Highest Regeneration Frequency Research Articles

Assessment of clonal fidelity and phytomedicinal potential in micropropagated plants of Bulbophyllum odoratissimum - An endangered medicinal orchid of Indo Burma megabiodiversity hotspot

Bulbophyllum odoratissimum (Sm.) Lindl. ex Wall. is widely used as traditional medicine for the treatment of tuberculosis, chronic inflammation and is listed as an endangered orchid due to over-exploitation and habitat destruction. The present study was carried out to establish a genetically stable in vitro regeneration protocol for mass multiplication using nodal segments and to determine phytochemical constituents, the antioxidant capacity of different parts (leaf, pseudobulb and root) of mother plants and acclimatized in vitro raised plants of B. odoratissimum. The synergistic effect of BA (4.0 mgl−1) and IBA (0.5 mgl−1) was found to be optimum for producing maximum shoot proliferation. IBA was found to be more effective for root proliferation than IAA, NAA and an average (3.08 roots/shoot) was obtained in 0.5 mgl−1 IBA. The well rooted plantlets were successfully acclimatized (more than 90% survival rate) in greenhouse, exhibiting normal development. The ISSR banding profiles confirmed high degree of genetic homogeneity among the clones. Extraction of all plant material from both mother plant and in vitro raised plant possess relevant TPC, TFC, radical scavenging activity (DPPH and ABTS) as well as total reducing power ability. HPLC chromatogram exhibited that phytochemical compounds detected in regenerated plants were similar to those present in mother plant and there was no remarkable difference. High regeneration frequency along with its genetic stability and secondary metabolite production validate the efficiency of this micropropagation protocol of B. odoratissimum for species recovery program as well as a potential source of commercial application.

Interspecific hybridization between Ganoderma lingzhi and G. applanatum through protoplast fusion.

Interspecific hybridization between Ganoderma lingzhi and G. applanatum was attempted through polyethylene glycol (PEG) induced fusion technique. The protoplast isolation procedure was simplified, and we obtained a significant number of protoplasts from both Ganoderma species. The number of protoplasts obtained was 5.27 ± 0.31 × 107/mL in G. lingzhi and 5.57 ± 0.49 × 106/mL in G. applanatum. Osmotic stabilizer NaCl (0.4M) at pH 5.8 and enzymolysis time 3.5h have supported high frequency of protoplast regeneration. G. lingzhi and G. applanatum regeneration frequency was 1.73 ± 0.04% and 0.23 ± 0.02%, respectively. 40% of PEG induced high number of protoplast fusion the regeneration frequency was 0.09% on a minimal medium. Two hundred fifty-two fusant colonies were isolated from the following four individual experiments. Among them, ten fusants showed the mycelial morphological difference compared to their parents and other fusant isolates. The fruiting body could be generated on oak sawdust and wheat bran substrate, and a few of them showed recombined morphology of the parental strains. The highest yield and biological efficacy (BE) were recorded in GF248, while least in GF244. The hybridity of the fusant was established based on mycelia, fruiting morphology, and PCR fingerprinting. ISSR and RAPD profile analysis of ten fusants and parents depicted that fusants contained polymorphic bands, which specified the rearrangement and deletion of DNA in the fusants. A Dendrogram was constructed based on the RAPD profile, and the clustering data exhibited two major clusters: cluster I included the G. lingzhi and Cluster II, including the G. applanatum and fusant lines. Total polysaccharide (α, β and total glucan) content was compared with fusants and parental strains. The present study highlighted the efficient methods for protoplast isolation from Ganoderma species. PEG-induced fusants showed high polymorphic frequency index, while the phenotypic characters showed high similarity to G. applanatum. A significant difference was observed in the mushroom yield and its total polysaccharide between the fusants and parental strains.

IN VITRO CLEANING UP AND MICROPROPAGATION OF PERS PECTIVE CHRYSANTHEMUM CULTIVARS AND NEW HYBRID FORMS OF THE NIKITA BOTANICAL GARDENS' BREEDING

An in vitro micropropagation protocol has been developed for garden chrysanthemum cultivars Mokroe Serebro, Predrassvetnyiy Ayu-Dag, Eldorado, and Ermitage of the NВG's breeding. 10-20 mg/1 of ribavirin promoted the primary explants cleaning up. А high frequency of regeneration of adventitious buds and microshoots was observed in cultivars Eldorado and Ermitage (21.0 and 23.5 microshoots/explant) cultured on MS medium with 0.75 mg/1 kinetin and Mokroe Serebro - on MS medium with 0.75 mg/1 ВАР (24.5 microshoots/explant). Spontaneous root formation facilitated 100% rooting of microshoots.

