Sugarcane Tissue Culture: A Method for Rapid Multiplication

Introduction
3
• Sugarcane, Saccharum spp. (2n=40 to 2n=128) belongs to family Poaceae,
(Graminae). It is the main sugar producing crop that contributes more than
75% to the total sugar pool at the global level. Sugarcane is cultivated as a
commercial crop in nearly 60 countries spread over the world. India is the
largest producer of sugar and second largest producer of sugarcane in the
world.
• Quality seed plays an immense value to keep phase in productivity and
sugar recovery. Due to limitation of time taking process to mass
multiplication of elite popular varieties there is an importance of
substitutes methodology to fasten the multiplication rate and to evolve
desirable clones to the changed climatic condition.
• Tissue culture provide an alternative method for the crop improvement.
Plant regeneration from tissue culture of sugarcane has been successfully
applied to breeding programs for rapid screening of clones for disease
resistance, salt tolerance, drought tolerance, herbicide resistance and early
maturity and high sugar

Uses and importance of sugarcane
• Sugarcane is mainly an industrial crop as the cane is supplied to sugar
industries.
• Sugarcane's products like sugar and fermented products are very important
in making and preserving various kind of medicines like syrups, liquids;
capsules etc.
• Sugarcane provides a juice, which is used for making white sugar, and
jaggery (gur) and many by-products like bagasse and molasses.
• Green tops of cane are a good source of fodder for cattle. Its remains are
good manure in alkaline and saline soils.
4

Table 1 State wise area production, productivity and sugar recovery
of sugarcane during 2013-14
States Area
(‘000 ha)
Production
(‘000 tonnes)
Productivity
(tonnes / ha)
Sugar
recovery (%)
Andhra Pradesh 195 676 72.0 9.74
Bihar 298 591 50.0 8.97
Gujarat 182 1175 71.0 11.09
Haryana 118 540 69.0 9.44
Karnataka 476 4177 88.0 10.95
Madhya Pradesh 85 325 57.0 9.91
Maharashtra 9401 7712 80.0 11.40
Tamil Nadu 285 1413 90.0 8.97
Uttar Pradesh 2513 6495 53.0 9.60
INDIA
5341 345600 64.7 10.23
Anonymous (2015) 5

SPECIES OF SUGARCANE
Species Classification
Sugar
content
Chromosom
e number
Origin
S. spontaneum Wild species Nil 2n= 40-128 Southern Asia
S. robustum Wild species Nil 2n= 60-80 New Guinea
S. officinarum Noble canes High 2n= 80
New Guinea, derived from
S. Robustum
S. barberi
Ancient
hybrid
Low 2n= 111-120
North India, derived from
S spontaneum x S. officinarum
S. sinense
Ancient
hybrid
Low 2n= 81-124
India and China, derived
from
S spontaneum x S. officinarum
S. edule Wild species
Compact
inflorescence
eaten as a
vegetable
2n= 60-80
with
Aneuploid
forms
Malanesia and Indonesia,
derived from S. officinarum
6

Nobilization of sugarcane
S. officinarum
S. spontaneum
Tropical Cultivated species thick,
soft, juicy, high sucrose but
susceptible to biotic and abiotic
stresses .
Wild species having wide
adaptability, profuse tillering and
resistant to biotic and abiotic
stresses but thin and low sucrose
content.
7

PLANT TISSUE CULTURE
•The culture of plant seeds, organs, tissues, cells,
or protoplasts on nutrient media under sterile
and aseptic/ in-vitro conditions.
8

History of plant tissue culture
1902 The idea of Totipotency of the plant cell given by Harberlandt
1904 Hannig cultured embryos from several cruciferous species
1926 Went discovered first plant growth hormone – Indole acetic acid
1934 White introduced vitamin B as growth supplement in tissue culture
media for tomato root tip
1941 Overbeek was first to add coconut milk for cell division in Datura
1955 Skoog and Miller discovered kinetin as cell division hormone
1957 Skoog and Miller gave concept of hormonal control (auxin: cytokinin)
of organ formation
1962 Murashige and Skoog developed MS medium with higher salt
concentration
Con…
9

1964 Guha and Maheshwari produced first haploid plants from pollen
grains of Datura (Anther culture)
1966 Steward demonstrated totipotency by regenerating carrot plants from
single cells of tomato
1972 Carlson produced first interspecific hybrid of Nicotiana tabacum by
protoplast fusion
1981 Larkin and Scowcroft introduced the term somaclonal variation
10

