Abstract
The introduction of foreign genes into plant has made possible to bring out desired traits into crop of our own interest. With the advancement in cell biology, regeneration of plants from single cell and advent of different procedures for gene transformation to the plants have opened new avenues for the efficient and applicable implementation of biotechnology for the modifications of desired crop characteristics. Identifications and isolation of different genes for various traits from different organisms have made possible to get the crop plants with modified characters. Over time improvement has been made in transformation technology depending upon the crop of interest. The efficiency of plant transformation has been increased with advances in plant transformation vectors and methodologies, which resulted in the improvement of crops. A detailed discussion on advanced techniques for genetic modification of plants with their handy use and limitation has been focused in this chapter.
Keywords
- Agrobacterium
- biolistic gun
- infiltration
- microinjection
- transformation
1. Introduction
Cell theory of Schleiden established the framework of modern plant biotechnology [1], and Schwann’s cell theory [2] was based upon phenomenon that cell is the basic unit of living organisms.
The idea of totipotency has been originated from the concept of cell theory, which later on laid the foundation of plant biotechnology. Hernalsteens [3] further provided the evidence and forecasted the production of somatic embryos from vegetative cells.
Among the key discoveries of plant biotechnology, gene transformation in crop plants and regeneration of plants from callus are the most significant achievements. In 1980s, chimeric genes were produced, which resulted in further expansion of genetic modification technology of the plants [1]. It led to the development of new transformation vectors [2], which ultimately changed the ways of DNA delivery systems [3]. Genetic transformation of crop plant can be achieved by a number of ways like
2. Plant regeneration
A number of attempts were made during 1950 to provide clear understanding of the phenomenon of totipotency but it was 1954 when Muir et al. [7] demonstrated the possible aspects of culturing of single plant cell. The cell divisions obtained by him from callus of tobacco placed on a small piece of filter paper provided the basis for totipotency. Davidonis and Hamilton further confirmed the results by obtaining similar results from single cell and group of cells suspended in the agar medium. It was the extension of these experiments when Jones et al. compared with many other crops, reported that it is more difficult to obtain somatic embryogenesis and plant regeneration from cotton except Coker 312. Davidonis and Hamilton [8] first described that plant regeneration from 2-year-old callus of
Rao et al. [12] in 2009 were the first who provide the clear evidence of totipotency and its use during regeneration from callus of plants when they were able to regenerate isolated single cells into flowering plants of tobacco, cultured in microchambers, without the aid of nurse cells or conditioned media. Verdiel et al. [9] in 2007 demonstrated that stem cells in meristematic regions are pluripotent and are dependent, whereas those that are present in embryogenic regions are totipotent and hence are independent.
3. Embryo formation
Improvements of many plants, such as cereals, soybean, cotton, canola, cassava, and woody tree species, are dependent on the development of somatic embryos; therefore, the formation of competent embryogenic cultures is imperative for the success of plant biotechnology. Somatic embryogenesis in cotton species has been reported to be the most difficult to regenerate [10, 11]. Regeneration in cotton has not been achieved rapidly, with the report of regeneration of
4. Binary vectors
In 1984, Braybrook [14] constructed the first binary vector pBIN19, since then efforts are being made to modify binary vectors in order to amplify their efficacy and transformation effectiveness. Japonica rice was successfully transformed using
Complete sequencing of pBIN19 has been performed [19]. A modified version pBIN20 has more single restriction sites within the multiple cloning site (MCS) [20]. A newly developed types of pPZP vectors are small and are steady in
Plant expression vectors of pRT100 series with a polyA signal under CaMV 35S promoter were developed by Töpfer et al. [22]. Making single-restriction sites available in the expression cassette holding the target gene is usually a very difficult task. Hou and Guo [23] constructed a set of pART7 and pART27 plasmids to deal with this problem. A MCS is present between CaMV35S promoter and OCS polyA signal of the shuttle plasmid pART7. NofI sites (an infrequent 8 bp recognition site) are present on both sides of the expression cassette. In pART27, the coding sequence of a target gene has been cloned in its MCS. NotI is used to cut the expression cassette, which is then cloned in the Not I site of pART27, a binary vector. In pART27 vector,
5. Methods involved in transformation
Transformation methods can be divided in two main categories: (1) direct and (2) indirect transformations, which are detailed in below sections.
5.1. Indirect transformation
In these methods, plants are transformed using
5.2. Direct transformation
In direct methods of transformation, bacterial cells are not used. The most frequently used direct methods include microprojectile bombardment or protoplast transformation. Problems with plant regeneration low transient expression of transgenes arise as a result of protoplast transformation (mostly in monocots).