High-frequency regeneration of plants in vitro from seedling-derived apical bud explants of Tilia mandshurica Rupr. & Maxim

High-frequency regeneration of plants <i>in vitro</i> from seedling-derived apical bud explants of <i>Tilia mandshurica</i> Rupr. &amp; Maxim

Establishment of an efficient shoot regeneration system in vitro in Brassica rapa

To improve the genetic transformation system for Brassica rapa L., we established a high-efficiency shoot regeneration protocol. A double haploid line ‘CX001’ with strong regeneration ability was identified from among 25 genotypes. Cotyledons from 4-d-old ‘CX001’ exhibited high-frequency (77.14%) shoot regeneration when explants were cultured on Murashige and Skoog (MS) basal medium supplemented with 1.0 mg L−1 6-benzylaminopurine (BA) and 0.5 mg L−1 α-naphthaleneacetic acid (NAA). The highest regeneration frequency (85.40%) and the maximum shoots number per explant (1.79) were achieved on medium containing 2.0 mg L−1 BA and 0.5 mg L−1 NAA. The addition of 2.5 mg L−1 AgNO3 was critical to shoot regeneration of the cotyledonal explants. The highest rooting rate was obtained on MS medium supplemented with 0.1 mg L−1 NAA. Flow cytometric analysis indicated that there was no ploidy variation between the mother plant and the regenerated plants. Regenerated plantlets with healthy roots were domesticated and transplanted into pots under greenhouse conditions. A reliable and efficient shoot regeneration protocol was established, which lays the foundation for genetic transformation in Brassica rapa.

Development of Interspecific Hybrids (Abelmoschus Esculentus × A. Tetraphyllus) in Okra Using Embryo Rescue Approach

Undomesticated related species are the rich stock of genes providing resistance to various diseases, pests and unfavourable environmental conditions. The sexual incompatibilities are bottleneck in introgression of such genes from wild species to popular varieties. The present study was executed to standardize a protocol of embryo rescue so to obtain the hybrids of Abelmoschus esculentus × A. tetraphyllus IC141017. Crosses were made between four (Arka Anamika, Pusa Makhmali, Parbhani Kranti, Jammu Okra-05) cultivated varieties of okra (A. esculentus) and a wild species (A. tetraphyllus IC141017) in reciprocal manner. Out of the four popular cultivars used in crossing, fruit set was recorded in Arka Anamika, Pusa Makhmali, Parbhani Kranti only when A. tetraphyallus IC141017 was used as a male parent. Fruit showed distal cracking after 25 d of pollination. The seeds appeared healthy for up to 15 d after pollination and later shrivelled and became pale yellow. Cross combinations, viz., Parbhani Kranti × A. tetraphyllus IC141017, Arka Anamika × A. tetraphyllus IC141017 and Pusa Makhmali × A. tetraphyllus IC 141017 showed the highest shoot regeneration on MS media containing 0.5 mg l-1 NAA + 1.0 mg l-1 IBA. Greatest number of shoots per explant (0.92±0.12) as well as the highest frequency of shoot regeneration (86±0.12%) was reported in Parbhani Kranti × A. tetraphyllus IC141017. Arka Anamika × A. tetraphyllus IC 141017 (86±0.12%) and Pusa Makhmali × A. tetraphyllus IC 141017 (82±0.13%) resulted in high frequency of shoot regeneration on MS media containing 0.5 mg l-1 NAA + 1.0 mg l-1 IBA. It can be inferred that MS media containing on 0.5 mg l-1 NAA + 1.0 mg l-1 IBA showed positive results in regeneration of interspecific hybrids of okra.

