herbicide-tolerant sugarcane (saccharum officinarum l.) plants:...

439

httpjournalstubitakgovtrbiology

Turkish Journal of Biology Turk J Biol(2014) 38 439-449copy TUumlBİTAKdoi103906biy-1306-81

Herbicide-tolerant sugarcane (Saccharum officinarum L) plantsan unconventional method of weed removal

Idrees Ahmad NASIR1 Bushra TABASSUM1 Zahida QAMAR1 Muhammad Aslam JAVED2 Muhammad TARIQ1Abdul Munim FAROOQ1 Shahid Javed BUTT3 Abdul QAYYUM1 Tayyab HUSNAIN1

1National Centre of Excellence in Molecular Biology University of the Punjab Lahore Pakistan2Agriculture Biotechnology Institute Ayub Agriculture Research Institute Faisalabad Pakistan

3Department of Horticulture PMAS-Arid Agriculture University Rawalpindi Rawalpindi Pakistan

Correspondence bushracembgmailcom

1 IntroductionIn Pakistan sugarcane (Saccharum officinarum L) is a major cash crop after cotton as it shares 36 and 08 of value added in agriculture and the gross domestic product respectively (wwwfinancegovpk) Sugarcane is the main source of sucrose that is deposited in stalk internodes and is the source of many industrial products like alcohol dextrans furfural and so on It is also used in the preparation of some natural pharmaceutical products (Julian et al 2005) Similarly many industrial and agricultural by-products that are derived from sugarcane are used in the paper industry animal nutrition and food (httpwwwpakissancomenglishallaboutcropsugarcaneshtml)

In Pakistan the national average yield of sugarcane in 2011ndash2012 was about 580 tha compared to the world average production of 65 tha (wwwfinancegovpk) The

presence of undesirable plants in sugarcane plantations reduces crop yield Weeds can cause major losses in terms of quality and quantity Weeds can reduce cane yields by competing for moisture nutrients and light during the growing season (Rainbolt and Dusky 2006) Ahmad et al (2012) reported that 15ndash30 of the reduction in yield of sugarcane is due to weeds in Pakistan Furthermore according to NETAFIM (httpwwwnetafimcomarticlesugarcane--philippines) weeds are estimated to cause a 12ndash72 reduction in cane yield worldwide Weed control by traditional methods in a field of growing sugarcane requires a lot of manpower Moreover it tears the surface of the soil resulting in the uprooting of plants and causes injuries to the people involved in the mechanical uprooting of the weeds Furthermore many of the weeds have deep and extensive roots that cannot be uprooted by traditional methods and the majority of them

Abstract Unnecessary weed growth in sugarcane fields forces plants to compete for nutrients and sunlight for survival which most often leads to significant yield losses As chemical herbicides cannot differentiate between crop plants and weeds the development of herbicide-tolerant crops is anticipated Four sugarcane varieties CPF-234 CPF-213 HSF-240 and CPF-246 were used to develop glyphosate herbicide tolerance A glyphosate-tolerant gene of 1368 bp cloned directionally under the 35S promoter with the β-glucuronidase (GUS) reporter gene was used as the transgene Through the biolistic transformation of sugarcane calli of all cultivars were transformed with glyphosate-tolerant gene constructs Efficient regeneration conditions were optimized on 15ndash25 mgL kinetin 15ndash25 mgL 6-benzylaminopurine (BAP) and 1 mgL gibberellic acid (GA3) The transformants exhibited the best shoot regeneration on a medium containing 2 mgL kinetin 2 mgL BAP 2 mgL GA3 and 1 mgL indole-3-acetic acid Based on initial screenings through GUS assay the transformation efficiency was 22 32 17 and 13 for cultivars 246 234 213 and 240 respectively In transformed sugarcane plants the transgene of 1368 bp was amplified ELISA with gene-specific coated monoclonal IgG confirmed the transgene protein expression It was revealed that all acclimatized transgenic sugarcane plants survived the glyphosate spray application of 900 mL0404 ha except for the control nontransformed plants However at spray application of 1100 mL0404 ha transgenic plants having the transgene protein OD of 02 to 10 did not survive while those that had a transgene protein OD range of between 10 and 20 did In addition weeds growing alongside transgenic sugarcane plants turned brown and subsequently died at glyphosate spray applications of both 900 and 1100 mL0404 ha

Key words Biolistic transformation glyphosate-tolerant gene herbicide tolerance Saccharum officinarum

Received 27062013 Accepted 12022014 Published Online 11062014 Printed 10072014

Research Article

NASIR et al Turk J Biol

440

can regenerate The introduction of herbicide-resistant crops has dramatically changed weed management in crop production systems (Owen 2008 Vencill et al 2012) Using genetic engineering as a complement for traditional breeding methods it is possible to introduce herbicide-tolerant traits into the Saccharum germplasm The absence of herbicide-tolerant genes in the genetic pool of wild relatives of sugarcane makes traditional breeding programs difficult The varieties that are productive and at the same time have resistance to certain pathogens and diseases can improve yield Development of herbicide-resistant sugarcane varieties through transgenic technology will be cost-effective in controlling weeds which ultimately improves yield potential To date several crops have been genetically modified for resistance against herbicides The most widely adopted biotech crop the Roundup Ready soybean was developed and released commercially by Monsanto it contains a version of the EPSPS gene (glyphosate) from Agrobacterium tumefaciens

Currently weeds in sugarcane crops are being controlled by the application of certain organophosphorus herbicides like Roundup The active ingredient of Roundup is glyphosate which is a nonselective broad-spectrum herbicide that translocates symplastically to the meristems of the growing plants It is well known for its high effectiveness low toxicity low residues in organisms and soil and overall limited environmental impact However heavy use of glyphosate-based herbicides increases the emergence of glyphosate-resistant weeds It was also reported that once glyphosate travels to a plantrsquos roots it is released into the rhizosphere (the area immediately around the roots) where it is immobilized at the soil matrix (Tesfamariam et al 2009) which leads to disruption of soil and root microbial communities (Busse et al 2001) Studies have found that about 1ndash2 of glyphosate may run off in rainfall after glyphosate is applied (Giesy et al 2000) Another study indicated that low doses of glyphosate cause birth defects in frogs and chickens (Paganelli et al 2010)

In plants including weeds glyphosate inhibits chloroplast-localized EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase) (Pline et al 2002 Castle et al 2004) Overexpression of the EPSPS gene in cells renders glyphosate tolerance in transformed plants (Castle et al 2004 Pline-Srnic 2006) Two mechanisms have been reported underlying glyphosate tolerance The first is characterized by the overexpression of the EPSPS gene while the second is linked to a herbicide-insensitive enzyme (Ge et al 2010)

The genetic manipulation of sugarcane requires the availability of an efficient transformation system that directly depends on the embryogenic capability of the callus and its subsequent regeneration (Barampuram and Zhang

2011) In the present study 4 elite sugarcane varieties from Pakistan with good yield potential were selected to become herbicide (glyphosate)-tolerant The glyphosate-tolerant gene accession JF4452901 was modified for the codon belonging to sugarcane as identified through OptimumGene software and was submitted locally under patent application 7652010 in Karachi Pakistan Moreover a reliable and efficient transformation system was also developed based on the optimization of calli and regeneration conditions in sugarcane

