The International Board for Plant Genetic Resources has recently coordinated efforts to augment the world soybean collection. A key step to exploiting the genetic resources of the subgenus Glycine in soybean improvement is obtaining viable hybrids between these species and soybean. Several specific attributes present among the perennial Glycine, and of potential use in soybean improvement can be listed. In broad outline the perennial Glycine exhibits a striking diversity of geno-types and ecological adaptation. Typically, the perennial Glycine species are minor, inconspicuous components of semi-open Eucalyptus woodland. The most promising results of more recent hybridizations are with a newly described species, G. argyrea Tind. The development represents a significant new contribution from wild perennial germplasm. These developments are the assembly of a large and diverse collection, the achievement of hybrids between the perennials and soybean, and the uncovering of diverse sources of resistance to soybean leaf rust.

Grain legumes such as pea (Pisum sativum L.) are highly valued as a staple source of protein for human and animal nutrition. However, their seeds often contain limited amounts of high-quality, sulfur (S) rich proteins, caused by a shortage of the S-amino acids cysteine and methionine. It was hypothesized that legume seed quality is directly linked to the amount of organic S transported from leaves to seeds, and imported into the growing embryo. We expressed a high-affinity yeast (Saccharomyces cerevisiae) methionine/cysteine transporter (Methionine UPtake 1) in both the pea leaf phloem and seed cotyledons and found source-to-sink transport of methionine but not cysteine increased. Changes in methionine phloem loading triggered improvements in S uptake and assimilation and long-distance transport of the S compounds, S-methylmethionine and glutathione. In addition, nitrogen and carbon assimilation and source-to-sink allocation were upregulated, together resulting in increased plant biomass and seed yield. Further, methionine and amino acid delivery to individual seeds and uptake by the cotyledons improved, leading to increased accumulation of storage proteins by up to 23%, due to both higher levels of S-poor and, most importantly, S-rich proteins. Sulfate delivery to the embryo and S assimilation in the cotyledons were also upregulated, further contributing to the improved S-rich storage protein pools and seed quality. Overall, this work demonstrates that methionine transporter function in source and sink tissues presents a bottleneck in S allocation to seeds and that its targeted manipulation is essential for overcoming limitations in the accumulation of high-quality seed storage proteins.

Using pea as our model crop, we sought to understand the regulatory control over the import of sugars and amino acids into the developing seeds and its importance for seed yield and quality. Transgenic peas simultaneously overexpressing a sucrose transporter and an amino acid transporter were developed. Pod walls, seed coats, and cotyledons were analysed separately, as well as leaves subtending developing pods. Sucrose, starch, protein, free amino acids, and endogenous cytokinins were measured during development. Temporal gene expression analyses (RT-qPCR) of amino acid (AAP), sucrose (SUT), and SWEET transporter family members, and those from cell wall invertase, cytokinin biosynthetic (IPT) and degradation (CKX) gene families indicated a strong effect of the transgenes on gene expression. In seed coats of the double transgenics, increased content and prolonged presence of cytokinin was particularly noticeable. The transgenes effectively promoted transition of young sink leaves into source leaves. We suggest the increased flux of sucrose and amino acids from source to sink, along with increased interaction between cytokinin and cell wall invertase in developing seed coats led to enhanced sink activity, resulting in higher cotyledon sucrose at process pea harvest, and increased seed number and protein content at maturity.

