The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global population. However, the delivery and access of gene-editing tools to the host genome and subsequent recovery of successfully edited plants form significant bottlenecks in the application of new plant breeding technologies. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. Hence, it is of importance to develop and improve multiple strategies for delivery and regeneration in order to be able to approach each application from various angles. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. Here, we will discuss the use of protoplast regeneration in the application of new plant breeding technologies and review pertinent literature on successful protoplast regeneration.

Protoplast Regeneration and Its Use in New Plant Breeding Technologies.

Ranunculus wallichianus is a medicinally important plant and an endemic species to the Western Ghats of South India. An efficient and reliable indirect regeneration protocol system for R. wallichianus was developed from leaf explants in the present investigation. Leaf explants were cultured on both full-strength and half-strength MS (Murashige & Skoog) medium supplemented with different concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of 2,4-D and NAA. Among the different concentrations tested, the highest percentage of yellowish-green compact nodular callus formation was observed on a half-strength MS medium with 2.0 mg L− 1 of 2, 4-D. Then, the in vitro raised organogenic callus was cultured on a half-strength MS medium containing various concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of BA, KIN, and TDZ with 0.5 mg L− 1 NAA and 10 % CW for in vitro shoot regeneration. The highest percentage of regeneration response (97 %) and a maximum number of shoots formation (11.1 ± 0.13 shoots/culture with 9.2 ± 0.21 cm mean shoot length) were obtained from MS medium containing 2.5 mg L− 1 BA with 0.5 mg L− 1 NAA and 10 % CW. The well elongated in vitro raised shoots were effectively rooted in a half-strength MS medium with 2.5 mg L− 1 IBA + 250 mg L− 1 activated charcoal. The well-rooted plantlets were successfully hardened and acclimatized with a survival rate of 94 %. Clonal fidelity of in vitro raised plantlets was assessed by using DNA-based RAPD and ISSR molecular markers. A total of 56 and 47 monomorphic bands were obtained from RAPD and ISSR markers respectively. This present in vitro propagation protocol system could be effective for the conservation of R. wallichianus with their genetic purity and its further investigations. The present study develops a protocol for mass multiplication of Ranunuculus wallichianus through indirect organogenesis and evaluates the clonal fidelity of regenerants by using RAPD and ISSR markers.

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Efficient in vitro plant regeneration from leaf-derived callus and genetic fidelity assessment of an endemic medicinal plant Ranunculus wallichianus Wight & Arnn by using RAPD and ISSR markers

Allium jesdianum Boiss. & Buhse. is the most important species of the Amaryllidaceae family with various pharmacological properties. Three subsequent experiments (germination, callogenesis, and elicitation) were carried out as a completely randomized design with six replication. At the first study, the highest seed germination (78.33%) was achieved at chemical pre-treatment including the combination of α-naphthalene acetic acid (1 mg L−1) and benzylaminopurine (3 mg L−1) under in vitro condition. The highest callus induction (86.7%) was observed at MS/2 media, which was supplemented by NAA (1 mg L−1) and BAP (3 mg L−1) from hypocotyl explants. Then, two chemical elicitors including methyl jasmonate (MeJ) (0, 25, 50, and 100 µM) and putrescine (Pu) (0, 0.5, and 1 mM) were used to investigate their effects on different biochemical traits under callus culture. The results showed the superiority of MeJ over Pu for increasing the secondary metabolites and antioxidant activity in calluses of Allium jesdianum, compared to the control. The highest contents for total phenolics (6.02 mg GAE g−1 FW), total flavonoids (0.52 mg QE g−1 FW), and total flavonols (0.39 mg QE g−1 FW) were observed under 50 µM of MeJ. Meanwhile, the highest value for anthocyanin (8.99 µ mol g−1 FW) was achieved at 25 µM of MeJ. The highest 2,2-diphenyl-1-picrylhydrazyl activities were observed at 50 and 100 µM of MeJ. Putrescine (0.5 mM) elicitation showed only superiority for callus growth rate (0.53 mm day−1). Enhancement of desired secondary metabolites at 50 µM MeJ could be suitable for future studies in biotechnological aspects of this medicinal plant.

Elicitation Effects on Some Secondary Metabolites and Antioxidant Activity in Callus Cultures of Allium jesdianum Boiss. & Buhse.: Methyl Jasmonate and Putrescine.

Medium composition affects the tissue culture-induced variation in triticale regenerants

Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 °C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 × 107 protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 °C with the 40 µg of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.

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Optimization of Isolation and Transformation of Protoplasts from Uncaria rhynchophylla and Its Application to Transient Gene Expression Analysis

In this study we report the development of effective in vitro systems for a medicinal plant Nigella damascena L. comprising: (1) callus induction, (2) somatic embryogenesis in callus cultures with subsequent plant regeneration, and (3) isolation and regeneration of callus-derived protoplasts. Callus development was achieved on 83–100% of hypocotyl and cotyledon explants, whereby Murashige and Skoog medium (MS) supplemented with 3 mg L−1 6-benzylaminopurine and 0.5 mg L−1 α-naphthaleneacetic acid (NAA; BN medium) was more advantageous than MS with kinetin and NAA (KN medium). Histological observations of calli revealed the presence of embryogenic zones from which somatic embryos developed on the hormone-free medium. Plant regeneration was observed on 76–95% of calli. A high capacity to form somatic embryos and regeneration was maintained in long-lasting cultures, i.e. even in 2 year old callus. The obtained callus was also a good source tissue for protoplast isolation. By applying a mixture of cellulase and pectolyase, the acceptable yield of viable protoplasts was achieved, especially from hypocotyl-derived callus maintained on BN medium. Protoplasts embedded in an alginate matrix and cultured in modified Kao and Michayluk media re-constructed their cell wall and re-entered mitotic divisions. About 30% of small cell aggregates formed microcalli, which, after the release from alginate, proliferated continuously on KN and BN media, irrespective of the tissue variant used as the protoplast source. Somatic embryo formation and plant regeneration were successful on hormone-free media. An effective plant regeneration system of N. damascena protoplast cultures has been developed and is being reported for the first time. The main objective of this study was to develop in vitro systems utilizing N. damascena seedlings, as
an easily accessible explant source, for efficient callus induction and proliferation, and plant
regeneration via somatic embryogenesis. Moreover, we attempted to validate the usefulness of the
obtained callus as a source of protoplasts and their capability to develop into plants.

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Aneta Malec-Pala

Papers

Effective callus induction and plant regeneration in callus and protoplast cultures of Nigella damascena L.