Embryo Regeneration Research Articles

English

The present study was conducted with the aim of evaluating some of the factors that influence induction and regeneration of somatic embryos in Vitex doniana since there are no available reports on tissue culture of this tree species. Leaves from plants growing under temporary shed were cultured on Murashige and Skoog supplemented with silver nitrate and four amino acids (proline, tryptophan, lysine and leucine) at varying concentrations, 0.11 mg/l thidiazuron, 2% sucrose and 100 mg/l myo-inositol in separate experiments. The explants cultured on media supplemented with tryptophan at 30.6 mg/l produced the optimal (6.5) number of embryos per explant. This number was fivefold more than the number obtained in the control. On the other hand, it was observed that the explants on media supplemented with silver nitrate at 8.45 mg/l gave the same mean (6.5) number of embryos per explant. These first ever results on the induction of somatic embryo in V. doniana could be used for mass propagation and to select useful traits of this tree species at the cellular level. However, further work needs to be done on the conversion of the regenerated embryos.   Key words: Amino acids, leaf explant, silver nitrate, somatic embryogenesis, Vitex doniana.

Improvement of Regeneration of Pelargonium radula via Somatic Embryogenesis

In vitro stem segments of Pelargonium radula cultured for callusing then differentiated into somatic embryos and subsequently regenerated plantlets. Initiation of callus was observed in culture medium containing low concentrations of the plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) and/or α-naphthalene acetic acid (NAA). At 0.2 mg/L 2,4-D and 0.2 mg/L NAA was showing the highest rate (92%) of callus induction. The callus showed no sign of browning after sub-cultured. Sub-culturing the callus onto medium with 0.2 mg/L 2,4-D showed the highest in proliferation rate resulted 13.45g weight of callus. The presence of agar at 6 g/L and 0.5 mg/L Gibberellic acid (GA3) improved the regeneration of the somatic embryos, which produced maximum number of plantlets (15 plantlets). Agar with concentration of 9 to12 g/L increased the incidence of browning. The former medium was more successful in plantlet regeneration when the selected embryos were subsequently transferred to regeneration medium with 3 g/L agar, 0.5 mg/L GA3 and 0.5 mg/L Benzylaminopurine (BAP).

Effects of inter-culture, arabinogalactan proteins, and hydrogen peroxide on the plant regeneration of wheat immature embryos

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins (AGPs) and hydrogen peroxide (H2O2) have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199 (KN199) and Xinchun 9 (XC9), together with low-frequency regeneration wheat line Chinese Spring (CS), presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L−1 AGP or 0.005 to 0.01 ‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L−1 AGP application level in callus induction medium plant regeneration rates of 8.49, 409.06 and 283.16% were achieved for Jimai 22 (JM22), Jingdong 18 (JD18) and Yangmai 18 (YM18), respectively; whereas at 100 mg L−1 AGP level, CS (105.44%), Chuannong 16 (CN16) (80.60%) and Ningchun 4 (NC4) (62.87%) acted the best. Moreover CS (79.05%), JM22 (7.55%), CN16 (101.87%), YM18 (365.56%), Yangmai 20 (YM20) (10.48%), and CB301 (187.40%) were more responsive to 0.005 ‰ of H2O2, and NC4 (35.37%) obtained the highest shoot regeneration rates at 0.01 ‰ of H2O2. Overall, these two methods, inter-culture and AGP (or H2O2) application, can be further applied to wheat transgenic research.