2 Materials and methods21 Plant material and callus inductionFour elite sugarcane varieties from Pakistan namely SPE-234 CPF-246 CPF-213 and HSF-240 were selected for genetic modification with a glyphosate-tolerant gene The varieties were kindly provided by the Ayub Agriculture Research Centre Faisalabad A leaf roll of 20ndash30 cm in length containing the apical meristem was excised from the plants and sterilized using 001 mercuric chloride solution The sterilized leaf roll was further dissected to reveal a stage 3 leaf whose transverse sections were used subsequently as explants for callus induction

For callus induction MS basal medium (Murashige and Skoog 1962) supplemented with sucrose (3) 24-dichlorophenoxyacetic acid (24-D 15 to 4 mgL) myo-inositol (200 mgL) and casein (1 gL) in different combinations was used (Table 1) The medium was solidified with 25 gL Phytagel after adjusting the pH to 57 followed by autoclaving at 121 degC and 1034 kPa for 20 min Transverse segments from immature sugarcane leaves were used as explants and a response was observed on all combinations of media at repeated intervals Subculturing was done at a regular interval of 7 days After 15 days calli were observed under the microscope and nodular compact white calli were marked as embryogenic to be used subsequently for transformation All cultures were kept at 25 plusmn 2 degC under a 168 photoperiod Calli pieces were placed in the middle of petri plates having MS media for 3ndash4 h with additional 3 gL Phytagel added to keep calli pieces in place prior to bombardment22 Plasmid constructionThe glyphosate-tolerant gene (GTGene patent application 7652010 Karachi Pakistan) was cloned directionally in plant expression vector pCAMBIA1301 (Cambia Australia) which was driven by Cauliflower mosaic virus 35S promoter and the nopaline synthase 3rsquo-end terminator For directional cloning NcoI and BglII restriction enzymes were used In pCAMBIA1301 the β-glucuronidase (GUS) exon was used as a reporter gene in the cassette (Figure 1) 23 Transformation of CEMB-GT gene in sugarcaneOne milliliter of absolute ethanol was mixed with 60 mg of tungsten particles followed by centrifugation at 10000


441

rpm for 1 min Supernatant was discarded and washed with 05 mL of sterilized water Microcarriers were resuspended in 05 mL of sterile water The final mixture was made by adding 5 microg of plasmid DNA 25 M of CaCl2 and 01 M spermidine with 50 microL of tungsten particles The mixture was spun at 7000 rpm for 1 min the supernatant was discarded and the pellet was washed with 70 ethanol followed by a final resuspension of the pellet in 40 microL of absolute ethanol The bombardment was done by loading 20 microL of the particle suspension into a homemade biolistic gun [developed by the Centre of Excellence in Molecular Biology (CEMB) University of the Punjab Lahore Pakistan] Bombardment was done under vacuum using

helium pressure of 7584 kPa The distance of the calli placed in petri plates was adjusted to be 14 cm prior to bombardment 24 Regeneration and multiplicationBombarded calli were placed onto the regeneration media supplemented with various plant growth regulator combinations A total of 4 combinations of media were tested and the regeneration efficiency for the transformed calli of each cultivar on all 4 media combinations was measured Regenerated shoots from each calli piece were designated as a separate plantlet which were subsequently multiplied on multiplication media (Table 1)

Table 1 Media codes along with composition of each medium used for sugarcane callus induction and its maintenance regeneration multiplication and root induction (CM Callus media Reg Regeneration media)

Medium code Medium composition

Callus induction and maintenance Shoot regeneration

CM1 MS + myo-inositol (200 mgL) + casein (1 gL) + 24-D (15 mgL)




Reg 1 Kinetin (25 mgL) + BAP (25 mgL) + charcoal (1 gL) + MS + myo-inositol (200 mgL) + casein (0498 gL)

Reg 2Kinetin (2 mgL) + BAP (2 mgL) + GA3 (1 mgL) + charcoal (1 gL) + MS + myo-inositol (200 mgL) + casein (0498 gL)


Reg 4 Kinetin (3 mgL) + BAP (3 mgL) + charcoal (1 gL) + MS + myo-inositol (200 mgL)

Multiplication MS + kinetin (2 mgL) + BAP (2 mgL) + GA3 (2 mgL) + IAA (1 mgL) + charcoal (1 gL)Rooting MS + NAA (2 mgL) + IBA (1 mgL) + charcoal (1 gL)

CaMV 35S promoter

GTGene NOS Poly A

T-Border (L)

T-Border (R) NcoI BglII NheI heI BstEII

GUS intron

Figure 1 Construct map of CEMB-GTGene Plant binary vector pCAMBIA1301 was used and the transgene was cloned directionally between NcoI and BglII restriction sites under the influence of CaMV 35S promoter (GTGene glyphosate resistance gene with modified gene sequences)


442

25 GUS histological assayYoung emerging shoots from the transformed calli were initially screened through GUS assay For this purpose fine sections from transformed sugarcane leaves and stem portions were excised These sections were stained according to Jefferson (1987) with some modifications (Hiei et al 1994) X-Gluc solution (5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid Fermentas Pakistan) was prepared in a phosphate buffer and sections of the plants were incubated in this solution from 1 h to overnight Sections were destained subsequently with 70 ethanol and were observed under fluorescent microscope26 GTGene confirmation in putative transgenic plants of sugarcaneTransformed sugarcane plants of all 4 varieties were subjected to polymerase chain reaction (PCR) for transgene confirmation Genomic DNA was extracted from putative transgenic sugarcane plants along with nontransgenic plants as a control by using a genomic DNA extraction kit (Fermentas Pakistan) according to supplier instructions GTGene-specific primers 5rsquo-CATGCCATGGATGTCCCACGGTGCTT-3rsquo and 5rsquo-TCTCGGAGATCTCTAAGCAGCCTTAGTGTC-3rsquo were designed using Primer 3 software and were amplified using standard procedures (Sambrook et al 1989) The reaction mixture contained 10X PCR buffer (100 mM Tris HCl pH 83 500 mM KCl 25 mM MgCl2) 1 mM dNTPs forward and reverse primers 1 U of Taq polymerase 50 ng of template DNA and ultrapure autoclaved water in a final volume of 20 microL Cycling conditions were 94 degC for 4 min followed by 40 cycles of 94 degC for 45 s 59 degC for 45 s and 72 degC for 120 min with a final extension at 72 degC for 10 min27 Enzyme-linked immunosorbent assay (ELISA) and dipstick assayTotal crude protein was isolated from GTGene-transgenic sugarcane plants Initially a gene-specific monoclonal IgG dipstick assay was done using coated sticks (Envirologix Brazil) The assay was done by simply dipping the coated stick in crude protein dissolved in buffer for 15ndash30 min The quantification of the transgene protein was done by ELISA according to the manufacturerrsquos protocol (Envirologix)28 Multiplication of transgenic sugarcane plants Positive transgenic sugarcane plants were multiplied by placing them on shooting media supplemented with 2 mgL kinetin 2 mgL 6-benzylaminopurine (BAP) 2 mgL gibberellic acid (GA3) and 1 mgL indole-3-acetic acid (IAA) followed by root induction using 2 mgL α-naphthalene acetic acid (NAA) and 1 mgL indole-3-butyric acid (IBA) (Table 1) Multiplied transgenic plantlets having CEMB-GTG transgenes were transferred to soil in the field for the glyphosate spray assay after acclimatization Root formation in culture is beneficial for

