Conditions tested in transient transformation experiments.
Keywords
1. Introduction
However, unlike others Poaceae, sorghum transformation has been a challenge mainly due to recalcitrance in tissue culture and long periods of selection required for the recovery and regeneration of putative transgenic plants (Casas et al., 1993, Zhao et al., 2000; Jeoung et al., 2002; Howe et al., 2005). Since the earliest 90’s, laboratories around the world have generated improvements in sorghum regeneration and transformation that are ensuing in more consistent protocols. Transgenic sorghum plants have been generated via biolistic (Casas et al., 1993; Casas et al., 1997; Zhu
Even though the efficiency of sorghum transformation using the microprojectile bombardment had been improved, by all this studies, since the initial experiments (from 0,3 to 1,3%), it is still low if compared with the efficiency of sorghum transformation mediated by
2. Genetic transformation of Sorghum bicolor
2.1. In vitro regeneration of transgenic sorghum cells
Sorghum tissue culture is reported to be highly recalcitrant mainly because the release of toxic phenolics compounds in culture media, lack of regeneration in long term
The regeneration of sorghum
However, the main explants used in the transformation of sorghum are immature zygotic embryos between 1.5 and 2.0 mm in length (Casas et al, 1993; Emani et al, 2002; Jeoung et al, 2002; Gao et al, 2005, Howe et al, 2006; Gurel et al, 2009). One of the constrains in working with immature embryos is the intensive labor to generate large quantities of explants to be used in the transformation procedures. In this sense, immature inflorescences are easier to isolate, show very good regeneration rates in tissue culture and, morphogenetic competence over a wider size range (1–5 cm) than immature embryos. Besides, it is faster to grow donor plants for the production of immature inflorescence than for immature embryo (Cai and Butler, 1990; Kaepler and Pedersen, 1997; Jogeswar et al 2007; Brandão et al. 2007).
Even though outstanding studies aiming to identify sorghum genotypes able to produce high quality callus from immature inflorescence have been conducted, the efficiency to produce transgenic sorghum plants using this type of explant is still very low.
2.2. Genetic transformation of Sorghum bicolor via microprojectile bombardment
The bombardment of plant cells with the DNA of interest is a direct method of transformation designed (Taylor and Fauquet, 2002) in the late 80's to manipulate the genome of plants recalcitrant to transformation via
Some advantages of the microprojectile bombardment are related to its efficiency in the transformation of monocots, the use of simple vectors, easier to handle, as well as the possibility of inserting more than one GOI into cells efficiently (Chen et al. 1998; Wu et al. 2002). Although considered a very efficient method in cereals, one drawbacks of this technique is the occurrence of multiple copies of the GOI in the transgenic plant and complex integration patterns (Wang and Frame, 2004).
The biolistic has proved to be an efficient method for introducing new features in sorghum and a few transformation protocols are already available (Casas et al. 1993; Casas et al. 1997; Zhu et al. 1998; Able et al. 2001; Devi and Sticklen, 2002 ; Emani et al. 2002; Tadesse et al., 2003; Girijashankar et al., 2005). The optimization of physical and biological parameters was the subject of most of the work published about sorghum transformation via bombardment. The pioneer work was done by Casas and collaborators between 1993 and 1997. Initially, using anthocyanin (
Optimization of physical and biological parameters to produce transgenic sorghum plants was also the purpose of the work by Able et al. (2001) and Tadesse et al. (2003). Able and co-workers (2001) analyzed the transient expression of the reporter genes GUS and GFP over different physical bombardment parameters to identify the best conditions to generate transgenic sorghum plants using the particle inflow gun (PIG). Three transgenics events were confirmed by molecular analyses. Tadesse and associates (2003) also used reporter genes to test different acceleration pressures, target distances, gap widths and macroprojectile travel distances to bombard immature and mature embryos, shoot tips and embryogenic calli. The strength of four different promoters (
Currently few sorghum events expressing genes with agronomical interest were developed by the laboratories working with transformation of this specie via microprojectile bombardment. The gene
Another chitinase, the gene
The
To withstand toxic aluminum concentrations present in acidic soils, sorghum was genetically modified to express the
3. Genetic transformation of immature sorghum inflorescence
Here, we report improvements made in the transformation process via microprojectile bombardment that enable us to obtain a protocol where putative transgenic plants can be produced with an efficiency ranging from 1.01 to 3.33% using immature inflorescences of sorghum.