Achievements of India in plant tissue culture
11
Maheshwari and
Rangaswamy
1958
somatic embryos in vitro
from the nucleus of citrus
ovule
1964
Delhi University
Guha and
Maheshwari
first haploid plants
triploid plants1970

BASIS FOR PLANT TISSUE CULTURE
• Two hormones affect plant differentiation:
• Auxin: stimulates root development
• Cytokinin: stimulates shoot development
• Generally, the ratio of these two hormones can determine plant development:
•  Auxin ↓ cytokinin = root development & initiation
•  Cytokinin ↓auxin = shoot multiplication & development
• Auxin = cytokinin = callus development
12

TYPES OF TISSUE CULTURE
1) Meristem culture
2) Embryo culture
3) Anther and pollen culture
4) Tissue, cell, callus culture
5) Protoplast culture and somatic hybridization
6) ovary and ovule culture
13

TISSUE CULTURE APPLICATIONS
 Micropropagation
 Genetic engineering
 Plant protection
 Plant improvement
 Plant conservation
14

Factors affecting plant tissue culture
Nutrient/Growth media
 Minerals, vitamins, carbon source, hormones
Environmental factors
 Light, temperature, photoperiod, Humitity
Ex-plant source
 Usually, younger, less differentiated ex-plant is better for
tissue culture
Genetics
 Different species show differences in amenability to tissue
culture
 In many cases, different genotypes within a species show
variable response to hormonal substances.
15

Advantages of plant tissue culture Technology
• Plant tissue culture research is multi-dimensional and it has
direct commercial applications as well as value in basic
research in cell biology, genetics and biochemistry
• An alternating tool of breeding by selecting and testing for
useful variants
• Spontaneously arising changes (somaclonal variation) in tissue
culture have been recognized as rich source of variability.
• Mass multiplication of selected genotype to cover large area in a
short duration of time.
16

Basic Requirements of Plant tissue culture laboratory
• Media preparation room
• Inoculation Room or Transfer room
• Culture Room or incubation room or growth room
• Washing Room
• Green House
17

Types of Micropropagation techniques in sugarcane
1. Shoot tip culture
2. Meristem culture
3. Callus culture
18

What is shoot tip culture
•The culture of epical bud portion of shoot of a plant
in nutrient media under controlled condition or in
lab called shoot tip culture.
•Mostly the shoot tip culture used for obtain
disease free plant without genetically changes.
20

Fig 1 Stage of shoot tip culture
Sugarcane epical
bud used as
Explant
shoot Initiation
in MS
Shoot Elongation
Initiation of
multiple shooting
shoot
multiplication
Establishment
of shoot tip
RootingHardening
21

Application of shoot-tip culture
22
•Virus elimination
•Storage of genetic resources
•Use in plant breeding
•Quarantine

Table 2 Composition of nutrient media for shoot tip culture
MS media
(Major & Minor elements)
+
SAP Quantity /litre
medium
Multiplication Rooting
Meso inositol 100 mg 100 mg 50 mg
Glycine 2.0 mg 2.0 mg 1.0 mg
GA3 0.5 mg 0.5 mg -
6 BAP 0.5 mg - -
IBA 0.75 mg - 1.50
Coconut water 100 ml 100 ml -
Sucrose 20 gm 20 gm 20 mg
6-BAP 5 mg - -
Kinetin - 1.07 mg -
Nicotinic Acid - 0.5 mg 0.25 mg
Naphthalene Acetic Acid
(NAA)
- 0.5 mg 0.5 mg
Pyrodoxine Hcl - 0.5 mg 0.25 mg
Thiamine Hcl - 0.1 mg 0.05 mg
23Source: plant cell and tissue culture (S. Narayanswamy)

