Immature Cotyledons Research Articles

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM IMMATURE COTYLEDONS OF PRUNUS MUME 'NANKO'

This study describes a successful method for somatic embryogenesis and plant regeneration from immature cotyledons of Prunus mume 'Nanko'. It appeared that the developmental stage of cotyledons that were cultured in vitro was the most important factor for embryogenesis. The highest frequency of embryogenesis was obtained from immature cotyledons harvested about 80 days after flowering on WPM containing 3% sorbitol, 1 μM 2,4-D and 1 μM BA after 60 days of culture in the dark at 25°C. Embryos could be germinated and grown to shoots by culturing them to WPM containing 3% sorbitol, 5 μM BA and 0.7% agar under the light at 25°C. Secondary somatic embryogenesis was observed when the somatic embryos were cultured on MS containing 3% sucrose, 0.1 μM NAA and 5 μM BA, 0.7% agar in the dark at 25°C.

Effect of desiccation, cold storage, and gibberellic acid on germination of somatic embryos in walnut (Juglans regia)

The effect of desiccation, cold storage, gibberellic acid (GA3), and various combinations of desiccation with GA3 and cold storage treatments on root formation and germination of walnut (Juglans regia) somatic embryos originating from immature cotyledons of open‐pollinated and non‐pollinated seeds was investigated. Desiccation was effective on germination and cold storage (4 weeks at 4°C) or GA3 (2.9,8.6,14.3, 20.0, and 25.7μm) treatment together with desiccation improved the germination rate of walnut somatic embryos. The highest percentage of germination (69.1%) was determined with desiccated embryos originating from open‐pollinated seeds of ‘Bilecik’ genotype on ‘Driver’ and ‘Kuniyuki’ walnut (DKW) basal medium containing 8.6 μm GA3. The germination levels of embryos originating from both open‐pollinated and non‐pollinated seeds were found to be similar.

Adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (Crataegus pinnatifida Bge. var. major N.E.Br.)

Hawthorn (Crataegus spp.) is an important plant with a long history as an ornamental and a source of medicine. A protocol is outlined for adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (C. pinnatifida Bge. var. major N.E.Br.). Adventitious buds were induced on both the leaves of sprouting winter buds and the leaves of in vitro plants, but the percentage of bud regeneration from leaves of in vitro plants was very low—less than 6%. On N6 medium supplemented with 31.08 μM BA and 9.67 μM NAA, the percentages of bud regeneration from leaves of sprouting winter buds of cultivars “Liaohong” and “Qiujinxing” were 31.4% and 17.6%, respectively. The regeneration abilities of three kinds of cotyledon explants, immature cotyledon, mature cotyledon, and cotyledon leaf, were compared. The percentage of bud regeneration from cotyledon leaves was higher. On MS media supplemented with 4.44 μM BA and 4.54–9.08 μM TDZ, the percentages of bud regeneration from cotyledon leaves of cultivars “Qiujinxing” and “Xiajinxing” were 27.7 ± 7.8% and 20.1 ± 4.7%, respectively, and the numbers of buds per explant were 5.9 ± 1.6 and 3.2 ± 0.7, respectively. On B5 medium supplemented with 2.22 μM BA, 2.32 μM Kn, and 0.57 μM IAA, adventitious buds grew quickly and 80–100% of buds developed into shoots. The shoots rooted successfully with the two-step rooting method. Ninety days after transplantation, more than 80% plants were survived. This system of adventitious bud regeneration from leaf and cotyledon explants could be useful for the genetic transformation and polyploidization of Chinese hawthorn.