3.1. Material and methods
3.1.1. Plant material and explants preparation
Seeds from nine
For the biolistic experiments 30 calli pieces of approximately 3 mm diameter (Figure 2B) were uniformly distributed within a 35 mm diameter circle of 60 x 15 mm Petri dishes containing CIM media in which a higher osmotic value was achieved by the addition of 12% sucrose.
3.1.2. Plasmid constructs
The genetic cassettes p35S::
3.1.3. Particle bombardment
Embryogenic calli were bombarded with tungsten microprojectiles using a biolistic particle helium acceleration device (Biomics – Brasília / Brazil). For the transient experiments 3 μL of each plasmid (stock 1 μg/μL), p35S::
Treatments | Osmotic Media (hours) | Helium Pressure (psi) |
Microcarrier Flying Distance (cm) |
1 | 0 | 1 000 | 3 |
2 | 0 | 1 000 | 6 |
3 | 0 | 1 000 | 9 |
4 | 4 | 1 000 | 3 |
5 | 4 | 1 000 | 6 |
6 | 4 | 1 000 | 9 |
7 | 24 | 1 000 | 3 |
8 | 24 | 1 000 | 6 |
9 | 24 | 1 000 | 9 |
10 | 0 | 1 200 | 3 |
11 | 0 | 1 200 | 6 |
12 | 0 | 1 200 | 9 |
13 | 4 | 1 200 | 3 |
14 | 4 | 1 200 | 6 |
15 | 4 | 1 200 | 9 |
16 | 24 | 1 200 | 3 |
17 | 24 | 1 200 | 6 |
18 | 24 | 1 200 | 9 |
Eighteen treatments (Table 1) were designed to test the permanence of explants on osmoticum prior to bombardment (0, 4 and 24 hs), pressure of the accelerating helium pulse (1000 and 1200 psi), and microprojectile flying distance (3, 6 and 9 cm), in transient sorghum transformation. The distance between the high pressure chamber and the macro-carrier membrane (8 mm), the distance between the macro-carrier membrane and the retention screen (17 mm) and the vacuum pressure (27 mmHg) were maintained constant. For each treatment three plates containing 30 calli pieces were bombarded once.
3.1.4. Expression analysis
A
3.1.5. Selection procedures
Explants were cultured on solid CIM media at 25ºC in the dark for one week and transferred to selective SE media (modified CIM supplemented with 0.5 mg.L-1 kinetin and without DL-asparagine) containing 15 µL.L-1 of the herbicide Finale® (3 mg.L-1 4-hidroxi(methyl) phosphynol-DL-homoalanine ammonium salt) for one week. After that, the explants were transferred to a media containing 30 and 45 µL.L-1 herbicide every week. Growing calli were cultured for one more week in a SE media supplemented with 45 µL.L-1 herbicide, and subsequently transferred to a callus maturation media RM [MS salts and vitamins (Murashige and Skoog, 1962), 60 g.L-1 sucrose, 100 mg. L-1 myo-inositol, 0.2 mg.L-1 NAA, 3 g/L phytagel, pH 5.8) supplemented with 30 µL.L-1 herbicide and cultured in the dark at 25oC for somatic embryo maturation. Approximately 2 to 4 weeks later, mature somatic embryos showing a white and opaque coloration were transferred to Magenta boxes (Sigma, São Paulo, Brazil) containing germination media composed by MS media without plant growth regulators, supplemented with 15 µL.L-1 herbicide and placed in a lighted (16 h / 60 µmol m-2 s-1) growth room. Germinated plantlets (4-6 cm) were cultured in soil, for the first week under a plastic lid, in a greenhouse.
3.1.6. Plant DNA extraction, polymerase chain reaction (PCR) and Southern blot hybridization analysis
Total genomic DNA was isolated from leaf tissue of primary transformants using a CTAB protocol described by Saghai-Maroof et al. (1984). The presence of
For Southern blot analysis 10 µg of total genomic DNA from each T0 plant were completely digested with
3.1.7. Statistical data analysis
The data obtained from the transient experiment was collected in Microsoft Excel (Version 5).The experimental design was based on randomized blocks, factorial 2x3x3 (2 pressure of the accelerating helium pulse, 3 time of explants on osmoticum and 3 microprojectile flying distance), with triplicates, totalizing 54 experimental units. The data were subjected to ANOVA and means compared by Tukey test (p< 0.05), using the statistical program SISVAR 4.0 (Ferreira, 2000).