Table 3 Effect of different hormones on shoot formation from apical meristem in
sugarcane variety CP 77-400
Media Concentration
(mg/l)
No. of
explants
cultured
Days for shoot
formation
Frequency of
shoot formation
(%)
Number of
shoots/explant
MS + BAP 1.0 10 10.8 ± 0.8342c
90 1.8 ± 0.1897c
1.5 10 10.3 ± 0.6332c
100 1.8 ± 0.2366c
2.0 10 11.1 ± 0.4989bc
70 1.4 ± 0.1549bc
2.5 10 12.1 ± 0.5736abc
80 1.3 ± 0.1449abc
MS+(BAP
+ Kinetin)
0.25 + 0.25 10 12.4 ± 0.6197abc
90 1.4 ± 0.1549abc
0.50 + 0.25 10 12.3 ± 0.6935abc
100 1.5 ± 0.1581abc
0.50 + 0.50 10 13.9 ± 0.6395a
90 1.1 ± 0.1703a
1.00 + 0.50 10 13.2 ± 0.8461ab
70 1.1 ± 0.0948ab
LSD 1.8690 1.869
Means followed by different letters in the same column differ significantly at p=0.05 according to Duncan’s new multiple
range test
Ali et al. (2008)Pakistan 25

Table 4 Effect of BAP on establishment and regeneration of shoot tip explant of
sugarcane cultivar CoC 671
Treatments Concentration
of growth
regulator (mg/l)
No. of shoot tips
inoculated
No. of shoot tip
established
(%)
Average number
of shoots per
culture (multiple
shoot) 25 DAI
MS + BAP 0.00 25 0 (0.00) 0.52 (2.08)
MS + BAP 0.50 25 2 (8.00) 2.01 (8.04)
MS + BAP 1.00 25 4 (20.00) 5.46 (21.84)
MS + BAP 2.00 25 18 (72.00) 19.91 (79.64)
Mean 6.00
SEm 0.27
CD (0.01) 1.13
Biradar et al. (2009)Karnataka, India
** Values outside the parenthesis are transformed, where as in parenthesis are actual values
26

Table 5 Effect of Benzyl amino purine (BAP) on establishment and regeneration
of shoot tip explant of sugarcane variety CoN 05072
Treatments Concentration of
growth regulator
(mg/l)
No. of shoot tips
inoculated
No. of shoot tips
established
(%)
Average number
of shoots per
culture (multiple
shoots)
(25 DAI)
MS 0.00 25 0(0.00) 0.68 (2.72)
MS + BAP 0.50 25 4 (16.00) 2.94 (11.76)
MS + BAP 1.0 25 8 (32.00) 6.46 (25.84)
MS + BAP 2.0 25 22 (88.00) 20.50 (82.00)
Values outside the parentheses are transformed, whereas in parentheses are actual values
Patel et al. (2012)Navsari
27

Table 6 Response of BAP and IAA on in vitro shoot multiplication of varieties
B41-227 and N14
PGRs (mg/l) B41-227 N14
BAP IAA
Number of
shoots per
explant
Shoot length
(cm)
Number of
leaves per
shoot
Number of
shoots
Per explant
Shoot length
(cm)
Number of
leaves per
shoot
1.5
0.25 4.00n ± 0.69 4.25gh ± 1.02 3.52l ± 0.19 2.80t ± 0.88 2.35no ± 0.54 3.33m ± 0.42
0.5 15.50a ± 2.90 5.93b ± 0.57 6.40a ± 1.49 3.22qr ± 1.67 2.92l ± 1.27 3.50l ± 1.11
0.75 7.00c ± 1.37 5.17d ± 0.53 4.33h ± 0.11 4.50m ± 0.77 4.37g ± 1.19 4.45g ± 0.78
1 6.21d ± 0.21 4.74e ± 0.27 4.50fg ± 0.54 5.80f ± 1.11 5.23d ± 1.18 3.35m ± 0.68
2
0.25 5.83f ± 0.93 4.55f ± 0.19 3.81j ± 0.05 11.00b ± 0.00 6.30a ± 0.23 5.80b ± 0.06
0.5 10.90b ± 0.82 5.26d ± 1.35 5.20d ± 0.08 5.41hi ± 1.91 3.78i ± 1.01 5.22d ± 0.40
0.75 6.02e ± 1.26 5.85b ± 0.00 4.50fg ± 0.89 4.64l ± 0.71 3.42j ± 0.44 4 .00i ± 0.90
1 5.52gh ± 0.77 5.52c ± 1.30 4.33h ± 0.41 3.22rs ± 0.92 2.88l ± 0.83 3.54l ± 0.78
2.5
0.25 6.00e ± 0.38 4.17h ± 1.46 4.51f ± 0.91 3.33q ± 1.22 4.15h ± 0.64 2.90o ± 0.62
0.5 5.24j ± 0.65 3.82i ± 0.04 3.33m ± 0.60 3.80o ± 0.69 3.23k ± 0.00 4.82e ± 0.00
0.75 5.20j ± 0.82 3.52j ± 0.31 5.51c ± 1.27 4.10n ± 0.54 2.25o ± 0.47 3.00n ± 0.81
1 4.81k ± 0.21 4.38g ± 0.67 4.34h ± 1.11 4.43m ± 0.00 5.21d ± 0.66 2.42r ± 0.66
3
0.25 4.53lm ± 0.55 3.19k ± 1.02 3.50l ± 0.00 3.52p ± 0.00 4.53f ± 0.31 3.84j ± 0.11
0.5 4.50m ± 0.67 2.93l ± 0.79 3.50l ± 0.15 3.33q ± 0.48 2.65m ± 0.00 3.60k ± 0.00
0.75 3.17rs ± 1.20 3.00l ± 0.15 3.33m ± 0.97 2.80t ± 0.01 2.11p ± 0.19 2.82p ± 0.92
1 3.05s ± 0.55 2.44n ± 0.44 2.92o ± 0.24 2.52u ± 0.51 2.46n ± 0.00 2.50q ± 0.71
CV (%) 8.33 5.27 7.91 8.33 5.27 7.91
PGRs=Plant growth regulators. *Values for number of shoots per explant, average shoot length and number of leaves per shoot given as mean ± SD. *Numbers with in
the same column with different letter(s) are significantly different from each other at p = 0.05 according to REGWQ
Tolera et al. (2014)Ethiopia 28

