Dennis J. Gray

Initiation and maintenance of long term somatic embryogenesis from anthers and ovaries of Vitis longii ‘Microsperma’

Anthers and ovaries of Vitis longii ‘Microsperma’ produced embryogenic callus when cultured on solidified Murashige and Skoog medium with 5μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1μM benzyladenine (BA). The initial callus was short-lived. However, long-term embryogenesis from callus was maintained through serial transfers by careful selection of clustered embryos with subtending callus. Alternatively, long term culture maintenance was through secondary embryogenesis which occurred directly from previously formed embryos on medium lacking growth regulators. Somatic embryos were white, exhibited frequent pluricotyly and tended to be larger than zygotic embryos. Histology of embryogenic callus demonstrated the presence of lipid-like substances and abundant starch. Somatic embryos were attached to callus by narrow to wide suspensor-like structures and possessed typical epidermal, cortical, and vascular tissue. Embryo cells contained abundant lipid-like accumulations but no starch. Embryos germinated when placed on medium containing 1μM BA and produced plants of normal appearance.

Effects of dehydration and exogenous growth regulators on dormancy, quiescence and germination of grape somatic embryos

SummaryDormant grape somatic embryos from five genetically distinct culture lines were subjected either to dehydration or exogenous growth regulators (benzyladenine, gibberellin or abscisic acid). Of growth regulator treatments tested, benzyladenine resulted in the highest germination rate but postgermination growth was abnormal. Abscisic acid treatment resulted in the least germination. Dehydration for 21 d under 75–95% relative humidity was effective only for the culture line that produced well developed embryos. However, for this line, more embryos produced shoots after dehydration (34%) when compared to growth regulator treatments and the postgermination growth resembled that of a seedling. Moisture content of dehydrated somatic embryos was similar to that of seed at equivalent relative humidities. Because dehydrated embryos germinate after addition of water, they are considered to be quiescent or nondormant. Plant recovery rates of 34% after 21 d of dehydrated storage at 70% relative humidity suggests that dehydration of somatic embryos may eventually provide for the conservation of clonally propagated crops in seed gene banks.

In vitro micropropagation and plant establishment of muscadine grape cultivars (Vitis rotundifolia)

Shoot apical meristems were used to establish regenerative axillary bud cultures of 9 muscadine grape cultivars. Meristems taken from 10 cm long shoots had less contamination (3%) and a higher survival rate (94%) than those from shorter or longer shoots. Of media tested, MS, 1/2 MS, and C2D resulted in equivalent shoot proliferation rates, whereas, WPM produced stunted shoots. When pooling results for 3 cultivars, 5, 10 and 20 μM BA and 5 μM TDZ produced the highest average number of shoots per cultured apex (3.4–3.8). However, shoots produced with TDZ were stunted and did not root well. For rooting of shoots directly in potting mix, a rooting powder pretreatment significantly increased the number of roots per shoot but did not affect percent rooting or root length. For rooting in vitro, 1 μM NAA significantly increased all parameters measured. Although more shoots rooted in vitro than in vivo (77% vs. 46%), the latter was judged preferable since acclimatized plants were produced in less time and a major culture step was eliminated. Significant differences among cultivars were noted for measured responses in all experiments.

Desiccated quiescent somatic embryos of orchardgrass for use assynthetic seeds

SummarySomatic embryos of orchardgrass became quiesent when desiccated to 13% water. Twelve percent germinated after 21 d of desiccated storage at 23° C and 4% developed into green plants. During desiccation, embryos decreased in size, became yellowish and brittle, and their outer walls collapsed. Within 15 min after imbibition, they rapidly enlarged and were indistinguishalbe from nondesiccated embryos. These results suggest that somatic embryos may be engineered to function as synthetic seeds for mass propagation.

OPTIMIZING AGROBACTERIUM-MEDIATED TRANSFORMATION OF GRAPEVINE

SummaryA translational fusion between the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTH) genes was used to optimize parameters influencing Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless. The corresponding bifunctional protein produced from this EGFP/NPTH fusion gene allowed for a single promoter to drive expression of both green fluorescence and kanamycin resistance, thus conserving promoter resources and climinating potential promoter-promoter interactions. The fusion gene, driven by either a double cauliflower mosaic virus 35S (CaMV 35S) promoter or a double cassava vein mosaic virus (CsVMV) promoter, was immobilized into Agrobacterium strain EHA 105. Somatic embryos capable of direct secondary embryogenesis were used as target tissues to recover transgenic plants. Simultaneous visualization of GFP fluorescence and kanamycin selection of transgenic cells, tissues, somatic embryos, and plants were achieved. GFP expression and recovery of embryogenic culture lines were used as indicators to optimize transformation parameters. Preculturing of somatic embryos for 7 d on fresh medium prior to transformation minimized Agrobacterium-induced tissue browning/necrosis. Alternatively, browning/necrosis was reduced by adding 1 gl−1 of the antioxidant dithiothreitol (DTT) to post co-cultivation wash media. While combining preculture with antioxidant treatments did not result in a synergistic improvement in response, either treatment resulted in recovery of more stable embryogenic lines than did the control. A 48h co-cultivation period combined with 75 mgl−1 kanamycin in selection medium was optimal. DNA analysis confirmed stable integration of transgenes into the grape genome: 63% had single gene insertions, 27% had two inserts, and 7 and 3% had three and four inserts, respectively. Utilizing optimized procedures, over 1400 stable independent transgenic embryogenic culture lines were obtained, of which 795 developed into whole plants. Transgenic grapevines have exhibited normal vegetative morphology and stable transgene expression for over 5 yr.

