Evaluation of embryological potential of soybean cultivars zoned in forest, steppe, and marshy woodlands of Ukraine as essential stage for the further transformation

Seven soybean (Glycine max (L.) Merr.) cultivars zoned in forest-steppe and marshy woodlands of Ukraine: "Chernuatka", "Vasylkivska", "Kyivska-91", "Kyivska-27", "Marjana", "Chernobura" and "Altair" were established in vitro for somatic embryogenesis inducing. Cultivars "Marjana" (88%) and "Vasylkivska" (86%) demonstrated the highest embryogenic capacity among all the tested cultivars. During the further plant regeneration cultivars "Marjana" and "Kyivska-91" showed the best capacity to form adult plants despite the fact that embryogenic capability of the cultivar "Kyivska-91" was 71%. According to the obtained data three genotypes were selected for the further investigation of effective methods of biolictic transformation of ukrainian cultivars.     

Plant regeneration from embryo-derived tissue cultures of soybeans

Summary Routine regeneration of fertile plants from a tissue culture of soybean has been achieved. Serially propagated embryogenic cultures were initiated from immature embryos of many genotypes. Organized tissues developed only on the cotyledons of embryos. Genotypic variation in the frequency of initiation of embryogenic tissue was noted. However, embryogenic tissue cultures were generated from all genotypes tested. Embryogenic tissue was serially increased and underwent morphogenesis. Whole fertile plants were recovered. Cultures have been maintained for two years without loss of morphogenic competency. Editor's Statement This procedure for initiating embryogenic tissue cultures from commerical cultivars of soybean and the subsequent development of fertile plants establishes a framework for studying the processes of embryogenesis and embryogenyin vitro as well as providing a system for tissue culture propagation and in vitro modification of soybean. Robert B. Horsch     

Comparison of somatic embryogenesis and embryo conversion in commercial soybean cultivars

Six commercially important soybean cultivars and one control cultivar were compared for differences in induction-efficiency of somatic embryogenesis, primary embryo yield, and embryo conversion. Cotyledons from immature seeds of similar developmental stage for all soybean cultivars were used for embryo induction. The experiments utilized a Latin square design to exclude the effect of differential lighting and position due to plate location in the growth chamber on the embryogenesis process. Results indicated that the efficiency of embryo induction and yield of primary somatic embryos were genotype-dependent. In contrast, no dependence on genotype was observed for the conversion of embryos to form roots and shoots. The percentage of cotyledons that gave a positive embryogenic response ranged from 26 to 89% for the soybean cultivars tested. The average number of primary globular-stage embryos per responding cotyledon after one month on induction medium ranged from 6 to 13 among the seven cultivars. Conversion frequencies for all genotypes ranged from 27 to 45%.     

Embryogenic response of multiple soybean [Glycine max (L.) merr.] cultivars across three locations

Summary Nine soybean [Glycine max (L.) Merr.] cultivars representing midwestern, mid-south, and southern US growing regions were evaluated at each of three locations (Athens, GA; Lexington, KY; and Wooster, OH) using uniform embryogenic induction and proliferation protocols in order to evaluate the portability of soybean somatic embryogenic protocols to different locations. The experimental design minimized variation between locations by having all cultivars present at all locations on all days. A quantitative weighted score for primary embryo induction was developed on average embryo number per explant and was used to describe non-embryogenic, poorly embryogenic, moderately embryogenic, and highly embryogenic responses. Ranking of cultivars remained similar across all locations, indicating a uniform transportability of the protocol, at least as far as embryo induction is concerned. Continued proliferation of embryogenic cultures was also measured using a repetitive growth measure but few meaningful conclusions could be made due to the high level of variability including inconsistent growth of cultures between each subculture. Overall, several cultivars were identified as being uniformly embryogenic or non-embryogenic at the primary induction phase at all locations, and we predict that those embryogenic cultivars could be used by any laboratory for high-efficiency induction of embryogenesis. The best of these cultivars, ‘Jack’, was uniformly responsive across all locations and should be selected as the genotype most likely to yield positive results when attempting to culture and genetically engineer soybeans via embryogenic protocols.     