Table 7 Effect of Naphthalene Acetic Acid (NAA) on root induction
in sugarcane cultivar CoC 671
Treatment
No
Treatment Concentration of
growth regulator
(mg/l)
No. of healthy
roots
1 MS 0.00 5 (50.00)
2 MS + NAA 0.50 6 (60.00)
3 MS + NAA 1.0 7 (70.00)
4 MS + NAA 2.0 8 (80.00)
5 MS + NAA 3.0 7 (70.00)
Mean - 6.6
SEm - 0.32
CD (0.01) - 1.27
Biradar et al. (2009)Karnataka, India
Values in parenthesis are in percentage
30

Table 8 Effect of different concentrations of NAA & IBA
combinations on root formation in sugarcane variety CO
86032
Media
composition mg/l
Days to root
induction
(Mean value)
No. of plants
showing roots
(Mean value)
Average frequency of
roots
½ MS + 1 NAA+
1 IBA
16.7 5.6 Low (2-5 Roots)
½ MS + 3 NAA+
1 IBA
15.2 8.3 Medium (5-8Roots)
½ MS + 3 NAA+
3 IBA
13.56 9.5 High ( > 9 Roots)
Godheja et al. (2014)Lucknow
31

Table 9 Effects of indole-3-butryic acid and α -naphthalene acetic acid on rooting in
sugarcane.
PGRs (mg l-1) C86-12 C86-56
IBA NAA
Number of
roots per shoot
± SD
Root
length(cm)
± SD
Number of
roots per shoot
± SD
Root length
(cm)
± SD
0 0 0.00y ± 0.00 0.00u ± 0.00 0.00y ± 0.00 0.00u ± 0.00
0 1 6.7q-t ± 0.00 2.06i-m ± 0.134 5.42t-v ± 1.769 0.34st ± 0.055
0 3 14.1cd ± 0.223 2.5de ± 0.000 6.26s-u ± 1.545 0.62p-r ± 0.045
0 5 17.5a ± 0.00 3.2a ± 0.223 7.76o-r ± 0.750 0.84op ± 0.055
1 0 10.94h-k ± 1.009 1.76jk ± 0.167 7.04p-s ± 0.230 0.48q-t ± 0.045
1 1 14.00cd ± 1.00 2.06hi ± 0.114 8.00n-q ± 0.557 0.52q-s ± 0.277
1 3 13.82cd ± 0.853 3.04ab ± 0.167 7.12p-s ± 0.130 0.40r-t ± 0.071
1 5 12.18e-h ± 0.164 2.02hi ± 0.130 7.06p-s ± 0.151 1.26mn ± 0.089
2 0 11.56g-i ± 0.966 2.96ab ± 0.089 8.22n-p ± 0.044 1.36lm ± 0.089
2 1 11.80f-i ± 0.570 2.46d-f ± 0.057 14.88c ± 0.164 2.80bc ± 0.02
2 3 4.94u-w ± 0.467 1.98hij ± 0.130 6.72q-t ± 0.045 1.52k-m ± .0.084
2 5 10.12j-m ± 0.130 2.30d-g ± 0.000 12.28e-h ± 0.045 1.42lm ± 0.045
3 0 6.2s-u ± 0.071 2.38d-g ± 0.045 3.68wx ± 0.045 0.62p-r ± 0.045
3 1 12.38e-g ± 0.192 2.78bc ± 0.045 6.00s-u ± 0.00 1.10n ± 0.071
3 3 10.12j-m ± 0.327 2.26e-i ± 0.134 14.00cd ± 0.000 2.62cd ± 0.084
3 5 13.16d-f ± 0.422 1.58kl ± 0.476 13.00d-f ± 0.00 2.12g-i ± 0.045
CV (%) 6.63 7.15 6.63 7.15
Shimelis et al. (2015)Ethiopia
*PGRs=plant growth regulators. Values for number of roots per explant and root length given as mean ± SD. Numbers with in the same column with
different letter(s) are significantly different from each other according to REGWQ at p<0.05.
32

