μM Kinetin Research Articles

Micropropagation, genetic fidelity and chromatographic analysis in Evolvulus alsinoides (L.): A potent multipurpose medicinal plant

Evolvulus alsinoides (L.) is an annually growing medicinal plant used in many popular systems as a source of traditional medicine. However, fresh plant material for medicinal uses and research purposes is critically unavailable year-round due to its short life cycle. A micropropagation method for E. alsinoides was developed, and its genetic diversity was described. Surface sterilized nodal explants were inoculated onto Murashige and Skoog (MS) medium supplemented with different plant growth regulators; the best response of multiple shoot induction was observed on MS media augmented with 5 μM kinetin (Kn) and 0.1 μM Indole-3-acetic acid (IAA), which gave a maximum number of 10.27 ± 0.0118 shoots. Further, the addition of 30 μM of adenine sulfate to the MS media enhanced shoot multiplication (18). The cloned shoots were accomplished to in vitro rooting on MS medium with 0.1 μM Indole-3-butyric acid (IBA) and 10 μM phloroglucinol, resulting in 16 healthy roots. The rooted plantlets were acclimatized in the shade house. The genetic stability of micropropagated plants was confirmed through molecular marker analysis. Gas chromatography-mass spectrometry (GC-MS) analysis of the tissues of clones revealed the contents of secondary metabolites. High-Performance Thin Layer Chromatography (HPTLC) revealed that squalene levels were highest in plants cultured in MS medium supplemented with adenine sulfate (378.89 ng/ 15 μl). The methods developed and defined are highly reproducible and can be used for commercial production of E. alsinoides valued for human health.

Micropropagation of Dipcadi montanum (Dalz.) Baker (Asparagaceae): a rare scapigerous herb.

Dipcadi montanum (Dalz.) Baker (Asparagaceae) is a rare scapigerous herb endemic to the Western Ghats, a global biodiversity hotspot running parallel to the western coast of India. This study reports the development of a reproducible protocol for mass propagation of this underutilized geophyte using bulb scale and immature leaf base explants. Miniature bulblets were successfully induced from both types of explants after 4 and 8 weeks of culture on full-strength semisolid MS basal medium fortified with 3% sucrose and varying levels of BAP (4.4-17.7 μM) and TDZ (4.5-18.1 μM). The addition of 2.7 μM NAA further enhanced the rate of microbulb induction. Rooting of the 8-week-old bulblets, obtained from both explants, was achieved with more than 90% efficiency on liquid as well as agar-gelled half-strength MS basal medium fortified with varying levels of IBA (2.46-9.84 μM) and NAA (2.68-10.74 μM), with or without 2.32 μM Kinetin. More than 95% of the rooted plants survived the initial acclimatization process under controlled ex-vitro conditions, and a survival rate of over 80% was recorded after 4 weeks of transfer to greenhouse conditions. After a brief dormancy, the regenerants resumed growth in the postmonsoon season and exhibited morphological resemblance to the donor plant. Comparative cytological analysis between the donor and 15 randomly selected regenerants revealed a stable somatic count of 2n = 20.

Somatic Embryogenesis and Direct Shoot Bud Formation from in vitro Root Segments of Garlic (Allium sativum L.)

In vitro techniques are unconventionally used for garlic improvement. In garlic tissue culture, numerous roots are produced in vitro. Segments (1 cm) of those in vitro roots were cultivated on MS medium with 5.0 μM 2, 4-D for callus initiation followed by transfer to 0.5 μM 2, 4-D for somatic embryogenesis. Root segments had 51.67% callus and somatic embryo formation. Plantlet regeneration was obtained from 90% calli after being transferred to MS medium supplemented with 5.0 μM kinetin. Within 6 months, 54.8 plants per root segment and 42.2 plants per root tip explant were found. MS media supplemented with 1.0 or 5.0 μM NAA in combination with 10.0 or 50.0 μM BA were used for direct shoot bud induction from root segments. The highest percentage (23.33 %) of direct shoot bud regeneration was found in 5.0 μM NAA with 50.0 μM BA within 2 months. The number of shoots per explant was varied from 1-81 with the highest average of 15.75 shoots per explant. The shoots produced bulblets upon transfer to a medium containing higher amount of sucrose (12%). The largest bulblets weighed 356.57 g in the medium with 5.0 μM NAA and 50.0 μM BA. In both pathways, regeneration occurred from only the apical (distal) end of the root segments. The protocols are promising for continuous regeneration of propagules and genetic transformation study for improvement of garlic. Plant Tissue Cult. & Biotech. 33(2): 135-142, 2023 (December)

