Survival of <i>Renanthera imschootiana</i> plantlets grown on different substrates after transplanting for 60 days.

<p>Means ± SE of 300 replicates with the different letters within columns are significantly different at <i>P</i><0.05 (DMRT).</p><p>* Substrate mixture 1: commercial sand for orchids+shattered fir bark (2∶1; v/v).</p><p>** Substrate mixture 2: commercial sand for orchids+sieved peat+shattered fir bark (2∶1∶1; v/v).</p><p>Survival of <i>Renanthera imschootiana</i> plantlets grown on different substrates after transplanting for 60 days.</p

Effect of organic amendments on seed germination of <i>Renanthera imschootiana</i> at 150 DAP on 1/4 MS containing 0.5 mg l⁻¹ NAA, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ AC for 75 days in culture.

<p>For each treatment, approximately 300 seeds (in three replicates) were cultured in a 500-ml culture flask containing 90 ml of medium. All experiments consisted of three independent replicates with 10 culture flasks per replicate. Values followed by different letters within a column are significantly different at <i>P</i><0.05 (DMRT). Percentage data were arcsin transformed before being subjected to ANOVA. Each mean is based on microscopic observations. BH, banana homogenate; CW, coconut water; MS, Murashige and Skoog medium; NAA, α-naphthaleneacetic acid; PH, potato homogenate.</p><p>Effect of organic amendments on seed germination of <i>Renanthera imschootiana</i> at 150 DAP on 1/4 MS containing 0.5 mg l⁻¹ NAA, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ AC for 75 days in culture.</p

Effect of sucrose concentration on <i>in vitro</i> germination of <i>Renanthera imschootiana</i> on quarter-strength MS medium supplemented with 0.5 mg l⁻¹ NAA, 20% CW and 1.0 g l⁻¹ AC 75 days after culture.

<p>Different letters indicate significant differences between days at <i>P</i><0.05 (DMRT).</p

Seed germination and seedling developmental growth stages of <i>Renanthera imschootiana</i> (modified from Zeng et al., 2012).

<p>Seed germination and seedling developmental growth stages of <i>Renanthera imschootiana</i> (modified from Zeng et al., 2012).</p

Effect of NAA and banana homogenate (BH) concentration on <i>Renanthera imschootiana</i> plantlet growth on Hyponex N016 medium with 1.0 g l⁻¹ peptone, 20 g l⁻¹ sucrose, 10% CW and 1.0 g l⁻¹ AC after 60 days in culture.

<p>* +, ++, +++ represents poor, normal, and good growth, respectively (see text for detailed explanation). All experiments consisted of three independent replicates with 10 culture flasks per replicate and 20 PLBs per flask. Values followed by different letters within a column are significantly different at <i>P</i><0.05 (DMRT).</p><p>Effect of NAA and banana homogenate (BH) concentration on <i>Renanthera imschootiana</i> plantlet growth on Hyponex N016 medium with 1.0 g l⁻¹ peptone, 20 g l⁻¹ sucrose, 10% CW and 1.0 g l⁻¹ AC after 60 days in culture.</p

<i>In Vitro</i> Propagation and Reintroduction of the Endangered <i>Renanthera imschootiana</i> Rolfe

<div><p><i>Renanthera imschootiana</i> Rolfe is an endangered tropical epiphytic orchid that is threatened with extinction due to over-collection and the loss of suitable habitats. <i>In vitro</i> propagation is a useful way to mass produce plants for re-establishment in the wild and for commercial propagation. Seeds collected 150 days after pollination (DAP) were the optimum stage for <i>in vitro</i> culture. Seed germination reached 93.1% on quarter-strength MS (i.e., MS containing a quarter of macro- and micronutrients) medium containing 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 20% coconut water (CW), 1.0 g l⁻¹ peptone, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ activated charcoal (AC). Quarter-strength MS medium supplemented with 1.0 mg l⁻¹ BA, 0.5 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 10 g l⁻¹ sucrose and 20% CW was suitable for the sub-culture of protocorm-like bodies (PLBs) in which the PLB proliferation ratio was 2.88. Quarter-strength MS medium containing 1.0 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 100 g l⁻¹ banana homogenate (BH), and 1.0 g l⁻¹ AC was suitable for plantlet formation and 95.67% of plantlets developed from PLBs within 60 days of culture. Hyponex N016 medium <b>s</b>upplemented with 0.5 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 20 g l⁻¹ sucrose, 150 g l⁻¹ BH, and 1.0 g l⁻¹ AC was suitable for the <i>in vitro</i> growth of plantlets about 2-cm in height. Plantlets 3-cm in height or taller were transplanted to Chilean sphagnum moss, and 95% of plantlets survived after 60 days in a greenhouse. Three hundred transplanted of seedlings 360-days old were reintroduced into three natural habitats. Highest percentage survival (79.67%) was observed in Yuanjiang Nature Reserve two years after reintroduction, followed by Huolu Mountain forest park (71.33%). This protocol is an efficient means for the large-scale propagation and <i>in vitro</i> and <i>in vivo</i> germplasm conservation of <i>R. imschootiana</i>.</p></div

