Compact Embryogenic Callus Research Articles

Somatic embryogenesis from mature sorghum seeds: An underutilized genome editing recipient system

Somatic embryogenesis is a process of cell totipotency in vitro, whereby an embryogenic cell develops from vegetative tissues rather than from zygotes after double fertilization. Sorghum is a recalcitrant crop in genetic transformation; previous recipient systems have usually been from immature zygotic embryos, which needed more time and labors to prepare. Here, an efficient 2,4-dichlorophenoxyacetic acid (2,4-D)-induced somatic embryogenesis system from mature sorghum seeds was introduced. 2,4-D can induce two types of calli from a plumular axis section. Low-concentration 2,4-D (e.g., 2 mg/L) induces white and loose non-embryogenic calli (type 1), while high-concentration 2,4-D (e.g., 8 mg/L) induces yellow and compact embryogenic calli (type 2), which can be clearly distinguished by Sudan red staining. Germinating seeds have a long 2-day window for SE induction. Somatic embryogenesis can be enhanced by HDAC inhibitor, trichostatin A (TSA), a histone deacetylase treatment, which shows more SE productivity and a bigger size. Importantly, this easily prepared protocol does not show obvious genotype dependency in sorghum hybrids. In this study, a high-concentration 2,4-D-induced SE system was established from mature sorghum seeds. This finding provides a technical option for the genome editing recipient in sorghum.

Induction of somatic embryos in cultures of Asparagus racemosus Willd: an endangered medicinally important plant

BackgroundSomatic embryogenesis is one of the most popular in vitro regeneration methods for mass micropropagation. In the present study, somatic embryogenesis via zygotic embryos was studied in Asparagus racemosus Willd. Since the callus quality plays an important role in plantlet development, therefore, compact embryogenic callus was selected for further embryogenesis.ResultsSomatic embryos were induced by zygotic embryos germinating on callus induction medium (MS media with 1.54 mg L−1 2,4-D and 0.43 mg L−1 kinetin) in dark. Thereafter, the compact embryogenic callus differentiated up on MS media with 0.1 mg L−1 NAA and 0.5 mg L−1 kinetin supplemented with various concentrations of ancymidol. An addition of 0.75 mg L−1 ancymidol resulted in significant enhancement of somatic embryo formation and no malformed embryos were observed. Scanning electron micrographs and thin sections confirmed the structures as somatic embryos. Furthermore, these embryos were transferred to the same medium supplemented with glutamine, casein hydrolysate, and 3% sucrose for conversion into green shoots. These shoots could be multiplied in vitro using BAP-supplemented medium.ConclusionsAn effective method for conservation of an overexploited threatened medicinally important species Asparagus racemosus has been developed. This is the first report of the formation of somatic embryos from zygotic embryos in A. racemosus. Ancymidol in combination with kinetin and NAA was found to be most efficient for somatic embryo maturation and germination. The established protocol would certainly advance the efficient somatic embryo induction, maturation, and germination which could be utilized for large-scale propagation of Asparagus species.

Optimizing callus induction and proliferation for Agrobacterium-mediated transformation of Brachypodium distachyon

Brachypodium distachyon has emerged as the model species for important temperate grass crops such as wheat and barley and the genome of the B. distachyon community inbred line Bd21 has been sequenced. Methods for tissue culture and Agrobacterium-mediated transformation have been developed for this model grass as a resource for reverse genetics and functional genomic analyses. In order to obtain a high quantity and quality of compact embryogenic callus (CEC) in B. distachyon, it is important to examine and optimize the optimal concentration of the auxin 2,4-D (dichlorophenoxyacetic acid) to use in both callus induction and callus proliferation media. Here, we investigated the effects of different concentrations of 2,4-D on callus induction and callus proliferation of B. distachyon Bd21. Our results showed that 2.5 mg l−1 2,4-D is an optimal concentration to use for both callus induction and proliferation, although 5.0 mg l−1 may also be used for callus proliferation. Additionally, the suitability of hygromycin or bialaphos as selectable markers was examined and results indicated that hygromycin is significantly more efficient than bialaphos when using the Agrobacterium-mediated transformation system.

