Artigo sobre biotecnologia vegetal

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Acta Hortic. 1224. ISHS 2018. DOI 10.17660/ActaHortic.2018.1224.16 
Proc. VII Int. Symp. on Production and Establishment of Micropropagated Plants 
Eds.: R. Paiva et al. 
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In vitro establishment and anatomical analysis of 
Bauhinia holophylla (Bong.) V.C.	 Steina,	 M.D.	 Cardoso	 Junior,	 M.C.	 Coimbra,	 I.	 Rodrigues-Brandão,	 M.S.	 Pádua	 and	A.H.F.	Castro	Universidade	Federal	de	São	João	del-Rei,	Campus	Centro-Oeste	Dona	Lindú,	Divinópolis,	Minas	Gerais,	Brasil.	
Abstract 
Bauhinia holophylla	 (Bong.)	 Steud.	 is	 an	 endemic	 legume	 of	 the	 Brazilian	
Savanna	that	has	economic	and	medicinal	importance.	The	natural	and	conventional	
propagation	occurs	through	seeds,	whose	production	occurs	during	a	few	months	per	
year.	 Currently,	 the	 Brazilian	 Savanna	 agricultural	 expansion	 aggravates	 the	
propagation	 and	 conservation	 of	B. holophylla	 species.	 Considering	 the	 few	 studies	
about	 the	environmental	 factors	effects	on	germination	and	growth	of	B. holophylla,	
micropropagation	 is	 a	 promising	 alternative	 for	 seedlings	 production	 and	 species	
conservation.	 The	 aim	 of	 this	 work	 was	 to	 establish	 a	 protocol	 for	 in	 vitro	 seed	
germination,	shoots	 induction	and	elongation	of	B. holophylla	explants.	To	obtain	 in	
vitro	seedlings,	seeds	were	germinated	on	culture	medium	WPM	50	and	100%,	MS	50	
and	100%	of	salts	concentration	or	water	+	agar.	After	30	days,	nodal	segments	of	B. 
holophylla	 from	 seedlings	were	 isolated	 and	 inoculated	 into	WPM	 culture	medium	
supplemented	with	the	6-benzylaminopurine	(BAP)	at	concentrations	of	0,	0.48,	8,	16	
μM.	After	 induction	of	shoots,	the	explants	were	 inoculated	 in	WPM	culture	medium	
containing	different	concentrations	of	gibberellic	acid	(GA3):	0,	5,	10	and	20	μM.	In	the	
experiments	 the	 following	 parameters	 were	 evaluated:	 germination	 percentage,	
oxidation,	 stem	 length,	 leaf	 number	 and	 number	 of	 nodes.	 At	 the	 end	 of	 the	
multiplication	experiment,	leaves	were	collected	for	histological	analysis.	For	in	vitro	
seeds	 germination,	 the	 water	 +	 agar	 substrate	 showed	 higher	 percentages	 of	
germination	 (91%),	not	differing	 from	 the	 culture	media	MS	50%	and	WPM	100%.	
However,	for	growth	parameters,	the	best	culture	medium	was	WPM	100%.	For	nodal	
segments	multiplication,	 there	was	 a	 significant	 increase	 on	 shoot	 number	 related	
with	BAP	concentration.	For	shoots	elongation,	there	was	reduction	on	stem	size	with	
the	 increase	 of	 GA3	 concentration.	 In	 the	 anatomical	 analyses,	 the	 development	 of	
palisade	and	spongy	parenchyma	and	vascular	bundles	was	observed	in	B. holophylla	
in	vitro	leaves.	
Keywords:	Cerrado,	micropropagation,	germination,	6-benzylaminopurine	
INTRODUCTION	Tropical	 savanna	 (Cerrado)	 is	 the	 second	 large	 biome	 in	 Brazil,	 covering	 25%	 of	national	 territory.	Cerrado	communities	are	remarkably	complex,	and	are	characterized	by	tree	species	diversity	far	higher	than	in	any	other	neotropical	savanna	region.	This	biome	is	characterized	 by	 seasonal	 rainfall	 with	 a	 five-month	 dry	 season,	 oligotrophic	 soils,	 and	frequent	 fires	 (Jackson	 et	 al.,	 1999).	 As	 an	 ecosystem	with	 limited	 resources,	 their	 native	plants	produce	and	accumulate	more	bioactive	compounds	(Ferreira-Rodrigues	et	al.,	2016).	
Bauhinia	holophylla	 (Bong.)	 Steud.	 is	 a	Cerrado	 endemic	 species,	 known	 as	 “pata	de	vaca”,	 and	 widely	 used	 in	 the	 treatment	 of	 several	 conditions,	 such	 as	 infections,	 pain,	diabetes,	and	gastric	ulcer	(Silva	and	Cechinel	Filho,	2002;	Rozza	et	al.,	2015).	The	increasing	deforestation	 of	 Cerrado,	 mainly	 due	 to	 agricultural	 expansion,	 has	 generated	 negative	impact	 on	 its	 biodiversity	 and	 there	 is	 an	 awareness	of	 the	 necessity	 of	 studies	 regarding	native	plants	including	those	with	medicinal	properties.	
 
