Efficient Plant Regeneration via Somatic Embryogenesis in Alocasia reginula cv. ‘Black Velvet’

doi:10.11983/CBB22106

Abstract

Abstract: In this study, a plant regeneration system via somatic embryogenesis was established in Alocasia genus. We obtained embryogenic cell suspension cultures of A. reginula through embryogenic calli induced from petioles, and achieved a high frequency of plant regeneration using embryogenic cell aggregates. Efficiency of embryogenic calli induced from petiole explants was highest (81.3%) on a Murashige and Skoog (MS) medium supplemented with 2.0 mg·L^-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg·L^-1 thidiazuron (TDZ). The embryogenic calli were crushed into cell aggregates and then transferred to liquid MS media supplemented with 2.0 mg·L^-12,4-D and 1.0 mg·L^-1TDZ for suspension cultivation. By subculturing biweekly, lots of cell aggregates were gained from embryogenic suspension cultures after 12 weeks. The cell aggregates within 28 weeks of suspension culture were transferred to solid 1/2MS media without plant growth regulator for differentiation culture, with an average of 2.3-2.5 plantlets regenerated from each cell aggregate. The formation and germination of somatic embryos were observed by optical microscopy and scanning electron microscopy (SEM). After the regenerated plantlets were transplanted to a greenhouse for 4 months, the achieved survival ratio was 95.3%. Flow cytometry (FCM) demonstrated that there was no chromosome ploidy variation in randomly selected 50 surviving plants. In addition, the nuclear DNA content was estimated at 10.94 pg·(2C)^-1, and the genome size was 5 290.12 Mb·C^-1. There was no significant variation in their phenotypes from the time the plants were transplanted to the greenhouse until they bloomed spontaneously. These results provide good technical support for the commercial production of seedlings and biotechnological breeding of A. reginula.

Key words: Alocasia reginula, embryogenic calli, embryogenic cell suspension, plant regeneration

Liu Xiaofei, Sun Yingbo, Huang Lili, Yang Yuchai, Zhu Genfa, Yu Bo. Efficient Plant Regeneration via Somatic Embryogenesis in Alocasia reginula cv. ‘Black Velvet’[J]. Chinese Bulletin of Botany, 2023, 58(5): 750-759.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22106

https://www.chinbullbotany.com/EN/Y2023/V58/I5/750

Figures/Tables 6

Figure 1 Induction of embryogenic calli from Alocasia reginula (A) Embryogenic calli at the wound site of petiole explants on MS solid medium containing 2.0 mg·L-1 2,4-D and 1.0 mg·L-1 TDZ after 8 weeks of incubation (bar=3 mm); (B) Calli were visible as light yellow dense grains (bar=2 mm).

Table 1 Percentage of embryogenic calli formed on MS medium with 2,4-D and TDZ from petiole segments of Alocasia reginula after 8 weeks of culture

2,4-D (mg·L^-1)
2,4-D (mg·L^-1)	0.5	1.0	2.0	4.0
0	0	0	0	0
0.5	14.7±2.3 f	45.3±6.1 c	38.7±4.6 cd	12.0±4.0 f
1.0	37.3±2.3 d	58.7±2.3 b	32.0±4.0 d	10.7±4.6 fg
2.0	38.7±4.6 cd	81.3±6.1 a	22.7±4.6 e	4.0±4.0 gh
4.0	10.6±4.6 fg	33.3±6.1 d	13.3±2.3 f	2.7±2.3 h

Figure 2 Suspension culture of embryogenic calli, the effect of suspension cultivation time on average number of plants originating from each cell aggregate, and plant regeneration of Alocasia reginula (A) Cell aggregates obtained by suspension cultivation in liquid MS medium (bar=5 mm); (B) Light yellow cell aggregates (bar= 1 mm). (C) Effect of suspension cultivation time on average number of plants originating from each cell aggregate (means with different lowercase letters are significantly different at the 0.05 level); (D) Cell aggregates were transferred onto MS medium to form spherical embryos (bar=0.5 mm); (E) Pear-shaped embryos (bar=0.5 mm); (F) Shield-shaped embryos (bar=0.5 mm); (G), (H) Cotyledonary embryos (bar=10 mm, bar=1 mm); (I) Integral cotyledonary embryo (bar=1 mm); (J) The separated integral plantlet sprouted young leaves and roots (bar=1 cm); (K) After 3 months of transplantation, leaves showed the unique blackish green velvet luster (bar=5 cm); (L) After 10 months of transplantation, the area of young leaves increased significantly (bar=5 cm); (M) Spadices sprouted from plant top (bar=4 cm).

Table 2 Influence of MS medium strength on plant regeneration of Alocasia reginula

Medium strength	Plant regeneration rate (%)	Average number of rege- nerated plants of each cell aggregate
1/8MS	56.4±3.9 d	0.5±0.1 d
1/4MS	70.3±5.7 c	1.1±0.1 c
1/2MS	100±0.0 a	2.5±0.2 a
MS	83.0±5.5 b	1.6±0.2 b

Figure 3 Stereo microscopy (A, B) and scanning electron microscope (SEM) (C, D) images showing developmental stages of somatic embryos (A) Spherical embryos (bar=250 μm); (B) Pear-shaped embryos (bar=250 μm); (C) Shield-shaped embryo with coleoptiles (red arrow) (bar=200 μm); (D) Complete cotyledonary embryo with coleoptiles (red arrow), shoot tip (blue arrow) and root tip (yellow arrow) (bar=250 μm).

Figure 4 Flow cytometry analysis of regenerated plant of Alocasia reginula 1: Nuclei at G0/G1 phase of Zea mays; 2: Nuclei at G0/G1 phase from regenerated plant of A. reginula; 3: Nuclei at G2 phase from regenerated plant of A. reginula.

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