Pollen storage of Fragaria and Potentilla

Summary Low temperature and humidity were used for the storage of pollen of four species of Fragaria (2n=14, 42 and 56) and two species of Potentilla (2n=14). The stainability of Fragaria pollen was assessed over a number of years by use of aceto-carmine stain and its viability by cross-pollinations; stainability of Potentilla pollen was assessed by acetocarmine. Fragaria pollen so stored remained stainable for seven years and showed the ability to set seed for three years. over the latter period, aceto-carmine staining gave a reasonable prediction of seed set produced by the stored pollen of Fragaria and so is of value in estimating pollen viability in that genus. Potentilla pollen remained stainable for three years.     

Low temperature storage of pistachio pollen

Summary Pollen from four male pistachio (Pistacia vera L.) clones was stored at –196°C and –20°C for up to 12 months and tested for ability to germinate in vitro following a period of hydration at high humidity. Germination of fresh pollen was high (>80%) for each clone. At –196°C, pollen of cv. Peters survived freezing, storage and thawing with no loss of germinability; pollen of the other three clones had sharp declines in germination possibly attributable to cellular lesions incurred during freezing or thawing. When the relative humidity of the –20°C storage environment was maintained at or near 33%, Peters pollen had high rate of germination through 12 months storage. Without control of relative humidity, Peters pollen germination was high at 4 months, but declined at 12 months. Germination requirements became more exacting for pollen stored at –20°C for 12 months at suboptimal humidity conditions. Pollen of the other three clones did not tolerate storage at –20°C as well as Peters pollen regardless of the storage humidity environment.     

Viability and storage of bromeliad pollen

Several bromeliad species from two different subfamilies, were used to develop a reliable method to evaluate pollen viability. Pollen germination on a medium containing 20% sucrose, 0.001%H₃BO₃ and 0.5% agar was comparable to germination on a compatible stigma. Maximum germination was reached within 2 to 10 hours depending on the species. Based on this test, six species were considered as being good pollen donors with germination percentages between 49%and 83%. Furthermore, pollen from these species and cultivars could be stored in liquid nitrogen (–196 ^°C) without a considerable loss of viability. For all species, a dehydration period of 4 hours prior to cryopreservation and a rehydration period of 1 hour after cryostorage were essential. Greenhouse humidity influenced anther moisture content and cryostorability. This revised version was published online in July 2006 with corrections to the Cover Date.     

Epicuticular wax content and morphology as related to ethylene and storage performance of ‘Navelate’ orange fruit

The effect of ethylene (2 μL L⁻¹) on total and soft epicuticular wax content and wax morphology has been investigated in mature ‘Navelate’ (Citrus sinensis, L. Osbeck) oranges held under non-stressful environmental conditions (22 °C and constant high relative humidity (90–95% RH)). In addition, the objective of the study was to understand whether the ethylene-induced changes in epicuticular wax might participate in the beneficial effect of ethylene reducing non-chilling peel pitting, by modifying peel water, osmotic or turgor potential, or disease incidence caused by Penicillium digitatum (Pers.:Fr.) Sacc. Ethylene increased total and soft epicuticular wax content in ‘Navelate’ fruit and induced structural changes in surface wax that might be related to the formation of new waxes. Changes in epicuticular wax morphology, but not in its content, might be involved in the protective role of ethylene reducing non-chilling peel pitting, although the beneficial effect of the hormone is not related to water stress. Cell water and turgor potentials in freshly harvested fruit and fruit stored in air under non-stressful conditions suggest that water stress is not a limiting factor leading to the development of this physiological disorder. In addition, the results indicated that formation of new waxes in fruit treated with ethylene may partially cover stomata, cracks or areas lacking wax occurring in stored fruit and is likely to improve physical barriers to P. digitatum penetration.     