Gene editing is becoming the plant breeding tool of choice, but prior to targeting a gene for editing, a knowledge of the gene family members (GFMs) controlling yield is required in the specific crop plant. Critical to yield are components from senescing leaves. We targeted genes controlling senescence in Pisum sativum and the release and transport of carbohydrates and amino acids from the source leaves to the pods and seeds. The expression of GFMs for cytokinin biosynthesis (IPT) and destruction (CKX), sucrose transporters (SUT), Sugar Will Eventually be Exported Transporters (SWEET), amino acid permeases (AAP), and cell wall invertases, was determined using RT-qPCR. GFMs were differentially expressed in leaves of different ages. The expression of many gene family members was lower in the expanding sink leaves compared with the senescing leaves, with the exception of two PsAAP GFMs and PsCKX5, which were highly expressed. Expression of specific PsSWEETs, SUTs, and AAPs increased in the mature and/or senescing leaves. Expression of PsIPTs was least in the mature source leaves, but as strong in the senescing leaves as in the young source leaves. PsCKX7 was expressed in source and senescing leaves. We discuss the potential impact of the targeted reduction of specific PsCKX GFMs on source-sink relationships.

Seed development largely depends on the long-distance transport of sucrose from photosynthetically active source leaves to seed sinks. This source-to-sink carbon allocation occurs in the phloem and requires the loading of sucrose into the leaf phloem and, at the sink end, its import into the growing embryo. Both tasks are achieved through the function of SUT sucrose transporters. In this study, we used vegetable peas (Pisum sativum L.), harvested for human consumption as immature seeds, as our model crop and simultaneously overexpressed the endogenous SUT1 transporter in the leaf phloem and in cotyledon epidermal cells where import into the embryo occurs. Using this 'Push-and-Pull' approach, the transgenic SUT1 plants displayed increased sucrose phloem loading and carbon movement from source to sink causing higher sucrose levels in developing pea seeds. The enhanced sucrose partitioning further led to improved photosynthesis rates, increased leaf nitrogen assimilation, and enhanced source-to-sink transport of amino acids. Embryo loading with amino acids was also increased in SUT1-overexpressors resulting in higher protein levels in immature seeds. Further, transgenic plants grown until desiccation produced more seed protein and starch, as well as higher seed yields than the wild-type plants. Together, the results demonstrate that the SUT1-overexpressing plants with enhanced sucrose allocation to sinks adjust leaf carbon and nitrogen metabolism, and amino acid partitioning in order to accommodate the increased assimilate demand of growing seeds. We further provide evidence that the combined Push-and-Pull approach for enhancing carbon transport is a successful strategy for improving seed yields and nutritional quality in legumes.

A study on the Nezara viridula male gonad cells was undertaken to compare normal development and development of insect cells after irradiation of 4th instar nymphs with a dose of 40 Gray. Visually, morphologically, biochemically and cytologically the insects were not all uniformly affected by the radiation. In all aspects of development there was a range from severely affected to nearly normal. Irradiated insects appeared to move slowly and were unable to mate with non-irradiated females. The testes of the males varied from bright orange to grey in colour and all were smaller in size than non-irradiated testes. The ultrastructure of the developing sperm showed abnormalities the axonemes, the mitochrondrial derivatives, nebenkern and centrioles. Cytochemically, the main difference observed was the presence of granules heavily stained with acid phosphatase in between mitochrondrial derivatives. The chromosomes of these irradiated insects were highly fragmented. Although a few sperm in irradiated insects appeared normal no progeny were produced as insect did not mate. The sterile insect technique (SIT) requires a balance between the effective radiation dose to achieve partial or full sterility, while maintaining physical fitness. The observation that abnormalities varied from almost none to severe at 40Gy could help the development of SIT for control of N. viridula and other Pentatomidae.

AbstractKey MessageAgrobacterium tumefacienswas used to transform radiata pine shoots and to efficiently produce stable genetically modified pine plants.AbstractMicropropagated shoot explants fromPinus radiataD. Don were used to produce stable transgenic plants byAgrobacterium tumefaciens-mediatedtransformation. Using this method any genotype that can be micropropagated could produce stable transgenic lines. As over 80% ofP. radiatagenotypes tested can be micropropagated, this effectively means that any line chosen for superior characteristics could be transformed. There are well-established protocols for progressing such germplasm to field deployment. Here we used open and control pollinated seed lines and embryogenic clones. The method developed was faster than other methods previously developed using mature cotyledons. PCR positive shoots could be obtain within 6 months ofAgrobacteriumco-cultivation compared with 12 months for cotyledon methods. Transformed shoots were obtained using either kanamycin or geneticin as the selectable marker gene. Shoots were recovered from selection, were tested and were not chimeric, indicating that the selection pressure was optimal for this explant type. GFP was used as a vital marker, and thebargene, (for resistance to the herbicide Buster®) was used to produce lines that could potentially be used in commercial application. As expected, a range of expression phenotypes were identified for both these reporter genes and the analyses for expression were relatively easy.

