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Sugarcane ( Saccharum spp. hybrids) is an important commodity field crop in tropical and subtropical countries providing sugar and biofuel feedstock and occupying a critical and strategic position in the global economy. This study was conducted to evaluate, compare, and optimize a rapid direct regeneration tissue culture system from immature leaf whorl and pre-emergent floral explants for three elite US sugarcane genotypes: CP84-1198, CP88-1762, and CP89-2143. Direct regeneration of adventitious shoot buds from the immature leaf roll explants and subsequent elongation and rooting of shoot buds was successfully obtained on modified Murashige and Skoog salt medium supplemented with 5 mg l –1 α-naphthaleneacetic acid and 0.5 mg l −1 kinetin. Significant genotype-specific differences in the morphogenetic potential of leaf roll explants were discernible with the explant developmental stage (explant position along the leaf roll axis) and orientation during in vitro culture. The highest number of shoots was regenerated from CP88-1762, followed by CP89-2143 and CP84-1198 from explants closest to the meristem that were oriented horizontally (CP88-1762) or vertically (CP89-2143 and CP84-1198) on the culture medium. Immature inflorescence-derived explants from all three genotypes when cultured on the above medium for 2 wk rapidly produced shoots, followed by rooting on medium supplemented with 4 mg l −1 indole-3-butyric acid. The regeneration protocols yielded robust rooted plantlets from immature leaf roll explants within 4 to 6 wk, which were readily acclimatized under greenhouse conditions. [ABSTRACT FROM AUTHOR]

WRKY proteins constitute a family of transcription factors involved in many plant processes, including responses to biotic and abiotic stress. A constitutive HvWRKY38 expression cassette was introduced into apomictic bahiagrass cultivar ‘Argentine’ by biolistic gene transfer. HvWRKY38 integration and expression was detected in transgenic bahiagrass plants and their apomictic seed progeny by Southern blot, PCR and quantitative real-time RT-PCR analysis, respectively. Transgenic and wildtype plants were grown hydroponically to allow uniform dehydration and rehydration treatments and measure whole plant relative water content and biomass. Transgenic bahiagrass plants retained water better during dehydration, recovered faster and produced more biomass following rehydration and survived severe dehydration stress under controlled environment conditions in contrast to non-transgenic plants. The observed dehydration tolerance is very desirable in perennial grasses like bahiagrass, where seasonal droughts affect establishment, persistence or productivity. Our results confirm the regulatory role of HvWRKY38 in dehydration tolerance. [ABSTRACT FROM AUTHOR]

Stomata regulate CO2 and water vapor exchange between leaves and the atmosphere. Stomata are a target for engineering to improve crop intrinsic water use efficiency (iWUE). One example is by expressing genes that lower stomatal density (SD) and reduce stomatal conductance (g sw). However, the quantitative relationship between reduced SD, g sw, and the mechanisms underlying it is poorly understood. We addressed this knowledge gap using low-SD sugarcane (Saccharum spp. hybrid) as a case study alongside a meta-analysis of data from 10 species. Transgenic expression of EPIDERMAL PATTERNING FACTOR 2 from Sorghum bicolor (SbEPF2) in sugarcane reduced SD by 26–38% but did not affect g sw compared with the wild type. Further, no changes occurred in stomatal complex size or proxies for photosynthetic capacity. Measurements of gas exchange at low CO2 concentrations that promote complete stomatal opening to normalize aperture size between genotypes were combined with modeling of maximum g sw from anatomical data. These data suggest that increased stomatal aperture is the only possible explanation for maintaining g sw when SD is reduced. Meta-analysis across C3 dicots, C3 monocots, and C4 monocots revealed that engineered reductions in SD are strongly correlated with lower g sw (r 2=0.60–0.98), but this response is damped relative to the change in anatomy. [ABSTRACT FROM AUTHOR]