Peel tissue α-farnesene and conjugated trienol concentrations during storage of ‘White Angel’בRome Beauty’ hybrid apple selections susceptible and resistant to superficial scald

In a 2-year study, fruit from eight red- and eight yellow-skinned ‘White Angel’בRome Beauty’ hybrid selections were stored for 21 weeks at 0.5°C plus 1 week at 20°C and evaluated for the incidence and severity of superficial scald. Five red-skinned (R-03, R-20, R-22, R-48 and R-85) and three yellow-skinned (Y-26, Y-55 and Y-65) selections were examined in both seasons. Peel-tissue samples taken at 0, 7, 14 and 21 weeks of storage were analyzed for concentrations of α-farnesene and its conjugated trienol (CTol) oxidation products by HPLC with UV detection. Three red-fruited (R-44, R-48 and R-85) and five yellow-fruited (Y-38, Y-40, Y-55, Y-65 and Y-67) lines exhibited scald symptoms. The remaining lines (R-01, R-03, R-16, R-20, R-22, Y-07, Y-26 and Y-28) were free of scald. Overall, production of α-farnesene and accumulation of CTols were not closely correlated with scald susceptibility. Data for the selections most prone to scald, Y-65, Y-40 and R-44, were consistent with the proposed role of α-farnesene oxidation products in scald induction, but for Y-55 and R-48, which developed mild to moderate scald and accumulated very little CTols, the data conflicted with the α-farnesene oxidation–scald induction hypothesis. Also, scald-resistant lines Y-07 and R-22 produced high levels of α-farnesene and reached CTol concentrations comparable to those in several scald-susceptible lines. We conclude that if CTol do play a role in scald induction, there must be other mitigating factors of at least equal importance. Moreover, our findings support the proposal that oxidation products of α-farnesene are not essential for scald development in fruit with severely compromised antioxidative defenses, but free radicals and/or toxic volatiles generated by α-farnesene oxidation can exacerbate scald symptoms.     

Hybrid Tea-rose pollen. I. Germination and storage

Summary Germination and storage trials were carried out with pollen of several rose varieties. The pollen grains germinated well in a 15% sucrose solution with 40 ppm boric acid. Staining the pollen with a 0.1% tetrazolium solution and standardizing the degree of colour at which the pollen grains are counted as viable, provided a good viability estimate, simpler to carry out than in vitro germination. Germination capacity and staining ability of the pollen were greatly impeded-about halved-by dehydration during storage in desiccators at low humidity. This effect could be corrected by humidifying the pollen beforehand for about one hour, though this pre-treatment increased the percentage of germinated pollen grains more than the percentage stained. There was no difference between the two percentages in fresh or in deep-frozen pollen.Pollen stored at 1°C and high relative humidity soon lost its germination capacity: between 0 and 20% humidity a considerable proportion of the pollen remained viable for 9 months and longer. Storage for the same period in vacuum-sealed glass tubes at –24°C maintained viability as well or better and would probably prolong it further. Some of the cold-stored pollen induced a reasonable seed set after one year, a low seed set was obtained even after two years of storage at 1°C and low humidity.     

Identification of Vigna spp. through specific seed storage polypeptides

Summary Seed globulins of ten Vigna species were separated by means of SDS electrophoresis. Both inter and intraspecific variation were observed.Groups of specific bands which do not vary in the species being present in all the accessions of the species were identified. These species-specific bands, allow the identification of the 10 Vigna spp. analyzed.     

Long-term storage of Narcissus anthers and pollen in liquid nitrogen

Summary Three methods for the long-term storage of narcissus pollen were compared; anthers in glass vials held in a desiccator with calcium chloride at 2°C, and polypropylene straws containing either anthers or naked pollen immersed in liquid nitrogen. Pollen from all storage treatments showed 15–16% germination in vitro after 3 days, compared with 27.4% for fresh pollen. Seed set per pod using pollen stored for 3 days was comparable to that of fresh pollen. However, after 351 days, pollen from anthers at 2°C exhibited only 0.1% germination and failed to set seed whereas no further change in germination rate was recorded for pollen from the two liquid nitrogen treatments and seed set was still equivalent to fresh pollen.     

