Gale H. McGranahan

Somatic embryogenesis and plant regeneration from cotyledons of walnut, Juglans regia L.

Abstract Immature cotyledons of open-pollinated seeds from five walnut (Juglans regia L.) cultivars were excised from fruits at 6–11 weeks after full pistillate bloom and grown on a sequence of media to induce somatic embryogenesis. Globular, heart, cotyledonary and complete somatic embryos were obtained. Embryogenic cultures were maintained for more than a year by repetitive embryogenesis in which the roots, cotyledons and hypocotyls of somatic embryos formed additional adventive somatic embryos. Mature somatic embryos required a cold treatment of 8–10 weeks at 2–4°C to overcome apical dormancy. Selected plantlets derived from these somatic embryos were grown to young plants in soil. In addition, somatic embryogenesis was induced in J. hindsii (Jeps.), Jeps., and in Pterocarya sp., another member of the Juglandaceae.

Improved efficiency of the walnut somatic embryo gene transfer system.

SummaryAnAgrobacterium-mediated gene transfer system which relies on repetitive embryogenesis to regenerate transgenic walnut plants has been made more efficient by using a more virulent strain ofAgrobacterium and vectors containing genes for both kanamycin resistance and beta-glucuronidase (GUS) activity to facilitate early screening and selection. Two plasmids (pCGN7001 and pCGN7314) introduced individually into the disarmedAgrobacterium host strain EHA101 were used as inoculum. Embryos maintained on medium containing 100 mg/l kanamycin after co-cultivation produced more transformed secondary embryos than embryos maintained on kanamycin-free medium. Of the 186 GUS-positive secondary embryo lines identified, 70% were regenerated from 3 out of 16 primary embryos inoculated with EHA101/pCGN7314 and grown on kanamycin- containing medium, 28% from 4 out of 17 primary embryos inoculated with EHA101/ pCGN7001 and grown on kanamycin medium, and 2% from one out of 13 primary embryos inoculated with EHA101/pCGN7001 but not exposed to kanamycin. Because kanamycin inhibits but does not completely block new embryo formation in controls, identification of transformants formerly required repetitive selection on kanamycin for several months. Introduction of the GUS marker gene allowed positive identification of transformant secondary embryos as early as 5–6 weeks after inoculation. DNA analysis of a representative subset of lines (n=13) derived from secondary embryos confirmed transformation and provided evidence for multiple insertion events in single inoculated primary embryos.

Origin of somatic embryos from repetitively embryogenic cultures of walnut (Juglans regia L.): Implications forAgrobacterium-mediated transformation

Early stages of somatic embryo development from embryogenic cultures ofJuglans regia (Persian or English walnut) are described. Histological examination reveals that secondary somatic embryos arise from cotyledons and hypocotyls of primary embryos cultured in the dark. The embryos originate by transverse to oblique divisions of surface cells. Single-cell origin of the secondary embryos confirms the potential of the repetitive embryogenesis system forAgrobacterium-mediated transformation and regeneration of non-chimeric, transgenic walnut plants.

Silencing crown gall disease in walnut (Juglans regia L.)

Crown gall disease can severely limit the growth and productivity of many tree crops and ornamental plants. We have developed a novel crown gall disease resistance strategy that is based upon the silencing of two highly conserved Agrobacterium tumefaciens oncogenes which are required for crown gall development. The tryptophan monooxygenase (iaaM) and isopentenyl transferase (ipt) genes are horizontally transferred from A. tumefaciens to the plant cell, mediating the de novo auxin and cytokinin production that initiates tumorigenesis. By expressing inducers of post-transcriptional gene silencing homologous to iaaM and ipt in plant cells, the corresponding oncogene mRNA transcripts are degraded in planta, providing functional resistance to crown gall disease. This strategy is highly efficacious in English (Persian) walnut (Juglans regia L.), as several transformed lines expressing self-complementary iaaM and ipt transgenes display a highly specific accumulation of small-interfering RNAs and a broad-spectrum suppression of tumorigenesis. Potential applications and limitations of this technology in the future development of crown gall-resistant fruit and nut crop are also discussed.

High levels of expression of full-length cryIA(c) gene from Bacillus thuringiensis in transgenic somatic walnut embryos

Abstract A full-length synthetic version of the cry IA(c) gene, expressing a Bacillus thuringiensis (Bt) insecticidal crystal protein (ICP), was transferred into walnut somatic embryos. Sixty one transgenic embryo lines or clones were obtained and bioassayed with first instar codling moth larvae. In 34% of these lines, designated as ‘class A’, expression was high enough to obtain 80–100% mortality. A total of 20% were designated ‘class B’, which produced a mortality between 25 and 70% and also caused a decreased rate of larval development. Insect mortality and development from the remaining 46% of the lines were indistinguishable from that of the control. Expression of a chimeric gene encoding β -glucuronidase (GUS) was evaluated to serve as a linked but unselected marker gene. About 62% of the class A embryo lines showed correspondingly high activity for the GUS gene. However, 38% of the class A would have been missed if the level of GUS activity was used as the sole indicator. Detectable levels of protein corresponding to cry IA(c) could be found only in class A, but not in class B or C clones. Southern analysis of border regions revealed single inserts for class A clones and multiple inserts for classes B and C.

