Embryogenic Callus Induction Research Articles

Protocol for Callus Induction and Somatic Embryogenesis in Moso Bamboo

Moso bamboo [Phyllostachys heterocycla var. pubescens (Mazel ex J. Houz.) Ohwi] is one of the most important forest crops in China and the rest of Asia. Although many sympodial bamboo tissue culture protocols have been established, there is no protocol available for plantlet regeneration as indicated by callus induction for monopodial bamboos, such as Moso bamboo. In the present report, embryogenic callus induction, embryoid development, and germination were established for Moso bamboo from zygotic seed embryos. Callus was initiated from zygotic embryos after 10–20 d culture on MS media supplemented with 4.0 mg/L 2, 4-D and 0.1 mg/L zeatin (ZT). About 50% of the explants produced calli, and nearly 15% of the calli were found to be embryogenic in nature. These embryogenic calli can be subcultured for proliferation in the Murashige and Skoog media (MS) supplemented with 0.5–2.0 mg/L 2, 4-D. These calli were found to have maintained their capacity for regeneration even after one year of subculture. The viable somatic embryoids regenerated in medium containing 5.0–7.0 mg/L ZT. Nearly 5% of the calli were found capable of regenerating into plantlets directly in MS medium containing 5.0–7.0 mg/L ZT. Root growth was more pronounced when the plantlets were transferred to medium containing 2.0 mg/L NAA. After 30 days of subculture, the plantlets were transferred to a greenhouse.

Plant regeneration via somatic embryogenesis from solid and suspension cultures of Vitis vinifera L. cv. ‘Manicure Finger’

Vitis vinifera L. cv. ‘Manicure Finger’ is one of the major table grape varieties in China. To provide a strong foundation for genetic transformation with potential for crop improvement, we undertook plant regeneration via somatic embryogenesis. Anthers and gynoecia were harvested from immature flowers and used as explants to induce embryogenic calli. Explants cultured in MS1 medium (based on Murashige and Skoog basal salts), supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4-μM 6-benzylaminopurine (6-BA) showed the highest rates of embryogenic callus induction (3.7% ± 1.3% for anthers and 4.8% ± 2.5% for gynoecia). After several months, somatic embryos were produced from embryogenic calli cultured in plant growth regulator-free MS2 medium (with reduced sucrose). Somatic embryos (SE) at the cotyledonary stage were isolated and cultured on three different media (MS2, MS3, or B) for conversion into plantlets, the efficiency of which ranged from 63.9% ± 4.8% to 83.9% ± 8.4%. After 1 mo of in vitro culture, 80% of plants with at least six leaves were successfully transplanted into soil. SE was repeatedly induced from previously induced somatic embryos for up to 1.5 yr. Using embryogenic calli as starting material, suspension cultures containing embryogenic cell aggregates were also established in liquid MS medium supplemented with 4.5-μM 2,4-D. The embryogenic cell aggregates continued to proliferate without differentiating for successive subculture cycles. After transfer to 2,4-D-free liquid medium for 4 wk, an average of 63.7% ± 9.0% mature SEs were produced per 20 mL of liquid medium. More than 40% of somatic embryos at cotyledonary stage, derived from the suspension cultures, successfully germinated into plants using solid medium.

Optimization of somatic embryogenesis protocol in Lycopersicon esculentum L. using plant growth regulators and seaweed extracts

In the present study a simple and efficient somatic embryogenesis system was developed from leaf explants of Lycopersicon esculentum L. The protocol has been developed by using plant growth regulators and seaweed extracts a natural biostimulant. The leaf sections were initially cultured on to leaf embryogenic callus induction medium fortified with various concentration and combinations of 2,4-dichlorophenoxy acetic acid (0.2–1.0 mg L−1), picloram (0.2–1.0 mg L−1), and kinetin (0.1–0.5 mg L−1). The best responding concentration in induction of friable embryogenic callus was tested for the proliferation. The friable cultures were detached from the mother culture and inoculated in three different media supplemented with plant growth regulators, plus 0–25 % Caulerpa scalpelliformis or 0–25 % Gracilaria corticata extracts for embryo development. A twofold increase in maturation and germination of somatic embryos was observed in the media containing seaweed extracts (MSMG2 and MSMG3) than the control (MSMG1). The plantlets transferred from plant growth chamber to greenhouse conditions exhibited higher survival rate (90 %) than directly shifted plantlets.

