Propagation Protocol Research Articles

Propagation of Medicinal Plants for Sustainable Livelihoods, Economic Development, and Biodiversity Conservation in South Africa.

South Africa is blessed with vast plant resources and unique vegetation types. Indigenous South African medicinal plants have been well-harnessed to generate income in rural communities. Many of these plants have been processed into natural products to heal a variety of diseases, making them valuable export commodities. South Africa has one of the most effective bio-conservation policies in Africa, which has protected the South African indigenous medicinal vegetation. However, there is a strong link between government policies for biodiversity conservation, the propagation of medicinal plants as a source of livelihood, and the development of propagation techniques by research scientists. Tertiary institutions nationwide have played a crucial role in the development of effective propagation protocols for valuable South African medicinal plants. The government-restricted harvest policies have also helped to nudge natural product companies and medicinal plant marketers to embrace the cultivated plants for their medicinal uses, and thus have helped support the South African economy and biodiversity conservation. Propagation methods used for the cultivation of the relevant medicinal plants vary according to plant family and vegetation type, among others. Plants from the Cape areas, such as the Karoo, are often resuscitated after bushfires, and propagation protocols mimicking these events have been established through seed propagation protocols with controlled temperatures and other conditions, to establish seedlings of such plants. Thus, this review highlights the role of the propagation of highly utilized and traded medicinal plants in the South African traditional medicinal system. Some valuable medicinal plants that sustain livelihoods and are highly sought-after as export raw materials are discussed. The effect of South African bio-conservation registration on the propagation of these plants and the roles of the communities and other stakeholders in the development of propagation protocols for highly utilized and endangered medicinal plants are also covered. The role of various propagation methods on the bioactive compounds' composition of medicinal plants and issues of quality assurance are addressed. The available literature, media online news, newspapers, and other resources, such as published books and manuals, were scrutinized for information.

Micropropagation of Strawberry Crop (Fragaria ananassa): A Review

The objective of this review paper is to summarize on the history, advantages, disadvantages of in vitro micropropagation of strawberry crop. Since the traditional method of propagation, that is, the use of runners as propagating material, is not that efficient because it leads to the spread of viral diseases. We have to go for in vitro micropropagation, the crops also yield more when compared to traditional methods of propagation. The paper also reviews a study done to develop a protocol for large-scale propagation of strawberry cv. ‘Sweet Charlie’ and ‘Winter Dawn’ using the micropropagation technique. Shoot cultures were obtained from shoot tips on Murashige and Skoog (MS) medium with 4% table sugar, 0.75% agar, 5 mg L-1 6-benzyladenine and 0.01 mg L-1 kinetin. These shoots were multiplied and maintained on MS medium with 1 mg L-1 6-benzyladenine and 0.1 mg L-1 kinetin. Rooting of in vitro raised shoots was successfully conducted by pulse treatment with 500 mg L-1 indole-3-butyric acid for 30 s and subsequent culturing in MS medium with 1 mg L-1 indole-3-butyric acid, 0.1 g L-1 activated charcoal and 6% table sugar. Plants were successfully acclimatized and survived in field conditions.

Peasants' perceptions on the socioeconomic importance and cultivation practice of Canarium schweinfurthii Engl. (Black olive tree) in the Western Highlands of Cameroon

