Cryopreservation of shoot tips of Caryophyllaceae ornamentals

Summary Shoot tips of 5 genera 38 species and cultivars of the Caryophyllaceae ornamentals were cryopreserved. Excised shoot tips were cooled at a rate of 0.5°C/min down to -40°C in the presence of 10% dimethyl sulphoxide and 3% glucose prior to immersion into liquid nitrogen. The shoot tips of all species and cultivars were viable after thawing. Little variation among species was observed in shoot regeneration. High viability of the cryopreserved shoot tips was maintained for up to 4 years. The plants derived from the cryopreserved shoot tips grew and flowered normally.Abbreviations BA 6-benzylaminopurine - DMSO dimethyl sulphoxide - MS Murashige & Skoog - NAA 1-naphtalenacetic acid     

Cryopreservation of axillary shoot tips of in vitro-grown grape (Vitis) by a two-step vitrification protocol

Axillary shoot tips of in vitro grape(Vitis vinifera L. cv. CabernetSauvignon) were successfully cryopreservedby vitrification. Axillary shoot tipswere excised from 4- and 5-month oldplantlets were cultured onsolidified 1/2 MS medium with 1 mg l^-1BA at 25 ^°C. Shoot tips wereprecultured on solidified mediumsupplemented with 0.3 M sucrose for 3 daysand then treated with a mixture of 2 Mglycerol plus 0.4 M sucrose (LS solution)for 20 min at 25 ^°C. They were thendehydrated with PVS2 for 80 min at0 ^°C before being plunged into LN for1 hr. Samples were then warmed rapidly inwater at 40 ^°C. The recovery of theshoot tips amounted to approximately 60%.To enhance further recovery, a two-stepdehydration procedure by vitrification wasexamined. Osmo-protected shoot tips werefirst dehydrated with a 50% PVS2 (halfstrength of the solution) for 30 min,followed by PVS2 for 50 min at 0 ^°C.This revised dehydration procedure improvedshoot recovery from 60 to 80%. Thisprocedure by vitrification was applied toten other species/cultivars of Vitiswith an average recovery of 64%. Thismethod incorporates a simple and reliableapproach for providing high levels ofgrowth recovery. It also appears promisingfor the cryopreservation of Vitisgermplasm.     

Cryopreservation of English walnut (Juglans regia L.) pollen

Summary Pollen from eight clones of English walnut (Juglans regia L.) was stored in liquid nitrogen (LN₂) at-196°C for one year. Pollen was monitored for percent germination in vitro before being frozen. Pollen was frozen within two hours of anther dehiscence and subsequently at 24 hr intervals until germination assays indicated that the ability to germinate was lost. Survival after one year was evaluated by determining percent germination in vitro. Freshly collected pollen of only five of the eight clones survived LN₂. By contrast, when the same pollen was held for 24 and 72 hr before freezing, pollen of all eight clones survived. Survival is correlated with moisture content (MC) of the pollen. Pollen samples with MC greater than 7.5% were killed by freezing in LN₂. All pollen samples with MC between 4 and 7.5% survived as did most of the samples with MC between 3.2 and 4%.     

In vitro induction of tetraploids in Phlox subulata L.

Tetraploid plants of Phlox subulata L. were induced successfully by treating shoot tips in vitro with colchicine. Shoot tips excised from in vitro shoots were treated with four different concentrations of colchicine (0.005, 0.01, 0.02, 0.04%) in solid MS medium supplemented with 4.54 μM TDZ and 0.49 μM IBA for 10, 20 or 30 days, respectively. The survival rates of shoots tips were affected by the concentration of colchicine and the duration of treatment. High concentration and longer duration reduced survival of the shoot tips, but the effect of duration of colchicine was more than that of concentration. Tetraploid plants were obtained in all of the treatments, but the percentages of tetraploids varied among different treatments, from 25.0% to 75.0%. The most efficient condition for inducing tetraploids was to treat shoot tips with 0.005% colchicine for 20 days, with 30.0% survival rate of shoot tips and 6 tetraploid plants out of 10 plants examined. The rooted tetraploid plants were transplanted successfully in a solar greenhouse. Under the same growing condition, significant varieties in flower bud and flower sizes were detected between 2x and 4x plants. The flower diameters of tetraploid and diploid plants were 2.91 cm and 2.24 cm, respectively.     