Effect of 2,4-D on Embryogenic Callus Induction of Malaysian indica Rice (Oryza sativa L.) Cultivars MR123 and MR127

The aim of this study is to study the effect of various concentrations of 2,4-D on embryogenic callus induction of indica rice MR123 cultivar and MR127 cultivar using mature seeds. Optimal media for induction of callus of both cultivars was MS basal media supplemented with 30g/L sucrose, 500mg/L glutamine and 500mg/L proline supplemented with 2.5 mg/L 2,4-D. The highest percentage of callus induction was 70% and 76% for MR123 and MR127 respectively. Both cultivars produced an embryogenic callus from scutellum after a week in culture with white-yellowish in color. The viability tested using Evans blue show callus obtained was embryogenic. This simple protocol using staining method could be used for screening embryogenic callus before further experiments can be conducted.

Somatic embryogenesis of Neoglaziovia variegata (Arruda) Mez, an important source of fiber from native Brazilian bromeliads

Neoglaziovia variegata (Arruda) Mez, a Bromeliad endemic to the Brazilian Caatinga and source of fiber for many different products, is considered an endangered species. Leaf and stem segments were cultivated on Murashige and Skoog (MS) medium with combinations of 2,4- dichlorophenoxyacetic acid (2,4-D) (0, 9, 18 and 27 µM) and glutamine (0, 50, 100, 150, 200 mg L-1) for the induction of embryogenic calli. Leaf segments did not present a favorable response for the combinations analyzed. The best response for embryogenic calli frequency with the best distribution of somatic embryos on the surface and better conversion rate was obtained using the stems explants in the medium containing 18 µM 2,4-D and 100 mg L-1 glutamine. The embryogenic calli obtained from the different induction media were transferred to MS medium supplemented with 30 g.L-1 sucrose, 2.4 g.L-1 Phytagel®, 0.53 µM α-naphthalene acetic acid (NAA) and 0.88 µM of 6-benzylaminopurine (BAP). The regenerated plants presented normal growth; however, many embryos did not fully develop.

Efficient callus induction and plantlets regeneration in durum wheat using mature embryos

The use of mature embryos as explants to initiate cultures is a best alternative to save time and costs, especially for producing somatic embryos for genetic transformation of durum wheat. However, plantlets regeneration from cultures derived from matured embryos is usually low. In this study, we tested matured embryos as explants from eight Moroccan durum wheat varieties (‘Irden’, ‘Marzak’, ‘Kyperounda’, ‘Isly’, ‘Amria’, ‘Karim’, ‘Marouane’ and ‘Tomouh’) to define suitable culture media for obtaining high frequencies of somatic embryogenesis and in vitro plantlets regeneration. For this purpose, we tested five induction and maintenance media (M1 to M5) based on MS media (macro and oligo-elements) which differed with respect to concentrations of plant hormones (2,4-D and BA), vitamins, sucrose, maltose, L-asparagine, and solidifying agents. All tested media induced embryogenic callus for the varieties and regenerate plantlets. However, a significant effect of variety, medium and variety × medium interaction were observed for callus induction and regeneration. Average callus growth as measured by relative fresh weight growth rate (RFWGR) across different media was the highest for ‘Amria’ (7215.4%) and the lowest for ‘Tomouh’ (2088.2%). M1 (2 mg/L 2,4-D) and M5 (3 mg/L 2,4-D) media gave highest RFWGR(6892.1% and 6332%, respectively) and M3 (1 mg/L 2,4-D) was the lowest (3708.8%), across different varieties. However, the embryogenic callus from M3 media regenerated the highest percentage of plantlet, upon transfer to regeneration medium, for most of the varieties. For the varieties ‘Marouane’, ‘Kyperounda’, ‘Marzak’, ‘Karim’, and ‘Tomouh’, the favourable medium was M3, whereas, for ‘Isly’, ‘Irden’ and ‘Amria’, both M2 (2.5 mg/L BA and 2.5 mg/L 2,4-D) and M3 were the favourable media for embryogenic callus induction. In this study, for the first time, favourable media for induction and regeneration from mature embryo of Moroccan durum wheat varieties were identified. These media will be used for callus induction and genetic transformation.

