Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification

 Routine cryopreservation of shoot tips from sweet potato [Ipomoea batatas (L.) Lam] has been hampered by their survival variability after cryogenic exposure. We examined the effects of light conditions on stock plants, sucrose preculture and cryoprotectant loading on survival after vitrification using PVS2 solution. The survival of vitrified sweet potato shoot tips cooled to approximately –208  °C was increased by preculturing with 0.3 M sucrose for 24 h at 22  °C. Survival was also enhanced by excising shoot tips immediately after the 8-h dark photoperiod. The best survival after cryogenic exposure was obtained using 2 M glycerol +0.4 M sucrose for 1 h at 22  °C followed by dehydration with PVS2 for 16 min at 22  °C. Rapid cooling was used and achieved by the immersion of foil strips into partially solidified nitrogen. Successfully vitrified and warmed shoot tips directly developed shoots on a medium containing 1 μM NAA, 0.5 μM BA and 0.1 μM kinetin with only minimum callus formation. Shoot formation occurred in all surviving shoot tips. This procedure shows promise for cryopreserving sweet potato shoot tips. Received: 2 March 1999 / Revision received: 21 September 1999 / Accepted: 29 September 1999     

Cryopreservation of in vitro sugar beet shoot tips using the encapsulation-dehydration technique: influence of abscisic acid and cold acclimation

It has been previously shown that shoot tips of in vitro plantlets of sugar beet (Beta vulgaris L. clone SES1) can be cryopreserved using the encapsulation-dehydration technique (survival rate of 37% after freezing). This article reports the influence of abscisic acid (ABA) and cold acclimation on survival after cryopreservation. When ABA was added to the multiplication medium of the plants, the survival rate of shoot tips after cryopreservation was not increased (45%). After cold acclimation of the plants, their growth pattern differed (plants became apically dominant) and the survival rate of the shoot tips after cryopreservation clearly increased (70% survival and 50% plant regeneration after freezing). This improved protocol was successfully applied to three other clones. Received: 28 October 1996 / Revision received: 28 January 1997 / Accepted: 15 March 1997     

Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips

 Shoot tips from in vitro-grown, cold-hardened stock plants of white poplar (Populus alba L.) were successfully cryopreserved at –196  °C by one-step vitrification. After preculturing at 5  °C for 2 days on hormone-free MS medium containing different sucrose concentrations, and loading for 20 min with 2 m glycerol and 0.4 m sucrose, shoot tips were treated with the PVS2 vitrification solution and plunged directly into liquid nitrogen. Best survival rate (90%) was obtained when shoot tips were precultured on 0.09 m sucrose, hormone-free MS medium, vitrified by exposure to PVS2 solution for 60 min at 0  °C and, following cryopreservation, rewarmed at 40  °C and washed in 1.2 m sucrose solution for 20 min. Regrowth was improved by plating shoot tips on a gelled MS medium containing 1.5 μm N⁶-benzyladenine plus 0.5 μm gibberellic acid, while shoot rooting was achieved on MS medium containing 3 μm indole-3-butyric acid. Following this procedure, almost 60% rooted shoots were obtained from cryopreserved shoot tips. Received: 1 February 1999 / Revision received: 3 May 1999 · Accepted: 21 May 1999     

Evaluation of nematode-resistant sugar beet (Beta vulgaris L.) lines by molecular analysis