Genetic fidelity of regenerated plants via shoot regeneration of muskmelon by inter simple sequence repeat and flow cytometry

A new and efficient method of direct shoot proliferation was developed here in two muskmelon genotypes ( Cucumis melo L.), including ‘Khatooni’ and ‘Sooski Sabz’ (100%) using 4-day-old cotyledonary explants in vitro . The regeneration of muskmelon was evaluated on Murashige and Skoog (MS) medium supplemented separately with 17 treatments as combinations of two auxins (1-Naphthaleneacetic acid [NAA] and Naphthoxyacetic acid [NOA]) and four cytokinins (Benzylaminopurine [BAP], Thidiazuron [TDZ], Kinetin [KIN], and N-(2-Chloro-4-pyridyl)-N′-phenylurea [CPPU]). The treatments were experimented on 4- and 8-day-old cotyledon explants. A significant degree of organogenesis occurred in 4-day-old cotyledons (P < 0.01) than in 8-day-old cotyledons and shoots grew significantly (P < 0.01) by increasing the concentration of cytokinins in the media. The highest regeneration frequency was obtained in media supplemented with 0.6 mg/l BAP and 0.025 mg/l NOA. Derivatives were transferred to the greenhouse after complete adaptation. For the first time in muskmelon, genetic fidelity was evaluated in mother plants and in regenerated plants by inter simple sequence repeat (ISSR) markers. Ten primers amplified 112 well-resolved bands with an average of 11 bands per primer and ranged from 7 to 13 bands. According to the results of molecular analysis, no genetic variation was observed among the ex vitro and mother plants. The stability of the ploidy level through plant regeneration was verified by a flow cytometry analysis which indicated monomorphic patterns in the regenerated and mother plants. More than other treatments, the combination of BAP and NOA appeared more suitable for the tissue culture of muskmelon.

Variability in somatic embryo-forming capacity of spinach

High variability in somatic embryo (SE)-forming capacity has previously been observed in several spinach cultivars. Such variability frequently accounted for more variation in embryogenic response of the explants than the factor being investigated. Hence, the variability in embryogenic capacity was examined in the present study at both the population and the single-seedling level, using seeds of spinach cultivar Matador obtained from nine European seed companies. Seed population obtained from Slovenia (Sl) was superior to others, with the highest regeneration frequency (100%) and the highest mean SE number (14.4). A total of 82% of these seedlings had 80–100% of regenerating explants, while in populations with intermediate embryogenic capacity approximately 40% of seedlings had 20–60% of regenerating explants. The explants from the majority of seedlings (52–100%) in the least responsive populations were irresponsive. Furthermore, the explants from Sl seedlings regenerated from 10–20 (43.5%) up to > 20 (27.6%) SEs on average, while the explants from the majority of seedlings belonging to other populations regenerated 1–10 SEs. The present study strongly indicates that the variability of plant material must not be overlooked, because choosing more responsive individuals for one treatment and less responsive ones for another may lead to misinterpretation of the data.

Expression of some Epigenetic-Related Genes in Regenerated Shoots of Prunus persica

Establishment of an efficient adventitious shoot regeneration system for mature embryo of Prunus persica via shoot organogenesis has been achieved. Three different combinations of plant growth regulators (PGRs) have been applied in the current study; 6-Benzyl adenine (BA) or thidiazuron (TDZ) with Indole acetic acid (IAA) or Indole butyric acid (IBA). The high regeneration frequency was illustrated by 1.5 mg/l BA with 0.5 mg/l IAA combinations. Quantitative expression of eight epigenetic regulatory genes; Chromomethylase 3 (CMT3), Domains Rearranged Methyltransferase2 (DRM2), Fertilization Independent Endosperm (FIE2) and Chromodomain Protein Like Heterochromatin Protein1 (LHP1), Pickle (PKL), Proliferating Cell Nuclear Antigen (PCNA), Shoot Meristemless (STM) and DICER-LIKE1 (DCL1) have been estimated in the peach regenerated shoots. We found that the auxins and cytokinins in different combinations especially BA effect on the expression of some epigenetic genes in relation to the mature embryo.

Direct organogenesis mediated improvised mass propagation of Pogostemon cablin: A natural reserve of pharmaceutical biomolecules

