Adaptation of <i>in vitro</i> regeneration protocol for Brazilian wheat genotypes

Roesler, Eduardo André; Manfroi, Ernandes; Morás, Andréa; Didoné, Dielli Aparecida; Grando, Magali Ferrari; Lau, Elene Yamazaki

doi:10.1590/0103-8478cr20170806

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BIOLOGY • Cienc. Rural 48 (11) • 2018 • https://doi.org/10.1590/0103-8478cr20170806 copy

Adaptation of in vitro regeneration protocol for Brazilian wheat genotypes

Adaptação do protocolo de regeneração in vitro para genótipos de trigo brasileiros

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT:

The availability of an efficient protocol for in vitro regeneration is imperative for genetic transformation. Using genotypes adapted to the target region as a transgenic platform accelerates the development of cultivars. Therefore, this study aimed to adapt an in vitro regeneration protocol for Brazilian wheat genotypes. For this purpose, the in vitro regeneration capacity of immature embryos from six Brazilian wheat genotypes using two protocols of regeneration of somatic embryos was analysed. Furthermore, combinations of 2,4-D and picloram in the callus induction medium were tested in order to improve regeneration efficiency. Genotypes with higher regeneration efficiency were BR18-Terena and PF020037, yielding 0.42 and 1.13 shoots per explant using the Hu and the Wu protocol, respectively. Adding 1mgL^-1 2,4-D in the callus induction medium was the most favourable, producing 3.73 and 3.07 shoots per explant for PF020037 and BR18-Terena, respectively. In conclusion, a protocol for regeneration for two Brazilian wheat genotypes recommended and developed to be cultivated at the Cerrado region has been adapted.

Key words:
Triticum aestivum; tissue culture; growth regulators

-----------------------------------------------------Culture medium Protocol according to Hu et al. (2003HU, T. et al Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Reports, v.21, p.1010-1019, 2003. Available from: <Available from: https://link.springer.com/article/10.1007/s00299-003-0617-6 >. Accessed: Oct. 30, 2017. doi: 10.1007/s00299-003-0617-6. https://link.springer.com/article/10.100... )-----------------------------------------------
CM4C: MS macro and micronutrients, MS FeNa₂.EDTA, MS vitamins, 100mgL^-1inositol, 100mgL^-1 ascorbic acid, 40gL^-1 maltose, 0.5mgL^-1 2,4-D (AcrosOrganics), 2.2mgL^-1picloram (Sigma), 7gL^-1agar (Sigma), pH 5.8
Resting: same components of CM4C, except using 2mgL^-1 2,4-D
MMS0.2C: same components of CM4C, except using 0.2mgL^-1 2,4-D and absence of picloram
MMS0C: same components of CM4C, except using 0.1µM L-tryptophan, 0.1µM L-tyrosine, 0.1µM L-phenylalanine, and absence of 2,4-D and picloram
---------------------------------------------------Culture medium according to Wu et al. (2009WU, H. et al. Agrobacterium-mediated transformation of bread and durum wheat using freshly isolated immature embryos. In: Jones DH and Shewry RP (eds) Transgenic Wheat, Barley and Oats: Production and Characterization Protocols. Humana Press, Totowa , 2009, p. 93-103. Available from: <Available from: https://link.springer.com/protocol/10.1007%2F978-1-59745-379-0_5 >. Accessed: Oct. 30, 2017. doi: 10.1007/978-1-59745-379-0_5. https://link.springer.com/protocol/10.10... )-----------------------------------------------------------
Inoculation/cocultivation: MS macronutrients, L7 micronutrients, MS vitamins, MS FeNa₂.EDTA, 100mgL^-1inositol, 1gL^-1glutamine, 100mgL^-1caseinhydrolysate, 1.95gL^-1 MES, 2mgL^-1 2,4-D, 2mgL^-1picloram, 10gL^-1 glucose, 40gL^-1 maltose, 2gL^-1phytagel, pH 5.7
Induction: MS macronutrients, L7 micronutrients, MS vitamins, MS FeNa₂.EDTA, 100mgL^-1inositol, 1gL^-1glutamine, 100mgL^-1casein hydrolysate, 1.95gL^-1 MES, 0.5mgL^-1 2,4-D, 2mgL^-1picloram, 40gL^-1 maltose, 7gL^-1agar, pH 5.7
Regeneration: L7 macro and micronutrients, MS FeNa₂.EDTA, L vitamins/inositol (200mgL^-1inositol, 10mgL^-1thiamine-HCl, 1mgL^-1 pyridoxine-HCl, 1mgL^-1ascorbic acid, 1mgL^-1calcium pantothenate, 1mgL^-1ascorbic acid), 100mgL^-1inositol, 30gL^-1 maltose, 0.1mgL^-1 2,4-D, 5mgL^-1zeatin (Sigma), 7gL^-1agar, pH 5.7
Selection: same components of Regeneration, except absence of 2,4-D and zeatin.

---------------------------------------------------A) Explants with shoots regeneration (%)---------------------------------------------------
		-----------------BR18-Terena-----------------			-----------------PF02003-----------------
		-----------------2,4-D (mgL^-1)-----------------			-----------------2,4-D (mgL^-1) -----------------
		0	1	2	0	1	2
Picloram (mgL^-1)	0	0.0cB	60.8aA	40.8aA	0.0bB	61.2aA	55.1aA
	1	7.3bB	26.2bA	12.3bA	54.6aA	59.4aA	44.5aA
	2	20.1aA	21.6bA	8.9bA	43.4aAB	27.1bB	48.5aA
---------------------------------------------------B) Number of shoots per explant---------------------------------------------------
		-----------------BR18-Terena-----------------			-----------------PF020037-----------------
		-----------------2,4-D (mgL^-1) -----------------			--------------2,4-D (mgL^-1) ---------------
		0	1	2	0	1	2
Picloram (mgL^-1)	0	0.00bC	3.07aA	1.63aB	0.00bB	3.73aA	2.68aA
	1	0.20bB	1.09bA	0.26bAB	3.12aA	1.95abA	1.53aA
	2	0.57aA	0.77bA	0.22bA	2.54aA	1.61bA	1.47aA

Universidade Federal de Santa Maria Universidade Federal de Santa Maria, Centro de Ciências Rurais , 97105-900 Santa Maria RS Brazil , Tel.: +55 55 3220-8698 , Fax: +55 55 3220-8695 - Santa Maria - RS - Brazil
E-mail: cienciarural@mail.ufsm.br

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[1] E-mail: elene.yamazaki-lau@embrapa.br. ^*Corresponding author

	Explants with shoots regeneration (%)			Number of shoots per explant
Wheat genotype	Hu⁽¹⁾	Wu⁽²⁾
Bobwhite SH9826	27.1abA	42.0aA	1.00a
BR18-Terena	47.6aA	22.5abB	0.42ab
BRS 264	8.2bcA	11.4bcA	0.24bc
MGS1 Aliança	0.9bcA	8.8bcA	0.10bc
MGS3 Brilhante	8.8cA	2.5cA	0.03c
PF020037	24.0abA	42.7aA	1.13a
PF020062	9.4bcA	8.1bcA	0.16bc
			Wu	Hu
			0.58 A	0.32 B