Plantlet Regeneration of Somatic embryos from Leaf Explants of Mentha arvensis(L.) A medicinally important Plant

The present study was conducted with the aim of evaluating some of the factors that influence induction and plantlet regeneration of somatic embryos in Mentha arvensis ( L) var piperascens Holmes (menthol or Japanese mint)since there are no available reports on Somatic embryogenesis and regeneration of plantlet in this medicinally important plant species. Leaves from plants growing under temporary shed were cultured on Murashige and Skoog medium fortified with (0.5-5.0mg/L) Napthlene acetic acid (NAA)+(0.5 mg/L)Thidizuron(TDZ). High frequency of somatic embryo formation was found at (2.5mg/L) NAA+ (0.5mg/L) TDZ in leaf explants respectively, Secondary somatic embryogenesis was also observed when primary somatic embryos were sub cultured same somatic embryo induction medium well developed cotyledonary stage embryos were germinated on MS medium supplemented with (0.5mg/L) (NAA) + (0.5-5.0mg/L)TDZ maximum 80% of somatic embryos germination and plant let formation was found at (2.5 mg/L) NAA+ (0.5 mg/L) (TDZ). The post translation survival rate of plants was 80% plants and flowers formation were morphological similar to the mother plants.

Performance of peanut mutants and their offspring generated from mixed high-energy particle field radiation and tissue culture

To develop new ways to breed peanut, we irradiated seeds of the Luhua 11 cultivar with a mixed high-energy particle field at different doses. The embryonic leaflets were extracted as explants and incubated on somatic embryo induction medium and then on somatic embryo germination and regeneration medium. After being grafted, the M1-generation plants were transplanted, and seeds from each M1-generation plant were harvested. In the following year, the M2-generation seeds were planted separately. Some M2-generation plants showed distinct character segregation relative to the mutagenic parent in terms of vigor, fertility, plant height, branch number, and pod size and shape. M2-generation plants that had a high pod weight per plant tended to produce M3-generation offspring that also had a high pod weight per plant, much higher than that of the mutagenic parent, Luhua 11. M4-generation seeds varied greatly in quality, and 35 individuals with an increased fat content (>55%) were obtained. Overall, the results indicate that the combination of mutagenesis via mixed high-energy particle field exposure and tissue culture is promising for peanut breeding.

Study of senescence in old cultures of the Bactris gasipaes Kunth in vitro

The in vitro propagation is a technique that is used for the large-scale generation of stable genotypes that are free of disease. Satisfactory propagation has been obtained using Bactris gasipaes Kunth through the direct in vitro regeneration of adventitious buds and somatic embryos, which is likely because of the pluri—and totipotency of the preprocambial cells, which reduces the probability of genetic instability. The consequences of the prolonged period of in vitro cultivation of perennial species such as the peach palm are not clear, as well as the senescence process has only been investigated in species originating from callus structures. The senescence of peach palm clones established and cultivated in vitro for 8 years through direct morphogenic methods (in vitro regeneration of adventitious buds without callus formation) was investigated in the leaves, roots and stem bases using histological, histochemical and ultrastructural analyses, in addition to the TUNEL reaction, to detect programmed cell death. These data from old cultures (8 years cultured) were compared to similar data from plants of the same species that had been established and cultivated in vitro for 1 year (young cultures). These analyses demonstrated the existence of intensive programmed cell death in the various tissues of the old cultures. The in vitro cultivation of peach palm for long periods promoted generalized senescence and probable aging of clones.

An efficient protocol for somatic embryogenesis of garlic (Allium sativum L.) using root tip as explant

Genetic improvement of garlic through conventional breeding is very difficult due to sterile nature of its flower. Hence, an alternative system is desirable to induce genetic variation. Tissue culture could be a good opportunities and somatic embryogenesis is one of the potential techniques of tissue culture for in vitro regeneration of garlic plant. The successes and production of somatic embryo depends on several factors such as optimization of media components, genotypes and explant type. Therefore, in the present investigation, garlic root tips were used as explant for callus and somatic embryo induction under different plant growth regulator combination. It was found that MS+1.0 mg l-1 2,4-D was the most favorable (86.10% regeneration with 2.19 cm callus diameter) for callus induction. This concentration also induced and produced good quality somatic embryo. In addition, MS+2.0 mg l-1 Kinetin gave better regeneration of somatic embryo and yielded the highest number (4.670) and longest length (7.0 cm) of shoots per callus. The procedure used a single hormonal signal for callus and somatic embryo induction as well as hormone free medium for further development of plantlet. Besides, maximum duration for callus induction and somatic embryo production was 17 and 10.67 days respectively. Thus, it appears that the protocol is cheap and time bound and particularly useful for conducting experiment for genetic improvement of garlic. Furthermore, as the protocol is cost effective, it can be further tested for commercial feasibility. DOI: http://dx.doi.org/10.3329/jbau.v12i1.20747 J. Bangladesh Agril. Univ. 12(1): 1-6, June 2014