Canarium schweinfurthii is one of the agro-forestry tree species with multiple uses in the subtropical countries which is still exploited wild. The purpose of this study was to get insights related to peasants' perceptions on the socio-economic importance and cultivation practice of the species in the Western Highlands of Cameroon. The methodology consisted of surveys among people spread over 7 villages. Two hundred (200) people who were involved in C. schweinfurthii exploitation were selected in the study area following a random approach and interviewed using a structured questionnaire. In addition to the identification of respondents, the information collected from interviewees focused on: 1) knowledge, uses, harvesting methods and the impact of the exploitation of the species on the well-being of people; 2) the trend of the population’s dynamics of the species and, 3) the know-how and practices in the cultivation of the plant. The results showed that almost all the interviewees (99%) knew the species that they exploit in six categories of use (food, medicine, handicraft, construction, timber industry and firewood supply), with a broad consensus (Cs = 0.98) for the use in food. The degree of consensus remained low for its use in medicine (Cs= -0.18), firewood supply (Cs= -0.21), timber industry (Cs= -0.65), construction (Cs= -0.77) and handicrafts (Cs= -0.84). According to 80% of respondents, income from the sale of the fruits significantly contributes to household welfare. The most cited harvesting method was fruits picking, which does not endanger the species. Only 37% of the interviewees perceived a regressive trend in the dynamics of the species' population over the last decade. For 71% of the respondents, the cultivation practice by farmers is rare or even non-existent. The main reasons for non-cultivation of the species are the uncertainty that the planted trees will bear fruits (80% of respondents) and the long duration of the vegetative growth phase (55% of respondents). 90% of respondents expressed their willingness to adopt the cultivation of the species if these constraints are circumvented. These findings showed that C. schweinfurthii would be an interesting candidate for participatory domestication in the Western Highland of Cameroon, for the intensification of agro-forestry systems. For this purpose, the establishment of an efficient protocol for asexual propagation of fruit-producing individuals is needed.

Micropropagation and assessment of genetic stability of Dendrobium transparens Wall. Ex Lindl. using RAPD and ISSR markers

IntroductionDendrobium species have been widely used for many health disorders since ancient times. However, due to unrelenting collection to meet the increasing demand for their use in medication and other health products, the natural habitats of medicinal Dendrobium transparens have been devastated and are on the verge of extinction.MethodsAn efficient in-vitro propagation protocol for Dendrobium transparens using seed derived protocorms was established and genetic homogeneity of the in-vitro regenerants and the wild plant was studied. ResultsThe maximum seed germination was observed in Full strength Murashige and Skoog medium (FMS). Induction of protocorms were achieved on basal as well as half-strength MS medium. The highest number of shoot (11.9 shoots/explant) was achieved in half MS medium fortified with 100 mL/L coconut water in addition with Benzyl amino purine (BAP) 1 mg/L and Kinetin 2 mg/L. Further, elongated shoots were transferred to full and half strength MS root initiating medium supplemented with different concentration of auxins. However, a maximum of (8.3 ± 0.6, 4.9 ± 0.1 cm) roots were achieved in full MS medium fortified with 100 mL/L coconut water and Napthalene acetic acid (NAA) 1.5 mg/L. Ten rapid Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) primers were used to analyze genetic stability among in-vitro and mother plant. RAPD primers produced a total of 23 fragments while ISSR primers produced a total of 16 fragments. ConclusionThe amplified bands of all the samples of in-vitro plants were similar to bands of mother plant. The present research reported here is indicating the applicability of tissue culture for true-to-type plant production and conservation of D. transperens.

In vitro propagation of Codonopsis pilosula (Franch.) Nannf. using apical shoot segments and phytochemical assessments of the maternal and regenerated plants