enhancing acclimatization of transgenic sugarcane plants The plant is said to be acclimatized when new leaves and roots develop in the field environment Each transformed cultivar was grown in separate lanes along with the control nontransgenic sugarcane plants in the field for analysis29 Glyphosate spray assayHerbicide glyphosate which is commercially available as Roundup was prepared in water at final concentrations of 900 mL and 1100 mL 80 L waterndash1 0404 handash1 and was sprayed in the morning hours when the temperature was about 20 degC onto transgenic sugarcane plants from the 3- to 7-leaf stage with the help of a spray machine to ensure an equal spray application at a height of 46 cm All plants of each transformed sugarcane cultivar were grown in separate lines along with the control nontransformed sugarcane plants which were grown on the sides of the plot area that was intended for transgenic sugarcane field trails Two-month-old field-grown sugarcane plants were initially sprayed with glyphosate at a concentration of 900 mL80 L of water and observations were made routinely for up to 15 days Subsequently the next spray dose of glyphosate at 1100 mL80 L of water was applied The effects on weedsherbs and transgenic plants were observed routinely for up to 15 days

3 Results31 Optimization of regeneration for sugarcaneIn sugarcane cultivars CPF-234 CPF-213 HSF-240 and CPF-246 calli appeared on explant edges or cut surfaces within 6ndash7 days while the whole explant turned into callus within about 15 days Calli were observed on all media tested in the study however the difference was in the embryogenic capability of each callus formed On CM1 and CM2 media the callus was morphologically friable green and embryogenic with the appearance of somatic embryos as shown in Figures 2a and 2b However the calli obtained on CM3 and CM4 media were whitish and powdery in appearance and did not have regeneration potential as revealed later in further regeneration studies

Regeneration response for the callus induction varied for each sugarcane variety 24-D in concentrations ranging from 15 to 4 mgL resulted in prolonged maintenance of the callus the best response was achieved at 1ndash2 mgL concentration with the best regeneration after a 3-month period An increase in the concentration of 24-D resulted in a decrease or loss of regeneration potential The best regeneration response was exhibited by cultivars 234 and 246 on Reg 4 (3 mgL kinetin + 3 mgL BAP) and Reg 2 (2 mgL kinetin + 2 mgL BAP + 1 mgL GA3) media respectively The best regeneration in cultivars 213 and 240 was observed on Reg 2

In summary cultivar 234 gave the best calli response on CM1 media with subsequent regeneration on Reg 4


443

media Similarly for cultivar 246 the best callus response was on CM1 media while successful regeneration was achieved on Reg 2 media Cultivar 213 exhibited friable callus response on CM2 media with regeneration on Reg 2 media For cultivar 240 calli were achieved on CM2 media while successful regeneration was observed on Reg

2 media However all cultivars gave excellent shooting and rooting response on one medium as shown in Table 132 Transformation of glyphosate-tolerant gene in sugarcaneOne hundred plates of calli of each sugarcane cultivar were used to create the glyphosate-tolerant gene construct Each

Figure 2 Tissue culturing of sugarcane a b) In vitro induction of friable embryogenic callus from a growing sugarcane leaf tip explant c d) Regeneration of small shoots emerging as a bunch from calli masses e f) Multiplication from a single shoot in sugarcane and complete plantlets with growing root


444

plate contained 8 pieces of calli mass The bombarded calli started regeneration and microshoots became visible after 2 weeks of culture on the regeneration media Figures 2c and 2d depict small shoots emerging from callus mass Regenerated plantlets were subjected to histochemical GUS assay for the initial screening the presence of blue color in cross-sections of leaf and stem explants confirmed putative transgenic plants (Figure 3) On the basis of the GUS screening transformation efficiency was calculated for each sugarcane cultivar Cultivars 246 and 234 showed 22 and 32 efficiency while transformation efficiency was 17 and 13 for cultivars 213 and 240 respectively (Figure 4)

The putative transgenic shoots were further dissected and single shoots were shifted to rooting media and then transferred to soil in the field Putative transgenic plants of each sugarcane variety were shifted to soil for acclimatization and were later subjected to molecular analysis Survival efficiency of each transgenic sugarcane cultivar varied from 78 for cultivar 246 to 647 72 and 88 for cultivars 240 213 and 234 respectively as compared to control nontransformed sugarcane plants which showed a 90 survival rate when shifted to soil in the field

33 Detection of the glyphosate-tolerant gene From the 2-month-old acclimatized putative transformed sugarcane plants the transgene was detected through PCR by using gene specific primers A 1368-bp amplification was obtained in almost all GUS-positive transgenic plants of varieties 234 213 240 and 246 (Figure 5) No amplification was observed in negative control plants but amplification was observed in the positive control plasmid

Furthermore PCR-positive plants were screened for their protein expression initially via dipstick assays The presence of a band at the place of the test line along with the control confirmed the expression of the transgene The quantification was done by ELISA and optical density for the transgene protein was found in the range of 02 to 1957 It was clear from the results that a varied expression of the transgene was seen in transgenic sugarcane plants as shown in Figure 6 and Table 2 34 Herbicide spray trialsRoundup was used as the herbicide against the transgenic sugarcane plants In total 60 transgenic sugarcane plants of each cultivar were grown in the field which were all homogeneous with respect to size and other morphological characters as were the nontransformed control plants After 2 months of field growth transgenic

Figure 3 GUS histological assay of transgenic sugarcane plants Greenish blue patterns of GUS expression as indicated by arrows are clearly visible in a c) leaf explant and b d) stem explant Exemplary transformed plants were taken at random and GUS staining was performed visualized under a fluorescent microscope


445

plants were sprayed in 2 separate and subsequent doses of Roundup at concentrations of 900 mL and 1100 mL80 L of water The initial Roundup spray application was of 900 mL80 L of water where nearly 10ndash12 of the sugarcane plants died 6ndash7 days after spraying including the control nontransgenic plants After the second spray application of 1100 mL80 L almost 30 of the transgenic sugarcane plants that exhibited a transgene protein OD range of 02 to 09 turned brown and died subsequently while the plants having transgene optical density ranges of above 10 recovered later and remained healthy (Figure 7) This observed correlation between the transgene protein expression and the glyphosate tolerance in transgenic plants is depicted in Table 2 It was observed that the maximum tolerance to glyphosate was exhibited by transgenic plants with a transgene protein OD range of 19ndash20 whereas

transgenic plants having low transgene protein (OD of less than 10) did not survive the second spray dose of the herbicide Weeds growing in the field turned brown with necrotic margins 5ndash6 days after herbicide application and died completely after 12ndash15 days The transgenic sugarcane plants surviving the second glyphosate application recovered completely 7ndash10 days after spray application These findings strongly support the hypothesis that with the increase in transgene expression resistance to herbicides in genetically modified plants increases Table 2 demonstrates that as the transgene protein expression increases tolerance to glyphosates increases proportionally Thus in these plants integrated EPSPS has strong protein expression they are in a better position to make a quick recovery after herbicide treatments

