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Bioscience Methods 2012, Vol.3, No.2, 7-20
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Research Article Open Access
Establishment of an in vitro Regeneration System as a Milestone for Genetic
Transformation of Sugarcane (Saccharum officinarum . L) against Ustilago
scitaminea
Naweed Anjum , Siddra Ijaz , Iqrar Ahmad Rana , Tariq Manzoor Khan , Iqrar Ahmad Khan , Muhammad
Naeem Khan , Ghulam Mustafa , Faiz Ahmad Joyia , Ahsan Iqbal
Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad
Corresponding author email: chinanoahl@163.com; Author
Bioscience Methods 2012, Vol.3, No.2 doi: 10.5376/bm.2012.03.0002
Received: 18 Feb., 2011
Accepted: 01 Mar., 2011
Published: 21 Mar., 2012
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article as:
Anjum et al., 2012, Establishment of an in vitro Regeneration System as a Milestone for Genetic Transformation of Sugarcane (Saccharum officinarum. L)
against Ustilago scitaminea, Bioscience Methods, Vol.3, No.2 7-20 (doi: 10.5376/bm.2012.03.0002)
Abstract Optimization of stable in vitro regeneration system is indispensible to apply molecular approaches in crops. Due to its
profound impact on genetic transformation studies, we established a reproducible and effectual in vitro regeneration system, in two
whip smut (Ustilago scitaminea) susceptible genotypes viz., S-2003- us-127 and S- 2003-us- 371. Twelve callus formation media
(CFM) were investigated for callus formation, in which four levels of 2,4-D (1 mg/L, 2 mg/L, 3 mg/L and 4 mg/L), two levels of
BAP (0 mg/L and 0.5 mg/L) and two levels of kinetin (0 and 0.1 mg/l) were used in different combinations with basal MS Salt.
CFM3 (3 mg/L 2,4- D), CFM4 (4 mg/L 2,4- D), CFM11 (3 mg/L 2,4-D and 0.1 mg/L kinetin) and CFM12 (4 mg/L 2,4 -D and 0.1
mg/L kinetin) were proved to be the best for callus formation response in genotype S- 2003-us- 127. But in case of genotype
S-2003- us-371, CFM3, CFM11 and CFM12 showed good response for callus induction. Among good responsive CFM, we selected
CFM with low dose of 2,4- D (CFM3 and CFM11) for our regeneration experiment. For regeneration study, four regeneration media
(RM) with different plant growth regulators viz., 2,4- D (0.1 mg/L), BAP (0.5 mg/L and 1 mg/L), kinetin (0.25 mg/L) and proline
(250 mmg/L) plus MS salt were used. Calli of three (3) different ages, viz., 21 days, 28 days and 35 days from CFM3 and CFM11
were shifted on four regeneration media (RM). Among these four regeneration media, RM2 (0.1 mg/L 2,4 -D and 1 mg/L BAP) gave
an excellent regeneration response for genotype S-2003- us-127, when 28 days old calli from CFM11 were transferred to this. This
combination was selected as combination of choice in genotype S- 2003-us- 127 for genetic transformation studies. Genotype
S-2003- us-371, showed maximum regeneration, when 35 days old calli from CFM11 were kept on RM4 (0.1 mg/L 2,4- D, 1 mg/L
BAP, 0.25 mg/L kinetin and 250 mg/L proline). Genetic stability of regenerated plants of selected media combination was confirmed
with RAPD (PCR) analysis by using 5 RAPD primers.
Keywords Biotechnology; Tissue culture; Plant Transformation; Disease resistance
Background
Sugarcane (Saccharum officinarum L.) belongs to genus
Saccharum, family Poaceae and characterized by high
levels of polyploidy (2n=80~270) and frequently
aneupolidy (Heinz and Mee., 1969). It accounts for
approximately 80% of world sugar production FAO,
(2009). This grass is the most suitable promising crop
which could be utilized mainly for sugar production
and then for power generation, paper making, live
stock feed, chipboard, cane wax, fertilizer, bioethanol,
syrup and mulch (Chaudhry and Naseer, 2008).
Sugarcane is the second major cash crop in Pakistan
after cotton. Many factors are involved in low cane
and sugar yields in which drought or low rainfall,
salinity, insect pests and diseases are remarkable
especially whip smut ( Ustilago scitaminea) a fungal
disease which causes 30%~100% economic damage
(Rangashawami, 1996; Nasir et al., 2000; Gururaj, 2001;
Khaliq et al., 2005; Ajit, 2006). Gene introduction by
conventional breeding becomes more difficult due to
limited flower production, environmental interactions,
large complex genome, slow breeding advances, back
crossing, low fertility, susceptibility to insect, pest
and diseases especially whip smut (Gururaj, 2001).