Maturation of Anthurium andraeanum cv. Eidibel somatic embryos from nodal segments

Maturation of somatic embryos of Anthurium andraeanum cv. Eidibel from embryogenic callus was evaluated. Following induction of embryogenic calli from nodal segments, tissues were transferred to 125-mL Erlenmeyer flasks containing 25 mL liquid medium, with 0, 4.52, or 9.05 μM 2,4-dichlorophenoxyacetic acid and 0, 0.47, or 2.32 μM kinetin. Callus cultures were maintained in a dark growth room at 25 ± 2°C. At 45 d, the mass of embryogenic calli, number of primary and secondary somatic embryos, and percentage browning were evaluated. Nonparametric tests were used to evaluate color, texture, and somatic embryo development. The highest yield of somatic embryos was in the medium with 0.47 μM kinetin. Calli were friable, with a lower yield of secondary somatic embryos, and have minimal browning. Histology revealed polar globular somatic embryos and mature somatic embryos with defined apical and root meristematic zones, axillary buds, and primary leaves. These are important features for converting somatic embryos into plantlets.

Influence of auxins on somatic embryogenesis and alkaloid accumulation in Leucojum aestivum callus

Abstract In vitro cultures of Leucojum aestivum are considered as an alternative for the production of galanthamine, which is used for the symptomatic treatment of Alzheimer’s disease. We studied the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro-o-anisic acid (dicamba) at concentrations of 25 and 50 µM on the induction of embryogenic callus and its capacity to induce somatic embryogenesis and alkaloid accumulation. The embryogenic response of the explants was from 30% for 25 µM of dicamba to 100% for picloram (for both 25 and 50 µM). 2,4-D (50 µM) stimulated greater callus proliferation and somatic embryo induction as compared to the other auxins. Polyethylene glycol (PEG) stimulated somatic embryo maturation. Callus grown on media containing 50 µM of auxins produced fewer phenolic compounds as compared with callus grown on media containing 25 µM of auxins. GC-MS analyses showed seven alkaloids in the in vivo bulbs and two to four in callus culture. Galanthamine was detected in callus cultivated with 2,4-D (25, 50 µM), picloram (25 µM), and dicamba (50 µM). Other alkaloids, trisphaeridine, tazettine, and 11-hydroxyvittatine were accumulated only in callus growing on medium with picloram (50 µM).

New approaches to improve the efficiency of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) from mature zygotic embryos

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from mature zygotic embryos of oil palm. Embryogenic calli were induced from mature zygotic embryos of oil palm on modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid or picloram, alone or in combination with activated charcoal. The greatest frequency of embryogenic callus induction (97.5%) was obtained by culturing mature zygotic embryos on callus induction medium with 450 μM picloram and 2.5 g L−1 activated charcoal. Embryogenic calli proliferated on a medium with a reduced concentration of picloram. Embryogenic calli were then subcultured on a medium supplemented with 12.3 μM 2-isopentenyladenine and 0.54 μM naphthaleneacetic acid, with subcultures at 4-wk intervals. Somatic embryos were regenerated on a medium with Murashige and Skoog macro- and micronutrients at half-strength concentrations supplemented with 20 g L−1 sucrose, 2.5 g L−1 activated charcoal, and 2.5 g L−1 Phytagel. Detailed histological analysis revealed that somatic embryogenesis followed an indirect pathway. Primary calli were observed after 4–6 wk of culture and progressed to embryogenic calli at 12 wk. Embryogenic cells exhibited dense protoplasm, a high nucleoplasmic ratio, and small starch grains. Proembryos, which seemed to have a multicellular origin, formed after 16–20 wk of culture and successive cell divisions. Differentiated somatic embryos had a haustorium, a plumule, and the first and second foliar sheaths. In differentiated embryos, the radicular protrusion was not apparent because it generally does not appear until after the first true leaves emerge.