Key Message Agrobacterium tumefaciens was used to transform radiata pine shoots and to efficiently produce stable genetically modified pine plants. Abstract Micropropagated shoot explants from Pinus radiata D. Don were used to produce stable transgenic plants by Agrobacterium tumefaciens-mediated transformation. Using this method any genotype that can be micropropagated could produce stable transgenic lines. As over 80% of P. radiata genotypes tested can be micropropagated, this effectively means that any line chosen for superior characteristics could be transformed. There are well-established protocols for progressing such germplasm to field deployment. Here we used open and control pollinated seed lines and embryogenic clones. The method developed was faster than other methods previously developed using mature cotyledons. PCR positive shoots could be obtain within 6 months of Agrobacterium co-cultivation compared with 12 months for cotyledon methods. Transformed shoots were obtained using either kanamycin or geneticin as the selectable marker gene. Shoots were recovered from selection, were tested and were not chimeric, indicating that the selection pressure was optimal for this explant type. GFP was used as a vital marker, and the bar gene, (for resistance to the herbicide Buster®) was used to produce lines that could potentially be used in commercial application. As expected, a range of expression phenotypes were identified for both these reporter genes and the analyses for expression were relatively easy.

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic-active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane-localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element-companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.

The potential movement of transgenes from genetically modified crops to non-genetically modified crops via insect-mediated pollen dispersal has been highlighted as one of the areas of greatest concern in regards to genetically modified crops. Pollen movement depends sensitively on spatial and temporal variation in the movement of insect pollinators between crop fields. This study tested the degree of variation in the diversity and relative abundance of flower-visiting insects entering versus leaving pak choi, Brassica rapa var. chinensis L. (Brassicales: Brassicaceae), crops throughout different stages of the flowering cycle. The relative abundance of flower-visiting insects varied significantly with Brassica crop phenology. Greater numbers of flower-visiting insects were captured inside rather than outside the crop fields, with the highest capture rates of flower-visitors coinciding with the peak of flowering in both spring-flowering and summer-flowering crops. Moreover, the ratio of flower-visiting insects entering versus leaving crop fields also varied considerably with changing crop phenology. Despite high variation in relative capture rates, the data strongly indicate non-random patterns of variation in insect movement in relation to crop phenology, with early-season aggregation of flower-visiting insects entering and remaining in the crop, and then mass emigration of flower-visiting insects leaving the crop late in the flowering season. Although pollen movement late in the flowering cycle might contribute relatively little to total seed set (and hence crop production), the findings here suggest that extensive late-season pollinator redistribution in the landscape could contribute disproportionately to long-distance gene movement between crops.

