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.

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.

Old Catharanthus roseus culture (14 years) produced somatic embryos and plants and showed normal genome size; demonstrated an increased antioxidant defense mechanism; and synthesized stress proteins as biochemical, proteomics, and flow-cytometry studies reveal.

Various strategies have been developed globally to conserve germplasm by propagating plants. One important technique is in vitro propagation and preservation through tissue culture. In many investigated plants, the long in vitro conservation is plagued with several limitations like genetic variations, developmental errors in cells or tissues due to induced stress. This provoked us to conduct a study of Catharanthus roseus culture maintained for over fourteen long years and a newly established 8-month-old culture. The present study investigated and compared the two tissue types differing by their age. The biomass accumulation, the biochemical differences of the two, dead cell analysis with aging via confocal microscopy, and liquid chromatography-mass spectroscopy (LC-MS)-based proteomic differences were studied in old and newly established Catharanthus culture. The proteomic study reveals more than 120 upregulated or high abundance proteins in old culture as compared to newly established Catharanthus. The identified upregulated proteins are stress protein 69, heat shock proteins (HSP), isocitrate dehydrogenase, pyruvate dehydrogenase, and others. These proteins had an association with antioxidant activities, related to stress, and a few are linked to respiration. Our study reveals the presence of a robust antioxidant defense mechanism, i.e., 51.94%, 78.8%, and 61% higher SOD, APX, and CAT activities in older cultures (O) as compared to newly established tissues (N), which perhaps act against stress and may play a key role in ameliorating negative impacts of long-term in vitro conditions. The inherent strong antioxidant defense system in old cultures added resilience and enabled the culture to revive growth quickly (within 1-2 days) following transfer to new medium as compared to new culture (7-10 days). The biomass accumulation was more (37.08 %) in old tissues as compared to new culture. The 2C DNA or genome size of C. roseus especially the 14-year-old culture-derived regenerated plant was measured by flow cytometry. The 2C DNA size of this Catharanthus (old culture) plant is 1.516 pg, which is very similar to new culture-derived plants' and field-grown plants' genome size. No anomaly in genome size was noted in plants of old culture, as opposed to common perception.

Genes, proteins and other networks regulating somatic embryogenesis in plants.

BACKGROUND: Somatic embryogenesis (SE) is an intricate molecular and biochemical process principally based on cellular totipotency and a model in studying plant development. In this unique embryo-forming process, the vegetative cells acquire embryogenic competence under cellular stress conditions. The stress caused by plant growth regulators (PGRs), nutrient, oxygenic, or other signaling elements makes cellular reprogramming and transforms vegetative cells into embryos through activation/deactivation of a myriad of genes and transcriptional networks. Hundreds of genes have been directly linked to zygotic and somatic embryogeneses; some of them like SOMATIC EMBRYOGENESIS LIKE RECEPTOR KINASE (SERK), LEAFY COTYLEDON (LEC), BABYBOOM (BBM), and AGAMOUS-LIKE 15 (AGL15) are very important and are part of molecular network. MAIN TEXT (OBSERVATION): This article reviews various genes/orthologs isolated from different plants; encoded proteins and their possible role in regulating somatic embryogenesis of plants have been discussed. The role of SERK in regulating embryogenesis is also summarized. Different SE-related proteins identified through LC-MS at various stages of embryogenesis are also described; a few proteins like 14-3-3, chitinase, and LEA are used as potential SE markers. These networks are interconnected in a complicated manner, posing challenges for their complete elucidation. CONCLUSIONS: The various gene networks and factors controlling somatic embryogenesis have been discussed and presented. The roles of stress, PGRs, and other signaling elements have been discussed. In the last two-to-three decades' progress, the challenges ahead and its future applications in various fields of research have been highlighted. The review also presents the need of high throughput, innovative techniques, and sensitive instruments in unraveling the mystery of SE.

Cadmium chloride (CdCl2) elicitation improves reserpine and ajmalicine yield in Rauvolfia serpentina as revealed by high-performance thin-layer chromatography (HPTLC).