A simple protocol for somatic embryogenesis in Rosa hybrida L. cv. Apollo

SummaryA successful regeneration system from in vitro-derived leaves of Rosa hybrida cv. Apollo, via somatic embryogenesis, has been established. The influence of different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and zeatin on the induction of embryogenic calli was evaluated. The highest percentage of embryogenic callus induction (48.3%) was obtained on full-strength MS medium containing 1.0 mg l–1 2,4-D, while at higher concentrations (4.0 or 5.0 mg l–1 2,4-D) no embryogenic calli were observed. The induction of embryogenic calli also occurred on MS media containing 0.5, 1.0, 2.0, or 4.0 mg l–1 naphthaleneacetic acid (NAA). The lowest concentration of NAA (0.5 mg l–1) showed the highest rate of production of embryogenic calli. For maturation, embryogenic calli were transferred onto MS media containing different concentrations of 6-benzyladenine (BA), thidiazuron (TDZ), or abscisic acid (ABA) alone, or in various combinations. The highest rate of embryo maturation (85.0%) was observed on MS medium supplemented with 1.0 mg l–1 ABA alone. Mature embryos showed the highest rate of germination on full-strength MS medium containing 2.0 mg l–1 BA alone. After rooting, well-developed plantlets were maintained under greenhouse conditions. The morphological characteristics and flower colours of somatic embryo-derived plants were the same as the donor parent.

Studies on Embryogenic Competence and Regeneration Potential With Relation to Anthocyanin Biosynthesis in Cotton (<i>G.hirsutum</i>)

Varietal genotype is very important in determining which varieties of cotton will produce embryos because most of the commercial varieties are recalcitrant to regeneration. Identification of genes involved in the induction of competence for embryogenesis and subsequent embryo development presents a challenge for modern molecular biology. Certain varieties of cotton, especially Coker 310 exhibit anthocyanin pigmentation during embryogenic calli induction and form somatic embryos readily, while other genotypes MCU 12 and KC3 does not exhibit anthocyanin pigmentation and are recalcitrant to regeneration. To find out the relationship between anthocyanin synthesis and somatic embryogenesis if any, candidate genes for anthocyanin synthesis and somatic embryogenesis were tested for its presence through PCR analysis in genomic DNA samples of Coker 310, MCU 12 and KC3. The results of this study revealed that the candidate gene sequences for the enzymes involved in anthocyanin biosynthesis (chalcone synthase, flavanone 3 hydroxylase, dihydroflavanol reductase, leucoanthocyanidin deoxygenase and anthocyanidin synthase) and transcription factors that regulate the expression of anthocyanin biosynthesis genes (bHLH and MYB) and the candidate gene sequences encoding the proteins associated with somatic embryogenesis (the arabinogalactan proteins, lipid transfer proteins and high mobility group proteins) were found in all the samples of Coker 310 while none of them were present in the samples of MCU 12 and KC 3. It may be inferred from this study that apart from the genes associated with somatic embryogenesis, genes associated with anthocyanin biosynthesis and regulation may also be involved in somatic embryogenesis and regeneration in cotton. Also, these genes can be used as reliable markers to screen cotton varieties for their embryogenic potential.

Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination <i>Artemisia annua</i> Progeny and Artemisinin Formation in Regenerated Plants

To enhance the understanding of artemisinin biosynthesis, we have successfully bred self-pollination Artemisia annua plants. Here, we report efficient somatic embryogenesis and organogenesis of self-pollination plants and artemisinin formation in regenerated plants. The first through sixth nodal leaves of seedlings are used as explants. On agar-solidified MS basal medium supplemented with TDZ (0.6 mg/l) and IBA (0.1 mg/l), all explants after inoculation of less than 3 weeks start to form embryogenic calli, which further produce globular, torpedo, heart and early cotyledon embryos. In all six positional leaves, explants from the sixth leaf show the rapidest responses to induction of embryogenic calli and somatic embryos. On this medium, somatic embryos continuously develop into adventitious buds, which can form adventitious roots on a rooting medium containing NAA (0.5 mg/l). Meanwhile, on agar-solidified MS basal medium supplemented with BAP (1 mg/l) and NAA (0.05 mg/l), approximately 100% of explants from leaves #3-6 form calli in less than 3 weeks of inoculation and adventitious buds via organogenesis in 3-4 weeks. In all six positional leaves, explants from the sixth leaf exhibit the rapidest response to induction of calli and adventitious buds. Nearly 100% adventitious buds can form adventitious roots on the rooting medium. Regenerated plants from both somatic embryogenesis and organogenesis complete self-pollination to produce seeds in 80-90 days of growth in growth chamber. LC-ESI-MS analysis demonstrates that regenerated plants biosynthesize artemisinin. These results show the highly efficient regeneration capacity of self-pollination A. annua plants that can form a new platform to enhance the understanding of artemisinin biosynthesis and metabolic engineering.

Analysis of retrotransposition and DNA methylation in barley callus culture

Mature barley (Hordeum vulgare cv. Zafer-160) embryos were cultured on callus induction medium (MS+ 4 mg l-1 Dicamba) for 30 days and embryogenic calli were transferred onto regeneration medium (MS+ 0.5 mg l-1 trans-zeatin riboside). Callus induction percentage was 67.2%; embryogenic callus induction percentage was 42.3% and their regeneration percentage was 63.8%. Retrotransposon movements and methylation alterations in 15-day-old, 30-day-old calli and 4-day-old barley seedling (control) were investigated with Inter-Retrotransposon Amplified Polymorphism (IRAP) and Methylation-Sensitive Restriction Fingerprinting (MSRF), respectively. IRAP patterns were quite monomorphic however MSRF indicated increase in cytosine methylation during callus formation. Changes in retroelement movements and methylation alterations were evaluated and discussed in the light of literature.

PEMBENTUKAN KALUS REMAH DARI EKSPLAN DAUN RAMIN (Gonystylus bancanus (Miq) Kurz.)

Ramin (Gonystylus bancanus (Miq.) Kurz) is the most favourite and the most over exploited woody species. This species has been listed in CITES APPENDIX II from 2004. The purpose of this experiment is to obtain the best treatment for friable and embryogenic callus formation that can develop to somatic embryo. Basal Murashige and Skoog (MS) media was used as growth medium. The experiment was conducted in three stages : callus induction and propagation stage, friable callus induction stage and embryogenic callus induction stage. The treatment of 3.0 – 5.0 mg/l 2,4-D was used for callus induction. The best of callus was propagated in 5.0 mg/l 2,4-D + 1.0 – 2.0 mg/l thidiazuron. The best of callus was subcultured for callus friable induction used 6.0 mg/l 2,4-D + 1.0 – 2.0 mg/l thidiazuron + 1.0 – 2.0 mg/l biotin. The best friable callus was subcultured for embryogenic callus induction used 7.0 – 8.0 mg/l 2,4-D + 1.0 – 2.0 mg/l biotin. The observation was made on texture, percentages, performance and color of friable callus. The results showed that callus can be induced on 5.0 mg/l 2,4-D. The treatment of 6.0 mg/l 2,4-D + thidiazuron produced friable callus yellowish on color. 2,4-D + biotin treatment produced very friable callus with yellowish on color and with no embryogenic callus development.

Multiplication of embryogenic calli in Coffea arabica L.