 Thirty sugar beet (Beta vulgaris) lines conferring complete resistance to the beet cyst nematode (BCN, Heterodera schachtii) originating from interspecific crosses with wild beets of the section Procumbentes (B. procumbens, B. webbiana and B. patellaris) were investigated by morphology and wild beet-specific molecular markers. The beet lines carrying chromosome mutations consisted of monosomic additions (2n=18+1), fragment additions (2n=18+fragment) and translocations (2n=18) from the wild beets. Genome-specific single-copy, satellite and repetitive probes were applied to study the origin, chromosomal assignment and presence of nematode resistance genes. Within the wild beet species at least three different resistance genes located on different chromosomes were distinguished: Hs1 on the homoelogous chromosomes I of each species, Hs2 on the homoelogous chromosomes VII of B. procumbens and B. webbiana and Hs3 on chromosome VIII of B. webbiana. A clear distinction between the three chromosomes was possible by morphological and molecular means. The translocation lines were separated into two different groups: one containing the resistance gene Hs1 from chromosome I and the other carrying a different nematode resistance gene. The molecular data combined with sequence analyses of Hs1 of the three wild beet species revealed a clear distinction between B. procumbens and B. webbiana. The evolutionary and taxonomical relationship of these species supporting the idea of three different species originating from a common ancestor is discussed. Received: 6 April 1998 / Accepted: 22 April 1998     

Cryopreservation of invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure

Invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott.) were successfully cryopreserved by vitrification. Excised shoot tips precultured on solidified MS supplemented with 0.3M sucrose and maintained under a 16 h phtoperiod at 25°C for 16 h were loaded with a mixture of 2M glycerol plus 0.4M sucrose for 20 min at 25°C. The shoot tips were then sufficiently dehydrated with a highly concentrated vitrification solution (PVS2) for 20 min at 25°C prior to immersion into liquid nitrogen. Successfully vitrified and warmed shoot tips resumed growth within 7 days and developed shoots directly without intermediate callus formation. The average rate of shoot recovery amounted to around 80%, and the vitrification protocol appeared to be very promising for the cryopreservation of taro germplasm.Abbreviations DMSO Dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS Murashige & Skoog medium (1962) - TDZ thidiazuron     

Chromosomal assignment of the nine linkage groups of sugar beet (Beta vulgaris L.) using primary trisomics

 Twenty-four marker loci representing each of the nine linkage groups of sugar beet (Beta vulgaris) have been assigned to the nine primary trisomics of Butterfass (1964). Single-copy RFLP probes were hybridized with filter-bound DNA of the trisomics. The autoradiographs were scanned and analyzed by densitometric methods. Statistics on the integrated optical densities of the RFLP bands revealed a clear relationship of each linkage group to a distinct trisomic type. For the first time each of the linkage groups could unequivocally be assigned to one sugar beet chromosome. A standard nomenclature of the 9 chromosomes of sugar beet is suggested and discussed with respect to previous numbering systems. Received: 27 February 1997/Accepted: 7 March 1997     

Mapping quantitative trait loci (QTLs) for resistance to Cercospora leaf spot disease (Cercospora beticola Sacc.) in sugar beet (Beta vulgaris L.)

The breeding of sugar beet varieties that combine resistance to Cercospora and high yield under non-diseased conditions is a major challenge to the breeder. The understanding of the quantitative trait loci (QTLs) contributing to Cercospora resistance offers one route to solving this problem. A QTL analysis of Cercospora resistance in sugar beet was carried out using a linkage map based on AFLP and RFLP markers. Two different screening methods for Cercospora resistance (a field test at Copparo, Italy, under natural infection, and a newly-developed leaf disc test) were used to estimate the level of Cercospora resistance; the correlation between scores from the field (at 162 days after sowing) and the leaf disc test was significant. QTL analysis was based on F₂ and F₃ (half-sib family) generations derived from crosses between diploid single plants of 93164P (resistant to Cercospora leaf spot disease) and 95098P (susceptible). Four QTLs associated with Cercospora resistance (based on Lsmean data of the leaf disc test) on chromosomes III, IV, VII and IX were revealed using Composite interval mapping. To produce populations segregating for leaf spot resistance as a single Mendelian factor, we selected for plants heterozygous for only one of the QTLs (on chromosome IV or IX) but homozygous for the others. Received: 1 September 1999 / Accepted 7 October 1999     

Cryopreservation of in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation vitrification