4. Results
4.1. Explant preparation and selection of transformed calli in vitro
Nine
Embryogenic calli from immature inflorescence of sorghum were bombarded three to four weeks after cultivation in CIM media (Fig. 2B), with the plasmids containing the
Growth of bombarded callus was slightly inhibited and some of them turned brown on selection medium supplemented with 3.0 mg.L-1 of PPT, compared to non-bombarded ones. After one week of cultivation the concentration of PPT on selection medium was increased to 6 mg.L-1 and most of the bombarded calli turned black. In order to reduce escapes within chimerical clusters, surviving bombarded clumps were carefully divided and cultured, at one week intervals, onto selection medium supplemented with 9 mg.L-1 PPT, during four weeks. At this herbicide concentration complete inhibition of non-transgenic calli growth was observed, most calli turned dark, necrotic and died (Fig. 2D). After six weeks of selection, surviving calli were transferred onto maturation media supplemented with 6 mg.L-1 PPT. On this medium, as soon as the yellowish calli become white and opaque, between 2 to 4 weeks of cultivation, they were transferred onto germination medium (Figure 2E). A concentration of 3 mg.L-1 PPT was used for the differentiation and germination of mature calli that occurs around 20 d of cultivation. Control non-bombarded explants did not survive on selection medium containing 6 or 9 mg.L-1 PPT. This selection procedure has been used successfully in different transformation experiments with calli derived from immature inflorescence of sorghum. The overall time for selection and regeneration of putative transgenic plants using this protocol is around 16 weeks.
4.2. Transient and stable transformation
The optimization of DNA delivery parameters was initially performed by using the transient expression of maize
Statistical analyses of the transient anthocyanin expression (Table 2) identified interactions among the different factors studied. Embryogenic calli submitted to 1000 psi of helium accelerating pressure, cultivated during 4 h in a higher osmotic medium and positioned at 3 cm from the micro-carrier launch platform (Treatment 4) presented a larger number of cells expressing anthocyanin than when the explants were positioned at 6 or 9 cm (Treatment 5 and 6). Without the pre-cultivation of explants in an osmotic medium, there were no differences among the positions (3, 6 or 9 cm) of explants (Treatments 1 to 3) and the overall amount of anthocyanin spots was lower. At 1200 psi acceleration pressure, treatments 10 to 18, the best results obtained were when calli were cultivated in osmotic medium for 24 h and positioned at 3 or 6 cm from the micro-carrier launch platform (Treatments 16 and 17).
TARGET DISTANCE | |||
Time in osmotic medium | Pressure 1000 psi | ||
3 cm | 6 cm | 9 cm | |
Without pre-treatment | (1) 30,283 aA | (2) 47,246 aA | (3) 37,013 aA |
4 hours | (4) 91,356 bB | (5) 52,786 aAB | (6) 31,170 aA |
24 hours | (7) 74,930 aA | (8) 79,240 aA | (9) 42,813 aA |
Time in osmotic medium | Pressure 1200 psi | ||
Without pre-treatment | (10) 43,183 aA | (11) 41,686 aA | (12) 44,626 aA |
4 hours | (13) 44,736 aA | (14) 74, 263 aA | (15) 44,370 aA |
24 hours | (16) 88,576 bB | (17) 59,086 aB | (18) 18,343 aA |
The highest number of cells expressing the anthocyanin genes was obtained with embryogenic calli cultivated in osmotic medium during 4 h before the bombardment, positioned at 3 cm distant from the microcarrier release platform and using 1000 psi accelerating pressure. Therefore, these conditions were used in six independent experiments to test stable transformation of embryogenic calli obtained from immature sorghum inflorescences, with a cassette containing the
Experiment number | Number of transgenic events | Number of calli bombarded | % Efficiency |
RLB3962008 | 1 | 93 | 1.07 |
RLB5420703 | 3 | 120 | 2.5 |
RLB5430703 | 2 | 60 | 3.33 |
RLB5222202 | 2 | 180 | 1.11 |
RLB020106 | 3 | 296 | 1.01 |
RLB120705 | 2 | 150 | 1.33 |
4.3. Evaluation of transgenic material
To estimate the transgene copy number and the inheritance of the
Transgenic Lines Number |
Herbicide-resistant plants | Herbicide-sensitive plants | Segregation ratio | Chi-square |
1 (RLB3962008) | 28 | 16 | 3:1 | χ2 = 3,03; P"/0,05 |
2 (RLB5222202) | 07 | 01 | 3:1 | χ2 = 0,67; P"/0,05 |
