Phytocompounds and Regulation of Flavonoids in In Vitro-Grown Safflower Plant Tissue by Abiotic Elicitor CdCl2.

In this study, a Gas chromatography-mass spectrometry (GC-MS) investigation of embryogenic callus and somatic embryo regenerated shoots of Carthamus tinctorius revealed the presence of a variety of sugars, sugar acids, sugar alcohols, fatty acids, organic acids, and amino acids of broad therapeutic value. The in vitro developed inflorescence contained a wide range of active compounds. In embryogenic calluses, important flavonoids like naringenin, myricetin, kaempferol, epicatechin gallate, rutin, pelargonidin, peonidin, and delphinidin were identified. To augment the synthesis of active compounds, the effect of cadmium chloride (CdCl2) elicitation was tested for various treatments (T1-T4) along with a control (T0). Varying concentrations of CdCl2 [0.05 mM (T1), 0.10 mM (T2), 0.15 mM (T3), and 0.20 mM (T4)] were added to the MS medium, and flavonoid accumulation was quantified through ultra-high-pressure liquid chromatography-tandem mass spectroscopy (UHPLC-MS/MS). The flavonoids naringenin, kaempferol, epicatechin gallate, pelargonidin, cyanidin, and delphinidin increased by 6.7-, 1.9-, 3.3-, 2.1-, 1.9-, and 4.4-fold, respectively, at T3, whereas quercetin, myricetin, rutin, and peonidin showed a linear increase with the increase in CdCl2 levels. The impacts of stress markers, i.e., ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD), on defense responses in triggering synthesis were also evaluated. The maximum APX and SOD activity was observed at T3, while CAT activity was at its maximum at T2. The impact of elicitor on biochemical attributes like protein, proline, sugar, and malondialdehyde (MDA) content was investigated. The maximum protein, proline, and sugar accumulation was noted at high elicitor dose T4, while the maximum MDA content was noted at T3. These elevated levels of biochemical parameters indicated stress in culture, and the amendment of CdCl2 in media thus could be a realistic approach for enhancing secondary metabolite synthesis in safflower.

Genome size and gas chromatography-mass spectrometry (GC-MS) analysis of field-grown and in vitro regenerated Pluchea lanceolata plants.

Pluchea lanceolata is a threatened pharmacologically important plant from the family Asteraceae. It is a source of immunologically active compounds; large-scale propagation may offer compounds with medicinal benefits. Traditional propagation method is ineffective as the seeds are not viable; and root sprout propagation is a slow process and produces less numbers of plants. Plant tissue culture technique is an alternative, efficient method for increasing mass propagation and it also facilitate genetic improvement. The present study investigated a three-way regeneration system in P. lanceolata using indirect shoot regeneration (ISR), direct shoot regeneration (DSR), and somatic embryo mediated regeneration (SER). Aseptic leaf and nodal explants were inoculated on Murashige and Skoog (MS) medium amended with plant growth regulators (PGRs), 2,4-dichlorophenoxy acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), and 6-benzyl amino purine (BAP) either singly or in combinations. Compact, yellowish green callus was obtained from leaf explants in 1.0 mg/l BAP (89.10%) added medium; ISR percentage was high, i.e., 69.33% in 2.0 mg/l BAP + 0.5 mg/l NAA enriched MS with 4.02 mean number of shoots per callus mass. Highest DSR frequency (67.15%) with an average of 5.62 shoot numbers per explant was noted in 0.5 mg/l BAP added MS medium. Somatic embryos were produced in 1.0 mg/l NAA fortified medium with 4.1 mean numbers of somatic embryos per culture. On BAP (1.0 mg/l) + 0.5 mg/l gibberellic acid (GA3) amended medium, improved somatic embryo germination frequency (68.14%) was noted showing 12.18 mean numbers of shoots per culture. Histological and scanning electron microscopic (SEM) observation revealed different stages of embryos, confirming somatic embryogenesis in P. lanceolata. Best rooting frequency (83.95%) of in vitro raised shootlets was obtained in 1.0 mg/l IBA supplemented half MS medium with a maximum of 7.83 roots per shoot. The regenerated plantlets were transferred to the field with 87% survival rate. The 2C genome size of ISR, DSR, and SER plants was measured and noted to be 2.24, 2.25, and 2.22 pg respectively, which are similar to field-grown mother plant (2C = 2.26 pg). Oxidative and physiological events suggested upregulation of enzymatic activities in tissue culture regenerated plants compared to mother plants, so were photosynthetic pigments. Implementation of gas chromatography-mass spectrometry (GC-MS) technique on in vivo and in vitro raised plants revealed the presence of diverse phyto-chemicals. The yields of alpha amyrin and lupeol (medicinally important triterpenoids) were quantified using high-performance thin-layer chromatography (HPTLC) method and enhanced level of alpha amyrin (2.129 µg g-1 dry wt) and lupeol (1.232 µg g-1 dry wt) was noted in in vitro grown leaf tissues, suggesting in vitro conditions act as a potential trigger for augmenting secondary metabolite synthesis. The present protocol represents a reliable mass propagation technique in producing true-to-type plants of P. lanceolata, conserving 2C DNA and ploidy successfully without affecting genetic homogeneity.

