Factors affecting minimal growth conservation of potato microplants in vitro

Combined effects of sucrose, mannitol and photoperiod on microplant conservation were studied in four potato genotypes belonging to two different groups viz., Tuberosum and Andigena. Minimal growth medium was based on Murashige and Skoog (MS) supplemented with 6 different concentrations of sucrose (30, 40, 50, 60, 70 and 80 gl^-1) with 4 different concentrations of mannitol (0, 20, 40 and 60 gl^-1). The cultures were conserved under two photoperiod conditions i.e. continuous illumination and 16-h photoperiod at 6 ± 1 °C. There were significant interactions between photoperiod and sucrose, and between photoperiod and mannitol. Maximum microplant survival and desirable microplant growth were observed under 16-h photoperiod. Sucrose alone did not improve culture viability over 30 months of storage. Inclusion of mannitol in the conservation medium increased microplant survival. Sucrose x mannitol interaction showed that sucrose was effective in enhancing microplant survival in combination with 20 or 40 gl^-1 mannitol, but not with 60 gl^-1 mannitol. Combined effect of sucrose, mannitol and photoperiod showed that optimum microplant growth and maximum culture viability were obtained when the cultures were grown in MS medium containing 40 gl^-1 sucrose and 20 gl^-1 mannitol under 16-h photoperiod. Potato microplants can be conserved in this medium and cultural conditions up to 30 months without subculturing. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis of,and selection for,factors affecting quantitative resistance to Xanthomonas campestris pv. oryzae in rice

Summary This study assessed 46 potato cultivars, breeding lines and Solanum spp. for heat-tolerance using short-term growth rates and carbon assimilation measurements of young in-vitro-derived plants. Plants of the 46 clones and species were transferred from greenhouse conditions to controlled conditions set at 33/25°C day/night with 12 h photosynthetic photon flux density (PPFD) at 430–450 mol m^-2s^-1 and an 8 h daylength extension (6 mol m^-2s^-1), to inhibit tuberization. Twenty eight accessions were also grown in a 20/10°C controlled environment. Plants were harvested after 10 and 20 days and dry weights of the plant components were measured for plant growth analysis. Gas exchange (leaf net photosynthesis and maintenance dark respiration) and leaf chlorophyll fluorescence parameters (O, P, and T) were measured at 30°C. Amongst the 30 accessions grown at both hot and cool temperatures, only two accessions (Yungay and AVRDC 1287.19) produced more dry weight in the hot chamber than the cool chamber. Hot/cool ratioss for net assimilation rate (NAR) and relative growth rate (RGR) exceoded unity in five and six accessions, respectively. For the 46 accessions grown under hot conditions, none had significantly greater values than those of the control clones for RGR and NAR. Differences between clones in maintenance respiration and net photosynthesis were more closely related to RGR, NAR, and total dry weight (TDWT) in clones which invested more dry weight in leaves and less in stems. Attributes of the chlorophyll fluorescence curve did not explain more of the clonal variation in RGR, NAR, and TDWT than did gas exchange parameters. No single gas exchange or fluorescence character explained more than 50% of the variability among clones for NAR, RGR, or TDWT, but combination of favourable attributes could improve potato heat tolerance in the future.     

Effects of salicylic acid on cold preservation and cryopreservation of encapsulated embryonic axes of Persian lilac (Melia azedarach L.)

Embryonic axes of Persian lilac (Melia azedarach L.)encapsulated into calcium alginate beads with sucrose (0.75 M) and salicylicacid (0, 50 M, 200 M) were subjected to cryopreservationtechnique with dehydration and freezing in liquid nitrogen or to coldpreservation by stocking alginate beads in empty petri dishes for 4 monthsat 4 °C. In these two cases 200 M salicylic acid enhancedsignificantly the percentage of viability of encapsulated embryonic axes andthe role of salicylic acid in increasing tolerance to dehydration is discussed.     

Influence of genotype and culture conditions on the production of embryos from anthers of tetraploid wheat (Triticum turgidum)

Summary Anther culture of 10 tetraploid wheat (Triticum turgidum) genotypes and two backcross lines representing a wide range of genetic variation was studied in a randomized block design with three replications. Each replication consisted of 2 pots with 3 plants. The day length was 16h and temperature 25° C/15°C for day/night in a controlled greenhouse where the anther donor plants were grown. Two different treatments were used for anther culture. The first one was potato 2 medium (Chuang et al., 1978) modified by adding 0.5 mg/l glutamine and solidified by gelrite (4g/l) (Henry & De Buyser, 1981). Cultures were incubated in light (15 E m^–2 S^–1) at 26°C at 16h day length. The second medium was described by Fadel & Wenzel (1990), differing from the first by the nature of the sugar (maltose) and consistency of the medium (semiliquid by ficoll). Anther cultures were incubated in the dark at 28°C. The study of about 1300 anthers per genotype and treatment showed that both genotype and treatment affected embryo formation of tetraploid wheat. The backcross lines exhibited significant differences for androgenic abilities when compared to their common parent. Most of the genotypes were medium dependent for androgenesis and revealed significant interactions with the two treatments. Five green plantlets were regenerated and fertile doubled haploid plants were obtained from three out of the 12 studied genotypes.     