A simple and reliable multi-gene transformation method for switchgrass

A simple and reliable Agrobacterium -mediated transformation method was developed for switchgrass. Using this method, many transgenic plants carrying multiple genes-of-interest could be produced without untransformed escape. Switchgrass (Panicum virgatum L.) is a promising biomass crop for bioenergy. To obtain transgenic switchgrass plants carrying a multi-gene trait in a simple manner, an Agrobacterium-mediated transformation method was established by constructing a Gateway-based binary vector, optimizing transformation conditions and developing a novel selection method. A MultiRound Gateway-compatible destination binary vector carrying the bar selectable marker gene, pHKGB110, was constructed to introduce multiple genes of interest in a single transformation. Two reporter gene expression cassettes, GUSPlus and gfp, were constructed independently on two entry vectors and then introduced into a single T-DNA region of pHKGB110 via sequential LR reactions. Agrobacterium tumefaciens EHA101 carrying the resultant binary vector pHKGB112 and caryopsis-derived compact embryogenic calli were used for transformation experiments. Prolonged cocultivation for 7 days followed by cultivation on media containing meropenem improved transformation efficiency without overgrowth of Agrobacterium, which was, however, not inhibited by cefotaxime or Timentin. In addition, untransformed escape shoots were completely eliminated during the rooting stage by direct dipping the putatively transformed shoots into the herbicide Basta solution for a few seconds, designated as the 'herbicide dipping method'. It was also demonstrated that more than 90 % of the bar-positive transformants carried both reporters delivered from pHKGB112. This simple and reliable transformation method, which incorporates a new selection technique and the use of a MultiRound Gateway-based binary vector, would be suitable for producing a large number of transgenic lines carrying multiple genes.

Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21 with two binary vectors containing hygromycin resistance and GUS reporter genes

Brachypodium distachyon (Brachypodium) is a novel model plant for structural and functional genomic studies of temperate grasses. Brachypodium as a model plant has many favorable features, such as small size, small genome, short life cycle, self-fertility, and simple growth requirements. The genome sequence of the standard line Bd21 has been released and genomic resources have been developed.It is imperative to develop a method for efficient Agrobacterium-mediated Brachypodium transformation. Yellowish and compact embryogenic calli derived from immature embryos of the Bd21 were transformed with the Agrobacterium strain AGL1. Seven- and nine-week-old calli were used for transformation with Agrobacterium carrying either pCAMBIA 1301 and pCAUGH. Transformation efficiencies were assessed through histochemical GUS assay. The efficiency of transformation with pCAMBIA 1301 (based on the number of callus lines producing GUS-detected plantlets and the number of calli used for transformation) reached 20.1% (7-week-old calli) and 1.7% (9-week-old calli), and with pCAUGH (based on the number of GUS-detected plantlets and the number of regenerants) 90 and 87% for 7- and 9-week-old calli, respectively. High selection pressure was obtained by using pCAUGH, which is preferred for saving labor and time consumption during the callus selection.

Morphogenetic routes of long-term embryogenic callus culture of Areca catechu

Early morphogenetic events and repetitive embryogenesis from callus culture of betel nut palm (Areca catechu L.) were studied using scanning electron microscopy. On Murashige and Skoog (MS) medium supplemented with 2 mg dm-3 dicamba, callus culture has capacity to form plantlets via somatic embryogenesis and to form secondary embryos for about 4 years. However, various abnormal embryos without differentiation of the leaf sheath and shoot apical meristem were observed, which showed bell-shaped and then cup-shaped or mushroom-shaped structures. These abnormal embryos contained distinctive structures, including a disk-shape interior region, surfaces with grooves and a stalk-like posterior region. During subculture, these abnormal embryos enlarged, became deformed and gradually lose their shape and then converted into nodular, compact embryogenic callus. It was also found that secondary embryos originated from interior surfaces or posterior regions of abnormal embryos, and gave rise to the next cycle of normal and abnormal embryos.

Somatic embryogenesis and regeneration of Cenchrus ciliaris genotypes from immature inflorescence explants

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.