aE-mail: vanessastein@ufsj.edu.br 
 
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In	 this	 context,	 native	 plants	 with	 ecological	 and	 medical	 potential,	 as	 Bauhinia	
holophylla,	 are	 categorized	 as	 specific	 targets	 for	 biotechnological	 improvement.	 The	establishment	of	a	tissue	culture	system	is	an	alternative	for	conservation,	propagation	and	production	 of	 secondary	 metabolites	 with	 pharmaceutical	 interest.	 Tissue	 culture	techniques	 comprise	 efficient	 tools	 in	 order	 to	 preserve	 genetic	 resources,	 especially	 for	endemic	species	that	are	vulnerable	(Izgü	et	al.,	2016).	Therefore,	 studies	 on	 morpho-	 and	 histodifferentiation	 may	 be	 additional	 tools	 by	which	 it	 is	 possible	 to	 obtain	 a	 better	 understanding	 of	 the	 in	 vitro	 regenerative	 system.	Indeed,	the	analysis	of	events	during	morpho-	and	histodifferentiation	has	already	improved	tissue	culture	systems	by	highlighting	the	structural	and	ultrastructural	changes	that	occur	along	the	morphogenesis	and	development	(de	Souza	et	al.,	2016).	The	aim	of	this	work	was	to	establish	a	protocol	for	in	vitro	seed	germination,	shoots	development	and	elongation	of	
B.	holophylla	explants.	
MATERIAL	AND	METHODS	
B.	holophylla	seeds	were	harvested	on	Cerrado,	located	in	the	south	of	Minas	Gerais,	at	835	m	altitude,	21°09’97”S	latitude	and	44°55’65”W	longitude	GRW.	Plant	material	samples	were	collected	and	herborized,	identified	and	deposited	in	the	PAMG	Herbarium,	belonging	to	the	Agricultural	Research	Company	of	Minas	Gerais	(EPAMIG),	located	in	Belo	Horizonte	(MG).	Approximately	 200	 fruits	 containing	 1500	 seeds	were	 harvested	 and	 treated	 for	 10	min	with	Captan	(1	g	kg-1	of	seeds),	dried	at	room	temperature	and	stored	in	a	cold	room.	
In	vitro	germination	Seeds	were	previously	washed	in	tap	water	and,	in	laminar	flow	chamber,	disinfested	with	 25%	 NaOCl	 (commercial	 sodium	 hypochlorite)	 for	 10	 min,	 washed	 five	 times	 with	sterile	 water.	 After	 disinfestation,	 seeds	 teguments	 were	 lightly	 excised	 with	 a	 scalpel	 to	allow	the	radicle	to	emerge.	Seeds	were	 inoculated	 in	 test	 tubes	containing	10	mL	WPM,	½WPM	medium	(Lloyd	and	McCown,	1980),	MS,	½MS	(Murashige	and	Skoog,	1962)	or	7	g	L-1	agar.	All	media	had	100	mg	L-1	Derosal	500SC.	The	pH	was	adjusted	to	5.7±0.1	before	autoclaving.	After	 inoculation,	 the	 seeds	 were	 transferred	 to	 growth	 room	 at	 27±1°C	 with	photoperiod	of	16	h	and	photon	flux	density	of	45	μmol	m-2	s-1	for	30	days.	For	the	experiment	50	seeds	were	inoculated	per	treatment.	The	parameters	analyzed	were	leaves	number,	nodes	number	and	stem	size.	
Shoots	induction	For	in	vitro	shoots	induction,	segments	with	approximately	1	cm	were	obtained	from	in	vitro	germinated	seedlings	with	30	days.	On	 laminar	chamber	the	nodal	segments	were	isolated	and	inoculated	on	WPM	culture	medium,	supplemented	with	6-benzylaminopurine	(BAP)	at	concentrations	of	0,	2,	4,	8	and	16	μM.	The	culture	media	were	solidified	with	0.7%	agar	and	the	pH	adjusted	to	5.8	before	autoclaving	at	121°C	for	21	min.	Twenty	repetitions	were	performed	per	treatment.	After	 inoculation,	 the	 seeds	 were	 transferred	 to	 a	 growth	 room	 at	 27±1°C	 with	photoperiod	of	16	h	and	photon	flux	density	of	45	μmol	m-2	s-1	for	30	days.	For	 the	 experiment	 20	 explants	 were	 inoculated	 per	 treatment.	 The	 parameters	analyzed	were	number	of	leaves,	number	of	nodes	and	stem	size.	
Shoots	elongation	(GA3)	After	 in	 vitro	 multiplication,	 shoots	 were	 transferred	 to	WPM	 culture	 medium	 and	maintained	for	30	days	in	a	growth	room	at	27±1°C	with	photoperiod	of	16	h.	After	 30	 days	 on	 WPM	 medium,	 the	 explants	 were	 transferred	 to	 WPM	 medium	supplemented	with	gibberellic	acid	(GA3)	at	concentrations:	0,	5,	10	and	20	μM.	The	culture	medium	was	 solidified	 with	 0.7%	 agar	 and	 the	 pH	 adjusted	 to	 5.8	 before	 sterilization	 at	121°C	for	21	min.	
 