3 (RLB5430703) | 34 | 12 | 3:1 | χ2 = 0,029; P"/0,05 |
4 (RLB5420703) | 37 | 8 | 3:1 | χ2 = 1,25; P"/0,05 |
5 (RLB2109056A) | 23 | 25 | 1:1 | χ2 = 0,83; P"/0,05 |
6 (RLB2109053B) | 28 | 20 | 1:1 | χ2 = 1,33; P"/0,05 |
Wild-type plant (CMSXS102B) | 0 | 50 | NDa | NDa |
Segregation data obtained from six T1 progenies sprayed with herbicide is presented in Table 4. Among the progenies of self -pollinated T0 transgenic plant lines, Chi-square tests showed a Mendelian segregation ratio of 3:1 in four lines. This ratio indicated that the
Leaves of all T0 transgenic events tested negative for the β-glucuronidase expression. However, GUS expression could be detected in germinated seeds (T1) of event RLB5420703 (Fig. 2J).
The presence of
The stable integration of the
5. Discussion
Immature inflorescence proved to be an excellent organ to increase considerably the quantity of tissue competent of embryogenic callus production. A large number of high quality calli is relatively easier and faster to produce from immature inflorescence.
Transient expression of anthocyanin allows us to detect in a rapid and precise manner the most efficient combination of biolistics parameters that rendered a higher transient expression. The frequency of transient activity expression as an indicator of stable transformation efficiency has already been used, successfully, by Christiansen et al. (2005) to optimize the transformation conditions of
An important step in the transformation via biolistics is the wound suffered by the explant during the microparticle entry into the cell. Usually to minimize this type of problem and to increase the capacity for somatic embryogenesis and plant regeneration, the target cells are plasmolised by an osmotic treatment (Vain et al. 1993). In this study all treatments where sorghum explants were incubated in an osmotic media a few hours before bombardment produced a higher number of anthocyanin spots, confirming that plasmolysis of cells can reduce damage and increase the efficiency of bombardments.
Acceleration pressure and microcarrier flying distance are parameters that influence the ability to deliver DNA into various explants. Analyzing the transient expression of anthocyanin, it was observed that a helium gas pressure of 1000 psi combined with a distance of 3 cm rendered the higher number of anthocyanin spots. This combination of biolistic physical parameters when tested in stable transformation experiments showed an efficiency of up to 3.33% of transgenic sorghum events production. Even though, biolistic parameters should be optimized for each equipment and explant used, other authors found optimal bombardment conditions similar to our results. Casas et al. (1993) and Tadesse et al. (2003) were able to generate transgenic sorghum plants via biolistic using a macro-carrier flying distance of 6 cm and a pressure of 1100 psi with an efficiency ranging from 0,3% to 1,3%.
We introduced the
The analysis of PPT-resistance showed that the trait was expressed by all of the transgenic events recovered, probably because of the herbicide selection pressure. In addition, it was inherited by the T1 progenies with a typical Mendelian segregation pattern in four out of six transgenic lines studied. Two lines showed a 1:1 segregation ratio; this type of transgene segregation had already been reported in wheat and maize (Cheng et al., 1997; Ishida et al. 1996). This abnormal segregation pattern might be partially caused by gene silence or non-detectable gene expression in the transgenic plants (Cheng et al., 1997; Vaucheret et al., 1998).
We report a transformation methodology for calli derived from immature inflorescence of sorghum, via biolistics; these transformation conditions are already being used at Embrapa Maize and Sorghum to introduce genes of agronomical interest into the sorghum genome.
Acknowledgments
We thank the McKnight Foundation, Fapemig “Fundação de Apoio a Pesquisa do Estado de Minas Gerais” and CNPq “Conselho Nacional de Desenvolvimento Científico e Tecnológico”-Brazil for financial support. We also acknowledge Dr. Vicki Chandler from the Department of Plant Science, University of Arizona, Tucson, Arizona /USA for the donation of genetic cassettes p35S::C1 and p35S::Bperu. This publication has been funded by “Fundo MP2 Embrapa / Monsanto”.
