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Protoplast is a versatile system for conducting cell-based assays, analyzing diverse signaling pathways, studying functions of cellular machineries, and functional genomics screening. Protoplast engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. This system allows the direct delivery of DNA, RNA, or proteins into plant cells and provides a high-throughput system to validate gene-editing reagents. It also facilitates the delivery of homology-directed repair templates (donor molecules) into plant cells, enabling precise DNA edits in the genome. There is a great deal of interest in the plant community to develop these precise edits, as they may expand the potential for developing value-added traits which may be difficult to achieve by other gene-editing applications and/or traditional breeding alone. This chapter provides improved working protocols for isolating and transforming protoplast from immature soybean seeds with 44% of transfection efficiency validated by the green fluorescent protein reporter. We also describe a method for gene editing in soybean protoplasts using single guide RNA molecules., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Soaking and maceration of dry soybean seeds induce the formation of aliphatic volatile compounds that impact the flavor properties of food products prepared from soybean. Most aliphatic volatile compounds are formed through oxygenation of unsaturated fatty acids by lipoxygenases; however, lipoxygenases are not responsible for the formation of 1-octen-3-ol. 1-Octen-3-ol in soybean products is in general an off-flavor compound; thus, a procedure to manage its formation is required. In this study, we show that the formation of 1-octen-3-ol after hydration of soybean seed powder is independent of oxygen, suggesting that 1-octen-3-ol is not formed de novo from unsaturated fatty acids but instead from its derivative. When crude methanol extract of soybean seeds was reacted with β-glycosidases, 1-octen-3-ol was rather liberated from its glycoside. We purified the parent glycoside from soybean seeds and confirmed it as ( R)-1-octen-3-yl β-primeveroside [( R)-1-octen-3-yl 6- O-β-d-xylopyranosyl-β-d-glucopyranoside]. Green immature soybean fruits (pericarp and seeds) contain a high amount of 1-octen-3-yl β-primeveroside. Its amount decreases after hydration of dry soybean powder. The results indicate that management of 1-octen-3-ol levels in soybean products requires a different strategy than that applied to off-flavor compounds formed de novo.

The incorporations of oleate (endogenous) and ricinoleate (nonendogenous) into phosphatidylcholine (PC) and acylglycerol (AG) in immature soybean microsomes were compared. [(14)C]Oleate and [(14)C]ricinoleate were incubated individually with soybean microsomal preparations for up to 4 h, and molecular species of PC and AG incorporated were identified and quantified by HPLC. The activities of acyl CoA:lysoPC acyltransferase and phospholipase A(2) are in general not affected by the fatty acid (FA) chain at the sn-1 position. However, comparison between oleate and ricinoleate revealed that different FA incorporated at sn-2 of PC showed some different selection of the molecular species of lysoPC. The incorporation of [(14)C]ricinoleate into triacylglycerols (TAG) was slightly better than that of [(14)C]oleate and indicated that soybean was capable of incorporating ricinoleate into TAG when ricinoleate can be produced endogenously in a transgenic soybean. The incorporation of FA into TAG in soybean microsomes was much slower than that in castor microsomes.

We have examined the processing and subcellular localization of a chimeric gene consisting of the bovine milk protein, beta-casein, under the control of a soybean seed lectin promoter and its 32 amino acid signal sequence in the seeds of transgenic soybean plants. The beta-casein expressed in developing soybean seeds is a doublet with apparent molecular weight slightly smaller than the bovine beta-casein and expression of the protein was highest in immature cotyledons. The casein proteins were purified from the immature soybean seeds by immunoaffinity chromatography and were analyzed by two-dimensional gel electrophoresis, blotting, and amino terminal sequencing. The N-terminal sequences of both of the doublet soybean casein polypeptides were identical to the N-terminal sequence of the bovine beta-casein indicating that the 32 amino acid lectin signal sequence was cleaved precisely from the chimeric protein in developing soybean seeds. Analysis of the purified soybean beta-casein polypeptides by mass spectrometry (MALDI-MS) showed that they are not phosphorylated. Absence of added phosphate groups is the cause of the size difference between the soybean beta-casein and native bovine beta-casein protein. Immunolocalization experiments showed that the casein protein was found in the protein storage vacuoles (PSV) in developing and mature soybean seeds. The precise removal of the 32 amino acid lectin amino terminal sequence from the chimeric lectin-casein fusion suggests that the lectin expression cassette can be used for production of pharmaceutical or other recombinant proteins of added value in the developing soybean seed.