 
 
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After	 inoculation,	 the	 explants	were	 transferred	 to	 the	 growth	 room	at	 27±1°C	with	photoperiod	of	16	h	and	photon	flux	density	of	45	μmol	m-2	s-1	for	30	days.After	 30	 days	 of	 cultivation,	 the	 following	 parameters	 were	 evaluated:	 length	 and	number	 of	 leaves.	 Twenty	 replicates	 were	 performed	 per	 treatment.	 Each	 replicate	 was	composed	of	one	test	tube.	
Light	microscopy	(LM)	For	anatomical	analysis,	leaves	of	plants	grown	in	vitro	were	collected	and	fixed	in	FAA	70	(70%	ethyl	alcohol	+	formaldehyde	+	acetic	acid	18:1:1	v/v)	for	72	h	and	then	stored	in	70%	 alcohol.	 After	 fixation	 the	 leaves	 were	 dehydrated	 in	 a	 gradual	 ethanol	 series	 and	infiltrated	 with	 Historesin	 (Leica®,	 Heidelberg,	 Germany)	 in	 accordance	 with	 Gerrits	 and	Smid	(1983).	Sections	of	5	µm	wide	were	obtained	with	a	RM	2125	RT	rotating	microtome	(Leica®,	Nussloch,	 Germany)	 and	 stained	 with	 toluidine	 blue	 (O’Brien	 et	 al.,	 1964).	 Images	 were	captured	 with	 an	 Opticam	 5.1	 MP	 digital	 camera	 attached	 to	 a	 MPS	 30	 DMLS	 light	microscope	(Leica®,	Wetzlar,	Germany).	In	the	cross	sections	the	morphogenesis	parameters	were	analyzed.	
Statistical	analysis	The	data	were	analyzed	using	analysis	of	variance	(ANOVA)	and	the	significance	of	the	differences	among	treatments	of	each	experiment	was	evaluated	by	Tukey	test	at	P=0.05.	
RESULTS	AND	DISCUSSION	The	 germination	 of	 B.	 holophylla	 seeds	 to	 obtain	 axillary	 segments	 for	 the	multiplication,	was	higher	on	½	MS,	WPM	and	water	+	agar,	respectively	(94,	91,	and	90%),	compared	with	MS	 (65%)	 and	½	WPM	 (65%)	 culture	medium	 (Figure	 1).	 Seeds	 start	 to	germinate	5	days	after	inoculation	and	the	maximum	germination	percentage	was	observed	at	20	days	of	culture.	
	Figure	1.	 In	vitro	germination	percentage	of	Bauhinia	holophylla	(Bong.)	Steud.	on	different	culture	media	after	30	days.	Water	+	agar	medium	showed	germination	rate	similar	to	½	MS	and	WPM.	For	seeds	germination	 sufficient	 water	 is	 necessary	 for	 activation	 of	 chemical	 reactions,	 related	 to	metabolism	 and	 process	 of	 resumption	 of	 embryo	 development	 (Bewley	 et	 al.,	 2013).	Therefore,	 seed	 germination	 results	 of	 a	 sequence	 of	 biochemical	 events,	 activated	 and	influenced	 by	 environmental	 factors	 such	 as	 temperature,	 light	 and	 water	 (Ferreira	 and	Borghetti,	 2004).	 Seed	 hydration	 depends	 on	 its	 chemical	 composition,	 tegument	permeability,	water	availability,	contact	area	and	temperature	(Martins	et	al.,	2003).	Water	 +	 agar	 medium,	 since	 it	 does	 not	 contain	 salts	 and	 sucrose,	 has	 good	 water	availability,	 an	 important	 factor	 for	 B.	 holophylla	 seeds	 germination.	 Once	 the	 imbibition	
 
