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Efficient plantlet regeneration via somatic embryogenesis and shoot organogenesis in Dichanthium annulatum (Forssk.): a halophyte C 4 apomictic forage crop
Abstract
Dichanthium annulatum is a popular halophyte C 4 perennial forage crop of semi-arid tropics that reproduces apomictically. In vitro plant regeneration in apomictic species helps in producing new germplasm that can supplement existing breeding program. Field-collected seeds and in vitro shoot apices were tested for response to 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) for initiation of somatic embryogenesis (SE) and direct shoot organogenesis (DSO). Explants produced callus on Murashige and Skoog (MS) medium with varying level of 2,4-D and 0.5 mgL-1 BAP. After 4th sub-culture, yellowish brown turns into white nodular callus tissue with a friable appearance, which exhibited the highest percentage of embryogenic callus induction on MS medium with 3.0 mgL-1 2,4-D and 0.5 mgL-1 BAP, was transferred to MS with BAP or kinetin and 0.125 mgL-1 2,4-D to promote somatic embryo germination into plantlet. The developmental stages of SE were identified using scanning electron microscopy and histology. Multiple shoots were observed with the combinations of BAP or kinetin with 2,4-D. The highest number of shoots (27) occurred on MS medium comprising 4.0 mgL-1 BAP and 0.125 mgL-1 2,4-D. In vitro plantlet was rooted on MS medium with 0.4% charcoal. The rooted 80% plants could be acclimatized and established in soil, as they showed healthy growth and fertility.
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Projections indicate that current plant breeding approaches will be unable to incorporate the global crop yields needed to deliver global food security. Apomixis is a disruptive innovation by which a plant produces clonal seeds capturing heterosis and gene combinations of elite phenotypes. Introducing apomixis into hybrid cultivars is a game-changing development in the current plant breeding paradigm that will accelerate the generation of high-yield cultivars. However, apomixis is a developmentally complex and genetically multifaceted trait. The central problem behind current constraints to apomixis breeding is that the genomic configuration and molecular mechanism that initiate apomixis and guide the formation of a clonal seed are still unknown. Today, not a single explanation about the origin of apomixis offer full empirical coverage, and synthesizing apomixis by manipulating individual genes has failed or produced little success. Overall evidence suggests apomixis arise from a still unknown single event molecular mechanism with multigenic effects. Disentangling the genomic basis and complex genetics behind the emergence of apomixis in plants will require the use of novel experimental approaches benefiting from Next Generation Sequencing technologies and targeting not only reproductive genes, but also the epigenetic and genomic configurations associated with reproductive phenotypes in homoploid sexual and apomictic carriers. A comprehensive picture of most regulatory changes guiding apomixis emergence will be central for successfully installing apomixis into the target species by exploiting genetic modification techniques.
South Texas is home to a high diversity of species due to its location at the confluence of subtropical, desert, and coastal ecoregions. Historical overgrazing of South Texas rangelands transformed the savanna and prairie to a landscape dominated by woody plants and shrubs interspersed with low seral grass species and bare ground. During the first half of the twentieth century, exotic grass species, coupled with the application of industrial agricultural practices appeared to be the future of forage production in South Texas and elsewhere. Several of these exotic species, namely King Ranch bluestem [Bothriochloa ischaemum (L.) Keng var. ischaemum], Kleberg bluestem [Dichanthium annulatum (Forssk.) Stapf,], Angelton bluestem [Dichanthium aristatum (Poir.) C. E. Hubbard], buffelgrass [Pennisetum ciliare (L.) Link], guineagrass [Megathyrsus maximus (Jacq.) B. K. Simon & S. W. L. Jacobs], Lehmann lovegrass (Eragrostis lehmanniana Nees), and bermudagrass [Cynodon dactylon (L.) Pers.] have escaped pasture cultivation. Additionally, the native grass tanglehead [Heteropogon contortus (L.) P. Beauv. ex Roem. & Schult.] has begun displaying invasive behaviors. Their monoculture growth habit simplifies vegetation structure, reduces biodiversity, and decreases habitat for many species of wildlife. They also alter natural fire regimes and nutrient cycling. This landscape-level transformation of vegetation composition and structure requires monitoring to quantify and assess the spatial and temporal distributions of invasive species as a basis to inform management practices. Current advances in remote sensing technologies, such as very high spatial resolution coupled with daily satellite imagery and unmanned aerial vehicles, are providing tools for invasive vegetation monitoring. We provide a synthesis of the natural history of these grasses, including their introductions, an overview of remote sensing applications in South Texas, and recommend future management practices.
Soil salinity is one of the important abiotic
stresses affecting plant growth and development. Halophytes
can be one of the options to explore the salt tolerance
potential and to identify the potential gene(s) which
can be used in crop improvement programs. In view of this,
the present experiment was conducted on grass halophyte,
Dichanthium annulatum, which can tolerate soil salinity up
to EC 30 dS/m (* 300 mM NaCl) to identify the
gene(s) for salt tolerance. The de novo assembly generated
267,196 transcripts and these assembled transcripts were
further clustered into 188,353 transcripts. An average of
64.47% of the transcripts was functionally annotated
against the viridiplantae databases since no genomic reference
is available for Dichanthium. Gene ontology and
pathways analysis using KAAS database identified that
48.13% transcripts were involved in molecular function,
37.21% in cellular component and 14.66% in biological
processes. The annotation of these genes provides a pathway
analysis for their putative functions under salt stress
conditions.
