Cryopreservation of shoot tips of Chrysanthemum morifolium and related species native to Japan

Summary The shoot tips of Chrysanthemum morifolium (syn. Dendranthema grandiflorum) and related species native to Japan were cryopreserved using preculture for 2 days, slow cooling (0.2°C/min) until –40°C with 10% dimethyl sulphoxide and 3% glucose prior to immersion into LN₂ and rapid thawing. High survival rates were observed in 3 cultivars of chrysanthemum, 12 species and 2 interspecific hybrids, and slightly low survival rates in 3 species. The shoot regeneration rates of the frozen shoot tips varied from 9.4 to 100% depending on species. Shoot tips of chrysanthemum showed high viability even after a storage of 8 months in LN₂. The thawed chrysanthemum shoot tips grew and flowered normally in a greenhouse under natural conditions.Abbreviations BA 6-benzylaminopurine - DMSO dimethyl sulphoxide - NAA 1-naphtalenacetic acid     

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     

Plant regeneration from in vitro cultures of cotyledon explants and anthers of Sinapis alba and its implications on breeding of crucifers

Summary Protocols for plant regeneration from cotyledon explant and anther cultures of Sinapis alba have been developed for creating doubled-haploids and somaclonal variation. Among the several cultivars tested in this study, only Arda responded well to in vitro plant regeneration both from anther-as well as cotyledoncultures. Multiple shoot formation in cotyledon explants, which always followed a brief callusing phase, was found to be the best on MS medium with ZEA (1.0mg/l) and NAA (0.1mg/l). Regeneration frequency declined sharply in the absence of auxin or presence of other cytokinins and/or auxin. The frequency of shoot regeneration also declined with reduction in the photoperiod to 16h. On MS + BAP (1.0mg/l) + NAA (1.0mg/l) medium, cotyledonary explants showed profuse callusing, which could regenerate shoots on high ZEA + low NAA/IAA medium. However, it declined with progressing time in culture. Anthers, excised from fresh as well as cold pretreated buds, cultured on 10% sucrose containing MS media with different hormonal constitution, developed calli and/or embryos. Initial culture temperature was important with embryogenesis occurring only in anthers cultured at 30°C for 3 weeks. A high temperature (35°C) treatment was lethal for both callus as well as embryo formation. While BAP + NAA and ZEA + NAA/IAA supported embryogenesis, further plant regeneration from anther-or embryo-callus could be achieved in ZEA + NAA/IAA media. Some of the regenerants flowered already in vitro and had small and sterile flowers. Cytological examination of some of the root differentiating calli indicated the presence of haploid as well as diploid cells. Shoots were rooted during prolonged incubation on the same medium or on transfer to MS (reduced)/ B5 + ZEA + NAA media.     

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.     

Interspecific hybrids of Cyclamen persicum and C. graecum

Summary Interspecific crosses were made to introduce the disease resistance of Cyclamen graecum into C. persicum cultivars and the abortive hybrid embryos were rescued by ovule culture. Diploid and tetraploid cultivars of C. persicum (CPD, 2n=2x=48; CPT, 2n=4x=96) were the pistillate parents and wild form of C. graecum (CG, 2n=84) were the staminate parents. After pollination, crossed ovaries were periodically collected and examined using paraffin sections. Histological observations suggested that both hybrid ovules of CPD × CG and CPT × CG should be transferred to the culture medium 35 days after pollination. Based upon this observation, crossed ovaries were collected 35 days after pollination, then ovules with placenta were explanted on culture medium and cultured in the dark at 25°C. Plantlets were induced from ovules cultured in MS medium containing 3% sucrose and 10% coconut milk. The hybrids (2n=66) derived from CPD × CG failed to yield viable seeds by self-pollination, although they showed some pollen fertility. In contrast, the hybrids (2n=90) derived from CPT × CG showed high pollen fertility and yielded viable seeds by self-pollination. Furthermore, they were resistant to disease caused by Fusarium oxysporum f. sp. cyclaminis, Erwinia herbicola pv. cyclamenae and Pseudomonas marginalis pv. marginalis.Abbreviations CPD C. persicum diploid - CPT C. persicum tetraploid - CG C. graecum     