Elicitation Induced α-Amyrin Synthesis in Tylophora indica In Vitro Cultures and Comparative Phytochemical Analyses of In Vivo and Micropropagated Plants.

Tylophora indica (Burm. f.) Merrill is an endangered medicinal plant that possesses various active agents, such as tylophorinine, kaempferol, quercetin, α-amyrin and beta-sitosterol, with multiple medicinal benefits. α-amyrin, a triterpenoid, is widely known for its antimicrobial, anti-inflammatory, gastroprotective and hepatoprotective properties. In this study, we investigated the metabolite profiling of tissues and the effects of cadmium chloride and chitosan on in vitro accumulation of alkaloids in T. indica. First, the callus was induced from the leaf in 2,4-D-, NAA- and/or BAP-fortified MS medium. Subsequent shoot formation through organogenesis and in vitro roots was later induced. Gas chromatography-mass spectrometry (GC-MS)-based phytochemical profiling of methanolic extracts of in vivo and in vitro regenerated plants was conducted, revealing the presence of the important phytocompounds α-amyrin, lupeol, beta-sitosterol, septicine, tocopherol and several others. Different in vitro grown tissues, like callus, leaf and root, were elicited with cadmium chloride (0.1-0.4 mg L-1) and chitosan (1-50 mg L-1) to evaluate the effect of elicitation on α-amyrin accumulation, measured with high-performance thin layer chromatography (HPTLC). CdCl2 and chitosan showed improved sugar (17.24 and 15.04 mg g-1 FW, respectively), protein (10.76 and 9.99 mg g-1 FW, respectively) and proline (7.46 and 7.12 mg g-1 FW), especially at T3 (0.3 and 25 mg L-1), in the leaf as compared to those of the control and other tissues. The antioxidant enzyme activities were also evaluated under an elicitated stress situation, wherein catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) displayed the highest activities in the leaf at T4 of both of the two elicitors. The α-amyrin yield was quantified with HPTLC in all tested tissues (leaf, callus and root) and had an Rf = 0.62 at 510 nm wavelength. Among all the concentrations tested, the T3 treatment (0.3 mg L-1 of cadmium chloride and 25 mg L-1 of chitosan) had the best influence on accumulation, irrespective of the tissues, with the maximum being in the leaf (2.72 and 2.64 µg g-1 DW, respectively), followed by the callus and root. Therefore, these results suggest future opportunities of elicitors in scaling up the production of important secondary metabolites to meet the requirements of the pharmaceutical industry.

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.

Internal and External Regulatory Elements Controlling Somatic Embryogenesis in Catharanthus: A Model Medicinal Plant.

Somatic or in vitro embryogenesis is a unique embryo producing process from vegetative cells observed in plants since 1958. Even over 60 years of research, the transition of somatic cells into embryonic fate is still not elucidated fully. Various networks and signaling elements have been noted to play important role in this "vegetative to reproductive" transition process. The networks include genotypes, explant types, the sugar/carbohydrate sources, cultural/environmental conditions like light quality and intensity, dissolved oxygen (DO) level, cell density, plant growth regulator (PGR) (auxin and cytokinin) signaling, PGR-gene interplay, stresses are important and cause new cellular reprogramming during embryonic acquisition. A wide array of genes, specific to zygotic embryogenesis, also express during somatic embryogenesis. A few embryogenesis-specific genes such as SOMATIC EMBRYOGENESIS LIKE RECEPTOR KINASE, LEAFY COTYLEDON, AGAMOUS-LIKE 15, and BABY BOOM are crucial and have been discussed. The chapter focuses the importance of these gene products, e.g., proteins, enzymes, and transcription factors in regulating embryogenesis. Many of these encoded proteins act as potential somatic embryogenesis markers. Besides, important elements such as genotype, herbaceous/woody plants' response in culture in inducing embryos have been discussed. All these elements are connected and form network in complex fashion thus difficult to unfold fully; some of the current progress and developments have been presented in this chapter.

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill.

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP + 1.4 µM GA3 enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica. Supplementary Information: The online version contains supplementary material available at 10.1007/s11240-022-02254-z.