Bi-directional duplex promoters with duplicated enhancers significantly increase transgene expression in grape and tobacco.

Novel bi-directional duplex promoters (BDDP) were constructed by placing two identical core promoters divergently on both upstream and downstream sides of their duplicated enhancer elements. Estimates of promoter function were obtained by creating versions of CaMV 35S and CsVMV BDDPs that contained reporter marker genes encoding β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP) interchangeably linked either to the upstream or downstream core promoters. GUS was used for quantitative analysis of promoter function, whereas, EGFP allowed visual qualitative evaluation. In addition, the GUS and EGFP genes placed in downstream positions were modified by translational fusion with neomycin phosphotransferase (NPTII) to allow simultaneous monitoring of promoter activity and selection of stable transformants. These versions of BDDP were compared with each other and with equivalent unidirectional constructs by evaluating their expression in grape and tobacco. For 35S promoter constructs tested in grape somatic embryos (SE), BDDP exhibited transient GUS expression 206- and 300-fold greater in downstream and upstream configurations, respectively, compared to a unidirectional 35S core promoter. Compared with a unidirectional double enhanced 35S promoter, BDDPs exhibited 0.5- and 3-fold increased GUS expression from downstream and upstream core promoters, respectively. The same differences in expression levels determined quantitatively with GUS were distinguished qualitatively with EGFP. Constructs using CsVMV core promoters yielded results relative to those obtained with 35S promoter. For example, the upstream BDDP CsVMV core promoter provided a 200-fold increase in GUS expression compared to a unidirectional core promoter. However, CsVMV promoter was found to have higher promoter activity than 35S promoter in both BDDP and unidirectional constructs. Incorporation of an additional duplicated enhancer element to BDDPs resulted in increased expression. For example, a 35S BDDP with two divergently arranged duplicated enhancer elements resulted in over a 6-fold increase in GUS expression in stably transformed tobacco plants compared to a BDDP with one duplicated enhancer element. Data demonstrate that BDDP composed of divergently-arranged core promoters separated by duplicated enhancers, all derived from a single promoter sequence, can be used to significantly enhance transgene expression and to direct synchronized expression of multiple transgenes.

Towards a More Sustainable Agriculture

Critical Reviews in Plant Sciences is pleased to devote these issues to research in Sustainable Agriculture. The general topic of “sustainability” has been discussed in many regards—from housing to population growth, land usage to the effects of pollution on the environment, and so on. Intertwined among all the issues encompassed by sustainability is that of a sustainable food source, without which global society would certainly crumble. These very timely and thoughtful reports take a careful look at issues confronting conversion of our agricultural base into a truly sustainable model. In particular, organic approaches are mentioned and discussed. But also importantly, uses of the wild landscape as food sources are examined and education of the populace on the needs and methods of sustainability are discussed. Throughout the issue, the authors make a case for the need to achieve more sustainability of our food and fiber supply, as well as the consequences for not doing so. We are especially indebted to Guest Editor Professor Tiziano Gomiero for taking the lead on this project, along with David Pimentel and Maurizio G. Paoletti for their contributions. It is important to note that Professors Pimentel and Paoletti are long-time members of the CRPS editorial board and the Guest Editors’ collective talents can be seen throughout the issue.