Specificity of expression of the GUS reporter gene ( uidA) driven by the tobacco ASA2 promoter in soybean plants and tissue cultures

Twelve independent lines were transformed by particle bombardment of soybean embryogenic suspension cultures with the tobacco anthranilate synthase (ASA2) promoter driving the uidA (beta-glucuronidase, GUS) reporter gene. ASA2 appears to be expressed in a tissue culture specific manner in tobacco (Song H-S, Brotherton JE, Gonzales RA, Widholm JM. Tissue culture specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase. Plant Physiol 1998;117:533-43). The transgenic lines also contained the hygromycin phosphotransferase (hpt) gene and were selected using hygromycin. All the selected cultures or the embryos that were induced from these cultures expressed GUS measured histochemically. However, no histochemical GUS expression could be found in leaves, stems, roots, pods and root nodules of the plants formed from the embryos and their progeny. Pollen from some of the plants and immature and mature seeds and embryogenic cultures initiated from immature cotyledons did show GUS activity. Quantitative 4-methylumbelliferyl-glucuronide (MUG) assays of the GUS activity in various tissues showed that all with observable histochemical GUS activity contained easily measurable activities and leaves and stems that showed no observable histochemical GUS staining did contain very low but measurable MUG activity above that of the untransformed control but orders of magnitude lower than the constitutive 35S-uidA controls used. Low but clearly above background levels of boiling sensitive GUS activity could be observed in the untransformed control immature seeds and embryogenic cultures using the MUG assay. Thus in soybean the ASA2 promoter drives readily observable GUS expression in tissue cultures, pollen and seeds, with only extremely low levels seen in vegetative tissues of the plants. The ASA2 driven expression seen in mature seed was, however, much lower than that seen with the constitutive 35S promoter; less than 2% in seed coats and less than 0.13% in cotyledons and embryo axes. The predominate tissue culture specific expression pattern of the ASA2 promoter may be useful for genetic transformation of crops.

Production of cotyledon-type somatic embryos directly from immature cotyledonary explants of Camellia sinensis (L.)

SummaryAttempts were made to produce cotyledon-type somatic embryos from immature cotyledonary explants of Camellia sinensis (tea). Seeds of the tea cultivar ‘TRI 2043’ were collected at successive developmental stages, and separated into groups depending on the morphological complexity of the seed coat. Further separation was based mainly on the size (diameter) of the cotyledons in each seed (early, mid and late immature: stages 1–3; intermediate: stage 4; mature: stage 5). To determine the most suitable stage for direct somatic embryogenesis, sterilised cotyledons at various developmental stages were placed on MS basal medium containing 2 mg l–1 6-benzyl aminopurine (BAP) and 0.2 mg l–1 naphthalene acetic acid (NAA). The results indicated that immature cotyledons, 10 mm in diameter (stage 3), were most responsive for the production of typical (cotyledon-type) somatic embryos. Cytological examination revealed that typical somatic embryos exhibited gradual tissue differentiation. Immature (stage 3) cotyledon segments were placed on different media containing 0 – 3 mg l–1 NAA, in combination with 2 mg l–1 BAP, to select a suitable NAA concentration for efficient production of typical somatic embryos. The optimum concentration of NAA was in the range 0.2 – 1 mg l–1. Cotyledon-type and seed-like somatic embryos were developed in 0.2 and 1 mg l–1 NAA, respectively.

Evaluation of somatic embryogenesis from immature cotyledons of Japanese soybean cultivars

To examine the capacity for plant regeneration through somatic embryogenesis, Japanese soybean cultivars were investigated by 3-step-somatic embryogenesis: 1) induction of somatic embryos (SEs) from immature cotyledons, 2) proliferation of SEs in liquid medium and 3) differentiation of SEs into cotyledon-stage embryos. At each step, properties of the cultures varied among cultivars. Notably, significant differences were observed on 1) efficiencies of SE induction, 2) compactness of individual SEs during proliferation, and 3) yields of differentiated embryos (numbers of cotyledon-stage embryos differentiated from 100 mg globular SEs). We found that the compactness of SEs in liquid medium was very important factor for the recovery of well differentiated embryos. In fact, Yuuzuru and Yumeyutaka which indicated high ratios of compact SEs at proliferation step showed high yields of well differentiated embryos. The abilities of these Japanese cultivars to yield cotyledon-stage embryos were superior or comparable to North American cultivar 'Jack', which is used as one of the most preferable genotypes for somatic embryogenesis, indicating their high potentials for plant regeneration.