The goal of this project was to evaluate the embryogenic callus induction of two Coffea arabica clones selected for their characteristics of rust resistance and high yield, as well as to compare their multiplication in two different media under both solid and liquid cultivation conditions. The protocol described by Teixeira et al. (2004) was used for callus induction in a randomized block design in which each clone was considered a treatment. Evaluation of callus induction was carried out 180 days after initiation by counting embryogenic calli. For callus multiplication, the treatments consisted of two different media (stage two of Albarran et al. (2004) and the multiplication medium described by Teixeira et al. (2004)) and two cultivation systems (solid and liquid). Evaluations were conducted by weighing calli 21, 42 and 63 days after initiation of the experiment. The two studied clones exhibited the same potential for embryogenic callus induction. The potential for embryogenic callus multiplication was influenced by the plant's genotype. When compared with the liquid system, the solid system displayed the highest level of embryogenic callus multiplication for the clones studied.

English

  Genetic transformation is considered as one of the most promising options for improvement of crop traits. Current transformation methods for sweetpotato depend on plant regeneration through organogenesis or somatic embryogenesis. Somatic embryogenesis and plant regeneration at a high frequency has been restricted to a few sweetpotato varieties. Three auxins namely:  2,4-dichlorophenoxyacetic acid (2,4-D), 4-fluoroamphetamine (4-FA) and 4,5-trichlorophenoxyacetic acid (2,4,5-T)were investigated in this study for enhancing somatic embryogenesis from various plant organs of recalcitrant African sweetpotato cultivars. 2,4-D was found to be the best (p ≤ 0.05) for induction of embryogenic callus. Cultivar Bwanjule had the highest (20.2%) embryogenic callus frequency among the five African cultivars tested. The highest number of plants in this study was regenerated from the non-African cultivar variety Jonathan on media supplemented with 0.2 mg Zeatin. The emergence of roots from callus of recalcitrant Ugandan cultivars and the comparable high embryogenic responses in this work demonstrate the potential for regenerating plants from African cultivars that have not been regenerated before. The regeneration of roots in this work could be useful for the initiation of root cultures. The most important application of this work is in genetic transformation of sweet potato, particularly for improvement of resistance to weevils.   Key words: Embryogenesis, plant growth regulators, plant regeneration, Ipomoea batatas

Propagação in vitro de genótipos de alface via embriogênese somática

A propagação in vitro via embriogênese somática é uma alternativa eficiente para a propagação em larga escala do material vegetal. No entanto, não há relatos do desenvolvimento de protocolos completos de embriogênese somática, com produção de plantas de alface. O presente trabalho teve como objetivo estabelecer a propagação in vitro de genótipos de alface, Paris White e Red Salad Bowl, avaliando a indução dos calos embriogênicos, regeneração dos embriões somáticos e posterior conversão em plantas. Para a indução de embriogênese somática, duas fontes de explante (folhas cotiledonares inteiras e seccionadas) foram cultivadas em meio MS+10,75µM de ANA+0,89µM de BA. A proliferação dos calos embriogênicos foi realizada em meio MS+24µM de AIA+0,15µM de BA. Para a maturação dos embriões somáticos e conversão em plantas, utilizou-se meio MS sem regulador de crescimento (semi-sólido sem carvão ativado e com carvão ativado e meio líquido). A fonte do explante seccionada foi estatisticamente superior apenas para o genótipo Paris White. Para a regeneração dos embriões somáticos, observou-se que, no genótipo Paris White, o meio líquido foi superior estatisticamente, quando comparado aos meios semi-sólidos e, para o genótipo Red Salad Bowl, foi o meio semi-sólido com acréscimo de carvão ativado. Tendo em vista o potencial da aplicabilidade comercial da embriogênese somática para a produção em larga escala de plântulas de alface do genótipo Paris White, os protocolos de indução, proliferação, maturação dos embriões somáticos e conversão em plantas, foram adequados, em especial, em sistema líquido.

An efficient regeneration system via somatic embryogenesis in mango ginger (Curcuma amada Roxb.)

