Transgenic sugarcane plants via microprojectile bombardment

Transgenic sugarcane plants were produced by bombardment of embryogenic callus with high-velocity DNA-coated microprojectiles, followed by a selection and regeneration procedure designed for this target tissue. Optimal bombardment conditions for embryogenic callus required microprojectile velocities higher than those previously found effective for sugarcane suspension culture cells. Bombardment of target tissues twice increased the number of transiently expressing cells in regenerable callus regions, to more than 300 per treated plate. Stable transformants were obtained following bombardment with the neomycin phosphotransferase (npt-II) gene under the control of the Emu strong monocot promoter. Stepped increases in antibiotic concentration during selection and regeneration allowed recovery of actively growing callus and plants on media containing geneticin concentrations completely inhibitory to untransformed controls. NPT-II levels in transformed plants were 20–50 times the background levels in control plants in ELISA assays, and Southern analysis revealed integration of one to three copies of the introduced gene in the sugarcane genome. The procedures described yield one to three transgenic plants per treated plate within 16 weeks of bombardment and provide a simple, efficient and broadly applicable system for genetic transformation of sugarcane. A similar approach should be applicable to other members of the Poaceae able to form embryogenic callus.     

小麦组织培养和基因枪轰击影响因素探讨

本研究就基因枪法转化小麦过程中的组织培养和轰击参数等影响因素进行了探讨。结果表明 ,小麦幼胚是比幼穗或成熟胚更理想的转化受体。因供试小麦品种基因型不同 ,幼胚愈伤组织再生频率差异明显 ,辽 - 1 0为 7.84% ,91 B5 6 9为 1 3 .6 8% ,东农 7742为 5 4 .90 %。小麦本身对选择剂卡那霉素有较高的天然抗性 ,采用 G41 8对小麦幼胚愈伤组织进行筛选效果明显。G41 8的毒性作用有滞后特点 ,3个小麦品种对 G41 8的敏感性依次为辽 - 1 0 >91 B5 6 9>东农 7742。用 G41 8做选择剂筛选辽 - 1 0、91 B5 6 9和东农 7742抗性愈伤的适合浓度分别为2 5 mg/L、3 0 mg/L和 3 5 mg/L。此外 ,不同轰击参数影响金粉分布的范围和密度。轰击距离为 6 cm或 9cm时 ,内部金粉密度大而外围金粉密度小 ,差异极大。轰击距离为 1 2 cm时 ,内部和外围金粉密度差异小 ,均匀度好     

基因枪介导小麦成熟胚遗传转化的影响因素

小麦成熟胚作为转化受体可克服小麦幼胚存在的受季节和幼胚发育阶段限制的缺点。以湖北省小麦品种‘鄂麦12’和模式品种‘Bobwhite’为材料,成熟胚为转化受体,优化基因枪转化法的轰击压力、轰击距离、选择剂等因素,建立以小麦成熟胚为转化受体的高效转化系统。结果表明:小麦成熟胚作为转化受体时,适宜轰击压力和轰击距离组合是900 psi、6 cm;成熟胚对选择剂G418的敏感性强于幼胚,轰击后需要延长恢复时间,选择剂G418的适合浓度为20～40 mg/L。在以上优化条件下小麦成熟胚转化频率达0.3%～0.9%,已初步建立基因枪介导的小麦成熟胚遗传转化系统。     

Development of a highly regenerable elite Acala cotton (Gossypium hirsutum cv. Maxxa) – a step towards genotype-independent regeneration

A step towards genotype-independent regeneration of cotton (Gossypium hirsutum L.) was achieved by selection for regeneration potential (RG) in commercial seed of elite cultivars. A callus induction medium (MCIM) empirically determined for the cultivar `Maxxa' paved the way for RG selection among individual genotypic variants within a cultivar. MCIM consists of a basal Murashige-Skoog medium, supplemented with a unique combination of two synthetic auxins. Hypocotyl explants of `Coker 312', `Maxxa' and `Riata' seedlings cultured on MCIM successfully produced a high quality, friable callus as defined by its color, texture, size, and organization. Based on the number of fertile plants regenerated on a per seedling basis, RG was estimated as 17.4%, 44.4% and 80% in Acala cotton cultivars `Maxxa', `Ultima', and `Riata', respectively. The high RG of the cultivar Riata, a Round-up Ready® transgenic cultivar in a Maxxa genetic background, is likely due to additional RG alleles introgressed from the transgenic parent. Genotypic differences between cultivars for RG was reflected by the need for supplemental kinetin to efficiently regenerate `Ultima' plantlets via somatic embryogenesis. RG selection pressure through two cycles of selection resulted in development of advanced highly regenerable `Max-R' lines in an elite genetic background with immediate potential as suitable germplasm for breeding and biotechnology applications. Based on the results presented here, strategies for genotype-independent transformation and regeneration of cotton are proposed that integrate selection and introgression of regeneration potential in improvement programs.     