Production of Benzyl Isothiocyanate from Moringa peregrina (Forssk.) Fiori via Suspension Cultures

Benzyl isothiocyanate (BITC) is one of the main constituents of Moringa peregrina, an endangered species in the flora of Egypt. The purpose of this study was to establish a procedure to enhance the in vitro production of BITC in Moringa peregrina suspension cultures. This could then aid in the mass production of this useful pure active compound and avoid the exploitation of natural resources of this valuable plant. Suspension cultures of Moringa peregrina was established from callus cells in liquid MS medium supplemented with 10.74 μM naphthalene acetic acid, 2.325 μM kinetin, in addition to salicylic acid as an elicitor and/or L-phenylalanine as a precursor of BITC. The combination of both salicylic acid and L-phenylalanine gave the highest accumulation of BITC after two weeks with 10.42-fold increase, compared to its content in the mother plant. This study is promising for the large-scale production of BITC, which has been identified as an anticancer agent with possible applications in the pharmaceutical industry. Plant Tissue Cult. & Biotech. 33(2): 85-95, 2023 (December)

Molecular identification and in vitro propagation of the endangered medicinal Cleome droserifolia plant of Saint Katherine

The protectorate of Saint Katherine considers one of the biggest protected areas in Egypt characterized by a unique location and environment. It supports a high number of rare and endangered species. Cleome droserifolia (Rorida droserifolia) is an endangered plant found in Saint Katherine, Sinai, Egypt. It has been known for its medicinal uses so it becomes rare due to over-grazing and over-collection for use in traditional medicine, research and trade home use. DNA barcoding analysis and in vitro culture tools were conducted for saving the plant. This research supports the goal number 15 of the United Nations Sustainable Development (SDGs); aimed at preserving, restore and reinstate sustainable usage of terrestrial ecosystems and halting biodiversity deterioration. Two pairs primers of three DNA markers: [ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL), maturase K (matK) and trnH-psbA] were used for PCR amplification and sequencing. BLAST results and phylogenetic analysis proved a correct authentication of Cleome droserifolia on both; species and genus levels and confirms the involvement of Cleome droserifolia in Cleome genus. For in vitro propagation, cotyledonary node explants of germinated seedlings were cultured on Murashige and Skoog (MS) medium fortified with 0.27 µM α-naphthaleneacetic acid (NAA), and 2.32 µM of kinetin (Kin). The highest mean number of axillary shoots was 8.16 shoots/explant. 85% of multiple shoots were successfully rooted on ¼ MS medium fortified with 7.42 µM indole-3-butyric acid (IBA). The successfully rooted plants were transferred to a mixture of sand, soil, and peat moss (1: 1: 1) and efficiently acclimatized in the greenhouse.

In vitro effect of Colchiploidy on andrographolide enhancement in Andrographis paniculata (Burm.f.) Wall. ex. Nees.

