Pereira, Carla Gomes; Carvalho-Silva, Micheline; Pereira, Luiz Alfredo Rodrigues; Silveira, Conceição Eneida Santos

Carla Gomes Pereira ⁶6Author for correspondence: carla_ghomes@yahoo.com.br

University of Brasilia, Department of Botany, Campus Darcy Ribeiro, Asa Norte, Brasilia, DF, Brazil.

https://orcid.org/0000-0002-9088-4879

Micheline Carvalho-Silva

University of Brasilia, Department of Botany, Campus Darcy Ribeiro, Asa Norte, Brasilia, DF, Brazil.

https://orcid.org/0000-0002-2389-3804

Luiz Alfredo Rodrigues Pereira

University of Brasilia, Department of Botany, Campus Darcy Ribeiro, Asa Norte, Brasilia, DF, Brazil.

https://orcid.org/0000-0001-8204-2586

Conceição Eneida Santos Silveira

University of Brasilia, Department of Botany, Campus Darcy Ribeiro, Asa Norte, Brasilia, DF, Brazil.

https://orcid.org/0000-0002-2854-2835

6Author for correspondence: carla_ghomes@yahoo.com.br

Area Editor: Dr. Nelson Augusto Santos Jr

Figuras (4)
Tabelas (3)

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Figure 1
Explant development rate of ten Cerrado mosses species in different treatments (1% for 5 min; 1% for 10 min; and 2% for 2 min), after 10 days of in vitro cultivation. The bars represent the standard error. * Precultivated material.

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Figure 2
a-g. Establishment and in vitro propagation of mosses. a-c. direct establishment (without contamination) - a. developed explants were transferred to new plates; b. at the end of the 1^st subculture, the established explants were fragmented and transferred to new plates; c. propagation of the species. a, d-g and c. indirect establishment (with contamination) - a. developed explants were transferred to new plates; d-e. the explants were subcultivated until they had produced uncontaminated regions; f. sufficiently developed explants were fragmented and subcultivated; g. presence of established explants with no visible contamination in the plates; c. explants, free of contamination, were used to establish in vitro aseptic cultures and used for propagation of the species.

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Figure 3
a-l. Indirect establishment of Bryum argenteum (a-h) and Isopterygium tenerifolium (i-l) - a. contaminated colony of explants in the 3^rd subculture (arrow); b. partial contamination (arrow) in the 4^th subculture; c. partial contamination (arrow) in the 5^th subculture after 24 days of cultivation; d. partial contamination (arrow) in the 5^th subculture after 35 days, showing the growth of protonemata external to the medium with contamination (asterisk); e. growth erect gametophytes, externally to the medium with contamination (arrow) in the 5^th subculture after 24 days; f. growth of gametophytes near contaminated areas (arrows); g. apical fragment of gametophyte without contamination in the 6^th subculture after 11 days of cultivation; h. established Bryum argenteum culture during the 6^th subculture; i. Isopterygium tenerifolium with contamination (arrow) in the 3rd subculture with 40 days; j. growth of gametophyte near the contamination (arrows); k. apical fragment of gametophyte without contamination in the 4th subculture after 40 days; l. established Isopterygium tenerifolium culture free of contamination in the 5^th subculture after 40 days. Bars: k. = 0.1 mm; f, g, j, l = 0.5 mm; c, d, e, h, i = 1 mm; a, b = 2 mm.

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Figure 4
a-p. Structures developed in Cerrado mosses grown in vitro - a. Bryum argenteum shoots (7); b. apical protonemata in Fissidens flaccidus (5); c. Protonemata at the stem section site (arrow) in Vitalia cuspidifera (20); d. Archegonium (arrow) of Bryum argenteum (10); e. Antheridium (arrow) of Isopterygium tenerifolium (13); f. apical growth from tip of the apex (arrow) in Bryum coronatum (13); g. Protonemata on the leaf lamina of Leucobryum crispum (20); h. Protonemata colony of Bryum argenteum with gametophytic buds (24); i. Protonemata colony of Pogonatum pensilvanicum with young gametophores (130); j. Gametophytes of Sphagnum platyphylloides (55); k. Tubers (arrow) on the axil of leaf of Bryum coronatum (100); l. Tubers (arrow) on the caulonemas of Bryum densifolium (150); m. Protonemal gemmae of Bryum argenteum in segmentation (arrow) (300); n. Protonemata colony formed from the germination of protonemal gemmae of Bryum argenteum (32); o. Gemmae on the apex of the protonema of Barbula indica (100); p. Regeneration of Leucobryum crispum from the apex of the leaf. (Numbers in parentheses = days of cultivation, without exchange of culture medium). Bars: d, e, m = 50 µm; a, b, c, k, l, o = 0.5 mm; f, g, h, p = 1 mm, i, j, n = 2 mm.

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Table 1
Disinfestation treatments with commercial bleach (2% active chlorine) – 1% concentration for 5 min (1% - 5'), 1% for 10 min (1% - 10'), and 2% for 2 min (2% - 2'), bryophyte species and respective collection data and number of explants per treatment.

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Table 2
Percentage of moss species developed with or without contamination after disinfestation treatments (solution of Sodium hypochlorite x exposure time) and cultivated in vitro for 10 days in Knop medium.

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Table 3
Characterization of moss species and responses observed during in vitro establishment in Knop medium. (GA = gametophytes apex; FG = female gametangium; MG = male gametangium; LA = leaf axillary; SS = place of the stem section; ALd = axillary of the leaf detached; ABd = axillary of the branch detached; LL = leaf lamina; AG = apical growth; B = branching.