4 DiscussionThe main purpose of the current study was to develop herbicide tolerance in elite sugarcane varieties in Pakistan Many herbicide-resistant crops have been developed previously around the world including nonglyphosate herbicide-resistant transgenes like 24-D (Bisht et al 2004) Dicamba (Herman et al 2005) HPPD inhibitors (Matringe et al 2005) and bar gene (Aasim et al 2013) However while they were all primarily developed for a single type of herbicide glyphosate-tolerant crops comprise a broad spectrum and are not limited to a single type of herbicide To date 9 glyphosate-tolerant crops have been developed including soybean cotton corn canola polish canola alfalfa sugar beet creeping bentgrass and wheat (httpwwwagbioscomdbasephp) All except creeping bentgrass and wheat have been grown commercially However this is the first report of development of glyphosate-tolerant sugarcane

0

10

20

30

40

Perc

enta

ge

246 234 213 240 Sugarcane cultivars

Percentage transformation


Figure 4 Transformation efficiency in different varieties of sugarcane It was 22 for cultivar 246 32 for 234 17 for 213 and 13 for 240

Figure 5 PCR amplification of GTGene from transformed sugarcane plants Sharp amplification at 1368 bp was clearly visible in some plants whereas no amplification was observed in nontransformed control plants Samples 1ndash9 belong to the transformed sugarcane plants that were taken at random for PCR amplification of transgene


446

0

05

1

15

2

25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Opt

ical

den

sity

Plant number Figure 6 Optical density of GTG protein quantified in transgenic sugarcane plants Range of OD was 02ndash1957 Optical density was calculated after EPSP-specific ELISA antibodies

Table 2 Correlation of transgene protein OD with the level of glyphosate tolerance conferred by transgenic sugarcane plants (+ Slightly tolerant ++ Moderately tolerant +++ Tolerant ++++ Highly tolerant)

SNo Transgeneprotein OD cv 234

Glyphosate tolerance

Transgene proteinOD cv 213


Transgene protein OD cv 240

Glyphosatetolerance


Glyphosatetolerance

Positive 0258 - 0258 - 0258 - 0258 -

Negative 000 - 000 - 000 - 000 -

1 0778 ++ 1271 +++ 0789 ++ 196 ++++

2 1336 +++ 1642 +++ 1731 +++ 183 +++

3 1148 +++ 1189 +++ 0748 ++ 0375 +

4 1188 +++ 0789 ++ 0818 ++ 0439 +

5 1957 ++++ 1731 +++ 2004 ++++ 098 ++

6 1853 +++ 0748 ++ 1326 +++ 179 +++

7 0375 + 0462 + 1853 +++ 117 +++

8 0439 + 1217 +++ 0375 + 165 +++

9 0984 +++ 0818 ++ 0439 + 177 +++

10 0816 +++ 2004 ++++ 1188 +++ 04 +

11 1928 ++++ 1326 +++ 195 ++++ 0375 +

12 0691 ++ 1906 ++++ 0375 + 0439 +

13 186 +++ 189 ++++ 0439 + 1188 ++++

14 186 +++ 1297 +++ 0984 ++ 1336 +++

15 1336 +++ 0375 + 1336 +++ 1148 +++

16 1148 +++ 0439 + 1148 +++ 1188 +++

17 1336 +++ 0984 ++ 1336 +++ 032 +

18 1148 +++ 1336 +++ 1777 +++ 05 +

19 179 +++ 1148 +++ 04 + 1326 +++

20 1157 +++ 1336 +++ 0267 + 1906 ++++

21 1685 +++ 1148 +++ 145 +++ 189 ++++

22 1777 +++ 1336 +++ 0439 + 098 ++

23 04 + 1148 +++ 0984 ++ 032 +

24 0267 + 032 + 0375 + 05 +

25 145 +++ 05 + 0439 + 135 +++


447

The agroclimatic conditions of Pakistan are quite favorable for sugarcane cultivation Pakistan has the fourth largest area in the world under sugarcane cultivation but the yield per unit area is one of the lowest (wwwpakissancom) The lack of a reproducible methodology for the stable transformation of sugarcane has been an important obstacle to its genetic manipulation for many years In the present study an attempt was made to develop an efficient method of regeneration for sugarcane calli and strong tolerance in transgenic plants against herbicide For this study 4 sugarcane varieties were selected namely cultivars CPF-234 CPF-213 HSF-240 and CPF-246 and their young immature leaves were used as explants to induce calli Snyman et al (2006) also suggested that immature leaves of sugarcane prove to be an excellent explant for production of an embryogenic friable callus Optimization for friable embryogenic calli is not only mandatory for genetic manipulation of sugarcane but also strengthens the development of the transformation system in sugarcane (Fitch et al 2001) In our study friable and embryogenic calli of all 4 cultivars were obtained on media containing 24-D casein was added to enhance embryogenic potential of the callus Similar studies were reported by Weng et al

(2006) and Joyce et al (2010) In several other studies 24-D was found to be mandatory for production of friable and embryogenic calli in sugarcane (Snyman 2004 Ramanand et al 2005 Franklin et al 2006 Basnayake et al 2011 Nawaz et al 2013) Our aim was to induce microshoots on calli masses rather than to pursue somatic embryogenesis Therefore we regenerated the calli into microshoots and subsequently root induction was initiated The degree of callogenesis and regeneration varied among the 4 sugarcane varieties which according to Joshi et al (2011) might be due to their genotypic variability It was also found in the present study that if the concentration of 24-D in callus media increased above 4 mgL the regeneration efficiency of the respective calli was seriously affected At higher concentrations a complete loss of regeneration potential was the outcome Our finding are supported by Chengalrayan et al (2005) who found that 24-D at up to 6 mgL reduced the fresh mass of the calli

For this purpose a glyphosate-tolerant gene was introduced in local elite sugarcane varieties CPF-234 CPF-213 HSF-240 and CPF-246 It is clear from the results (Figure 7 Table 2) that transgenic sugarcane showed an increased tolerance to glyphosate application (1100 mL

Figure 7 Glyphosate spray assay trial in sugarcane field a) Two-month-old growing transgenic sugarcane plants in fields weeds flourishing well as clearly visible b) Glyphosate spray application at dose of 900 mL80 L water Weeds died 15 days after glyphosate application and some transgenic plants that exhibited GTG protein OD below 10 also turned brown as marked by arrows c d) Spray dose application of 1100 mL80 L water Weeds died 15 days after spray application whereas transgenic sugarcane plants recovered 7ndash10 days after spray dose and remained green and healthy subsequently