Mukundpur forest range is situated in Amarpatan Tahsil of Satna district of Madhya Pradesh India. Thisstudy area is under high ecological stress for mining purposes. For assessment of biodiversity vegetation sampling was done. The calculation of frequency and IVI (Important value Index) for the species of grasses have been done. The threat and conservation status is assessed by Normal Distribution Principle. In the present study the 19 grass species have been found. The 6 species are in category-4 which requires highest degree of protection. These species are Dichanthium annulatum, Phaseolus trilobus, Paspa lidium, Adaintum spp, Grewia hirsuta and Zizyphus oeroplia. The 3 species are in the category-2. These are Setaria intermedia, Ipomoea reniformis and Saccharum spontaneum. Setaria intermedia is important species of food security for human life for future.
Background:
Oxidative stress as well as bacterial and fungal infections are common source of diseases while plants are source of medication for curative or protective purposes. Hence, aim of study was to compare the pharmacological potential of seven grass species in two different solvents i.e. ethanol and acetone.
Methods:
Preliminary phytochemical tests were done and antioxidant activities were evaluated using ELISA and their IC50 values and AAI (%) were recorded. ANOVA was used for statistical analyses. DNA damage protection assay was done using p1391Z plasmid DNA and DNA bands were analyzed. Antimicrobial activity was done via disc diffusion method and MIC and Activity Index were determined. Cytotoxic activity was carried out using the brine shrimps' assay and LC50 values were calculated using probit analysis program.
Results:
Phytochemical studies confirmed the presence of secondary metabolites in most of the plant extracts. Maximum antioxidant potential was revealed in DiAEE, DiAAE (AAI- 54.54% and 43.24%) and DaAEE and DaAAE (AAI- 49.13% and 44.52%). However, PoAEE and PoAAE showed minimum antioxidant potential (AAI- 41.04% and 34.11%). SaSEE, DiAEE and ElIEE showed very little DNA damage protection activity. In antimicrobial assay, DaAEE significantly inhibited the growth of most of the microbial pathogens (nine microbes out of eleven tested microbes) among ethanol extracts while DaAAE and ImCAE showed maximum inhibition (eight microbes out of eleven tested microbes) among acetone plant extracts. However, PoAEE and PoAAE showed least antimicrobial activity. F. oxysporum and A. niger were revealed as the most resistant micro-organisms. ImCEA and ImCAE showed maximum cytotoxic potential (LC50 11.004 ppm and 7.932 ppm) as compared to the other plant extracts.
Conclusion:
Fodder grasses also contains a substantial phenols and flavonoids contents along with other secondary metabolites and, hence, possess a significant medicinal value. Ethanol extracts showed more therapeutic potential as compared to the acetone extracts. This study provides experimental evidence that the selected species contains such valuable natural compounds which can be used as medicinal drugs in future.
An efficient callus induction and plant regeneration system has been standardized for an ethnomedicinal plant, Elephantopus scaber Linn. Two explants i. e. seeds and leaf segments were used for callus induction. Murashige and Skoog (MS) medium supplemented with 5.0 μM 2, 4-dichlorophenoxy acetic acid (2, 4-D) and 0.5 μM kinetin (Kn) gave the optimum frequency (89 %) of callus induction from seed explant. The results showed that the highest response in terms of percent callus regenerating (91 %) and number of shoots (56) per culture was recorded on MS medium supplemented with 6.0 μM N6-benzylaminopurine (BA) and 1.5 μM α naphthalene acetic acid (NAA). The best rooting of regenerated shoots was obtained on half strength MS medium supplemented with 6.0 μM indole-3- butyric acid (IBA). On this medium, 100 % of the shoots produced roots with a mean number of 3.2 roots per shoot. The positive role of vesicular arbuscular mycorrhizae (VAM) along with potting mix has been well established in the present study. Of the various potting mix employed for plant acclimatization, the highest response of 100 % plant survival was noticed when autoclaved garden soil, sand (2:1) and VAM was utilized as potting mix. Inter-simple sequence repeats (ISSR) were used to establish the clonal fidelity of regenerated plantlets and the banding profiles from callus derived plants were monomorphic and similar to those of mother plant, thus ascertaining the true-to-type nature of these plants.