BackgroundCodonopsis pilosula (Franch.) Nannf. is a medicinal plant traditionally used in China, Korea, and Japan to treat many diseases including poor gastrointestinal function, low immunity, gastric ulcers, and chronic gastritis. The increasing therapeutic and preventive use of C. pilosula has subsequently led to depletion of the natural populations of this species thus necessitating propagation of this important medicinal plant. Here, we developed an efficient and effective in vitro propagation protocol for C. pilosula using apical shoot segments. We tested various plant tissue culture media for the growth of C. pilosula and evaluated the effects of plant growth regulators on the shoot proliferation and rooting of regenerated C. pilosula plants. Furthermore, the tissues (roots and shoots) of maternal and in vitro-regenerated C. pilosula plants were subjected to Fourier-transform near-infrared (FT-NIR) spectrometry, Gas chromatography-mass spectrometry (GC–MS), and their total flavonoids, phenolics, and antioxidant capacity were determined and compared.ResultsFull-strength Murashige and Skoog (MS) medium augmented with vitamins and benzylaminopurine (1.5 mg·L−1) regenerated the highest shoot number (12 ± 0.46) per explant. MS medium augmented with indole-3-acetic acid (1.0 mg·L−1) produced the highest root number (9 ± 0.89) and maximum root length (20.88 ± 1.48 mm) from regenerated C. pilosula shoots. The survival rate of in vitro-regenerated C. pilosula plants was 94.00% after acclimatization. The maternal and in vitro-regenerated C. pilosula plant tissues showed similar FT-NIR spectra, total phenolics, total flavonoids, phytochemical composition, and antioxidant activity. Randomly amplified polymorphic DNA (RAPD) test confirmed the genetic fidelity of regenerated C. pilosula plants.ConclusionsThe proposed in vitro propagation protocol may be useful for the rapid mass multiplication and production of high quality C. pilosula as well as for germplasm preservation to ensure sustainable supply amidst the ever-increasing demand.

MULTIPLICACIÓN in vitro DE Phalaenopsis sp. USANDO REGULADORES DE CRECIMIENTO VEGETAL, AGUAMIEL Y PULQUE (COMPLEJOS ORGÁNICOS)

<p><strong>Background:</strong> <em>Phalaenopsis</em> is an orchid of economic importance worldwide. Its natural reproduction is slow due to its monopodial growth (a single apical meristem). Therefore, it is necessary to make <em>in vitro</em> propagation protocols more efficient. <strong>Objectives:</strong> 1. To evaluate three concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with three of 6-Benzyl Adenine (BA) in the <em>in vitro</em> induction of protocormic like bodies (PLBs) at from leaf explants of <em>Phalaenopsis</em> sp. var. Dudu, under three periods of darkness, 2. To test the effect of four concentrations of honey water and fermented sap, as organic complexes (CO), on the <em>in vitro</em> multiplication rate of PLBs. <strong>Methodology:</strong> Two tests were carried out: Test 1. Three concentrations of 2,4-D (3, 4, 5 mgL<sup>-1</sup>) and three of BA (1, 2, 3 mgL<sup>-1</sup>) were evaluated in leaf explants of <em>Phalaenopsis</em> sp., combined with three periods of darkness 14, 21, 28 days in a completely randomized design with a trifactorial arrangement (27x10). Test 2. Four concentrations of honey water and fermented sap (0, 10, 50, 100 mlL<sup>-1</sup>) were evaluated, arranged in a completely random design (8x10). <strong>Results:</strong> The best combination of plant growth regulators (PGR) for the formation of PLBs (14 PLBs/explant) was with 5 mgL<sup>-1</sup> of 2,4-D and 2 mgL<sup>-1</sup> of BA, during 21 days of darkness. Regarding the use of CO, the addition of 10 mlL<sup>-1</sup> of fermented sap and 50 mlL<sup>-1</sup> of honey water induced the highest rates of multiplication of PLBs/explant (41.4 and 39.6 PLBs). The regeneration of <em>Phalaenopsis</em> sp. from PLBs occurred in 225 days. <strong>Implications:</strong> The results obtained in the two trials are the basis for establishing a protocol for the multiplication of <em>Phalaenopsis</em> sp. <strong>Conclusions:</strong> The combination of 5 mgL<sup>-1</sup> of 2,4-D and 2 mgL<sup>-1</sup> of BA produced the highest number of PLBs/explant (14) at 21 days of darkness and subsequent light exposure for six weeks. On the other hand, the addition of 10 mlL<sup>-1</sup> of fermented sap and 50 mlL<sup>-1</sup> of honey water as CO induced the highest rates of PLBs multiplication (41.4 and 39.6 PLBs, respectively). 100% survival of seedlings was observed after 21 days of acclimatization, under greenhouse conditions.</p>

STANDARDIZATION OF MICROPROPAGATION TECHNIQUE OF MULBERRY (MORUS ALBA L.)