Pogostemon cablin (Blanco) Benth. or “Patchouli” is a prized medicinal herb used in various ethno-medicinal preparations and perfume industry. However due to over-collection and nematode infections in the roots of P. cablin, the wild populations are facing a steady decline and are at risk. In the present research, we report a fast clonally stable regeneration protocol for P. cablin with the utilization of meta-topolin (mT), a prized aromatic cytokinin (CK) for the first time. The highest rate of shoot proliferation i.e. 45 shoots/explant was observed after 4 weeks from leaf segments on MS media fortified with 1.0 mg/l mT. The highest rate of rooting was observed when the regenerated micro shoots were cultured in MS medium supplemented with 1.5 mg/l IBA. Direct root regeneration was tested upon with the applications of three conventional auxins i.e., Naphthalene acetic acid (NAA), Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA) using the same explants. The genetic homogeneity of the regenerated clones was ascertained, using two gene targeted molecular marker systems namely Start Codon Targeted Polymorphism (SCoT) and Inter-Retrotransposon Amplified Polymorphism (IRAP) ascertaining genetic stability amongst the regenerates and the donor plant. In precise, the present research provides vital insights into the role of aromatic CKs like mT in the high frequency regeneration of medicinally important plants like P. cablin which might serve as important baseline information for the pharma industry where supply of Quality Planting Material (QPM) is a real challenge.

Development of Interspecific Hybrids (Abelmoschus Esculentus × A. Tetraphyllus) in Okra Using Embryo Rescue Approach

ABSTRACT: Undomesticated related species are the rich stock of genes providing resistance to various diseases, pests and unfavourable environmental conditions. The sexual incompatibilities are bottleneck in introgression of such genes from wild species to popular varieties. The present study was executed to standardize a protocol of embryo rescue so to obtain the hybrids of Abelmoschus esculentus × A. tetraphyllus IC141017. Crosses were made between four (Arka Anamika, Pusa Makhmali, Parbhani Kranti, Jammu Okra-05) cultivated varieties of okra (A. esculentus) and a wild species (A. tetraphyllus IC141017) in reciprocal manner. Out of the four popular cultivars used in crossing, fruit set was recorded in Arka Anamika, Pusa Makhmali, Parbhani Kranti only when A. tetraphyallus IC141017 was used as a male parent. Fruit showed distal cracking after 25 d of pollination. The seeds appeared healthy for up to 15 d after pollination and later shrivelled and became pale yellow. Cross combinations, viz., Parbhani Kranti × A. tetraphyllus IC141017, Arka Anamika × A. tetraphyllus IC141017 and Pusa Makhmali × A. tetraphyllus IC 141017 showed the highest shoot regeneration on MS media containing 0.5 mg l-1 NAA + 1.0 mg l-1 IBA. Greatest number of shoots per explant (0.92±0.12) as well as the highest frequency of shoot regeneration (86±0.12%) was reported in Parbhani Kranti × A. tetraphyllus IC141017. Arka Anamika × A. tetraphyllus IC 141017 (86±0.12%) and Pusa Makhmali × A. tetraphyllus IC 141017 (82±0.13%) resulted in high frequency of shoot regeneration on MS media containing 0.5 mg l-1 NAA + 1.0 mg l-1 IBA. It can be inferred that MS media containing on 0.5 mg l-1 NAA + 1.0 mg l-1 IBA showed positive results in regeneration of interspecific hybrids of okra.

Direct regeneration and genetic fidelity analysis of regenerated plants of Andrographis echioides (L.) - An important medicinal plant

There are several studies about the phytochemical investigations and pharmacological activities of Andrographis echioides (L.), but there is scanty information about its in vitro propagation. Hence the present study is undertaken for in vitro micropropagation of Andrographis echioides (L.) through direct organogenesis. Initially, aseptic leaf explants of in vitro seedlings were excised and inoculated on to plant growth regulators (PGR’s) supplemented in the Murashige and Skoog (MS) media. Among the individual and combinations of PGR’s tested, the combination of thidiazuron (TDZ) along with indole-3-acetic acid (IAA) at 0.5 and 0.2 mg/L concentrations respectively, was proved to be best for high frequency (24.48 ± 1.54) of direct shoot regeneration of A. echioides. Rooting of in vitro raised shoots with the highest rooting percent of 90.42 was carried on IAA (1.0 mg/L), and indole-3-butyric acid (IBA) (1.0 mg/L) amended MS media. in vitro plantlets of A. echioides were hardened in poly cups possessing a 1:2 ratio of compost and garden soil. Initially, hardened plantlets were maintained in a greenhouse for four weeks by irrigating with half-strength MS salt solution and later transferred to field conditions with 80 % survivability. Genetically clonal nature of regenerated plantlets was confirmed by carrying polymerase chain reaction (PCR) based molecular analysis using inter simple sequence repeats (ISSR) and ribulose bisphosphate carboxylase large chain (rbcLa) primers.

