In Vitro Cell. Dev. Biol.ÐPlant 37:434±439, July±August 2001 DOI:10.

1079/IVP2001178
q 2001 Society for In Vitro Biology
1054-5476/01 $10.0010.00

EFFECT OF VARIOUS GROWTH REGULATORS ON SHOOT REGENERATION OF SUGARCANE

K. CHENGALRAYAN and M. GALLO-MEAGHER*

Agronomy Department, University of Florida, Gainesville, Florida 32611-0300

(Received 2 October 2000; accepted 13 February 2001; editor M. Horn)

Summary
Sugarcane (Saccharum spp. hybrid cv. CP 84-1198) embryogenic calluses were induced from young leaves cultured on
modified Murashige and Skoog basal medium supplemented with 13.6 mM 2,4-dichlorophenoxyacetic acid. Five
concentrations, 0.5, 1.0, 2.5, 5.0, and 10.0 mM, of five different growth regulators, 6-benzylaminopurine, kinetin, 6-g,g-
(dimethylallylamino)purine, zeatin, and thidiazuron, were tested with or without 22.5 mM a-naphthaleneacetic acid to
compare their ability to induce regeneration from embryogenic callus. After 4 wk on medium, the percentage of shoot
meristem induction was evaluated, and after 10 wk the total number of shoots produced, as well as the percentage of
shoots greater than 1 cm in length, was obtained. Although it had the lowest percentage of elongated shoots, medium
containing thidiazuron alone performed better than all other growth regulators tested, with the highest percentage of shoot
induction and the largest number of shoots, particularly at a concentration of 2.5 mM.
Key words: Saccharum; tissue culture; thidiazuron.

Introduction
Global raw sugar production in 1999/2000 is estimated to be
133.9 million metric tons, a 3% increase over the previous year and
a 22% increase over the past 6 yr (International Trade Data System,
2000). Sugarcane accounts for approximately 70% of the world's
sugar and is an economically important cash crop in the tropical
and subtropical regions of many countries (International Trade Data
System, 2000). Due to its global importance, much research has
focused on sugarcane crop improvement through plant breeding,
and more recently through biotechnology. The first report on
transgenic sugarcane plants was in 1992 (Bower and Birch, 1992).
Subsequently, there have been several reports on sugarcane
transformed with marker genes (Bower et al., 1996; Arencibia
et al., 1998; Elliott et al., 1998), as well as transgenic sugarcane
containing genes controlling such characters as herbicide resis-
tance (Chowdhury and Vasil, 1992; Gallo-Meagher and Irvine,
1996; EnrõÁquez-ObregoÂn et al., 1998; Falco et al., 2000), insect
resistance (Arencibia et al., 1997, 1999), and sugarcane mosaic
virus resistance (Ingelbrecht et al., 1999). Sugarcane mosaic virus
resistant plants have also been obtained as somaclonal variants
derived from tissue culture (Oropeza et al., 1995; Oropeza and
GarcõÂa, 1996).
Effective utilization of biotechnological approaches such as the
isolation of somaclonal variants, protoplast fusion and genetic
transformation, rely on efficient and reliable regeneration systems.
Sugarcane tissue culture was first initiated in Hawaii in 1961 (for
review see Liu, 1984). Subsequently, several protocols for somatic
embryogenesis and organogenesis have been developed using callus
*Author to whom correspondence should be addressed: Email
mgmea@gnv.ifas.ufl.edu
derived from various explants. Rapid callus formation has been
obtained mostly from young expanding leaves or immature
inflorescences. Somatic embryogenesis was reported from young
leaves (Ahloowalia and Maretzki, 1983; Ho and Vasil, 1983; Chen
et al., 1988; Brisibe et al., 1994), immature inflorescences (Liu,
1993; Blanco et al., 1997), and apical meristems (Ahloowalia and
Maretzki, 1983), as well as organogenesis from young leaves (Chen
et al., 1988; Fitch and Moore, 1990). In most of these studies, callus
was induced in the presence of auxin, either 2,4-dichlorophenoxy-
acetic acid (2,4-D) or picloram. To promote regeneration, callus was
then transferred to medium with either a reduced auxin concentra-
tion or containing no auxin (Ahloowalia and Maretzki, 1983). Callus
has also been transferred to 9.3 mM kinetin and 22.3 mM a-
naphthaleneacetic acid (NAA) to obtain rapid regeneration (Irvine
and Benda, 1987; Irvine et al., 1991).
More recently, rapid shoot regeneration from embryogenic callus
was obtained using modified MS medium (Murashige and Skoog,
1962) containing thidiazuron (TDZ) (Gallo-Meagher et al., 2000).
All TDZ treatments resulted in faster shoot regeneration than the
kinetin/NAA treatment described above and more shoot production
than either the standard 2,4-D or kinetin/NAA treatments.
However, it is not known whether TDZ is superior to other
cytokinins for sugarcane regeneration from callus, or whether TDZ
in combination with an auxin would be better than TDZ alone.
Therefore, the objective of the present study was to conduct
detailed, systematic studies to determine the most efficient
sugarcane regeneration system. Five different growth regulators,
6-benzylaminopurine (BA), kinetin, 6-g,g-(dimethylallyl-
amino)purine (2iP), zeatin and TDZ with or without NAA
(22.5 mM) were evaluated for their ability to induce sugarcane
regeneration from embryogenic callus. To our knowledge, this is
the first report in which a number of different cytokinins and