What is callus
• Plant callus (plural calluses or calli) is a mass of unorganized
and undifferentiated cells derived from plant tissue (explants)
for use in biological research and biotechnology
• In plant biology, callus cells are those cells that cover a plant
wound
• Plant growth regulators, such as auxins, cytokinins,
and gibberellins, are supplemented into the medium to initiate
callus formation or somatic embryogenesis
34

Fig 2 Stages of callus
Callus induction and proliferation (1-2); Differentiation/(regeneration of
shoots (3-4), and the establishment of plantlet rooting induction (5-6).
35

Application of callus culture
 The whole plant can be regenerated in large number from callus
tissue through manipulation of the nutrient and hormonal
constituents in the culture medium which is called as
organogenesis or morphogenesis.
 callus can be induced to form somatic embryos which can gives
rise to whole plant.
 Callus tissue is good source of genetic or karyotypic variability
 Cell suspension culture in moving liquid medium can be initiated
from callus culture.
36

Table 10 Composition of nutrient media for callus formation
MS media + Quantity/ litre medium
Meso inositol 100mg
Glycine 2.0 mg
Nicotinic Acid 0.5 mg
Pyrodoxine Hcl 0.5 mg
Thiamine Hcl 0.1 mg
2,4 - D 3 mg
Coconut water 100 ml
Sucrose 20 gm
Agar Agar 8 gm
37
Source: plant cell and tissue culture (S. Narayanswamy)

Table 11 Effect of different concentrations of auxins on callus induction from leaf
sheath explants of sugarcane
Name of the hormone Concentration (mg/l) No. of explants
inoculated
No. of explants
showed callus
% of explants with
callus induction
IBA 0.5 20 - -
1.0 20 - -
2.0 20 - -
3.0 20 - -
4.0 20 - -
5.0 20 - -
NAA 0.5 20 - -
1.0 20 - -
2.0 20 1 5
3.0 20 2 10
4.0 20 - -
5.0 20 - -
2,4-D 0.5 20 3 15
1.0 20 9 45
2.0 20 15 75
3.0 20 17 85
4.0 20 8 40
5.0 20 - -
Gopitha et al. (2010)Tamilnadu, India 39

0
10
20
30
40
50
60
70
80
90
1.5 + 0.1 2.0 + 0.15 3.0 + 0.2 3.5 + 0.25 4.0 + 0.3
Phytohormone 2,4-D and Kinetin in different concentration (mg/l)
Fig 3 Effect of different concentrations of 2,4-D and Kinetin on callus
induction from shoot tip explant of sugarcane variety 018%ofexplantwithcallusinduction
Satpal et al. (2011)Ganjam, India 40

Fig 4 Effect of different concentrations of BAP and IBA on shoot regeneration
from callus tissue of sugarcane variety 018
0
10
20
30
40
50
60
70
80
90
3.0 + 0.2 4.0 + 0.5 5.0 + 1.0 6.0 + 1.5 7.0 +2.0
Satpal et al. (2011)Ganjam, India
Phytohormone BAP and IBA in different concentration (mg/l)
%ofexplantproducedshoot
41