Summary: Sugarcane (Saccharum spp. hybrid) is a prime feedstock for commercial production of biofuel and table sugar. Optimizing canopy architecture for improved light capture has great potential for elevating biomass yield. LIGULELESS1 (LG1) is involved in leaf ligule and auricle development in grasses. Here, we report CRISPR/Cas9‐mediated co‐mutagenesis of up to 40 copies/alleles of the putative LG1 in highly polyploid sugarcane (2n = 100–120, x = 10–12). Next generation sequencing revealed co‐editing frequencies of 7.4%–100% of the LG1 reads in 16 of the 78 transgenic lines. LG1 mutations resulted in a tuneable leaf angle phenotype that became more upright as co‐editing frequency increased. Three lines with loss of function frequencies of ~12%, ~53% and ~95% of lg1 were selected following a randomized greenhouse trial and grown in replicated, multi‐row field plots. The co‐edited LG1 mutations were stably maintained in vegetative progenies and the extent of co‐editing remained constant in field tested lines L26 and L35. Next generation sequencing confirmed the absence of potential off targets. The leaf inclination angle corresponded to light transmission into the canopy and tiller number. Line L35 displaying loss of function in ~12% of the lg1 NGS reads exhibited an 18% increase in dry biomass yield supported by a 56% decrease in leaf inclination angle, a 31% increase in tiller number, and a 25% increase in internode number. The scalable co‐editing of LG1 in highly polyploid sugarcane allows fine‐tuning of leaf inclination angle, enabling the selection of the ideotype for biomass yield. [ABSTRACT FROM AUTHOR]

Metabolic engineering for hyperaccumulation of lipids in vegetative tissues of high biomass crops promises a step change in oil yields for the production of advanced biofuels. Energycane is the ideal feedstock for this approach due to its exceptional biomass production and persistence under marginal conditions. Here, we evaluated metabolically engineered energycane with constitutive expression of the lipogenic factors WRINKLED1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1), and OLEOSIN1 (OLE1) for the accumulation of triacylglycerol (TAG), total fatty acid (TFA), and biomass under field conditions at the University of Florida‐IFAS experiment station near Citra, Florida. TAG and TFA accumulation were highest in leaves (up to 9.9% and 12.9% of DW, respectively), followed by juice from crushed stems, stems, and roots. TAG and TFA accumulation increased up to harvest time and correlated highest with OLE1 and DGAT1 expression. Biomass dry weight, TAG, and TFA content differed greatly depending on DGAT1 and OLE1 expression in transgenic lines with similar WRI1 expression. Biomass did not significantly differ between WT and line L2 with DAGT1 and OLE1 expressed at low levels and TAG and TFA accumulating to 12‐ and 1.6‐fold that of WT leaves, respectively. In contrast, line L13, with intron‐mediated enhancement of DGAT1 expression, displayed a 245‐ to 330‐fold increase in TAG and a 4.75‐ to 6.45‐fold increase in TFA content compared with WT leaves and a biomass reduction of 52%. These results provide the basis for developing novel feedstocks for expanding plant lipid production and point to new prospects for advanced biofuels. [ABSTRACT FROM AUTHOR]

Background: Metabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG. Results: In this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED1 (WRI1) and the TAG protect factor oleosin (OLE1). Including an intron in the codon-optimized TmDGAT1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI1 (W), DGAT1 with intron (Di), and OLE1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic "push pull and protect" factors correlated with biomass reduction. Conclusions: Intron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels. [ABSTRACT FROM AUTHOR]

The recalcitrant structure of lignocellulosic biomass is a major barrier in efficient biomass-to-ethanol bioconversion processes. The combination of feedstock engineering via modification in the lignin synthesis pathway of sugarcane and co-fermentation of xylose and glucose with a recombinant xylose utilizing yeast strain produced 148% more ethanol compared to that of the wild type biomass and control strain. The lignin reduced biomass led to a substantially increased release of fermentable sugars (glucose and xylose). The engineered yeast strain efficiently co-utilized glucose and xylose for fermentation, elevating ethanol yields. In this study, it was experimentally demonstrated that the combined efforts of engineering both feedstock and microorganisms largely enhances the bioconversion of lignocellulosic feedstock to bioethanol. This strategy will significantly improve the economic feasibility of lignocellulosic biofuels production. [ABSTRACT FROM AUTHOR]

The carbon neutral potential of bioenergy relies in part on the ability of feedstocks to sequester carbon (C) in the soil. Sugarcane is one of the most widely used bioenergy crops, yet there remain unknowns about how it impacts soil C dynamics. In addition, Oilcane, a genetically modified version of Sugarcane has been produced to accumulate more energy‐dense oils and less soluble lignin, which enhances conversion efficiency but may also impact soil C cycling. Thus, our objectives were to examine the impact of Sugarcane litter decomposition on soil C formation and losses and determine if the genetic modifications to produce Oilcane alter these dynamics. To do this, we incubated bagasse (processed stem litter) and leaf litter from Sugarcane and Oilcane in microcosms with forest soil for 11 weeks. We used differences in natural abundance δ13C between C3 forest soil and C4 litter to trace the fate of the litter into respiratory losses as well as stable and unstable soil C pools. Our results show that genetic modifications to Oilcane did not substantially alter soil C dynamics. Sugarcane and Oilcane litter both led to net soil C gains dominated by an accumulation of the added litter as unstable, particulate organic C (POC). Oilcane litter led to small but significantly greater net soil C gains than Sugarcane litter due to greater POC formation, but the formation of stable, mineral associated organic matter (MAOC) did not differ between crop types. Sugarcane and Oilcane had opposing effects on tissue type where Sugarcane bagasse formed more MAOC, while Oilcane leaves preferentially remained as POC which may have important management implications. These results suggest that genetic modifications to Sugarcane will not significantly impact soil C dynamics; however, this may not be universal to other crops particularly if modifications lead to greater differences in litter chemistry. [ABSTRACT FROM AUTHOR]

