<i>In vitro</i> propagation and root anatomical features of <i>Phragmipedium kovachii</i> J.T. Atwood, Dalström &amp; Ric.Fernández

Hussien, Muthab; Molkanova, Olga Ivanovna; Koval, Vladimir

doi:10.1590/1413-7054202549002125

Phragmipedium kovachii is a renowned orchid for its striking flowers and ecological significance. This study aimed to develop an efficient protocol for its micropropagation and conservation. Shoots measuring 0.7-1.0 cm were used as explants and cultured on half-strength Murashige and Skoog (MS) medium with various combinations of 6-benzylaminopurine (6-BAP) and indole-3-acetic acid (IAA), with or without 100 mL L^-¹ coconut water. The highest protocorm-like bodies (PLBs) formation rate (96.7%) and the maximum PLBs per explant (12.58 ± 0.33) were obtained on medium containing 2.0 mg L^-¹ 6-BAP, 0.6 mg L^-¹ IAA, and coconut water. During the regeneration phase, PLBs regenerated on media supplemented with either 0.5 mg L^-¹ 6-BAP and coconut water, as well as those on media containing 0.5 mg L^-¹ meta-Topolin (mT) and banana homogenate, produced optimal plantlets for subsequent rooting stages. Rooting was enhanced using 1.0 mg L^-¹ IAA with coconut water or 0.5 mg L^-¹ indole-3-butyric acid (IBA) with banana homogenate. Plantlets grown on coconut water-supplemented media developed longer roots with a pentarch stele, well-differentiated sclerenchyma tissue, and numerous metaxylem vessels, exhibiting the highest survival rate. In contrast, those cultured on banana homogenate produced thicker roots with a polyarch stele. Acclimatization was successfully achieved using substrates consisting of equal proportions of bark, perlite, and peat, as well as a mixture of soil, sand and perlite (2:1:1), ensuring high ex vitro survival rates (87 and 80%, respectively). This study provides an effective protocol for the ex situ conservation and large-scale production of P. kovachii plants.

Index terms:
Orchidaceae; growth regulators; organic additives; plant anatomy

[TFN1] *Values are mean ± SD; the letters “a”, “b” and “c” denote groups by Duncan’s multiple range test at the 5% level.

Organic additives	Cytokinin type	Plantlet length (cm)	Number of leaves	Number of shoots	Number of roots	Rooting%	Necrosis %	PLBs proliferation %
Without organic additives	Control	0.96 ± 0.19 d *	1.14 ± 0.19 e	0.62 ± 0.15 e	0 d	0 h	57.00± 3.33 a	20.00 ± 0 cde
	6-BAP	0.96 ± 0.07 d	1.51 ± 0.25 de	1.75 ± 0.14 d	0.56 ± 0.30 d	26.67 ± 3.33 ef	37.00 ± 3.33 cd	26.67 ± 3.33 cd
	KIN	0.93 ± 0.04 d	1.24 ± 0.04 de	0.85 ± 0.08 e	0.23 ± 0.10 d	13.00 ± 3.33 g	47.00 ± 8.81 ab	53.33 ± 3.33 b
	mT	0.94 ± 0.19 d	1.60 ± 0.21 de	1.37 ± 0.36 de	0.51 ± 0.26 d	20.00 ± 5.77 fg	30.00 ± 0 c	67.00 ± 3.33 a
Coconut water	Control	1.53 ± 0.03 b	2.50 ± 0.14 b	2.17 ± 0.22 d	1.08 ± 0.08 cd	43.00 ± 3.33 bc	0 d	30.00 ± 5.77 c
	6-BAP	1.98 ± 0.06 a	3.50 ± 0.14 a	5.58 ± 0.22 a	1.25 ± 0.14 c	51.67 ± 1.67 b	0 d	6.67 ± 3.33 f
	KIN	1.40 ± 0.06 b	2.67± 0.17 b	4.08 ± 0.36 b	0.33 ± 0.17 d	46.67 ± 3.33 bc	5.00 ±3.33 d	13.33 ± 3.33 ef
	mT	0.99 ± 0.07 d	1.50 ± 0.14 de	3.17 ± 0.22 c	0.67 ± 0.08 d	40.00 ± 5.77 cd	7.00 ± 3.33 d	26.67 ± 3.33 cd
Banana homogenate	Control	1.07 ± 0.07 cd	2.33 ± 0.33 bc	1.83 ± 0.17 d	1.07 ± 0.33 cd	30.00 ± 0 def	15.00 ± 3.33 d	16.67 ± 3.33 def
	6-BAP	1.48 ± 0.16 b	2.50 ± 0.29 b	1.92 ± 0.08 d	1.47 ± 0.29 b	36.67 ± 3.33 cde	10.00 ± 5.77 d	13.33 ± 3.33 ef
	KIN	1.33 ± 0.03 bc	2.33 ± 0.17 bc	2.17 ± 0.44 d	1.67 ± 0.33 ab	43.33 ± 3.33 bc	14.00 ± 6.67 d	6.67 ± 3.33 f
	mT	1.23 ± 0.03 bcd	1.83 ± 0.17 cd	3.33 ± 0.22 c	2.33 ± 0.33 a	63.00 ± 3.33 a	5.00 ± 3.33 d	13.33 ± 3.33 ef