Transgenic Pinus radiata from Agrobacterium tumefaciens–mediated transformation of cotyledons

A method for Agrobacterium tumefaciens-mediated transformation of Pinus radiata cotyledon explants was developed using commercially available open-pollinated seed. Pinus radiata is the most widely planted commercial conifer species in the Southern Hemisphere. Reports on transformation of this species have relied on particle bombardment of embryogenic callus derived from immature embryos. The main drawback to the method is the small number of genotypes that are amenable to transformation and regeneration. Since more than 80% of genotypes of radiata pine can be regenerated using cotyledons from mature seed, cotyledon explants were cocultivated with A. tumefaciens strain AGL1 containing a plasmid coding for the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (GUS) gene (uidA). Transformed shoots were selected using either geneticin or kanamycin. Critical factors for successful transformation were survival of the cotyledons after cocultivation and selection parameters. Of the 105 putative transformants that were recovered from selection media, 70% were positive for integration of the nptII gene when analysed by PCR. GUS histochemical assay for uidA expression was unreliable because of reaction inhibition by unidentified compounds in the pine needles. Further, only 4 of the 26 independent transformants characterised by PCR and Southern analysis contained an intact copy of both genes. The remaining 22 transformants appeared to have a truncated or rearranged copy of the T-DNA. It is possible that the truncation/rearrangements are due to the Cauliflower mosaic virus (CaMV) 35S promoter. Analysis of the T-DNA junction sites and sequencing of the introduced DNA will help elucidate the nature of T-DNA insertion so that genetic modification of radiata pine can be targeted effectively.Communicated by P. Debergh     

Adventitious shoot regeneration from seven commercial strawberry cultivars (<Emphasis Type="Italic">Fragaria </Emphasis>×<Emphasis Type="Italic"> ananassa</Emphasis> Duch.) using a range of explant types

The parameters for optimal regeneration of seven commercial strawberry cultivars were tested using a range of explants and culture conditions. Efficient levels of regeneration--those needed to carry out transformation experiments--with the cultivars Calypso, Pegasus, Bolero, Tango and Emily were achieved with leaf discs, petioles, roots and stipules. Regeneration from cv. Elsanta proved to be difficult from all explant material, although unpollinated ovaries proved to be a promising explant source, with 12% of the explants regenerating shoots. In cv. Eros, regeneration occurred only from root tissue. A comparison of the genetic background suggests that there is a strong genetic component amongst the different cultivars determining their regeneration capacity. The development of these regeneration systems provides a means to use almost the whole stock plant for the efficient genetic transformation of commercial strawberry varieties.     

Minimal Handling and Super-Binary Vectors Facilitate Efficient, Agrobacterium-Mediated, Transformation of Sugarcane (Saccharum spp. hybrid)

Agrobacterium-mediated transformation (AMT) of sugarcane has been limited by low transformation efficiency, high variability between experiments and genotype specificity. We tested combinations of parameters that have been useful in other recalcitrant plant systems, aiming to develop an efficient and reproducible method. Applied to elite sugarcane cultivar Q117, key parameters were (i) minimal handling of callus near the time of co-cultivation, (ii) use of a super-binary helper vector with additional virB,C,G gene copies, and (iii) use of Agrobacterium strain AGL1. Transformation efficiency was in the range 0.5 to 3.5 stably transformed, embryogenic-callus-forming lines per gram fresh weight of co-cultivated callus, over six independent callus batches. Addition of 5 μM copper sulphate to the callus-growth medium appeared beneficial in a single further test. Following selection for aminoglycoside resistance conferred by PUbi-aphA, 87 % of transformed lines that formed embryogenic callus were regenerable to plants. Southern blot analysis of 24 transgenic lines showed 21 % with a single-copy insertion of an intact T-DNA without vector backbone, and a mean transgene copy number of 2.5. Over multiple batches, the AMT protocol approached the transformation efficiency from our routine conditions for particle bombardment of Q117. However, the same parameters were ineffective for AMT of cultivars Q208 and Q172, and yielded a lower transformation efficiency (0.02) with KQ228. As experienced in other systems such as rice, high-efficiency transformation of one recipient genotype may provide useful starting parameters for work towards AMT of additional genotypes.     