Efficient resynthesis of oilseed rape (Brassica napus L.) from crosses of winter types B. rapa × B. oleracea via simple ovule culture and early hybrid verification

Resynthesis of Brassica napus is an important tool for the broadening of genetic diversity as well as crop cultivar improvement in oilseed rape. To the best of our knowledge, this is the first study focusing on the crossability, regeneration, and early verification of hybrids between diploid winter turnip rapes (Brassica rapa ssp. oleifera f. biennis), as the female component, and diploid winter curly kales (Brassica oleracea convar. acephalla var. sabellica). Pollination efficiency, mean embryo regeneration per bud and siliqua, as well as their relationships were evaluated in ovule cultures of twenty-four different one-sided crosses between six accessions of B. rapa and four accessions of B. oleracea. Successful germination of the embryos was achieved in 23 combinations. The mean number of embryos per bud reached 0.34. The most productive cross, Svalofs Duro × Kapral, yielded 2.7 embryos per bud in the best replication. A significant and positive correlation (r = 0.26*) was detected between the pollination efficiency (i.e., setting of siliquae) and the number of embryos per siliqua. In the majority of crosses, procedures implemented exhibited better results than in similar published experiments on spring Brassica genotypes, and provides a simplified and less laborious method. The hybrid nature of all regenerants was verified via flow cytometry. The use of bulked samples for analysis was reliable due to highly significant differences in relative DNA content between all hybrid combinations and their respective parental components. Chromosome counting and morphological assessment further corroborated the results of the flow cytometric analysis.

Obtaining calli and regenerated plants in anther cultures of pea

Pea (Pisum sativum L.) is a species for which there is no efficient method for the recovery of haploid plants yet. This research investigated the influence of various genotypes, nutrient media, and stress treatments on callus formation, embryogenesis and plant regeneration in anther cultures of pea. A wide range of pea genotypes and nutrient media was studied. Morphogenic calli were initiated on media supplemented with α-naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and 2,4-dichlorophenoxyacetic acid (2,4-D) without application of stress treatments. Embryogenic calli and embryos were regenerated on media with low sucrose content in the presence of 2,4-D or indole-3-butyric acid (IBA) after cold stress (4°C) of isolated buds, alone or in combination with in vitro treatment of isolated anthers at higher temperatures (35–38°C). The efficiency of regeneration via shoot morphogenesis on different nutrient media and the peculiarities of regeneration from embryogenic calli were investigated. Green embryogenic calli initiated on 2,4-D were able to develop through shoot morphogenesis on a medium supplemented with BA and NAA. This process led to regeneration of hypertrophic embryos at various developmental stages. The origin of regenerated plants (i.e. from microspores or somatic anther cells) was estimated using marker alleles determining morphological traits. Almost all R<sub>0</sub> regenerants derived from morphogenic calli originated from anther somatic cells.