Production of non-chimeral colchiploids in Rubus species by tissue culture

Summary Diploid Rubus allegheniensis Porter and R. rusticanus Merc. shoot tips were proliferated in tissue culture and treated with 0.02 to 0.08% colchicine to induce non-chimeral polyploidy. About 20 shoot tips of each treatment were transferred to test tubes containing modified Anderson's medium for shoot proliferation. Shoots at the 3- or 4-leaf stage were transplanted into Jiffy-7 peat pellets and placed under intermittent mist for rooting. Root tip squashes were made for chromosome counts. Thirteen R. allegheniensis and 7 R. rusticanus plants proved to be autotetraploids (28 chromosomes). Stomata of the autotetraploids were significantly larger than those of the diploids but stomatal conductance of CO₂ and photosynthesis rate were reduced significantly as compared to diploids. Pollen grains in tetraploid were larger than those in diploid R. allegheniensis plants. These results indicate that tissue culture can facilitate the production of non-chimeral colchiploids in Rubus spp.     

The response of a range of genotypes of Vitis vinifera to sequential shoot tip cultures at high temperatures

Summary The evaluation of genetic variation and the release of virus-free cultivars are important in Vitis breeding. Sequential shoot tip cultures and high temperatures (32°, 35°, 38°C) were used to study their effects on the rate of shoot elongation during the proliferation phase (initial stage) of the in vitro culture in some Vitis vinifera cultivars. In the first culture the shoot elongation was more evident at 32°C than at 25° or 35°C; in addition at 35°C it was observed the shoot tip death in some cvs. while at 38°C in all cvs. In the sequential shoot tip cultures we noted a decline on shoot elongation and also an inhibition of shoot morphogenesis in the 4th and 5th recultures.     

Low temperature tolerance of biomass and pollen germination in potato clones of Andean and European origin

Summary The temperature-related performances of six tetraploid potato clones, two Andean, three European, and one hybrid, were compared by cultivating them in growth chambers under one of two temperature regimes: 10°C day/4°C night or 20°C day/10°C night. Preformance was measured in terms of biomass production and pollen germinability.For dry matter yield at second harvest, the temperature-related performance ratios (performance at 10°C/4°C divided by performance at 20°/10°C) were highest for the Andean clones, intermediate for the Andean/European hybrid clone 201₅×S₁₂, and lowest for the European cultivars. The ability of the European cultivars to maintain their normal rates (i.e. rates at 20/10°C) of biomass production when cultivated at low temperatures varied greatly, with clone S₃ performing most poorly at 10/4°C.For pollen germinability in vitro, performance ratios were highest for the hybrid clone 201₅×S₁₂ and lowest for the European clones.The high tuber yield of hybrid 201₅×S₁₂ at 10/4°C can be attributed to its low-temperature tolerance and it being daylength neutral.     

Storage of sugarbeet pollen

Summary To develop the technology for long-term pollen preservation, sugarbeet pollen was collected from plants grown in the greenhouse and in the field, and was stored 1 day to 1 year at 5, -18, and -196°C. Pollen containing about 12% moisture was successfully stored in liquid nitrogen (LN₂) up to 1 year; this pollen effected fertilization of male-sterile flowers as well as freshly collected pollen. Germination of the resultant seed was good and not different from seed from fresh pollinations. Pollen stored at -18°C for 1 year did not result in as much seed set as fresh pollen, and 1 year at 5°C was essentially lethal. In vitro pollen germination served as a post-storage viability measure, provided the pollen was hydrated before germination. The methods tested in these experiments provided a relatively simple, reliable, and inexpensive means for preservation of sugarbeet pollen for breeding purposes and for preservation of genetic resources.Joint contribution of the Agricultural Research Service, USDA, and the Beet Sugar Development Foundation.     