Refining the application of direct embryogenesis in sugarcane: effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency

A rapid in vitro protocol using direct somatic embryogenesis and microprojectile bombardment was investigated to establish the developmental phases most suitable for efficient sugarcane transformation. Immature leaf roll disc explants with and without pre-emergent inflorescence tissue were compared. It was shown that for effective transformation to occur, explants should be cultured for several days to allow initiation of embryo development prior to bombardment. Leaf roll discs with pre-emergent inflorescences showed a higher degree of embryogenic competence than non-flowering explants, and transformation efficiency was higher when explants containing floral initials were bombarded. Despite the occurrence of high numbers of phenotypically negative plants, combining the use of inflorescent leaf roll discs with direct embryogenic regeneration has the potential to improve the speed and efficiency of transgenesis in sugarcane.     

The effect of auxin on plant regeneration of wheat,barley and triticale

One of the basic components of a medium influencing somatic embryogenesis of cereals from immature embryos is the type of auxin. According to some researchers, phytohormones can also play an important role during Agrobacterium-mediated transformation. In this first part of research, the influence of three types of auxins used alone or in combination of two on somatic embryogenesis and plant regeneration in three cereal species has been tested. Eight cultivars of barley, five cultivars of wheat and three cultivars of triticale have been used. Efficiency of plant development on two regeneration media, with and without growth regulators has been compared. Efficiency of regeneration characterized by frequency of explants that form embryogenic callus ranged from 25% for wheat cultivar Torka to 100% for two barley cultivars. Mean number of plantlets regenerating per explant differed significantly (from 2 to 58) depending on the type of auxin in inducing media, the type of regenerating media as well as cultivar. The biggest differences in regeneration efficiency were observed between barley cultivars, however regeneration of plants occurred in all combinations tested. The best regeneration coefficients for most barley cultivars were obtained after culture on dicamba or dicamba with 2,4-D. However, in the case of highly regenerating cv Scarlett, the most effective culture media contained picloram or 2,4-D alone. The highest values of regeneration coefficients for two triticale cultivars (Wanad and Kargo) were obtained on picloram (26.1 and 21.4, respectively) and for `Gabo' on picloram with dicamba (12.6). The range of mean number of regenerated plantlets was from 12 to 30. Dicamba alone or lower concentrations of picloram with 2,4-D were the best media influencing embryogenic callus formation in five wheat cultivars. However, the highest values of regeneration coefficients ranging from 10.6 to 26.8 were obtained at lower concentrations of picloram with 2,4-D or picloram with dicamba. R2 regeneration medium containing growth regulators was significantly better for plantlet development in several combinations (cultivar and induction medium) than the one without growth regulators. Generally, regeneration coefficients for all tested cultivars of three cereal species on the best media were high, ranging from 5.5 for barley cultivar Rodion to 51.6 for another barley cultivar Scarlett. Plantlets developed normally, flowering and setting seed.     

Screening wheat genotypes for high callus induction and regeneration capability from anther and immature embryo cultures

Plant regeneration via tissue culture varies with the genotype and is an important factor in establishing cell selection and genetic transformation systems. To select genotypes – especially Japanese ones – with a high regeneration capability, we screened 107 wheat genotypes (78 domestic, 29 foreign) for callus induction and regeneration capability from anther and immature embryo cultures. For anther culture, 83 of 107 genotypes tested induced calli and 45 regenerated plants. Only 9 genotypes, however, produced green plants, 25 produced only albino plants, and 11 produced both green and albino plants. Glennson 81 was the highest in callus induction, followed by Orofen, Danchi–komugi and Chris. The genotypes with a relatively high regeneration capability were Framala 80 at 24% and Glennson 81 at 19%, these two genotypes produced only green plants. For immature embryo culture, 97 genotypes showed a 90% callus induction rate and 74 genotypes regenerated plants. Very few genotypes produced albino plants. The genotypes with a high regeneration capability were Genaro 81 at 90%, Chinese Spring at 80%, and Norin 75 at 75%. This revised version was published online in June 2006 with corrections to the Cover Date.     