A standardization of protocol for rapid multiplication of mulberry (Morus alba L.) was established in vitro using nodal explant excised from hydroponically grown mature mulberry plants. Shoot initiation was induced on Murashige and Skoog (MS) medium supplemented with different concentrations of (BAP) 6- Benzyl amino purine. Effect of different concentration of 6- Benzyl amino purine was studied on multiplication of shoot, revealed that elongated shoots (2-3 cm) were cultured on half strength MS medium supplemented with (IBA) indole-3-butyric acid. Higher percentage of shoot initiation (90%) was observed on MS medium supplemented with 2 mg/l 6-bezyl amino purine. Initiated shoots gave a higher average number of shoots and elongation was observed when it was subcultured on MS medium supplemented with 1.4 mg/l 6-bezyl amino purine. A maximum percentage of root formation (70%) was observed when elongated shoots were subcultured on half strength MS medium containing 1.3 mg/l indole-3-butyric acid. Rooted plants transferred to pots containing sterile FYM, garden soil and sand (1:1:1) for acclimatization in green house showed (75%) survival capacity.

MICROPROPAGATION OF PHARMACEUTICAL CROP RUBIA CORDIFOLIA

Plants are cherished as the chief resource for healthcare products attributed to synthesizing diverse array of bio-molecules through various metabolic alterations. Over exploitation of such resources can intimidate its existence. Developing medicinal plants into crops and the use of micropropagation as technique to mass produce high-demand biomass can make a continuous supply of natural therapeutic products. Rubia cordifolia is one such plant, so an improved, efficient, reproducible and reliable in vitro clonal propagation protocol is to be adopted for the conservation of germplasm. MS medium fortified with phytohormons viz., BA, Kin, 2-iP, TDZ, mT, IAA, IBA and NAA singly or in combinations were used for the study. After one month of inoculation, 2.5 μM of BA and TDZ (9.37) endorsed the instigation of multiple shoots through axillary bud proliferation in a short span of time. In vitro produced microshoots were subjected to ex vitro rooting by treating 2.5 mM IBA for 5 min and planting in plastic pots containing potting mixture, to produce maximum rooting (79.17%) response. It was found to be an efficient and effective mode of rooting, since it combines both hardening and rooting. Under green house condition, all the rooted plantlets showed more than 90% survival. So direct regeneration protocol from nodal explants without the intervention of callus and thus ensure production of clonally uniform plants of R. cordifolia.

Carrot hairy roots (Daucus carota L.) characterisation and optimisation for high β‐carotene extraction

Hairy roots are widely known as a biological system for the production of highly diverse biomolecules. β‐carotene – a precursor for vitamin A – is known to be an anti‐oxidant and anti‐gastric cancer and protection agent against cardiovascular disease, heart disease and stroke. β‐carotene has been chemically synthesised and consumed by humans. However, the chemical process often produces a by‐product that may be harmful to human health. Therefore, this study established a protocol to induce hairy roots (HRs) from a Vietnamese carrot variety and produce natural β‐carotene. The Rhizobium rhizogenes ATCC15834 harbouring Ri plasmid and a Vietnamese carrot variety were used as materials for genetic transformation and HR induction studies. The result showed that approximately 50 HR lines were obtained. Culture medium supplemented with 30 mg/L of sucrose that gave the highest biomass of HR was shown in carrot HR line 30, which had a doubling time of 6.5 days. The highest content of β‐carotene extraction, at 128 mg/100g hairy roots, was achieved with a ratio volume (v/v) of 2‐propanol and plant samples of 20:1, followed by two hours’ incubation with 2‐propanol at 60 °C. Our study reveals a highly efficient protocol for Vietnamese carrot hairy root establishment and multiplication. A very efficient protocol for β‐carotene extraction from the hairy root was established to produce natural β‐carotene that achieves the same β‐carotene quantity as that produced by normal roots. This study provides new insight into the production of high‐content and natural β‐carotene for therapeutic application.