High frequency regeneration of plants via callus-mediated organogenesis from cotyledon and hypocotyl cultures in a multipurpose tropical tree (Neolamarkia Cadamba)

In this works, a simple, efficient and repeatable protocol was developed for in vitro regeneration via callus-mediated organogenesis of Neolamarkia Cadamba using cotyledonary petioles and hypocotyls. Effects of basal medium, plant growth regulators, the types and age of explant on the formation of adventitious buds/shoots were studied. Meanwhile, histological analysis for early ontogenic stages and genetic stability assessment by flow cytometry were investigated. Our investigation demonstrated that, compared with 6-benzyladenine (BA), N6-(2-isopentenyl) adenine (2-ip), Thidiazuron (TDZ) was the optimal cytokinin for buds/shoots induction on cotyledon and hypocotyl explants. Douglas-fir and sugar pine medium (DCR) supplemented with 22.7 μM TDZ and 0.27 μM α-naphthalene acetic acid (NAA) was most effective on bud induction, with the highest bud-induction rate and numbers of buds on cotyledon and hypocotyl explants. The available shoot per explant hit 35.2 when the induced callus sub-cultured to a medium without TDZ. It was found that TDZ could promote induction of the callus and the buds, however, continuous exposure beyond 4 weeks of supplemented high concentration (exceed 11.35 μM), TDZ was harmful to the proliferation and growth of buds/shoots. DCR appeared more efficiency than Murashige and Skoog medium (MS), Woody Plant medium (WPM), anther culture of cereal crops medium (N6) on bud induction. Age of cotyledon and hypocotyl explants in 20-day to 25-day was most beneficial to adventitious buds/shoots formation. Histological investigation confirmed that the buds originated from the wounded incisions of cotyledonary petiole and hypocotyl fragments, with callus formation. The regeneration plantlets were successfully acclimatized in greenhouse, yielded above 95% survival rate in field, exhibited normal morphology and growth characteristics. The analysis of flow cytometry on N. cadamba indicated no variation in the ploidy levels between the regenerated plantlets and the donor trees. The developed procedure can be used for mass production, germplasm exchange and transgenic studies to improve the resistance of the species via Agrobacterium-mediated.

Feasible regeneration and agro bacterium-mediated transformation of Brassica juncea with Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene.

In the present study an effort has been made to optimize the in vitro regeneration protocol for Agrobacterium-mediated transformation of Brassica juncea, because of its importance as oilseed crops. The highest callus induction frequency of 87% was observed on MS (Murashige and Skoog, 1962) medium supplemented with 4 µM 6-benzyladenine (BA) after four weeks of culture period. Subculturing of organogenic calli in MS media with a similar hormonal composition resulted in shoot organogenesis after six weeks of culture cultivation. The highest shoot induction frequency (92%) was recorded on MS medium containing 4 µM BA in combination with 1 µM of α-naphthalene acetic acid (NAA). Further, well-developed roots were formed in MS media augmented with 6 µM of Indole acetic acid (IAA) in combination with 1 µM Kinetin (Kn). Cotyledon explants were exploited in vitro for the successful transformation of B. juncea. A binary vector comprised of the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene under the transcriptional control of a glycinin promoter and with a basta selection marker was introduced into A. tumefaciens strain GV3101 via electroporation. EaDAcT gene is responsible for unusual triacylglycerol’s production where the sn-3 position is esterified with acetate instead of the long-chain fatty acid found in the triacylglycerol’s. The highest regeneration frequency (100%) of transgenic shoots was observed on MS medium supplemented with 4 µM BA plus 1 µM NAA in the presence of 25 mg l−1 basta and 160 mg l−1 timintin. The efficiency of stable transformation was found to be approximately 7% in the transgenic plants. Moreover, the transformed regenerated shoots were confirmed by PCR analysis using EaDAcT gene-specific primers.