Table 12 Effect of combination of BAP and IBA in MS medium on shoot
regeneration from the callus tissue.
Hormonal
supplements, mg/l
% of explant
produced shoots
No .of shoot/ explant Average length of the
shoots (cm)
BAP+IBA
0.5+0.1 40 3.0 ± 0.81 2.2 ± 0.47
0.5+0.2 65 4.0 ± 0.94 3.1 ± 0.47
0.5+0.5 25 3.2 ± 0.47 3.0 ± 0.94
0.5+1.0 15 3.5 ± 0.62 2.8 ± 0.89
1.0+0.1 50 4.2 ± 0.47 3.2 ± 0.47
1.0+0.2 69 5.2 ± 0.37 3.5 ± 0.16
1.0+0.5 40 3.2 ± 0.43 2.1 ± 0.04
1.0+1.0 30 3.0 ± 0.47 2.0 ± 0.23
2.0+0.1 45 3.3 ± 0.61 3.4 ± 0.29
2.0+0.2 61 3.4 ± 0.65 2.9 ± 0.28
2.0+0.5 92 12.4 ± 1.90 6.2 ± 0.37
2.0+1.0 75 10.5 ± 1.31 4.0 ± 0.61
Behera & Sahoo (2009)Bhubaneswar, India 42

Table 13 Effect of BAP and NAA combination on morphogenesis of the
callus tissues in sugarcane varieties Co 99004 and CoN 05071
Co 99004 CoN 05071
Growth
regulators levels
(mg/l)
Days required
for
morphogenesis
Average
no. of
shoots per
culture
Length of
usable
shoots
(cm)
Days required
for
morphogenesis
Average no. of
shoots per
culture
Length of
usable
shoots (cm)
1 BAP +1 NAA 47 4.9 3.8 42 6.2 3.9
2 BAP +2 NAA 39 6.8 6.2 31 7.8 5.0
3 BAP +3 NAA 35 10.8 7.5 27 11.9 6.8
4 BAP +4 NAA 28 12.5 4.8 30 13.5 4.8
Patel et al. (2012)Navsari
43

Table 14 Effect of auxins (NAA+IBA) combination on root formation of the
In vitro grown micro-shoots cultured on ½ strength MS medium.
Auxin
NAA+IBA
(mg/l )
% of micro
shoots rooted
No of
roots/micro
shoots
Average
length of
roots(cm)
Days to
emergence of
roots
0.5+0.5 0 0 0 0
0.5+1.0 40 5.2 ± 0.61 2.3 ± 0.37 10-17
1.0+1.0 52 5.8 ± 0.61 3.2 ± 0.89 10-15
1.5+0.5 60 6.4 ± 0.71 1.4 ± 0.28 15-17
0.5+1.5 48 5.3 ± 0.74 1.2 ± 0.33 10-17
2.0+0.5 50 6.4 ± 0.92 1.9 ± 0.14 10-12
0.5+2.0 75 10.4 ± 0.67 3.5 ± 0.47 12-14
2.5+0.5 60 6.7 ± 0.96 2.5 ± 0.89 10-12
0.5+2.5 82 11.3 ± 1.08 3.9 ± 0.47 10-15
3.0+0.5 40 5.2 ± 1.01 3.2 ± 0.61 15-17
3.0+1.0 35 4.2 ± 0.37 3.0 ± 0.80 15-17
1.0+3.0 30 3.3 ± 0.47 2.8 ± 0.49 15-17
Behera & Sahoo (2009)Bhubaneswar, India 45

Table 15 Effect of different auxin on formation of root in the in vitro grown micro
shoots cultured
Name of the
Hormone
Concentration
mg/l
% of micro
shoots Rooted
Average No. of
roots/micro
shoots
Average length
of roots (cm)
IBA 0.5 33 8.2 1.5
1.0 65 10.5 2.4
3.0 88 12.6 3.7
NAA 0.5 42 10.4 1.0
1.0 63 11.6 1.4
3.0 95 15.1 4.9
IAA 0.5 - - -
1.0 20 5.5 1.3
3.0 55 7.4 1.6
NAA + IBA 0.5 + 0.5 - - -
0.5 + 1.0 45 6.3 2.2
1.0 + 0.5 52 7.2 2.6
1.0 + 1.0 63 9.1 3.9
3.0 + 0.5 80 12.3 3.5
3.0 + 1.0 85 14.5 4.1
Gopitha et al. (2010)Tamilnadu, India 46

