Production of synthetic seeds has unraveled new vistas in in vitro plant propagation technology, because it offers many useful advantages on a commercial scale for the propagation of a variety of crop plants. These tools provide methods for production of synthetic seeds for conversion into plantlets under in vitro and in vivo conditions. This technology is useful for multiplying and conserving the elite agricultural and endangered medicinal plant species, which are difficult to regenerate through conventional methods and natural seeds. The synthetic seed technology was developed in different economically important plant species such as vegetable crops, forage legumes, industrially important crops, cereals, spices, plantation crops, fruit crops, ornamental plants, orchids, medicinal plants and wood yielding forest trees etc. All these aspects are presented in this review.

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Synthetic seeds: A review in agriculture and forestry

Artificial seeds are artificially encapsulated somatic embryos (usually) or other vegetative parts such as shoot buds, cell aggregates, auxiliary buds, or any other micropropagules which can be sown as a seed and converted into a plant under in vitro or in vivo conditions. An improved artificial seed production technique is considered a valuable alternate technology of propagation in many commercially important crops and a significant method for mass propagation of elite plant genotypes. The production of plant clones multiplied by tissue culture and distributed as artificial seeds could be a useful alternative to the costly F1 hybrids for different plant crops. The delivery of artificial seeds also facilitates issues such as undertaking several ways for scaling up in vitro cultures and acclimatization to ex vitro conditions. The development of an artificial seed technique also provides a great approach for the improvement of various plant species such as trees and crops.

https://www.mdpi.com/2073-4395/7/4/71/pdf

Artificial Seeds (Principle, Aspects and Applications)

A protocol for in vitro production of genetically uniform populations of the medicinal plant Eryngium planum, rich in selected phenolic acids, has been established. Shoot-tips were collected from axenic seedlings and grown on a Murashige and Skoog basal medium supplemented with 6-Benzyladenine (BA) and Indole-3-acetic acid (IAA). The highest shoot proliferation efficiency (17 shoots per explant) was obtained when 1.0 mg L−1 BA and 0.1 mg L−1 were added. Proliferating shoots were rooted and transferred to soil (89 % frequency of survival). Flow cytometric analysis of intact (field-grown) and microrpropagated plants revealed that all plants were uniform in genome size and had similar DNA contents. Thin-layer chromatography (TLC) analysis indicated that multiple shoots and roots from in vitro-derived plants produced high amounts of phenolic acids, primarily of rosmarinic acid (RA). Levels of phenolic acids in in vitro-derived plants were similar to those of intact plants. Furthermore, high-performance liquid chromatography revealed that root cultures in liquid medium accumulated substantial levels of RA. Thus, rapid establishment of in vitro-grown organ cultures of E. planum can also serve as reliable sources for bioactive compounds.

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Phenolic acid and DNA contents of micropropagated Eryngium planum L.

Eryngium maritimum L. is a valuable medicinal species, but since it is protected plant, collection from natural populations is forbidden. Therefore, establishing an efficient system for micropropagation of this species is desirable. To determine the optimal nutritional factors needed for shoot multiplication, root development and secondary metabolites accumulation, different media and plant growth regulators were tested. The highest plant regeneration efficiency (over 96 %), with 4.4 shoots per explant was induced on Murashige and Skoog (MS) medium supplemented with 1.0 mg L−1 benzyladenine (BA) and 0.1 mg L−1 indole-3-acetic acid (IAA). The in vitro-regenerated shoots were rooted (83.3–100 %) and transferred to an experimental plot with 62 % efficiency. Flow cytometric analysis revealed no variation in nuclear DNA content in field- and in vitro-delivered plant material. Ultra high performance liquid chromatography (UHPLC) indicated that multiple shoots and roots from in vitro-regenerated plantlets and adventitious root cultures maintained the production of rosmarinic (RA) and chlorogenic (CGA) acids and triterpenoid saponins found in the rosette leaves and roots of E. maritimum intact plants. UHPLC revealed a 12-fold increase of RA and CGA and 3.2-fold higher accumulation of triterpenoid saponins in roots of in vitro-derived plantlets in comparison to roots from field-grown plants. Adventitious root cultures allowed continuous growth of excised root in liquid media with or without exogenous auxins. The roots grown in liquid medium supplemented with 0.1 mg L−1 IAA showed higher (227-fold) phenolic acids accumulation than those without auxin. Obtained results confirmed that micropropagation is a useful strategy in the protection of endangered species and a renewable source of a high quality plant material for secondary metabolites production.

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The Effect of Nutritional Factors and Plant Growth Regulators on Micropropagation and Production of Phenolic Acids and Saponins from Plantlets and Adventitious Root Cultures of Eryngium maritimum L.