Genotype specificity of the somatic embryogenesis response in cotton

Summary Thirty eight cultivars, strains, and races ofGossypium were screened for somatic embryogenesis with the protocols developed as a model forG. hirsutum L. cv. Coker 312. Four classes of response were identified; high, moderate, low, and non-embryogenic. Four cultivars were further screened with 13 growth regulator regimes to determine if culture environment could change the classification or induce a higher level of response. The classification or level of response did not change. Screening of individual seedlings within a cultivar indicated that genotypic variation for embryogenesis existed. Highly embryogenic individuals were selected from cvs. Coker 312 and Paymaster 303 for use as germplasm sources for transfer of the embryogenic trait to other cultivars and genetic stocks. Only genetically responsive genotypes are amenable to the model developed for Coker 312.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid (Carolina Biological) - 2iP N₆-(2-iso-pentenyl) adenine (Sigma) - NAA A-naphthaleneacetic acid (Sigma) - K kinetin (Sigma) - IE Index of embryogenesis     

Future area expansion outweighs increasing drought risk for soybean in Europe

The European Union is highly dependent on soybean imports from overseas to meet its protein demands. Individual Member States have been quick to declare self-sufficiency targets for plant-based proteins, but detailed strategies are still lacking. Rising global temperatures have painted an image of a bright future for soybean production in Europe, but emerging climatic risks such as drought have so far not been included in any of those outlooks. Here, we present simulations of future soybean production and the most prominent risk factors across Europe using an ensemble of climate and soybean growth models. Projections suggest a substantial increase in potential soybean production area and productivity in Central Europe, while southern European production would become increasingly dependent on supplementary irrigation. Average productivity would rise by 8.3% (RCP 4.5) to 8.7% (RCP 8.5) as a result of improved growing conditions (plant physiology benefiting from rising temperature and CO₂ levels) and farmers adapting to them by using cultivars with longer phenological cycles. Suitable production area would rise by 31.4% (RCP 4.5) to 37.7% (RCP 8.5) by the mid-century, contributing considerably more than productivity increase to the production potential for closing the protein gap in Europe. While wet conditions at harvest and incidental cold spells are the current key challenges for extending soybean production, the models and climate data analysis anticipate that drought and heat will become the dominant limitations in the future. Breeding for heat-tolerant and water-efficient genotypes is needed to further improve soybean adaptation to changing climatic conditions.     

Enhancement of microspore culture efficiency of recalcitrant barley genotypes

The culture response of isolated microspores of seven recalcitrant cultivars of barley has been largely improved by identifying an appropriate pretreatment and utilizing ovary co-cultivation. After comparison of three pretreatment media, medium B was shown to be most efficient for inducing microspore embryogenesis, while 0.3 M mannitol frequently used for the responsive cv. Igri was found to be ineffective for recalcitrant genotypes. A further significant improvement of embryogenesis was achieved by using ovary co-culture, which resulted in an overall 2.1-fold increase in embryo formation and 2.4-fold increase in green plant regeneration from all cultivars compared with the control. Optimal co-culture conditions were identified as 5 ovaries/ml medium kept over 20 days in induction culture. Microspore plating densities in cultures with and without co-culture were found to be optimal at 4᎒⁴/ml and 8-12᎒⁴/ml, respectively. The most effective and reproducible method for culturing microspores of recalcitrant genotypes appeared to be the combination of medium B pretreatment with ovary co-culture. By using this procedure, the genotypic difference in microspore embryogenesis could be reduced. It was found that medium B mainly enhanced percent live embryogenic microspores, and ovary co-culture subsequently improved cell division and embryogenic development. The method described here is important for the application of the microspore culture technique to barley breeding and biotechnology.     

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Summary Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.Abbreviations LSEA low somatic embryogenesis ability - HSEA high somatic embryogenesis ability - NAA 1-naphthaleneacetic acid     

Mature embryo axis-based high frequency somatic embryogenesis and plant regeneration from multiple cultivars of barley (Hordeum vulgare L.)