An in vitro method to enhance the andrographolide production in Andrographis paniculata was developed using colchiploidy. Andrographis paniculata (Burm.f.) Wall. ex. Nees. is an annual herbaceous diploid medicinal plant with 2n=50, belonging to the Acanthaceae family. Andrographolide is the chief secondary metabolite present in plant and has significant commercial value due to its pharmacological properties. The objective of this study was to investigate the in vitro impact of colchiploids on the production of andrographolide. Additionally, we aimed to compare this effect in calli that were in vitro-raised from colchiploid plants with those derived from diploid plants. Colchiploidy was induced with seedlings of A. paniculata treated with 0.1% (w/v) colchicine for 12 h under dark conditions. Callus culture of Andrographis paniculata established from colchiploid leaf explants on 0.8% agar gelled Murashige and Skoog (MS) medium fortified with 5.0 µM 2,4-D + 2.3 µM Kinetin and 3% sucrose. A significant increase in andrographolide production (2.4%) was achieved in the colchiploid calluses compared to the diploid calluses (0.015%) quantified by high-performance liquid chromatography (HPLC) analysis. Plants with higher amounts of physiologically active secondary metabolites arise as a result of polyploidy. This method of andrographolide production can be used to harvest andrographolide on a large scale.

Efficient shoot regeneration using leaf explant and simulated microgravity effect on shoot development in Solanum nigrum L.

Solanum nigrum L. is an important medicinal plant of India and in the present study, leaves were used as an explant to establish shoot cultures. Murashige and Skoog’s18 (MS) medium supplemented with 20 μM kinetin (Kn) regenerated optimum 93.80 ± 0.89 shoots (100% response) within eight weeks through indirect organogenesis. Further, leaves were placed under simulated microgravity conditions and shoot regeneration was achieved within 16th days in both horizontal and vertical conditions. But horizontal position was better as compared to vertical position and it formed 11.60 ± 0.46 shoots. The in vitro shoots were successfully rooted in ½MS static medium fortified with sucrose (1%) and indole-3-butyric acid (IBA, 2 μM) which induced 39.40 ± 1.08 roots (100% response) within four weeks. This study can be used for mass-propagation of the species, extraction of valuable metabolites and to observe the effect of microgravity on plants.

Revisiting In vitro Micropropagation Protocols of Mimosa pudica for Enhanced Seed Germination, Shoot Multiplication, and Root Initiation

Mimosa pudica is a medicinal plant worthy of therapeutic properties. It is often overlooked as one of the weed species, and the potential was underappreciated despite its abundance in nature, particularly in tropical climate countries. Considering the aptitude of this species, the micropropagation protocol of M. pudica was revisited and enhanced. The seed surface sterilization and germination were assessed, followed by shoot multiplication rate and root initiation efficiency. Seeds of M. pudica were best surface sterilized with 35% of Clorox and recorded the highest germination rate at 65.55% in media of three-quarter strength Murashige and Skoog (MS) combined with 30 g of sucrose. The explants were harvested and subcultured into multiplication media fortified with 6-benzylaminopurine, kinetin, and coconut water. The highest number of new shoots and leaves were obtained at 3 shoots and 3.27 cm in media of MS combined with 17.6 μm kinetin. Half-strength MS media devoid of hormone was recommended for root induction, resulting in the 5 healthy roots at 2.06 cm after 4 weeks of culture. Overall, the developed micropropagation protocol of M. pudica could facilitate its large-scale cultivation, indicating its potential as a medicinal crop for the extraction of bioactive compounds.

Species-Specific Secondary Metabolites from Primula veris subsp. veris Obtained In Vitro Adventitious Root Cultures: An Alternative for Sustainable Production