The chemical substances used to disintegrate the cell walls and electrical field protoplasts lose their viability and ability to divide. In cotton, some attempts have been made to transform cells directly in the shoot apex either through the gene gun or
6. Overview of transformation techniques
6.1. Protoplasts and somatic hybridization
In 1970 and 1971, two major advances were made, which proved the beneficial role of protoplasts in the enhancement of plants: (i) somatic hybrid cells and novel hybrid plants are developed by inducing fusion of protoplasts of different species having no taxonomic relationships between them [26], and (ii) use of cultured protoplasts in regeneration of plants [27].
Geerts et al. [28] initiated the micropod culture and Schryer et al. [29] improved it [29]. In grain legumes, protoplast fusion is not well studied [30], but today, a variety of plants can be regenerated from protoplasts. Likewise, vast ranges of somatic hybrids are developed among related as well as unrelated plant species. However, functional hybrids have been developed in case of small number of plants such as Citrus
6.2. Direct gene transformation through imbibitions
Desiccated plant tissues can uptake foreign DNA through the process of imbibition [34]. Numerous asserts and disproof can be found in literature regarding this method. During the process of desiccation, various physiological and substantial alterations take place such as bursting of the cell wall, leakiness and changes in structure of the cell membrane, quick expansion of the cell, and development of a huge water flow among the peripheral solution and the dehydrated tissue. Uptake and transitory expression, by the cereal and legume seed embryos, of the DNA plasmid bearing the
Permeability of the membrane is a key factor in this process. This was proved by an increase in uptake and expression of the DNA as a result of using 20% DMSO during the process. Successive studies have led to 70% transitory
6.3. Agrobacterium -mediated transformation
Chilton et al. [39] introduced the idea of virulent strain of
Ti plasmid is an integrated part of the plant genome during tumor formation (transformation), suggesting that the plasmid could be used as a vector to transfer other genes. It was reported that various methods were tested to insert genes into the Ti plasmid. Transformed crown-gall tumor tissues, which were grown on hormone-free media, only formed highly aberrant shoots in culture [40]. This was related to the presence of genes controlling the expression of auxin and cytokinin synthesis. Deletion of these genes resulted in the production of transformed tissues that required media supplemented with auxin and cytokinin for continued growth and regeneration of normal shoots and plants. Nadolska-Orczyk et al. [43] showed that efficient
As
6.4. Biolistic transformation
In early 1980s, direct DNA delivery methods for protoplast were developed [44], especially for the economically important cereal crops, which were not subjected to the umbrella
6.5. Biolistic with Agrobacterium
Limitations of
6.6. Chemical method
To enhance the uptake of DNA, a combination of polybrene-spermidine treatment is used to obtain non-chimeric transgenic cotton plants. Polybrene-spermidine combination treatment for plant genetic transformation has the advantage because it is less toxic than the other chemicals; furthermore, it also protects condensation effect, DNA shearing, and integration of the plasmid with host genome [35]. To deliver plasmid DNA into cotton suspension culture obtained from cotyledon-induced callus, polybrene and/or spermidine treatments were used. Researchers have also regenerated and analyzed the cotton plants containing
6.7. Microinjection
Microinjection technique is based on introducing DNA in the cells using injection pipette of microcapillary glass [51]. This operation requires a micromanipulator. In this case, cells are immobilized by holding glass and gentle suction. Both pipettes are filled with mineral oil.
Microinjection is mostly used for animal cells, while with plants, a cell wall causes hindrance for transformation by microinjection as it works as barrier for microglass tools. Using microinjection technique for protoplast, there is a risk of toxic compounds to be released, which may cause sudden death of the protoplast. It is also possible to remove vacuoles before microinjection but regeneration and division may be decreased [52].
Protoplast microinjection involves different methods for immobilization, in which instead of using sucking poly-L-lysine is coated to the protoplasts. One of the major advantages of the microinjection is that it not only allows the transformation of the DNA plasmid but also the whole chromosome [53]. This technique is being used for the cellular mechanism and functions of the plant cells and to study the physiology of the plastids especially for tobacco [54] (Figure 3). Major limitation of microinjection method involves the use of expensive micromanipulator and it is a time-consuming procedure. Furthermore, frequency of transformation is very low and dependent on the species, i.e., proved to be successful in tobacco [55], Petunia [53], Rape [56], and Barley [57].
