposed, 3.0 mg/L has become standard usage of 2,4-D in callus
induction stage in sugarcane tissue culture (Yadav and Ahmad, 2013). This is also
supported by Smaiullah et al. (2013), Bisht et al. (2011), Ali et al. (2008), and Gopitha
et al. (2010) which used 3.0 mg/L of 2,4-D to induce optimum callus growth. Although
exogenous auxin such as 2,4-D has an essential role for cell differentiation in callus
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induction stage, but 2,4-D also causes somaclonal variation. As it is mentioned by Roy
et al. (2010), the cause of the morphological, agronomical and biochemical variations
may be linked with the use of synthetic auxin (2,4-D) and inherent chromosomal
instability in callus culture. It is also supported by Dolozel and Novak (1984) who
indicated that somaclonal variation caused by 2,4-D is appeared in Trandescantia
stamen hair system which increased the frequency of blue to pink mutation.
5.1.3 The roles of sucrose as a source of carbohydrate in callus growth
In callus induction stage, the amount of sucrose used as a source of carbohydrate
was 2%. Using sucrose as a source of carbohydrate has been performed in many tissue
culture studies. This is due to its efficiency to uptake across the plasma membrane
(Swamy et al., 2010) and its properties as the main sugar product of photosynthesis in
most plants (Scott, 2008). Another reason of sucrose selected as a source of
carbohydrate in in vitro culture is because of its physical and chemical properties which
is highly soluble in water. Sucrose also has little apparent effect on plant metabolic
processes, even at high concentrations. It is stable and its metabo