<i>In vitro</i> organogenesis and genetic transformation of mandarin cultivars

The in vitro organogenesis of Fremont (Citrus clementina x ), Citrus reticulataThomas (Citrus reticulata), and Nules (Citrus clementina) mandarins was evaluated aiming to optimize a regeneration protocol that could be applied in genetic transformation. The use of epicotyl-derived explants resulted in higher explant responsiveness and number of shoots developed per explant when compared with the use of internodal-derived explants. The highest efficiency in shoot regeneration was observed in the presence of 1 mg L-1 of BAP, regardless of the explant type and cultivar. The in vitro organogenesis protocol produced transgenic plants from three mandarin cultivars expressing attA gene under the control of phloem-specific promoters.

Index terms
antimicrobial peptide; cultivar improvement; cytokinin; huanglongbing; transgenic plants

Authorship

Leonardo Soriano

Forestry Eng. – Dr – University of São Paulo / Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab. – Piracicaba, SP – Brazil. E-mail: soriano84@gmail.com University of São PauloBrasilPiracicaba, SP, Brasil Forestry Eng. – Dr – University of São Paulo / Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab. – Piracicaba, SP – Brazil. E-mail: soriano84@gmail.com

http://orcid.org/0000-0002-8958-4000

Eveline Carla da Rocha Tavano

Biologist – Dr – University of São Paulo / Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab. – Piracicaba, SP – Brazil. E-mail: ectavano@gmail.com University of São PauloBrasilPiracicaba, SP, Brasil Biologist – Dr – University of São Paulo / Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab. – Piracicaba, SP – Brazil. E-mail: ectavano@gmail.com

http://orcid.org/0000-0001-5385-3267

Marcelo Favaretto Correa

Biologist – Lab. technician – University of São Paulo / Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab. – Piracicaba, SP – Brazil. E-mail: mfcorrea@cena.usp.br University of São PauloBrasilPiracicaba, SP, Brasil Biologist – Lab. technician – University of São Paulo / Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab. – Piracicaba, SP – Brazil. E-mail: mfcorrea@cena.usp.br

http://orcid.org/0000-0002-9871-9149

Ricardo Harakava

Biologist – Dr – Researcher – State Department of Agriculture and Supply / Biological Institute –Biochemical Plant Pathology Lab. – São Paulo, SP – Brazil. E-mail: harakava@biologico.sp.gov.br State Department of Agriculture and SupplyBrasilSão Paulo, SP, Brasil Biologist – Dr – Researcher – State Department of Agriculture and Supply / Biological Institute –Biochemical Plant Pathology Lab. – São Paulo, SP – Brazil. E-mail: harakava@biologico.sp.gov.br

http://orcid.org/0000-0003-1431-2665

Beatriz Madalena Januzzi Mendes

Agr. Eng. – Associate Professor 2 (Retired) – University of São Paulo / Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab. – Piracicaba, SP – Brazil. E-mail: bmendes@cena.usp.br University of São PauloBrasilPiracicaba, SP, Brasil Agr. Eng. – Associate Professor 2 (Retired) – University of São Paulo / Center for Nuclear Energy in Agriculture – Plant Biotechnology Lab. – Piracicaba, SP – Brazil. E-mail: bmendes@cena.usp.br

Francisco de Assis Alves Mourão Filho

Agr. Eng. – Full Professor – University of São Paulo / Luiz de Queiroz College of Agriculture – Crop Science Department – Piracicaba, SP – Brazil. E-mail: francisco.mourao@usp.br University of São Paulo / Luiz de Queiroz College of AgricultureBrasilPiracicaba, SP, Brasil Agr. Eng. – Full Professor – University of São Paulo / Luiz de Queiroz College of Agriculture – Crop Science Department – Piracicaba, SP – Brazil. E-mail: francisco.mourao@usp.br

http://orcid.org/0000-0001-8950-9513

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (3)
Tables (2)

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Figure 1
In vitro organogenesis of epicotyl and internodal-derived explants from Fremont, Thomas and Nules mandarin cultivars under the influence of the different concentration of BAP a-b) Mean of percentage of responsive explants derived from epicotyl (a) and internodal (b) segments for Fremont, Thomas, and Nules. c-d) Mean of number of shoots per explants derived from epicotyl (c) and internodal (d) segments for Fremont, Thomas, and Nules.