6.8. The pollen tube pathway method
Transformation by pollen tube pathway has got great intention in molecular breeding [58]. After pollination, a foreign DNA/plasmid is applied to the styles. To reach ovule, DNA uses the pollen tube pathway. This method of transformation was first used by Luo et al. [60] in rice [59]. In case of rice, a high frequency of transgenic plants was obtained, and this method was then applied to the other commercially important crops, such as wheat [60], soybean [61],
6.9. Liposomes
Direct transformation of the foreign DNA into the plant cells using liposomes was Employed in the 1980s. Liposomes are phospholipids with spherical shape, carry nucleic acid, and internally aqueous. Liposomes were put in a nutshell with the DNA fragments to get attached to the cell membranes. Thus, in this way DNA enters the cell and then to the nucleus. For the transfer of the bacterial, plant, and animal genes, lipofection has been a very competent technique. Lipofection takes place by fusion through membrane, and it has improved transformation efficiency because the genetic material used for lipofection is not naked as used in conventional techniques [64, 65]. In spite of cheap and less equipment demanding technique, liposome transformation is not so common. Major limitations in this technique are its low efficiency and being so hectic. Therefore, success story for liposome-mediated transformation have been published so far only for tobacco [66] and wheat [67].
6.10. Shoot apex method of transformation
Transformation by shoot apex method is a rapid method of transformation in cotton, and it is a genotype independent method. In this method, shoots are isolated from the plant and subjected to a virulent strain of
6.11. Sonication-assisted Agrobacterium -mediated transformation (SAAT)
SAAT method is based on principle of causing thousands of wounds by ultrasound. These wounds allow the
6.12. Infiltration
This simple procedure in which plant at the early stages is placed upside down in the beaker containing 5% sucrose solution with
Vacuum infiltration was applied for the first time in 1993 for transformation of
Infiltration method is most suitable to plants which have smaller genome. Vacuum infiltration is excluded for plants having genome greater than
6.13. Silicon carbide-mediated transformation (SCMT)
SCMT is less complicated method of plant transformation. Silicon carbide fibers are added to a suspension containing plant tissue (cell clusters, immature embryos, and/or callus) and plasmid DNA; it is mixed and then vortexed. Kaeppler et al. [73] demonstrated that DNA-coated fibers penetrate the cell wall in the presence of small holes created by collisions between the plant cells and fibers. The fibers mostly used in this procedure are single crystals of silica organic minerals like silicon carbide, elongated in shape having a length of 10–80 mm, and a diameter of 0.6 mm and show a high resistance to expandability.
The factors controlling the efficiency of SCMT are fiber size, vortexing parameters, shape of the vessels used, the plant material, and the characteristics of the plant cells, especially the thickness of the cell wall. The main advantages of this procedure are the low expenses and its usefulness for various plant materials. Disadvantages of this method are low transformation efficiency, damage to cells negatively influencing their further regeneration capability, and the necessity of obeying extraordinarily rigorous precaution protocols during laboratory work, as breathing the fibers in, especially asbestos ones, can lead to serious sicknesses [74].
Transgenic forms, cell colonies, or plants were derived from maize [75], and rice [76], from wheat [77], from tobacco [78], and from
6.14. Electroporation of intact plant cells and tissues
The principles of electroporation are the same for plant cells and protoplasts. However, difference may exist in other plant tissues such as pollen, microspores, leaf fragments, embryos, callus, seeds, or buds. During electroporation, the material used can be in the form of plasmid DNA and
Protocols were established for successful electroporation in cell suspensions, e.g., in tobacco [82], rice [24], and in wheat [83]. In early 1990s, experiments were performed to obtain transgenic plants. It was reported that the best results were obtained for maize. Researchers transformed immature embryos and embryogenic callus type I, which were treated by a solution of pectolytic enzymes, and then transferred into electroporating cuvettes [66]. The electroporation efficiency was relatively high when compared with micro bombardment conducted for same species, and about 90 transgenic plants were regenerated from 1440 embryos (6.25%) and 31 plants from 55 callus clusters (54.6%).
Laursen et al. [86] obtained similar results for this species. The authors estimated that the integration of transgenes took place approximately in one per 10,000 cells. Sorokin et al. [87] reported that much lower efficiency, i.e., about three transgenic plants from 1080 immature embryos (0.28%), was observed in the case of wheat electroporation. The transformation efficiency could be increased by the post-pulse addition of ascorbic acid or another ascorbate without any negative influence on cell viability [69]. Tissues were electroporated in liquid media containing 8 mg/L benzyl adenine that showed maximal regeneration through secondary somatic embryogenesis. DaSilva et al. [88] reported that the secondary somatic embryos regenerated from electroporation were positive for
6.15. Electrophoresis
At the end of the 1980s, Songstad et al. [78] developed a method employing electrophoresis for the transformation of immature embryos, especially in monocotyledonous plants. This method was adopted as an alternative for transformation, but it is very expensive and yield poor results when compared with micro-projectile bombardment [53]. Transfected embryos were placed between the tip of two pipettes and connected to electrodes. The pipette connected to the anode was filled with agar / agarose followed by an EDTA containing electrophoresis buffer.