Abstract Flow cytometry (FCM) has been used for plant DNA content estimation since the 1980s; however, presently, the number of laboratories equipped with flow cytometers has significantly increased and these are used extensively not only for research but also in plant breeding (especially polyploid and hybrid breeding) and seed production and technology to establish seed maturity, quality and advancement of germination. A broad spectrum of horticultural and medicinal species has been analyzed using this technique, and various FCM applications are presented in the present review. The most common application is genome size and ploidy estimation, but FCM is also very convenient for establishing cell cycle activity and endoreduplication intensity in different plant organs and tissues. It can be used to analyze plant material grown in a greenhouse/field as well as in vitro. Due to somaclonal variation, plant material grown in tissue culture is especially unstable in its DNA content and, therefore, FCM analysis is strongly recommended. Horticultural species are often used as internal standards in genome size estimation and as models for cytometrically studied cytotoxic/anticancer/allelopathic effects of different compounds. With the growing interest in genome modification, increased application of FCM is foreseen.

/pdf/flow-cytometry-a-modern-method-for-exploring-genome-size-and-3k1ldf81qt.pdf

Flow cytometry – a modern method for exploring genome size and nuclear DNA synthesis in horticultural and medicinal plant species

Flow cytometry was used to study the genome size and the cell cycle in different plant materials of Plantago asiatica (2n = 2C = 4x) cultured in vitro, including long-term callus lines. The nuclear DNA content varied from 2.97 to 3.45 pg/2C, depending on the plant material analyzed. However, in most of the cases it was similar to that of the seedling (3.3 pg), the source material. This suggests that in vitro cultures of P. asiatica produce genetically stable material. It seems that a significantly higher or lower than in a seedling leaf DNA content in callus can be an indication of the lack of its organogenic potential. Analysis of the cell cycle showed that there was no endopolyploidization in any of the seedling organs; thus P. asiatica is likely a non-polysomatic species. A high proportion of 2C nuclei (>80%; tetraploid ploidy level) was maintained in almost 70% of the calli but did not ensure their high organogenic capacity. Two out of 82 calli were octoploid and they were non-organogenic. In 30% of calli endoreplication up to 16C occurred. Since endopolyploid cells were present in organogenic as well as in non-organogenic calli, the polysomaty level of P. asiatica callus cannot be considered as an indication of its totipotency.

Nuclear DNA content in different plant materials of Plantago asiatica L. cultured in vitro

Somatic seeds of Plantago asiatica L. were produced for the first time. Shoot-tips isolated from in vitro obtained 4-week shoots were encapsulated using sodium alginate and calcium chloride. Capsules with or without sucrose and with and without cytokinin - indole-3-butyric acid (IBA) were used. Sucrose presence in capsules very distinctly influences somatic seeds of Plantago asiatica germination and their conversion into plants. However, addition of IBA to capsules has not clear influence on the ability of plant regrowth. Plantlets transplanted to soil grew to phenotypically normal plants.

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Somatic seeds of Plantago asiatica L.

Shoot-tip multiplication of the medicinal species - Plantago asiatica was carried on MS medium with IAA and BAP or kinetin. Best results in micropropagation were achieved by adding 0.1 mg/dm 3 IAA and 1 mg/dm 3 BAP. After 6 weeks shoots were transferred to MS medium for rooting. The resulting plantlets were transferred after 8 weeks into pots and after a period of adaptation into the ground (field culture). The species Plantago asiatica was propagated in vitro by shoot-tip multiplication for the first time.

/pdf/micropropagation-of-plantago-asiatica-l-through-culture-of-4xptx8yu18.pdf

Micropropagation of Plantago asiatica L. through culture of shoot-tips

A vanishing species in Poland - Plantago maritima L. was regenerated in vitro from tips of shoots (obtained in vitro) and from different explants of 4-week-old seedlings: seedling tips, hypocotyls, cotyledons, roots. Murashige and Skoog basal medium, supplemented with 0.6 pM indole-3-acetic acid in combination with cytokinins 6-benzyladenine, zeatin or kinetin, was used. The plants obtained in the result of micropropagation were normal in appearence. It was proved that Plantago maritima species was amenable to propagation from different kinds of explants. The method may be of significance for protection of sea plantain.

/pdf/micropropagation-of-plantago-maritima-l-a-vanishing-species-41fpsxh4w2.pdf

Micropropagation of Plantago maritima L. - a vanishing species in Poland

The Far East medicinal plant - Plantago camtschatica was propagated in vitro from tips of shoots (obtained in vitro) and from different explants of 4-week-old seedlings: seedling tips, hypocotyls, cotyledons, roots, first leaves To our knowledge there is no information in literature about in vitro culture of this plantain MS basal medium, supplemented with 06 pM IAA in combination with various cytokinins (BA, KIN, ZEA), was used After 6 weeks of culture, micropropagation rate (MR) - mean number of buds and shoots per explant - was calculated Our study proved that P camtschatica species was amenable to propagation in vitro from different kinds of explants However, multiplication by adventitious shoot regeneration from hypocotyl explants was found to be the most suitable method for the propagation of this plant Adventitious shoots could root without stimulation what allows to omit the stage of rooting The plants obtained as a result of micropropagation were not phenotypically changed

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E. Andrzejewska-Golec

Papers

Nuclear DNA content in different plant materials of Plantago asiatica L. cultured in vitro

Somatic seeds of Plantago asiatica L.

Micropropagation of Plantago asiatica L. through culture of shoot-tips

Micropropagation of Plantago maritima L. - a vanishing species in Poland

Micropropagation of Plantago camtschatica Link