We describe the construction of new helper Ti plasmids forAgrobacterium-mediated plant transformation. These plasmids are derived from three differentAgrobacterium tumefaciens Ti plasmids, the octopine plasmid pTiB6, the nopaline plasmid pTiC58, and the L,L-succinamopine plasmid pTiBo542. The T-DNA regions of these plasmids were deleted using site-directed mutagenesis to yield replicons carrying thevir genes that will complement binary vectorsin trans. Data are included that demonstrate strain utility. The advantages ofAgrobacterium strains harbouring these ‘disamed’ Ti plasmids for plant transformation viaAgrobacterium are discussed.

New Agrobacterium helper plasmids for gene transfer to plants

Plant tissue culture has become one of the fundamental tools of plant science research. It is extensively employed in the production, conservation and improvement of plant resources. The presence of somaclonal variation in populations derived from tissue culture is affecting the use of tissue culture negatively and has remained a major problem. Conversely, it is a source of new desirable clones/variants with better agronomic traits. In this review, we summarize the possible causes, detection methods and desirability of variants. Somaclonal variation is one of the most researched and reviewed topics. Hence, we restricted ourselves to outlining various examples which may be used as important references for researchers who intend to identify and/or characterize somaclonal variants while using tissue culture for research and production. Emphasis is placed on the negative effects of somaclonal variation. However, this review also includes examples of some useful variants generated as a result of somaclonal variation.

Somaclonal variation in plants: causes and detection methods

A chimeric gene was constructed consisting of the promoter of the nopaline synthase gene of Agrobacterium and the structural gene of the aminoglycoside phosphotransferase of Tn5. This chimeric gene was recombined into the T-DNA of Agrobacterium. When this strain was used in a cocultivation experiment, transformed calli could be selected out of a pool of non-transformed calli when kanamycin was added into the growing medium

Chimeric genes as dominant selectable markers in plant cells

The broad applications of somatic embryogenesis, both in basic and applied research, have stimulated studies on the determination of in vitro conditions for the induction of somatic embryos and their conversion into plants. As a result, efficient protocols on SE induction and plant regeneration have recently become available for many plant species, including Arabidopsis thaliana (L.) Heynh., a model plant in genetics and embryogenesis. Studies on factors controlling in vitro plant morphogenesis are highly desirable not only for the development of improved regeneration systems, but also for the analysis of molecular mechanisms underlying plant embryogenesis. This review focuses on the conditions influencing the induction of embryogenic potential in in vitro cultured plant cells. The roles of explant type, endo- and exogenous plant growth regulators and stress factors in the induction of somatic embryogenesis are especially emphasized. Possible mechanisms by which different factors induce or modify embryogenic competence in cultured plant cells are also discussed. Since the production of genetically solid and true-to-type plants is desired, especially for transformation and micropropagation practice, the problem of the genetic characteristics of regenerants, in terms of their chimerism and somaclonal variation, is discussed in some detail. Special consideration is given to A. thaliana– a major model plant species for classical genetics and genomics. Recent availability of efficient embryogenic cultures in this organism makes it possible to benefit from advanced genomic research of Arabidopsis to study plant embryogenesis on the molecular level.

Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Heynh

The commercial multiplication of a large number of diverse plant species represents one of the major success stories of urilizing tissue culture technology profitably. Micropropagation has now become a multibillion dollar industry, practised all over the world. Of the various methods used to micropropagate plants, somatic embryogenesis and enhanced axillary branching have become the principal methods of multiplication. Long-term benefits of this enterprise, however, lie in the production of clonally uniform plants. The concept of genetic uniformity among micropropagated plants derived through organized meristems was exploded by several convincing reports of the incidence of somaclonal variation at morphological, cytological (chromosome number and structure), cytochemical (genome size), biochemical (proteins and isozymes), and molecular (nuclear and organellar genomes) levels. Somaclonal variation is not limited to any particular group of plants; it has been reported, for example, in ornamentals, plantation crops, vegetable and food crops, forest species and fruit trees. The upsurge of these reports, facilitated to a large extent by the technical developments made in molecular biology, is a matter of great concern for any micropropagation system. The economic consequences of somaclonal variation can be enormous in forest trees and woody plants, as they have long life cycles. Therefore, somaclonal variation has to be dispensed with if large-scale micropropagation of diverse plant species is to become not only successful but also accepted by end-users. In the light of the various factors (genotype, ploidy level, in vitro culture age, explant and culture type, etc.) that lead to somaclonal variation of divergent genetic changes at the cellular and molecular levels, genetic analysis of micropropagated plants using a multidisciplinary approach, especially at the DNA sequence level, initially and at various cultural stages, is essential. The results obtained at early multiplication stages from these tests could help in modifying the protocol/s for obtaining genetically true-to-type plants, and ultimate usage by entrepneneurs without any ambiguity.

