Development of transgenic tobacco lines with pyramided insect resistant genes
Insect pests are among the major constraints rendering drastic decreases in crop yield. The expression of stacked insecticidal
genes in crops can lead to resistance durability and can delay the development of resistance in target insect pests. The present study
was designed to introduce an insect resistance trait in locally cultivated Turkish tobacco cultivars (Basma and Nail) with pyramided
insecticidal genes. Agrobacterium strain LBA4404 harboring plasmid pKGH4 with
cry1Ac
and
cry2A
genes under the control of 35S
promoter was used to infect leaf discs of both cultivars; plasmid also contained
uidA
within the T-DNA region for earlier screening
of putative transformants. The overall transformation efficiency was calculated as 30.7% and 18.8% in Basma and Nail, respectively.
PCR results confirmed the integration of
cry1Ac
,
cry2A
,
uidA
, and
nptII
genes in 40 plants of Basma and 16 plants of Nail. ELISA
results showed variation in expression of cry1Ac protein among transgenic plants varying from 0.017 to 0.607 μg/g of fresh tissue.
Bioassay results with potato tuber moth (
Phthorimea operculella
Zeller) showed significant mortality of the targeted pest on primary
transformants. Furthermore, T1 transgenic progeny exhibited the inheritance of T-DNA in Mendelian as well as non-Mendelian fashion.
The results revealed that lines can serve as a source of germplasm in tobacco breeding programs.
___
- Adamczyk JJ, Perera JO, Meredith WR (2009). Production of mRNA
from the
Cry1Ac
transgene differs among Bollgard
lines which
correlate to the level of subsequent protein. Transgenic Res 18:
143-149.
- Altman DW, Benedict JH, Sach ES (1996). Transgenic plants for
the development of durable insect resistance, a case study for
cotton and
Bacillus thuringiensis.
Ann NY Acad Sci 792: 106-
114.
- Anayol E, Bakhsh A, Karakoç ÖC, Onarıcı S, Köm D, Aasim
M, Özcan SF, Barpete S, Khabbazi SD, Önol B et al. (2016).
Towards better insect management strategy: restriction of
insecticidal gene expression to biting sites in transgenic cotton.
Plant Biotechnol Rep 10: 83-94.
- Ansari MR, Shaheen T, Bukhari SA, Husnain T (2015). Genetic
improvement of rice for biotic and abiotic stress tolerance.
Turk J Biol 39: 911-919.
- Bakhsh A (2010). Expression of two insecticidal genes in cotton.
PhD, University of the Punjab, Lahore, Pakistan: pp. 112-113.
- Bakhsh A, Siddique S, Husnain T (2012). A molecular approach
to combat spatio-temporal variation in insecticidal gene
(Cry1Ac) expression in cotton. Euphytica 183: 65-74.
- Bakhsh A, Emine A, Sancar F (2014). Comparison of transformation
efficiency of five
Agrobacterium tumefaciens
strains in
Nicotiana
tabacum
L. J Food Agric 26: 259-264.
- Bakhsh A, Khabbazi SD, Baloch FS, Demirel U, Çalişkan ME,
Hatipoğlu R, Özcan S, Özkan H (2015). Insect resistant
transgenic crops: retrospects and challenges. Turk J Agric For
39: 531-548.
- Bravo A, Soberon M (2008). How to cope with insect resistance to Bt
toxins? Trends Biotechnol 26: 573-579.
- Chetty VJ, Ceballos N, Garcia D, Narvaez-Vasquez J, Lopez W,
Orozco-Cardenas ML (2013). Evaluation of four Agrobacterium
tumefaciens strains for the genetic transformation of tomato
(
Solanum lycopersicum
L.) cultivar Micro-Tom. Plant Cell Rep
32: 239-247.
- Daud MK, Variath MT, Ali S, Jamil M, Khan MT, Shafi M, Shuijin Z
(2009). Genetic transformation of
bar
gene and its inheritance
and segregation behavior in the resultant transgenic cotton
germplasm (br001). Pak J Bot 41: 2167-2178.
- Day S, Aasim M, Bakhsh A (2016). Effects of preconditioning, plant
growth regulators and KCl on shoot regeneration of peanut
(
Arachis hypogea
). J Anim Plant Sci 26: 294-300.
- Duan X, Li W, Xue Q, Abo-El-Saad M, Xu D, Wu R (1996). Transgenic
rice plants harboring an introduced potato proteinase inhibitor
II gene are insect resistant. Nature Biotechnol 14: 494-498.