Effects of different combinations of growth regulators on the in vitro growth parameters of Poncirus trifoliata L. (Raf.)

In this study, an in vitro propagation protocol of Poncirus trifoliata L. (Raf.) rootstock, which is one of the main rootstocks used in citrus, was created. Monocular node parts of the plant, which is the source of explant, were cultured in MS nutrient media containing BAP at dissmilar concentrations (0.5-1.0-2.0 mg/l). Afterward, it was observed that shoot appearance and tillering were achieved in the node explants cultured in MS nutrient medium comprising 1.0 mg/l BAP. The shoots were removed to a new culture in MS nutrient media containing NAA and IBA for rooting. While 30% rooting was achieved in the rooting medium, 62% of the rooted plantlets were acclimated to external conditions. 2 mg/l BAP and 2 mg/l silver nitrate (AgNO3) doses were supplied to the MS medium. The results of this study were obtained and a compound was found to the yellowing and leaf fall problem. As a result, in vitro propagation of Poncirus trifoliata, which is among the important citrus rootstocks, was tried, and its rooting ability was investigated in different nutrient media concentrations after shoots were obtained from different explants.

Development of In vitro Mass Propagation Protocol for Gerbera (Gerbera jamesonii Bolus) var. Orange

An in vitro mass propagation protocol was developed for Gerbera (Gerbera jamesonii Bolus) var. orange using flower bud explants. The young (7 days) explants were found to be suitable for culture and highest (100%) callus induction was obtained in MS medium supplemented with 5.0 mg/l BAP with 1.0 mg/l NAA. The maximum shoot regeneration (95%) and the number of shoots (10) were observed in MS medium supplemented with 6.0 mg/l BAP with 0.3 mg/l NAA. The highest shoot multiplication frequency (95%) and the number of shoots (60) were obtained in MS medium supplemented with only 2.0 mg/l BAP. The well-developed highest number of roots (5.0) per plant and average root length (5.0 cm) with 100% root induction efficiency without callusing was recorded in the half strength of MS medium devoid of any hormonal supplement. Acclimatized in vitro grown plantlets were successfully transferred into the field condition. Plant Tissue Cult. & Biotech. 32(2): 217-226, 2022 (December)

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

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.

Highly effective organogenesis and somatic embryogenesis of Clivia

• Regeneration of Clivia via somatic embryogenesis was reported for the first time. • A reproducible and simple protocol for Clivia propagation was established. • The reproduction coefficient of Clivia has been greatly improved. Clivia miniata Regel. is a world-famous ornamental and medicinal plant species. Expanding the demand for commercial Clivia cultivation requires a reliable, highly efficient, stable system for reproduction. In this study, an efficient protocol for Clivia in vitro regeneration through somatic embryogenesis via a TCL technique was established. Clivia regeneration systems were optimized for commercial application. Under complete darkness, MS media supplemented with 0.5 mg/L NAA and 1.0 TDZ induced callus production from hypocotyls, and the callus induction rate was 25.58%. The calli were then transferred to MS media + 0.5 mg/L NAA + 0.5 mg/L TDZ media and allowed to grow for 60 days, and the callus proliferation rate reached 217%. Organogenic calli inoculated in MS media + 0.5 mg/L NAA + 2.0 mg/L BAP produced adventitious shoots, and the induction rate was 81.11%. The shoots were transferred to rooting media (1/2-strength MS media+0.5 mg/L NAA); they produced roots after 45 days, and the rooting rate was 100%. Moreover, somatic embryogenesis of Clivia could be directly induced by the use of 1 mm root TCLs of in vitro plants. In the presence of 2.0 mg/L PIC, the formation of globular embryos could be directly induced from the vascular bundles of TCLs at 45 days after inoculation. An average of 10∼16 globular embryos were induced per TCL. After approximately 4 months, the somatic embryos developed into plantlets. Our in vitro propagation protocol could be used for sustainable commercial utilization as well as conservation of Clivia bioresources.