A highly reproducible regeneration system through somatic embryogenesis from the excised mature embryos (MEs) of dry seeds of a range of European barley cultivars was developed. By minimizing the germination of plated MEs, primary callus could be obtained with high frequency which permitted efficient embryogenesis and regeneration of a large number of green plants. Different approaches were tested to reduce or prevent normal germination: (i) the use of a well defined balance of maltose and 2,4-D in the induction medium, (ii) soaking of seeds in water containing 2,4-D solution, (iii) direct culture of excised embryonic axes, (iv) longitudinally bisected MEs giving two halves, and (v) complete removal of the elongated main shoot including any roots within a week of culture initiation. Culturing of bisected MEs and whole embryonic axes gave the best responses with respect to large amounts of callus combined with minimal germination. The incorporation of BAP at low levels in the medium was found to be most effective for embryogenesis and the maintenance of long-term morphogenic capacity (more than 11 months up to now). This procedure allows the complete regeneration of plants in 16-20 weeks, from the initial isolation of MEs through all the steps to the development of plants ready to be transferred to the soil. The protocol was first developed for cv. Golden Promise and successfully applied to commercial cultivars. All cultivars tested formed embryogenic callus, with overall rates ranging from 22-55% and an average number of green plants per embryogenic callus from 1.5 to 7.5 across the genotypes.     

Genotype effects on proliferative embryogenesis and plant regeneration of soybean

Summary Proliferative somatic embryogenesis is a regeneration system suitable for mass propagation and genetic transformation of soybean [Glycine max (L.) Merr.]. The objective of this study was to examine genotypic effects on induction and maintenance of proliferative embryogenic cultures, and on yield, germination, and conversion of mature somatic embryos. Somatic embryos were induced from eight genotypes by explanting 100 immature cotyledons per genotype on induction medium. Differences in frequency of induction were observed among genotypes. However, this step was not limiting for plant regeneration because induction frequency in the least responding genotype was sufficient to initiate and maintain proliferative embryogenic cultures. Six genotypes selected for further study were used to initiate embryogenic cultures in liquid medium. Cultures were evaluated for propagation of globular-stage tissue in liquid medium, yield of cotyledon-stage somatic embryos on differentiation medium, and plant recovery of cotyledon-stage embryos. Genotypes also differed for weight and volume increase of embryogenic tissue in liquid cultures, for yield of cotyledon-stage embryos on differentiation medium, and for plant recovery from cotyledon-stage embryos. Rigorous selection for a proliferative culture phenotype consisting of nodular, compact, green spheres increased embryo yield over that of unselected cultures, but did not affect the relative ranking of genotypes. In summary, the genotypes used in this study differed at each stage of plant regeneration from proliferative embryogenic cultures, but genotypic effects were partially overcome by protocol modifications.     

Histology of embryogenic responses in soybean anther culture

In order to clarify the embryogenic responses in soybean anther culture, anthers of four cultivars were cultured under known conditions to trigger androgenic response. A histological study was performed with anthers in vivo and with approximately 100 explants sampled after 9, 12, 15, 18, 21, 30 and 45 days of culture. In vitro culture triggered the frequent accumulation of phenolic compounds on the locular and anther surfaces, and also caused the destruction of cells and tissues in complex structure such as the tapetum, microspores and pollen grains. Somatic embryogenesis of unicellular origin was observed from the epidermis and the middle layer, and of multicellular origin from connective calluses. No androgenic response could be observed in the anthers of these four soybean genotypes, in the medium and conditions indicated. We point out to the need of changing the approach to the study of androgenesis in soybean, either by using culture conditions unfavourable to the proliferation of diploid tissues, or by culturing isolated microspores.     