The pipette connected to the cathode contained agar that was mixed with DNA and an electrophoresis buffer. This pipette was in contact with the apical meristem of the embryo, whereas the second one was located near basal apical part of embryo. Electrophoresis-mediated transformation efficiency depends on various factors, such as electrical field parameters, duration of electrophoresis, contents of electrophoresis buffer, and physicochemical properties of the embryonic tissues. Voltage of 25 mV and an amperage of 0.5 mA for 15 minutes are mostly used for electrophoresis program [74]. In spite of its simplicity, electrophoresis is not considerable method in plant transformation, and the reason behind is less viability of treated embryos. Ahokas et al. [84] showed that none of the plants showed expression of transgene inserted. Griesbach et al. [85] obtained successfully transformed plants of
7. Conclusion
Plant transformation is an essential tool for incorporating new characteristics in crop plant like cotton. Cotton is recalcitrant crop hence a reproducible regeneration is not available in local cotton varieties. Among all strategies developed by different researchers, a little success in cotton (
References
- 1.
Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP Bittner ML, Brand LA, Fink CL, Fry JS. Expression of bacterial genes in plant cells. Proceedings of the National Academy of Sciences. 1983; 80 :4803-4807. DOI: 6308651 - 2.
Hoekema A, Hirsch P, Hooykaas P, Schilperoort R. A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature. 1983;303 :179-180. DOI: 10.1038/303179a0 - 3.
Hernalsteens J, Van Vliet F, De Beuckeleer M, Depicker A, Engler G, Lemmers M, et al. The Agrobacterium tumefaciens Ti plasmid as a host vector system for introducing foreign DNA in plant cells. 1980. Biotechnology (Reading, Mass). 1992;24 :374. DOI: 10.1038/287654a0 - 4.
Dai S, Zheng P, Marmey P, Zhang S, Tian W, Chen S, et al. Comparative analysis of transgenic rice plants obtained by Agrobacterium -mediated transformation and particle bombardment. Molecular Breeding. 2001;7 :25-33. DOI: 10.1023/A:1009687511633 - 5.
Skoog F, Miller C, editors. Chemical regulation of growth and organ formation in plant tissue cultured in vitro. Symposia of the Society for Experimental Biology. 1957; 11 :118-131. - 6.
Azam S, Samiullah TR, Yasmeen A, ud Din S, Iqbal A, Rao AQ, et al. Dissemination of Bt cotton in cotton growing belt of Pakistan. Advancements in Life Sciences. 2013; 1 :18-26. - 7.
Muir W, Hildebrandt A, Riker A. Plant tissue cultures produced from single isolated cells. Science. 1954; 119 :877-878. DOI: 10.1126/science.119.3103.877-a - 8.
Davidonis GH, Hamilton RH. Plant regeneration from callus tissue of Gossypium hirsutum L. Plant Science Letters. 1983;32 :89-93. DOI: 10.1016/0304-4211(83)90102-5 - 9.
Verdeil J-L, Alemanno L, Niemenak N, Tranbarger TJ. Pluripotent versus totipotent plant stem cells: dependence versus autonomy? Trends in Plant Science. 2007; 12 :245-252. DOI: 10.1016/j.tplants.2007.04.002 - 10.
Larkin P, Ryan S, Brettell R, Scowcroft W. Heritable somaclonal variation in wheat. Theoretical and Applied Genetics. 1984; 67 :443-455. DOI: 10.1007/BF00263410 - 11.
Rao AQ, Hussain SS, Shahzad MS, Bokhari SYA, Raza MH, Rakha A, et al. Somatic embryogenesis in wild relatives of cotton ( Gossypium Spp.). Journal of Zhejiang University Science B. 2006;7 :291-298. DOI: 10.1631/jzus.2006.B0291 - 12.
Rao AQ, Bakhsh A, Kiani S, Shahzad K, Shahid AA, Husnain T, et al. The myth of plant transformation. Biotechnology Advances. 2009; 27 :753-763. DOI: 10.1016/j.biotechadv.2009.04.028 - 13.
Vasil IK. A history of plant biotechnology: from the cell theory of Schleiden and Schwann to biotech crops. Plant Cell Reports. 2008; 27 :1423-1440. DOI: 10.1007/s00299-008-0571-4 - 14.
Braybrook SA, Stone SL, Park S, Bui AQ, Le BH, Fischer RL, et al. Genes directly regulated by LEAFY COTYLEDON2 provide insight into the control of embryo maturation and somatic embryogenesis. Proceedings of the National Academy of Sciences of the United States of America. 2006; 103 :3468-3473. DOI: 10.1073/pnas.0511331103 - 15.
Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice ( Oryza sativa L.) mediated byAgrobacterium and sequence analysis of the boundaries of the T‐DNA. The Plant Journal. 1994;6 :271-282. DOI: 10.1046/j.1365-313X.1994.6020271.x - 16.
Kumria R, Waie B, Rajam M. Plant regeneration from transformed embryogenic callus of an elite indica rice via Agrobacterium . Plant Cell, Tissue and Organ Culture. 2001;67 :63-71. DOI: 10.1023/A:1011645315304 - 17.
Zheng S-J, Khrustaleva L, Henken B, Sofiari E, Jacobsen E, Kik C, et al. Agrobacterium tumefaciens -mediated transformation ofAllium cepa L.: the production of transgenic onions and shallots. Molecular Breeding. 2001;7 :101-115. DOI: 10.1023/A:1011348229189 - 18.
Jaiwal PK, Kumari R, Ignacimuthu S, Potrykus I, Sautter C. Agrobacterium tumefaciens -mediated genetic transformation of mungbean (Vigna radiata L. Wilczek)—a recalcitrant grain legume. Plant Science. 2001;161 :239-247. DOI: 10.1016/S0168-9452(01)00352-1 - 19.
Frisch DA, Harris-Haller LW, Yokubaitis NT, Thomas TL, Hardin SH, Hall TC. Complete sequence of the binary vector Bin 19. Plant Molecular Biology. 1995; 27 :405-409. DOI: 10.1007/BF00020193 - 20.
Hennegan KP, Danna KJ. pBIN20: an improved binary vector for shape Agrobacterium -mediated transformation. Plant Molecular Biology Reporter. 1998;16 :129-131. DOI: 10.1023/A:1007444100898 - 21.
Hajdukiewicz P, Svab Z, Maliga P. The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Molecular Biology. 1994;25 :989-994. - 22.
Töpfer R, Gronenborn B, Schell J, Steinbiss H-H. Uptake and transient expression of chimeric genes in seed-derived embryos. The Plant Cell. 1989; 1 :133-139. DOI: 10.1105/tpc.1.1.133 - 23.
Hou W-S, Guo S-D, Lu M. Development of transgenic wheat with a synthetical insecticidal crystal protein gene via pollen-tube pathway. Acta Agronomica Sinica. 2003; 29 :806-809. - 24.
Zapata C, Park S, El-Zik K, Smith R. Transformation of a Texas cotton cultivar by using Agrobacterium and the shoot apex. Theoretical and Applied Genetics. 1999;98 :252-256. DOI: 10.1007/s001220051065 - 25.
Wilkins TA, Rajasekaran K, Anderson DM. Cotton biotechnology. Critical Reviews in Plant Sciences. 2000; 19 :511-550. - 26.
Power J, Cummins S, Cocking E. Fusion of isolated plant protoplasts. Nature (London). 1970; 225 :1016-1018. DOI: 19701607260 - 27.
Takebe I, Labib G, Melchers G. Regeneration of whole plants from isolated mesophyll protoplasts of tobacco. Naturwissenschaften. 1971; 58 :318-320. DOI: 10.1007/BF00624737 - 28.
Geerts P, Sassi K, Mergeai G, Baudoin J-P. Development of an in vitro pod culture technique for young pods of Phaseolus vulgaris L. In Vitro Cellular & Developmental Biology-Plant. 2000;36 :481-487. DOI: 10.1007/s11627-000-0086-3 - 29.
Schryer P, Lu Q, Vandenberg A, Bett K. Rapid regeneration of Phaseolus angustissimus andP. vulgaris from very young zygotic embryos. Plant Cell, Tissue and Organ Culture. 2005;83 :67-74. DOI: 10.1007/s11240-005-2586-7 - 30.
Ochatt S, Abirached-Darmency M, Marget P, Aubert G. The Lathyrus paradox:“poor men’s diet” or a remarkable genetic resource for protein legume breeding. Breeding of Neglected and Under-Utilised Crops, Spices and Herbs. 2007. INRA, C.R, de Dijon, URLEG, B.P.86510, 21065 Dijon cedex, France pp. 41-60. - 31.
Davey M, Cocking E, Freeman J, Pearce N, Tudor I. Transformation of Petunia protoplasts by isolated Agrobacterium plasmids. Plant Science Letters. 1980;18 :307-313. DOI: 10.1016/0304-4211(80)90121-2 - 32.
Vasil IK. Molecular improvement of cereals. Plant Molecular Biology. 1994; 25 :925-937. - 33.