Environmental Modulation of Mini-Clonal Gardens for Cutting Production and Propagation of Hard- and Easy-to-Root Eucalyptus spp.

Clonal Eucalyptus propagation is essential for various industry sectors. It requires cuttings to successfully develop adventitious roots (ARs). Environmental conditions are influential on AR development and may be altered to modulate the productivity of hard-to-root clones. The current knowledge gap in research on the physiological patterns underlying commercial-scale propagation results hinders the design of novel strategies. This study aimed to identify patterns of variation in AR-relevant parameters in contrasting seasons and species with distinct rooting performances. E. dunnii and E. ×urograndis (hard- (hardR) and easy-to-root (easyR), respectively) mini-stumps were subjected to light modulation treatments and to mini-tunnel use (MT) for a year. The treatment impact on the branching and rooting rates was recorded. The carbohydrate content, AR-related gene expression, and mineral nutrition profiles of cuttings from the control (Ctrl) and treated mini-stumps were analyzed. Light treatments were often detrimental to overall productivity, while MTs had a positive effect during summer, when it altered the cutting leaf nutrient profiles. Species and seasonality played large roles in all the assessed parameters. E. ×urograndis was particularly susceptible to seasonality, and its overall superior performance correlated with changes in its gene expression profile from excision to AR formation. These patterns indicate fundamental differences between easyR and hardR clones that contribute to the design of data-driven management strategies aiming to enhance propagation protocols.

In vitro Propagation of Curcuma caesia Roxb. via Bud Culture Technique and ISSR Profiling of the Plantlets for Genetic Homogeneity

An in vitro propagation protocol has been developed for Curcuma caesia Roxb., an endangered medicinal plant by Foundation for Revitalisation of Local Health Traditions and Central Forest Department of India. The plant bears poorly germinated seeds and produces two-storage organs: rhizomes and multiple root tubers. Only rhizomes have medicinal-economic values. They serve as propagules too, which results in a shortage of planting material. Therefore, a complete one-year production cycle of C. caesia has been standardized through in vitro propagation including explants establishment (one month), subculture cycles (seven months), rooting (one month) followed by primary hardening (one month) and secondary hardening (two months). Dormant shoot buds on rhizome served as explants for culture initiation on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA) and kinetin (KIN) in combination with citric acid (CA), adenine sulfate (AdS) and indole- 3-acetic acid (IAA). Maximum bud break (70%) was obtained on MS with 8 mg L-1 BA, 8 mg L-1 KIN, 100 mg L-1 CA, 200 mg L-1 AdS and 2 mg L-1 IAA (standard medium). Shoot production potential continued on this medium during the subsequent seven-month-long subculture cycle. The in vitro raised shoots rooted best on ½-strength MS containing 1 mg L-1 indole-3-butyric acid. Plantlet survival rate was >95% after acclimatization. The genetic homogeneity of plantlets with the mother plant was analyzed using Inter Simple Sequence Repeats which generated a monomorphic banding pattern to confirm the uniformity of in vitro raised plantlets of C. caesia.

Encapsulation of Brunfelsia pauciflora microprogated shoots as a suitable conservation protocol