Species	Vouchers	Collection Date	Numeber of Explants	NaClO Tratment Time
Barbula indica	503	September / 2015	40	1% - 10'
Barbula indica ^() ()** precultivated species	503	September / 2015	88	1% - 10'
Bryum argenteum	499	April / 2015	200	1% - 5'
			200	1% - 10'
			200	2% - 2'
Bryum argenteum ^() ()** precultivated species	587	December / 2017	200	1% - 5'
Bryum coronatum	501	June / 2015	56	1% - 10'
Bryum densifolium	31B	May / 2017	96	1% - 5'
			96	1% - 10'
			96	2% - 2'
Fissidens flaccidus	501	June / 2015	32	1% - 10'
Isopterygium tenerifolium	509	September / 2015	200	1% - 5'
			200	1% - 10'
			200	2% - 2'
Leucobryum crispum	583	July / 2016	200	1% - 5'
			200	1% - 10'
			200	2% - 2'
Pogonatum pensilvanicum ^() ()** precultivated species	526	May / 2016	32	2% - 2'
Sphagnum platyphylloides	541	May / 2016	88	1% - 10'
Vitalia cuspidifera	560	July / 2016	200	1% - 5'
			200	1% - 10'
			200	2% - 2'

	Treatment (active chlorine-min)	Number of species	Number of species developed	Average percentage of developed explants
Development without contamination	1% - 5	6	3 (50%)	3.6%
	1% - 10	10	4 (40%)	2.4%
	2% - 2	6	5 (80%)	7.7%
Development with contamination	1% - 5	6	3 (50%)	5.6%
	1% - 10	10	9 (90%)	17.7%
	2% - 2	6	6 (100%)	15.5%

Species	Natural Growth Factors		Sample Type	Establishment	Growth Form (in 30 days)	Form of initial development / Structure of development	Growth (in 30 days) ^c c average diameter and standard deviation of colony in millimeters; / ^d d number and standard deviation of shoots developed or branches developed; / ^e e average height and standard deviation of gametophytes in millimeters;	Subcultures / Time for IE ^f f IE = indirect establishment.	Propagule / Period of Emergence	Maximum culture period
Species	Environment	Habit	Sample Type	Establishment	Growth Form (in 30 days)	Form of initial development / Structure of development		Subcultures / Time for IE ^f f IE = indirect establishment.	Propagule / Period of Emergence	Maximum culture period
Barbula indica	Urban, cement, shaded	Acrocarpous/Dioicous	Fresh and Pre-cultivated (120 days)	Indirect	Only protonemata	Protonemata: GA	10(1.9)/0/0	5/105 days	Gemmae / 3 months	< 1 year
Bryum argenteum ^a a species collected in 2015;	Urban, cement, shaded	Acrocarpous/Dioicous	Fresh	Direct and Indirect	Predominantly protonemata	Protonemata: GA, FG, LA, SS; Shoots: GA, ALd	10(3)/15(4.5)/ 1(0.1)	6/135 days	protonemal gemmae / 4 months	< 1 year
Bryum argenteum ^b b species collected in 2017;	Urban, cement, shaded	Acrocarpous/Dioicous	Pre-cultivated (7 days)	Direct and Indirect	Predominantly protonemata	Protonemata: GA, Ald; Shoots: GA, Ald	10(2.5)/15(4.4)/ 1(0.3)	6/135 days	protonemal gemmae / 4 months	< 1 year
Bryum coronatum	Urban, soil, shaded	Acrocarpous/Dioicous	Fresh	Direct and Indirect	Protonemata and gametophytes	Protonemata: GA, SS; Shoots: GA, AG	9(1.3)/20(3.6)/ 2(0.2)	4/80 days	Tubers / 3 months	< 1 year
Bryum densifolium	Forest, soil, shaded	Acrocarpous/Dioicous	Fresh	Indirect	Predominantly protonemata	Protonemata: ALd	15(1.6)/6(2.5)/ 5(2.7)	6/135 days	Tubers / 4 months	< 1 year
Fissidens flaccidus	Urban, soil, shaded	Acrocarpous/Monoicous	Fresh	Indirect	Predominantly protonemata	Protonemata: GA	14(0.9)/10(2.4)/ 1.5(0.1)	5/105 days	-	9 months
Isopterygium tenerifolium	Open field, soil, sunny	Pleurocarpous/Monoicous	Fresh	Direct and Indirect	Predominantly, gametophytes	Protonemata: GA, FG, MG, ALd, ABd, SS; Shoots: LA	12(1.3)/8(B) (2.7)/5(0.8)	5/135 days	-	< 1 year
Leucobryum crispum	Forest, soil, Shaded	Acrocarpous/Dioicous	Fresh	Direct and Indirect	Protonemata and gametophytes	Protonemata: GA, LA, ALd, LL	10(2.4)/7(2)/ 3(0.5)	5/105 days	-	8 months
Pogonatum pensilvanicum	Forest, soil, sunny	Acrocarpous/Dioicous	Pre-cultivated (9 months)	Direct and Indirect	Protonemata and gametophytes	Protonemata: LA	10(1)/15(2.1)/ 2(0.3)	3/50 days	-	8 months
Sphagnum platyphylloides	Lake, humid soils, sunny	Acrocarpous/Dioicous	Fresh	Indirect	Predominantly, gametophytes	Shoots: GA, AG	16(2.4)/3(B) (0.9)/10(0.3)	6/135 days	-	10 months
Vitalia cuspidifera	Forest, rock, shaded	Pleurocarpous/Monoicous	Fresh	Direct and Indirect	Protonemata and gametophytes	Protonemata: GA, ALd, ABd, SS; Shoots: LA	8(0.5)/0/0	5/105 days	-	6 months

[1] 6Author for correspondence: carla_ghomes@yahoo.com.br

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Indirect establishment increases the chances of in vitro propagation of mosses occurring in the Cerrado - a new method

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