Genetic fidelity of organized meristem-derived micropropagated plants: A critical reappraisal

Expiants of five plant species (Allium cepa, Antirrhinum majus, Brassica campestris. Glycine max, and Nicotiana tabacum) were co-cultivated with three Agrobacterium tumefaciens strains under different conditions to assess the effects of acetosyringone and medium pH on strain virulence. Tumours were incited on all dicotyledonous species by strains N2/73 and A281. The presence of acetosyringone during co-cultivation generally enhanced the virulence of these strains, most markedly N2/73 on A. majus and G. max, and A281 on G. max. Strain Ach5 was virulent only on N. tabacum in the absence of acetosyringone, which, when present, extended the host range to include A. majus. There was evidence to suggest that acetosyringone may suppress virulence in some strain/plant species interactions. Virulence was affected in some cases by medium pH, but there was no general effect across plant species.

The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species

Plants were regenerated by somatic embryogenesis from long-term callus cultures derived from five garlic (Allium sativum L.) cultivars. Thirty-five of these plants were subjected to RAPD analysis. The frequency of variation was found to be cultivar dependent: approximately 1% in the two clones Solent White and California Late and around 0.35% in another three clones, Chinese, Long Keeper and Madena. Certain band changes were found in regenerants of different cultivars, suggesting the existence of a mutation-sensitive part of the garlic genome. The karyotypes of another 75 regenerants derived from the same callus cultures of three parental garlic clones were examined. Of these plants, 9.3% were found to be tetraploids, 4% aneuploid and 2.6% showed a change in the position of the secondary constriction. No association could be shown between the rate of variation for molecular and cytological characters either by comparing cultivars or examining individual regenerants.

Detection of somaclonal variation in garlic (Allium sativum L.) using RAPD and cytological analysis

We report the encapsulation of in vitro-derived nodal cuttings or shoot tips of cassava in 3% calcium alginate for storage and germplasm exchange purposes. Shoot regrowth was not significantly affected by the concentration of sucrose in the alginate matrix while root formation was. In contrast, increasing the sucrose concentration in the calcium chloride polymerisation medium significantly reduced regrowth from encapsulated nodal cuttings of accession TME 60444. Supplementing the alginate matrix with increased concentrations of 6-benzylaminopurine and α-naphthaleneacetic acid enhanced complete plant regrowth within 2 weeks. Furthermore, plant regrowth by encapsulated nodal cuttings and shoot tips was significantly affected by the duration of the storage period as shoot recovery decreased from almost 100% to 73.3% for encapsulated nodal cuttings and 94.4% to 60% for shoot tips after 28 days of storage. The high frequency of plant regrowth from alginate-coated micropropagules coupled with high viability percentage after 28 days of storage is highly encouraging for the exchange of cassava genetic resources. Such encapsulated micropropagules could be used as an alternative to synthetic seeds derived from somatic embryos.

Encapsulation of nodal cuttings and shoot tips for storage and exchange of cassava germplasm

Plantlets were regenerated by adventitious shoot budding in tissue culture from leaf explants of a single genotype of sugar beet. DNA was extracted from the parental plant and from 120 regenerants. RAPD analysis was carried out using five decanucleotide primers; 4,557 RAPD marker bands were examined and two polymorphisms were observed. Thirty secondary regenerants were then derived, using the same tissue culture technique, from thirty of the primary regenerants. Again RAPD analysis was employed and a single band polymorphism was observed out of 1,050 bands examined. The overall frequency of detection of somaclonal polymorphisms using RAPD (3 in 5,607 = 0.05%) is similar to frequencies previously reported using isozyme and RFLP technologies.

The detection of somaclonal variants of beet using RAPD.

A simplified technique which simultaneously induces and cryoprotects embryogenic calli using sucrose followed by dehydration was developed for the cryopreservation of cassava genetic resources. An initial experiment to optimise the sucrose concentration needed for both embryo production and cryoprotection showed that higher concentrations of sucrose—between 0.4 M and 0.5 M—significantly reduced the viability as well as the number of embryos produced by the embryogenic clumps in the absence of freezing. Post-thaw viability as well as embryogenic competence of clumps depended on the percentage moisture lost, duration of exposure to higher sucrose concentrations and the duration of induction of embryogenic clumps. Extending the period of cryoprotection to 21 days coupled with increased moisture loss (greater than 75%) significantly increased both post-thaw viability and the embryogenic competence of cryopreserved clumps to 95%, while reducing the duration decreased post-thaw viability. Cryopreserved callus clumps developed secondary and cyclic embryos similar to those of the non-cryopreserved controls. The optimised protocol was successfully applied to SM1-2075-1 Line 1 somatic embryos. The rate of plant recovery from cryopreserved embryos of both TME 9 and SM1-2075-1 Line 1 was comparable to that of the non-cryopreserved embryos. Successful cryopreservation of embryogenic clumps of cassava can be used to establish in vitro genebanks for long-term conservation of cassava genetic resources to complement field genebanks and other in vitro methods already being used.

B. V. Ford-Lloyd

Papers

The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species

Detection of somaclonal variation in garlic (Allium sativum L.) using RAPD and cytological analysis

Encapsulation of nodal cuttings and shoot tips for storage and exchange of cassava germplasm

The detection of somaclonal variants of beet using RAPD.

Cryopreservation of embryogenic calli of cassava using sucrose cryoprotection and air desiccation