The percent work aimed to establish an efficient in vitro propagation and conservation protocol for Brunfelsia pauciflora plant using encapsulation techniques that allow the genetic stability of conserved material. The effect of plant growth regulators types (BAP, Kin, 2ip, and NAA) and concentrations at 0.0, 1.0, and 2.0 mg/l were stud-ied during the establishment phase in comparison with a hormonal-free medium. In vitro proliferated shootlets were encapsulated using various levels of sodium alginate at 1, 2, and 3% (W/V) and calcium chloride at 2.5,5 and 10 %. The regrowth ability of encapsulated explants was evaluated after conservation for 3, 5, and 7 weeks and compared with non-encapsulated buds. The findings of the current study proved that during the establish-ment phase, applying BAP at 2.0 mg/l gave the best results during the three successive subcultures. The best treatment 2ip (2.0mg/l) + BAP (2.0mg/l) + NAA (2.0mg/l) that was considered ideal propagation protocol for shooting and rooting of Brunfelsia. Successful plant recovery from encapsulated explants using low concentra-tions of sodium alginate (1%) and calcium chloride at 2.5% proved that the explants could to survive at the high-est percent (100%) during conservation periods 3, 5, and 7weeks. Increasing calcium chloride to 5% with the same concentration of sodium alginate (1%) caused a gradually reduced percent of survival to 53.33% with an increasing conservations period (from 3 to 7weeks). It is the first paper on the application of the syndetic seed technique on Brunfelsia for the shortest period of conservation and successive renovation of shootlets whose genetic stabilization was evaluated by analysis of RAPD that proved that the genetic similarity ranged from 56.1 to 80%. These results were confirmed by anatomical sections made in Brunfelsia leaf blade.

Long-Term Field Evaluation of Conventional vs. Micropropagated Plants of Chrysanthemum cinerariifolium

Pyrethrum is a perennial herbaceous plant endemic to the eastern coast of the Adriatic Sea, and introduced in large areas of nearly all continents, where it is cultivated for the industrial extraction of pyrethrins. Pyrethrins are a group of six closely related monoterpene esters, widely used as natural insecticides. The world production of natural pyrethrins is lower than the market demand, and a wider introduction of this crop within the Mediterranean agrosystems could be an appealing opportunity for farmers and manufacturers. The availability of adequate amounts of selected plant material to bring into cultivation is, however, one of the major issues. Therefore, the in vitro propagation of elite pyrethrum genotypes could be a suitable alternative to conventional propagation methods. In this paper, we present the results of a 9-year field comparison between pyrethrum plants coming from an in vitro propagation protocol and plants obtained by cutting from the same mother plants. Furthermore, since plantlets derived from in vitro regeneration may experience ploidy changes, we evaluated the stability of the ploidy level of pyrethrum-micropropagated plants by flow cytometry (FCM) analysis. FCM screening revealed no differences among the morphotypes and between them and the mother plant. Likewise, the field evaluation of plants gave no significant differences between flower yields in both groups. Hence, micropropagation was confirmed as an easy, efficient and reproducible method to obtain large quantities of selected pyrethrum genotypes.

Rapid In Vitro Propagation of Fig (Ficus carica L.) ‘Violette de Solliès’ Supported by Molecular and Microscopy Analyses

Ficus carica L. is a common fig that is an incredibly nutritional fruit, well-known for its medicinal and economic values. This study aims to establish an efficient protocol for the mass propagation of fig plantlets (Ficus carica L.) for the cultivar ‘Violette de Solliès’. Surface-sterilized shoot-tip explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of cytokinins (6-benzylaminopurine, BAP; thidiazuron, TDZ; kinetin, Kn; and zeatin, Zea). Induced shoots were rooted on Woody Plant Medium (WPM) with different concentrations of auxins (naphthalene-acetic acid, NAA; indole-3-acetic acid, IAA; and indole-3-butyric acid, IBA). Rooted explants were acclimatized in eight different soil substrates prior to cultivation in a commercial plot. The propagated plantlets were analyzed for genetic stability and clonal fidelity using RAPD and SCoT molecular markers, whereas scanning electron microscopy (SEM) was performed to observe the stomata morphology of post-acclimatized plants. MS media supplemented with 5.0 mg/L BAP was the optimal treatment for multiple shoot induction (15.20 ± 1.03 shoots), whereas the highest percentage of rooting (93.33%) was achieved in WPM supplemented with 3.0 mg/L IBA. Plantlets were successfully acclimatized in biochar soil with a survival rate of 100%. RAPD and SCoT analysis showed no polymorphism occurrences across six subculture cycles, whereas observations via SEM indicated normal stomata structures on the leaves of acclimatized plantlets. This study documents an efficient micropropagation protocol for Ficus carica cv. ‘Violette de Solliès’ for the production of uniformed and true-to-type plant stocks suitable for commercial propagation.