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 beta-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.

Induction and cryopreservation of embryogenic cultures from nucelli and immature cotyledon cuts of mango (Mangifera indica L. var Zihua)

In this paper, we described the direct somatic embryogenesis from both immature cotyledon cuts and nucelli in the same mango cultivar (Mangifera indica L. var Zihua), studied the effect of growth conditions of embryogenic cultures (EMs) on cryopreservation and compared the cryopreservation response of EMs induced from these two different explants. Histological studies demonstrated that EMs derived from nucelli could be induced directly from epidermal cells of both sides of nucelli, whereas EMs derived from cotyledon cuts were induced only from epidermal cells of the adaxial side of the cotyledons. EMs from either nucelli or cotyledon cuts could be maintained in liquid medium or on solid medium and cryopreserved using a vitrification procedure. Success of cryopreservation of EMs depended on the dehydration treatment and the defined growth conditions during culture but not on their origins. When EMs were sampled during their exponential growth phase in liquid medium and dehydrated with PVS(3) solution for 5 min, survival of the EMs induced from cotyledon cuts and nucelli reached 77.7 and 80%, respectively, after cryopreservation in liquid nitrogen for 24 h. Furthermore, when dehydrated with PVS(3) solution for 30 min, all EMs induced from cotyledon cuts and 96.7% of EMs induced from nucelli could survive after cryopreservation. Cryopreservation did not affect the plant regeneration potential of EMs through somatic embryogenesis. The protocols of somatic embryogenesis and cryopreservation of mango EMs established in this study may offer potential ways to improve mango germplasm conservation and genetic improvement.

In vitro plant regeneration from immature cotyledon explants of macadamia (Macadamia tetraphylla L. Johnson)

The macadamia tree, an Australian native, is highly valued for its nuts. Macadamia improvement programs so far have relied on conventional breeding and selection. The production of improved cultivars required to meet future demands could be accelerated by the application of modern biotechnological techniques, but this requires an efficient and reproducible regeneration system that has not yet been established for macadamia. We report here shoot regeneration from immature cotyledon explants of macadamia. Adventitious buds were induced on the cotyledon explants from fruits collected at 140 and 190 days after full bloom (DAFB) on MS medium supplemented with either 10 or 15 microM TDZ. The addition of 2% coconut milk (CM) to 10 microM TDZ containing media resulted in enhanced adventitious bud induction from 190 DAFB explants. Further shoot development from the induced buds was depressed in media containing TDZ + CM; the addition of 0.001 microM IAA to this combination doubled shoot development, from 1.9-3.9 shoots per explant. The transfer of bud clumps to media supplemented with 8.8 microM BA alone or in combination with either 0.14 microM GA(3) or 0.001 microM IAA significantly increased shoot production from the previously induced explants by 1.5-2 times of that observed in TDZ + CM medium. Histological examinations revealed that shoot regeneration was primarily by organogenesis originating from cells on or just below the cut surfaces of explants.

Molecular Control of Gene Co-suppression in Transgenic Soybean via Particle Bombardment

Molecular co-suppression phenomena are important to consider in transgene experiments. Embryogenic cells were obtained from immature cotyledons and engineered with two different gene constructs (pHV and pHVS) through particle bombardment. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V3-1) as a target. sGFP(S65T) as a reporter gene was, however, inserted into the flanking region of the V3-1 gene (pHVS). Fluorescence microscopic screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Stable integration of the transgenes was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Seeds of transgenic plants obtained from the pHV construct frequently lacked an accumulation of endogenous glycinin, which is encoded by homologous genes to the target gene V3-1. Most of the transgenic plants expressing sGFP(S65T) showed highly accumulation of glycinin. The expression of sGFP(S65T) and V3-1 inherits into the next generations. sGFP(S65T) as a reporter gene may be useful to increase the transformation efficiency of transgenic soybean with avoiding gene co-suppression.