Direct somatic embryogenesis and flow cytometric assessment of ploidy stability in regenerants of Caladium × hortulanum 'Fancy'.

Caladium × hortulanum 'Fancy' is an important ornamental plant grown in pots and landscapes and known for its colorful leaves often used for interior decorations. In this work, we present a method of in vitro regeneration from three explants source through direct somatic embryogenesis (DSE) wherein the regenerated plants were screened for ploidy changes through flow cytometry analysis. Tuber, leaf and petiole explants were cultured on MS basal medium supplemented with 1-napthalene acetic acid (NAA), 6-benzyl amino purine (BAP) and N-phenyl-N'-1, 2,3-thiadiazol-5-ylurea (TDZ) concentrations. Tuber explants induced highest direct somatic embryos on NAA (1 mg L- 1) + BAP (0.5 mg L- 1) with 55.6 mean number of embryos per explant while as leaf and petiole explants amended with 1 mg L- 1 TDZ developed 18.7 and 12.27 mean number of embryos per explant respectively. The highest embryo conversion frequency was achieved on BAP (2 mg L- 1) + NAA (0.2 mg L- 1) with 44.2, 18.7 and 7.5 mean number of plantlets produced per tuber, leaf and petiole explant respectively after 4 weeks of culture. Plantlets were later rooted and maximum number of roots (6.33) per shoot was achieved on 2 mg L- 1 indolebutyric acid amended medium. Description of the process of DSE is presented through the histological and SEM evidences. The 2C DNA content of field grown plants and the DSE regenerants evaluated under flow cytometric analysis were 8.06 pg and 8.28 pg respectively showing no ploidy changes. Hence, a successful protocol of inducing direct somatic embryos from three explant types with efficient embryo conversion frequency was obtained with regenerants showing similar DNA ploidy as that of their parent plants.

Shotgun label-free proteomic and biochemical study of somatic embryos (cotyledonary and maturation stage) in Catharanthus roseus (L.) G. Don.

Somatic embryogenesis is an important and wonderful biotechnological tool used to develop whole plant from a single or a group of somatic cells. The differentiated somatic cells become totipotent stem cells by drastic reprogramming of a wide range of cellular activities, leading to the acquisition of embryogenic competence. After acquiring competence, the cells pass through globular, heart, torpedo and cotyledonary stages of embryo; however, all advanced embryos do not convert into full plant, produce adventive embryos or callus instead, thus reverses the programming. This is a big limitation in propagation of many plants. Understanding and unraveling the proteins at this 'embryo to plantlet' transition stage will help to get more numbers of plants. Thus, our study was aimed at an identification of differentially abundant proteins between two important advanced stages, i.e. cotyledonary-(T1) and maturation stage (T2) of somatic embryos in Catharanthus roseus. A total of 2949 and 3030 proteins were identified in cotyledonary and maturation stage, respectively. Of these, 1129 proteins were common to both. Several proteins were found to be differentially accumulated in two different embryo stages in which over 60 proteins were most accumulated during somatic embryo maturation time. More chlorophyll accumulation was noted at this time under the influence of gibberellic acid (GA3). Proteins like Mg-protoporphyrin IX chelatase, chlorophyll a-b-binding protein, photosystem I iron-sulfur center, photosystem II Psb, photosystem II subunit P-1, P-II domain-containing protein, RuBisCO large chain, RuBisCO small chain, RuBisCO activase, RuBisCO large subunit-binding proteins were synthesized. Some of the identified proteins are linked to chlorophyll synthesis, carbohydrate metabolism and stress. The identified proteins are categorized into different groups on the basis of their cellular location, role and other metabolic processes. Biochemical attributes like protein, sugar, proline, antioxidant enzyme (APX, SOD and CAT) activities were high in T2 as compared to T1. The proteins like peroxidases, pathogenesis-related proteins, the late-embryogenesis abundant proteins, argonaute, germin and others have been discussed in C. roseus somatic embryo maturation process.

Mass propagation through direct and indirect organogenesis in three species of genus Zephyranthes and ploidy assessment of regenerants through flow cytometry.

