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. 2022 Dec 11;13(12):2335.
doi: 10.3390/genes13122335.

Ploidy Status, Nuclear DNA Content and Start Codon Targeted (SCoT) Genetic Homogeneity Assessment in Digitalis purpurea L., Regenerated In Vitro

Yashika Bansal  1 , A Mujib  1 , Zahid H Siddiqui  2 , Jyoti Mamgain  1 , Rukaya Syeed  1 , Bushra Ejaz  1
Affiliations

Ploidy Status, Nuclear DNA Content and Start Codon Targeted (SCoT) Genetic Homogeneity Assessment in Digitalis purpurea L., Regenerated In Vitro

Yashika Bansal et al. Genes (Basel). .

Abstract

Digitalis purpurea L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient propagation protocol for D. purpurea by optimizing various PGR concentrations in Murashige and Skoog (MS) medium. The genetic homogeneity of in vitro regenerants was assessed by the flow cytometric method (FCM) and Start Codon Targeted (SCoT) marker technique. Firstly, the seeds inoculated in full MS medium added with 0.5 mg/L GA3 produced seedlings. Different parts such as hypocotyl, nodes, leaves and apical shoots were used as explants. The compact calli were obtained on BAP alone or in combinations with 2, 4-D/NAA. The hypocotyl-derived callus induced somatic embryos which proliferated and germinated best in 0.75 mg/L BAP-fortified MS medium. Scanning electron microscopic (SEM) images confirmed the presence of various developmental stages of somatic embryos. Shoot regeneration was obtained in which BAP at 1.0 mg/L and 2.0 mg/L BAP + 0.5 mg/L 2,4-D proved to be the best treatments of PGRs in inducing direct and indirect shoot buds. The regenerated shoots showed the highest rooting percentage (87.5%) with 24.7 ± 1.9 numbers of roots/shoot in 1.0 mg/L IBA augmented medium. The rooted plantlets were acclimatized in a greenhouse at a survival rate of 85-90%. The genome size and the 2C nuclear DNA content of field-grown, somatic embryo-regenerated and organogenic-derived plants were estimated and noted to be 3.1, 3.2 and 3.0 picogram (pg), respectively; there is no alteration in ploidy status and the DNA content, validating genetic uniformity. Six SCoT primers unveiled 94.3%-95.13% monomorphic bands across all the plant samples analyzed, further indicating genetic stability among in vitro clones and mother plants. This study describes for the first time successful induction of somatic embryos from hypocotyl callus; and flow cytometry and SCoT marker confirmed the genetic homogeneity of regenerated plants.

Keywords: SCoT marker; SEM; flow cytometry; genetic homogeneity; indirect somatic embryogenesis; nuclear DNA content; shoot organogenesis.

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Conflict of interest statement

The authors have no conflict of interest in the current research investigation.

Figures

Figure 3
Figure 3
Callus induction and indirect somatic embryogenesis from hypocotyl callus in D. purpurea. (A,B) Callus induction and proliferation (bars (A) = 0.3 cm, (B) = 0.5 cm), (C) Somatic embryos formation with different developmental stages (bar = 0.5 cm), (D) Isolated bipolar stages of early and late globular embryos with shoot and root ends (arrowheads) (bar = 0.3 cm).
Figure 1
Figure 1
In vitro germination of seeds and direct shoot regeneration in D. purpurea. (A) Germinated seedlings having elongated hypocotyls (bar = 0.5 cm). (B,C) In vitro direct shoot proliferation (bars = 1.0 cm).
Figure 2
Figure 2
Callus induction and callus-mediated shoot organogenesis from leaf explant in D. purpurea. (A) Callus initiation (bar = 1.0 cm), (B) Callus proliferation after postweeks (bar = 0.5 cm), (C,D) Shoot regeneration from leaf derived callus (bars = 0.5 cm).
Figure 4
Figure 4
Effect of BAP-fortified MS medium on somatic embryo germination after a three- and six-week period, respectively. Values indicate the Mean ± SE of three replicates per treatment. Mean values with different superscripts within a column are significantly different from each other as per DMRT at p ≤ 0.05.
Figure 5
Figure 5
Scanning electron microscopic (SEM) images of somatic embryos in D. purpurea. (A) Globular shaped somatic embryo (bar = 20 μm), (B) Cluster of globular shaped somatic embryos (bar = 2 μm), (C) Globular shaped embryo with suspensor (bar = 2 μm), (D) Heart shaped somatic embryo (bar = 2 μm).
Figure 6
Figure 6
Effect of various PGRs concentration on root induction frequency and mean number of roots/shoot. Values indicate the mean ± SE of 3 replicates per treatment. Mean values with different superscripts within a column are significantly different from each other as per DMRT at p ≤ 0.05.
Figure 7
Figure 7
(A,B) Root induction of in vitro regenerated D. purpurea plants (bars = 1.0 cm). (C) Tissue culture raised D. purpurea plants in pots (bar = 3 cm).
Figure 8
Figure 8
Flow cytometric histogram peaks of relative 2C DNA content of nuclei obtained from (A) Digitalis lanata (standard), (B) Field-grown (mother) plant, (C) somatic embryo regenerated plant, and (D) organogenic-derived plant of Digitalis purpurea.
Figure 9
Figure 9
SCoT banding profiles of in vitro propagated plants with mother plant (DP_M) of D. purpurea with SCoT 14 primer. (A) DP-1_1 to DP-1_6 (somatic embryo-derived plants), (B) DP-2_1 to DP-2_6 (organogenic-derived plants).
Figure 10
Figure 10
Dendrogram of SCoT analysis depicting the genetic similarity between the mother plant (DP-M) and the in vitro regenerated plants of D. purpurea.
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