Oilcane is a metabolically engineered sugarcane (Saccharum spp. hybrid) that hyper-accumulates lipids in its vegetable biomass to provide an advanced feedstock for biodiesel production. The potential impact of hyper-accumulation of lipids in vegetable biomass on microbiomes and the consequences of altered microbiomes on plant growth and lipid accumulation have not been explored so far. Here, we explore differences in the microbiome structure of different oilcane accessions and non-modified sugarcane. 16S SSU rRNA and ITS rRNA amplicon sequencing were performed to compare the characteristics of the microbiome structure from different plant compartments (leaf, stem, root, rhizosphere, and bulk soil) of four greenhouse-grown oilcane accessions and non-modified sugarcane. Significant differences were only observed in the bacterial microbiomes. In leaf and stem microbiomes, more than 90% of the entire microbiome of non-modified sugarcane and oilcane was dominated by similar core taxa. Taxa associated with Proteobacteria led to differences in the non-modified sugarcane and oilcane microbiome structure. While differences were observed between multiple accessions, accession 1566 was notable in that it was consistently observed to differ in its microbial membership than other accessions and had the lowest abundance of taxa associated with plant-growth-promoting bacteria. Accession 1566 is also unique among oilcane accessions in that it has the highest constitutive expression of the WRI1 transgene. The WRI1 transcription factor is known to contribute to significant changes in the global gene expression profile, impacting plant fatty acid biosynthesis and photomorphogenesis. This study reveals for the first time that genetically modified oilcanes associate with distinct microbiomes. Our findings suggest potential relationships between core taxa, biomass yield, and TAG in oilcane accessions and support further research on the relationship between plant genotypes and their microbiomes. [ABSTRACT FROM AUTHOR]

Wheat (Triticum aestivum L.) is a key crop for the global food supply. Its complex polyploid genome and recalcitrance to tissue culture pose challenges for genetic transformation, especially for elite cultivars. This study aimed to improve transformation efficiency in elite Korean wheat cultivars by integrating morphogenic regulator genes (GRF4–GIF1), using an engineered Agrobacterium tumefaciens strain. By integrating speed breeding to accelerate donor plant growth, we also reduced the time required to generate transgenic plants. Modifying the resting period and incorporating GRF4–GIF1 significantly improved the outcomes, leading to a transformation efficiency of 4.46% ± 0.93% in Bobwhite cultivar. Application of these methods to Korean elite cultivars also successfully yielded transformations with Keumkang and Baekkang cultivars. Furthermore, using a T3SS AGL-1 strain expressing the type III effector AvrPto enhanced the transformation efficiency in Baekkang cultivar by 2.27 times (17.7%) compared to the standard AGL-1 strain. Using the protocol as described here, we successfully developed stable transgenic lines of the Korean wheat cultivar, marking a pioneering achievement in Korean wheat molecular breeding. The modified protocol can contribute to wheat genetic research and breeding programs in Korea. [ABSTRACT FROM AUTHOR]

We recently generated oilcane, a metabolically engineered sugarcane with hyper-accumulation of energy dense triacylglycerol in vegetative tissues. Refinement of this strategy in high biomass crops like sugarcane may result in elevated lipid yields that exceed traditional oilseed crops for biodiesel production. This is the first report of agronomic performance, stable co-expression of lipogenic factors, and TAG accumulation in transgenic sugarcane under field conditions. Co-expression of WRI1; DGAT1, OLE1, and RNAi suppression of PXA1 was stable during the 2-year field evaluation and resulted in TAG accumulation up to 4.4% of leaf DW. This TAG accumulation was 70-fold higher than in non-transgenic sugarcane and more than 2-fold higher than previously reported for the same line under greenhouse conditions. TAG accumulation correlated highest with the expression of WRI1. However, constitutive expression of WRI1 was negatively correlated with biomass accumulation. Transgenic lines without WRI1 expression accumulated TAG up to 1.6% of leaf DW and displayed no biomass yield penalty in the plant cane. These findings confirm sugarcane as a promising platform for the production of vegetative lipids and will be used to inform strategies to maximize future biomass and lipid yields. The main conclusion is that constitutive expression of WRI1 in combination with additional lipogenic factors (DGAT1-2, OLE1, PXA1) in sugarcane under field conditions leads to hyper-accumulation of TAG and reduces biomass yield. [ABSTRACT FROM AUTHOR]