Leaf-based induction of protocorm-like bodies, their encapsulation, storage and post-storage germination with genetic fidelity in Mokara Sayan × Ascocenda Wangsa gold

• First report on direct induction of PLBs Mokara Sayan × Ascocenda Wangsa gold. • Earliest and maximum PLB induction occurred on MS medium fortified with 3 mg L –1 TDZ. • Synthetic seeds were developed encapsulating PLBs with 3 % Na-alginate and 75 mM CaCl 2 . • Better storage of synthetic seeds up to 180 days was attained at 25°C in comparison to 4°C. • RAPD marker-based analysis ensured genetic integrity of synthetic seed-derived plantlets. An innovative and accelerated in vitro mass propagation protocol via direct induction of protocorm-like bodies (PLBs) from leaf sections of orchid hybrid Mokara Sayan × Ascocenda Wangsa gold was developed for the first time. Initially, leaf sections were transferred into Murashige and Skoog (MS) medium supplemented with two cytokinin sources namely, thidiazuron (TDZ) and zeatin with variable concentrations. The earliest (∼8 days) induction of PLBs with maximum number (34 PLBs cm –2 leaf section) and induction frequency (82.8 %) were recorded in MS medium fortified with 3 mg L –1 TDZ. The same medium formulation resulted in the highest growth rate (∼93.7 mg day –1 ) with maximum diameter of PLBs (3.4 mm) when compared with other treatments. Observations on proliferating PLBs under variable pressure scanning electron microscopy illustrated their distinctive growth phases wherein proliferating PLBs exhibited globular shape, typical epidermal texture with a rough surface that steadily became more wrinkled, consisting of basal absorbing hair-bodies and the apical zone forming shoot primordia. Stereo microscopic observations revealed gradual maturation of PLBs, initiation of leaf and shoot primordia. PLBs were encapsulated with calcium-alginate for short-term storage and germplasm exchange. Lower concentrations of sodium alginate (1 and 2 %) produced synthetic seeds that were soft to grip and extremely irregular in shape, whereas higher concentration (4 %) resulted in stiffness of synthetic seeds and reduced germination frequency. The most suitable gel matrix to form uniform spherical beads was optimized at 3 % sodium alginate and 75 mM calcium chloride. Synthetic seeds were effectively stored at 25 °C for up to 180 days. The germination efficiency of synthetic seeds gradually declined after storage at both 4 °C and 25 °C, with synthetic seeds stored at 25 °C being more tolerant than those stored at 4 °C. Even after 180 days of storage, the synthetic seeds that were stored at 25 °C exhibited 71.2 % germination frequency and ∼14 days to germination. Synthetic seeds stored at 4 °C deteriorated rapidly and degenerated completely within 180 days. Random amplified polymorphic DNA marker-based analysis carried out with randomly selected synthetic seed-derived plantlets showed 100 % monomorphism with a total 512 bands of molecular size ranging between 130—2100 bp, ensuring post-storage genetic fidelity.

SRRS: A blockchain fast propagation protocol based on non-Markovian process

The blockchain technology that underlies many cryptocurrencies, such as Bitcoin and Ethereum, is demanded to raise for more efficient transactions. Thus, in the last decade, many solutions have been proposed to improve the propagation of blocks. Motivated by SRRS (social reinforcement rumor spreading), we propose a blockchain fast propagation protocol named SRRS protocol based on a non-Markovian process, where the verification and transmission of a block depend on its propagation history. The simulated experimental results show that our protocol can significantly accelerate the propagation of blocks. Compared with existing major solutions, our protocol does not change the main procedure of Bitcoin and the block data structure, which is a huge advantage for practical applications. Moreover, employing the theories of complex networks and percolation, we develop a method of theoretical analysis, which can find the optimal setting of our protocol.