448

80 Lndash1 0404 handash1) as compared to control nontransgenic sugarcane plants We applied glyphosate at 2 rates (800 mL and 1100 mL0404 ha) to get the estimated tolerance that the transgenic plants acquired At the first application of glyphosate only the weeds and nontransgenic plants died None of the transgenic plants died although a few plants did have brown leaf symptoms To further evaluate the level of glyphosate tolerance in transgenic sugarcane plants a second application of spray at the rate of 1100 mL was applied As a result necrotic symptoms developed on the control nontransformed sugarcane plants which ultimately lead to their death It was also found that transgenic sugarcane plants exhibited variable concentrations of transprotein in them Plants having an OD range of transprotein of less than 10 turned brown and did not survive a glyphosate dose of 1100 mL80 L while the plants having an OD range of above 10 did survive It can be concluded that with the increase in transgene expression tolerance to glyphosate increases in transgenic plants while variability in transgene expression could be dependent on the integration position of the transgene in the plant Moreover transgenic sugarcane plants initially showed necrotic symptoms and turned light brown but then later recovered 7ndash10 days after glyphosate application The explanation behind this recovery is that the mode of action of the herbicide glyphosate is to inhibit the enzyme EPSPS which is involved in the synthesis of aromatic amino acids like tyrosine phenylalanine and tryptophan When we apply glyphosate spray to the sugarcane plants the naturally present EPSPS enzyme is inhibited However for plants with the glyphosate-tolerant transgene no inhibition will be effective as the transgene has modified the natural sequence of EPSPS which remains active and triggers the transgenic plant towards recovery from glyphosate spray toxicity Therefore we have transgenic sugarcane plants that start recovering from the toxic effects of the glyphosate

The results were reproducible however minor differences in transformation efficiency for each variety were observed As the presence of undesirable plants in sugarcane field reduces yield significantly the development of herbicide-tolerant sugarcane plants will prove advantageous in this respect Furthermore the development of an efficient transformation system will allow biotechnologists to improve sugarcane against pest attacks fungal resistance insect resistance and other related diseases Studies with glyphosate-resistant wheat by Feng et al (2005) found that glyphosate provided both preventive and curative activities against rust diseases of wheat caused by Puccinia striiformis f sp tritici and Puccinia triticina Growth-chamber studies demonstrated wheat rust control at multiple plant growth stages using a glyphosate spray dose typically recommended for weed control Mitchell (2003) reported successful control of weeds in glyphosate-tolerant sugar beet crops (Beta vulgaris) Three weed control trials were carried out with sugar beet and it was found that the variety used in 2 subsequent years was fully tolerant no plant loss was observed even at the highest glyphosate dose Similarly herbicide-resistant sugarcane was successfully developed by Zambrano et al (2003) and Manickavasagam et al (2004) The stability of gene expression and agronomic performance of a transgenic herbicide-resistant sugarcane was evaluated by Leibbrandt et al (2003) From this study it was concluded that a glyphosate-tolerant transgene is functional in transgenic sugarcane plants at a level that can tolerate the glyphosate application at a dosage that all the weeds and control plants were unable to survive

AcknowledgmentsWe extend our thanks to the Punjab Agriculture Research Board for providing funds and to the Ayub Agriculture Research Institute Faisalabad for providing the plant material to conduct these experiments

References

Aasim M Khawar KM Oumlzcan S (2013) Production of herbicide-resistant cowpea (Vigna unguiculata L) transformed with the bar gene Turk J Biol 37 472ndash478

Ahmad S Saleem S Zubair M Khalil IA Sohail K Rehman Z (2012) Farmersrsquo practices and yield response of sugarcane in Jhang and Sargodha districts Pakistan Sarhad J Agric 28 201

Barampuram S Zhang ZJ (2011) Recent advances in plant transformation Plant chromosome engineering Methods Mol Biol 701 1ndash35

Basnayake SW Moyle R Birch RG (2011) Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars Plant Cell Rep 30 439ndash348

Bisht NC Burma PK Pental D (2004) Development of 24-D-resistant transgenics in Indian oilseed mustard (Brassica juncea) Curr Sci 87 367ndash370

Busse MD Ratcliffe AW Shestak CJ Powers RF (2001) Glyphosate toxicity and the effects of long-term vegetation control on soil microbial community Soil Biol Biochem 33 1777ndash1789

Castle LA Siehl DL Gorton R Patten PA Chen YH Bertain S Cho HJ Duck N Wong J Liu D et al (2004) Discovery and directed evolution of a glyphosate tolerance gene Science 304 1151ndash1154

Chengalrayan K Abouzid A Gallo-Meagher M (2005) In vitro regeneration of plants from sugarcane seed-derived callus In Vitro Cell Dev 41 477ndash482


449

Chengalrayan K Gallo-Meagher M (2001) Effect of various growth regulators on shoot regeneration of sugarcane In Vitro Cell Dev 37 434ndash439

Feng PCC Baley GJ Clinton WP Bunkers GJ Alibhai MF Paulitz TC Kidwell KK (2005) Glyphosate inhibits rust diseases in glyphosate-resistant wheat and soybean P Natl Acad Sci USA 102 17290ndash17295

Fitch MMM Lehrer AT Komor E Moore PH (2001) Elimination of Sugarcane yellow leaf virus from infected sugarcane plants by meristem tip culture visualized by tissue blot immunoassay Plant Pathol 50 676ndash680

Franklin G Arvinth S Sheeba CJ Kanchana M Subramonian N (2006) Auxin pretreatment promotes regeneration of sugarcane (Saccharum spp hybrids) midrib segment explants Plant Growth Regul 50 111ndash119

Ge X Davignon DA Ackerman JHJ Sammons RD (2010) Rapid vacuolar sequestration the horseweed glyphosate resistance mechanism Pest Manag Sci 66 345ndash348

Giesy JP Dobson S Solomon KR (2000) Ecotoxicological risk assessment for Roundupreg herbicide Rev Environ Contam Toxicol 167 35ndash120

Herman P Behrens LMS Chakraborty BM Barycki CJ Weeks DP (2005) A three-component dicamba O-demethylase from Pseudomonas maltophilia Strain DI6 gene isolation characterization and heterozygous expression J Biol Chem 280 24759ndash24767

Hiei Y Ohta S Komari T Kumashiro T (1994) Efficient transformation of rice (Oryza sativa) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA Plant J 6 271ndash282

Jefferson RA (1987) Assaying chimeric genes in plants the genes fusion system Plant Mol Biol Rep 5 387ndash405

Joshi R Shukla A Sairam RK (2011) In vitro screening of rice genotypes for drought tolerance using polyethylene glycol Acta Physiol Plant 33 2209ndash2217

Joyce P Kuwahata M Turner N Lakshmanan P (2010) Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane Plant Cell Rep 29 173ndash183

Julian KC Chikwamba MR Sparrow P Fischer R Mahoney RR Richard M Twyman RM (2005) Plant-derived pharmaceuticals - the road forward Trends Plant Sci 10 580ndash585

Leibbrandt NB Snyman SJ (2003) Stability of gene expression and agronomic performance of a transgenic herbicide-resistant sugarcane line in South Africa Crop Sci 43 671ndash677

