Adrian Slater

RAPD ANALYSIS OF A VARIANT OF BANANA (MUSA SP.) CV. GRANDE NAINE AND ITS PROPAGATION VIA SHOOT TIP CULTURE

SummaryA morphological variant obtained from in vitro corm-derived plants of banana (Musa sp.) cv. Grande Naine (AAA) was evaluated up to harvest and the genetic basis of variation was confirmed by the random amplified polymorphic DNA (RAPD). The corms formed during the multiplication phase of shoot tip-derived cultures of the cv. Grande Naine grown on Murashige and Skoog (MS) medium enriched with 13.3 μM N6-benzyladenine (BA) developed numerous morphological variants after transfer to MS medium with 6.66 μM BA. The variant designated as CUDBT-B1, with distinct morphological features, was further evaluated. The morphological features of CUDBT-B1 were variegated leaf, pseudostem, bracts, ovary of the male flower and fruits, reduced height, decreased lamina length and breadth, and early flowering. These features were also manifested in the second-cycle progeny of CUDBT-B1. RAPD assay showed a marker DNA band of 1650 bp, and differential band intensity between the CUDBT-B1 and normal clone. CUDBT-B1 was multiplied using shoot tip culture, and the shoots were rooted on half-strength MS medium supplemented with 2.69 μM α-naphthaleneacetic acid. All plantlets showed variegated leaves under field conditions.

THIDIAZURON-INDUCED ORGANOGENESIS AND SOMATIC EMBRYOGENESIS IN SUGAR BEET (BETA VULGARIS L.)

SummaryImproved in vitro tissue culture systems are needed to facilitate the application of recombinant DNA technology to the improvement of sugar beet germplasm. The effects of N6-benzyladenine (BA) and thidiazuron (TDZ) pretreatment on adventitious shoot and somatic embryogenesis regeneration were evaluated in a range of sugar beet breeding lines and commercial varieties. Petiole explants showed higher frequencies of direct adventitious shoot formation and produced more shoots per explant than leaf lamina explants. TDZ was more effective than BA for the promotion of shoot formation. The optimal TDZ concentrations were 2.3–4.6 μM for the induction of adventitious shoot regeneration. Direct somatic embryogenesis from intact seedlings could be induced by either BA or TDZ. TDZ-induced somatic embryogenesis occurred on the lower surface of cotyledons at concentrations of 0.5–2μM and was less genotype-dependent than with Ba. A high frequency of callus induction could be obtained from seedlings and leaf explants, but only a few of the calluses derived from leaf explants could regenerate to plants via indirect somatic embryogenesis. These results demonstrated that TDZ could prove to be a more effective cytokinin for in vitro culture of sugar beet than BA. Rapid and efficient regeneration of plants using TDZ may provide a route for the production of transgenic sugar beet following Agrobacterium-mediated transformation.

Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.)

Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate “escapes” and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3–11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents. Green fluorescent shoots were obtained at frequencies of 2–5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.

The plant cell cycle in context.

Biological scientists are eagerly confronting the challenge of understanding the regulatory mechanisms that control the cell division cycle in eukaryotes. New information will have major implications for the treatment of growth-related diseases and cancer in animals. In plants, cell division has a key role in root and shoot growth as well as in the development of vegetative storage organs and reproductive tissues such as flowers and seeds. Many of the strategies for crop improvement, especially those aimed at increasing yield, involve the manipulation of cell division. This review describes, in some detail, the current status of our understanding of the regulation of cell division in eukaryotes and especially in plants. It also features an outline of some preliminary attempts to exploit transgenesis for manipulation of plant cell division.

Assessment of polysomaty, embryo formation and regeneration in liquid media for various species of diploid annual Medicago.

To avoid polyploidy in regenerants the source of explant material should be monosomatic. Therefore, the leaf and petiole tissue of five diploid Medicago species (Medicago ciliaris, Medicago murex, Medicago orbicularis, Medicago polymorpha and Medicago truncatula cv. Jemalong, and the ecotype R108-1) was assessed for polysomaty by flow cytometry. For the species studied the frequency of 2C nuclei was about 90% in leaves compared with that in petioles. Embryos were readily formed from tissue of leaves in liquid media containing 1 mg l(-1) or 4 mg l(-1) dichlorophenoxyacetic acid (2,4-D). For embryo development two procedures were tested - prolonged use of induction medium and treatment with polyethylene glycol Mw 6000 (PEG). The highly regenerable genotypes M. truncatula cv. Jemalong and R108-1 showed efficient conversion of embryos after maturation in liquid medium. The regenerated plants were diploid and with normal phenotype.