Primula veris subsp. veris L. is a perennial herbaceous and medicinal plant species the roots and flowers of which are a source of valuable pharmaceutical raw materials. The plant tissues are used to produce expectorant and diuretic drugs due to their high content of triterpene saponins and phenolic glycosides. Underground roots of P. veris can be obtained only through a destructive process during the plant’s harvesting. In the present study, an in vitro adventitious root production protocol was developed as an alternative way of production, focused on four species-specific secondary metabolites. Root explants were cultured in Murashing & Skoog liquid medium supplemented with 5.4 μM α-naphthaleneacetic acid, 0.5 μM kinetin, L-proline 100 mg/L, and 30 g/L sucrose, in the dark and under agitation. The effect of temperature (10, 15 and 22 °C) on biomass production was investigated. The content of two flavonoid compounds (primeverin and primulaverin), and two main triterpene saponins (primulic acid I and II) were determined after 60 days of culture and compared with 1.5-year-old soil-grown plants. The accumulated content (mg/g DW) of bioactive compounds of in vitro adventitious roots cultured under 22 °C was significantly higher than the other two temperatures of the study, being 9.71 mg/g DW in primulaverin, 0.09 mg/g DW in primeverin, 6.09 mg/g DW in primulic acid I, and 0.51 mg/g DW in primulic acid II. Compared to the soil-grown roots (10.23 mg/g DW primulaverin, 0.28 mg/g DW primeverin, 17.01 mg/g DW primulic acid I, 0.09 mg/g DW primulic acid II), the in vitro grown roots at 22 °C exhibited a 5.67-fold higher content in primulic acid II. However, primulic acid I and primeverin content were approximately three-fold higher in soil-grown roots, while primulaverin content were at similar levels for both in vitro at 22 °C and soil-grown roots. From our results, tissue culture of P. veris subsp. veris could serve not only for propagation but also for production of species-specific secondary metabolites such as primulic acid II through adventitious root cultures. This would therefore limit the uncontrolled collection of this plant from its natural environment and provide natural products free from pesticides in a sustainable way.

Rosmarinic Acid Production from Origanum dictamnus L. Root Liquid Cultures In Vitro.

In the present work Origanum dictamnus L. was studied as a suitable in vitro adventitious root culture system for the production of important bioactive molecules, such as rosmarinic acid (RA). Callus culture was initiated from leaf, petiole and root explants on solid MS medium supplemented with either 5 μM NAA + 5 μM kinetin (ODK3) or 5 μM NAA + 0.5 μM kinetin (ODK4). New roots formed from leaf, petiole and root calluses were aseptically transferred into Erlenmeyer flasks containing 100 mL liquid medium and shaken at 120 rpm in the dark. The liquid medium used was the MS supplemented either with 35 μM IBA + 2.5 μM kinetin (ODY1) or 5 μM NAA + 0.5 μM kinetin (ODY2). Biomass production parameters, RA content (%) and yield index (YI) were recorded for each treatment explant type, medium composition and incubation period. Results showed, in every case, the production of RA in vitro. Between the two liquid media (ODY1, ODY2) and the different culture periods, the ODY1 medium and the longest 200-day-culture period were more effective for RA and biomass production, regardless of the initial explant type used. The combination of ODK4-ODY1 resulted in higher RA (5.1% and 4.7%), fresh biomass production (19.0 g and 11.6 g), mean YI (93.7 mg and 51.4 mg) and YI per explant (3.75 mg and 2.06 mg) for roots derived from leaf calluses and root calluses, respectively. However, the solid ODK3 (200 days)-liquid ODY1 (40 days) transition treatment was more beneficial for roots derived from petiole calluses leading to an 18.8-fold increase in fresh biomass growth rate. RA accumulation and YIs were also significantly influenced by explant type, with the highest value produced from root petiole calluses (6.6% RA dry weight, 115.3 mg mean YI and 4.61 mg YI per explant) after 240 days.

Cretan Dittany (Origanum dictamnus L.), a Valuable Local Endemic Plant: In Vitro Regeneration Potential of Different Type of Explants for Conservation and Sustainable Exploitation

Origanum dictamnus L. is a medicinal local endemic to the Island of Crete, Greece. Its propagation through biotechnological tissue culture techniques is essential due to its augmented multi-industrial sector demand. For direct organogenesis, among different culture media variants (MS, Gamborg B5), and cytokinins [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyl adenine (2-iP)], the MS + added with BA (2.2 μM) was the most effective treatment for shoots and roots formation. For indirect organogenesis, all explant types (leaves, petioles, roots) showed a 100% callusing rate after 2 months in all media variants tested; ODK1: 20 μM thidiazuron (TDZ) + 5 μM indole-3-butyric acid (IBA) or ODK2: 0.5 μM kinetin + 5 μM 2,4-dichlorophenoxy acetic acid (2,4-D). The leaves and petiole explants assured a low rate of shoot regeneration (20%) in ODK1. Afterwards, leaf-, petiole-and root-callus derived from both media were transferred to four new media plant growth regulators-free or with BA + IBA + gibberellic acid (GA3). After 10 months from callus transferring, the petiole callus gave rise to roots (20-75%) while the leaf callus exhibited 10-30% shoot or 30% root regeneration. In this study, indirect organogenesis of O. dictamnus was carried out for the first time, thus various organs can be used for plant regeneration, and the developed protocol may be applicable in the horticulture industry.