影响农杆菌介导的木薯基因转化因素的研究

Factors influencing agrobacterium-mediated cassava transformation were investigated. Among the four Agrobacterium strains tested, LBA 4404 (pTOK 233) and LBA 4404 (pBin9GusInt) gave higher transient expression than C 58 C1 (pIG121Hm) and EHA 105 (pBin9Husint). Pretreatment of explants by bombardment or vaccum had no significant effect on transient expression while preinduction of Agrobacterium with acetosyringone showed better effects, and preculture of explants showed worse effects. All the cultivars tested were susceptible to Agrobacterium infection, while the types of explants and the physiological state of the explants had a strong influence on the transient expression efficiency. The 15-day-old somatic cotyledons and the fully expanded leaves from in vitro plantlets were the most susceptible to Agrobacterium infection. The results also showed that all the four selective reagents (hygromycin, geneticin, PPT, and kanamycin) synchronously suppressed the growth of callus, shoot organogenesis and shoot rooting in a dose dependent manner.     

The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation

Doubled haploid (DH) genotypes from a genetic mapping population of Brassica oleracea were screened for ease of transformation. Candidate genotypes were selected based on prior knowledge of three phenotypic markers: susceptibility to Agrobacterium tumefaciens, shoot regeneration potential and mode of shoot regeneration. Mode of regeneration was found to be the most significant of the three factors. Transgenic plants were successfully obtained from genotypes that regenerated multiple shoots via a distinct swelling or callus phase. The absence of tissue culture blackening (associated with genotypes that formed callus) was found to be critical for transformation success. Transgenic shoots were obtained from genotypes that regenerated via an indirect callus mode, even when susceptibility to Agrobacterium was low. The most efficient genotype (DH AG1012) produced transgenic shoots at an average rate of 15% (percentage of inoculated explants giving rise to transgenic plants). The speed and efficiency of regeneration enabled the isolation of transgenic shoots 5–6 weeks after inoculation with A. tumefaciens. This line was also self-compatible, enabling the production of seed without the need for hand-pollination. A genetically uniform DH genotype, with an associated genetic map, make DH AG1012 highly desirable as a potential model B. oleracea genotype for studying gene function. The possibility of applying the same phenotypic tissue culture markers to other Brassica species is discussed.     

影响农杆菌介导的木薯基因转化因素的研究

本文研究了影响农杆菌介导的木薯基因转化的因素。结果表明,供试的4个菌种中,LBA4404(pBin9GusInt)及LBA4404(pTOK)瞬时表达效果较好。对农杆菌的诱导处理能增强瞬时表达效果,外植体的预处理对瞬时表达无影响,而外植体的预培养显著降低瞬时表达。所有供试的木薯品种都能被农杆菌侵染,但外植体的类型及生理状况对农杆菌的侵染力影响很大,成熟胚状体的子叶(萌发15d)及试管苗完全展开的叶片对农杆菌亲和性最高。四种筛选剂(kanamycin、hygromycin、phosphinothricin及geneticin)均表现出剂量效应且能同步抑制芽器官发生、愈伤生长及芽切段生根。     

Plant genotype and growth regulators interaction affecting in vitro morphogenesis of blackberry and raspberry

The morphogenic response of somatic (leaf and petiole) and de-differentiated tissue (callus) of two blackberry (Rubus fruticosus) and one raspberry (Rubus idaeus) cultivars have been studied in vitro. With the aim to induce regeneration the effect of two sets of plant growth regulator (PGR) combinations (high cytokinin/auxin ratios and high auxin/cytokinin ratios) in Murashige and Skoog basal medium, were analysed. The three cultivars were characterised by a qualitatively different morphogenic response to the PGR combinations. Raspberry adventitious shoot regeneration from somatic tissue was improved by the 6-benzylaminopurine (BAP)/indol-3-butyric acid (IBA) combinations. On the contrary, shoot regeneration of both blackberry cultivars was reduced by high concentrations of BAP and completely inhibited by BAP/IBA combination. Media supplemented with high auxin/cytokinin ratios promoted callus production and root differentiation according to genotype and type of auxin. All the genotypes responded to media supplemented with IBA. 2,4-dichlorophenoxyacetic acid induced good callus formation in blackberry, but was toxic to raspberry. Indirect shoot formation was observed only in callus of blackberry cultivar Hull Thornless cultivated on medium with 10 μM BAP, the same concentration able to trigger efficient direct shoot regeneration from leaf explants of the same cultivar. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transformation of different barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) cultivars by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> infection of in vitro cultured ovules