In vitro cultures play a promising role for production of pharmaceutically important plant secondary metabolites and the use of elicitation can mitigate the low productivity of active compounds. In the present study, the influence of cadmium chloride (CdCl2) elicitation on alkaloid yield was investigated in Rauvolfia serpentina. This heavy metal was employed to enhance the yield of reserpine and ajmalicine in leaf derived callus, leaves, stems and roots of in vitro grown cultures. Different concentrations [0.05 mM (C1), 0.10 mM (C2), 0.15 mM (C3) and 0.20 mM (C4)] of CdCl2 were added to the MS medium. The elicitor's influence on callus biomass, biochemical attributes and the yield of alkaloids was monitored at regular intervals. The amendment of CdCl2 improved growth and maximum callus biomass (1.29 g fresh weight and 0.16 g dry weight) was noted at 0.15 mM (C3) after 6 days of elicitation. The addition of elicitor in medium caused cellular stress and to analyse the role of CdCl2 in plant defence responses various antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities were measured in treated and non-treated cultures. The antioxidant enzyme activity increased linearly with elevated levels of CdCl2 in medium; highest APX (0.88 EU min-1 mg-1protein), SOD (5.40 EU min-1 mg-1protein) and CAT (4.21 EU min-1 mg-1protein) activity were observed in leaves of in vitro regenerated plants at C4. The quantitative analyses of reserpine and ajmalicine were conducted in different elicitated tissues using high-performance thin-layer chromatography (HPTLC) method. The study reveals enriched level of reserpine and ajmalicine in cultivated tissues and the enhancement was noted up to C3 (0.15 mM) elicitor level. Reserpine yield was maximum (0.191 mg g-1 DW) in roots of in vitro regenerated plants. The accumulation of ajmalicine was, however, better in leaf derived callus at C3 (0.131 mg g-1 DW). Higher elicitor dose (0.20 mM) inhibited callus biomass growth and subsequent alkaloid accumulation. The present study indicates the use of CdCl2 as a propitious method in enhancing reserpine and ajmalicine yield in R. serpentina.

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."

Essential oil yield estimation by Gas chromatography-mass spectrometry (GC-MS) after Methyl jasmonate (MeJA) elicitation in in vitro cultivated tissues of Coriandrum sativum L.

Coriandrum sativum is an important spice plant known for its unique fragrance. Coriander oil is also one of the major essential oils in world global market. The oil yield varies with different coriander varieties; and the content and quality of oil is governed by several factors. In recent times, a variety of technologies have been exploited to improve phyto-compounds including essential oils. In this present study, Methyl jasmonate (MeJA) was amended in medium and the yield of essential oil was measured and compared in different cultivating tissues. The cultured tissues were nonembryogenic callus and embryogenic tissues (induction, proliferation and maturation stages of embryos). MeJA acts as a signaling molecule in accumulating secondary metabolites. Four different MeJA treatments i.e. T1 = 50, T2 = 100, T3 = 150 and T4 = 200 µM, along with a control (T0) were used and the yield of coriander essential oil was estimated in different in vitro cultivating tissues by using Gas chromatography-mass spectrometry (GC-MS). The addition of MEJA enriched essential oil yield, maximum oil being in maturation stage of embryos at T3 (150 µM). Other added treatments also had varied stimulatory role. The addition of MeJA induced stress as the stress marker enzymes like superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) content were high compared to non treated tissue (T0). In T4, the CAT activity was maximum i.e. 5.83 and 6.28 mg-1 protein min-1 in Co-1 and RS respectively in matured somatic embryos. The SOD activity was also high at maturation stage of embryos at T4 (5.3 mg-1 protein min-1 in RS). The APX activity on the other, was high (3.32 mg-1 protein min-1) in induction stage of embryogenesis at T3. The comparative biochemical (sugar, protein and proline) analyses of tissues were performed and presented that had high and low essential oil. MeJA induced stress may help in accumulating essential oils in C. sativum.