References
- 1.
Able, J.A., Rathus, C., Godwin, I.D. : The investigation of optimal bombardment parameters for transient and stable transgene expression in sorghum.- In Vitro Cell Dev Biol.37 341 348 2001 - 2.
Bai Z. L. Wang L. Q. Zheng L. P. Li A. J. Wang F. L. A study on the callus induction and plant regeneration of different sorghum explants.Acta Agric Boreali Sinica10 60 63 1995 - 3.
Bhat S. Kuruvinashetti M. S. 1995. - 4.
Bhat S. Kuruvinashetti M. S. Bhat S. 1994 Callus induction and plantlet regeneration from immature inflorescence in some maintainer (B) lines of kharif sorghum ( (L.) Moench.). Karnataka J Agric Sci7 387 390 . - 5.
Brandão R. L. 3 Al+3. Tese doutorado. Universidade Federal de Lavras, UFLA, Lavras, Brazil, 1272007 - 6.
Cai T. Butler L. G. 20 101 110 1990 . - 7.
Carvalho C. H. S. Zehr U. B. Gunaratna N. Anderson J. Kononowicz H. H. Hodges T. K. D. Axtell J. D. Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency . . 27, 259-2692004 - 8.
Casas A. M. Kononowicz A. K. Zehr U. B. Tomes D. T. Axtell J. D. Buttler L. G. Bressan R. A. Hasegawa P. M. 90 11212 11216 1993 - 9.
Casas A. M. Kononowicz A. K. Haan T. G. Zhang L. Tomes D. T. Bressan R. A. Hasegawa P. M. 33 92 100 1997 . - 10.
Chen C. Meyermans H. Van Doorsselaere J. Van Montagu M. Boerjan W. 117 117 719 1998 - 11.
Cheng M. Fry J. E. Pang S. Zhou H. Hironaka C. M. Duncan D. R. Conner T. W. Wan Y. 115 971 980 .1997 - 12.
Christiansen P. Andersen C. H. Didion T. Folling M. Nielsen K. K. 2005 - 13.
Devi, P., Sticklen, M. : In vitro culture and genetic transformation of sorghum by microprojectile bombardment.- Plant Biosystems.137 137 249 254 2003 . - 14.
Emani C. Sunilkumar G. Rathore K. S. 162 181 192 2002 . - 15.
Ferreira, D.F.: SISVAR.exe: sistema de análise de variância. UFLA, Lavras,2000 [SISVAR.exe: variance analysis system] - 16.
Gao Z. Xie X. Ling Y. Muthukrishnan S. Liang G. H. 3 591 599 2005 . - 17.
Girijashankar V. Sharma H. C. Sharma K. K. Swathisree V. Sivarama Prasad. L. Bhat B. V. Royer M. Secundo B. S. Narasu M. L. Altosaar I. Seetharama N. 24 513522 513 522 2005 . - 18.
Goff S. A. Klein T. M. Roth A. B. Fromm M. E. Cone K. C. Radicella J. P. Chandler V. L. 9 2517 2522 .1990 - 19.
Gupta S. Khanna V. K. Singh R. Garg G. K. Strategies for overcoming genotypic limitations of in vitro regeneration and determination of genetic components of variability of plant regeneration traits in sorghum . ular Tiss. Organ. Cult.,86 379 388 ,2006 . - 20.
Gurel, S., Gurel, E., Kaur, R., Wong, J., Meng, L., Tan, H-Q., Lemaux, P. : Efficient, reproducible Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos.- Plant Cell Rep, 28:429-444,2009 . - 21.
Howe A. Sato S. Dweikat I. Fromm M. Clemente T. 784 791 .2006 - 22.
Ishida Y. Saito H. Ohta S. Hiei Y. Komari T. Kumashiro T. 14 745 750 .1996 - 23.
Jeoung J. M. Krishanaveni S. Muthukrishnana S. Trick H. N. Liang G. H. 137 20 28 2002 . - 24.
Jogeswar, G., Ranadheer, D., Anjaiah, V., Kishor, P.B.K. : High frequency somatic embryogenesis and regeneration in different genotypes of Sorghum bicolor (L.) Moench from immature inflorescence explants.- In Vitro Cell Dev. Biol. Plant.,43 159 166 2007 . - 25.