434
 SHOOT REGENERATION OF SUGARCANE 435

Fig. 1. Comparison of sugarcane shoot regeneration. (A) Embryogenic, yellow, nodular callus developed after 12 wk in culture on
modified MS basal medium supplemented with 13.6 mM 2,4-D. (B±F) Regenerating callus cultured on modified MS basal medium
supplemented with: B, 5 mM TDZ; C, 10 mM zeatin; D, 5 mM kinetin; E, 5 mM BA; F, 10 mM 2iP, respectively.

cytokinin-like compounds have been compared for their effects on
sugarcane regeneration.
Materials and Methods
Callus initiation. Sugarcane, Saccharum spp. hybrid cv. CP 84±1198,
an important parent in US breeding programs (USDA-ARS, Canal Point, FL)
was selected in this study. Embryogenic calluses were induced from young
leaves as described by Irvine et al. (1983). Apical portions of healthy shoots
were stripped to the terminal bud and attached immature leaf rolls were
immersed in a solution of 10% commercial bleach (0.5% sodium
hypochlorite) for 20 min. Excess bleach was removed by repeated washings
(four or five times) with sterile deionized and distilled water. Leaf rolls were
peeled under sterile conditions to cylinders approximately 5 mm in
diameter. Serial slices, 3 mm thick were removed from immediately above
the apical meristem. Three to six slices were taken from each cylinder and
cultured on callus induction medium (CI3) consisting of modified MS basal
medium (Heinz et al., 1977), supplemented with 20 g l21 sucrose and
13.6 mM 2,4-D. Cultures were transferred onto fresh CI3 medium every 2±
3 wk for long-term maintenance.
Shoot regeneration. A completely randomized design was used with ca.
500 mg of 9-month-old callus per Petri dish …100  20 mm† with three
replicates per treatment. Calluses were cultured on modified MS medium
supplemented with either BA, kinetin, 2iP, zeatin, or TDZ at five
concentrations: 0.5, 1.0, 2.5, 5.0, and 10.0 mM, with or without 22.5 mM
NAA for a total of 50 treatments. All cultures were transferred to fresh
medium every 2±3 wk. After 4 wk of culture initiation, the percentage of
shoot meristem induction was calculated as the weight of green callus over
the total weight of the callus produced and compared among the different
treatments. Each treatment was also visually inspected to verify results.
Total number of shoots and percentage of shoots greater than 1 cm in length
were determined 10 wk after culture initiation. Statistical analysis was
carried out using analysis of variance, and treatment means were separated
using Duncan's Multiple Range Test (PROC GLM, SAS Institute, 1996).
Culture conditions. The pH of all media was adjusted to 5.6±5.8 and
they were gelled with 0.7% Agargele (Sigma-Aldrich Co., St. Louis, MO).
All cultures were incubated in a growth chamber at 25 ^ 18C; under cool
white fluorescent light at 60 mmol m22 s21 with a 16-h photoperiod.
Results and Discussion
Shoot meristem induction. Young sugarcane leaves are known
to be good explant sources for callus production (Ahloowalia and
Maretzki, 1983; Ho and Vasil, 1983; Chen et al., 1988; Brisibe
et al., 1994), and 2,4-D has been used to induce callus from various
sugarcane explants (Heinz and Mee, 1969; Chen et al., 1988; Fitch
and Moore, 1990; Oropeza and GarcõÂa, 1996; Gallo-Meagher et al.,
2000). Therefore, young leaves of sugarcane variety CP 84±1198
436 CHENGALRAYAN AND GALLO-MEAGHER