Improved somatic embryogenesis of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) with focus on induction parameters and efficient plant regeneration

A study of four parameters (induction medium, floral explant, developmental stage and year) was carried out to determine the best combination for the embryogenesis induction of eight grapevine (Vitis vinifera L.) cultivars. Anthers and ovaries were extracted from flower buds at three developmental phases and incubated in two induction media over two consecutive years. As average, the percentage of embryogenesis on Nitsch and Nitsch-derived medium (9.1%) was higher than in Murashige and Skoog-derived medium (5.9%) and embryogenesis from ovaries (10.1%) was 2-fold higher than from anthers (4.9%). Earlier flower developmental stages (II–III) favored embryogenic induction from anthers, while later stages (III–V) did it from ovaries. Induction of embryogenic cultures was genotype dependent. Two years after the establishment of the embryogenic lines, an average of 48.0% of the pro-embryogenic masses were viable and suitable to initiate cell suspensions. Embryogenic cultures of four genotypes showed a high percentage of conversion from embryos to plants: Albariño (61.8%), Garnacha (48.8%), Tempanillo (71.0%) and Sultanina (69.0%). Moreover, cell suspensions were competent for transient transformation based on β-glucuronidase assay, as up to 6,387 blue spots per Petri plate after Biolistic bombardment were obtained. Here, we present the advantage of ovaries over anthers for the embryogenesis induction of several grapevine cultivars. This is the first report of embryogenesis from the cultivars Albariño, Verdejo and Muscat Hamburg as well as transient transformation of Albariño and Tempranillo.     

Genetic improvement of the somatic embryogenesis and regeneration in soybean and transformation of the improved breeding lines

Somatic embryos of soybean [Glycine max (L.) Merrill] have been used to generate transgenic plants by particle bombardment. The induction and proliferation of somatic embryos from immature cotyledons are dependent on the genotype of the cultivar. Whereas somatic embryogenesis and plant regeneration are inefficient in most cultivars, they are efficient in the cultivar Jack. We previously established a breeding line, QF2, by the integration of null mutations of each subunit of the major seed storage proteins glycinin and β-conglycinin, but the embryogenic response of this line is insufficient to allow efficient transformation. We have now backcrossed QF2 to cultivar Jack in order to combine the null traits with competence for somatic embryogenesis. The backcrossed breeding lines selected on the basis of the absence of the major storage proteins exhibited an improved capacity for the induction and proliferation of somatic embryos compared with that of QF2. The induced somatic embryogenic tissue of these breeding lines was successfully used for the production of transgenic plants by particle bombardment. These results also indicate that somatic embryogenesis in soybean is genetically controlled and inherited in a manner independent of the null traits of the major seed storage proteins.     

Somatic embryogenesis and plant regeneration in Chinese soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)—impacts of mannitol,abscisic acid,and explant age

From a preliminary experiment on 98 Chinese soybean varieties, 12 varieties with somatic embryogenesis frequency ranging from 0.0% to 85.7% were selected for further study in order to enhance the efficiency of somatic embryogenesis and plant regeneration. The effects of different mannitol concentrations, abscisic acid (ABA) concentrations, and embryo explant ages (sizes) were investigated. Significant differences in somatic embryogenesis were found among the 12 soybean varieties, with initiation frequencies varying from 22.1% to 89.0% under suitable mannitol concentration, and with N25281, N25263, and N06499 having the highest somatic embryogenic capacity. The results showed that all three factors were relevant for raising rates of callus initiation and somatic embryogenesis, but with differential responses among the genotypes. The treatment of 3.0% (w/v) mannitol, 5 mg l⁻¹ ABA, and a 4- to 5-mm-sized explant was found to be optimal for somatic embryogenesis, generating the highest explant-based regeneration rate at 83.0%. The greatest average number of plantlets regenerated per explant (1.35) was observed in N25281. The above results provide a basis for efficient regeneration of soybean and are informative for the development of genetic transformation systems in Chinese soybean germplasm.     