Effect of cytokinins and light quality on adventitious shoot regeneration from leaflet explants of peanut (Arachis hypogaea)

ABSTRACT Plant tissue culture and novel breeding techniques such as genome editing in the economically important crop Arachis hypogaea constitute potential for genetic improvement. Therefore, in this study, high-frequency in vitro regeneration via formation of adventitious shoots was described for different A. hypogaea lines. The leaflets of the primary leaves of 5-day-old seedlings were cultured for 16 weeks on Murashige and Skoog’s medium supplemented with equimolar concentrations (22.19 µM) of the cytokinins 6-benzylaminopurine (BAP); thidiazuron (TDZ); zeatin (Zea) or meta-topolin (MT), which was reduced to 7.40 µM after eight weeks plus 2.3 µM kinetin. Highest shoot regeneration percentages of 89.1% and 81.3% for BAP and TDZ containing media, respectively, were achieved in the line ’Jimmy’s pride’ with the maximum shoot number obtained on BAP. The regeneration percentages for three further lines were lower than 50%. Furthermore, the effect of different light qualities, applied using LEDs, and compared to fluorescent lamps was investigated. Shoot regeneration percentages did not improve significantly under the different LEDs, instead a pronounced effect on the regeneration efficiency of the culture temperature was observed which differed among the light treatments. However, fluorescent lamps can be replaced by red or blue LEDs without negative effects on shoot regeneration.

Preliminary Study of <i>Pleurotus ostreatus</i> (Jacq.) P. Kumm. Spent Mushroom Compost as Nutrient Supplement on the Shoot Induction of Fig Tissue Culture

Spent mushroom compost is identified as waste substrate. As the mushroom agriculture industry is growing rapidly, the production also increases and contributes to large amounts of spent mushroom compost annually. The conversion of spent mushroom compost as waste to wealth is a great concept that can give value to agricultural industry and solid waste management of mushroom cultivation. The use of SMC as nurient supplement may reduce the production cost for in vitro culture of fig plant. This research is to study the use of Pleurotus ostreatus (Jacq.) P. Kumm. spent mushroom compost (SMC) as a nutrient supplement of the media in shoot induction of fig plant. Nodal segment of fig’s stem was cultured in MS media supplemented with different concentrations of SMC. With presence of SMC, 10% SMC have the highest shoot proliferation (2.167 ± 1.169) and length of shoot (0.154 ± 0.060 cm). The study revealed that in presence of 10% of SMC could produce new shoots at a frequency which was comparable to the control (p<0.05; n=3; Tukey’s multiple range test). Among the different media combinations of plant growth regulators with SMC, 15 μM Kinetin with 10% SMC is the most effective treatment which gave maximum number of shoot proliferation (3.00 ± 1.27). 30 μM IAA with 10% SMC media have the highest leaves regenerated which were 1.33 ± 0.84. In conclusion, the SMC added in media for plant tissue culture had positive effect on shoot induction thus highlighted the potential of waste substrate of SMC to act as nutrient supplement for plant tissue culture.

In Vitro Cultures and Volatile Organic Compound Production in Chiliadenus montanus (Vhal.) Brullo.