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begins,	the	digested	reserves	starts	to	translocate	from	cotyledons,	especially	sucrose,	amino	acids	 and	 phosphorus	 compounds	 (Marcos	 Filho,	 2015)	 to	 the	 embryonic	 axis	 and	consequently	stimulating	the	respiratory	activity.	MS	medium	has	a	high	total	ionic	concentration,	with	a	high	concentration	of	nitrogen,	potassium	(Braga	et	al.,	2015),	zinc	and	chlorine;	in	comparison	to	other	media.	However,	for	woody	species,	media	with	more	diluted	mineral	balance	have	been	more	used,	such	as	the	WPM	culture	medium	which	has	 lower	 salt	 concentrations,	 in	particular,	 the	nitrogen	and	potassium	(Madke	et	al.,	2014).	Salt	reduction	is	generally	beneficial	for	the	culture	growth	of	woody	species.	This	was	confirmed	in	B.	holophylla	by	reducing	the	salt	concentration	of	the	½	MS	medium	or	by	using	the	WPM	medium,	thus	hindering	the	imbibition	and	embryo	metabolism	resumption	(Reis	et	al.,	2015).	However,	the	½	WPM	medium	was	not	effective	for	supplying	the	seeds’	needs	and	promoting	satisfactory	germination.	Regarding	 seedling	 growth	 after	 germination,	 WPM	 and	 ½	 MS	 growth	 medium	promoted	 greater	 stem	 length	 (Figure	 2A).	 Reduction	 in	 salt	 concentration,	 especially	ammonium,	nitrate	and	sulfate,	is	important	for	B.	holophylla	in	vitro	culture	since,	when	MS	medium	 is	 50%	 diluted	 the	 seedlings’	 growth	 was	 similar	 to	 WPM	 culture	 medium.	Moreover,	 although	 the	water	 +	 agar	medium	provides	 germination,	 addition	 of	 salts	 and	sucrose	on	culture	medium	is	essential	for	growth	and	maintenance	of	in	vitro	seedlings.	For	the	other	analyzed	parameters,	nodes	and	leaves	number,	there	was	no	difference	between	the	 treatments	 (Figure	 2B).	 Higher	 nutrient	 concentrations	 on	 culture	 medium	 produced	more	vigorous	seedlings	which	is	important	for	acclimatization.	
	Figure	2.	 In	 vitro	 growth	 of	 Bauhinia	 holophylla	 (Bong.)	 Steud.	 seedlings	 on	 different	culture	media	at	30	days.	Stem	length	(A),	nodes	and	leaves	number	(B).	For	 in	vitro	 shoots	multiplication,	nodal	 segments	 isolated	 from	 in	 vitro	 germinated	seedlings	were	cultured	 for	30	days	 in	culture	media	with	different	concentrations	of	BAP.	There	 was	 a	 positive	 correlation	 between	 the	 BAP	 concentration	 for	 number	 of	 shoots	(Figure	 3A)	 and	 number	 of	 leaves	 (Figure	 3D),	with	 the	 highest	 buds	 and	 leaves	 number	verified	in	the	concentration	of	16	μM.	The	higher	concentration	of	cytokinin	than	auxin	may	stimulate	 shoots	 and	 leaves	 formation	 (Rineksane	 et	 al.,	 2017).	 Regarding	 the	 number	 of	nodes	(Figure	3B)	and	stem	length	(Figure	3C)	there	was	no	difference	between	treatments.	The	presence	of	BAP	in	media	was	a	key	contributing	factor	for	plant	shoot	production	from	meristems.	Research	has	shown	that	when	the	hormone	BAP	is	used	in	tissue	culture,	it	promotes	physiological	processes	that	aid	plant	development.	For	instance,	the	presence	of	BAP	 has	 been	 linked	 to	 enhanced	 protein	 synthesis	 and	 heightened	 polysome	 function	(Mongi	 et	 al.,	 2012).	 BAP	 growth	 regulator	 is	 a	 stable	 synthetic	 cytokinin	 and	 less	susceptible	 to	 oxidative	 degradation	 than	 its	 natural	 counterparts	 (Daffalla	 et	 al.,	 2011).	Nevertheless,	B.	holophylla	 in	vitro	multiplication	on	BAP	was	 low,	with	a	maximum	of	1.4	shoots	explant-1.	 For	B.	racemosa	 the	maximum	shoots	proliferation,	 from	cotyledon	node,	was	obtained	in	MS	culture	medium	enriched	with	BAP	and	Kinetin	(Rajanna	et	al.,	2011).	
 