Trueness-To-Type and Yield Components of the Banana Hybrid Cultivar FHIA-18 Plants Regenerated Via Somatic Embryogenesis in a Bioreactor

Summary A population of 1,500 plants of the banana hybrid ‘FHIA-18’ (AAAB), regenerated from somatic embryos, which were multiplied in bioreactors, showed similar characteristics to plants propagated from shoot tip cultures both in the acclimatization stage and in field experiments carried out in Cuba. The plants originating from somatic embryos were similar to the plants obtained from shoot tips with respect to plant height, diameter of the pseudostem and number of suckers. Both groups of plants obtained from in vitro cultures were significantly different to the plants obtained from suckers during the flowering period of the mother plants, which was shortened by two months. The greater plant height and diameter of the pseudostem in the plants coming from somatic embryos and shoot tip is due to the effect of in vitro culture, and this was observed in different banana and plantain cultivars. During the second cycle of evaluation, the plants coming from the three propagation methods studied in this work had similar growth habits without significant differences in the majority of the morphological parameters evaluated. These results confirm that the difference obtained during the first cycle between the distinct populations is attributed to temporary changes. The original characteristics of the cultivar were evident from the second cycle of culture. Only 0.13% somaclonal variant was observed in the plants coming from somatic embryogenesis. These percentages are low taking into consideration that other propagated methods accept up to 5% variants in field conditions.     

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.     

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.     

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     

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.     

The effect of pre- and post-inoculation temperature on resistance in certain cultivars of poplar to races of Melampsora larici-populina kleb.

Summary Four cultivars of Populus spp., compatible to varying degrees with four races of M. larici-populina Kleb., were raised in a controlled environment on a high (28°/20°C, day/night) and low (20°/10°C) temperature regime. Leaf discs cut from the plants were inoculated separately with four individual races of M. laricipopulina and subsequently incubated at either low (20°C) or high (25°C) temperature for 14 days when disease development on the discs was assessed using three parameters (Incubation period to flecking, uredia per leaf disc and uredospores per mm²). The degree of resistance in all cultivar/race combinations was high on cultivars cultured at a high temperature regime compared to those cultured on a low temperature regime. Analysis of variance demonstrated that the major components: pre-inoculation temperature regime, post-inoculation temperature regime, race and cultivar, and most second and third order interactions between these were highly significant (P<0.001) for most disease parameters. The variance of the temperature components and all interactions involving these were usually higher than those for the cultivar and race components and those interactions lacking temperature components.These results emphasize the importance of the temperature regime at which plants are raised and the temperature of incubation, following the inoculation in determining the relative degree of resistance of these cultivars of poplar to races of M. larici-populina. The implications of these results in the epidemiology of leaf rust and the stability of the host-parasite relationship are discussed.     

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.     

Characterisation and origin of rust and powdery mildew resistance genes in VPM1 wheat

Summary The expression of rust resistances conferred by closely linked genes derived from VPM1 varied with environmental conditions and with genetic backgrounds. Under low light and low temperature conditions seedlings carrying Yr17 showed susceptible responses. Stem rust and leaf rust resistance genes Sr38 and Lr37 tended to confer more resistance at 17±2° C than at normal temperatures above > 20° C. These studies supported the hypothesis that Yr17, Lr37 and Sr38 were derived from Aegilops ventricosa, whereas Pm4b was probably derived from T. persicum. Studies on certain addition lines and parental stocks indicated that wheat cytoplasm may enhance the expression of Sr38.     