Background: The metabolic engineering of high-biomass crops for lipid production in their vegetative biomass has recently been proposed as a strategy to elevate energy density and lipid yields for biodiesel production. Energycane and sugarcane are highly polyploid, interspecific hybrids between Saccharum officinarum and Saccharum spontaneum that differ in the amount of ancestral contribution to their genomes. This results in greater biomass yield and persistence in energycane, which makes it the preferred target crop for biofuel production. Results: Here, we report on the hyperaccumulation of triacylglycerol (TAG) in energycane following the overexpression of the lipogenic factors Diacylglycerol acyltransferase1-2 (DGAT1-2) and Oleosin1 (OLE1) in combination with RNAi suppression of SUGAR-DEPENDENT1 (SDP1) and Trigalactosyl diacylglycerol1 (TGD1). TAG accumulated up to 1.52% of leaf dry weight (DW,) a rate that was 30-fold that of non-modified energycane, in addition to almost doubling the total fatty acid content in leaves to 4.42% of its DW. Pearson's correlation analysis showed that the accumulation of TAG had the highest correlation with the expression level of ZmDGAT1-2, followed by the level of RNAi suppression for SDP1. Conclusions: This is the first report on the metabolic engineering of energycane and demonstrates that this resilient, high-biomass crop is an excellent target for the further optimization of the production of lipids from vegetative tissues. [ABSTRACT FROM AUTHOR]

Sorghum is a versatile crop with great potential as a sustainable food, feed, and bioenergy source. To mitigate the severely negative impact of climate change and population growth on food and energy security, further elevation of the crops stress tolerance is urgently needed. Genome editing technologies such as CRISPR/Cas have great potential to accelerate functional genomics and crop improvement by supporting targeted modification of almost any crop gene sequence. We describe the recent progress in genome editing of sorghum. In addition, we review remaining challenges and prospects of emerging gene editing technologies for rapid precision breeding of this crop. [ABSTRACT FROM AUTHOR]

[Display omitted] • Commercially viable alternative feedstock for drop-in fuel and bioproduct production. • Sustainable and dedicated supply of feedstock without competing for food or land. • Proof-of-concept for bioprocessing at an industrially relevant scale. • Field grown energycane-oilcane hyperaccumulated 0.42 MT/ha of vegetative lipids. • 0.38 MT/ha of vegetative lipid was recovered with a recovery efficiency of 88.7% • Green pretreatment prevented lipid degeneration and improved recovery by 18.7% Development of sustainable and scalable technologies to convert lignocellulosic biomass to biofuels is critical to achieving carbon neutrality. The potential of transgenic bioenergy crops as a renewable source of sugars and lipids has been demonstrated at bench-scale. However, scaling up these processes is important for holistic analysis. Here proof-of-concept for chemical-free hydrothermal pretreatment of transgenic energycane-oilcane line L13 at an industrially relevant scale to recover vegetative lipids along with cellulosic sugars is presented. Pilot-scale processing of 97 kg of transgenic energycane-oilcane L13 stems and high solids pretreatment of bagasse enhanced the recovery of cellulosic glucose and xylose by 5-fold as compared to untreated bagasse and helped in the enrichment of vegetative lipids in the biomass residues which allowed its recovery at the end of the bioprocess. Palmitic and oleic acids were the predominant fatty acids (FAs) extracted from stems and leaves. The processing did not affect lipid composition. The efficiency of lipid recovery from untreated biomass was 75.9% which improved to 88.7% upon pretreatment. The vegetative tissues of transgenic energycane-oilcane L13 contained 0.42 metric tons/hectare of lipids. Processing vegetative tissues yielded 0.38 metric tons/hectare of lipids. This approaches an oil yield similar to soybean (global average 0.44 metric tons/hectare) and is almost twice as high as the oil yield from sugarcane engineered to hyperaccumulate lipids (0.20 metric tons/hectare). The study suggests that further optimization by state-of-the-art metabolic engineering and biomass processing can establish transgenic bioenergy crops for commercial drop-in fuel production. [ABSTRACT FROM AUTHOR]