Based on recent molecular and morphological studies we present a modern worldwide phylogenetic classification of the ±12,074 grasses and place the 771 grass genera into 12 subfamilies (Anomochlooideae, Aristidoideae, Arundinoideae, Bambusoideae, Chloridoideae, Danthonioideae, Micraioideae, Oryzoideae, Panicoideae, Pharoideae, Puelioideae, and Pooideae), six supertribes (Andropogonodae, Arundinarodae, Bambusodae, Panicodae, Poodae, Triticodae), 52 tribes (Ampelodesmeae, Andropogoneae, Anomochloeae, Aristideae, Arundinarieae, Arundineae, Arundinelleae, Atractocarpeae, Bambuseae, Brachyelytreae, Brachypodieae, Bromeae, Brylkinieae, Centotheceae, Centropodieae, Chasmanthieae, Cynodonteae, Cyperochloeae, Danthonieae, Diarrheneae, Ehrharteae, Eragrostideae, Eriachneae, Guaduellieae, Gynerieae, Hubbardieae, Isachneae, Littledaleae, Lygeeae, Meliceae, Micraireae, Molinieae, Nardeae, Olyreae, Oryzeae, Oryzoideae, Paniceae, Paspaleae, Phaenospermateae, Phareae, Phyllorachideae, Poeae, Steyermarkochloeae, Stipeae, Streptochaeteae, Streptogyneae, Thysanolaeneae, Triraphideae, Tristachyideae, Triticeae, Zeugiteae, and Zoysieae), and 81 subtribes (Aeluropodinae, Agrostidinae, Airinae, Ammochloinae, Andropogoninae, Anthephorinae, Anthistiriinae, Anthoxanthinae, Arthraxoninae, Arthropogoninae, Arthrostylidiinae, Aveninae, Bambusinae, Boivinellinae, Boutelouinae, Brizinae, Buergersiochloinae, Calothecinae, Cenchrinae, Chionachninae, Chusqueinae, Coicinae, Coleanthinae, Cotteinae, Cteniinae, Cynosurinae, Dactylidinae, Dichantheliinae, Dimeriinae, Duthieinae, Eleusininae, Eragrostidinae, Farragininae, Germainiinae, Gouiniinae, Guaduinae, Gymnopogoninae, Hickeliinae, Hilariinae, Holcinae, Hordeinae, Ischaeminae, Loliinae, Melinidinae, Melocanninae, Miliinae, Monanthochloinae, Muhlenbergiinae, Neurachninae, Olyrinae, Orcuttiinae, Oryzinae, Otachyriinae, Panicinae, Pappophorinae, Parapholiinae, Parianinae, Paspalinae, Perotidinae, Phalaridinae, Poinae, Racemobambosinae, Rottboelliinae, Saccharinae, Scleropogoninae, Scolochloinae, Sesleriinae, Sorghinae, Sporobolinae, Stipinae, Torreyochloinae, Traginae, Trichoneurinae, Triodiinae, Tripogoninae, Tripsacinae, Triraphidinae, Triticinae, Unioliinae, Zizaniinae, and Zoysiinae). In addition, we include a radial tree illustrating the hierarchical relationships among the subtribes, tribes, and subfamilies. We use the subfamilial name, Oryzoideae, over Ehrhartoideae because the latter was initially published as a misplaced rank, and we circumscribe Molinieae to include 13 Arundinoideae genera. The subtribe Calothecinae is newly described and the tribe Littledaleeae is new at that rank.
The present study conducted in district Bahawalpur of Southern Punjab province, Pakistan to enlist the
medicinal plants and their uses among local people. Previous studies focus primarily on the exploration of medicinal plants of Cholistan desert while rest of the area remained un-explored. The ethno-medicinal survey was conducted regularly for a period of 10 years and tries to eradicate the errors in the utilizations of the plants and to finally to document ethno-medicinal uses of plant species through questionnaire and personal interviews during field trips. Plants with their correct nomenclature were arranged by family name, vernacular name, parts used and ethno-medicinal uses. For the identification of plants we used field guides and flora of Pakistan and as a result 123 plant species currently under utilization by local people were identified. Previously we collected all the plant specimens, after careful identification we preserved and mounted on herbarium sheets, were placed in the department of Botany, Govt. Sadiq Egerton College, Bahawalpur, Pakistan. The study will provide a baseline for future studies relating to pharmacological, chemical isolations, taxonomic and well as biochemical studies by giving a quick approach to the
specific plant species.
A world-wide phylogenetic classification of Poaceae (Gramineae)
It is concluded from a review of the literature that plant cell culture itself generates genetic variability (somaclonal variation). Extensive examples are discussed of such variation in culture subclones and in regenerated plants (somaclones). A number of possible mechanisms for the origin of this phenomenon are considered. It is argued that this variation already is proving to be of significance for plant improvement. In particular the phenomenon may be employed to enhance the exchange required in sexual hybrids for the introgression of desirable alien genes into a crop species. It may also be used to generate variants of a commercial cultivar in high frequency without hybridizing to other genotypes.
Apomixis is an asexual reproductive process in plants whereby seeds that are exact genetic replicas of the maternal genetic material are produced. Vielle Calzada, Crane, and Stelly argue that this process, if understood and harnessed, will revolutionize genetic engineering and seed production for agriculturally important crops.
A critical step in the development of a reproducible Agrobacterium tumefaciens mediated transformation system for a recalcitrant species, such as pearl millet, is the establishment of optimal conditions
for efficient T-DNA delivery into target tissue from which plants can be regenerated. A multiple shoot regeneration system,
without any intervening callus phase, was developed and used as a tissue culture system for Agrobacterium-mediated transformation. Agrobacterium super virulent strain EHA105 harboring the binary vector pCAMBIA 1301 which contains a T-DNA incorporating the hygromycin
phosphotransferase (hpt II) and β-glucuronidase (GUS) genes was used to investigate and optimize T-DNA delivery into shoot apices of pearl millet. A
number of factors produced significant differences in T-DNA delivery; these included optical density, inoculation duration,
co-cultivation time, acetosyringone concentration in co-cultivation medium and vacuum infiltration assisted inoculation. The
highest transformation frequency of 5.79% was obtained when the shoot apex explants were infected for 30min with Agrobacterium O.D.600=1.2 under a negative pressure of 0.5×105 Pa and co-cultivated for 3days in medium containing 400μM acetosyringone. Histochemical GUS assay and polymerase chain
reaction (PCR) analysis confirmed the presence of the GUS gene in putative transgenic plants, while stable integration of
the GUS gene into the plant genome was confirmed by Southern analysis. This is the first report showing reproducible, rapid
and efficient Agrobacterium-mediated transformation of shoot apices and the subsequent regeneration of transgenic plants in pearl millet. The developed
protocol will facilitate the insertion of desirable genes of useful traits into pearl millet.