English

The aim of this study was to establish an in vitro system for the induction, maturation and regeneration of somatic embryo in sugarcane from buds of cultivar RB 867515. Embryogenic calluses were obtained on semi-solid MS medium supplemented with 4.42 mg L-1 2,4-D. After four weeks of culture of explants on the callus induction medium, globular structures were obtained. At the end of 20 days in maturation medium, somatic embryos were observed. Histological analysis showed somatic embryos with caulinar and root apex, protodermal tissue, and the vascular system, which apparently has no connection with the vascular tissue explant that gave rise to it confirming the presence of the somatic embryo. The embryos were transferred to regeneration medium containing 1 mg L-1 GA3 and BAP, and after 1 to 2 weeks of culture, green points were observed, indicating the beginning of the formation of shoots.   Key words: Saccharum spp, bud culture, 2.4-D, morphogenetic pathway, embryogenesis, plant regeneration.

The use of 2D‐DIGE to understand the regeneration of somatic embryos in avocado

Avocado embryogenic cell cultures can be classified into two groups based on their morphology when cultured on a medium containing auxin: somatic embryo (SE) and proembryonic masses (PEM) type cultures. The calli of SE-type cell lines are able to go through the maturation process, whereas the calli of PEM cell lines rarely mature. We have investigated four independent avocado cell cultures (two SE and two PEM). The aim of this study was to link the differential regeneration capacity of the four cell cultures to a proteomic pattern and to gain insight into the regeneration capacity. A 2D-DIGE analysis followed by a blind multivariate analysis was able to separate the two SE lines from the PEM lines indicating that the protein profiles of SE and PEM calli are different. Based on the variable importance, that is, the differential protein pattern, we hypothesize that the regeneration capacity in avocado is correlated to the ability to overcome the physicochemical stress stimuli associated with the in vitro culture. Our identical culture conditions do not seem to trigger an appropriate response in PEM lines.

Effects of whole seed priming on the in vitro germination of hybrid banana embryos (Musa spp.)

Summary Trials were conducted to determine the effects of hydro- and hormonal priming on in vitro germination and regeneration of hybrid banana embryos. Fully-mature seeds of Pisang Jajee × M. acuminata ssp. burmannicoides were hydroprimed (for 1, 2, 3, 4 or 5 days) or hormone primed (gibberellic acid (GA3), 6-benzylaminopurine (BAP), indole-3-acetic acid (IAA)) at different concentrations (2, 5, 10, 15 and 20 ppm) for three days prior to in vitro culture. The embryos were extracted and initiated on basal MS medium devoid of growth regulators. Hydropriming for three days resulted in 40.8% germination compared with 20.8% germination of non-primed seeds. Priming in GA3 at 10 ppm for three days doubled the germination (82.4%) and also enhanced growth parameters. Early shoot emergence was recorded with BAP priming (5 ppm), while priming with IAA (10 ppm) enhanced root length and root number.

Enhancing somatic embryogenesis in avocado (Persea americana Mill.) using a two-step culture system and including glutamine in the culture medium

The development of a more efficient in vitro regeneration system for somatic embryos (SEs) of avocado (Persea americana) would facilitate the development of new superior cultivars for this valuable horticultural crop. In this study, we report a new and efficient method for maintenance and regeneration of avocado SEs. Avocado SEs of four cultivars remained healthy and viable in vitro for 11 months on a medium used for mango somatic embryogenesis, compared with 3–4 months on Murashige and Skoog medium. Various supplements and media modifications were investigated to improve the low conversion rate of regenerated plants from avocado SEs reported previously. The one-step system for regeneration of white-opaque somatic embryos (WOSEs) used solid medium only over a period of 12–14 weeks (subculturing every 6 weeks). Addition of proline and glutamine improved the total regeneration from 0 to 17.5% and 10.5%, and plant/shoot recovery from 0 to 12.5% and 5%, respectively. A two-step culture system involving the transfer of WOSEs of cultivar ‘Reed’ after 6 weeks on solid to liquid medium for 12–15 days as an intermediate step, followed by subculturing again onto solid medium for 6 weeks improved total regeneration to 29% and plant/shoot recovery to 18.3 from 0% when regenerated by subculturing on solid medium only. Supplementation with proline in the solid as well as liquid medium in the two-step culture system at 0.4g/L increased total regeneration to 35% and plant/shoot recovery to 20%. We were able to achieve highest regeneration using glutamine at 1g/L in the two-step culture system in terms of both total regeneration (58.3%, including 43.3% bipolar regeneration) and plant/shoot recovery (36.7%) rates, which were significantly higher than in any other treatment investigated.