Influence of high temperature on the performance of some Phaseolus species at different developmental stages

Summary The effect of short term high temperature exposure on the performance of five Phaseolus species and of long term (continuous) exposure on the performance of P. vulgaris was studied at three growth stages. Phaseolus species subjected to 26.7, 32.2 or 37.3°C for two days showed small differences in the number of pods produced and in visual leaf damage, but large differences in leaf heat killing time, as measured by conductivity. P. coccineus had the shortest heat killing time (20–60 minutes) and P. acutifolius and P. lunatus the longest times (180 and 153 minutes), respectively. The P. vulgaris genotypes were intermediate in killing times to P. acutifolius and P. coccineus. Species response was not consistent with temperature within developmental stage. On average, the number of pods decreased as temperature increased from 32.2 to 37.3°C. Heat killing time and leaf damage also increased with temperature. CO₂ exchange rates of plants grown at prolonged high temperatures (30–40°C/20–30°C, day/night) decreased with the age of the plant. Shoot lenght was decreased as high temperature. P. vulgaris genotypes differed on the basis of either short term exposure or of continuous exposure. These results suggest that there may be useful germplasm in Phaseolus for improving heat tolerance.Scientific Journal Series Paper Number 13,8000 of the Minnesota Agricultural Experiment Station, USA.     

Regenerative trait and cold hardiness in highly productive cultivars of alfalfa and red clover

Summary In earlier work on improvement of persistance in forage legumes, we selected genotypes from highly productive cultivars of alfalfa, Algonquin and Apica (Euphytica 45: 105–112, 1990) and cv. Florex red clover (Plant Cell Reports 8: 395–398, 1989) capable of in vitro regeneration from callus and cell culture. The alfalfa germplasm and its F₁ progeny as well as an F₂ red clover population were tested for cold stress tolerance. Plantlets were hardened in culture tubes at 2 or 5°C, 8h photoperiod, for at least four weeks and then subjected to freezing temperatures, –16 or –10°C for alfalfa and red clover, respectively. Survival of regenerative genotypes was significantly higher than of the non-regenerative ones in both species. A strong oositive correlation (r=0.78) between the regenerative trait and plant survival was found in alfalfa. The experiments indicate that in vitro selection for regenerative trait may improve cold stress tolerance of alfalfa and red clover.     

A new method for selecting cytochimeras by the maximum number of nucleoli per cell in Allium wakegi Araki and A. fistulosum L.

Summary A new method for determining ploidy levels in cytochimeral plants was developed by examining the maximum number of nucleoli per cell. Colchicine-treated plants of Allium wakegi Araki and A. fistulosum L., which have different ploidy levels in the first (LI), the second (LII), and the third (LIII) germ layer, were used together with untreated 2-2-2 plants of the same species. Nucleoli in guard cells for LI and in mesophyll cells for LII were stained in a 50% AgNO₃ aqueous solution at 55° C for three hours under dark, humid conditions. In both species the ploidy levels of LI determined by the maximum number of nucleoli per guard cell accorded well with those determined by guard cell length. In A. fistulosum the ploidy levels of LII determined by the maximum number of nucleoli per mesophyll cell clearly agreed with those determined by pollen size. This method provided more precise and efficient identification for LI and LII ploidy than the conventional methods of using guard cell length for LI and pollen size for LII.     

Clonal propagation of Acacia saligna by shoot tip culture

Summary Multiple shoots buds were obtained successfully from shoot tips of Acacia saligna by placing explants into solidified Murashighe & Skoog (1962) medium (MS medium) supplemented with 5.0 to 9.0 mg/L BAP. Sequential culture treatment was highly effective for shoot elongation using MS medium containing 0.3 mg/L BAP and 0.2 mg/L IAA. The shoots rooted best on MS medium supplemented with 2.0 mg/L IBA. Plantlet survival after transfer to soil was more than 90%. The shoot proliferation method described could be used for the mass clonal propagation of selected genotypes.     