KeywordsPearl millet–Multiple shoot induction–
Agrobacterium tumefaciens
–GUS expression–Genetic transformation–Shoot organogenesis
An efficient, highly reproducible system for plant regeneration via somatic embryogenesis was developed for Cenchrus ciliaris genotypes IG-3108 and IG-74. Explants such as seeds, shoot tip segments and immature inflorescences were cultured on Murashige
and Skoog (MS) medium supplemented with 2.0–5.0 mg dm−3 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg dm−3 N6-benzyladenine (BA) for induction of callus. Callus could be successfully induced from all the three explants of both the
genotypes. But the high frequency of embryogenic callus could be induced only from immature inflorescence explants. Somatic
embryos were formed from nodular, hard and compact embryogenic calli when 2,4-D concentration was gradually reduced and BA
concentration increased. Histological studies of somatic embryos indicated the presence of shoot apical meristem with leaf
primordia. Ultrastructural details of globular and scutellar somatic embryos further validated successful induction and progression
of somatic embryogenesis. Shoots were differentiated upon germination of somatic embryos on MS medium containing 2,4-D (0.25
mg dm−3) and BA or kinetin (1–5 mg dm−3). Roots were induced on ½ MS medium containing charcoal (0.8 %), and the regenerated plants transferred to pots and established
in the soil showed normal growth and fertility.
Under appropriate in vivo or in vitro conditions, certain somatic plant cells have the capability to initiate embryogenic development (somatic embryogenesis). Somatic embryogenesis provides an unique experimental model to understand the molecular and cellular bases of developmental plasticity in plants. In the last few years, the application of modern experimental techniques, as well as the characterization of Arabidopsis embryogenesis mutants, have resulted in the accumulation of novel data about the acquisition of embryogenic capabilities by somatic plant cells. In this review, we summarize relevant experimental observations that can contribute to the description and definition of a transitional state of somatic cells induced to form totipotent, embryogenic cells. During this somatic-to-embryogenic transition, cells have to dedifferentiate, activate their cell division cycle and reorganize their physiology, metabolism and gene expression patterns. The roles of stress, endogenous growth regulators and chromatin remodelling in the coordinated reorganization of the cellular state are especially emphasized.
Cotyledon explants of Korean ginseng (Panax ginseng C. A. Meyer) produced somatic embryos directly on growth regulator-free medium. Somatic embryos developed as either multiple
or single-state forms, depending on the degree of maturity of the cotyledons. Cotyledon explants from midmature zygotic embryos
formed multiple embryos, while cotyledons from fully mature zygotic embryos formed single embryos. Somatic single embryos
regenerated into normal plantlets with both roots and shoots, while multiple embryos did not produce roots but regenerated
only into multiple shoots. In full-strength MS basal medium, the root growth of plantlets derived from single embryos was
weak compared to that of shoots. Deletion of ammonium nitrate from the MS medium promoted the root growth of the plantlets.
The ginseng plants with well-developed shoots and roots regenerated from single embryos were successfully acclimatized in
a greenhouse when they were planted in soil.
The goals of this study were to identify habitats occupied by the Eurasian grass Bothriochloa ischaemum (King Ranch bluestem) on the eastern Edwards Plateau of central Texas, USA, and to measure the effects of this invasive species
on plant diversity. A set of descriptive field studies were conducted to determine the relationships between habitat characteristics
(woody cover, slope, presence of roads and trails, history of prescribed burning, and presence of grazing) and the presence
and abundance of this species. Species richness and diversity of perennial herbaceous species were compared between plots
in which B. ischaemum was absent and plots that it dominated.
The only habitat in which B. ischaemum was never found was under the canopies of woody plants. B. ischaemum grew in plots of all inclinations (flat sites to steep hillsides), with little evidence of habitat preference. B. ischaemum was more often, although not exclusively, found in plots near roads, probably because roads facilitate seed dispersal. There
was no significant relationship between either grazing or fire history and the distribution of this species. B. ischaemum-dominated plots had lower species diversity and species richness than plots in which no B. ischaemum was found. The results of this study confirm that B. ischaemum is very common on the eastern Edwards Plateau and suggest that it will continue to spread throughout this region and to reduce
native herbaceous plant diversity there.
Modern plant breeding programs depend heavily on germplasm resources composed of closely related breeding lines and cultivars. Asexual introduction of recombinant DNA offers novel opportunities for crop improvement, but most transformation methods rely on tissue culture systems which are mutagenic. The resultant transgenic plants frequently contain undesirable genetic changes (somaclonal variation), in addition to the introduced transgene. Such plants may have reduced agronomic performance, which complicates their use as parents in a breeding program. The development of tissue culture systems that are less mutagenic should enable the production of transgenic plants with superior performance. In this study, agronomic traits were measured for plants regenerated from cultures of two barley (Hordeum vulgare L.) genotypes, using three different tissue culture systems, and compared with the performance of uncultured controls. Plants derived from all three systems were shown to have reduced performance for one or more agronomic traits, but there were clear differences attributable to the culture system. Plants derived from standard embryogenic callus tissues were shown to have the greatest reductions in agronomic performance. Two other tissue culture systems, which had been developed for increased regenerability, showed better performance. Plants derived from highly differentiated, meristematic tissues showed the least reductions in agronomic performance. Plants derived from a modification of the embryogenic callus system-which is characterized by an intermediate level of differentiation-showed intermediate levels of agronomic performance. These results demonstrate that modifications of methodology, in addition to improving plant regenerability, can reduce the level of somaclonal variation in regenerated plants.
Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 microM) or high (10 microM) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mM) in the presence of 1 microM 2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 microM 2,4-D-containing culture medium by 10 mM 2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.
Eleven Dichanthium annulatum (Forssk) plants were regenerated from embryogenic callus co-transformed with two plasmids encoding either the hygromycin phosphotransferase gene (hph) or the beta-glucuronidase (GUS) gene (uidA). Analysis of these putative transformants showed that three plants were transformed with the hph gene, showed the presence of the hph transcript and expressed hygromycin resistance after transfer to soil. Two of these also contained the uidA gene but did not express GUS and were shown to be the same transformation event. All three of the transformants set seed. Hygromycin resistance varied from 68-100% in the progeny of the three transformants. Transgene transmission appeared to have been mainly through apomixis.
Sustained post-embryonic organ initiation and development in plants depends on coordinating the formation and differentiation of pluripotent stem cells in apical meristems. Transcriptional regulation and intercellular signalling appear to play key roles in this coordination process. Here we discuss the current knowledge about the molecular regulation of stem cell maintenance in the shoot apical meristem and recent attempts to delineate the molecular signatures of "stemness" in flowering plants. We also outline contemporary molecular approaches for deciphering the process of stem cell renewal in the shoot apical meristem.
Rice (Oryza sativa ssp. indica) is an important economic crop in many countries. Although a variety of conventional methods have been developed to improve this plant, manipulation by genetic engineering is still complicated. We have established a system of multiple shoot regeneration from rice shoot apical meristem. By use of MS medium containing 4 mg L(-1) thidiazuron (TDZ) multiple shoots were successfully developed directly from the meristem without an intervening callus stage. All rice cultivars tested responded well on the medium and regenerated to plantlets that were readily transferred to soil within 5-8 weeks. The tissue culture system was suitable for Agrobacterium-mediated transformation and different factors affecting transformation efficiency were investigated. Agrobacterium strain EHA105 containing the plasmid pCAMBIA1301 was used. The lowest concentration of hygromycin B in combined with either 250 mg L(-1) carbenicillin or 250 mg L(-1) cefotaxime to kill the rice shoot apical meristem was 50 mg L(-1) and carbenicillin was more effective than cefotaxime. Two-hundred micromolar acetosyringone had no effect on the efficiency of transient expression. Sonication of rice shoot apical meristem for 10 s during bacterial immersion increased transient GUS expression in three-day co-cultivated seedlings. The gus gene was found to be integrated into the genome of the T(0) transformant plantlets.
Phytochemical investigation of Dichanthium annulatum (Forrsk), herbs revealed the isolation and identification of two pairs of lignoflavone derivatives stereoisomers; an epimers of tricin 4`-O-(threo-βguaiacylglyceryl) ether (Salcolin A) and tricin 4`-O-(erythro-β-guaiacylglyceryl) ether (Salcolin B) (1) and an epimer of tricin 4`-O-[threo-β-guaiacyl-(7``-O-methyl-9``-O-acetyl)-glyceryl] ether and tricin 4`-O-[erythro-βguaiacyl-(7``-O-methyl-9``-O-acetyl)-glyceryl] ether (2), one flavone; tricin (3), two flavone glycosides; tricin 7-O-β-D-glucopyranoside (4) and tricin 7-O-neohesperidoside (5), one flavone C-glucoside; isoorientin (6), one phenolic acid; p-coumaric acid (7), one lignan; 4-ketopinoresinol (8) and two sterols; stigmasterol (9) and β-sitosterol-3-O-β-D-glucoside (10). The structural elucidations of isolated compounds were established on the basis of NMR and MS spectral analyses. All isolated compounds and biological activities for different extracts, n-hexane, ethyl acetate and n-butanol fractions (antiviral, antimicrobial and cytotoxic activities) of Dichanthium annulatum were done for the first time.
Pennisetum pedicellatum Trin. is a C4 tropical perennial forage grass known as Deenanath grass holding great potential to address vital, puzzling issues such as bio-economy, human health and the environment. Genetic complexity not only is a big barrier in selection and breeding practices but also restricts harnessing of the full potential of this multipurpose fodder crop. By optimizing a rapid, effective and consistent in vitro regeneration protocol through callus mediated and direct shoot organogenesis, we can multiply these plants for producing a large amount of biomass. Calli were induced from mature seeds and shoot apices of two genotypes on Murashige and Skoog (MS) medium containing different levels of 9.0–22.5 µM 2,4-Dichlorophenoxyacetic acid (2,4-D) and a constant level of 2.22 µM N6-benzyladenine (BA). Seventy-one percent of the calli turned embryogenic when 2,4-D (2.25 µM) was reduced in the medium. Somatic embryogenesis was accomplished on MS medium with an increased level of either BA (4.4–17.6 µM) or kinetin (4.6–18.4 µM) along with 0.56 µM 2,4-D. Ultra-structural and histological studies indicated globular, scutellar and coleoptilar somatic embryos. A maximum of 82.7±1.6% plant regeneration was accomplished from seed explants on MS medium containing 8.8 µM BA and 0.56 µM 2,4-D. Multiple shoots were induced on MS media containing either BA (4.4–18.0 µM) or kinetin (4.6–18.4 µM) along with 0.56 µM 2,4-D using shoot apices. A maximum of 22.1±0.45 shoots per shoot apex was produced on MS medium containing 8.8 µM BA and 0.56 µM 2,4-D. Successful rooting of healthy shoots generated through both the pathways could be optimized on ½MS medium containing 0.4% charcoal. Complete plants could be hardened in plastic pots and were well established on field conditions. In vitro regeneration was established for the first time successfully, which would be very useful for genetic improvement of this apomictic species through transgenic approach.