Manickavasagam M Ganapathi A Anbazhagan VR Sudhakar B Selvaraj N Vasudevan A Kasthurirengan S (2004) Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds Plant Cell Rep 23 134ndash143

Matringe M Sailland A Pelissier B Roland A Zind O (2005) p-Hydroxyphenylpyruvate dioxygenase inhibitor-resistant plants Pest Manag Sci 61 269ndash276

Mitchell BJ (2003) Control of weeds in glyphosate-tolerant sugar-beet crops (Beta vulgaris) Irish J Agri Food Sci Res 42 265ndash274

Murashige T Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol Plantarum 15 473ndash497

Nawaz M Ullah I Iqbal N Iqbal MZ Javed MA (2013) Improving in vitro leaf disk regeneration system of sugarcane (Saccharum officinarum L) with concurrent shootroot induction from somatic embryos Turk J Biol 37 726ndash732

Owen MDK (2008) Weed species shifts in glyphosate-resistant crops Pest Manag Sci 64 377ndash387

Paganelli A Gnazzo V Acosta H Lopez SL Carrasco AE (2010) Glyphosate-based herbicides produce teratogenic effects on vertebrates by impairing retinoic acid signaling Chem Res Toxicol 23 1586ndash1595

Pline WA Viator R Wilcut JW Edmisten KL Thomas J Wells R (2002) Reproductive abnormalities in glyphosate-resistant cotton caused by lower CP4-EPSPS levels in the male reproductive tissue Weed Science 50 438ndash447

Pline-Srnic W (2006) Physiological mechanisms of glyphosate resistance Weed Technol 20 290ndash300

Rainbolt CR Dusky JA (2006) Weed Management in Sugarcane ndash 2007 Electronic Data Information Source (EDIS) WG004 Gainesville FL USA Agronomy Department University of Florida

Ramanand KN Subhanand N Lal M Singh SB (2005) Plantlet regeneration through leaf callus culture in sugarcane Sugar Tech 8 85ndash87

Sambrook J Fritsh EF Maniatis T (1989) Molecular Cloning A Laboratory Manual 2nd ed New York NY USA Cold Spring Harbor Laboratory Press

Snyman SJ (2004) Sugarcane Transformation In Curtis I editor Transgenic Crops of the World Amsterdam the Netherlands Springer pp 103ndash114

Snyman SJ Meyer GM Richards JM Haricharan N Ramgareeb S Huckett BI (2006) Refining the application of direct embryogenesis in sugarcane effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency Plant Cell Rep 25 1016ndash1023

Tesfamariam TS Bott I Cakmak V Roumlmheld V Neumann RG (2009) Glyphosate in the rhizosphere ndash role of waiting times and different glyphosate binding forms in soils for phytotoxicity to non-target plants Eur J Agron 31 126ndash132

Vencill WK Nichols RL Webster TM Soteres JK Mallory-Smith C Burgos NR Johnson WG McClelland MR (2012) Herbicide resistance toward an understanding of resistance development and the impact of herbicide-resistant crops Weed Sci 60 2ndash30

Weng LX Deng H Xu JL Li Q Wang L Jiang Z Zhang HB Li Q Zhang L (2006) Regeneration of sugarcane elite breeding lines and engineering of stem borer resistance Pest Manag Sci 62 178ndash187

Zambrano AY Demey JR Gonzalez V (2003) In vitro selection of a glyphosate-tolerant sugarcane cellular line Plant Mol Biol Rep 21 365ndash373

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can regenerate The introduction of herbicide-resistant crops has dramatically changed weed management in crop production systems (Owen 2008 Vencill et al 2012) Using genetic engineering as a complement for traditional breeding methods it is possible to introduce herbicide-tolerant traits into the Saccharum germplasm The absence of herbicide-tolerant genes in the genetic pool of wild relatives of sugarcane makes traditional breeding programs difficult The varieties that are productive and at the same time have resistance to certain pathogens and diseases can improve yield Development of herbicide-resistant sugarcane varieties through transgenic technology will be cost-effective in controlling weeds which ultimately improves yield potential To date several crops have been genetically modified for resistance against herbicides The most widely adopted biotech crop the Roundup Ready soybean was developed and released commercially by Monsanto it contains a version of the EPSPS gene (glyphosate) from Agrobacterium tumefaciens

Currently weeds in sugarcane crops are being controlled by the application of certain organophosphorus herbicides like Roundup The active ingredient of Roundup is glyphosate which is a nonselective broad-spectrum herbicide that translocates symplastically to the meristems of the growing plants It is well known for its high effectiveness low toxicity low residues in organisms and soil and overall limited environmental impact However heavy use of glyphosate-based herbicides increases the emergence of glyphosate-resistant weeds It was also reported that once glyphosate travels to a plantrsquos roots it is released into the rhizosphere (the area immediately around the roots) where it is immobilized at the soil matrix (Tesfamariam et al 2009) which leads to disruption of soil and root microbial communities (Busse et al 2001) Studies have found that about 1ndash2 of glyphosate may run off in rainfall after glyphosate is applied (Giesy et al 2000) Another study indicated that low doses of glyphosate cause birth defects in frogs and chickens (Paganelli et al 2010)

In plants including weeds glyphosate inhibits chloroplast-localized EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase) (Pline et al 2002 Castle et al 2004) Overexpression of the EPSPS gene in cells renders glyphosate tolerance in transformed plants (Castle et al 2004 Pline-Srnic 2006) Two mechanisms have been reported underlying glyphosate tolerance The first is characterized by the overexpression of the EPSPS gene while the second is linked to a herbicide-insensitive enzyme (Ge et al 2010)

The genetic manipulation of sugarcane requires the availability of an efficient transformation system that directly depends on the embryogenic capability of the callus and its subsequent regeneration (Barampuram and Zhang

2011) In the present study 4 elite sugarcane varieties from Pakistan with good yield potential were selected to become herbicide (glyphosate)-tolerant The glyphosate-tolerant gene accession JF4452901 was modified for the codon belonging to sugarcane as identified through OptimumGene software and was submitted locally under patent application 7652010 in Karachi Pakistan Moreover a reliable and efficient transformation system was also developed based on the optimization of calli and regeneration conditions in sugarcane

2 Materials and methods21 Plant material and callus inductionFour elite sugarcane varieties from Pakistan namely SPE-234 CPF-246 CPF-213 and HSF-240 were selected for genetic modification with a glyphosate-tolerant gene The varieties were kindly provided by the Ayub Agriculture Research Centre Faisalabad A leaf roll of 20ndash30 cm in length containing the apical meristem was excised from the plants and sterilized using 001 mercuric chloride solution The sterilized leaf roll was further dissected to reveal a stage 3 leaf whose transverse sections were used subsequently as explants for callus induction