A non-destructive selection method for faster growth at suboptimal temperature in common bean (Phaseolus vulgaris)

Summary A non-destructive method has been developed to select common bean (Phaseolus vulgaris L.) plants whose growth is less effected at a suboptimal temperature. Shoot weight was determined at a suboptimal (14°C) and optimal temperature (20°C), 38 days after sowing and accessions identified with a significantly lower than average weight reduction at 14°C compared to their weight at 20°C. Weight of primary leaves and of the shoot was correlated with seed weight at both temperatures, but no correlation was found between shoot weight reduction at 14°C and seed weight.     

Selection for fast germination in rapeseed (Brassica napus L. and B. Campestris L.)

Summary Eleven populations of Brassica napus L. and twelve populations of B. campestris L. were subjected to three cycles of selection for fast germination at 2°C and at 25°C. The seeds from the selected populations, and unselected control populations grown in the same environment as the selected populations, were examined for germination behaviour at 2°C and 25°C, and for growth behaviour at 10°C. The populations in both species responded differently in terms of germination behaviour to selection for fast germination. In most of the populations that did respond positively to selection, selection practised at 2°C was superior to selection at 25°C in improving percent germination at 2°C, and was as good as the selection at 25°C in improving germination rate at the higher temperature. Selection for fast germination had no effect on growth characteristics of B. napus and B. campestris populations grown at 10°C. Thus, selection for fast germination at one low temperature may lead to improvement in germination characteristics over a range of temperatures, but will not necessarily lead to improved growth performance of the selected populations.     

Genotypic and exogenous factors affecting shoot regeneration from anther callus of linseed (Linum usitatissimum L.)

Summary The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. Hella. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l^-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l^-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.Abbreviations B5 Gamborg's (1975) medium - BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - N6 Chu's (1978) medium - NAA -naphthaleneacetic acid - MS Murashige & Skoog's (1962) medium - ZEA zeatin     

Interspecific hybrid of Helianthus annuus × H. simulans: Characterization and utilization in improvement of cultivated sunflower (H. annuus L.)

The first success at interspecific hybridization between cultivated sunflower(H. annuus) and a diploid perennial species, H. simulans is reported. The F₁s from both direct and reciprocal directions exhibited dominance of the wild species phenotype and were pollen sterile. Meiosis was irregular in the F₁ hybrids and both univalents and multivalents were observed. Multiplication of the interspecific hybrids was achieved through in vitro culture of nodal sections on Murashige & Skoog medium supplemented with 0.5 mg l^-1 benzyladenine. Fertility of the interspecific hybrids was improved by subjecting the in vitro proliferating shoots to 0.001% colchicine incorporated in the shoot multiplication medium. The amphiploids serve as fertile bridges and facilitate interspecific gene transfer through conventional breeding. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interspecific hybrids of Cyclamen persicum Mill. and C. purpurascens Mill. produced by ovule culture

Summary Interspecific crosses were made to introduce the scent of flowers of C. purpurascens into C. persicum cultivars and ovule culture was used to rescue the abortive hybrid embryos. Cultivars of C. persicum diploid (CPD, 2n=2×=48) and C. persicum tetraploid (CPT, 2n=4×=96) were the pistillate parents and wild species of C. purpurascens (CP, 2n=34) were staminate parents. After pollination, crossed ovaries were collected periodically and examined using paraffin sections. Histological observations suggested that both hybrid ovules of CPD x CP and CPT x CP should be transferred to culture medium 35 days after pollination. Based up on this observation, crossed ovaries were collected 28 days after pollination and ovules with placenta were transferred to MS (1962) medium containing 3% sucrose. These ovules were cultured in the dark at 25° C. The hybrids (2n=41) derived from CPD x CP had the scent of C. purpurascens, whereas the hybrids (2n=65) derived from CPT x CP had the scent of C. persicum. Although both hybrids had complete genomes from the parents and produced a few viable pollen grains, they failed to yield viable seeds by self- and cross-pollination with fertile pollen grains of C. persicum cultivars.Abbreviations CPD C. persicum diploid - CPT C. persicum tetraploid - CP C. purpurascens     