Seeds of grain legumes are important energy and food sources for humans and animals. However, the yield and quality of legume seeds are limited by the amount of sulfur (S) partitioned to the seeds. The amino acid S-methylmethionine (SMM), a methionine derivative, has been proposed to be an important long-distance transport form of reduced S, and we analyzed whether SMM phloem loading and source-sink translocation are important for the metabolism and growth of pea (Pisum sativum) plants. Transgenic plants were produced in which the expression of a yeast SMM transporter, S-Methylmethionine Permease1 (MMP1, YLL061W), was targeted to the phloem and seeds. Phloem exudate analysis showed that concentrations of SMM are elevated in MMP1 plants, suggesting increased phloem loading. Furthermore, expression studies of genes involved in S transport and metabolism in source organs, as well as xylem sap analyses, support that S uptake and assimilation are positively affected in MMP1 roots. Concomitantly, nitrogen (N) assimilation in root and leaf and xylem amino acid profiles were changed, resulting in increased phloem loading of amino acids. When investigating the effects of increased S and N phloem transport on seed metabolism, we found that protein levels were improved in MMP1 seeds. In addition, changes in SMM phloem loading affected plant growth and seed number, leading to an overall increase in seed S, N, and protein content in MMP1 plants. Together, these results suggest that phloem loading and source-sink partitioning of SMM are important for plant S and N metabolism and transport as well as seed set.

The generation of novelty is a key focus for breeders of ornamental crops. This paper reviews outcomes of the application of in vitro breeding techniques to generate novel genetic combinations from which new varieties can be selected in a range of genera. Interspecific hybridisation between Limonium sinuatum and L. perezii resulted in range of hybrids and back-cross hybrids. All F1 hybrids were sterile and through oryzalin-induced chromosome doubling fertility was restored. The tetraploid forms of the interspecific hybrids were used to generate back-cross hybrids with a low level of fertility. Sandersonia is a flower crop well known to New Zealand growers. Very little variation has been observed despite the crop being entirely seed propagated. Attempts to increase variation in this crop have included hybridisation with Littonia and Gloriosa. There are considerable differences in the growth and agronomic performance of the various Sandersonia hybrids, with some lines almost impossible to grow and others extremely vigorous. Hybrids have all been infertile and attempts to double chromosomes have not been successful in any of the lines tested. Gentiana is a relatively poorly known cut flower crop. Ten years ago the colour range was blue, pink and white. Red-flowered varieties are now available to commerce. Yellow-flowered interspecific hybrids have been developed between G. triflora and G. lutea. These hybrids have proven difficult to grow out of in vitro culture. Chromosome doubling has been carried out to overcome the anticipated infertility of these hybrids, and work is underway to develop a strategy for producing back-cross hybrids.

This chapter describes the transformation of Pinus radiata using organogenic cotyledon explants rather than the more common somatic embryogenesis methods for conifers. The advantages of our method are the year round availability of seed and that over 80% of genotypes can be easily regenerated from the mature cotyledon explants. The transformation efficiency (i.e., the number of transformed shoots regenerated from excised cotyledons) is 1.7% and, as with other Agrobacterium tumefaciens transformation methods, the majority of transgene integrations are single copy. Critical factors for success are survival of the cotyledons, Agrobacterium strain, and selection pressure after cocultivation.

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 beta-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.

Triploid plants of Sandersonia aurantiaca were produced by crossing diploid and tetraploid forms of S. aurantiaca. Enlarged ovules were transferred to in vitro culture 14-30 days after pollination. The triploid nature of the embryo derived plants was determined by, flow cytometry and chromosome counts both of which showed that the triploid plants had features that were midway between those of the two parents. The mean nuclear DNA contents of 2C nuclei from diploid, triploid and tetraploid forms of S. aurantiaca were 6.86pg, 10.04pg and 13.55pg, respectively. The nuclear DNA content of 1C nuclei of sperm cells from pollen grains was 2.94pg. Mitotic chromosome counts from the three plants gave 2n = 24, 36 and 48 chromosomes for the diploid, triploid and tetraploid forms, respectively. Meiotic chromosome counts for the diploid and tetraploid plants were n = 12 and n = 24, respectively. The triploid showed mainly bivalents, but lagging chromosomes led to micronuclei and infertility in gametes. The morphological features of the various plants corroborated other evidence indicating that the triploid plants were the result of a cross between diploid and tetraploid plants.