A milestone in the doubled haploid pathway of cassava

This study was aimed at inducing androgenesis in cultured anthers of cassava (Manihot esculenta Crantz) to develop a protocol for the production of doubled haploids. Microspore reprogramming was induced in cassava by cold or heat stress of anthers. Since the anthers contain both haploid microspores and diploid somatic cells, it was essential to verify the origin of anther-derived calli. The origin of anther-derived calli was assessed by morphological screening followed by histological analysis and flow cytometry (FCM). Additionally, simple sequence repeat (SSR) and amplified fragmented length polymorphism (AFLP) assays were used for the molecular identification of the microspore-derived calli. The study clearly demonstrated the feasibility of producing microspore-derived calli using heat- or cold-pretreated anthers. Histological studies revealed reprogramming of the developmental pathway of microspores by symmetrical division of the nucleus. Flow cytometry analysis revealed different ploidy level cell types including haploids, which confirmed their origin from the microspores. The SSR and AFLP marker assays independently confirmed the histological and FCM results of a haploid origin of the calli at the DNA level. The presence of multicellular microspores in the in vitro system indicated a switch of developmental program, which constitutes a crucial step in the design of protocols for the regeneration of microspore-derived embryos and plants. This is the first detailed report of calli, embryos, and abnormal shoots originated from the haploid cells in cassava, leading to the development of a protocol for the production of doubled haploid plants in cassava.

Somatic embryogenesis and plant regeneration from immature cotyledons of European chestnut (Castanea sativa Mill.)

Here, we established a protocol for induction of somatic embryogenesis and plant regeneration from immature cotyledons of open-pollinated seeds of European chestnut (Castanea sativa Mill.) cultivars ‘Osmanoglu’ and ‘Sariaslama’. Basal media, Murashige and Skoog medium (MS), Driver and Kuniyuki Walnut medium (DKW), and Woody Plant Medium (WPM) supplemented with l-glutamine or casein hydrolysate, with or without silver nitrate, agar or gelrite, and various plant growth regulator (PGR) combinations were tested in initial cultures for induction of somatic embryos. The effects of initial cultures on the percentage of somatic embryos and average number of embryos per cotyledon explant, subcultured monthly, were determined at the end of 4 mo. Interactions were observed among the different treatments for ‘Osmanoglu’ cultivar, with the highest rates of somatic embryogenesis (4.7–9.7%) being obtained in MS, DKW, or WPM basal media supplemented with (1) 6-benzyladenine (BA; 1 mg/L) + kinetin (KIN; 2 mg/L) + indole-3-butyric acid (IBA; 0.01 mg/L); (2) BA (1 mg/L) + 1-phenyl-3-(1,2,3-thiadiazol-5-yl; TDZ 0.1 mg/L) + IBA (0.01 mg/L), and (3) KIN (2 mg/L) + TDZ (0.1 mg/L) + IBA (0.01 mg/L) PGR combinations plus l-glutamine or casein hydrolysate, with or without silver nitrate, and with either gelrite or agar. The highest percentages (12.0% and 11.2%) of somatic embryogenesis for ‘Sariaslama’ were obtained in DKW supplemented with PGR combinations of (1) BA (1 mg/L) + KIN (2 mg/L) + IBA (0.01 mg/L), (2) BA (1 mg/L) + TDZ (0.1 mg/L) + IBA (0.01 mg/L), respectively. The average number of somatic embryos ranged between 0 and 0.65 per explant for ‘Osmanoglu’ and between 0 and 0.49 per ‘Sariaslama’ explant. For germination of somatic embryos, root, shoot, and plantlet regeneration, different treatments included desiccation, cold and gibberellic acid (GA3), and BA alone or in combination with auxins (IBA or α-naphthaleneacetic acid, NAA; 0.1 mg/L). The highest rate of somatic embryos regeneration (27.5%) occurred using MS basal media with half-strength microelements containing 0.1 mg/L BA + 0.1 mg/L NAA, after treatments of desiccation, or desiccation plus cold or GA3 (3 mg/L).