Efficient somatic embryogenesis in sugar beet ( Beta vulgaris L.) breeding lines

Efficient regeneration via somatic embryogenesis (SE) would be a valuable system for the micropropagation and genetic transformation of sugar beet. This study evaluated the effects of basic culture media (MS and PGo), plant growth regulators, sugars and the starting plant material on somatic embryogenesis in nine sugar beet breeding lines. Somatic embryos were induced from seedlings of several genotypes via an intervening callus phase on PGo medium containing N6-benzylaminopurine (BAP). Calli were mainly induced from cotyledons. Maltose was more effective for the induction of somatic embryogenesis than was sucrose. There were significant differences between genotypes. HB 526 and SDM 3, which produced embryogenic calli at frequencies of 25–50%, performed better than SDM 2, 8, 9 and 11. The embryogenic calli and embryos produced by this method were multiplied by repeated subculture. Histological analysis of embryogenic callus cultures indicated that somatic embryos were derived from single- or a small number of cells. 2,4-dichlorophenoxyacetic acid (2,4-D) was ineffective for the induction of somatic embryogenesis from seedlings but induced direct somatic embryogenesis from immature zygotic embryos (IEs). Somatic embryos were mainly initiated from hypocotyls derived from the cultured IEs in line HB 526. Rapid and efficient regeneration of plants via somatic embryogenesis may provide a system for studying the molecular mechanism of SE and a route for the genetic transformation of sugar beet.

EFFICIENT PROCEDURES FOR CALLUS INDUCTION AND ADVENTITIOUS SHOOT ORGANOGENESIS IN SUGAR BEET (BETA VULGARIS L.) BREEDING LINES

SummaryImproved in vitro tissue culture systems are needed to facilitate the application of transgene technology to the improvement of sugar beet germplasms. Several commercially important sugar beet breeding lines (SDM, 3, 5, 8, 9, 10, 11, HB 526, and CMS 22003) and commercial varieties (Roberta and Gala) were tested for their regeneration capacity through adventitious shoot organogenesis from cotyledons, hypocotyls, root/hypocotyl/shoot transition zone tissues, and leaf lamina and petiole via an intervening callus phase. Callus induction and adventitious shoot regeneration was dependent on genotype and combinations of plant growth regulators. With cotyledon or hypocotyl explants, SDM 3 and 10 showed a better response on adventitious shoot regeneration in medium containing benzyladenine (BA) and 2,3,5-triiodobenzoic acid or 1-naphthaleneacetic acid (NAA) than SDM 11, 5, and 9. Shoot regeneration was obtained from hypocytyl-root or hypocotyl-shoot transition zone tissue in SDM 9, 10, and HB 526 grown on PGo medium supplemented with BA to induce callus, and the regeneration frequency was 25%. Adventitious shoots were also regenerated from leaf explants of SDM 3 and 9 cultured on medium containing NAA for callus induction and BA and NAA to induce shoot regeneration, and in SDM 10 and CSM 22003 cultured on medium containing BA for callus induction and to induce shoot regeneration.

EFFECT OF AUXINS ON INDIRECT IN VITRO MORPHOGENESIS AND EXPRESSION OF gusA TRANSGENE IN A LECTINACEOUS MEDICINAL PLANT, EUPHORBIA NIVULIA BUCH.-HAM.

