Somatic embryogenesis from young inflorescences of the giant bamboo Dendrocalamus asper (Schult f.) Backer ex Heyne

Bamboos are an important worldwide non-timber forest product with current rising interest due to their environmentally friendly applications. Besides the consolidated uses of the sweet shoots and culms for structural uses, Dendrocalamus asper is an imposing ornamental bamboo for horticulture. The present work aimed to establish in vitro calli culture and plant regeneration through somatic embryogenesis starting from young inflorescences of the giant bamboo, D. asper. Pre-anthesis inflorescences were collected, disinfested, and subjected to callus induction on MS basal medium supplemented by 0 µM, 9 µM, 18 µM, 27 µM, and 36 µM of 2,4-D in combination with 9 µM of 2-iP or 9 µM Kin. The different obtained calli types were characterized and subcultured in 0 µM, 4.5 µM, 9 µM, and 18 µM of 2,4-D in combination with 9 µM of both cytokinins for multiplication and differentiation. Additionally, the explant incision and its inoculation orientation onto culture media were tested for callus induction improvement. The 2,4-D was essential for callus induction, and its combination with both cytokinins resulted in embryogenic callus induction and further somatic embryos regeneration. The subsequent reduction of this auxin to 4.5 µM resulted in somatic embryo maturation. Somatic embryos transferred to a plant growth regulator-free medium resulted in plantlet conversion. The present work showed the feasibility of using inflorescences as explants and the efficiency of using the 2-iP in combination with 2,4-D to callus induction and in vitro bamboo plant regeneration through somatic embryogenesis.

Global Transcriptomic Analysis Reveals Differentially Expressed Genes Involved in Embryogenic Callus Induction in Drumstick (Moringa oleifera Lam.)

The plant embryogenic callus (EC) is an irregular embryogenic cell mass with strong regenerative ability that can be used for propagation and genetic transformation. However, difficulties with EC induction have hindered the breeding of drumstick, a tree with diverse potential commercial uses. In this study, three drumstick EC cDNA libraries were sequenced using an Illumina NovaSeq 6000 system. A total of 7191 differentially expressed genes (DEGs) for embryogenic callus development were identified, of which 2325 were mapped to the KEGG database, with the categories of plant hormone signal transduction and Plant-pathogen interaction being well-represented. The results obtained suggest that auxin and cytokinin metabolism and several embryogenesis-labeled genes are involved in embryogenic callus induction. Additionally, 589 transcription factors from 20 different families were differentially expressed during EC formation. The differential expression of 16 unigenes related to auxin signaling pathways was validated experimentally by quantitative real time PCR (qRT-PCR) using samples representing three sequential developmental stages of drumstick EC, supporting their apparent involvement in drumstick EC formation. Our study provides valuable information about the molecular mechanism of EC formation and has revealed new genes involved in this process.

Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9

To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium-mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD600 of the infection solution(suspension of A. tumefaciens) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.

MicroRNA Zma-miR528 Versatile Regulation on Target mRNAs during Maize Somatic Embryogenesis

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.

Improved method for regeneration and Agrobacterium-mediated transformation of Indian short-day onion (Allium cepa L.)

A high-auxin medium, usually used for callus induction, was not effective for Indian short-day onion cv. Bhima super. In this study, we found that the onion seedling radicle was a better explant than shoot tip for embryogenic callus induction, and induction efficiency up to 85.33% along with high embryogenic calli weight was obtained in routinely used medium containing 1.0 mg/L 2,4-D, but specifically supplemented with 0.5 mg/L kinetin. MS medium supplemented with 1.5 mg/L kinetin and 0.125 mg/L ABA showed 73.15% shoot regeneration efficiency from the calli induced from seedling radicle. Geneticin and hygromycin B at 50 mg/L showed optimal selection pressure for 8-week-old onion calli. Agrobacterium-mediated transformation of 8-week-old friable embryogenic calli induced from seedling radicle resulted in phenotypically normal transgenic plants with 1% transformation efficiency. In this study, regeneration and transformation protocols were developed for a widely used Indian short-day onion cultivar, which is instrumental for the development of stable transgenics in this crop.

Genotypic variation in the response to embryogenic callus induction and regeneration in Saccharum spontaneum

Embryogenic callus induction and regeneration are useful in many aspects of plant biotechnology, especially in the functional characterization of economically important genes. However, in sugarcane, callus induction and regeneration vary across genotypes. Saccharum spontaneum is an important wild germplasm that confers disease resistance and stress tolerance to modern sugarcane cultivars, and its genome has been completely sequenced. The aim of this study was to investigate the effect of genetic variations on embryogenic callus induction and regeneration in S. spontaneum and to screen genotypes having high tissue culture susceptibility. The study was performed using nine genotypes of S. spontaneum and the following five parameters were assessed to determine the response of genotypes to embryogenic callus induction and regeneration: callus induction, embryogenic callus ratio, embryogenic callus induction, embryonic callus regeneration and regeneration capacity. All the genotypes varied significantly (P < 0.01) in all the parameters, except for embryonic callus regeneration, which was high (>80%) for all the genotypes. High broad-sense heritability (86.1–96.8%) indicated that genetic differences are the major source of genotypic variations. Callus induction was found to be strongly positively correlated with embryogenic callus induction (r = 0.890, P < 0.01) and regeneration capacity (r = 0.881, P < 0.01). Among the nine tested genotypes, VN2 was found to be the most responsive to tissue culture and could therefore be used to characterize functional genes in S. spontaneum. We also suggested an approach with potential applications in facilitating the rapid identification of sugarcane genotypes susceptible to tissue culture.

INDUKSI KALUS EMBRIOGENIK DAN EMBRIO SOMATIK DARI EKSPLAN DAUN KULIM (Scorodacarpus borneensis Becc.)

Kulim is one of woody plant that have multifunction as wood source and for spice and medicinal. Generative propagation of this plant have trouble because seed use limited. The use of leaf segment through somatic embryogenesis to solve the problem. The objective of this study is to obtain the best treatment to embryogenic callus induction. The modification of basal medium of Murashige and Skoog was used as growth medium. The experiment was conducted in three stages are callus induction, embryogenic callus and somatic embryo induction. The treatment of 2,4-D (3,0 – 12 mg/l) used for callus induction. For embriogenic callus induction used 2,4-D (3,0 – 12,0 mg/l) combined with NAA 0,5 mg/l. The treatment of thidiazuron (0,1 – 0,7 mg/l) used for somatic embryo induction. The result showed that the treatment of 2,4-D 6,0 mg/l is the best for callus induction with compact of texture, green, dry and non embryogenic. The treatment of combination 2,4-D 12.0 mg/l with NAA 0.5 mg/l is the best for friable callus induction. The treatment of 2,4-D 6.0 mg/l combined with NAA 0,5 mg/l is the best for embryogenic callus induction with very friable of texture, easy to separate, dry, smooth and glossy. Thidiazuron of 0,1 mg/l treatment is the best for somatic embryos induction with the average number of 7,8 somatic embryos.