Initiation, long-term cryopreservation, and recovery of Abies alba Mill. embryogenic cell lines

Somatic embryogenesis of Abies alba (Mill.) has significant potential to become an effective method for vegetative propagation of this species. To induce somatic embryogenesis in A. alba, the influence of the mother tree, sampling dates, and cold treatment storage of cones were examined. The initiation frequencies ranged from 1.7% to 16.6%. The sampling date and cone storage, but not the mother tree, had a significant effect on the initiation of embryogenic cultures. Storage of embryogenic cell lines was tested through cryopreservation for 6 yr. Four out of 12 cryostored embryogenic cell lines recovered, and the regeneration of cotyledonary embryos was obtained with two cell lines. The ability of embryogenic cell masses to produce somatic embryos and the mean number of cotyledonary embryos were higher when the maturation protocol was based on embryogenic suspensions dispersed on filter paper. The properly developed germinants were obtained only from maturation media where 32 μM abscisic acid was used, being 16.2% when polyethylene glycol (PEG) was not present and 1.8% when supplemented with 10% (w/v) PEG, respectively. The present study provides evidence that it is possible to cryopreserve A. alba embryogenic cultures while maintaining their maturing ability for the lengthy period (6 yr) needed for progeny testing of field-grown trees. Therefore, our findings are important for further studies and advanced breeding work of the species; however, the conversion of germinants into ex vitro conditions still remains a significant challenge.

Transfer of auxinic herbicide resistance from Brassica kaber to Brassica juncea and Brassica rapa through embryo rescue

Auxinic herbicides are widely used in agriculture to selectively control broadleaf weeds. Prolonged use of auxinic herbicides has resulted in the evolution of resistance to these herbicides in some biotypes of Brassica kaber (wild mustard), a common weed in agricultural crops. In this study, auxinic herbicide resistance from B. kaber was transferred to Brassica juncea and Brassica rapa, two commercially important Brassica crops, by traditional breeding coupled with in vitro embryo rescue. A high frequency of embryo regeneration and hybrid plant establishment was achieved. Transfer of auxinic herbicide resistance from B. kaber to the hybrids was assessed by whole-plant screening of hybrids with dicamba, a widely used auxinic herbicide. Furthermore, the hybrids were tested for fertility (both pollen and pistil) and their ability to produce backcross progeny. The auxinic herbicide-resistant trait was introgressed into B. juncea by backcross breeding. DNA ploidy of the hybrids as well as of the backcross progeny was estimated by flow cytometry. Creation of auxinic herbicide-resistant Brassica crops by non-transgenic approaches should facilitate effective weed control, encourage less tillage, provide herbicide rotation options, minimize occurrence of herbicide resistance, and increase acceptance of these crops.

Transformation of tef (Eragrostis tef) by Agrobacterium through immature embryo regeneration system for inducing semi-dwarfism