 
 
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	Figure	3.	 Bauhinia	holophylla	(Bong.)	Steud.	in	vitro	grown	at	different	concentrations	of	6-benzylaminopurine	 (BA)	 at	 30	 days.	 Regression	 analysis	 of	 shoot	 number	 (A),	nodes	number	(B),	stem	length	(C),	and	leaves	number	(D).	On	 shoot	 elongation	 experiment,	 GA3	 addition	 proportionally	 inhibited	B.	holophylla	growth.	The	highest	shoots	length	(Figure	4A),	number	of	nodes	(Figure	4B)	and	number	of	leaves	(Figure	4C),	were	verified	on	treatment	without	the	growth	regulators,	differing	from	GA3	 treatments	 (Figure	 4).	 Responses	 of	 GA	 over	 stem	 elongation	 vary	 according	 to	 the	analyzed	species	and	are	well-established	 in	cultivated	species	at	 temperate	environments	(Rineksane	et	al.,	2017).	UV-B	also	affects	cell	division	and	cell	elongation	which	is	likely	to	be	 at	 least	 partly	mediated	 through	 reduction	 in	 bioactive	GA	 (Roro	 et	 al.,	 2017).	GAs	 are	thought	 to	 elicit	 biological	 responses	 via	 a	 specific	 GA-receptor	 interaction,	 and	 the	structural	 limitations	 on	 biological	 activity	 described	 above	 suggest	 that	 the	 interaction	receptor:GAs	must	be	highly	specific	(Richards	et	al.,	2001).	
	Figure	4.	 Bauhinia	 holophylla	 (Bong.)	 Steud.	 shoots	 elongation	 cultivated	 in	 vitro	 at	different	concentrations	of	gibberellic	acid	(GA3)	at	30	days.	Mean	values	of	shoot	length	(A),	number	of	nodes	(B),	and	number	of	leaves	(C).	
 