Peas (Pisum sativum L.).

In this chapter we describe a robust method for transformation of peas that has been successfully used in our laboratory since 1992. Using immature pea seed collected from field- or greenhouse-grown plants, we have produced transgenic lines for over 30 genotypes including named pea cultivars and advanced breeding lines. This method uses immature cotyledons as the explant, and the transformation efficiency is in the range 0.2 to 13.5% of cotyledons producing at least one independently transformed line. Agrobacterium tumefaciens strains AGL1 and KYRT1 are the most successful in our procedure, and kanamycin, phosphinothricin, and hygromycin are reliable selectable markers. Potentially useful genes have been introduced for pest and disease resistance, altering quality traits, and investigating metabolic pathways and are being studied in transgenic pea lines.

Efficient production of transgenic soybean (Glycine max [L] Merrill) plants mediated via whisker-supersonic (WSS) method

The present study was designed to evaluate the transformation efficiency and proof the capability of whisker supersonic (WSS) method as an alternative option for soybean (Glycine max [L] Merrill) transformation. We compared soybean transformation efficiency obtained by WSS-mediated with that of particle bombardment transformation by carrying out molecular analysis of the T0 plants in two independent experiments. For this, we used for both transformation techniques the same genotype, the same plasmid and the same selection method. To assess the efficiency of soybean genetic transformation, we evaluated the efficiency of multi gene transformation by the selection with hygromycin and the expression of green fluorescent protein [sGFP (S65T)] resulted from both techniques. Regenerable embryogenic cells were induced from immature cotyledons of soybean c.v Jack on MSD40 media within 3 weeks then proliferated on FN lite liquid media and engineered with pUHG gene construct through both WSS and particle bombardment-mediated transformation. The pUHG was constructed with pUC 19 and contain the hpt gene conferring resistance to hygromycin as a selective marker and sGFP(S65T) as a reporter gene. Fluorescence microscopy screening after the selection of hygromycin, identified the clearly expression of sGFP(S65T) in the transformed soybean embryos. Stable integration of the transgenes was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. The average transformation efficiency achieved with WSS was higher than that obtained by particle bombardment and hence it may represent an alternative method for soybean transformation.

In vitro plant regeneration from immature embryo axis and cotyledons of common bean (Phaseolus vulgaris L.)

Phaseolus vulgaris L. is the most important economic species within the genus Phaseolus. It is grown in all parts of the world. Genetic improvement by conventional breeding has met considerable success, although production of hybrids between species within the genus has been limited due to sexual incompatibility. Recent advances in tissue culture have offered the opportunity to produce cultivars, which could not be obtained by conventional breeding methods. The use of tissue culture and genetic engineering is viewed as a logical approach to improve bean production. Gene transfer techniques will have a great impact on legumes. Although the concept of cell totipotency is widely proved, in vitro morphogenesis has not yet been achieved for a large number of cultivated beans. Regeneration protocols are strongly influenced by the genotype. In tissue and cell culture of beans, the factors controlling shoot morphogenesis and somatic embryogenesis are still unknown. The reported data suggest a possible way for future research.