Production and performance of potato mini-tubers

Summary Production of mini-tubers as a source for seed potato was investigated by growing in soil micropropagated plants and micro-tubers produced from micropropagated plants. Cultures of several cultivars were initiated from indexed tubers and multiplied on modified MS medium. Cultures were micropropagated by using a modular system which allowed batch handling. Micropropagated plants produced mini-tubers in glasshouse after 70–115 days of growth in soil. A large proportion of the mini-tubers produced were between 9 and 15 mm diameter. Several factors, e.g., explant number, duration of in vitro culture and genotype influenced mini-tubers production. Micropropagated plants after culture of 86 days or longer produced micro-tubers ca. 2 to 10 mm diameter. Plants, which formed micro-tubers in vitro, produced less number of mini-tubers in soil. Micro-tubers produced 1 to 3 mini-tubers when grown in soil in chain-type paper pots, but produced conventional sized tubers when grown in soil under plastic polytunnel. Mini-tuber number varied widely between potato cultivars; cvs. Bintje and British Queen produced more mini-tubers than the other cultivars. Mini-tubers developed green hard skins when kept in light for 3 weeks, and could be stored in dark at 4° C upto 6 months. In a field trial, small mini-tubers ca. 5–10 mm diameter produced more but smaller tubers than mini-tubers ca. 15–20 mm diameter. The micropropagated plants and the plants grown from mini-tubers were genetically stable, and did not show any morphological aberrations except for one variegated plant among the several thousand produced. It is concluded that the production of mini-tubers by soil planting of micropropagated plants is a rapid and efficient method for producing seed potato tubers.The use of any trade mark and other commercial products mentioned in this paper does not constitute any recommendation or advertisement for such products on behalf of the Agriculture and Food Development Authority, Dublin, Ireland     

Introgression of in vitro regeneration capability of Lycopersicon pimpinellifolium Mill. into recalcitrant tomato cultivars

The goal of this research was to study the introgression of the high regeneration capacity of Lycopersicon pimpinellifolium Mill line WV-700, in recalcitrant tomato cultivars (L. esculentum Mill cvs. Petomech, Santa Rita and VFN-8) using backcrossing. Hypocotyl explants of in vitro germinated seeds were cultivated in half strength MS medium supplemented with 5mg/l 6-BA to assess their shoot regeneration capacity. The apical shoot of the in vitro germinated seeds were cultured on a separate medium. Apical shoots from genotypes showing high regeneration rates were acclimated in a glasshouse and used as pollen donors for the next backcrossing. After four backcrossings, the material showed a similar mean fruit weight for the cultivated tomato and a high regeneration capacity similar to the wild species. It is shown that L. pimpinellifolium can be used with success as donor parent to introgress in vitro regeneration capacity to recalcitrant tomato cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

Additional evidence for oligogenic inheritance of durable host resistance to coffee berry disease (<Emphasis Type="Italic">Colletotrichum kahawae</Emphasis>) in arabica coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

Breeding for host resistance to coffee berry disease (CBD) in arabica coffee (Coffea arabica) was initiated some 35–40 years ago in Kenya, Ethiopia and Tanzania in response to severe CBD epidemics. The release of CBD resistant cultivars to the coffee growers has been in progress since 1985. The resistance of cultivars like Ruiru 11 (Kenya) and Ababuna (and other cvs in Ethiopia) appears to be of a durable nature, since confirmed cases of a breakdown of host resistance under field conditions have not been reported over the past 20 years. Host resistance to the hemibiotrophic fungus Colletotrichum kahawae is of a quantitative nature, but nevertheless can be practically complete in some genotypes of arabica coffee. There is still no consensus on the genetics of CBD resistance, some claiming convincing evidence for oligogenes (1–3 major genes) and others for polygenes determining CBD resistance. Results from genetic studies with germplasm from the centre of genetic diversity for C. arabica in Ethiopia are presented here. These together with the recent identification of molecular markers associated with and the mapping of one major gene, provides additional evidence for oligogenic inheritance of CBD resistance. The development of cultivars combining yield and quality with durable host resistance to CBD has contributed greatly to increased sustainability of arabica coffee production in Africa. It has also considerable relevance to arabica coffee in Latin America and Asia, where CBD is still a quarantine disease but with a risk of becoming endemic one day, just as has happened earlier with coffee leaf rust (Hemileia vastatrix).     

Embryo rescue in Rosa hybrida L.