Fig. 2. Effect of 2iP, zeatin, kinetin, BA, and TDZ on the percentage of shoot meristems produced from yellow, embryogenic
sugarcane callus after 4 wk of culture. A, Without NAA; B, supplemented with 22.5 mM NAA.

were used as starting material to induce callus on modified MS
medium containing 13.6 mM 2,4-D, and initial culturing produced
a white non-regenerative callus. However, 2±3-wk subculturing of
the white callus on the same medium over a period of 3 mo. resulted
in the production of callus which was compact, yellow and
embryogenic (Fig. 1A; Ho and Vasil, 1983). Yellow callus is
typically produced from 2,4-D cultures following the fourth
subculture with 3±11-wk intervals for each subculture (Fitch and
Moore, 1990).
The effect of various growth regulators on green shoot meristem
induction was evaluated after 4 wk using this yellow, embryogenic
callus as starting material. The type of growth regulator used in the
culture medium had a significant effect on the induction of green
shoot meristems …P , 0:0001; Figs. 1B±F and 2A). Medium
containing TDZ had the highest percentage of shoot meristems
across the five concentrations …88:6 ^ 10:5† with essentially 100%
shoot meristem induction occurring at concentrations of 1.0 mM
and above. Similar results were also observed on sugarcane callus
derived from inflorescences when cultured on 1 mM or higher TDZ
(Gallo-Meagher et al., 2000). Therefore, nearly all explants
initiated shoot meristems in response to TDZ. Growth regulators
BA …76:2 ^ 2:8†; kinetin …72:5 ^ 6:2†; and zeatin …63:7 ^ 8:8†
had similar intermediate effects, whereas medium containing
2iP had the lowest percentage of shoot meristem induction
…37:0 ^ 6:0†: For any given growth regulator, there was no
significant effect on the percentage of shoot meristems formed
over the tested concentrations.
Earlier reports showed that combining NAA with kinetin promoted
rapid sugarcane regeneration from callus (Irvine and Benda, 1987;
Irvine et al., 1991). However, at this early stage of regeneration,
addition of 22.5 mM NAA to the media did not alter shoot meristem
induction when combined with TDZ, BA, kinetin, or zeatin (Fig. 2B).
Only NAA added to medium containing 2iP caused an improve-
ment in the percentage of shoot meristems formed …P , 0:0001†:
However, the higher percentages of shoot meristems produced for
the 2iP plus NAA media were still lower than those obtained for the
other growth regulators. Thus, at the shoot meristem induction stage
of sugarcane regeneration and at the concentrations tested, TDZ
was the most effective growth regulator, and there was no advantage
to adding 22.5 mM NAA to the medium.
 SHOOT REGENERATION OF SUGARCANE
Fig. 3. Effect of 2iP, zeatin, kinetin, BA, and TDZ on total number of shoots produced from sugarcane embryogenic callus after 10 wk
of culture. A, Without NAA; B, supplemented with 22.5 mM NAA.
Shoot regeneration. After 10 wk of culture, shoot regeneration
was dependent on growth regulator and concentration (Fig. 3A, B;
P , 0:0001†: Similar to the results for the production of shoot
meristems, TDZ was superior to the other growth regulators, across
the concentrations used …608:2 ^ 221:3† (Fig. 3A). All other growth
regulators induced much fewer shoots (zeatin, 232:2 ^ 125:6; BA,
185:5 ^ 53:2; kinetin, 180:4 ^ 89:9; 2iP, 79:1 ^ 58:9†: The
number of shoots produced across BA concentrations was
unchanged. However, the effect of concentration was sig-
nificant for kinetin …P ˆ 0:0016†; 2iP …P ˆ 0:0012†; and
zeatin …P , 0:0001†; with the trend being more shoots produced
with increasing hormone concentrations except at 10 mM. Compar-
isons between the best individual concentrations for each growth
regulator revealed that 2.5 mM TDZ …925:5 ^ 96:9† was the best for
shoot production, followed by 10 mM zeatin …455:7 ^ 50:5†: A
lower number of shoots was produced on 5 mM kinetin …284:0 ^
45:1†; 5 mM BA …231:0 ^ 119:2† and 10 mM 2iP …173:3 ^ 11:8;
Fig. 3A). Thidiazuron has been shown to be highly effective in shoot
induction for a variety of plants (for review see Murthy et al., 1998)
and these data confirm results of our earlier work which suggested
that the use of TDZ for sugarcane regeneration is an advance over
437
the current protocols (Gallo-Meagher et al., 2000). In that report,
1.0 mM TDZ produced a greater number of shoots than 2.5 mM.
However, that evaluation was made after a 4-wk period, compared
to the 10-wk time frame used in the present study.
Adding NAA to media containing kinetin, zeatin, or TDZ did not
affect the number of shoots produced (Fig. 3B). In media containing
BA and 2iP, addition of NAA significantly increased the number of
shoots produced …P ˆ 0:0029; 286:9 ^ 79:2; P , 0:0001; 207:1 ^
50:7; respectively). However, even with their positive effect on
shoot production, these treatments still produced far fewer shoots
than TDZ (Fig. 3B). So, it appears that NAA may enhance the
number of shoots produced depending upon the cytokinin used, but
TDZ alone remains the most effective treatment.
Shoot elongation. The length of shoots produced was dependent
on growth regulator and concentration …P ˆ 0:0003; Fig. 4A).
Although TDZ produced the highest percentage of shoot meristems
and the largest number of shoots, it had the lowest percentage of
shoots that were more than 1 cm in length …16:0 ^ 2:7†: It has been
reported previously that TDZ can reduce shoot elongation (Bates
et al., 1992; Murthy et al., 1998) and our results confirm these
observations. However, with more time in culture, these shoots do
438 CHENGALRAYAN AND GALLO-MEAGHER