Callus and microshoot cultures were established for Chiliadenus montanus (Vhal.) Brullo. (Asteraceae), a medicinal plant known for producing volatile organic compounds (VOCs). Callus induction was achieved successfully by culturing leaf explants on full-strength Murashige and Skoog medium (MS) supplemented with 2.2 µM 2, 4-dichlorophenoxy acetic acid (2,4-D) and 6.9 µM kinetin (Kin). Successful direct shoot regeneration was achieved using nodal explants cultured onto half-strength MS media supplemented with 1.4 μM Gibberellic Acid (GA3) and 4.4 μM 6-Benzylaminopurine (BAP). Indirect microshoots were successfully regenerated using callus cultured on MS media supplemented with 8.8 μM BAP, 2.2 μM Zeatin, and 1.4 μM GA3 followed by culturing on MS media supplemented with 8.8 μM BAP and 0.5 μM naphthalene acetic acid (NAA). Using wild plant aerial parts, callus and microshoots samples, VOCs were extracted successfully using Headspace Solid-Phase Micro-Extraction (HS-SPME) and analyzed by gas chromatography–mass spectrometry (GC-MS). In wild plant extracts, sesquiterpene hydrocarbons were found to be predominant with the following principal components: Alloaromadendrene (11.92%), trans-Cadina-1(6),4-diene (7.54%), and α-caryophyllene (6.77%). The analysis of in vitro microshoots revealed high levels of oxygenated monoterpenes with cis-Myrtanol (16.62%), and β-Cyclocitral (14.3%) as the main components. Callus extract was dominated by monoterpene hydrocarbons and the main compounds identified were (Z)-β-Ocimene (22.27%), p-Cymene (15.13%), and α-pinene (13.78%). In conclusion, an efficient in vitro production system of VOCs in C. montanus was established that can be used in the future for boosting their production without endangering wild plants.

Micropropagation and Acclimatization of Common Oregano (<i>Origanum vulgare</i> L. Subsp. <i>vulgare</i>) by Shoot Tip Culture

Origanum vulgare L. is a commercially valued species with remarkable biological properties. It is subject to over-exploitation practices that seriously threaten its sustainability for future generations. Thus, micropropagation serves as a tool for the protection and domestication of this species. In this study, we established an in vitro vegetative propagation protocol for Origanum vulgare. This is done through the axillary bud technique by carrying out various tests. Six culture media (MS, MSm, N30K, SD, SH and B5) were tested. Therefore, SD was chosen for the following experiments. Seven cytokinins (adenine (Ad), N6-(2-isopentenyl) (2ip), zeatin (Zeat), kinetin (Kin), benzyladenine (BAP), 1,3-diphenylurea (DPU) and thidiazuron (TDZ) at 5 concentrations (0.44, 1.33, 2.22, 3.11 and 4.44 μM/L) were evaluated. Thus, Kin at 3.11 μM allowed high regeneration of vitroplants, optimal elongation, total rooting of explants, maximum bud multiplication, and absence of hyperhydric explants. In fact, the integration of auxins (indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthaleneacetic acid (NAA)) into the culture medium and their combinations with 3.11 μM Kinetin contributed to the optimization of the root part. Thus, it was improved in particular in the case of 3.11 μM Kin and 6.27 μM IBA. Three polyamines (Putrescine, Spermidine and Spermine) at different concentrations (1.134, 3.402, 5.67, 7.938 and 11.34 μM/L) combined at 3.11 μM Kin and 6.27 μM IBA were tested. In fact, 1.304 μM putrescine was considered to be the most suitable for in vitro culture of explants, since it allowed optimal propagation of buds and roots, also a high rate of regeneration and rhizogenesis. GA3 at 1.15 μM combined with 3.11 μM Kin and 6.27 μM IBA permitted maximum bud multiplication. The acclimatization was carried out successfully using vitroplants showing good foliar and root development. Thus, three months after acclimatization, the seedlings were transferred into large pots under natural light and temperature conditions. Almost all acclimatized plants developed flowers in the first year between May and July.

6-Benzylaminopurine Alleviates the Impact of Cu2+ Toxicity on Photosynthetic Performance of Ricinus communis L. Seedlings.