Summary Two crosses between Rosa hybrida L. cultivars, that fail to produce progenies by conventional means, were carried out. In one of them, total hip abscision occured seven weeks after pollination; in the other one, only 2% of the fertilized hips remained on the plants after eleven weeks. Four- and five-week-old fertilized ovules were isolated in the first cross and six-week-old embryos in the second. The ovules were cultured on six media which differed in their mineral salt and sucrose concentrations while the embryos were cultured on only one of these media and eventually cold treated for one month at 4° C before being placed in a culture room set at 23° C. The embryos that had been isolated in ovulo when they were still not visible under a binocular lens developed atypically. The embryos isolated in ovulo when they were heart-shaped and on average 0.27 mm long performed in ovulo germination on some media and/or enlargement on all of them after two weeks; after another two week culture, once isolated from the ovule, all of them germinated. The cultured isolated embryos, that were exposed to cold, enlarged and germinated more rapidly when placed at 23° C than those not exposed to cold. Furthermore, the plantlets resulting from untreated embryo germination were characterized by large cotyledons which were only partially green. These results are discussed in regard to embryogenesis, precocious germination and dormancy.     

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 simple,rapid and reliable bioassay for evaluating seedling vigor under submergence in <Emphasis Type="Italic">indica</Emphasis> and <Emphasis Type="Italic">japonica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Submergence is a major stress causing yield losses particularly in the direct-seeded rice cultivation system and necessitates the development of a simple, rapid and reliable bioassay for a large scale screening of rice germplasms with tolerance against submergence stress. We developed two new bioassay methods that were based primarily on the seedling vigor evaluated by the ability of fast shoot elongation under submerged conditions, and compared their effectiveness with two other available methods. All four bioassay methods using cultivars of 7 indica and 6 japonica types revealed significant and consistent cultivar differences in seedling vigor under submergence and/or submergence tolerance. Japonica cultivars were more vigorous than indica cultivars, with Nipponbare being the most vigorous. The simplest test tube method showed the highest correlations to all other methods. Our results suggest that seedling vigor serves as a submergence avoidance mechanism and confers tolerance on rice seedlings to flooding during early crop establishment. A possible relationship is discussed between seedling vigor based on fast shoot elongation and submergence tolerance defined by recovery from submergence stress.     

Analysis of durable resistance to stem rust in barley

Summary Since the mid-1940's, barley cultivars grown in the northern Great Plains of the USA and Canada have been resistant to stem rust caused byPuccinia graminis f. sp.tritici. This durable resistance is largely conferred by a single gene,Rpg1, derived from a single plant selection of the cultivar Wisconsin 37 and an unimproved Swiss cultivar. At the seedling stage, barley genotypes withRpg1 generally exhibit low mesothetic reactions at 16–20° C and slightly higher mesothetic reactions at 24–28° C to many stem rust pathotypes. This resistance is manifested by a low level of rust infection and mostly incompatible type uredia on adult plants.Rpg1 reacts in a pathotype-specific manner since some genotypes ofP. g. f. sp.tritici are virulent on cultivars carrying this gene in the field. Several factors may have contributed to the longevity of stem rust resistance in barley, a) since barley is planted early and matures early, it can sometimes escape damage from stem rust inoculum carried from the south; b) one or more minor genes may augment the level of resistance already provided byRpg1; c) the cultivation of resistant wheat cultivars and eradication of barberry have reduced the effective population size and number of potential new pathotypes ofP. g. f. sp.tritici, respectively; and d) virulent pathotypes ofP. g. f. sp.tritici andP. g. f. sp.secalis have not become established. This situation changed in 1989 when a virulent pathotype (Pgt-QCC) ofP. g. f. sp.tritici became widely distributed over the Great Plains. However,Rpg1 may still confer some degree of resistance to pathotype QCC because stem rust severities have been low to moderate and yield losses light on barley cultivars carrying the gene during the last four seasons (1989–1992). Several sources of incomplete resistance to pathotype QCC have been identified in barley. To facilitate the transfer of resistance genes from these sources into advanced breeding lines, molecular marker assisted selection is being employed.