Waldron C, Malcolm S, Murphy E, Roberts J. A method for high-frequency DNA-mediated transformation of plant protoplasts. Plant Molecular Biology Reporter. 1985; 3 :169-173. DOI: 10.1007/BF02886753 - 34.
Hess D. Transformation experiments in higher plants-reproducibility of anthocyanin induction in petunia and preliminary characterization of transforming principle. Zeitschrift Fur Pflanzenphysiologie. 1969; 61 :286-298. - 35.
Muzaffar A, Kiani S, Khan MAU, Rao AQ, Ali A, Awan MF, et al. Chloroplast localization of Cry1Ac and Cry2A protein-an alternative way of insect control in cotton. Biological Research. 2015; 48 :1. DOI: 10.1186/s40659-015-0005-z - 36.
Yoo J, Jung G. DNA uptake by imbibition and expression of a foreign gene in rice. Physiologia Plantarum. 1995; 94 :453-459. DOI: 10.1111/j.1399-3054.1995.tb00953.x - 37.
Pett A, Delhaye S, Tempe J, Morel G. Recherches sur les guanidines des tissus de crown gall. mise en evidence d'une relation biochimique specifique entre le souches d' Agrobacterium tumefaciens et les tumeurs qu'elles induisent. Physiologie Végétale. 1970. - 38.
Chilton M-D, Drummond MH, Merlo DJ, Sciaky D, Montoya AL, Gordon MP, et al. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell. 1977; 11 :263-271. DOI: 10.1016/0092-8674(77)90043-5 - 39.
Chilton M-D, Saiki RK, Yadav N, Gordon MP, Quetier F. T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells. Proceedings of the National Academy of Sciences. 1980;77 :4060-4064. - 40.
Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J. Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens . The EMBO Journal. 1983;2 :411. - 41.
Bevan M, Flavell R, Chilton M. A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation. 1983. Biotechnology (Reading, Mass). 1992; 24 :367. DOI: 10.1038/304184a0 - 42.
Thomas JC, Adams DG, Keppenne VD, Wasmann CC, Brown JK, Kanost MR, et al. Protease inhibitors of Manduca sexta expressed in transgenic cotton. Plant Cell Reports. 1995; 14 :758-762. DOI: 10.1007/BF00232917 - 43.
Nadolska-Orczyk A, Orczyk W, Przetakiewicz A. Agrobacterium -mediated transformation of cereals—from technique development to its application. Acta Physiologiae Plantarum. 2000;22 :77-88. DOI: 10.1007/s11738-000-0011-8 - 44.
Shillito R. Methods of genetic transformation: electroporation and polyethylene glycol treatment. Molecular Improvement of Cereal Crops. Springer Netherlands; 1999. p. 9-20. DOI: 10.1007/978-94-011-4802-3_2 - 45.
Vasil IK. The story of transgenic cereals: the challenge, the debate, and the solution—a historical perspective. In Vitro Cellular & Developmental Biology-Plant. 2005; 41 :577-583. DOI: 10.1079/IVP2005654 - 46.
Sailaja M, Tarakeswari M, Sujatha M. Stable genetic transformation of castor ( Ricinus communis L.) via particle gun-mediated gene transfer using embryo axes from mature seeds. Plant Cell Reports. 2008;27 :1509-1519. DOI: 10.1007/s00299-008-0580-3. - 47.
Finer JJ, McMullen MD. Transformation of cotton (Gossypium hirsutum L.) via particle bombardment. Plant Cell Reports. 1990; 8 :586-589. DOI: 10.1007/BF00270059 - 48.
Zhao T-J, Zhao S-Y, Chen H-M, Zhao Q-Z, Hu Z-M, Hou B-K, et al. Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling. Plant Cell Reports. 2006;25 :1199-1204. DOI: 10.1007/s00299-006-0184-8 - 49.
Sanford JC, Klein TM, Wolf ED, Allen N. Delivery of substances into cells and tissues using a particle bombardment process. Particulate Science and Technology. 1987; 5 :27-37. DOI: 10.1080/02726358708904533 - 50.
Altpeter F, Baisakh N, Beachy R, Bock R, Capell T, Christou P, et al. Particle bombardment and the genetic enhancement of crops: myths and realities. Molecular Breeding. 2005; 15 :305-327. DOI: 10.1007/s11032-004-8001-y - 51.
Crossway A, Oakes JV, Irvine JM, Ward B, Knauf VC, Shewmaker C. Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts. Molecular and General Genetics MGG. 1986; 202 :179-185. DOI: 10.1007/BF00331634 - 52.
Lörz H, Paszkowski J, Dierks‐Ventling C, Potrykus I. Isolation and characterization of cytoplasts and miniprotoplasts derived from protoplasts of cultured cells. Physiologia Plantarum. 1981; 53 :385-391. DOI: 10.1111/j.1399-3054.1981.tb04517.x - 53.