Analysis of the genotypic effect on different developmental pathways in wheat gametophyte cultures

Seven wheat cultivars and one wild subspecies Triticum aestivum ssp. spelta were compared for their in vitro fertility and androgenetic capacity by studying their anther culture response and in vitro seed production. Both haploid embryogenesis and in vitro seed set showed very wide genotype dependent variability in accordance with previous observations. At the same time, an analysis of the data showed a significant negative correlation between the in vitro androgenetic ability and the in vitro fertilization potential, which was especially obvious in the case of highly embryogenic genotypes. The reason for this inverse correlation might be the different genetic regulation of the two quantitative traits. Presumably, alleles which increase the stability of the gametogenetic developmental programme hinder the initiation of haploid embryogenesis, while alleles with the opposite effect promote the sporophytic type of growth.Abbreviations 2,4-D 2,4-diclorophenoxyacetic acid     

Somatic embryogenesis induction system for cloning an adult Cyphomandra betacea (Cav.) Sendt. (tamarillo)

Somatic embryogenesis is a valuable tool for plant breeding. In recent years, different aspects related to somatic embryogenesis (SE) induction in tamarillo have been studied at our laboratory. In this work, results concerning the establishment of a protocol for cloning an adult tamarillo tree through SE are presented. Attempts to induce SE in tamarillo from various explants directly taken from an adult tree were unsuccessful and only calli with no embryogenic potential were initiated. To overcome the lack of potential of adult tissues for SE, an indirect approach was attempted in which shoots from an adult tree were first established in vitro and then wounded leaves were used for SE induction. A low rate of embryogenic tissue formation was obtained (19.4%), but it was in the range of initiation rates from leaf explants of in vitro cloned plantlets of different tamarillo cultivars (red, orange and yellow) that originated from a single seedling (13.3–54.4%). High variation in SE initiation among juvenile controls could not be explained by different organogenetic potential, as no significant differences in shoot proliferation or rooting ability during micropropagation could be detected. Subcultures of embryogenic lines from the adult tree allowed us to obtain a large amount of embryogenic tissue that, after 8 weeks on a PGR-free medium, gave an average of 111 plants per gram of fresh mass of embryogenic tissue. A RAPD comparative analysis of somatic embryo-derived plantlets and the donor tree confirmed that the plantlets had no variation in the DNA regions amplified by 12 primers. These results open the way for large-scale cloning of elite tamarillo trees through SE.     

Protocol for in vitro somatic embryogenesis and regeneration of rice (Oryza sativa L.)

Development of highly efficient and reproducible plant regeneration system has tremendous potential to provide improved technology to assist in genetic transformation of indica rice cultivars for their further exploitation in selection. For the development of a highly reproducible regeneration system through somatic embryogenesis, mature embryos of highly popular rice cultivars i.e., Govind (for rainfed areas), Pusa Basmati-1 (aromatic basmati) and Jaya (for irrigated areas) were used. Optimum callus formation (%) to MS medium supplemented with 2, 4-D was obtained at 12.0 microM in Govind, 14.0 microM in Jaya and 15.0 microM in Pusa Basmati-1. All the cultivars showed good proliferation on MS medium without hormone. In Govind, highest embryogenic response was observed in MS medium supplemented with 2, 4-D (0.4 microM) + kinetin (0.4 microM), while in Pusa Basmati-1 with 2, 4-D (0.4 microM) + kinetin (2.0 microM) and in Jaya on hormone-free MS medium. Excellent embryo regeneration in Govind was observed on MS medium supplemented with low concentrations (1.1 microM) of BAP or hormone-free MS medium, while in Pusa Basmati-1 and Jaya embryogenesis was observed on MS medium supplemented with higher concentration of BAP (2.2 microM). Similarly, maximum plantlets with proliferated roots were observed in Govind on hormone-free MS medium, while in Pusa Basmati-1 and Jaya on MS medium supplemented with high concentration of NAA (4.0 microM). Developed plantlets were further successfully acclimatized and grown under pot culture up to maturity. Further the yield potential of in vitro developed plants was accessed at par to the direct seeded one under pot culture. Present, protocol standardizes somatic embryogenesis and efficient regeneration of agronomically important, high yielding and diverse indica rice cultivars which can be utilized as an efficient tool for molecular studies and genetic transformation in future.     

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.