Callus Induction and Regeneration from Anther Cultures of Indonesian Indica Black Rice Cultivar

The assembly of superior varieties and collection of rice germplasm involves the process of selecting and storing elders that have superior genotypic properties and phenotypes. The anther culture techniques on indica black rice cultivar have a high difficulty factor to get plants, because of the low regeneration ability at the plant formation phase from the anther callus. This study aimed to investigate the influence of the cold-pretreatment time on anther, the combination of plant growth regulators (PGR’s) concentrations, and putrescine concentrations in media for the increase callus induction and plant regeneration of indica black rice. The optimization of the cold pre-treatment time was important to obtain the high-frequency callus induction, which showed that anther at the 4°C for 8 days formed the high callus induction (20%). To accelerate the callus induction, the application of 20 µM putrescine in the MS medium could produce more friable embryogenic callus for 24 days with 27% of callus formation. Generally, the optimal medium for the high frequency of callus induction contained 2 mgL−1 NAA+0.5 mgL−1 Kinetin+20 µM putrescine. Especially indica black rice cultivars, the best media to get a high plant regeneration frequency were N6 media containing the combination of 2 mgL−1 IAA and 2,5 mgL−1 Kinetin. The total callus regenerated to plantlet about 12.5%. The study of the callus induction and in-vitro plant regeneration medium for indica black rice were still important to develop to get the best result for other cultivars.

In vitro Plant Regeneration from Mature Seed Explants of Withania somnifera (L.) Dunal, an Important, Rare and Endangered Medicinal Plant

Withania somnifera (L.) Dunal a member of the Solanaceae family, is a traditional medicinal plant commonly known in India as Ashwagandha. It is used for different diseases such as hiccup, cough, rheumatism, tuberculosis, and exhibits excellent antitumor and anti-bacterial activities as well. Direct organogenesis of plants using mature seeds provides faster response and is also a time saving approach, thus the present study was conducted to investigate the optimal concentrations and combinations of plant growth regulators with MS medium for the establishment of an efficient regeneration system in W. somnifera using mature seed as an explant. Therefore, an efficient in vitro protocol for high frequency regeneration has been developed using mature seeds as explant. In the present study, the multiple shoots along with embryogenic callus induction was best seen in MS medium supplemented with BAP (1.5 mg/L) and IAA (0.5 mg/L). Furthermore, MS medium fortified with GA3 (0.3 mg/L) and IBA (3.0 mg/L) alone was suited for shoot elongation and rhizogenesis respectively. The rooted plantlets were hardened and successfully established in the soil. The establishment of a highly reproducible regeneration system would greatly influence the efforts of improvement of the hereby studied medicinal plant species through useful gene transfer technology.

Analysis of tornado and near-ground turbulence using a hybrid meteorological model/engineering LES method

A new hybrid meteorological model/engineering LES method was used to analyze a tornado and near-ground turbulence under realistic conditions. The hybrid method estimates high frequency turbulence regeneration in an actual severe storm that has complex thermal conditions in order to alleviate the problem of high frequency component dissipation in meteorological models and generate appropriate field data to investigate near-ground flow and wind loading on buildings. The high frequency regeneration method was validated in the convective boundary layer and the energy spectrum of the velocity field was successfully extended to higher frequency region in good agreement with −5/3 Kolmogorov law.Multi-scale simulations of a tornado were conducted based on meteorological model outputs with three different terrain conditions, including an actual urban building geometry and terrain, and the changes in the main tornado vortex and the interactions with near-ground turbulent field were examined. On roughened surfaces with uniform blocks and buildings, the main tornado vortex was connected to near-ground vertical vortices arising due to the urban geometry. The structure was significantly deformed, splitting into numerous finer vortices below several times the building height. These near-ground vortices create localized sharp pressure drop patterns and also affect the movement of the tornado. These effects of low pressure on buildings were quantitatively analyzed.

Standardization of Regeneration, Agrobacterium-Mediated Transformation, and Introduction of Nucleocapsid Gene of Watermelon Bud Necrosis Virus in Watermelon

Seven types of explants (proximal cotyledon, distal cotyledon, distal cotyledon leaves, proximal cotyledon leaves, distal hypocotyls, proximal hypocotyls, and basal petioles) of watermelon (Citrullus lanatus) cv. Sugar Baby were tested for their regeneration capacity on MS medium supplemented with different concentrations and combinations of hormones (BAP 0–3 mg/l alone and IAA 0–0.5 mg/l and BAP 0–3 mg/l together). The results showed that the proximal petiole cultured in MS + BAP (3 mg/l) showed the highest percentage of callus induction (28%) and shoot production (24%). The proximal cotyledons cultured in MS + BAP (2 mg/l) + IAA (0.1 mg/l) showed the highest regeneration frequency (76%). Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pBI121 containing the GUS gene (s-glucuronidase) and kanamycin-resistance gene, nptII, was used to transform petiole explants of watermelon cv. Sugar Baby. Various conditions such as the agrobacterial concentration of OD600 0.6, infection time of 20 min, co-cultivation duration of 2 days and selection at 100 mg/l kanamycin were found as important parameters for the successful Agrobacterium-mediated transformation of watermelon. Further, a transgene construct using the nucleocapsid protein (NP) gene from watermelon bud necrosis virus (genus Tospovirus family Peribunyaviridae) was developed in pBI121 and used to transform watermelon. The successful transformation of petiole explant of watermelon with the GUS gene as well as the NP gene was confirmed by molecular assays, which showed a transformation efficiency of 14.2% and 0.375% with the GUS and NP gene, respectively.

