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

ii

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

iii

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

iv

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

v

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.

vi

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

vii

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

viii

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

ix

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

x

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

xi

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

xii

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

xiii

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

1

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

2

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

3

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.

4

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.

5

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).

6

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

7

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

8

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.

9

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,

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).

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).

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).

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

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

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,

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

17

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).

18

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,

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).

20

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.

21

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.

22

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).

23

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.

24

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

25

Figure 2 Callus induction of 6 sugarcane cultivars from innermost spindle leaf explant

after 2 months inoculation.

26

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

27

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

28

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.

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).

30

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

31

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.

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.

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.

34

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 %

35

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.

36

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

37

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.

38

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

39

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

40

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.

41

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.

42

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%

43

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.

44

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

45

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.

46

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.

47

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

48

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