Copper (Cu) is an essential element involved in various metabolic processes in plants, but at concentrations above the threshold level, it becomes a potential stress factor. The effects of two different cytokinins, kinetin (KIN) and 6-benzylaminopurine (BAP), on chlorophyll a fluorescence parameters, stomatal responses and antioxidation mechanisms in castor (Ricinus communis L.) under Cu2+ toxicity was investigated. Ricinus communis plants were exposed to 80 and 160 μM CuSO4 added to the growth medium. Foliar spraying of 15 μM KIN and BAP was carried out on these seedlings. The application of these cytokinins enhanced the tissue water status, chlorophyll contents, stomatal opening and photosynthetic efficiency in the castor plants subjected to Cu2+ stress. The fluorescence parameters, such as Fm, Fv/Fo, Sm, photochemical and non-photochemical quantum yields, energy absorbed, energy trapped and electron transport per cross-sections, were more efficiently modulated by BAP application than KIN under Cu2+ toxicity. There was also effective alleviation of reactive oxygen species by enzymatic and non-enzymatic antioxidation systems, reducing the membrane lipid peroxidation, which brought about a relative enhancement in the membrane stability index. Of the various treatments, 80 µM CuSO4 + BAP recorded the highest increase in photosynthetic efficiency compared to other cytokinin treatments. Therefore, it can be concluded that BAP could effectively alleviate the detrimental effects of Cu2+toxicity in cotyledonary leaves of R. communis by effectively modulating stomatal responses and antioxidation mechanisms, thereby enhancing the photosynthetic apparatus’ functioning.

Plant regeneration via somatic embryogenesis in diploid cultivated cotton (Gossypium arboreum L.)

The diploid cotton species G. arboreum offers a better opportunity to elucidate gene structure and function as opposed to the allotetraploid cotton species. As a prerequisite for this, a reliable and efficient method of high frequency plant regeneration in G. arboreum must be established. In this study, callus was induced from hypocotyl, root and cotyledon of G. arboreum seedlings on MSB medium (MS salts and B5 vitamins) with 0.09 µM 2, 4-D (2, 4-dichlorophenoxyacetic acid) and 2.32 µM KT (kinetin). During propagation, callus was effectively selected for subculture on different media based on cell morphology to induce embryogenic callus and somatic embryos. Embryogenic callus was induced on MS5 medium (MSB, 37.59 mM KNO3, 62.47 µM NH4NO3, 3% (w/v) glucose, 6.8 mM glutamine, 3.8 mM asparagine) from suspended cultures after several cycles of alternate solid MS3 medium—liquid MS4 medium culture, a key step in somatic embryogenesis. Solid MS3 medium was supplemented with 2.46 µM IBA (īndole-3-butyric acid), 0.93 µM KT, 6.8 mM Gln, 3.8 mM Asn; liquid MS4 medium was supplemented with 0.1 g/L NaCl, 0.1 g/L KCl and 0.1 g/L CuSO4. The solid–liquid alternate culture was effective for embryogenic callus induced in G. arboreum. MS5 medium with maltose (3% w/v) or glucose (1.5% w/v) + maltose (1.5% w/v) performed better than that added single glucose (3% w/v) for somatic embryo maturation and germination. The regenerated plants with well-developed roots were directly transferred to the soil or grafted onto germinated cotton plants. Plant regeneration via somatic embryogenesis in diploid cultivated species was established, but there is a need for improvement and optimization for gene functional analysis and gene editing in the diploid cotton species. Regenerated plants of G. arboreum, a cultivated diploid cotton, were obtained via somatic embryogenesis by adjusting the culture mode and medium compositions.

Micropropagation and Cryopreservation of Yukon Draba (Draba yukonensis), a Special Concern Plant Species Endemic to Yukon Territory, Canada.