A reproducible protocol for somatic embryogenesis was established for mango ginger (Curcuma amada Roxb.)—an important horticultural aromatic rhizomatous plant. Embryogenic callus induction was obtained from leaf sheath explants of in vitro raised plants on Murashige and Skoog (MS) agar medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid and 0.5 mg/L 6-benzyladenine (BA). Embryogenic callus proliferation, somatic embryo (SE) formation and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose and BA on SE formation were also evaluated. Half strength MS liquid medium necessary for SE formation and optimal sucrose concentration was found to be 3.0 %. BA at 0.3 mg/L produced the highest number (84.71 %) of SEs from leaf sheath explants. Secondary somatic embryos originated from primary somatic embryos on the same medium supplemented with 0.4–0.6 mg/L BA. Stereo microscopic and scanning electron microscopic observation revealed that the globular and torpedo shaped somatic embryos resulted in suspension culture during development. Mature somatic embryos germinated readily and developed into normal plantlets after 3 weeks on half strength MS basal agar medium under dark condition. Well rooted plantlets were successfully acclimatized at the survival rate of 70 %.

Genetic transformation and full recovery of alfalfa plants via secondary somatic embryogenesis

A genetic transformation method via secondary somatic embryogenesis was developed for alfalfa (Medicago sativa L.). Mature somatic embryos of alfalfa were infected by Agrobacterium strain GV3101 containing the binary vector pCAMBIA2301. pCAMBIA2301 harbors the uidA Gus reporter gene and npt II acts as the selectable marker gene. Infected primary embryos were placed on SH2K medium containing plant growth regulators to induce cell dedifferentiation and embryogenesis under 75 mg/L kanamycin selection. The induced calli were transferred to plant medium free of plant growth regulators for embryo formation while maintaining selection. Somatic embryos germinated normally upon transfer to a germination medium. Plants were recovered and grown in a tissue culture room before transfer to a greenhouse. Histochemical analysis showed high levels of GUS activity in secondary somatic embryos and in different organs of plants recovered from secondary somatic embryos. The presence and stable integration of transgenes in recovered plants were confirmed by polymerase chain reaction using transgene-specific primers and Southern blot hybridization using the npt II gene probe. The average transformation efficiency achieved via secondary somatic embryogenesis was 15.2%. The selection for transformation throughout the cell dedifferentiation and embryogenic callus induction phases was very effective, and no regenerated plants escaped the selection procedure. Alfalfa transformation is usually achieved through somatic embryogenesis using different organs of developed plants. Use of somatic embryos as explants for transformation can avoid the plant development phase, providing a faster procedure for introduction of new traits and facilitates further engineering of previously transformed lines.

Efficient Regeneration Potential is Closely Related to Auxin Exposure Time and Catalase Metabolism During the Somatic Embryogenesis of Immature Embryos in Triticum aestivum L

Regeneration of cultured tissue is a prerequisite of Agrobacterium- and biolistic-mediated plant transformation. In this study, an efficient protocol for improving wheat (Triticum aestivum L.) immature embryo regeneration was developed. Based on the statistical analysis of embryogenic callus induction efficiency, green spot differentiation efficiency, and plant regeneration efficiency from five wheat accessions, improved culture conditions were found to be more effective for embryogenic callus production than the traditional conditions. Using semi-quantitative reverse transcription polymerase chain reaction, a candidate gene, designated as TaCAT1, which encodes a catalase was identified to have a significant correlation with high-regeneration trait of wheat immature embryos. Three amino acid substitutions were found in TaCAT1 protein between high- and low-regeneration wheat accessions. Hydrogen peroxide content in the cultured calli increased from day 5 to 15, and then decreased sharply on day 20, followed by a second peak on day 25 during regeneration stage. Furthermore, a 3,500-bp 5' flanking region upstream of the first codon ATG of TaCAT1 was isolated using inverse polymerase chain reaction. In silico, analysis revealed that the TaCAT1 promoter contained two regulatory motifs associated with responses to auxin.