 Alginate-coated meristems from in vitro-grown axillary buds of mint (Mentha spicata L.) were successfully cryopreserved by vitrification. Excised meristems from nodal segments cold hardened at 4  °C for 3 weeks were encapsulated and osmoprotected by a mixture of 2 M glycerol plus 0.4 M sucrose. These meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 h at 0  °C prior to a plunge into liquid nitrogen. Successfully encapsulated vitrified meristems developed shoots within a week after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 90%. This procedure was successfully applied to other Mentha species. It was also confirmed that encapsulated vitrified meristems produced a much higher rate of shoot formation than the encapsulated dried meristems. Thus, this revised encapsulation vitrification method appears promising for the cryopreservation of mint and other germplasm. Received: 24 November 1998 / Revision received: 8 February 1999 / Accepted: 26 February 1999     

Doubled haploid plant production from unpollinated ovules of sugar beet (Beta vulgaris L.)

 The effects of cold pretreatment, AgNO₃ and activated charcoal on haploid plant production from unpollinated sugar beet ovules were investigated. Both cold pretreatment and the addition of charcoal increased the frequency of embryo formation, whereas AgNO₃ decreased or completely inhibited it. Colchicine (50, 100, 150 or 500 mg l^–1) and trifluralin (1.7, 3.4 or 5.0 mg l^–1) for 12, 24, 36 or 48 h were compared in agar-solidified, agarose-solidified or liquid media. Although colchicine gave a higher doubling rate (25.3%) than trifluralin (18.2%), the difference was not significant. Both agents were more effective when used in liquid (29.1%) than agarose-solidified medium (20.7%) and agar-solidified medium (15.4%). A treatment duration of 48 h was significantly more effective (27.5%) than 12 h (13.6%) but it was not different from 24 h (16.3%) or 36 h (18.6%). Received: 25 October 1999 / Revision received: 18 May 2000 / Accepted: 29 May 2000     

切花百合离体茎尖玻璃化法超低温保存研究

以切花百合西伯利亚试管苗离体茎尖为试材,通过正交设计试验对预培养培养基中蔗糖浓度、预培养时间和PVS2处理时间等影响超低温保存存活率的主要因素进行了分析,初步建立了切花百合种质玻璃化法超低温保存的技术方案。通过形态观察、可溶性蛋白和同工酶检测,冻存前后材料的遗传稳定性没有发生改变,表明该方法对切花百合的种质保存具有较强的实用意义。     

Selection in vitro for UV-tolerant sugar beet (Beta vulgaris) somaclones

With a reduced stratospheric ozone concentration, the generation of UV-tolerant plants may be of particular importance. Among different crop plants there is large variation in sensitivity to UV-B radiation. This study was undertaken to investigate the possibilities of using somaclonal variation and selection in vitro for improving UV-B tolerance in sugar beet (Beta vulgaris L.). Sugar beet callus was exposed to UV radiation (280–320 nm, 0.863–5.28 kJ m^-2 day^-1, unweighted) and resultant shoots were selected from surviving cells. After establishment of the plants, they were grown under either visible radiation (114 μmol m^-2 s^-1 PAR) or with the addition of UV radiation (6.3 kJ m^-2 day^-1 biologically effective UV-B). Screening of regenerants in vivo for tolerance to UV radiation was undertaken 10 months after termination of the UV selection pressure. Screening was done visually and by using a number of physiological parameters, including chlorophyll fluorescence induction, ultraweak luminescence, pigment analysis and total content of UV-screening pigments. A clear difference between the unselected and the UV-selected somaclones was observed when visually studying the UV damage and other leaf injury. The observations were supported by the ultraweak luminescence measurements. Unselected plants showed significantly greater damage when subjected to subsequent UV radiation as compared to the selected plants. The clones subjected to UV selection pressure displayed a significantly higher concentration of UV-screening pigments under subsequent UV radiation. The unselected plants under subsequent UV treatment showed a lower carotenoid concentration when compared to selected plants. However, no significant difference between treatments was found for chlorophyll a/b, or F/F_max, a measure of photosynthetic quantum yield.     