Genus Zephyranthes consists of economically important plant species due to their high ornamental value and presence of valuable bioactive compounds. However, this genus propagates by asexual division only which gives slow propagation rate. Plant tissue culture has the potential to provide efficient techniques for rapid multiplication and genetic improvement of the genus. In this work, a dual in vitro regeneration system through callus mediated shoot regeneration and direct shoot regeneration in species Zephyranthes candida, Zephyranthes grandiflora and Zephyranthes citrina was investigated. Bulb, leaf and root explants were cultured on Murashige and Skoog (MS) medium amended with different plant growth regulators (PGR's) viz. 2,4-dichlorophenoxyacetic acid (2,4-D), 1-Naphthalene acetic acid (NAA), 6-benzyl amino purine (BAP), N-phenyl-N'-1,2,3 -thiadiazol-5-ylurea (TDZ), 6-Furfuryl- aminopurine (KIN) alone or in combinations for callus induction and regeneration. Only bulb explants showed callus induction and regeneration response on different PGR combinations with a varied response in callus induction percentage, callus color and callus texture. Creamish compact callus (CC) was induced on 2 mg L[Formula: see text] 2,4-D, brown friable callus (BF) on 2 mg L[Formula: see text] NAA + 1 mg L[Formula: see text] BAP and green friable callus (GF) callus on 1 mg L[Formula: see text] KIN + 3 mg L[Formula: see text] NAA. The maximum shoot multiplication from different callus types (indirect organogenesis) was achieved on 2 mg L[Formula: see text] BAP alone without combinations. Bulb explants of Z. grandiflora induced maximum callus induction percentage (86.4%) and shoot regeneration percentage (83.5%) with the maximum 08 shoots per 150 mg callus mass. The induction and regeneration response was followed in the order of Z. grandiflora > Z. candida > Z. citrina. Similarly, maximum direct organogenesis from bulb explants was obtained in Z. grandiflora (93.3%) followed by Z. candida (91.5%) and Z. citrina (90.4%) on 3 mg L[Formula: see text] TDZ amended MS media. Adventitious root induction was achieved on 2 mg L[Formula: see text] IBA with a maximum of 8 roots per shoot. The in vitro raised plantlets were successfully acclimatized in the field with 85% survival efficiency. The genome size (2C DNA content) of the field-grown plants and in vitro regenerated plants, evaluated through flow cytometry technique, were similar and showed no ploidy changes. An efficient mass propagation protocol was established for obtaining plants with unaltered genome size in the three species of Zephyranthes.

Genome size analysis of field grown and somatic embryo regenerated plants in Allium sativum L.

In the present study, an efficient in vitro propagation protocol has been developed from clove explants of Allium sativum L., one of the oldest vegetable and medicinal plant used worldwide. Garlic is propagated vegetatively as cross-fertilization is strictly precluded due to sterile flowers. Due to a low rate of multiplication, limited genetic improvement possibility and increased germplasm degradation, plant tissue culture becomes an efficient and preferred tool for quality and rapid propagation of garlic. Here, the clove explants were cultured on Murashige and Skoog basal medium amended with different concentrations of Plant Growth Regulators (PGRs) namely 2,4-dichlorophenoxy acetic acid (2,4-D), 6-benzyl amino purine (BAP), and 1-naphthalene acetic acid (NAA). Within 2 weeks of inoculation, white compact callus was formed, maximum callus induction frequency (85.99%) was on 1.5 mg l-1 2, 4-D added MS medium. Induced callus transformed into an embryogenic callus on 2, 4-D and BAP amended MS medium with highest embryogenic frequency (77.7%) was noted on 0.25 mg l-1 2, 4-D and 1.0 mg l-1 BAP added medium. Embryogenic callus differentiated into progressive stages of somatic embryos starting from globular, scutellar, and finally to coleoptilar stage of the embryo. Histological and scanning electron microscopic study of embryogenic callus was conducted, showing different stages of embryos, their origin and development, re-confirming somatic embryogenesis incidence in A. sativum. Green and mature somatic embryos were germinated and converted into plantlets on 0.5 mg l-1 BAP amended MS medium. The in vitro regenerated plants were cultured separately in IBA and NAA supplemented media for root induction. The MS medium amended with 1.0 mg l-1 IBA proved to be the best PGR treatment in inducing roots. The rooted plants were acclimatized and transferred ex vitro with about 87% survival rate. Cytological and flow cytometric analyses were performed to assess the genetic stability of in vitro regenerated plants. Cytological studies of in vitro regenerated plants showed 2n = 16 chromosome number and did not reveal any numerical variation in chromosomes. Flow cytometry was employed to measure the 2C DNA content of somatic embryo regenerated A. sativum plants and compared with in vivo grown garlic. The histogram peaks of relative 2C DNA content of in vitro regenerated plantlets were similar to the corresponding 2C DNA peak of in vivo grown plants. Flow cytometric 2C DNA content of embryo regenerated and field-grown A. sativum plants were the same, i.e., 33.45 pg and 33.56 pg, respectively, confirming genetic similarity. In conclusion, the present cytological and flow cytometric study suggest that the in vitro culture conditions are quite safe, did not encourage genetic alterations, and regenerants were "true to type."