The aim of this work was to test a protocol for producing tetraploid plants of Gentiana. Tetraploid plants were produced by growing in vitro cultures of Gentiana triflora var. japonica 'Royal Blue' in the presence of oryzalin for 4 weeks. A population of plants was regenerated from these in vitro cultures and transferred to the greenhouse. The treated plants were screened for higher ploidy level on the basis of leaf thickness and further confirmation of polyploidy was provided by measuring guard cell lengths. The tetraploid nature of one plant with larger than normal guard cells was confirmed by flow cytometry and chromosome counts. The mean nuclear DNA contents of 2C and 4C nuclei were 9.26 pg and 17.77 pg DNA, respectively. Mitotic chromosome counts from diploid and tetraploid plants were 2n=26 and 2n=52 respectively.

Genetic engineering in peas (Pisum sativum L.) is discussed in relation to Agrobacterium tumefaciens-mediated transformation and direct gene transfer via electroporation. There are several methods of A. tumefaciens-mediated transformation applied routinely in a number of laboratories around the world. All have been used to transfer potentially useful genes into peas. Each of these methods are compared with regard to ease of transformation, efficiency and genotype independence. Some transgenic peas have been field tested and could be suitable for commercial release depending on the regulatory environment and patent claims on the genes and technologies. A number of useful genes that have been transferred into peas are discussed. In addition transgenic peas have been studied as 'model systems' for nodule development, and as bioreactors.

We compared the efficiency of two Agrobacterium tumefaciens strains, AGL 1 and KYRT1, for producing transgenic pea plants. KYRT1 is a disarmed strain of Chry5 that has been shown to be highly tumourigenic on soybean. The efficacies of the strains were compared using cotyledon explants from three pea genotypes and two plasmids. The peas were sourced from field-grown plants over three Southern Hemisphere summer seasons. Overall, KYRT1 was found to be on average threefold more efficient than AGL 1 for producing transgenic plants. We suggest that KYRT1 is sensitive to cocultivation temperature as the expected increase in efficiency was not achieved at high laboratory temperatures.

Transformed pea (Pisum sativum L.) lines were produced with two chimeric gene constructs encoding the coat protein (CP) of alfalfa mosaic virus (AMV) strain NZ1. To determine whether transformed lines have improved AMV resistance, progeny of independently derived transgenic lines were tested in the greenhouse and five lines with improved virus resistance were identified. Resistance was observed only in individual plants that accumulated detectable CP product from the transgene, suggesting that resistance is CP mediated. Plants that accumulated detectable amounts of transgene CP product yet were susceptible to AMV were found among the progeny of most lines, indicating that these lines are only partially resistant to the challenging AMV strains, 425 and NZ1(Lincoln). A field test was conducted with the progeny of four independently derived transgenic lines. To test for improved virus resistance under field conditions, plots of transgenic lines and nontransgenic controls were inoculated with two AMV strains, NZ1(Lincoln) and NZ34. Analysis of disease severity in seedlings in inoculated plots confirmed that partial virus resistance can be produced by genetically modifying peas with AMV CP sequences.

Intergeneric hybrids were obtained between Sandersonia aurantiaca and Littonia modesta using ovule culture. The embryos were rescued by culturing 14 to 30 day old ovules. The ovules were cultured on modified KM medium for varying lengths of time until they germinated. After germination the embryo-derived-plantlets were transferred to modified growth regulator-free MS medium on which they developed tubers and became quiescent. The quiescent tubers could be successfully transferred to soil. The hybrid nature of both the putative Sandersonia × Littonia and the Littonia × Sandersonia hybrids was indicated by flow cytometry that showed the hybrid plants had a DNA content midway between that of the two parents. Mitotic and meiotic chromosome counts from S. aurantiaca, L. modesta and the hybrids gave chromosome numbers of (2n=) 24, 22 and 23 respectively. Morphological analyses of the leaves and flowers showed that the hybrids displayed features that were intermediate between both parents. Hybrids were male and female sterile. No morphological differences were observed between the two hybrids.