Table 16 Effect of different auxins and auxin concentration on root formation of
shoots in variety Co 99004
Treatments
No
Treatments No. of roots Root length (cm)
T1 ½ MS + 0.5 mg/l NAA + 0.5 mg/l IBA 10.2 5.28
T6 ½ MS + 1 mg/l NAA + 0.75 mg/l IBA 10.2 5.12
T8 ½ MS +1 mg/l NAA + 1.5 mg/l IBA 9.0 3.38
T12 ½ MS +2 mg/l NAA + 1.5 mg/l IBA 4.0 2.02
T13 ½ MS + 0.5 mg/l NAA 10.4 5.20
T14 ½ MS + 0.5 mg/l IBA 1.0 0.46
T15 ½ MS 0.0 0.00
SEM 0.163 0.048
CD at 5% 0.436 0.137
CV % 4.68 3.03
47Gadakh et al. (2014)Navsari

GENETIC ENGINEERING
Figure 5: Flow diagram of Agrobacterium mediated transformation in sugarcane
48
.Normal
plant leaf
disc

Figure 6 Molecular analysis of alien gene (AVP 1) in transformed and
non transformed plants in sugarcane cultivar CP77- 400
Lane M= 1kb Marker, Lane1, 3= transgenic sugarcane
plants, lane 2,4= non transformed, lane 5=positive
control, NC= negative control
Kumar et al. (2014)Pakistan
Regeneration of profuse roots and
acclimatization of transgenic plants
49
630 BP
AVP 1 Arabidopsis Vacuolar pump

Current status of sugarcane transgenic
Sr.no Transgenic Method Reference
1 Transformation in callus
regeneration
Particle bombarment Franks and Birch
(1991)
2 First transgenic plants were
produced
High velocity DNA coated
microprojectile by
bombardment
Bower and Birch
(1992)
3 Herbicide resistant transgenic
plants of Saccharum spp.
hybrids variety NCo 310
- Gallo-Meagher and
Irvine (1993)
4 Transgenic sugarcane plants
using truncated Cry 1A gene
encoding the active region of
B.thuringiensis ð- endotoxin
CaMV 35S promoter Arencibia et al. (1997)
5 Resistance against the borer to
the sugarcane
Bombarding
their embryogenic calli with
tungsten particles coated.
Braga et al. (2003)
50Conti…

6 Most useful npt II
gene (selectable marker) for selection of
sugarcane callus transformation
A. tumefaciens
system
Zhangsun et al.
(2007)
7 Transgenic sugarcane plants expressing
transgene against borer mainly Cry1Ab
aprotinin gene and Cry 1Aa3 evaluated and
expression is checked through
serological and
molecular techniques
Arvinth et al.
(2009)
8 Transgenic sugarcane lines have been
developed with gene expressing Cry protein,
proteinase inhibitor or lectin resistance to
borers, sucking insects or grubs
- Srikant et al. (2011)
9 Creating sugarcane cultivar Phil 6607 resistant
to fungal diseases by genetic transformation
with a chitinase gene
Particle
bombardment
Khamrit et al.
(2012)
51

CONCLUSION…
• From the forgoing discussion, it can be concluded that MS medium
supplemented with 3 mg/l 2, 4- D is very useful for callus culture and 2 mg/l
of BAP is good for shoot formation and multiplication.
• NAA and IBA showed better response than IAA for producing roots,
However half strength MS medium with 3 mg/l of NAA is most suitable for
root induction, root number and root length.
• Combination and contribution of hormonal substances plays important role
for successful plant regeneration and multiplication.
• Because of variable performance of different varieties to nutrient
composition, standardization of protocol need to be done to ahead for mass
multiplication.
• Genetic engineering make possible the production of transgenic sugarcane
plants resistance to drought and salinity, herbicides, pathogens and pests.
• Thus, this is good technology for getting true to type plantlets in large scale
and to fulfil the demand of farmers and extension of the area under sugarcane.
52

FUTURE THRUST
•Need to find out substances/ elements for reducing
multiplication cycle.
•To adjust suitability of standard protocol for all the
varieties of a crop
•To remove undesirable characters from release
/promising variety
53

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