Plant Development and Biotechnology

Introduction Dennis J. Gray and Robert N. Trigiano HISTORY OF PLANT TISSUE CULTURE History of Plant Tissue and Cell Culture James D. Caponetti, Dennis J. Gray, and Robert N. Trigiano SUPPORTING METHODOLOGIES Getting Started with Tissue Culture: Media Preparation, Sterile Technique, and Laboratory Equipment Caula A. Beyl Histological Techniques Robert N. Trigiano, Kathleen R. Malueg, Kimberly A. Pickens, Zong-Ming Cheng, and Effin T. Graham Photographic Methods for Plant Cell and Tissue Culture Dennis J. Gray Elements of In Vitro Research Michael E. Compton A Brief Introduction to Plant Anatomy Robert N. Trigiano and Dennis J. Gray Plant Growth Regulators in Plant Tissue Culture and Development Victor P. Gab Software and Databases as Tools for Analyzing Nucleic Acid and Protein Sequences Zhijian T. Li Molecular Approaches to the Study of Plant Development Albrecht G. von Arnim PROPAGATION AND DEVELOPMENT CONCEPTS Shoot Culture Procedures Michael E. Kane Propagation from Nonmeristematic Tissues: Organogenesis Otto J. Schwarz, Anjuna R. Sharma, and Robert M. Beaty Molecular Aspects of In Vitro Shoot Organogenesis Shibo Zhang and Peggy G. Lemaux Propagation from Nonmeristematic Tissues: Nonzygotic Embryogenesis Dennis J. Gray Some Developmental and Molecular Aspects of Somatic Embryogenesis (Nonzygotic Embryogenesis) Andreas Mordhorst, Erika Charbit, and Sacco C. de Vries CROP IMPROVEMENT TECHNIQUES Use of Protoplasts for Plant Improvement Richard E. Veilleux, Michael E. Compton, and James A. Saunders Haploid Cultures Sandra M. Reed Embryo Rescue Sandra M. Reed Genetic Engineering Technologies Zhijian T. Li and Dennis J. Gray Genetically Modified Plant Controversies: Sensational Headlines versus Pragmatic Research Harry A. Richards, Laura C. Hudson, Matthew D. Halfhill, and Charles N. Stewart, Jr. Construction and Use of a Simple Gene Gun for Particle Bombardment Dennis J. Gray, Michael E. Compton, Ernest Hiebert, Chia Min Lin, and Victor P. Gaba A Simple Illumination System for Visualizing Green Fluorescent Protein Dennis J. Gray, Subramanian Jayasankar, and Zhijian T. Li Germplasm Preservation Leigh E. Towill Valuable Secondary Products from In Vitro Culture Mary Ann Lila In Vitro Plant Pathology Subramanian Jayasankar and D. J. Gray SPECIAL TOPICS Variation in Tissue Culture Subramanian Jayasankar Commercial Laboratory Production Gayle R. L. Suttle Indexing for Plant Pathogens Alan C. Cassells and Barbara M. Doyle Entrepreneurship for Biotechnology Ventures: From Bench to Bag David W. Altman

Comparative anatomy and morphology of Vitis vinifera (Vitaceae) somatic embryos from solid- and liquid-culture-derived proembryogenic masses

Ontogeny of somatic embryos of grapevine (Vitis vinifera) produced from solid- and liquid-culture-derived proembryogenic masses (PEM) was compared using light and scanning electron microscopy. Somatic embryos produced from solid-medium-derived PEM (SPEM) had large cotyledons, little or no visible suspensor structure, and a relatively undeveloped concave shoot apical meristem, whereas those from liquid-medium-derived PEM (LPEM) had smaller cotyledons, a distinct suspensor, and a flat-to-convex shoot apical meristem. The convex shoot apical meristem in LPEM-derived somatic embryos formed as early as the heart stage of development; it was 4-6 cell layers deep and rich in protein. Suspensors persisted in fully developed and mature LPEM-derived somatic embryos. The SPEM-derived somatic embryos exhibited dormancy, as do mature zygotic embryos, which also have a rudimentary suspensor, whereas LPEM-derived embryos were not dormant. We hypothesize that the presence of a persistent suspensor in LPEM-derived somatic embryos modulates development, ultimately resulting in rapid germination and a high plant-regeneration rate.

In vitro studies on the anatomy and morphology of bud regeneration in melon cotyledons

SummaryThe anatomy and morphology of bud regeneration were investigated in melon (Cucumis melo L.) cv. Galia, which regenerates in vitro only by direct organogenesis from the cotyledon explant. Explants were cut from the cotyledon proximal to the apex from 3-d-old in vitro seedlings. After 3 d on Murashige and Skoog medium with N6-benzyladenine, cell division can be observed in the epidermal layer on the adaxial side in the center of the explant, near the most proximal (wounded) cut edge. Over the next week, the area of the meristem increases laterally. Additional cell layers are added to the meristematic area by cell division in the epidermis. In places the epidermis remains active in cell division. Alongside those active areas there are zones where the epidermis has become inactive, although the subepidermal layers continue to divide. In transverse section, the explant now has small protuberances on the adaxial surface. After 10 d on cytokinin-containing medium, the first signs of development are visible on the adaxial surface adjacent to the proximal cut edge. The protuberances observed after 10 d are neither primordia nor buds, although some meristematic bulges are observed. The first regenerated shoot buds are observed histologically after 15 d, by which time the surface has many protuberances and some small leaves. The first shoot is found by histology after 22 d. By this time the surface is covered with protrusions and leaves, mostly without accompanying buds. The leaves may be produced from the protrusions initially visible after 10 d.