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Figure 2
Southern blot analyses of transgenic plants of mandarin cultivars Thomas, Fremont and Nules transformed with pCAtPP2/attA (a) or pCAtSuc2/attA (b-c) gene constructs. a) Plants of cv. Fremont (events 1-3), Thomas (events 4-10) and Nules (event 11). Events 2, 3, 4, 6, 7, 9, 10 and 11 have one insertion. Events 1, 5 and 8 have more than three insertions. b) Plants of cv. Fremont (events 1-8), Thomas (event 9) and Nules (events 10-11). Events 1, 2, 3, 4, 5, 7, 9 and 11 have one insertion. Events 6 and 8 have two insertions. Event 10 not transformed. c) Plants of cv. Thomas (events 1-11). Events 1, 4, 5, 6, 8 and 11 have one insertion. Events 2, 7, 9 and 10 have two insertions. Event 3 has three insertions. C+: positive control (725 bp fragment of attA gene). C-: negative control (DNA from non-transgenic plant). Total DNA from transgenic and non-transgenic plants was digested with BamHI (pCAtSuc2/attA) or HimdIII (pCAtPP2/attA) and hybridized with a probe of the attA gene (150 ng).

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Figure 3
Relative quantification of the attA gene expression in transgenic plants of Thomas, Fremont, and Nules mandarin cultivars. a) Transgenic lines of Fremont cv. containing pCAtPP2/attA (bars 1 - 2) or pCAtSuc2/attA (bars 3 - 6) gene constructs. b) Transgenic lines of Thomas cv. containing pCAtPP2/attA (bars 7 - 10) or pCAtSuc2/attA (bars 11 - 15) gene constructs. c) Transgenic lines of Nules cv. containing pCAtPP2/attA (bar 16) or pCAtSuc2/attA (bars 17-18) gene constructs. Relative expression of the target gene was normalized with the ubiquitin (UBQ) gene and calculated in relation to the transgenic line that presented the lowest ?Ct value (*).

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Table 1
In vitro organogenesis from epicotyl and internodal-derived segments from Fremont, Thomas, and Nules mandarins (mean of three experiments, totaling 108 explants per treatment).

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Table 2
Fremont, Thomas, and Nules mandarin genetic transformation experiments with gene constructs harboring the attacin A gene associated with phloem-specific promoters.

Figure 1 In vitro organogenesis of epicotyl and internodal-derived explants from Fremont, Thomas and Nules mandarin cultivars under the influence of the different concentration of BAP a-b) Mean of percentage of responsive explants derived from epicotyl (a) and internodal (b) segments for Fremont, Thomas, and Nules. c-d) Mean of number of shoots per explants derived from epicotyl (c) and internodal (d) segments for Fremont, Thomas, and Nules.

Figure 2 Southern blot analyses of transgenic plants of mandarin cultivars Thomas, Fremont and Nules transformed with pCAtPP2/attA (a) or pCAtSuc2/attA (b-c) gene constructs. a) Plants of cv. Fremont (events 1-3), Thomas (events 4-10) and Nules (event 11). Events 2, 3, 4, 6, 7, 9, 10 and 11 have one insertion. Events 1, 5 and 8 have more than three insertions. b) Plants of cv. Fremont (events 1-8), Thomas (event 9) and Nules (events 10-11). Events 1, 2, 3, 4, 5, 7, 9 and 11 have one insertion. Events 6 and 8 have two insertions. Event 10 not transformed. c) Plants of cv. Thomas (events 1-11). Events 1, 4, 5, 6, 8 and 11 have one insertion. Events 2, 7, 9 and 10 have two insertions. Event 3 has three insertions. C+: positive control (725 bp fragment of attA gene). C-: negative control (DNA from non-transgenic plant). Total DNA from transgenic and non-transgenic plants was digested with BamHI (pCAtSuc2/attA) or HimdIII (pCAtPP2/attA) and hybridized with a probe of the attA gene (150 ng).