Efficient plant regeneration and histological evaluations of regenerants through organogenesis and somatic embryogenesis in Spermacoce hispida L.—An underutilized medicinally important plant

Shaggy button weed (Spermacoce hispida L.) is a therapeutically important herb widely used in Indian System of medicine particularly Ayurvedic, Siddha, and Unani. S. hispida is recalcitrant during germination in natural habitat. Plant tissue culture is an alternate approach for mass multiplication of such vulnerable plants. Plant regeneration by organogenesis and somatic embryogenesis in S. hispida was attempted using cotyledonary leaf, shoot tip, hypocotyl, and nodal explants. Maximum shoot regeneration frequency (72%) and number of shoots (10.5 ± 0.3) through direct organogenesis was achieved from nodal explants cultured on Murashige and Skoog (MS) medium amended with 2.0 mg/dm3 of 6-benzylaminopurine (BAP). Further, MS medium containing 0.5 mg/dm3 2, 4-dichlorophenoxyacetic acid (2, 4-D) produced higher frequency of callus induction (86.25%) from hypocotyl segments. Optimum shoot regeneration response (69.5%) through indirect organogenesis was observed from hypocotyl-derived callus on MS medium fortified with 1.0 mg/dm3 kinetin with 6.1 shoots per callus. The highest regeneration frequency (68%) through somatic embryogenesis was recorded from nodal explants on MS medium supplemented with 2.0 mg/dm3 BAP and 0.8 mg/dm3 kinetin. The regenerated plantlets were acclimatized successfully under ex vitro conditions with normal phenotypic appearance. Shoot tips and nodes did not induce organogenesis (indirect), whereas nodal segments showed better regeneration via organogenesis (direct) and somatic embryogenesis in MS medium with either BAP or BAP with kinetin. Histological examinations confirmed the distinctive developmental stages and bipolar structure of somatic embryos. The described regeneration protocols can be effectively utilized for the large-scale multiplication, germplasm conservation, pharmacological and genetic manipulation studies.

REPEATED REGENERATION OF CRINOID SPINES IN THE UPPER PENNSYLVANIAN OF EASTERN OHIO: EVIDENCE OF ELEVATED PREDATION INTENSITY AND SIGNIFICANCE FOR PREDATOR-DRIVEN EVOLUTION OF CRINOID MORPHOLOGY

Regeneration of portions of the crinoid endoskeleton following loss to predation attempts or autotomy is a well-known phenomenon. To date, however, most effort has focused on patterns, frequencies, and evidence of regeneration of arms and portions of the calyx, with few reports of unusual or significant regeneration-related features preserved in isolated ossicles. Here we describe brachial spines belonging to undetermined pirasocrinid (cladid) crinoids from the Upper Pennsylvanian Ames Member of the Glenshaw Formation of east-central Ohio that display evidence for multiple episodes of breakage and regeneration. As evidenced by major size discontinuities along the length of single spines, some specimens regenerated at least two times during the lifespan of the individual; such a pattern of repeated regeneration of a single skeletal element has not previously been documented. Given the position of these spines on the elevated crown of a moderately long-stemmed crinoid, frequent snipping by predatory fishes seems the most likely interpretation for the observed pattern. The repeatedly regenerated specimens occur in an assemblage displaying an unusually high regeneration frequency among pirasocrinid brachial spines, with over 30% of spines displaying at least one plane of regeneration. Paradoxically, pirasocrinids are unfavorable as prey items relative to other organisms and co-occurring crinoids; hence, it is most likely that associated biota (e.g., commensals, parasites) were the true targets of predators. This assemblage supports the interpretation that the development of extreme spinosity in pirasocrinids was largely driven by predation. However, this morphological pattern may largely represent an evolutionary strategy rooted in minimizing collateral damage incurred by predation on other organisms rather than direct predation on the crinoids themselves.