The probable genome donors to Arachis hypogaea L. based on arachin seed storage protein

Summary Interrelationships among six diploid species (A. chacoense, A. villosulicarpa, A. batizocoi, A. correntina, A. duranensis and A. cardenasii), tetraploid wild groundnut A. monticola and four accessions of A. hypogaea were studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of total seed proteins and arachin immunoprecipitates. Arachin in A. monticola and A. hypogaea cv. Trombay Groundnut 9 (TG-9) was composed of four acidic subunits (47.5 kd, 45.1 kd, 42.6 kd and 41.2 kd) and one major basic subunit (21.4 kd). The arachin subunit pattern in cv. Spanish Improved (SP) and TG-1 was almost similar to the pattern observed in A. monticola with the exception that the SP lacked the 41.2 kd and TG-1 lacked the 42.6 kd acidic subunits of A. monticola. Seed extracts of all diploid species studied reacted with the anti-arachin antibodies raised against SP arachin. The electrophoretic analysis of immunoprecipitates of diploid species showed a range of acidic subunits from 46.2 kd to 41.2 kd and one or two basic subunits of 21.4 kd and 20.2 kd. None of the diploid species showed the 47.5 kd subunit found in A. monticola or A. hypogaea. Of the diploid species, A. duranensis and A. cardenasii demonstrated two acidic subunits of 45.1 kd and 41.2 kd and a basic subunit of 21.4 kd as found in A. monticola. Likewise A. batizocoi showed two acidic subunits of 45.1 kd and 42.6 kd and the basic subunit of 21.4 kd as was observed in A. monticola. Based on electrophoretic data, our research supports the earlier conclusion that the probable genome donors to A. monticola are A. batizocoi and A. duranensis or A. cardenasii.     

Digestive enzyme inhibitors and storage pest resistance in cowpea (Vigna unguiculata) seeds

Summary Amylase and trypsin inhibitors are proteins which inhibit digestive enzymes. The loss of the activity of these enzymes produces a reduction of starch and protein digestion. Amylase and trypsin inhibitor activity have been investigated in seeds of 20 cowpea lines in trying to establish their role in the storage pest resistance. A broad variation has been observed for both the inhibitors. Correlation analysis between inhibitor levels and extent of attack has shown that neither amylase nor trypsin inhibitors can separately explain the resistance to Callosobruchus maculatus (F.). Lines bruchid resistant in fact, have high levels of both inhibitors. Conversely lines with low levels of amylase and trypsin inhibitors are bruchid susceptible. The breeding for high contents of both amylase and trypsin inhibitors can be an effective way to obtain lines with resistance to storage pest.     

Pollen storage in cocoyam (Xanthosoma sagittifolium (L.) Schott)

Summary The effect of different storage conditions on cocoyam pollen viability was investigated. Two levels of temperature (0° and 5°C) and four levels of relative humidity (0, 10, 20 and 30 percent) represented the storage environments.Viable cocoyam pollen could be obtained after 28 days in storage. The best storage condition was 5°C and 30% relative humidity.     

Some consequences of pollen storage in the raspberry (Rubus idaeus L.)

Summary Raspberry pollen was stored at 5°C and –13°C for six months and then tested for its ability to induce pyrene set and the production of viable seedlings, and for its effect upon the segregation of a major gene s. Only pollen stored at –13°C gave a pyrene set and germination percentage adequate to produce sufficient seedlings for a breeding programme. It is suggested that tests of pollen viability in the raspberry should include studies of pollen germination, and the effect of this pollen on pyrene set and seed germination. Possible causes of the loss of viability in the pollen stored at 5°C are discussed.     