Symptoms associated with fungal damage, viral diseases, and immature soybean (Glycine max) seeds were characterized using image processing techniques. A Red, Green, Blue (RGB) color feature-based multivariate decision model discriminated between asymptomatic and symptomatic seeds for inspection and grading. The color analysis showed distinct color differences between the asymptomatic and symptomatic seeds. A model comprising six color features including averages, minimums, and variances for RGB pixel values was developed for describing the seed symptoms. The color analysis showed that color alone did not adequately describe some of the differences among symptoms. Overall classification accuracy of 88% was achieved using a linear discriminant function with unequal priors for asymptomatic and symptomatic seeds with highest probability of occurrence. Individual classification accuracies were asymptomatic 97%, Alternaria spp. 30%, Cercospora spp. 83%, Fusarium spp. 62%, green immature seeds 91%, Phomopsis spp. 45%, soybean mosaic potyvirus (black) 81%, and soybean mosaic potyvirus (brown) 87%. The classifier performance was independent of the year the seed was sampled. The study was successful in developing a color classifier and a knowledge domain based on color for future development of intelligent automated grain grading systems.

Somatic embryogenesis from immature soybean cotyledons was induced on Murashige & Skoog medium, supplemented with 2,4-dichlorophenoxyacetic acid. The full developmental analyses of primary and secondary somatic embryogenesis was monitored by scanning electron microscopy. The developmental stages of embryogenesis from the first unequal cell division through the three- and four-cell embryos, and multi-cell globular and heart shape embryos, to the fully developed plants were determined. When the globular stage embryos were incubated on the embryo maturation medium for 4 to 6 weeks a secondary embryogenesis was observed and scanned. Chimeric embryos with epidermis zones similar to mature leaf were characterized.

The expression of lipoxygenases (LOXs) is known to be developmentally regulated in soybeans (Glycine max. [L.] Merr.). Hormones have been firmly established as being involved in the growth and developmental processes of a number of plant species. Correlation between the expression of LOXs and the development and germination of soybean embryos suggests that plant hormones may affect the expression of LOXs. The present studies were conducted to investigate the effects of exogenous auxins on the expression of LOX isozymes and LOX activities in cultured cotyledon tissues of immature soybean seeds. The results revealed that at least one of the more acidic nonembryo LOX isozymes was induced by either alpha-naphthaleneacetic acid or indoleacetic acid but not by 2,4-dichlorophenoxyacetic acid after 4 days' exposure. Levels of LOX-1, -2, and -3 proteins and activities were significantly decreased by 2,4-dichlorophenoxyacetic acid 10 days after explanting. S1 analysis showed that embryo LOX messenger RNAs were detectable in the tissues treated with each of the auxins. The reduced levels of the embryo LOX proteins may, therefore, be regulated at the levels of translation, posttranslational modification, or degradation. The more acidic isozymes induced by alpha-naphthaleneacetic acid showed enzymatic activity and shared the same molecular mass and isoelectric point values as the germination-associated LOX isozymes found in hypocotyls and radicles, suggesting that those LOXs are involved in germination competency of soybean embryos.

Immature soybean (Glycine max L.) embryos from commercially important cultivars were the targets of rapidly accelerated, DNA-coated, gold particles. Protoplasts were prepared from these tissues and propagated in culture under selection conditions for the introduced neomycin phosphotransferase II gene. Kanamycin-resistant calli were obtained at a rate of approximately 10(-5). Enzyme assays and Southern blot hybridization confirmed the expression of the foreign gene and its stable integration into the soybean genome. Our results show that particle acceleration can be used for the introduction of foreign DNA into the soybean genome and indicate the technique may be useful in the recovery of engineered plants by transformation of regenerable tissues.