<i>In vitro</i> seed culture, seedling development and reintroduction of <i>Renanthera imschootiana</i> Rolfe.

<p>(A) Stage 0, seed under scanning electron microscopy, ungerminated. (B) Stage 1, testa ruptured. (C) Stage 2, appearance of rhizoids. (D) Stage 3, emergence and elongation of first leaf. (E) Stage 4, one leaf and root present. (F) Stage 5, presence of two or more leaves. (G) Proliferation of PLBs on quarter-strength MS (1/4 MS) medium supplemented with 1.0 mg l⁻¹ BA, 1.0 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone and 20% CW. (H) Differentiation of PLBs on 1/4 MS medium supplemented with 1.0 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 100 g l⁻¹ BH, and 1.0 g l⁻¹ AC. (I) Development of seedlings on Hyponex N016 medium supplemented with 0.5 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 150 g l⁻¹ BH, and 1.0 g l⁻¹ AC. (J) Transplanted plantlets 6 months after acclimatization in the greenhouse. (K) Reintroduced flowering plantlets from <i>in vitro</i>-derived seedlings on the trunk of <i>Pinus massoniana</i> on Huolu Mountain, Guangzhou. (L) Reintroduced plantlets from <i>in vitro</i>-derived seedlings at the Orchids Garden, South China Botanical Garden. Bars: (A) 50 µm, (B) 100 µm, (C) 0.05 mm, (D) 0.35 mm, (E) 0.40 mm, (F) 0.60 mm, (G, H) 3.0 cm, (I) 4.0 cm, (J, K) 5.0 cm, (L) 3.0 cm.</p

Survival rates of different ages of transplanted <i>Renanthera imschootiana</i> seedlings after 360- or 720-day reintroduction.

<p>Means ± SE of 300 replicates with the different letters are significantly different at <i>P</i><0.05 (DMRT).</p><p>Survival rates of different ages of transplanted <i>Renanthera imschootiana</i> seedlings after 360- or 720-day reintroduction.</p

Effect of basal medium supplemented with 0.5 mg l⁻¹ NAA, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ AC on germination and development of <i>Renanthera imschootiana</i> 150 DAP seed after 75 days in culture.

<p>For each treatment, approximately 300 seeds were cultured in a 500-ml culture flask containing 90 ml of medium. All experiments consisted of three independent replicates with 10 culture flasks per replicate. Values followed by different letters within a column are significantly different at <i>P</i><0.05 (DMRT). Percentage data were arcsin transformed before being subjected to ANOVA. Each mean is based on microscopic observations. DAP, days after pollination; KC, Knudson's C medium; MS, Murashige and Skoog medium; NAA, α-naphthaleneacetic acid; RE, Robert Ernst medium; VW, Vacin and Went medium.</p><p>*Lin et al. (2008).</p><p>Effect of basal medium supplemented with 0.5 mg l⁻¹ NAA, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ AC on germination and development of <i>Renanthera imschootiana</i> 150 DAP seed after 75 days in culture.</p

Effect of NAA and banana homogenate (BH) concentration on differentiation of <i>Renanthera imschootiana</i> PLBs on 1/4 MS medium containing with 1.0 g l⁻¹ peptone, 10 g l⁻¹ sucrose, 10% CW and 1.0 g l⁻¹ AC after 60 days in culture.

<p>All experiments consisted of three independent replicates with 10 culture flasks per replicate and 20 PLBs per flask. Values followed by different letters within a column are significantly different at <i>P</i><0.05 (DMRT).</p><p>Effect of NAA and banana homogenate (BH) concentration on differentiation of <i>Renanthera imschootiana</i> PLBs on 1/4 MS medium containing with 1.0 g l⁻¹ peptone, 10 g l⁻¹ sucrose, 10% CW and 1.0 g l⁻¹ AC after 60 days in culture.</p