Variation in the storage proteins of peas in relation to sulphur amino-acid content

Summary A cellulose acetate electrophoretic technique has been used to characterise the storage proteins present in pea seeds in order to try and determine the proportions of proteins present which are enriched in sulphur amino-acids. Some of the eight varieties examined were shown to differ in their proportions of the various storage proteins. When the proteins were separated and estimated quantitatively and also characterised by their sub-unit composition, it was shown that cellulose acetate electrophoresis of crude protein extracts or of whole globulins did not give the degree of resolution required. The procedures which have to be undertaken in order to obtain this information are considered, and the difficulties of incorporating them into a plant breeding programme discussed.     

Slow-growth conservation of potato microplants: efficacy of ancymidol for long-term storage in vitro

Ancymidol was investigated as an alternative mediumsupplement to mannitol for slow-growth conservation ofpotato microplants in vitro. Differentconcentrations of ancymidol (0, 5, 10, 15, 20, 25, 30,35 and 40 M) were tested in slow-growthmedia based on MS medium supplemented with either 30or 60 gl^-1 sucrose. The cultures were conservedunder a 16-h photoperiod at two temperature regimesi.e. 24 ± 1 ^°C and 6 ± 1 ^°C. Therewere significant interactions between ancymidol andother factors such as sucrose, temperature andgenotype for microplant survival, microshoot heightand overall microplant growth. Ancymidol did have abeneficial effect on culture viability after prolongedmaintenance in vitro. The growth-inhibitingeffect of ancymidol persisted through a 16-monthculture period. Combined effect of ancymidol, sucroseand temperature showed that optimum culture viabilityand desirable microplant growth were obtained when thecultures were grown in MS medium supplemented with 10M ancymidol plus 60 gl^-1 sucrose at6 ± 1 ^°C. Vitrification and flaccidity, whichare very frequently observed in potato microplantcultures during prolonged maintenance in vitrounder osmotic stress (mannitol), were not observedwhen the microplants were conserved in ancymidolmedia. Genetic stability of potato microplantsconserved in ancymidol media was evaluated usingrandomly amplified polymorphic DNA (RAPD)fingerprints. Ancymidol did not induce any detectablegenetic variation in genomic DNA as visualized by theabsence of either any additional RAPD fragment oralterations in RAPD fragment patterns.     

Effects of Agrobacterium tumefaciens-mediated transformation and tissue culture on storage lipids in Brassica napus

The variation obtained in storage fatty acids induced by the procedures of tissue culture and transformation with Agrobacterium tumefaciens was investigated and compared in rapeseed, Brassica napus, cv. Hanna. An increased variation in the fatty acid profiles was noted after tissue culture and transformation compared with plants derived directly from seeds. In the second generation of rapeseed transformants, T₂, the content of oleic acid ranged from 39–72%, 12–31% for linoleic acid and 7–16% for linolenic acid. This could be compared with the oleic acid content in the T₂ generation of tissue culture-derived plants which ranged between 47–76% and in seed-derived material where oleic acid ranged between 55–69%.In the T₃ generation the ranges in transgenic seeds were decreased but still larger than in the seed derived plants. The range in transgenic plants was 49–64% for oleic acid, 20–28% for linoleic acid and 9–18% for linolenic acid. The most extreme individuals, both highest and lowest in the common fatty acids, were found in the group of transformed plants independent of generation. The total lipid content was also affected by the two treatments and seeds with the lowest and highest lipid content were both found among the transformed plants. In conclusion, care should be taken to use proper controls when performing transformation experiments in order to distinguish variation in the fatty acid profiles induced by the transformation procedure and tissue culture treatments from the changes due to transgenic expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stability of cassava plants at the DNA level after retrieval from 10 years of in vitro storage

Summary Cassava (Manihot esculenta Crantz) germplasm collections are conventionally maintained by continuous vegetative propagation in the field. Tissue culture techniques provide a more convenient way to conserve germplasm. The cassava in vitro gene bank held in trust at CIAT comprises nearly 6000 accessions. A study was carried out to determine whether any DNA rearrangements resulting from in vitro storage under slow growth could be detected by molecular analysis in retrieved plants. RFLPs with homologous probes, RAPDs with twenty primers and DNA fingerprinting with M13 probe were tested to detect variation at DNA level in cassava plants after ten-years in vitro storage. The molecular marker data obtained in this study supports the stability of the cassava germplasm under the in vitro storage conditions described in this work.     