Yukon Draba (Draba yukonensis) is a small, short-lived perennial mustard species that is endemic to southwestern Yukon in Canada. This plant has been categorized as a species of Special Concern. It faces the threat of habitat loss due to natural and man-made causes and a population that is unevenly distributed to a few large and several small subpopulations in the area. It will therefore be judicious to undertake investigations on the conservation of this species to save it from further deterioration which may lead to its extinction. In this study, a protocol was developed for in vitro propagation and cryopreservation of Yukon Draba. The micropropagation protocol was optimized using shoot tips which enabled clonal propagation and in vitro storage of the species. Shoots grew best in the medium containing MS basal salts and had the highest multiplication with the addition of 2 µM 6-benzylaminopurine or 5 µM Kinetin with 3% sucrose. The addition of 10 µM Indole Butyric Acid (IBA) produced the highest number of adventitious roots on the shoots and the longest root length was observed at 2 µM IBA. The rooted plantlets were transferred to greenhouse and the highest survival (87.5%) was observed for the plantlets treated with a lower concentration of IBA (2 µM). Cryopreservation protocol was developed using the droplet-vitrification method for in vitro shoot tips. Two-week-old shoots had the highest survival and regrowth following exposure to plant vitrification solution 3 (PVS3) for 30 min, prior to direct immersion of the droplets into the liquid nitrogen. The optimized protocols for the micropropagation and cryopreservation may be useful for the long-term germplasm conservation and reintroduction of this species in its natural habitat.

In Vitro Regeneration of Muscari racemosum Mill. Using Twin Bulb Scales, Primary Bulbs, and Leaf Bases

Turkey is an important center of diversity for many plants species including bulbs, rhizomes, tubers, and other plants of high agricultural and horticultural importance. These species have a special importance as ornamental plants. However, due to urbanization and related factors, many of them are under threat. One of these species is the endemic Muscari racemosum Mill. The current study aimed to develop an efficient in vitro commercial bulblet propagation procedure using different explants. Twin-scale bulb explants were regenerated on MS medium having several doses of Kinetin+NAA (1-Naphthaleneacetic acid). The best regeneration was exhibited on 4.65 μM Kinetin+5.37 μM NAA at the end of 10 weeks with induction of 4.08 bulblets/explant with a mean diameter of 0.31 cm. The primary bulblets were cultured on MS medium having 18.60 μM Kinetin+5.37 μM NAA. About a 2.5-fold increase in the diameter of the bulbs (0.76 cm) was exhibited on the regenerated bulblets. The bulblets were regenerated on leaf bases taken from MS medium having several doses of BAP (6-Benzylaminopurine) + NAA. The regenerated bulbs were rooted on MS medium having 4.90 μM IBA (Indole-3-butyric acid) followed by their transference to a greenhouse for acclimatization. This study provided important information on commercial clonal propagation of M. racemosum and the importance of explants and growth regulators in plant regeneration.

Callus induction and chemical characterization of cell suspension cultures of jojoba (Simmondsia chinensis L.)

Jojoba (Simmondsia chinensis L.) oil is also known as liquid wax or fixed oil. It is an important metabolite of jojoba having commercial importance in cosmetics as well as a potential biofuel source. We presented an efficient system for in vitro establishment of cell suspension cultures (CSC) from proliferating friable calluses. For this purpose, cotyledon, internode, and leaf explants were cultured on MS medium + 1, 2, 4, 6, 8 or 10 µM 2, 4-Dichlorophenoxyacetic acid (2, 4-D), α-Naphthalene acetic acid (NAA) alone or in combination with 1 or 2 µM N6-benzylaminopurine (BAP) or Kinetin. Results demonstrated that 100% healthy, friable and variegated calluses were obtained on 8 µM, 10 µM 2, 4-D or 2, 4-D 10 µM + 2 µM BAP and represented as callus lines (CL) CL-1, CL-2 or CL-3, respectively, after 38 days. One-gram callus tissue per CL was then immersed in the respective liquid medium and agitated on an orbital shaker at 60-70 rpm under the growth room conditions (25 ± 2 °C, 16 h light period) for the preparation of CSC. After 15 days, CSC was sieved and large clumps were removed. Growth measurement of CSC was determined by cell counting, packed cell volume (PCV) and cell viability. The highest number of viable cells was obtained at 2.57 OD with CL-3, where PCV was highest (0.35 ml) on CL-1 of 38 days old calluses. 2,3,5-Triphenyltetrazolium chloride was a reliable approach for the determination of cell viability of CSC.