Immature soybean (Glycine max L. Merrill cv Provar) cotyledons were grown aseptically for 6 days in complete culture medium with zero, deficient (17 micromolar), sufficient (1.5 millimolar), or supraoptimal (7.5 millimolar) levels of sulfate. Some cotyledons at each sulfate concentration were supplemented with l-methionine. No sulfate or 17 micromolar sulfate were inadequate for growth and protein accumulation, but all the major subunits of the 7S and 11S storage protein fractions were detected. The ratio of 11S to 7S proteins was <1.0. Addition of 8.4 millimolar methionine overcame the restriction of cotyledon growth and protein accumulation in the sulfate-deficient media, and the ratio of 11S to 7S proteins was significantly increased. The amino acid compositions of the 7S and 11S fractions from sulfate-sufficient cotyledons and from sulfate-deficient cotyledons were not significantly different. There was no difference in fresh weight or total protein accumulation in cotyledons grown in 1.5 millimolar or 7.5 millimolar sulfate. At 7.5 millimolars sulfate, the 11S to 7S ratio was significantly increased, and the amount of beta-subunit in the 7S fraction decreased. At all sulfate levels supplemented with methionine, the 11S to 7S ratio was greater than 1, and no beta-subunit was detected in the 7S fraction. Supplemental methionine in media of any sulfate concentration increased growth and protein methionine content to a greater extent than high (7.5 millimolar) sulfate only. Adding supraoptimal sulfate is not equivalent to supplementing with methionine. Results of this study of in vitro growth are compared to results of studies of seed development on intact plants supplied with various sulfur concentrations.

Freezing of plant tissue adversely affects lipid composition. Immature soybean cotyledons (Glycine max L. Merr.) var. "Harosoy 63" were frozen with liquid N(2), dry ice, or stored in a freezer (-20 C) before lipid extraction. The effects of freezing temperature, thawing rate, and cold storage on the lipid composition of frozen tissue revealed significantly higher levels of phosphatidic acid, and diminished levels of phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine from the control. Regardless of freezing temperature, phosphatidic acid levels increased from 4.7 mole% to nearly 50 mole% of the total phospholipid when frozen tissues were stored 10 days at -20 C. During the same period, N-acylphosphatidylethanolamine decreased from 54.1 mole% to 6.6 mole% phospholipid. At least 8 mole% of the phosphatidic acid increase occurred during slow thawing of the frozen tissues. In autoclaved samples, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine levels were not different from the control. Labeling of the lipid-glycerol with (3)H, and fatty acids with (14)C, demonstrated the degradation product was primarily phosphatidic acid. Apparently enzymic destruction of the phospholipids occurred during freezing, cold storage, and thawing.

Messenger RNAs (mRNAs), isolated from immature soybean (Glycine max L., Merr.) seeds, that bound to oligo(dT)-cellulose were fractionated by centrifugation in sucrose density gradients containing dimethyl sulfoxide. mRNAs with sedimentation values between 21S and 25S coded for the in vitro translation of polypeptides with electrophoretic mobilities similar to those of the alpha' and alpha subunits of the 7S seed storage protein. High pressure liquid chromatographic analyses of the trypsin-induced fragments ("column fingerprinting") verified that the polypeptides produced in vitro were closely related to authentic alpha' and alpha subunits.The fractions of RNA that coded for the in vitro synthesis of these subunits contained three major species of nonribosomal RNA (molecular weights of 1.1, 0.84, and 0.75 x 10(6)) sufficiently large to code for the synthesis of the alpha' and alpha subunits.

The various nitrogenous solutes important to embryo development in symbiotic soybean plants were determined during the midpodfilling stage. Glutamine was the principal form of nitrogen, contributing 55% of the embryo nitrogen requirement. Asparagine was the second most important, contributing 20%. The ureides allantoin and allantoic acid directly contributed only insignificantly to the total nitrogen requirement of the embryo. These conclusions were based upon analyses of tissue extracts, translocation studies of radiolabeled solutes, analysis of in vivo seed coat exudate collected from the freespace of attached, surgically altered seeds, and the in vitro culture of isolated immature soybean embryos.

The time course profiles of C(2)H(2) reduction by intact Scirpus olneyi (bulrush), Oryza sativa (rice) and Spartina alterniflora (cordgrass) with roots in atmospheres of N(2) and 30-day-old Glycine max (soybean) in air were all immediately linear. This is the first report of immediately linear rates of C(2)H(2) reduction by grass roots removed from soil. The immediately linear profile of C(2)H(2) reduction by soil-free grass roots was achieved by preventing contact between the roots and air. Roots of soybeans and S. olneyi receiving pretreatments of O(2) above normal environmental levels for 15 min before assay exhibited a short delay in C(2)H(2) reduction. These initially nonlinear rates of C(2)H(2) reduction are attributable to transient O(2) inhibition of nitrogenase. Initial nonlinear rates of C(2)H(2) reduction were also observed with immature soybean plants and with intact plant assays of O. sativa and S. olneyi in which C(2)H(2) was injected into cylinders surrounding the plant tops. These results indicate that, apart from O(2) inhibition of nitrogenase, the diffusion of C(2)H(2) and C(2)H(4) between the nitrogen-fixing sites and the sampling ports may cause initial nonlinear rates of C(2)H(2) reduction. We conclude that in situ plant-associated nitrogenase activity should result in immediate reduction of C(2)H(2) and that linear rates are observed when the proper assay conditions are used. Our data suggest that nitrogen fixation is closely associated with the roots of S. olneyi, O. sativa, and S. alterniflora growing in salt marsh sediment.