Successful application of genetic transformation for integration of a transgene is much dependent upon availability of an efficient in vitro plant regeneration procedure and detection of transgene insertion and expression. Isolated immature embryos (IEs) of Eragrostis tef cultivar DZ-01-196 were used for embryogenic callus formation and the callus was transformed with GA inactivating gene PcGA2ox under the control of a triple CaMV 35S promoter using Agrobacterium transformation procedure. Embryogenic callus was induced from immature embryos in a medium containing KBP minerals in the presence of 2,4-dichlorophenoxiyacetic acid. The embryogenic calli were further inoculated with Agrobacterium and the calli were grown in co-cultivation medium (CCM) followed by selection in KBP and regeneration (K4NB) media. Putatively transformed E. tef embryogenic calli were tolerant to treatment with the selectable marker kanamycin, while 75mgl−1 geneticin inhibited growth of non-transformed shoots derived from matured embryos completely after 12days. A total of 55 plants were regenerated from all the embryogenic calli to fully viable plants setting seeds at maturity. Eight putatively transformed T0 plants were produced carrying the transgene in their genome which was detected by PCR. Sequence analysis confirmed amplified PCR products to have 97.2 and 99.8% sequence identity to PcGA2ox and nptII, respectively. However, detection of the transgene, PcGA2ox or nptII, in T1 plants was inconsistent although phenotypic analysis of T1 plants showed changes in pheno-morphic and agronomic characters such as plant height, number of internodes, tillering, panicle length, biomass, yield as well as GA content. Culm reduction was due to absence of elongation of the upper-most internodes. Panicle length in semi-dwarfed plants showed no relation with culm length. GA analysis showed plants with semi-dwarf phenotype to be associated with a low level of bioactive GA1 and its immediate precursors. Up to 3.7 fold increase in grain yield per plant was found in some semi-dwarfed plants.

Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis)

A simple and rapid protocol was established for repetitive somatic embryogenesis and subsequent plant regeneration in two important Brassica oleracea varieties, cabbage and cauliflower. Direct regeneration of somatic embryos (SEs) was achieved from immature zygotic embryos cultured on B5 plant growth regulator (PGR)-free (B5-0) induction medium and on B5 medium supplemented with 1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) (B5-D). Zygotic embryos of both cabbage and cauliflower at the cotyledonary (C) stage (1.8 mm long) incubated on B5-0 medium displayed the highest embryo-forming capacities (EFCs) of 11.84 and 11.95, respectively. Secondary somatic embryos (SSEs) appeared on the cabbage and cauliflower’s primary embryos at a high frequency (83.3 and 87.5 %, respectively), and this process continued in a repetitive way on PGR-free Murashige and Skoog (MS-0) medium. The embryogenic potential of the cultures with a gradual diminution was maintained for 10 months (ten cycles). A total of 20 % of the mature SSEs from cabbage and 55 % from cauliflower spontaneously regenerated plantlets on MS-0 medium. The addition of 1 mg l−1 6-benzyladenine (BA) or 6-furfurylaminopurine (Kin) in the regeneration medium significantly improved somatic embryo conversion into plantlets by up to 56 % in cabbage and 79 % in cauliflower. Regenerated plants acclimated successfully to ex vitro conditions and displayed morphological and reproductive characteristics similar to seed-derived plants. Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of cabbage and cauliflower.

Abscisic Acid Effect on Improving Horse Chestnut Secondary Somatic Embryogenesis

The effect of abscisic acid on the development of primary androgenic embryo and secondary somatic embryogenesis was investigated with the aim of improving multiplication rates and secondary somatic embryo quality in horse chestnut microspore and anther culture. The early embryo stage (globular) had a better response than late stages (heart, torpedo, and cotyledonary) in both types of cultures. Also, microspore culture had a high potential for mass secondary embryo production. The number of secondary somatic embryos was three times higher on hormone-free medium than on medium enriched with 0.01 mg·L−1 abscisic acid. However, most of the embryos on hormone-free medium had abnormal morphology. For this reason, abscisic acid was added to the media to improve embryo quality. The morphology of abscisic acid treated embryos was better than abscisic acid non-treated embryos. The optimal abscisic acid concentration for secondary somatic embryo induction and production of high-quality embryos was 0.01 mg·L−1. Overall, the effect of abscisic acid on the induction of secondary somatic embryogenesis and plant regeneration of androgenic embryos of this species may be helpful for the further synthesis of secondary metabolites in vitro and their application in the pharmaceutical industry.