Optimization of somatic embryogenesis in suspension cultures of horsegram [ Macrotyloma uniflorum (Lam.) Verdc.]—A hardy grain legume

Cell suspension cultures were established from immature cotyledon derived calli from drought tolerant legume horsegram [ Macrotyloma uniflorum (Lam.) Verdc.]. Embryogenic callus could be originated from cut slices of the immature cotyledons on MS solid medium [ Murashige, T. Skoog, K., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497] augmented with 1.0 μM zeatin and 4.5 μM NAA. Numerous somatic embryoids (26.4%) appeared on MS liquid basal nutrient medium with 5.6 μM NAA and with absence of zeatin during 3 weeks culture. Sustained cell division resulted in the formation of cell aggregates, and then progressed to globular, heart and further if they differentiate properly to torpedo and cotyledonary stages within 5 weeks. Transfer of individual embryos on to a fresh MS basal medium with no plant growth regulators was able to achieve complete maturation. Only a relatively few number of embryos developed into root/shoot when transferred to 0.9 μM GA 3, 15 g/l −1 sucrose and 2.4 g/l −1 gelrite containing medium. Substitution of sucrose associated with the use of l-glutamine gave, in the range of concentrations tested, the strongest enhancement of the embryo growth and development. About 5% of somatic embryos were converted into true-to-type fertile plants.

Direct differentiation of somatic embryos on cotyledons of Azadirachta indica

Somatic embryos regenerated in high-frequency (up to 100 %) on immature cotyledons of Azadirachta indica at a low concentration of thidiazuron (TDZ; 0.5 μM). Regeneration occurred exclusively from abaxial surface. Frequency of regeneration declined with the age of the cotyledons: on semimature cotyledons regeneration occurred only from some regions of the abaxial surface and on mature cotyledons it was confined to corners. However, an increase in concentration of TDZ to 1.0 μM improved the regeneration response. Higher regenerative capacity of immature cotyledons was due to presence of milk in immature fruits because regeneration response of immature cotyledons declined on washing of cotyledons for 24 h in liquid medium, and milk from immature fruits augmented the regeneration response of mature cotyledons. Somatic embryos regenerated readily on hormone-free medium and plantlets derived were able to survive after transfer to soil.

Enhancing the frequency of somatic embryogenesis following Agrobacterium-mediated transformation of immature cotyledons of soybean [Glycine max (L.) Merrill.

Abstract A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent cells/tissues on immature cotyledons of soybean \[Glycine max (L.) Merrill.\] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm in length, a 1:1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants o...

Microarrays for global expression constructed with a low redundancy set of 27,500 sequenced cDNAs representing an array of developmental stages and physiological conditions of the soybean plant.

BackgroundMicroarrays are an important tool with which to examine coordinated gene expression. Soybean (Glycine max) is one of the most economically valuable crop species in the world food supply. In order to accelerate both gene discovery as well as hypothesis-driven research in soybean, global expression resources needed to be developed. The applications of microarray for determining patterns of expression in different tissues or during conditional treatments by dual labeling of the mRNAs are unlimited. In addition, discovery of the molecular basis of traits through examination of naturally occurring variation in hundreds of mutant lines could be enhanced by the construction and use of soybean cDNA microarrays.ResultsWe report the construction and analysis of a low redundancy 'unigene' set of 27,513 clones that represent a variety of soybean cDNA libraries made from a wide array of source tissue and organ systems, developmental stages, and stress or pathogen-challenged plants.The set was assembled from the 5' sequence data of the cDNA clones using cluster analysis programs. The selected clones were then physically reracked and sequenced at the 3' end. In order to increase gene discovery from immature cotyledon libraries that contain abundant mRNAs representing storage protein gene families, we utilized a high density filter normalization approach to preferentially select more weakly expressed cDNAs. All 27,513 cDNA inserts were amplified by polymerase chain reaction. The amplified products, along with some repetitively spotted control or 'choice' clones, were used to produce three 9,728-element microarrays that have been used to examine tissue specific gene expression and global expression in mutant isolines.ConclusionsGlobal expression studies will be greatly aided by the availability of the sequence-validated and low redundancy cDNA sets described in this report. These cDNAs and ESTs represent a wide array of developmental stages and physiological conditions of the soybean plant. We also demonstrate that the quality of the data from the soybean cDNA microarrays is sufficiently reliable to examine isogenic lines that differ with respect to a mutant phenotype and thereby to define a small list of candidate genes potentially encoding or modulated by the mutant phenotype.