Upstream sequences of the gene encoding the alpha' subunit of beta-conglycinin were analyzed for interactions with nuclear proteins from immature soybean seeds. Two factors were identified that interact with specific sequence elements within 257 base pairs 5' of the transcription start site. One factor, SEF 3, binds exclusively to a region composed of two elements located at -183 to -169 base pairs and -153 to -134 base pairs relative to the start of transcription. Each of these sites includes the hexanucleotide sequence AACCCA, which may serve as a primary recognition sequence. During seed development, SEF 3 binding activity was found to increase in soybean embryos during the time of beta-conglycinin synthesis and to decrease as seeds neared maturity. The position of the SEF 3 binding sequence corresponds with a previously reported seed-specific enhancer region, and it seems likely that this factor may act as a positive regulator of transcription of the beta-conglycinin, alpha' subunit gene in developing soybean seeds. The second factor, SEF 4, also binds within the -257 to -77 region but also interacts with sites located further upstream.

The maturation of soybean (Glycine max L. Merr.) somatic embryos was characterized. Maturation was assayed by evaluating the ability of somatic embryos to make the transition to a plantlet through a germination-like process. Somatic embryos were organized from cotyledons of immature soybean embryos. Maturation of somatic embryos occurred on a Murashige-Skoog basal medium supplemented with activated charcoal and 0.28 molar sucrose. After 8 weeks on this medium, somatic embryos exhibited vigorous, high frequency development to plantlets. The "germination" frequency (conversion) of somatic embryos, and plantlet recovery frequency varied concurrently with maturation period. Conversion and plant recovery required no exogenous growth regulators. Desiccation of immature somatic embryos under controlled humidity regimes resulted in increased frequency of conversion of immature somatic embryos. Morphological abnormalities appeared in the somatic embryos, but few were detrimental to conversion velocity. There was little effect of genotype on conversion velocity or frequency.

The use of immunoaffinity columns containing anti-gibberellin (GA) antibodies for the selective purification of GAs in plant extracts is described. GA(1), GA(3), GA(4), GA(5), GA(7), and GA(9) conjugates to bovine serum albumin were synthesized and used to elicit anti-GA polyclonal antibodies (Abs) in rabbits. Protein A purified rabbit serum, containing a mixture of anti-GA Abs, was immobilized on matrices of Affi-gel 10 or Fast-Flow Sepharose 4B. Columns of these immunosorbents retained a wide range of C-19 GA methyl esters, but no C-20 GA methyl esters. Quantitative recovery of C-19 GA methyl esters was achieved from the columns, which, after reequilibration in buffer, could be reused up to 500 times. The immunosorbents were tested by examination of extracts from immature soybean and pea seeds. GAs were initially purified by passing the extracts through DEAE-cellulose and concentrating them on octadecylsilica. The extracts were methylated and further purified on the mixed anti-GA immunoaffinity columns. GAs were detected and quantified as methyl esters or methyl ester trimethylsilyl ethers by gas chromatography-mass spectrometry-selected ion monitoring. GA(7) was found in soybean seeds, 17 days after anthesis, at low levels (8.8 nanograms per gram fresh weight). C-19 GAs were examined in cotyledons, embryonic axes, and testae of G2 pea seeds harvested 20 days after anthesis. High levels of GA(20) and GA(29) were found in cotyledons (3580 and 310 nanograms per gram fresh weight, respectively) and embryonic axes (5375 and 1430 nanograms per gram) fresh weight, respectively). Lower levels of GA(9) were found in cotyledons and embryonic axes (147 and 161 nanograms per gram fresh weight, respectively). GA(9) was the major GA of testae at levels of 195 nanograms per gram fresh weight. Trace quantities of GA(20) and GA(51) were also observed in testae.