Low levels of expression of wild type Bacillus thuringiensis var. kurstaki cryIA (c) sequences in transgenic walnut somatic embryos

Abstract Insecticidal crystal protein fragments (ICPFs) of Bacillus thuringiensis encoded by cry IA (c) and cry IA (b) were previously shown to be lethal to key target walnut insect pests (codling moth, navel orangeworm and Indianmeal moth). One of these genes, cry IA (c), was used to transform walnut somatic embryos using a binary vector (pWB139) in which this gene was expressed as a protein fusion with the kanamycin resistance gene from bacteria. Transgenic embryos representing individual transformation events were germinated to produce shoots that were maintained as micropropagated shoot lines in the laboratory. Transgenic shoots were then grafted onto seedling rootstocks and transplanted into the field. DNA analysis (Southern blotting) demonstrated that these shoots were transformed by pWB139 or by the control construct pWB149 (which does not contain the cry IA (c) gene). Insect feeding trials of walnut embryos grown in vitro and infested with neonatal Indianmeal moth, codling moth or navel orangeworm larvae showed non-significant mortality; confirming insufficient or incomplete expression of ICPs in the transgenic embryos. Therefore, transformation of somatic walnut embryos with the vector pWB139 was ineffective in protecting the embryos from damage by lepidopteran insect larvae.

Transformation of pecan and regeneration of transgenic plants

A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of ‘Elliott’, ‘Wichita’, and ‘Schley’ were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line ‘Elliott’6, 3 from ‘Schley’5/3, and 3 from ‘Wichita’9. Transgenic embryos of ‘Wichita’9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis.

Regeneration by somatic embryogenesis of triploid plants from endosperm of walnut, Juglans regia L. cv. Manregian

Plants were regenerated by somatic embryogenesis from endosperm tissue of open-pollinated seeds of Juglans regia L. cv Manregian. These plants were obtained by growing endosperm tissue on media similar to those used for plant regeneration from walnut cotyledons (Tulecke and McGranahan 1985). The plants appear morphologically uniform and have a triploid chromosome number of 3n=48. Nine plants have been grown to a young sapling stage in soil. This embryogenic line from endosperm has been maintained in culture for two years by the process of repetitive somatic embryogenesis.

Defining the sources of Paradox: DNA sequence markers for North American walnut (Juglans L.) species and hybrids

Abstract One of the most important rootstocks in the California walnut industry is Paradox, which refers to the offspring of a California black walnut pollinized by a Persian walnut ( Juglans regia ). Paradox was developed by Luther Burbank, who did not distinguish between northern ( J. hindsii ) and southern ( J. californica ) California black walnut. Although it is generally accepted that Paradox designates hybrids between J. hindsii and J. regia , the name is commonly applied to any black walnut–Persian walnut hybrid. Moreover, due to gene flow among black walnut species, the genealogy of Paradox hybrids may also include species such as Arizona ( J. major ) and Eastern black ( J. nigra ) walnut. Since the nuts from which Paradox seedlings are grown are collected from wild trees, their genetic backgrounds are not generally known. In conjunction with a large study aimed at evaluating Paradox hybrids from different industry sources, we have been working to develop molecular markers that can be used to infer the parentage of individual Paradox seedlings. Representatives of the five black walnut species from North America were screened for variability in the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA and in three noncoding regions from the chloroplast genome, the trn T– trn L, trn L– trn F, and trn D– trn T spacers. Unique sequence markers were identified for each species, and total DNA extracts from 27 Paradox source trees were tested for those markers. Chloroplast DNA profiles were used to trace the maternal lineages of the Paradox source trees, while the ITS data provided evidence as to whether or not the source trees were themselves hybrids. Our results indicate that, among industry Paradox sources, there is considerable genetic contribution from species other than J. hindsii .

Agrobacterium-mediated transformation of somatic embryos as a method for the production of transgenic plants

Somatic embryos have been successfully used as a target tissue for transformation and regeneration of transgenic walnut plants. Walnut somatic embryos, initiated originally from developing zygotic embryos, proliferate numerous secondary embryos from single cells in the epidermal layer. These single cells in intact somatic embryos are susceptible to transformation by genetically engineeredAgrobacterium tumefaciens and provide a means to regenerate nonchimeric transgenic plants. This gene transfer system has been made more efficient using, a) vector plasmids containing two marker genes encoding β-glucuronidase (GUS) and aminoglycoside phosphotransferase (APH(3′)II) and B) a more virulent strain ofAgrobacterium. This system should be applicable to any crop that undergoes repetitive embryogenesis from singleAgrobacterium-susceptible cells.