Fig. 4. Effect of 2iP, zeatin, kinetin, BA, and TDZ on the percentage of shoots that were more than 1 cm in length produced from
sugarcane embryogenic callus after 10 wk of culture. A, Without NAA; B, supplemented with 22.5 mM NAA.

elongate and their rooting on 19.7 mM IBA proceeds normally (data
not shown). Kinetin …64:5 ^ 12:9† and 2iP …55:4 ^ 4:4† had the
highest percentages of elongated shoots, followed by zeatin …44:5 ^
13:9† and BA …30:3 ^ 10:0†: Shoot elongation decreased with
increasing concentrations for BA and kinetin …P ˆ 0:0012 and
0.0001, respectively). A similar trend also was observed for
1.0±10.0 mM zeatin …P ˆ 0:0086†: However, concentration did not
influence shoot length for 2iP and TDZ.
The addition of 22.5 mM NAA significantly affected shoot length
…P , 0:0001; Fig. 4B). With the exceptions of 0.5 mM 2iP, 0.5 mM
TDZ, and 10 mM zeatin, all other concentrations of growth
regulators caused a reduction in the percentage of shoots greater
than 1 cm when NAA was included in the culture medium (Fig. 4B).
The interaction of NAA and growth regulator concentration was
significant only for TDZ …P , 0:0001† and zeatin …P ˆ 0:0071†:
Therefore, it can be concluded that in most cases 22.5 mM NAA is
inhibitory to sugarcane shoot elongation.
In conclusion, our results indicate that shoots were induced in all
types and concentrations of the growth regulators tested. However,
TDZ was superior to other cytokinins tested for sugarcane
regeneration from callus, with 2.5 mM TDZ being the best
concentration for shoot production in this 10-wk study. Addition
of 22.5 mM NAA to the medium failed to increase shoot
regeneration for three of the five growth regulators, and reduced
the production of elongated shoots with all growth regulators.
For efficient regeneration of sugarcane, we recommend a protocol
based on these results and those from our previous study on
sugarcane regeneration with cv. CP 84±1198 (Gallo-Meagher et al.,
2000). Begin by culturing young leaf explants on CI3 until compact,
yellow embryogenic callus is formed. This callus should then be
placed on MS medium plus 2.5 mM TDZ, and once shoots have
reached a length greater than 1 cm, they should be transferred onto
the same MS basal medium containing 19.7 mM IBA for rooting
with subsequent transfer to the soil.
Acknowledgment
The authors wish to thank Drs K. H. Quesenberry and D. S. Wofford for
critical review of the manuscript. The authors also thank Dr J. D. Miller
(USDA-ARS, Canal Point, FL) for the young leaf rolls of CP 84±1198, Mr N.
 SHOOT REGENERATION OF SUGARCANE
Stevens for callus maintenance, and Dr R. C. Littell for help in the statistical
analyses. This research was supported by the Florida Agricultural
Experiment Station and a grant from the Florida Sugar Cane League, Inc.,
and approved for publication as Journal Series No. R-07809.
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