A protocol for Agrobacterium-mediated transformation of Brachypodium distachyon community standard line Bd21

Brachypodium distachyon is a novel model system for structural and functional genomics studies of temperate grasses because of its biological and genetic attributes. Recently, the genome sequence of the community standard line Bd21 has been released and the availability of an efficient transformation system is critical for the discovery and validation of the function of Brachypodium genes. Here, we provide an improved procedure for the facile and efficient Agrobacterium-mediated transformation of line Bd21. The protocol relies on the transformation of compact embryogenic calli derived from immature embryos using visual and chemical screening of transformed tissues and plants. The combination of green fluorescent protein expression and hygromycin resistance enables early identification of transformation events and drastically reduces the quantity of tissue to be handled throughout the selection process. Approximately eight independent fully developed transgenic Bd21 plants can be produced from each immature embryo, enabling the generation of thousands of T-DNA lines. The process--from wild-type seeds to transgenic T(1) seeds--takes approximately 8 months to complete.

Agrobacterium‐mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T‐DNA insertional mutagenesis

Brachypodium distachyon is a promising model system for the structural and functional genomics of temperate grasses because of its physical, genetic and genome attributes. The sequencing of the inbred line Bd21 (http://www.brachypodium.org) started in 2007. However, a transformation method remains to be developed for the community standard line Bd21. In this article, a facile, efficient and rapid transformation system for Bd21 is described using Agrobacterium-mediated transformation of compact embryogenic calli (CEC) derived from immature embryos. Key features of this system include: (i) the use of the green fluorescent protein (GFP) associated with hygromycin selection for rapid identification of transgenic calli and plants; (ii) the desiccation of CEC after inoculation with Agrobacterium; (iii) the utilization of Bd21 plants regenerated from tissue culture as a source of immature embryos; (iv) the control of the duration of the selection process; and (v) the supplementation of culture media with CuSO4 prior to and during the regeneration of transgenic plants. Approximately 17% of CEC produced transgenic plants, enabling the generation of hundreds of T-DNA insertion lines per experiment. GFP expression was observed in primary transformed Bd21 plants (T0) and their progeny (T1). The Mendelian inheritance of the transgenes was confirmed. An adaptor-anchor strategy was developed for efficient retrieval of flanking sequence tags (FSTs) of T-DNA inserts, and the resulting sequences are available in public databases. The production of T-DNA insertion lines and the retrieval of associated FSTs reported here for the reference inbred line Bd21 will facilitate large-scale functional genomics research in this model system.

Efficient Somatic Embryogenesis in Alstroemeria

In Alstroemeria high frequencies of compact embryogenic callus (CEC) induction (40%) and friable embryogenic callus (FEC) induction (15%) were obtained from nodes with axil tissue cultured first on a Murashige and Skoog (MS) medium supplemented with 10 μM thidiazuron and 0.5 μM indole-3-butyric acid and after that on a Schenk and Hildebrandt (SH) medium supplemented with 9.1 μM 2,4-dichlorophenoxy acetic acid and 2.2 μM benzylaminopurine (BA). Both types of callus were maintained on modified MS medium supplemented with 20.8 μM picloram. CEC and FEC formed somatic embryos and subsequently plants when transferred to MS medium supplemented with 2.2 μM BA. Plants were produced after 12 weeks (CEC) or after 16 weeks (FEC) of culture. Regenerated plants were established in the greenhouse and flowered normally.

Isolation of protoplasts, and culture and regeneration into plants in Alstroemeria

Summary An efficient system for the regeneration of plants from protoplasts was developed in Alstroemeria. Friable embryogenic callus (FEC) proved to be the best source for protoplast isolation and culture when compared with leaf tissue and compact embryogenic callus. Protoplast isolation was most efficient when FEC was cultured under vacuum for 5 min in an enzyme solution consisting of 4% cellulase, 0.5% Driselase and 0.2% Macerozyme, followed by culture for 12 – 16 h in the dark at 248C. Cell wall formation and colony formation were better in a liquid medium than on a semi-solid agarose medium. Micro-calluses were formed after 4 wk of culture. Ninety percent of the micro-calluses developed into FEC after 12 wk of culture on proliferation medium. FEC cultures produced somatic embryos on a regeneration medium and half of these somatic embryos developed shoots. Protoplast-derived plants showed more somaclonal variation than vegetatively propagated control plants.

Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye ( Secale cereale L.).

A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3-5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N6 regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.