Griesbach R. Chromosome-mediated transformation via microinjection. Plant Science. 1987; 50 :69-77. DOI: 10.1016/0168-9452(87)90032-X - 54.
Knoblauch M, Hibberd JM, Gray JC, van Bel AJ. A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes. Nature Biotechnology. 1999; 17 :906-909. DOI: 10.1038/12902 - 55.
Schnorf M, Neuhaus-Url G, Galli A, Iida S, Potrykus I, Neuhaus G. An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis. Transgenic Research. 1991; 1 :23-30. DOI: 10.1007/BF02512993 - 56.
Neuhaus G, Spangenberg G, Scheid OM, Schweiger H-G. Transgenic rapeseed plants obtained by the microinjection of DNA into microspore-derived embryoids. Theoretical and Applied Genetics. 1987; 75 :30-36. DOI: 10.1007/BF00249138 - 57.
Holm PB, Olsen O, Schnorf M, Brinch-Pedersen H, Knudsen S. Transformation of barley by microinjection into isolated zygote protoplasts. Transgenic Research. 2000; 9 :21-32. DOI: 10.1023/A:1008974729597 - 58.
Song X, Gu Y, Qin G. Application of a transformation method via the pollen-tube pathway in agriculture molecular breeding. Life Science Journal. 2007; 4 :77-79. - 59.
Luo Z, Wu R. A simple method for the transformation of rice via the pollen-tube pathway. Plant Molecular Biology Reporter. 1989; 7 :69-77. DOI: 10.1007/BF02669590 - 60.
Mu H-M, Liu S-J, Zhou W-J, Wen Y-X, Zhang W-J, Wei R-X: [Transformation of wheat with insecticide gene of arrowhead proteinase inhibitor by pollen tube pathway and analysis of transgenic plants] [In Process Citation]. Yi chuan xue bao= Acta Genetica Sinica. 1998; 26 :634-642. - 61.
Hu C-Y, Wang L. In planta soybean transformation technologies developed in China: procedure, confirmation and field performance. In Vitro Cellular & Developmental Biology-Plant. 1999; 35 :417-420. DOI: 10.1007/s11627-999-0058-1 - 62.
Tjokrokusumo D, Heinrich T, Wylie S, Potter R, McComb J. Vacuum infiltration of Petunia hybrida pollen with Agrobacterium tumefaciens to achieve plant transformation. Plant Cell Reports. 2000;19 :792-797. DOI: 10.1007/s002990050009 - 63.
Chen WS, Chiu CC, Liu HY, Lee TL, Cheng JT, Lin CC, et al. Gene transfer via pollen‐tube pathway for anti‐fusarium wilt in watermelon. IUBMB Life. 1998; 46 :1201-1209. DOI: 10.1080/15216549800204762 - 64.
Shou H, Palmer RG, Wang K. Irreproducibility of the soybean pollen-tube pathway transformation procedure. Plant Molecular Biology Reporter. 2002; 20 :325-334. DOI: 10.1007/BF02772120 - 65.
Murakawa T, Kajiyama S, Ikeuchi T, Kawakami S, Fukui K. Improvement of transformation efficiency by bioactive-beads-mediated gene transfer using DNA-lipofectin complex as entrapped genetic material. Journal of Bioscience and Bioengineering. 2008; 105 :77-80. DOI: 10.1263/jbb.105.77 - 66.
Dekeyser RA, Claes B, De Rycke RM, Habets ME, Van Montagu MC, Caplan AB. Transient gene expression in intact and organized rice tissues. The Plant Cell. 1990; 2 :591-602. DOI: 10.1105/tpc.2.7.591 - 67.
Zhu Z, Sun B, Liu C, Xiao G, Li X. Transformation of wheat protoplasts mediated by cationic liposome and regeneration of transgenic plantlets. Chinese Journal of Biotechnology. 1992; 9 :257-261. - 68.
Yaqoob A, Shahid AA, Samiullah TR, Rao AQ, Khan MAU, Tahir S, et al. Risk assessment of Bt crops on the non‐target plant‐associated insects and soil organisms. Journal of the Science of Food and Agriculture. 2016; 96 :2613-2619. DOI: 10.1002/jsfa.7661 - 69.
Hussain SS, Husnain T, Riazuddin S. Sonication assisted Agrobacterium mediated transformation (SAAT): An alternative method for cotton transformation. Pakistan Journal of Botany. 2007;39 :223. - 70.
Clough SJ, Bent AF. Floral dip: a simplified method forAgrobacterium ‐mediated transformation ofArabidopsis thaliana . The Plant Journal. 1998;16 :735-743. DOI: 10.1046/j.1365-313x.1998.00343.x - 71.