SummaryThe types of auxin in Murashige and Skoog (MS) medium containing N6-benzyladenine (BA) determined indirect morphogenesis, i.e. development to bipolar somatic embryos or monopolar shoots in Euphorbia nivulia Buch.-Ham. Indirect in vitro morphogenesis depended on growth regulators, explant excision period, and light. Calli induced from explants collected in March–April were superior in the induction of indirect morphogenesis to those collected in July–August. Light enforced in vitro morphogenesis, while darkness was inhibitory. The presence of kinetin in the medium also inhibited morphogenesis. Calli developed on explants collected in March–April grown on MS medium fortified with α-naphthaleneacetic acid (NAA) and BA facilitated indirect organogenesis, while those developed on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and BA underwent somatic embryogenesis. MS medium with 13.3 μM BA and 2.69 μM NAA was the best for induction of shoots from callus, which developed a mean of 15.7 shoots. Shoots were best rooted on half-strength MS medium enriched with 2.46 μM indole-3-butyric acid with a mean of 5.1 roots per shoot. MS medium supplemented with 2.26 μM 2,4-D and 4.44 μM BA induced the highest number (mean of 13.4) of somatic embryos. Of the embryos transferred on half-strength MS medium containing 2.89 μM gibberellic acid, 78% of embryos developed to the cotyledonary stage. Most cotyledonary embryos (80%) underwent conversion to plantlets upon being transferred to half-strength MS basal medium in light. The survival rate of organogenesis and embryo-derived plants was 80 and 90%, respectively. Calli transformed with Agrobacterium tumefaciens showed expression of the gusA transgene and resistance to kanamycin, but did not undergo morphogenesis.

Rapid transformation and regeneration of alfalfa (Medicago falcata L.) via direct somatic embryogenesis

Two simple, rapid and efficient protocols for theregeneration of transformed tetraploid lines ofalfalfa (Medicago falcata L.) have beendeveloped and compared. Leaf explants fromembryogenic lines 47/1-150 and 47/1-5 were inoculatedwith Agrobacterium tumefaciens containingconstructs carrying the nptII selectable markergene and promoter:gusA gene fusions under thecontrol of the CaMV 35S or Arabidopsis cdc2a,CycB1 and CycA2 promoters. In the firstregeneration system (the MSH system), inoculated leafexplants were incubated on MS medium supplemented with2,4-D and kinetin and then subcultured onto plantgrowth regulator-free MS medium in order to inducedirect somatic embryogenesis. In the secondregeneration system (the B5h system), the inoculatedexplants were incubated on B5h medium to induceindirect production of somatic embryos viaembryogenic callus. In both systems, an effectivekanamycin selection regime was employed and wasmaintained when the embryos were subcultured onto arecovery medium (Boi2Y) to promote further embryodevelopment. The use of Boi2Y medium was particularlyimportant for shortening the regeneration time andpromoting a higher frequency of healthy plantletproduction from the somatic embryos. The maturesomatic embryos were finally transferred to plantgrowth regulator-free MS medium for plantletformation. Transgenic plantlets were produced within10–14 weeks in the MSH system and 12–16 weeks in theB5h system. The MSH system appears to be the fastesttransformation system reported for leguminous speciesto date. Confirmation of transformation was obtainedusing a re-callusing assay on kanamycin and subsequentSouthern blot hybridisation and PCR analysis. Theability to induce expression of GUS activity in leafexplants containing the cell division cycle genepromoter:gusA constructs by 2,4-D treatment alsoproved to be a reliable indicator of transformation.

The application of a DNA-based identification technique to over-the-counter herbal medicines.

Reliable methods to identify medicinal plant material are becoming more important in an increasingly regulated market place. DNA-based methods have been recognised as a valuable tool in this area with benefits such as being unaffected by the age of the plant material, growth conditions and harvesting techniques. It is possible that the methods of production used for medicinal plant products will degrade or remove DNA. So how applicable are these techniques to processed medicinal plant products? A simple PCR-based identification technique has been developed for St. Johns Wort, Hypericum perforatum L. Thirteen St. Johns Wort products were purchased including capsules, tablets and tinctures. DNA was extracted from each product, and the species specific PCR test conducted. DNA was successfully extracted from all thirteen products, using a fast and efficient modified method for extracting DNA from tinctures. Only four products yielded the full length ITS region (850 bp) due to the quality of the DNA. All of the products tested positive for H. perforatum DNA. DNA-based identification methods can complement existing methods of authentication. This paper shows that these methods are applicable to a wide range of processed products, provided that they are designed to account for the possibility of DNA degradation.