Production and characterization of inter-sectional hybrids between <Emphasis Type="Italic">Kalanchoe spathulata</Emphasis> and <Emphasis Type="Italic">K. laxiflora</Emphasis> ( = <Emphasis Type="Italic">Bryophyllum crenatum</Emphasis>)

The genus Kalanchoe is currently divided into section Kalanchoe and section Bryophyllum, and there has been no successful report on the production of inter-sectional hybrids. Therefore, reciprocal crosses were made between Kalanchoe spathulata (sect. Kalanchoe) and K. laxiflora (sect. Bryophyllum) in order to obtain basic information on the reproductive barriers between these two sections. The seeds were aseptically germinated in vitro and the plants were grown in greenhouse till flowering. When K. spathulata was used as a maternal donor, 39 out of 80 plants showed intermediate characteristics between K. spathulata and K. laxiflora. In contrast, no plants were obtained in the reverse crosses. Hybridity of these plants was confirmed by flow cytometric analysis, chromosome numbers and RAPD analysis. Bulbil formation on the leaf margin as one of the conspicuous characteristics of K. laxiflora was not observed in the hybrids. Some of the hybrid lines showed some pollen fertility, but failed to yield viable seeds by self-pollination or backcross-pollination. Successful production of the inter-sectional hybrid between the two species suggests that they are not so distantly related as considered previously.     

Effects of low temperature storage on the pollen of Brassica campestris,B. oleracea and B. napus

Summary Four indica cultivars viz. Kalinga-I, Ptb. 10, IR 27280-13-3-3-3 and Co. 41 were found to possess male sterile cytoplasm with fertility restoring genes while the cultivar Krishna was found to maintain the male sterility in all the cases. All the plants in the F₁ of Kalinga-I × Krishna were observed to be completely male sterile and continued to show complete pollen sterility in subsequent backcross generations when backcrossed with recurring pollen parent, Krishna. Thus, it was posible to develop a new cytoplasmic-genetic male sterile line in indica rice (Krishna A) with Kalinga-I male sterile cytoplasm and this male sterile cytoplasm was found to be genetically different from others. Further, the newly developed male sterile line (Krishna A) was observed to be tolerant for low temperature at seedling stage.     

Predicting transgressive segregants in early generation using single seed descent method-derived <Emphasis Type="Italic">micro-macrosperma</Emphasis> genepool of lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medikus)

In a self-fertilised crop like lentil, the identification of transgressive segregants for economically important trait such as seed yield is an important aspect of any practical breeding programme. The prediction of expected transgressive segregants in F₁ generation obtained as a ratio of additive genic effect [d] and additive variance (D) i.e. [d]/√D was studied in 28 crosses of lentil generated in a diallel fashion involving four parents each of macrosperma (exotic) and microsperma (Indian) types, respectively, resulting in three hybridization groups. The seed material advanced to F₂, F₃ and F₄ generations through single seed descent method was evaluated to determine the observed transgressive segregants for seed yield/plant. The observed frequency of crosses showing more than 20% transgressive segregants in F₂ to F₄ generations were exhibited in 9(32%) crosses, of which 7(77%) crosses were of macrosperma × microsperma type. Genotypes Precoz and HPL-5 of the exotic group (macrosperma) produced maximum number of transgressive segregants with the genotypes L-259, L-4145 and PL-406 of the Indian origin (microsperma). Goodness of fit (non-significant χ² value) in F₂ generation was observed for 19(68%) crosses of the total genepool, out of which 9(56%) crosses each in F₃ and F₄ generation belonged to the macrosperma × microsperma group, depicting it as the gene pool of paramount importance to obtain maximum transgressive segregants, therefore establishing the efficacy of the method used.     

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.