Ultrastructural expression of cytoplasmic male sterility in sugar beet (Beta vulgaris L.)

Summary The development of microspore mother cells (MMC) and tapetum in male-fertile and male-sterile anthers of Beta vulgaris L. was compared at the electron microscope level. These studies were complemented by morphometric analyses of mitochondria in both tissues through successive stages of microsporogenesis. The earliest irregularities in the ultrastructure of male-sterile anthers were noted within the tapetum at the tetrad stage. These disturbances were initially expressed by a slight reduction in mitochondrial size and the appearance of concentric configurations of endoplasmic reticulum. As development proceeded, a further decrease in mitochondrial size become more conspicuous and was accompanied by a reduction in ribosome population and a failure of the tapetum to produce Ubisch bodies. This failure to produce Ubisch bodies is reflected in the underdevelopment of sterile microspore exine.     

Improvement of protoplast culture protocols for Beta vulgaris L. (sugar beet)

Summary The effects of NaCl, feeder cells and the embedding of protoplasts in calcium alginate have been investigated in an attempt to improve culture conditions of recalcitrant sugar beet (Beta vulgaris L.) mesophyll protoplasts. While the use of NaCl in all instances proved detrimental to protoplast development, the other two treatments had clear beneficial effects. Minimum plating densities, necessary to sustain cell division, could be reduced to <5% (<4000 protoplasts / ml) of the control levels and plating efficiencies could be significantly enhanced by approx. 10 fold. Plants could still be regenerated from soft calli derived from mesophyll protoplasts cultured under the modified conditions at a frequency of 20–30 %. In particular, the use of alginate is considered of potentially great importance for the further application of beet protoplasts for other aims e.g. asymmetric hybridization.     

An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.)

A transformation protocol, based on co-inoculation with two strains of Agrobacterium, Agrobacterium tumefaciens LBA4404 and A. rhizogenes 15834 containing a binary vector with the GUS gene, was established for the induction of transgenic hairy roots from sugar beet (Beta vulgaris L.) explants. It resulted in marked improvement in the formation of hairy roots and the integration of the binary vector T-DNA into the host genome. Of 250 inoculated sugar beet hypocotyls, 84% yielded hairy roots 5–7 days after inoculation, of which 70% were co-transformed with the binary vector T-DNA. To determine stable expression of alien genes in hairy roots, the nematode resistance gene Hs1 ^pro-1 was used as a reporter gene. In addition, molecular marker analysis was applied to monitor stable incorporation of a translocation from the wild beet B. procumbens. The molecular analysis and the nematode (Heterodera schachtii) resistance test in vitro demonstrated that the genomic structure and the expression of the Hs1 ^pro-1 -mediated nematode resistance were well-maintained in all hairy root cultures even after repeated sub-culture. Received: 25 November 1997 / Revision received: 26 May 1998 / Accepted: 15 June 1998     

A linkage map of sugar beet (Beta vulgaris L.)

Summary We have established a first linkage map for beets based on RFLP, isozyme and morphological markers. The population studied consisted of 96 F₂ individuals derived from an intraspecific cross. As was expected for outbreeding species, a relatively high degree of polymorphism was found within sugar beet; 47% of the DNA markers were polymorphic for the chosen population. The map consists of 115 independent chromosomal loci designated by 108 genomic DNA probes, 6 isozyme and one morphological marker. The loci cover 789 cM with an average spacing of 6.9 cM. They are dispersed over nine linkage groups corresponding to the haploid chromosome number of Beta species. Eighteen markers (15.4%) showed distorted segregation which, in most instances, can be explained by gametic selection of linked lethal loci. The application of the linkage map in sugar beet breeding is discussed.     

The genetics of selfing with concurrent backcrossing in breeding hybrid sugar beet (Beta vulgaris altissima L.)