A highly reproducible plant regeneration protocol through somatic embryogenesis and shoot organogenesis has been developed for Cenchrus ciliaris. Three explants (seeds, shoot apices, and immature inflorescences) of four genotypes (IG-3108, IG-718, IG-74, and DBC15-8/32/10) were used for callus induction and plant regeneration. The highest rate of callus formation was found using Murashige and Skoog (MS) medium supplemented with 0.5 mg L−1 benzylaminopurine (BA) and 3.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D). The largest number of somatic embryos was generated with the addition of 400 mg L−1 L-proline, 400 mg L−1 L-glutamine, and 300 mg L−1 casein hydrolysate. Somatic embryos were successfully germinated on MS medium with 3.0 mg L−1 BA and 0.25 mg L−1 2,4-D. In vitro plant regeneration was accomplished through somatic embryogenesis using all three explants. Ultra-structural features of somatic embryos confirmed proper formation and ontogeny.
The results of the experiments conducted to regenerate Pennisetum glaucum via organogenesis pathway, using Thidiazuron (TDZ), a urea-derived cytokinin, are described herein. Effect of six concentrations of TDZ (1, 3, 5, 7, 10 or 12 mg/l) was studied on shoot tip explants of four pearl millet genotypes viz., 843B, 7042-DMR, 7042-S and ICMP-451. Multiple shoot formation, without visible intervening callus phase could be obtained at all levels of TDZ across all the genotypes. The maximum number of shoots formed per shoot tip was 31.0 ± 1.8 on MS medium containing 10 mg/l TDZ in genotype 7042-S after 9 weeks of culture. Explants were subcultured after every 20 days in the same induction medium. As the levels of TDZ increased up to 10 mgll, the number of regenerated shoots formed per explant increased. On the contrary, percentage explant responded decreased as the level of TDZ increased. Maximum number of explant responded was 83.3 ± 3.3 on MS medium containing 1 mg/l TDZ, in genotype 7042-DMR. There was a significant effect of levels of TDZ and genotypes on number of shoots formed per shoot tip, whereas percentage explant response was dependent on levels of TDZ in the medium. The shoots regenerated on 1-5 mgll TDZ were large and healthy, whereas shoots regenerated on 7, 10 and 12 mg/l TDZ were short on MS basal medium. Multiplication rate varied from 2 to 31 shoots per explant in different concentrations of TDZ used in the medium. In vitro raised shoots were elongated on MS basal medium and 85 per cent shoots rooted on rooting media (V, strength MS medium containing 0.4% activated charcoal). All rooted shoots were hardened before transferring to soil where 75 per cent plantlets survived successfully. Total time taken from initiation of culture to transfer of plantlets to pots was 16 weeks. Fifty per cent of the shoots regenerated on 7 and 10 mg/l TDZ produced in vitro flowering within 90 days.
Traditional plant-breeding procedures are based on manipulation of genes and chromosomes through sexual reproduction in whole plants. The procedures were developed from Mendelian genetic principles and were expanded with developments in cytology, polyploidy, mutation induction, quantitative genetics, heterosis, male sterility, and related areas. In recent years a technology for genetic manipulation at the cellular level has emerged, which has a unique potential for supplementing traditional plant-breeding procedures. This technology has been developed from advances in knowledge of cell culture and molecular biology. The nature of the cell culture techniques, benefits to plant breeding from their use, and problems associated with their use are discussed in this chapter. This is an intensely researched area of science, from which new information is rapidly emerging.
To meet the ever-increasing demand for forages, genetic improvement of forage crops is imminent. Development of efficient breeding strategies in forage crops requires sound knowledge of their taxonomy, reproductive diversity, genome organization, and evolution of their cultivated as well as wild relatives. Dichanthium species are generally range grasses growing in hot and arid conditions. Four species of Dichanthium are popular as forage crops because of their high palatability and nutritive value. Dichanthium forms an agamic complex with two other genera, Bothriochloa and Capillipedium, where genetic exchange still takes place due to the occurrence of a few sexual or facultative apomictic types. This resulted in various intergrades, which made taxonomic positioning of different species of these three genera highly contentious. Dichanthium annulatum has been studied in detail for understanding the mechanism of apomixis for the ultimate aim of transferring this trait to crop plants. Various cytological features of Dichanthium complex have indicated the kind of genome organization. Several cultivars have been released for commercial cultivation from different countries. Owing to the predominantly apomictic mode of reproduction, the genetic improvement was restricted to selection methodologies. But the recent availability of advanced molecular and biotechnological tools enables gene transfer for desirable agronomic traits such as better forage quality and stress tolerance from the wild relatives.