Ye GN, Stone D, Pang SZ, Creely W, Gonzalez K, Hinchee M. Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation. The Plant Journal. 1999;19 :249-257. DOI: 10.1046/j.1365-313X.1999.00520.x - 72.
Chung M-H, Chen M-K, Pan S-M. Floral spray transformation can efficiently generate Arabidopsis . Transgenic Research. 2000;9 :471-486. DOI: 10.1023/A:1026522104478 - 73.
Kaeppler H, Somers D, Rines H, Cockburn A. Silicon carbide fiber-mediated stable transformation of plant cells. Theoretical and Applied Genetics. 1992; 84 :560-566. DOI: 10.1007/BF00232262 - 74.
Songstad D, Somers D, Griesbach R. Advances in alternative DNA delivery techniques. Plant Cell, Tissue and Organ Culture. 1995; 40 :1-15. DOI: 10.1007/BF00041112 - 75.
Petolino J, Hopkins N, Kosegi B, Skokut M. Whisker-mediated transformation of embryogenic callus of maize. Plant Cell Reports. 2000; 19 :781-786. DOI: 10.1007/s002999900180 - 76.
Nagatani N, Honda H, Shimada T, Kobayashi T. DNA delivery into rice cells and transformation using silicon carbide whiskers. Biotechnology Techniques. 1997; 11 :471-473. DOI: 10.1023/A:1018497529493 - 77.
Brisibe EA, Gajdosova A, Olesen A, Andersen SB. Cytodifferentiation and transformation of embryogenic callus lines derived from another culture of wheat. Journal of Experimental Botany. 2000; 51 :187-196. DOI: 10.1093/jexbot/51.343.187 - 78.
Kaeppler HF, Gu W, Somers DA, Rines HW, Cockburn AF. Silicon carbide fiber-mediated DNA delivery into plant cells. Plant Cell Reports. 1990; 9 :415-418. DOI: 10.1007/BF00224152 - 79.
Dalton S, Bettany A, Timms E, Morris P. Transgenic plants of Lolium multiflorum, Lolium perenne, Festuca arundinacea andAgrostis stolonifera by silicon carbide fibre-mediated transformation of cell suspension cultures. Plant Science. 1998;132 :31-43. DOI: 10.1016/S0168-9452(97)00259-8 - 80.
Bullock W, Dias D, Bagnal S, Cook K, Teronde S, Ritland J, et al., editors. A high efficiency maize “whisker” transformation system. Plant and Animal Genomes IX Conference, San Diego, CA; 2001. - 81.
Frame BR, Drayton PR, Bagnall SV, Lewnau CJ, Bullock WP, Wilson HM, et al. Production of fertile transgenic maize plants by silicon carbide whisker‐mediated transformation. The Plant Journal. 1994; 6 :941-948. DOI: 10.1046/j.1365-313X.1994.6060941.x - 82.
Abdul-Baki AA, Saunders JA, Matthews BF, Pittarelli GW. DNA uptake during electroporation of germinating pollen grains. Plant Science. 1990; 70 :181-190. DOI: 10.1016/0168-9452(90)90132-8 - 83.
Zaghmout O, Trolinder NL. Simple and efficient method for directly electroporating Agrobacterium plasmid DNA into wheat callus cells. Nucleic Acids Research. 1993;21 :1048. DOI: PMC309253 - 84.
Ahokas H. Transfection of germinating barley seed electrophoretically with exogenous DNA. Theoretical and Applied Genetics. 1989; 77 :469-472. DOI: 10.1007/BF00274265 - 85.
Griesbach R. An improved method for transforming plants through electrophoresis. Plant Science. 1994; 102 :81-89. DOI: 10.1016/0168-9452(94)03936-4 - 86.
Laursen, C. M., Krzyzek, R. A., Flick, C. E., Anderson, P. C., & Spencer, T. M. Production of fertile transgenic maize by electroporation of suspension culture cells. Plant Molecular Biology , (1994).24 (1), 51-61. DOI: 10.1007/BF00040573 - 87.
Sorokin AP, Ke XY, Chen DF, Elliott MC. Production of fertile transgenic wheat plants via tissue electroporation. Plant Science. 2000 28; 156 (2):227-33. dx.doi.org/10.1016/S0168-9452(00)00260-0 - 88.
Da Silva RF, Menéndez-Yuffá A. Transient gene expression in secondary somatic embryos from coffee tissues electroporated with the genes gus and bar. Electronic Journal of Biotechnology. 2003 6 (1):11-2. dx.doi.org/10.4067/S0717-34582003000100006