Kaeppler H. F. Pedersen J. F. 48 71 75 .1997 - 26.
Klein T. M. Wolf E. D. Wu R. Sanford J. C. High-velocity microprojectiles for delivering nucleic acids into living cells . .6117 70 73 1987 - 27.
Lu L. Wu X. Yin X. Morrand J. Chen X. Folk W. R. Zhang Z. J. Development of marker-free transgenic sorghum [Sorghum bicolor (L.) Moench] using standard binary vectors with bar as a selectable marker . Tiss Organ Cult.99 97 108 2009 - 28.
Murashige, T., Skoog, F.A. : Revised medium for rapid growth and bioassays with tobacco tissue culture.- ,15 473 497 1962 . - 29.
Mall T. K. Dweikat I. Sato S. J. Neresian N. Xu K. Ge Z. Wang D. Elthon T. Clemente T. 75 467 479 2011 . - 30.
Nahdi, S.; de Wet JMJ 1995 In vitro regeneration of lines from shoot apeses. Internation Sorghum and Millet News letter36 89 90 . - 31.
Nguyen, T-V., Thu, T. T., Claeys, M., Angenon, G. : Agrobacterium-mediated transformation of sorghum (Sorghum bicolor (L.) Moench) using an improved in vitro regeneration system.- ., 91:155-164,2007 . - 32.
Nwanze K. F. Seethrama N. Sharma H. C. Stenhouse J. W. Frederiksen R. Shantharam S. Raman K. 3 209 215 1995 . - 33.
Patil V. M. Kuruvinashetti M. S. 64 217 219 1998 - 34.
Rueb, S., Hensgens, L.A.M. : Improved histochemical staining for β-glucuronidase activity in monocotyledonous plants.- Rice Genet. Newsl.6 168 169 1998 . - 35.
Saghai-Maroof M. A. Soliman K. M. Jorgensen R. A. Allard R. W. 81 8014 8019 . - 36.
Sambrook, J., Fritsch, E.F., Maniatis, T. : Molecular Cloning: A Laboratory Manual. 2.nd.ed.- Cold Spring Harbor Laboratory London1989 . - 37.
Sanford J. C. Smith F. D. Russell J. A. Optimizing the biolistic process for different biological applications. In: WU, R. (dE.). Recombinant DNA- Part H. San Diego: Academic,1993 483 510 (, 217). - 38.
Sasaki T. Yamamoto Y. Ezaki B. Katsuhara M. Ahn S. J. Ryan P. R. Delhaize E. Matsumoto H. 37 645 653 ,2004 . - 39.
Shyamala D. Devi P. 41 1482 1486 2003 - 40.
Tadesse, Y., Sági, L., Swennen, R., Jacobs, M. : Optimisation of transformation conditions and production of transgenic sorghum (Sorghum bicolor) via microparticle bombardment- Rev. Plant Biotechnol. Appl. Genetics75 1 18 2003 . - 41.
Taylor NJ, Fauquet CM. Particle bombardment as a tool in plant science and agricultural biotechnology. DNA Cell Biol.12 21 963 977 2002 - 42.
Vain, P., Mcmullen, M.D., Finer, J.J. : Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize- Plant Cell Rep.12 84 88 1993 . - 43.
Van Nguyen, T., Thu,T.T., Claeys, M., Angenon, G. : Agrobacterium-mediated transformation of sorghum (Sorghum bicolor (L.) Moench) using an improved in vitro regeneration system.- Plant Cell Tiss Organ Cult. 91:155 164 2007 - 44.
Vaucheret H. Béclin C. Elmayan T. Feuerbach F. Godon C. Morel J. B. Mourrain P. Palauqui J. C. Vernhettes S. 16 651 659 1998 . - 45.
Wu L. Nandi S. Chen L. Rodriguez R. L. Huang N. 2002 Expression and inheritance of nine transgenes in rice. Transgenic Res.11 533 541 . - 46.
Zhao Z. Y. Cai T. Tagliani L. Miller M. Wang N. Pang H. Rudert M. Schroeder S. Hondred D. Seltzer J. Pierce D. 2000 . - 47.
Zhu H. Muhukrishnan S. Krishnaveni S. Wilde G. Jeoung J. M. Liang G. H. 243 252 1998 .
Notes
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