Perennial ryegrass (Lolium perenne L.) is an important grass species as turf and forage. The optimal combination of 2,4-D and BAP in the medium for callus induction and the optimal kinetin concentration in the medium for plant regeneration were investigated. Cultivar variation in the callus induction and plant regeneration of eleven perennial rye-grass was studied using the optimized culture medium. The results showed that the highest callus induction rate with the best callus quality assurance was obtained on MS medium containing 5 mg l -1 2,4-D and 0.05 mg l -1 BAP. MS medium containing 3 mg l -1 BAP, 1 mg l -1 NAA and 1 mg l -1 kinetin was most suitable for plant regeneration. Callus induction and plant regeneration of the eleven cultivars of perennial ryegrass were varied greatly, and higher overall plant regeneration rates were observed in cultivars Barlennium, Pace, Emerald and Premier.
Efficient plant regeneration has been achieved from immature inflorescence derived callus cultures of salt tolerant grass Leptochloa fusca (L.). Young inflorescence explants displayed wide-ranging responses for callus induction and plant regeneration when subjected to different cold treatment durations and without cold treatment exposure (control) prior to its inoculation to MS medium supplemented with different concentrations/combinations of plant growth regulators (PGRs). The PGRs included auxins: 2, 4-dichlorophenoxy acetic acid (2, 4-D), picloram (Pic), 3, 6-dichloro-2-methoxy benzoic acid (dicamba) and cytokinins: Kinetin (KN), N6-benzyl adenine (BA). These treatments promoted different callus induction frequencies as well as various callus types such as type 1, type 2 and type 3. Induction of type 2 callus (white and compact) with potential for regeneration was obtained from cold treated (3 days at 10 °C) immature inflorescence cultured on MS medium containing 2.0 mg/l dicamba and 0.25 mg/l BA. The study demonstrated that 2.0 mg/l dicamba and 0.25 mg/l BA induced callus promoted improved frequency compared to zilch shoot regeneration response with other combinations involving 2, 4-D, picloram, KN and BA. Full strength MS supplemented with 2.0 mg/l NAA and 0.5 mg/l BA was found to be optimal for plant regeneration. The regeneration frequencies ranged from 13.8 ± 1.366 to 55.5 ± 2.766 with highest number of shoots (19.1 ± 0.560) per 50-60 mg of callus as explants after 28 days of inoculation. Plant regeneration was also obtained on the dicamba callus induction medium itself within 21 days inoculation of immature inflorescence explants. Half strength MS medium both semisolid and liquid devoid of plant growth regulators promoted highest frequency (92.8 ± 4.099 and 100 ± 0.00) of rooting in regenerated shoots. Plants with well developed roots were successfully transferred to pots and grown to maturity with normal flowering and seed set. This is the first report on induction of callus and subsequent plant regeneration in kallar grass using immature inflorescence explants.
An efficient and reproduciblein vitroculture system for differentiation of multiple shoots was established from shoot apical meristems in oat. Shoot apices as initial explants were isolated from asepticallygerminated oat seedlings, and were culturedin vitro. After four to five weeks in culture, the apical meristems inside of the primary and axillary shoots were enlarged. Multiple shoots were produced via differentiation of adventitious shoots from the enlarged apical meristems. The multiple shoots were induced and maintained on MS basal medium with various combinations of 2,4-D and BA. All four oat cultivars tested differentiated multiple shoots from shoot apical meristems at a high frequency. Fertile oat plants were produced fromin vitromultiple shoots. These in vitro multiple shoots would be an alternative regenerable target tissue for genetic transformation of oat.
Cell proliferation and the formation of somatic embryos from the scutellum of cultured immature embryos of Pennisetum americanum (pearl millet) were examined by light and scanning electron microscopy. The initial region of cell-division activity was in the scutellar node area around the main procambial strand. Cells on the abaxial side of the scutellum, particularly in the coleorhizal half of the embryo, enlarged and became richly cytoplasmic with prominent nuclei. Some of these cells on the periphery of the scutellum underwent internal segmenting divisions and either directly formed embryoids or continued to proliferate and produced an embryogenic callus tissue which could be subcultured. The richly cytoplasmic peripheral cells of the callus, as well as the embryoids, proliferated again to form a secondary callus tissue, organized structures, embryoids, and plants. Increased cell-division activity in the shoot meristem region of some of the embryoids resulted in the formation of broad meristematic zgnes....
High frequency regeneration via somatic embryogenesis was achieved in the leaf-base cultures of wheat (Triticum aestivum L. cv Sonalika) by optimizing the concentration of the hormone, 2,4-dichlorophenoxy acetic acid, and selecting for the appropriate part of the leaf base as explant. It was possible to distinguish compact (morphogenic) and friable (non-morphogenic) calli by the naked eye, after about 60 days of culture on medium enriched with 2,4-dichlorophenoxy acetic acid. The fact that the compact calli are morphogenic, while the friable ones are not, was evident from the observation that only the former formed plantlets after transfer to the basal medium. The morphogenic and non-morphogenic cultures showed substantial difference in soluble protein content on a fresh weight basis. A comparison of silver-stained profiles of soluble polypeptides from morphogenic and non-morphogenic calli revealed many polypeptides specifically associated with either type of calli.