- Fearing PL, Brown D, Vlachos D, Meghji M, Privalle L (1997).
Quantitative analysis of CryIA(b) protein expression in Bt
maize plants, tissues, and silage and stability of expression over
successive generations. Mol Breed 3: 169-176.
- Gatehouse AMR, Hilder VA, Boulter D (1992). Potential of plant-
derived genes in the genetic manipulation of the crops for
insect resistance. In: Biotechnology in Agriculture N° 7: Plant
genetic manipulation for crop protection. CAB International,
pp. 155-181.
- Guo WZ, Sun J, Guo YF, Zhang TZ (2001). Investigation of different
dosage of inserted Bt genes and their insectresistance in
transgenic Bt cotton. Acta Genet Sin 28: 668-676.
- Hashmi JA, Zafar Y, Arshad M, Mansoor S, Asad S (2011).
Engineering cotton (
Gossypium hirsutum
L.) for resistance
to cotton leaf curl disease using viral truncated AC1 DNA
sequences. Virus Genes 42: 286-296.
- Hernandez-Martinez P, Ferre J, Escriche B (2008). Susceptibility of
Spodoptera exigua
to 9 toxins from
Bacillus thuringiensis.
J
Invertebr Pathol
97: 245-250.
- Hobbs SLA, Warkentin TD, Delong CMO (1993) Transgene copy
number can be positively or negatively associated with
transgene expression. Plant Mol Biol 2: 17-26.
- Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SGR, Fraley
T (1985). A simple and general method for transferring genes
into plants. Science 227: 1229-1231.
- Ivo NL, Nascimento CP, Vieira LS, Campos FAP, Aragão FJL (2008).
Biolistic-mediated genetic transformation of cowpea (
Vigna
unguiculata
) and stable Mendelian inheritance of transgenes.
Plant Cell Reports 27: 1475-1483.
- ISAAA (2016). Global Status of Commercialized Biotech/GM Crops:
2016. ISAAA Brief No. 52. Ithaca, NY, USA: ISAAA.
- Kos M, van Loon JJ, Dicke M, Vet LE (2009). Transgenic plants as
vital components of integrated pest management. Trends
Biotechnol 27: 621-627.
- Liu R, Zhang HH, Chen ZX, Shahid MQ, Fu XL, Liu XD (2015).
Drought-tolerant rice germplasm developed from an
Oryza
officinalis
transformation-competent artificial chromosome
clone. Genet Mol Res 14: 13667-13678.
- Lombardo L, Coppola G, Zelasco S (2016). New technologies for
insect-resistant and herbicide-tolerant plants. Trends in
Biotechnology 34: 49-57.
- Maqbool A, Abbas W, Rao AQ, Irfan M, Zahur M, Bakhsh A,
Riazuddin S, Husnain T 2010.
Gossypium arboreum
GHSP26
enhances drought tolerance in
Gossypium hirsutum
L.
Biotechnol Progress 26: 21-25.
- Manjunatha R, Pradeep S, Sridhar S, Manjunatha M, Naik MI,
Shivanna BK, Hosamani V (2009). Quantification of Cry1Ac
protein over time in different tissues of Bt cotton hybrids.
Karnataka J Agric Sci 22: 609-610.
- McCormick S, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley
R (1986). Leaf disc transformation of cultivated tomato (
L.
sculentum
) using
Agrobacterium tumefaciens
. Plant Cell Rep
5: 81-84.
- McHughen A, Jordan MC (1989). Recovery of transgenic plants from
“escape” shoots. Plant Cell Rep 7: 611-614
- Micallef MC, Austin S, Bingham ET (1995). Improvement of
transgenic alfalfa by backcrossing. In Vitro Cell Dev Biol Plant
31: 187-192.
- Murashige T, Skoog F (1962). A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol Plant 1962
15: 473-497.
- Nadeem MA, Nawaz MA, Shahid MQ, Doğan Y, Comertpay G,
Yıldız M, Hatipoğlu R, Ahmad A, Alsaleh A, Labhane N
et al. (2017). DNA molecular markers in plant breeding:
current status and recent advancements in genomic selection
and genome editingç Biotechnol Biotechnol Equip DOI:
10.1080/13102818.2017.1400401.
- Nester EW (2015).
Agrobacterium
: nature’s genetic engineer. Front
Plant Sci 5: 730.