Most cultivars of higher plants display poor regeneration capacity of explants due to yet unknown genotypic determined mechanisms. This implies that technologies such as transformation often are restricted to model cultivars with good tissue characteristics. In the present paper, we add further evidence to our previous hypothesis that regeneration from young barley embryos derived from in vitro-cultured ovules is genotype independent. We investigated the ovule culture ability of four cultivars Femina, Salome, Corniche and Alexis, known to have poor response in other types of tissue culture, and compared that to the data for the model cultivar, Golden Promise. Subsequently, we analyzed the transformation efficiencies of the four cultivars using the protocol for Agrobacterium infection of ovules, previously developed for Golden Promise. Agrobacterium tumefaciens strain AGL0, carrying the binary vector pVec8-GFP harboring a hygromycin resistance gene and the green fluorescence protein (GFP) gene, was used for transformation. The results strongly indicate that the tissue culture response level in ovule culture is genotype independent. However, we did observe differences between cultivars with respect to frequencies of GFP-expressing embryos and frequencies of regeneration from the GFP-expressing embryos under hygromycin selection. The final frequencies of transformed plants per ovule were lower for the four cultivars than that for Golden Promise but the differences were not statistically significant. We conclude that ovule culture transformation can be used successfully to transform cultivars other than Golden Promise. Similar to that observed for Golden Promise, the ovule culture technique allows for the rapid and direct generation of high quality transgenic plants.     

Development of Agrobacterium-mediated transformation technology for mature seed-derived callus tissues of indica rice cultivar IR64

Indica rice cultivar IR64 is most recalcitrant to regenerate, which affects the transformation efficiency especially when mature seed-derived callus tissues are used as explants. Therefore, a simple, rapid and improved genetic transformation protocol has been developed for the indica rice cultivar IR64 using Agrobacterium-mediated genetic transformation. With different hormonal combination tested, the maximum callus induction was observed on MS medium supplemented with 2.5 mg/l 2,4-D and 0.15 mg/l BAP from the scutellum explants. Three weeks old scutellum derived callus explants were immersed in Agrobacterium suspension (strain LBA4404, OD600=1.0) and co-cultured at 26±2°C in dark for 2 d. The maximum transformation efficiency (12%) was achieved with infection of callus explants for 20 min along with use of 150 μm acetosyringone. The maximum plant regeneration was observed on MS medium supplemented with 3 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA. The maximum root induction was observed on MS medium along with 10 g/l glucose and 20 g/l sucrose. The integration of the transgene in T1 transgenic plants was confirmed by polymerase chain reaction and Southern blot analyses. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants. By using this improved method we have successfully raised transgenic rice plants within 3 mo from seed inoculation to plant regeneration.     

Transformation of Recalcitrant Melon (Cucumis melo L.) Cultivars is Facilitated by Wounding with Carborundum

Transformation of the recalcitrant melon (Cucumis melo L.) cultivars Kιrka?aç 637 and Noi Yarok was accomplished by wounding cotyledon explants by vortexing with carborundum prior to inoculation with Agrobacterium tumefaciens. The addition of silver nitrate to the regeneration‐selection medium reduced the transformation efficiency, as the percentage of the explants forming putative transgenic calli and bud‐like protuberances was decreased and no transgenic shoots were produced. Chimeric transgenic plants were obtained after the regeneration of putatively transformed callus, bud‐like protuberances, buds and shoots on selective medium with kanamycin. The treatments producing the most buds or shoots from explants after 30–40 days of cultivation were the most successful for the production of transgenic plants. Only treatments where explants were vortexed with carborundum produced transgenic melon shoots of either cultivar. Subculture every 18–20 days on fresh regeneration‐selection medium containing 50 mg/L kanamycin after either a relatively high (100 mg/L) or low level (50 mg/L) of kanamycin in the first regeneration‐selection medium was necessary for the successful transformation of cultivar Kιrka?aç 637. These techniques are now being used in breeding programs for the production of melon lines bearing resistances to zucchini yellow mosaic virus and cucumber mosaic virus, important viruses limiting agricultural production.     