Embryo Rescue and Induction of Somatic Embryogenesis as a Method to Overcome Seed Inviability in Zea mays ssp. mays × ; Zea mays ssp. parviglumis Crosses

Zea mays ssp. mays (2n=40) and Z. mays ssp. parviglumis (2n=20) were crossed to obtain hybrid plants by embryo rescue. Hybrid embryos were isolated and cultured on GarcĂ­a et al. (1992) basic medium supplemented with 2,4-dichlorophenoxyacetic acid and/or kinetin in different concentrations. Caryopses harvested 23 d after pollination (DAP) were turgid, with 0.3 to 0.5 mm long embryos, while those harvested 30 DAP were shrunken, with 1 to 1.5 mm long embryos. Twenty days after plating, 100 % of the younger embryos gave rise to white, compact embryogenic calli. Subsequently, coleoptiles, leaf-like structures, shoots and roots originated from them and 35 hybrid plants were regenerated from 60 embryos. Embryogenic or organogenic calli frequencies did not differ among hormonal treatments, but they decreased, on average, from 90.5 to 44.3 %, comparing 50 and 120-d-old cultures. The older embryos regenerated plants only by germination, although they gave rise to organogenic callus with low frequencies. Regenerated plants showed a somatic chromosome number of 2n=30, pollen fertility of 40 to 80 % and 15 % viable naked caryopses.

Friable embryogenic callus and somatic embryo formation from cotyledon explants of African marigold (Tagetes erecta L.).

Embryogenic callus and somatic embryos were induced from cotyledonary explants of African marigold (Tagetes erecta L.). Cotyledons were first cultured on MS medium supplemented with 2.0 mg l-1 2,4-D and 0.2 mg l-1 kinetin. After 5 weeks, calli were transferred to MS medium supplemented with 0.02 mg l-1 thidiazuron where compact embryogenic callus developed. Friable embryogenic callus developed when the compact embryogenic callus was transferred to medium containing 2,4-D and subcultured every 2 weeks. Friable embryogenic callus has been maintained for more than 2 years without losing the capacity to generate embryos. Embryo development was obtained when friable embryogenic callus was transferred to MS medium supplemented with 3 mg l-1 ABA and 60 g l-1 sucrose. The addition of 10-30 mM L-glutamine improved embryo development.

Partial Organogenesis and Somatic Embryogenesis from Petiole Explants of Field-grown Papaya (Carica papaya L.)

Petiole discs from young leaves of female papaya (L-45) plants were cultured in MS or B5-based media containing 0, 2.25, 4.5, 11.25, and 22.5 μm 2,4-D. Compact embryogenic callus emerged from vascular tissue of petiole discs in about 3 weeks. In MS medium, 66% and 51% explants formed embryogenic callus with 11.25 and 22.5 μm 2,4-D, respectively. On the other hand, 79% explants formed embryogenic callus in B5-based medium with 4.50 μm 2,4-D. However, explants became necrotic in B5-based medium with 22.5 μm 2,4-D. Subculturing callus in auxin-free medium resulted in the development of roots or somatic embryos. Microscopic observations revealed that the roots were produced only by the callus that had retained its continuity with the vascular tissue. This investigation revealed that petioles from field grown papaya plants are potential explants for somatic embryogenesis and 2-week exposure to 2,4-D is adequate for inducing morphogenesis. Additionally, an interaction between 2,4-D and the components in the MS and B5-based media was observed.

Callus friability and somatic embryogenesis inHevea brasiliensis

The influence of plant growth regulators, sucrose, calcium and various macronutrient media on callus friability and somatic embryogenesis was investigated inHevea brasiliensis Mull. Arg. Friable and embryogenic calli were spontaneously formed in two rubber tree clones (PR 107 and RRIM 600) on the Medium for Hevea (MH), with 3,4-dichlorophenoxyacetic acid (3,4-d), kinetin and sucrose, while compact embryogenic calli were enhanced in three other clones (PB 260, PB 235 and GT1). Callus friability was enhanced in clone PB 260 when the concentration of one growth factor (3,4-d or kinetin) was reduced from 4.5 μLM to 0.45 μM during the first culture, or when high sucrose or calcium levels 351 mM and 12 mM, respectively) were maintained during subcultures. The different macronutrient media did not alter callus texture but only use of MH and Murashige and Skoog (MS) media led to somatic embryogenesis. Friable calli obtained by modifying the auxin/cytokinin balance lost their embryogenic potential. In contrast, those obtained on media with high sucrose or calcium concentrations were mainly composed of embryogenic cells embedded in a mucilaginous matrix. Such calli could be of potential interest for establishing embryogenic cell suspension cultures.