Organic additives	Auxin	Auxin concentration mg L^-1	Plantlets length (cm)	Number of roots	Rooting %	Root length (cm)	Number of leaves
Potato homogenate	IBA	control	2.85 ± 0.15 i *	1.22 ± 0.22 g	63 ± 3.00 ef	1.16 ± 0.11 i	3.11 ± 0.11 de
		0.5	3.41 ± 0.05 fg	2.15 ± 0.10 de	67 ± 5.00 def	1.88 ± 0.05 gh	3.78 ± 0.22 abcd
		1.0	3.64 ± 0.09 ef	2.22 ± 0.11 cde	60 ± 5.77 f	2.27 ± 0.05 efg	3.11 ± 0.11 de
	IAA	0.5	2.93 ± 0.03 hi	1.34 ± 0.19 fg	56.67 ± 3.33 f	1.16 ± 0.22 i	3 ± 0 e
	IAA	1.0	3.31 ± 0.07 g	1.44 ± 0.11 fg	60 ± 6.00 f	2.30 ± 0.11 ef	3.45 ± 0.22 cde
Coconut water	IBA	control	3.19 ± 0.09 gh	1.45 ± 0.22 fg	70 ± 4.33 cde	1.69 ± 0.05 h	3.56 ± 0.29 bcde
		0.5	4.01 ± 0.1 bcd	2.67 ± 0.19 bcd	80 ± 5.80 bc	3.19 ± 0.20 b	4.22 ± 0.10 ab
		1.0	4.22 ± 0.11 b	3.11 ± 0.11 b	87 ± 3.33 ab	3.1 ± 0.29 bc	4.00 ± 0.20 abc
	IAA	0.5	3.78 ± 0.12 cde	1.89 ± 0.29 ef	73.33 ± 3.33 cde	2.44 ± 0.16 de	3.78 ± 0.11 abcd
	IAA	1.0	5.2 ± 0.08 a	3 ± 0.19 b	86.67 ± 3.33 ab	4.27 ± 0.21 a	4.34 ± 0.20 a
Banana homogenate	IBA	control	2.51 ± 0.10 j	1.89 ± 0.11 ef	76.67 ± 5.00 bcd	1.19 ± 0.06 i	3.45 ± 0.29 cde
		0.5	4.01 ± 0.05 bcd	4.33 ± 0.19 a	96 ± 6.00 a	2.83 ± 0.07 bcd	4.21 ± 0.11 ab
		1.0	2.98 ± 0.01 hi	3.26 ± 0.38 b	86 ± 5.77 ab	2.02 ± 0.07 fgh	3.44 ± 0.30 cde
	IAA	0.5	3.75 ± 0.13 de	2.78 ± 0.11 bc	83 ± 3.33 bc	2.72 ± 0.05 cd	3.78 ± 0.45 abcd
	IAA	1.0	3.44 ± 0.14 fg	2.22 ± 0.22 cde	83.33 ± 4.33 bc	2.48 ± 0.11 de	3.78 ± 0.10 abcd

Parameters	Organic additives
Parameters	Banana homogenate	Coconut water	Potato homogenate
Root diameter (µm)	312.24 ± 2.20 a *	285.31 ± 0.50 b	264.42 ± 2.01 c
Velamen thickness (µm)	18.05 ± 0.39 a	13.94 ± 0.75 b	11.89 ± 0.84 b
Velamen % of ring root diameter	5.78 ± 0.16 a	4.88 ± 0.26 b	4.49 ± 0.31 b
Exodermis thickness (µm)	9.96 ± 0.19	9.34 ± 0.74	8.33 ± 0.34
Exodermis % of ring root diameter	3.22 ± 0.07	3.27 ± 0.26	3.15 ± 0.14
Cortex thickness (µm)	100.34 ± 1.03 b	104.95 ± 1.31 a	89.54 ± 0.75 c
Cortex % of ring root diameter	64.27 ± 0.25 b	73.56 ± 0.45 a	67.74 ± 0.38 c
Endodermis thickness (µm)	6.11 ± 0.31	5.24 ± 0.27	4.98 ± 0.23
Endodermis % of ring root diameter	1.96 ± 0.10	1.83 ± 0.09	1.88 ± 0.09
Stele diameter (µm)	57.06 ± 0.67 a	40.51 ± 0.37 b	40.45 ± 0.48 b
Stele % of ring root diameter	18.28 ± 0.29 a	14.2 ± 0.13 b	15.3 ± 0.25 b

In vitro propagation and root anatomical features of Phragmipedium kovachii J.T. Atwood, Dalström & Ric.Fernández

Propagação in vitro e características anatômicas das raízes de Phragmipedium kovachii J.T. Atwood, Dalström & Ric.Fernández