Morphogenic response of the alginate encapsulated nodal segment and antioxidative enzymes analysis during acclimatization of Ocimum basilicum L.

Synthetic seed technology is a potential tool for a more efficient and cost effective rapid clonal propagation system. In the present investigation, synthetic seeds were produced by encapsulating nodal segments of Ocimum basilicum in calcium alginate gel. For encapsulation of nodal segment, 3% (w/v) sodium alginate and 75 mM CaCl₂.2H₂O were found most suitable. The synthetic seeds when cultured on half-strength Murashige and Skoog (MS) medium supplemented with 5.0 μM benzyladenine (BA) and 0.5 μM Indole -3- acetic acid (IAA) produced maximum number of shoots (7.9 ± 0.54) after 8 weeks of culture exhibiting 80% in vitro conversion response. Further, synthetic seeds stored at 4 °C for 4 weeks resulted in maximum conversion response (90%) when placed back to regeneration medium. Both root and shoot formation took place in the same medium but the roots were thin and difficult to handle. Individual elongated shoots were rooted on MS medium supplemented with 1.0 μM Indole -3- butyric acid (IBA). Plants regenerated from the synseeds were hardened, acclimatized, and established in soil with 80% survival rate. Changes in antioxidative enzymes viz., Superoxide dismutase (SOD) and Catalase (CAT) in O. basilicum indicated the adaptation of micropropagated plants to ex vitro conditions.     

Differential cold sensitivity of pollen grain germination in two Prunus species

Summary Pollen grains from selected cutivars of almond [Prunus dulcis (Mill.) D. A. Webb] and peach (Prunus persica Batsch L.) were exposed to a range of temperatures between 1°C and 34°C on a thermogradient plate. Pollen germination at temperatures below 9°C was conspicuously greater in almond than peach. Miximal germination percentages were attained at about 16°C (almond) and 23°C (peach). The two species did not differ in their capacity for pollen tube elongation over a broad range of temperatures. Maximal pollen tube elongation occurred at temperatures 5°C to 8°C higher than maximal pollen germination. Species affiliation appeared to be of much greater consequence than chilling requirement or bloom date of the sporophyte in predicting gametophytic response to temperature.     

Combining ability analysis of Fusarium head blight resistance in western European wheat lines

The inheritance of Fusarium head blight (FHB) resistance was investigated in eight western European wheat lines using a half-diallel of F₁ crosses. The parents and F₁ crosses were point-inoculated, with a highly aggressive isolate of Fusarium graminearum, in replicated field and glasshouse trials. Type II resistance was assessed by measuring the % FHB spread and % wilted tips. There was a good correlation between the two disease parameters, % FHB spread area under the disease progress curve (AUDPC) and % wilted tips AUDPC (r = 0.86, P < 0.01). Correlation coefficients between the field and glasshouse environments were r = 0.46 (P < 0.01) for % FHB spread AUDPC and r = 0.40 (P < 0.05) for % wilted tips AUDPC. Both general combining ability (GCA) and specific combining ability (SCA) effects influenced the inheritance of FHB resistance, suggesting that in this set of parents both additive and non-additive (dominance or epistatic) effects influence the inheritance of type II FHB resistance. Highly significant GCA-by-environment (P < 0.0001) and SCA-by-environment (P < 0.005) interactions were also observed. Specific combinations of western European wheat varieties were identified with type II FHB resistance at a level equal to or more resistant than the winter wheat variety ‘Arina’.