 Sugar beet hybrids are produced by crossing a cytoplasmic male-sterile (CMS) line with a pollinator. New CMS lines are produced by crossing a fertile plant to an existing CMS line. The fertile plant is also selfed. In the following generation, one of the selfed, fertile progeny is paired and isolated with one of the crossed, CMS progeny, to give a second generation of selfing and crossing. Over a series of such crosses and selfs, a new fertile inbred line and its corresponding, near-isogenic CMS partner are produced. Selection among lines takes place at one or more stages of the backcrossing programme. A method is presented here for calculating the genetic variances and covariances within and between lines and generations based on a derivation of additive genetic relationships modified from an approach widely employed in animal breeding. The genetic variances and covariances are used to predict response to selection from varying strategies, from which optimum schemes can be determined. Results suggest that selection should generally take place after three generations of backcrossing when the fertile plant used to initiate the backcrossing process is not inbred, but can take place after generation two when the fertile plant is inbred. Doubled haploid production is unlikely to provide an extra advantage that would be worthwhile in such a system. The method developed here can be used to explore a wide range of more complex breeding systems. Received: 27 July 1998 / Accepted: 19 October 1998     

Cadmium effects on leaf transpiration of sugar beet (Beta vulgaris)

Seedlings of sugar beet ( Beta vulgaris L. cv Monohill) were cultivated for 4 weeks in nutrient solution containing different concentrations of CdCl₂ (0 to 10 μ M ). The effects of Cd on appearance and function of stomata and leaf cuticle were investigated by water loss measurements and microscopy. The leaf transpiration rate increased with increasing Cd concentrations while the sum total of stomatal aperture area per unit leaf area decreased. Already at low Cd levels. an increase of defective and undeveloped stomata was found in Cd treated plants. These stomata are closed or have small apertures and probably lack a functional closing mechanism. The number of intact stomata per unit leaf area was lower in leaves of Cd treated plants than in controls, and Cd induced closure of intact stomata. The total number of stomata per leaf area slightly increases with increasing Cd concentration. as does the percentage of small stomata. Furthermore. specific leaf area increased, while the density of leaf structure was decreased by Cd. From this observation we conclude that the increase in transpiration rate caused by Cd is primarily due to effects on the permeability of the leaf cuticle to water.     

Crop-weed interactions in the Beta vulgaris complex at a local scale: allelic diversity and gene flow within sugar beet fields

Crop-wild hybrids and weed beets are the main source of agronomic concern for sugar beet production all over Europe. In order to understand the dynamics of crop-wild interactions and the evolution of weediness in Beta vulgaris, we investigated genetic features of bolting individuals occurring at a local scale, i.e. within two sugar beet fields of the French northern area of sugar beet production. By analysing ploidy level, mitochondrial DNA and microsatellite polymorphism, the genetic diversity and the genetic relationships among three different classes of individuals (variety, in-row and out-row weed-beets) from a given field were examined. Such genetic analyses provide a unique opportunity to obtain evidence for the weeds origin and the evolutionary hypotheses previously stated. All the individuals shared in common the Svulg mitochondrial haplotype, and thus a common maternal origin. Conversely, the large genetic diversity at microsatellite loci highlighted the large diversity of the pollinator plants (cultivated and wild plants) during the-seed production process, as well as during the further evolution of weed beets in the sugar production area. Received: 23 April 2001 / Accepted: 15 June 2001     

Plant regeneration from protoplasts of sugar beet (Beta vulgaris)

Mesophyll protoplasts from two of five sugar beet lines tested were regenerated into plants. Mesophyll protoplasts of all lines showed high plating efficiencies up to 4.0% developed hard compact callus, and two of the lines also developed white, soft and friable callus consisting of starch grain-containing cells. Whereas the compact callus never regenerated into plants, the white friable ones frequently developed globular structures, which became green in the light and formed adventitious shoots after cytokinin (BAP or thidiazuron) treatment. Genetic analysis by PCR-fingerprinting and flow cytometry showed uniformity and unchanged ploidy levels in 15 independently regenerated plantlets in line NF. but altered ploidy level (from diploid to triploid) in a regenerated plantlet of clone VRB.