Figure 3 Relative quantification of the attA gene expression in transgenic plants of Thomas, Fremont, and Nules mandarin cultivars. a) Transgenic lines of Fremont cv. containing pCAtPP2/attA (bars 1 - 2) or pCAtSuc2/attA (bars 3 - 6) gene constructs. b) Transgenic lines of Thomas cv. containing pCAtPP2/attA (bars 7 - 10) or pCAtSuc2/attA (bars 11 - 15) gene constructs. c) Transgenic lines of Nules cv. containing pCAtPP2/attA (bar 16) or pCAtSuc2/attA (bars 17-18) gene constructs. Relative expression of the target gene was normalized with the ubiquitin (UBQ) gene and calculated in relation to the transgenic line that presented the lowest ?Ct value (*).

Table 1 In vitro organogenesis from epicotyl and internodal-derived segments from Fremont, Thomas, and Nules mandarins (mean of three experiments, totaling 108 explants per treatment).

cultivars	BAP (mg L^-1)	responsive explants (%)		number of shoots/explant
cultivars	BAP (mg L^-1)	epicotyl	internodal	epicotyl		internodal
Fremont	0.0	36.11	9.26	3.06		1.11
	0.5	56.48	23.15	4.61	2.17
	1.0	78.70	50.93	6.11	4.00
	1.5	57.41	15.74	4.56	1.78
	2.0	32.41	7.41	2.89	0.83
mean		52.22A	21.30a	4.24A	1.98b
SD		18.73	17.68	1.32	1.25
Thomas	0.0	34.26	7.41	3.28	1.39
	0.5	65.74	13.89	5.67	1.56
	1.0	73.15	40.74	5.89	3.89
	1.5	55.56	12.04	4.67	1.44
	2.0	36.11	5.56	3.39	0.72
mean		52.96A	15.93b	4.58A	1.80b
SD		17.40	14.27	1.23	1.21
Nules	0.0	30.56	16.67	3.22	2.06
	0.5	54.63	24.07	5.00	2.50
	1.0	82.41	54.63	6.56	5.17
	1.5	54.63	18.52	4.67	2.11
	2.0	31.48	12.96	3.17	1.83
mean		50.74A	25.37a	4.52A	2.73a
SD		21.28	16.84	1.41	1.38

^*
Means followed by the same letter in the column are not significantly different by the Tukey’s test (P = 0.05).

Table 2 Fremont, Thomas, and Nules mandarin genetic transformation experiments with gene constructs harboring the attacin A gene associated with phloem-specific promoters.

genotype	gene construct	no. PCR-positive plants/ total explants	genetic transformation efficiency (%)^a	no. Southern blot + plants
genotype	gene construct	no. PCR-positive plants/ total explants	genetic transformation efficiency (%)^a	no. Southern blot + plants
Fremont	pCAtPP2/attA	3/1378	0.22	3
Fremont	pCAtSuc2/attA	8/1874	0.43	8
Thomas	pCAtPP2/attA	10/1743	0.57	7
Thomas	pCAtSuc2/attA	12/1297	0.93	12
Nules	pCAtPP2/attA	3/963	0.31	1
Nules	pCAtSuc2/attA	2/709	0.28	1

^a
PCR-positive plants (x100) / total explants

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<i>In vitro</i> organogenesis and genetic transformation of mandarin cultivars

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[TFN0101] ^*
Means followed by the same letter in the column are not significantly different by the Tukey’s test (P = 0.05).