High frequency somatic embryogenesis and plant regeneration from zygotic embryo-derived callus cultures of three Allium species

The plant regeneration ability of zygotic embryo-derived callus cultures was studied for 12 A. cepa varieties and accessions, two A. fistulosum varieties, one A. fistulosum x A. cepa interspecific hybrid and two A. porrum varieties. Compact embryogenic callus was induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid. The embryogenic calluses of all three Allium species were similar in appearance. For all accessions tested plants could be regenerated at a high frequency from this compact callus through somatic embryogenesis, when using kinetin supplemented MS medium (regeneration medium). Addition of abscisic acid to the regeneration medium stimulated the formation of both somatic embryos and shoots for a number of varieties. Concerning shoot regeneration from callus cultures, significant differences existed between genotypes of all accessions except one.

Transient Gene Expression in Maize, Rice, and Wheat Cells Using an Airgun Apparatus

An airgun apparatus has been constructed for transient gene expression studies of monocots. This device utilizes compressed air from a commercial airgun to propel macroprojectile and DNA-coated tungsten particles. The beta-glucuronidase (GUS) reporter gene was used to monitor transient expression in three distinct cell types of maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). The highest level of GUS activity in cultured maize cells was observed when distance between stopping plate and target cells was adjusted to 4.3 centimeters. Efficiency of transformation was estimated to be 4.4 x 10(-3). In a partial vacuum of 700 millimeters Hg, velocity of macroprojectile was measured at 520 meters per second with a 6% reduction in velocity at atmospheric pressure. A polyethylene film placed in the breech before firing contributed to a 12% increase in muzzle velocity. A 700 millimeters Hg level of vacuum was necessary for maximum number of transfornants. GUS expression was also detected in wheat leaf base tissue of microdissected shoot apices. High levels of transient gene expression were also observed in hard, compact embryogenic callus of rice. These results show that the airgun apparatus is a convenient, safe, and low-cost device for rapid transient gene expression studies in cereals.

Somatic Embryogenesis and Plant Regeneration from Inflorescence Callus of Zizania latifolia Turcz.

Summary A compact embryogenic callus was obtained from young inflorescence explants of Zizania latifolia Turcz. (Gramineae) cultured on a medium containing Murashige and Skoog's (1962) salts and vitamins, 0.25 mg·1-1 calcium pantothenate, 100 mg·1-1 asparagine and 1 mg·1-1 2,4-dichlorophenoxyacetic acid (2,4-D) under illumination or in darkness. The embryogenic nature of the callus was retained throughout subcultures on media containing a lower concentration of 2,4-D spanning more than 3 years. These compact organized calli produced many embryoids after transfer to media based on B5 (Gamborg et al. 1968) supplemented with either spermine (20 mg·1-1), kinetin (1 mg·1-1) and naphthaleneacetic acid (NAA, 0.1 mg·1-1), or benzyladenine (1 mg·1-1), kinetin (1 mg·1-1) and NAA (0.1 mg·1-1). These embryoids germinated and produced plantlets in 30 days on the same medium or when transferred to MS or B5 medium without hormonal adjuvents.

Plant Regeneration by Somatic Embryogenesis From Parts of Cultured Mature Embryos of Pennisetum americanum (L.) K. Schum

Summary Whole and excised parts of mature embryos and seedlings of Pennisetum americanum (L.) K. Schum. were cultured in Murashige and Skoog's medium (MS) supplemented with 2,4-dichlo-rophenoxyacetic acid and coconut milk. A soft friable callus, which later developed only roots, was formed when whole embryos or isolated mesocotyl and radicle were cultured. A compact embryogenic callus was induced only from the shoot apex and the two youngest leaf primordia excised from mature embryos or 4–5 day old seedlings. The complete removal of mesocotyl tissue from the base of the shoot apex at the time of culture was critical for the formation of compact callus, and for inhibiting the development of the friable callus. The embryoids developed by a sequence of internal segmenting divisions from single embryogenic cells, and showed a well organized embryonic axis with a closed vascular system, a coleoptile, a coleorhiza, and a scutellum. Upon transfer to media which promoted shoot and root growth, normal plantlets were formed and transferred successfully to soil.