- Olsen KM, Daly JC, Holt HE, Finnegan EJ (2005). Season-long
variation in expression of
Cry1Ac
gene and efficacy of
Bacillus
thuringiensis
toxin in transgenic cotton against
Helicoverpa
armigera
(Lepidoptera: Noctuidae). J Econ Entomol 98: 1007-
1017.
- Puspito AN, Rao AQ, Hafeez MN, Iqbal MS, Bajwa KS, Ali Q, Rashid
B (2015). Transformation and evaluation of Cry1Ac+Cry2A
and
GTGene
in
Gossypium hirsutum
L. Front Plant Sci 6: 1-13.
- Rao AQ, Bakhsh A, Kiani S, Shahzad K, Shahid AA, Husnain Y
(2009). The myth of plant transformation. Biotechnol Adv 27:
753-763.
- Rao AQ, Bakhsh A, Nasir IA, Riazuddin S, Husnain T (2011).
Phytochrome B mRNA expression enhances biomass yield and
physiology of cotton plants. Afr J Biotechnol 10: 1818-1826.
Rashid B, Zafar S, Husnain T, Riazuddin S (2008). Transformation
and inheritance of Bt genes in
Gossypium hirsutum.
J Plant Biol
51: 248-254.
- Sayyed AH, Wright DJ (2001). Cross-resistance and inheritance
of resistance to
Bacillus thuringiensis
toxin Cry1Ac in
diamondback moth (
Plutella xylostella
L) from lowland
Malaysia. Pest Manag Sci 57: 413-421.
- Sohail MN, Karimi SM, Asad S, Mansoor S, Zafar Y, Mukhtar Z
(2012). Development of broad-spectrum insect-resistant
tobacco by expression of synthetic
cry1Ac
and
cry2Ab
genes.
Biotechnol Lett 34: 1553-1560.
- Sunilkumar G, Mohr L, Lopata-Finch E, Emani C, Kathore KS
(2002). Developmental and tissue-specific expression of a
CaMV 35S promoter in cotton as revealed by GFP. Plant Mol
Biol 50: 463-474.
- Tabashnik BE, Carrière Y (2017). Surge in insect resistance
to transgenic crops and prospects for sustainability. Nat
Biotechnol 35: 926-935.
- Tabashnik BE, Brévault T, Carrière Y (2013). Insect resistance to Bt
crops: lessons from the first billion acres. Nat Biotechnol 31:
510-521.
- Tabashnik BE, Gassmann AJ, Crowder DW, Carriere Y (2008). Insect
resistance to Bt crops: evidence versus theory. Nat Biotechnol
26: 199-202.
- Tang CM, Sun J, Zhu XF, Guo WZ, Zhang TZ, Shen JL, Gao CF,
Zhou WJ, Chen ZX, Guo SD (2000). Inheritance of resistance
to
Helicoverpa
armigera
of 3 kinds of transgenic
Bt
strains
available in upland cotton in China. Chinese Sci Bull 45: 363-
367.
- Turkish Statistical Institute (TUİK) (2016). Retrieved on March 8,
2017.
- Vaneva-Gancheva T, Dimitrov Y (2013). Chemical control of the
potato tuber moth
Phthorimaea operculella
(Zeller) on tobacco.
Bulg J Agric Sci 19: 1003-1008.
- Villégier AS, Blanc G, Glowinski J, Tassin JP (2003). Transient
behavioural sensitization to nicotine becomes long-lasting
with monoamine oxidases inhibitors. Pharmacol Biochem
Behav 76: 267-274.
- Wu G, Cui H, Ye G, Xia Y, Sardana R, Cheng X, Li Y, Altosaar I, Shu
Q (2002). Inheritance and expression of the
cry1Ab
gene in Bt
(
Bacillus thuringiensis
) transgenic rice. Theoret App Genet 104:
727-734.
- Xia LH, He YR, Chun LU, Yang ZY, Hong ZJ (2007). Inheritance and
resistance to insects in c
ryIA(c)
transgenic cabbage. Chinese J
Agric Biotechnol 4: 57-61.
- Zhang BH, Liu F, Liu ZH, Wang HM, Yao CB (2001). Effects of
kanamycin on tissue culture and somatic embryogenesis in
cotton. Plant Growth Reg 33: 137-149
- Zhu F, Lavine L, Neal OS, Lavine M, Foss C, Walsh D (2016).
Insecticide resistance and management strategies in urban
ecosystems. Insects 7: 2.