Sensitivity of random amplified polymorphic DNA analysis to detect genetic change in sugarcane during tissue culture

Random amplified polymorphic DNA (RAPD) analysis using 10-mer oligonucleotide primers efficiently differentiated sugarcane cultivars and proved suitable for detecting gross genetic change such as that which can occur in sugarcane subjected to prolonged tissue culture, for example in protoplast-derived callus. However, RAPD analysis was not sufficiently sensitive to detect smaller genetic changes that occur during sugarcane genetic transformation. The length of DNA scored for polymorphism per primer averaged 13.2 kb, or 0.0001% of the typical sugarcane genome size of 1.2 × 10₇ kb (2C). RAPD analysis of sugarcane plants regenerated from embryogenic callus revealed very few polymorphisms, indicating that gross genetic change is infrequent during this tissue culture procedure, although epigenetic effects result in transient morphological changes in regenerated plants. More sensitive variations on the RAPD technique may increase the practicality of DNA-based screening of regenerated plant lines to reveal somaclonal variants.     

High-efficiency,microprojectile-mediated cotransformation of sugarcane,using visible or selectable markers

Transient expression of the maize anthocyanin regulatory elements,R andC1, was used to optimise parameters for microprojectile-mediated delivery of DNA into sugarcane embryogenic callus. Osmotic treatment of target tissues and particle acceleration in a high-pressure helium pulse increased the frequency of transient expression to 5–8×10³ cells per bombardment, with minimal tissue damage. An average of 0.34% of transiently expressing cells developed into stably transformed, anthocyanin-pigmented proembryoids which subsequently regenerated into plantlets. However, constitutive expression ofR andC1 proved deleterious, and no anthocyanin-pigmented plant survived beyond 3 cm in height. We also compared selective subculture of callus portions showing luciferase activity with antibiotic selection on medium containing G418 or phosphinothricin, upon bombardment of callus with constructs driving strong expression ofluc, aphA orbar genes. Selective subculture based on luciferase activity enabled recovery of 1.4±0.5 independent transgenic plants per bombardment, compared to 19.8±3.7 independent transgenic plants per bombardment from an optimised G418 selection regimen, and no transformed plants from phosphinothricin selection. Whenluc andaphA on separate plasmids were coprecipitated onto microprojectiles before bombardment, 67–79% of callus lines selected for G418 resistance also showed luciferase activity detectable under a low-light camera. Southern analysis confirmed a very high cotransformation frequency, with variable copy numbers of introduced genes. The high efficiencies of gene transfer, selection and cotransformation in the optimised system, coupled with the simple initiation and regeneration of embryogenic callus, provide an effective tool for practical genetic transformation of sugarcane.     

Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane (Saccharum spp. interspecific hybrids) leaf culture

Rapid and efficient in vitro regeneration methods that minimise somaclonal variation are critical for the genetic transformation and mass propagation of commercial varieties. Using a transverse thin cell layer culture system, we have identified some of the developmental and physiological constraints that limit high-frequency regeneration in sugarcane leaf tissue. Tissue polarity and consequently the orientation of the explant in culture, size and developmental phase of explant, and auxin concentration play a significant role in determining the organogenic potential of leaf tissue in culture. Both adventitious shoot production and somatic embryogenesis occurred on the proximal cut surface of the explant, and a regeneration gradient, decreasing gradually from the basal to the distal end, exists in the leaf roll. Importantly, auxin, when added to the culture medium, reduced this spatial developmental constraint, as well as the effect of genotype on plant regeneration. Transverse sections (1-2 mm thick) obtained from young leaf spindle rolls and orienting explants with its distal end facing the medium (directly in contact with medium) are critical for maximum regeneration. Shoot regeneration was observed as early as 3 weeks on MS medium supplemented with alpha-naphthalenencetic acid (NAA) and 6-benzyladenine, while somatic embryogenesis or both adventitious shoot organogenesis and somatic embryogenesis occurred on medium with NAA and chlorophenoxyacetic acid. Twenty shoots or more could be generated from a single transverse section explant. These shoots regenerated roots and successfully established after transplanted to pots. Large numbers of plantlets can be regenerated directly and rapidly using this system. SmartSett, the registered name for this process and the plants produced, will have significant practical applications for the mass propagation of new cultivars and in genetic modification programs. The SmartSett system has already been used commercially to produce substantial numbers of plants of orange rust-resistant and new cultivars in Australia.