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GA	 also	 stimulates	 leaf	 expansion,	 which	 initially	 involves	 cell	 division	 and	 cell	expansion,	and	later,	cell	expansion	only	(Richards	et	al.,	2001).	Differences	in	B.	holophylla	leaves	cultured	at	different	concentrations	of	GA3	were	verified	in	histological	analysis.	The	leaves	maintained	in	culture	medium	without	growth	regulators	showed	simple	epidermis,	a	layer	of	palisades	parenchyma	and	lacunar	parenchyma.	The	greatest	differences	observed	in	anatomical	 analysis	were	mesophyll	development.	 Leaves,	 cultivatedin	 culture	medium	without	 growth	 regulator,	 had	 tabular	 cellular	 epidermis,	 greater	 mesophyll	 thickness	(10.73	μm),	well-developed	palisade	parenchyma	(3.75	μm)	formed	by	a	layer	of	turgid	cells	with	 thin	 cell	 wall,	 prominent	 nucleus	 and	 few	 intercellular	 spaces,	 and	 the	 lacunae	parenchyma	(3.92	μm)	composed	of	relatively	organized	isodiametric	cells	with	intercellular	spaces	(Figure	5A).	However,	explants	of	leaves	maintained	on	GA3	culture	medium	showed	uninform	epidermal	cells,	more	elongated	and	narrow	palisade	parenchyma	cells,	occupying	2.61	 μm	 of	 mesophyll	 and	 many	 intercellular	 spaces	 between	 these	 cells.	 Lacunar	parenchyma	(2.86	μm)	presented	flatter	cells	and	also	many	intercellular	spaces	(Figure	5B-D).	
	Figure	5.	 Cross-sectional	photomicrographs	of	leaves	of	Bauhinia	holophylla	(Bong.)	Steud.	grown	 in	 vitro	 at	 different	 concentrations	 of	 gibberellic	 acid	 (GA3)	 at	 30	 days.	Photomicrographs	of	leaves	grown	without	growth	regulators	(A),	at	5	μM	(B),	10	μM	(C),	and	20	μM	(D)	GA3.	5	μm	bars.	GA	 regulates	 cell	 elongation	 and	 cell	 wall	 relaxation	 by	 inducing	 the	 expression	 of	expansins,	 xyloglucan	 endotransglucosylase/hydrolases	 (XTHs),	 which	 cause	 cell	 wall	changes	 that	 allows	 turgor-driven	 cell	 expansion	 (Kutschera	 and	 Niklas,	 2013).	 More	elongated	and	narrow	palisade	parenchyma	cells	are	related	with	an	increase	in	cell	length	(cell	 elongation)	 induced	 by	 GA3.	 These	 results	 are	 related	 to	 the	 fact	 that	 mesophyll	elongation	consists	of	cell	division	and	posterior	elongation	(Richards	et	al.,	2001).	Leaf	optical	properties	can	be	altered	due	to	light	availability	responses	via	structural	leaf	modifications	and	concentration	of	photosynthetic	pigments	(Sanches	and	Válio,	2006)	in	 both	 shady	 or	 high-irradiance	 environments	 or	 by	 endogenous	 manipulation	 of	 the	gibberellin	 (GA)	metabolism	 (Lu	 et	 al.,	 2015).	 GA3	 induced	 a	 decrease	 in	 leaf	 thickness	 in	paclobutrazol	 supplemented	 plants	 independently	 of	 light	 level.	 The	 leaf	 area	 increase	induced	by	GA3	supplementation	provides	plants	with	a	higher	light	capture	surface,	forming	thinner	 leaves	 that	 increase	 light	 transmittance	 through	 the	 leaf	 lamina	 (mesophyll	 cells)	(Kutschera	and	Niklas,	2013).	
 
 
 
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CONCLUSION	
B.	holophylla	in	vitro	germination	is	possible	using	WPM,	½	MS	or	water	and	agar.	For	shoots	multiplication	the	addition	of	BAP	(16	µM)	is	important	in	increasing	the	number	of	shoots	and	number	of	leaves.	For	shoots	elongation	the	addition	of	GA3	proportionally	inhibited	explants	growth.	As	for	the	leaves	development	in	different	GA3	concentrations,	it	presented	mesophyll	reduction	and	parenchyma	cell	changes.	
ACKNOWLEDGEMENTS	CNPQ	 -	 National	 Council	 of	 Scientific	 and	 Technologic	 Development	 and	 Fapemig	 -	Research	Supporting	Foundation	of	Minas	Gerais.	
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