For callus induction MS basal medium (Murashige and Skoog 1962) supplemented with sucrose (3) 24-dichlorophenoxyacetic acid (24-D 15 to 4 mgL) myo-inositol (200 mgL) and casein (1 gL) in different combinations was used (Table 1) The medium was solidified with 25 gL Phytagel after adjusting the pH to 57 followed by autoclaving at 121 degC and 1034 kPa for 20 min Transverse segments from immature sugarcane leaves were used as explants and a response was observed on all combinations of media at repeated intervals Subculturing was done at a regular interval of 7 days After 15 days calli were observed under the microscope and nodular compact white calli were marked as embryogenic to be used subsequently for transformation All cultures were kept at 25 plusmn 2 degC under a 168 photoperiod Calli pieces were placed in the middle of petri plates having MS media for 3ndash4 h with additional 3 gL Phytagel added to keep calli pieces in place prior to bombardment22 Plasmid constructionThe glyphosate-tolerant gene (GTGene patent application 7652010 Karachi Pakistan) was cloned directionally in plant expression vector pCAMBIA1301 (Cambia Australia) which was driven by Cauliflower mosaic virus 35S promoter and the nopaline synthase 3rsquo-end terminator For directional cloning NcoI and BglII restriction enzymes were used In pCAMBIA1301 the β-glucuronidase (GUS) exon was used as a reporter gene in the cassette (Figure 1) 23 Transformation of CEMB-GT gene in sugarcaneOne milliliter of absolute ethanol was mixed with 60 mg of tungsten particles followed by centrifugation at 10000


441















CaMV 35S promoter

GTGene NOS Poly A

T-Border (L)


GUS intron



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Glyphosatetolerance


Glyphosatetolerance

Positive 0258 - 0258 - 0258 - 0258 -

Negative 000 - 000 - 000 - 000 -

1 0778 ++ 1271 +++ 0789 ++ 196 ++++

2 1336 +++ 1642 +++ 1731 +++ 183 +++

3 1148 +++ 1189 +++ 0748 ++ 0375 +

4 1188 +++ 0789 ++ 0818 ++ 0439 +

5 1957 ++++ 1731 +++ 2004 ++++ 098 ++

6 1853 +++ 0748 ++ 1326 +++ 179 +++

7 0375 + 0462 + 1853 +++ 117 +++

8 0439 + 1217 +++ 0375 + 165 +++

9 0984 +++ 0818 ++ 0439 + 177 +++

10 0816 +++ 2004 ++++ 1188 +++ 04 +

11 1928 ++++ 1326 +++ 195 ++++ 0375 +

12 0691 ++ 1906 ++++ 0375 + 0439 +

13 186 +++ 189 ++++ 0439 + 1188 ++++

14 186 +++ 1297 +++ 0984 ++ 1336 +++

15 1336 +++ 0375 + 1336 +++ 1148 +++

16 1148 +++ 0439 + 1148 +++ 1188 +++

17 1336 +++ 0984 ++ 1336 +++ 032 +

18 1148 +++ 1336 +++ 1777 +++ 05 +

19 179 +++ 1148 +++ 04 + 1326 +++

20 1157 +++ 1336 +++ 0267 + 1906 ++++

21 1685 +++ 1148 +++ 145 +++ 189 ++++

22 1777 +++ 1336 +++ 0439 + 098 ++

23 04 + 1148 +++ 0984 ++ 032 +

24 0267 + 032 + 0375 + 05 +

25 145 +++ 05 + 0439 + 135 +++


447






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441















CaMV 35S promoter

GTGene NOS Poly A

T-Border (L)


GUS intron



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Glyphosatetolerance


Glyphosatetolerance

Positive 0258 - 0258 - 0258 - 0258 -

Negative 000 - 000 - 000 - 000 -

1 0778 ++ 1271 +++ 0789 ++ 196 ++++

2 1336 +++ 1642 +++ 1731 +++ 183 +++

3 1148 +++ 1189 +++ 0748 ++ 0375 +

4 1188 +++ 0789 ++ 0818 ++ 0439 +

5 1957 ++++ 1731 +++ 2004 ++++ 098 ++

6 1853 +++ 0748 ++ 1326 +++ 179 +++

7 0375 + 0462 + 1853 +++ 117 +++

8 0439 + 1217 +++ 0375 + 165 +++

9 0984 +++ 0818 ++ 0439 + 177 +++

10 0816 +++ 2004 ++++ 1188 +++ 04 +

11 1928 ++++ 1326 +++ 195 ++++ 0375 +

12 0691 ++ 1906 ++++ 0375 + 0439 +

13 186 +++ 189 ++++ 0439 + 1188 ++++

14 186 +++ 1297 +++ 0984 ++ 1336 +++

15 1336 +++ 0375 + 1336 +++ 1148 +++

16 1148 +++ 0439 + 1148 +++ 1188 +++

17 1336 +++ 0984 ++ 1336 +++ 032 +

18 1148 +++ 1336 +++ 1777 +++ 05 +

19 179 +++ 1148 +++ 04 + 1326 +++

20 1157 +++ 1336 +++ 0267 + 1906 ++++

21 1685 +++ 1148 +++ 145 +++ 189 ++++

22 1777 +++ 1336 +++ 0439 + 098 ++

23 04 + 1148 +++ 0984 ++ 032 +

24 0267 + 032 + 0375 + 05 +

25 145 +++ 05 + 0439 + 135 +++


447






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Opt

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Glyphosatetolerance


Glyphosatetolerance

Positive 0258 - 0258 - 0258 - 0258 -

Negative 000 - 000 - 000 - 000 -

1 0778 ++ 1271 +++ 0789 ++ 196 ++++

2 1336 +++ 1642 +++ 1731 +++ 183 +++

3 1148 +++ 1189 +++ 0748 ++ 0375 +

4 1188 +++ 0789 ++ 0818 ++ 0439 +

5 1957 ++++ 1731 +++ 2004 ++++ 098 ++

6 1853 +++ 0748 ++ 1326 +++ 179 +++

7 0375 + 0462 + 1853 +++ 117 +++

8 0439 + 1217 +++ 0375 + 165 +++

9 0984 +++ 0818 ++ 0439 + 177 +++

10 0816 +++ 2004 ++++ 1188 +++ 04 +

11 1928 ++++ 1326 +++ 195 ++++ 0375 +

12 0691 ++ 1906 ++++ 0375 + 0439 +

13 186 +++ 189 ++++ 0439 + 1188 ++++

14 186 +++ 1297 +++ 0984 ++ 1336 +++

15 1336 +++ 0375 + 1336 +++ 1148 +++

16 1148 +++ 0439 + 1148 +++ 1188 +++

17 1336 +++ 0984 ++ 1336 +++ 032 +

18 1148 +++ 1336 +++ 1777 +++ 05 +

19 179 +++ 1148 +++ 04 + 1326 +++

20 1157 +++ 1336 +++ 0267 + 1906 ++++

21 1685 +++ 1148 +++ 145 +++ 189 ++++

22 1777 +++ 1336 +++ 0439 + 098 ++

23 04 + 1148 +++ 0984 ++ 032 +

24 0267 + 032 + 0375 + 05 +

25 145 +++ 05 + 0439 + 135 +++


447






448




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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