Screening Multiple Soybean Cultivars (MG 00 to MG VIII) for Somatic Embryogenesis Following Agrobacterium‐Mediated Transformation of Immature Cotyledons

A total of 15 soybean [Glycine max (L.) Merr.] cultivars representing maturity groups 00 to VIII were assessed for their embryogenic and transformation potentials. All cultivars were identified as embryogenic under hygromycin selection following Agrobacterium‐mediated transformation of immature cotyledons. Histochemical β‐glucuronidase (GUS) assays of induced hygromycin‐resistant somatic embryos (SEs) showed that 13 out of 15 cultivars were amenable for transformation. Wide variations among different genotypes were observed for their embryogenic capacity under hygromycin selection. Three cultivars, Cisne, Council, and Kunitz, were highly embryogenic yielding more than 50% responding explants and 1.5 to 2.4 hygromycin‐resistant SEs per responding explant. The transformation potential of multiple soybean cultivars was highly correlated with the embryogenic potential of immature cotyledons under hygromycin selection. It was possible to distinguish between highly and poorly embryogenic genotypes by visually observing the phenotype of cultured immature cotyledons. For highly embryogenic cultivars, the induction of somatic embryos mainly originated from actively dedifferentiating and browning/necrotic tissues along the margins of the abaxial side of cultured cotyledons.

Production of Transgenic Soybean Plants with Two Anti-Fungal Protein Genes Via Agrobacterium and Particle Bombardment

Utilizing either Agrobacterium-mediated transformation or particle bombardment we obtained transgenic soybean [Glycine max (L.) Merr.] plants expressing the chitinase gene (chi) and the barley ribosome-inactivating protein gene (rip). Six regenerated plants were grown to maturity and set seed. The identification of transgenic soybean plants that co-integrated the two anti-fungal protein genes was determined by polymerase chain reaction (PCR) and Southern blot analysis. Protein detection from the soybean leaves demonstrated the expression of the chitinase (CHI) and the ribosome-inactivating protein (RIP) in the six R0 transformants. Soybean cotyledonary nodes were transformed using the bivalent plant expression vector pBRC containing chi and rip both driven by the CaMV 35S double promoter. Following vacuum (0.06 MPa) infiltration treatment of the tissue for 5 min, Agrobacterium was co-cultivated with the cotyledonary nodes for 3 d on MSB medium (MS salts and B5 vitamins) (pH 5.2), the transformation frequency reached a maximum of 1.33 %. The chi and rip genes were present in all the transgenic plants. Co-bombardment of immature cotyledons with plasmids pBchE (encoding chi) and pARIP (encoding rip) resulted in a maximum transformation frequency of 0.52 % with a 50 % co-integration rate. Our results demonstrate efficient co-transformation of multiple genes in soybean.

Factors influencing regeneration of soybean from mature and immature cotyledons

Soybean (Glycine max L.) plantlets were e?ciently regenerated from mature and immature cotyledons of?ve different genotypes by studying various parameters affecting regeneration. Green organogenic noduleswere induced at the proximal end, which subsequently differentiated into shoot buds on modi?ed MS(Murashige T. and Skoog F. 1962. Physiol. Plant. 15: 473-497) medium. The presence of 6-benzylaminopurine(BAP) and thidiazuron (TDZ) in the medium exerted a synergistic effect, in that regenerationeffciency was higher than for either cytokin alone. The regenerated shoot buds elongated and rooted onMS medium containing 0.29 lM gibberellic acid (GA3) and 2.69 lM a-naphthaleneaceticacid (NAA),respectively. Rooted plants were established in the greenhouse with 87% success and produced viable seeds.Preliminary studies with Agrobacterium show great promise for soybean transformation based on theregeneration protocol reported here.