The cultivated strawberry, Fragaria ×ananassa , is a recently domesticated fruit species of economic interest worldwide. As such, there is significant interest in continuous varietal improvement. Genomics-assisted improvement, including the use of DNA markers and genomic selection have facilitated significant improvements of numerous key traits during strawberry breeding. CRISPR/Cas-mediated genome editing allows targeted mutations and precision nucleotide substitutions in the target genome, revolutionizing functional genomics and crop improvement. Genome editing is beginning to gain traction in the more challenging polyploid crops, including allo-octoploid strawberry. The release of high-quality reference genomes and comprehensive subgenome-specific genotyping and gene expression profiling data in octoploid strawberry will lead to a surge in trait discovery and modification by using CRISPR/Cas. Genome editing has already been successfully applied for modification of several strawberry genes, including anthocyanin content, fruit firmness and tolerance to post-harvest disease. However, reports on many other important breeding characteristics associated with fruit quality and production are still lacking, indicating a need for streamlined genome editing approaches and tools in Fragaria ×ananassa . In this review, we present an overview of the latest advancements in knowledge and breeding efforts involving CRISPR/Cas genome editing for the enhancement of strawberry varieties. Furthermore, we explore potential applications of this technology for improving other Rosaceous plant species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Vondracek, Altpeter, Liu and Lee.)

Key Message: Targeting dCas9 fused with the 3xSRDX effector to the 5'UTR leads to strong repression of magnesium chelatase in highly polyploid sugarcane., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination. Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools.

Genetic transformation of sugarcane has a tremendous potential to complement traditional breeding in crop improvement and will likely transform sugarcane into a bio-factory for value-added products. We describe here Agrobacterium tumefaciens-mediated transformation of sugarcane. Embryogenic callus induced from immature leaf whorls was used as target for transformation with the hypervirulent Agrobacterium strain AGL1 carrying a constitutive nptII expression cassette in vector pPZP200. Selection with 30 mg/L geneticin during the callus phase and 30 mg/L paromomycin during regeneration of shoots and roots effectively suppressed the development of non-transgenic plants. This protocol was successful with a commercially important sugarcane cultivar, CP-88-1762, at a transformation efficiency of two independent transgenic plants per g of callus.

Rye (Secale cereale L.) is one of the most recalcitrant plant species for tissue culture and genetic transformation. Embryogenic rye callus loses its ability to regenerate plants quickly in response to high density of Agrobacterium and other stressors. The cocultivation of Agrobacterium and rye immature embryos in liquid medium facilitated washing of the cultures to avoid Agrobacterium overgrowth and allowed a high throughput. More than 40 independent transgenic plants were regenerated with one to four Southern-positive, independent events from 100 inoculated immature embryos. Agrobacterium strain AGL0 supported stable integration of a constitutive nptII selectable marker expression cassette into the genome of rye inbred line L22, as indicated by regeneration of plantlets on paromomycin-containing culture medium, Southern blot, Western blot, and the analysis of T-DNA::plant DNA boundary sequences. Transgenic plants were phenotypically normal and fully fertile, which might be a consequence of the short time in tissue culture.

A protocol that facilitates rapid establishment of Agrobacterium-mediated transformation for perennial ryegrass is described. The synthetic green fluorescent protein (sgfpS65T) reporter gene is introduced in combination with the nptII selectable marker gene into axillary bud derived embryogenic calli of perennial ryegrass (Lolium perenne L.) by co-cultivation with Agrobacterium tumefaciens strain AGL0 harboring binary vector pYF132. Following the co-cultivation calli are cultured for 48 h in liquid callus medium containing timentin at 10 degrees C and 70 rpm, which reduces Agrobacterium overgrowth. Using green fluorescent protein (GFP) as a nondestructive visual marker allows identification of responsive genotypes and transgenic cell clusters at an early stage. GFP screening is combined with paromomycin selection to suppress wild type cells. Transgenic plantlets ready to transfer to soil are obtained within 4 mo of explant culture. Between 8 and 16% of the Agrobacterium-inoculated calli regenerate independent, Southern positive transgenic plants. Reproducibility and efficiency in this perennial ryegrass transformation protocols is controlled by multiple factors including genotype dependent tissue culture and gene transfer response, a short tissue culture-and-selection period and the efficient suppression of Agrobacterium following Agrobacterium-mediated gene transfer.