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Glyphosatetolerance


Glyphosatetolerance

Positive 0258 - 0258 - 0258 - 0258 -

Negative 000 - 000 - 000 - 000 -

1 0778 ++ 1271 +++ 0789 ++ 196 ++++

2 1336 +++ 1642 +++ 1731 +++ 183 +++

3 1148 +++ 1189 +++ 0748 ++ 0375 +

4 1188 +++ 0789 ++ 0818 ++ 0439 +

5 1957 ++++ 1731 +++ 2004 ++++ 098 ++

6 1853 +++ 0748 ++ 1326 +++ 179 +++

7 0375 + 0462 + 1853 +++ 117 +++

8 0439 + 1217 +++ 0375 + 165 +++

9 0984 +++ 0818 ++ 0439 + 177 +++

10 0816 +++ 2004 ++++ 1188 +++ 04 +

11 1928 ++++ 1326 +++ 195 ++++ 0375 +

12 0691 ++ 1906 ++++ 0375 + 0439 +

13 186 +++ 189 ++++ 0439 + 1188 ++++

14 186 +++ 1297 +++ 0984 ++ 1336 +++

15 1336 +++ 0375 + 1336 +++ 1148 +++

16 1148 +++ 0439 + 1148 +++ 1188 +++

17 1336 +++ 0984 ++ 1336 +++ 032 +

18 1148 +++ 1336 +++ 1777 +++ 05 +

19 179 +++ 1148 +++ 04 + 1326 +++

20 1157 +++ 1336 +++ 0267 + 1906 ++++

21 1685 +++ 1148 +++ 145 +++ 189 ++++

22 1777 +++ 1336 +++ 0439 + 098 ++

23 04 + 1148 +++ 0984 ++ 032 +

24 0267 + 032 + 0375 + 05 +

25 145 +++ 05 + 0439 + 135 +++


447






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Glyphosatetolerance


Glyphosatetolerance

Positive 0258 - 0258 - 0258 - 0258 -

Negative 000 - 000 - 000 - 000 -

1 0778 ++ 1271 +++ 0789 ++ 196 ++++

2 1336 +++ 1642 +++ 1731 +++ 183 +++

3 1148 +++ 1189 +++ 0748 ++ 0375 +

4 1188 +++ 0789 ++ 0818 ++ 0439 +

5 1957 ++++ 1731 +++ 2004 ++++ 098 ++

6 1853 +++ 0748 ++ 1326 +++ 179 +++

7 0375 + 0462 + 1853 +++ 117 +++

8 0439 + 1217 +++ 0375 + 165 +++

9 0984 +++ 0818 ++ 0439 + 177 +++

10 0816 +++ 2004 ++++ 1188 +++ 04 +

11 1928 ++++ 1326 +++ 195 ++++ 0375 +

12 0691 ++ 1906 ++++ 0375 + 0439 +

13 186 +++ 189 ++++ 0439 + 1188 ++++

14 186 +++ 1297 +++ 0984 ++ 1336 +++

15 1336 +++ 0375 + 1336 +++ 1148 +++

16 1148 +++ 0439 + 1148 +++ 1188 +++

17 1336 +++ 0984 ++ 1336 +++ 032 +

18 1148 +++ 1336 +++ 1777 +++ 05 +

19 179 +++ 1148 +++ 04 + 1326 +++

20 1157 +++ 1336 +++ 0267 + 1906 ++++

21 1685 +++ 1148 +++ 145 +++ 189 ++++

22 1777 +++ 1336 +++ 0439 + 098 ++

23 04 + 1148 +++ 0984 ++ 032 +

24 0267 + 032 + 0375 + 05 +

25 145 +++ 05 + 0439 + 135 +++


447






448




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Glyphosatetolerance


Glyphosatetolerance

Positive 0258 - 0258 - 0258 - 0258 -

Negative 000 - 000 - 000 - 000 -

1 0778 ++ 1271 +++ 0789 ++ 196 ++++

2 1336 +++ 1642 +++ 1731 +++ 183 +++

3 1148 +++ 1189 +++ 0748 ++ 0375 +

4 1188 +++ 0789 ++ 0818 ++ 0439 +

5 1957 ++++ 1731 +++ 2004 ++++ 098 ++

6 1853 +++ 0748 ++ 1326 +++ 179 +++

7 0375 + 0462 + 1853 +++ 117 +++

8 0439 + 1217 +++ 0375 + 165 +++

9 0984 +++ 0818 ++ 0439 + 177 +++

10 0816 +++ 2004 ++++ 1188 +++ 04 +

11 1928 ++++ 1326 +++ 195 ++++ 0375 +

12 0691 ++ 1906 ++++ 0375 + 0439 +

13 186 +++ 189 ++++ 0439 + 1188 ++++

14 186 +++ 1297 +++ 0984 ++ 1336 +++

15 1336 +++ 0375 + 1336 +++ 1148 +++

16 1148 +++ 0439 + 1148 +++ 1188 +++

17 1336 +++ 0984 ++ 1336 +++ 032 +

18 1148 +++ 1336 +++ 1777 +++ 05 +

19 179 +++ 1148 +++ 04 + 1326 +++

20 1157 +++ 1336 +++ 0267 + 1906 ++++

21 1685 +++ 1148 +++ 145 +++ 189 ++++

22 1777 +++ 1336 +++ 0439 + 098 ++

23 04 + 1148 +++ 0984 ++ 032 +

24 0267 + 032 + 0375 + 05 +

25 145 +++ 05 + 0439 + 135 +++


447






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_GoBack


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Opt

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Glyphosatetolerance


Glyphosatetolerance

Positive 0258 - 0258 - 0258 - 0258 -

Negative 000 - 000 - 000 - 000 -

1 0778 ++ 1271 +++ 0789 ++ 196 ++++

2 1336 +++ 1642 +++ 1731 +++ 183 +++

3 1148 +++ 1189 +++ 0748 ++ 0375 +

4 1188 +++ 0789 ++ 0818 ++ 0439 +

5 1957 ++++ 1731 +++ 2004 ++++ 098 ++

6 1853 +++ 0748 ++ 1326 +++ 179 +++

7 0375 + 0462 + 1853 +++ 117 +++

8 0439 + 1217 +++ 0375 + 165 +++

9 0984 +++ 0818 ++ 0439 + 177 +++

10 0816 +++ 2004 ++++ 1188 +++ 04 +

11 1928 ++++ 1326 +++ 195 ++++ 0375 +

12 0691 ++ 1906 ++++ 0375 + 0439 +

13 186 +++ 189 ++++ 0439 + 1188 ++++

14 186 +++ 1297 +++ 0984 ++ 1336 +++

15 1336 +++ 0375 + 1336 +++ 1148 +++

16 1148 +++ 0439 + 1148 +++ 1188 +++

17 1336 +++ 0984 ++ 1336 +++ 032 +

18 1148 +++ 1336 +++ 1777 +++ 05 +

19 179 +++ 1148 +++ 04 + 1326 +++

20 1157 +++ 1336 +++ 0267 + 1906 ++++

21 1685 +++ 1148 +++ 145 +++ 189 ++++

22 1777 +++ 1336 +++ 0439 + 098 ++

23 04 + 1148 +++ 0984 ++ 032 +

24 0267 + 032 + 0375 + 05 +

25 145 +++ 05 + 0439 + 135 +++


447






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herbicide-tolerant sugarcane (saccharum officinarum l.) plants:...

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