Twelve durum wheat cultivars were evaluated for their response to in vitro tissue culture. Zygotic immature embryos were used to induce callus formation using four different Murashige and Skoog-based media. Each contained 9.05 μM 2,4-dichlorophenoxy acetic acid but differed in their carbon source (sucrose or maltose) and the presence of NaCl (0 mM or 40 mM). The influence of both genotype and medium on the type and percentage of callus produced was observed. Calli were either compact and frequently embryogenic, or soft and watery. Percentages ranged from 54 to 100%, depending upon genotype and induction medium. All calli were then plated on a regeneration medium containing 20 g/l sucrose, 2.68 μM 1-naphthaleneacetic acid and 2.22 μ 6-benzylaminopurine. The regeneration of plantlets was higher from compact than from soft calli, with a strong dependence on genotype and type of induction medium used. MSm induction medium (30 g/l maltose) and MS40s (30 g/l sucrose plus 40 mM NaCl) were best for inducing compact calli, and gave the highest proportion of regenerated plants. The in vitro response (number of total shoots from a compact callus/number of embryos plated) was higher for immature embryos of ‘Baztan’, ‘Bradano’ and ‘Don Pedro’. These cultivars are a good starting material for experiments involving transformation of calli from zygotic immature embryos.
Buffel grass (Cenchrus ciliaris L.) is an important apomictic grass used as forage for ruminant livestock. Biotechnological methods provide opportunities for producing new germplasm. Mature embryos of fourteen buffel grass apomictic cultivars (2n = 4x = 36) were used to induce embryogenic callus formation using a basal medium supplemented with 3% sucrose and with the testing of five concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and four concentrations of 6-benzylaminopurine (BAP). The effects of cultivar and culture medium on callus induction and plant regeneration were evaluated. Significant differences were observed among the fourteen cultivars and the five concentrations of 2,4-D (P < 0·01). Values for embryogenic callus production varied from 0 to 86·7. Most cultivars showed the highest level of embryogenic callus production on the medium with the concentration of 3 mg L−1 2,4-D. The addition of different BAP concentrations in combination with 2,4-D in the medium inhibited embryogenic callus growth and did not permit plant regeneration. The data clearly demonstrated that the genotype and concentrations of 2,4-D had significant effects both on the frequency of embryonic callus formation from mature embryos and on the subsequent efficiency of plant regeneration of apomictic cultivars of buffel grass. Cultivars Biloela and Nunbank showed the greatest efficiency in in vitro culture response.
The effect of benzyladenine (BA) on the production of shoot-forming callus from seeds of two Poa pratensis cultivars was studied. Addition of low concentrations (0.1–0.3 mg l-1) of BA to Murashige & Skoog (MS) callus induction medium containing 1 or 2 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-d) stimulated somatic embryogenesis and strongly increased the percentage of seeds producing shoot-forming callus in both cultivars.
In this research, a medium was developed that would stimulate multiple shoot initiation from shoot apex explants of Hibiscus cannabinus L. (kenaf). Adventitious shoot formation on a shoot induction media supplemented with combinations of 2,4-dichlorophenoxyacetic
acid (2,4-D) (0, 0.5, 2.3 μmol·l–1) and thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea; TDZ) (0, 1, 5, 20 μmol·l–1) was evaluated. Multiple shoot induction medium with 1 μmol·TDZ l–1 resulted in the highest number of regenerated shoots per explant for all three kenaf cultivars tested (Tainung 1, Tainung 2,
and Everglades 71). The 2,4-D did not enhance multiple shoot formation. Additionally, kenaf cultivars 7N and SF459 also produced
multiple shoots on the induction medium with 1 μmol·TDZ l–1. Multiple shoot clumps formed after 2 weeks in culture without callus formation. Shoots elongated and rooted within 2 weeks
after subculture on a plant growth regulator-free medium. A histological study demonstrated the de novo regeneration of shoots
from the shoot apex.
Optimization of in vitro plant regeneration and genetic transformation of apomictic species such as Dichanthium annulatum would enable transfer of desirable genes. Seven genotypes of this grass species were screened through mature seed explant for embryogenic callus induction, callus growth and quality (color and texture), and shoot induction. Genotype IG-1999, which produced highly embryogenic, rapidly growing good-quality callus capable of regenerating at a high frequency, was selected for transformation experiments. Using a binary vector (pCAMBIA1305), frequency of GUS expression was compared between two methods of transformation. Bombardment of embryogenic calli with gold particles coated with pCAMBIA1305 at a distance of 11 cm, pressure of 4 bars, and vacuum of 27 Hg passing through 100 muM mesh produced maximum GUS expression (23%). Agrobacterium infection was maximum at an optical density of 2.0 when cocultured under vacuum for 15 min and cocultivated for 3 days at 28 degrees C in constant dark on MS medium of pH 5.8 with 3 mg/l 2,4-D, and 400 muM acetosyringone. Among two binary vectors used for Agrobacterium-mediated transformation, pCAMBIA1301 showed higher frequency of GUS expression while pCAMBIA1305 recorded more of the GUS spots per callus. Supplementation of acetosyringone in the cocultivation medium was found indispensable for Agrobacterium-mediated transformation. Injuring the calli through gold particle bombardment before their cocultivation with Agrobacterium improved the transformation efficiency. Several transgenic plants were developed using the PIG method, while stable GUS-expressing calli were multiplied during selection on MS medium containing 250 mg/l cefotaxime and 50 mg/l hygromycin, incubated in constant dark. A highly significant difference was observed between two methods of transformation for both frequency of GUS expression and GUS spots per callus. PIG-mediated transformation resulted in higher GUS expression compared to the Agrobacterium method. These results demonstrate that Dichanthium annulatum is amenable to Agrobacterium-mediated genetic transformation using a binary vector.
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