In vitro interspecific fertilization,embryo development and formation of hybrid seedlings between Gossypium hirsutum and G. arboreum

Summary In vitro hybridization between Gossypium hirsutum, and G. arboreum was carried out. Hybrid seedlings were obtained after successive use of the following five kinds of media: 1) pollen grain germination medium, 2) double-fertilization medium, 3) embryo development medium, 4) seedlings formation medium, 5) green seedlings growth medium. The factors affecting in vitro interspecific fertilization, embryogenesis and seedling formation were studied. The key factors were temperature and relative humidity (RH). The optimum RH for in vitro interspecific fertilization was 65%, and a suitable temperature range was 26–30°C. Pollen grain germination ratio decreased rapidly at a RH of 80%. When the temperature and the RH were higher than 32°C and 80%, respectively, the fertilization rate decreased to zero.Effects of petals and calyces of the maternal flowers on in vitro interspecific fertilization and ovule growth were identified. Even though the petals or calyces were excised, hybrid plantlets were also obtained after media were improved and a shake culture was used. In addition, the process of double-fertilization and embryo development were studied cytologically. The developmental characteristics of the hybrid embryos derived from in vitro interspecific fertilization were a later occurrence of the double-fertilization process and a much lesser division of endosperm cells. But the embryo development was not affected by these characteristics, and young hybrid embryos can develop to their cotyledon stage finally on artificial media.     

Interrelationships of allozymes and ribosomal DNA alleles in wild barley

Summary A genetic resources preservation program led to an in vitro germplasm collection of yam (Dioscorea spp.), obtained by nodal cutting and maintained under slow growth conditions with ((Knop, 1865) in George & Sherrington, 1984) modified medium. The collection comprises accessions of 14 species from Africa and Asia, including edible varieties from the humid intertropical areas, viz 10 wild species (D. abyssinica, D. bulbifera, D. burkilliana, D. dumetorum, D. hirtiflora, D. mangenotiana, D. minutiflora, D. praehensilis, D. schimperana, D. togoensis), 5 edible species (D. alata, D. bulbifera, D. cayenensis-D. rotundata complex, D. dumetorum and D. esculenta) and 1 interspecific hybrid (D. cayenensis-D. rotundata complex, cv. Krengle x D. praehensilis). Three factors that may influence the success in transfer from the in vivo to the in vitro conditions have been studied. These are: the type of introducted material (nodal cutting fragments, seeds and exchanged microplants), the introduction date and the genotype. Some significant differences in success were due to the type of introduced material, whereas the introduction date had no effect. On the other hand, some species showed a greater success in the transfer from the in vivo to the in vitro conditions than others. The three tuberization types (basal tuberization, aerial tuberization and boulage (tuberization without vegetative development) phenomena), according to species, are discussed.     

Somatic embryogenesis and plant regeneration of Tripsacum dactyloides L.

Summary This article reports the culture and plant regeneration of Tripsacum dactyloides. Mature embryos of Tripsacum dactyloides dactyloides were used to obtain embryogenic callus cultures. Currently, 180 normal plants have been regenerated from these cultures. Callus was initiated on MS medium supplemented with dicamba (10 mol or 20 mol) and sucrose (3% or 6%), and plants were regenerated on hormone free MS medium containing 2% sucrose. No significant differences were found in callus initiation frequency or in embryogenic response of cultures on the four combinations of sucrose and dicamba tested. The embryogenic cultures have been maintained for 9 months (12 subcultures) and have retained regeneration capacity. Plants regenerated from tissue culture of maize-by-Tripsacum hybrids could be useful in maize improvement.     

Micropropagation of Indica rice through proliferation of axillary shoots

Summary Week old seedlings of indica rice variety Jaya obtained on basal MS medium and further sub-cultured on agar solidified MS medium supplemented with cytokinins, sucrose (3% w/v) and mannitol (1% w/v) lead to development of multiple shoot buds. Shoot cultures were maintained and multiplied in liquid medium containing BAP 5 mg l^-1, sucrose (3% w/v) and mannitol (1% w/v). Profuse rooting was obtained on transfer to MS liquid medium containing IBA 1 mg l^-1 and sucrose (3% w/v). Complete plants were successfully transferred to soil and grown to maturity.     

Condensed tannins in birdsfoot trefoil: Genetic relationships with forage yield and quality in NC-83 germplasm

Summary Genetic relationships of condensed tannins (CT) with other forage quality parameters have not been adequately studied in birdsfoot trefoil (Lotus corniculatus L.). The objectives were to bidirectionally select for CT concentration in birdsfoot trefoil to determine selection response and to create populations for examination of genetic relationships of CT with forage quality parameters, principally lignin. High-and low-tannin parental clones were selected from NC-83 birdsfoot trefoil germplasm and intercrossed to produce Syn₁ populations. Herbage samples, harvested for two years at two locations, were analyzed from high-tannin, low-tannin, and parental populations for CT concentration, herbage yield, and forage quality parameters. The mean condensed tannin concentrations in the high-and low-tannin populations were 69.3±0.8 and 21.1±0.8 g catechin equivalent (CE) kg^-1 dry matter (DM) compared with 36.4±0.8 g CE kg^-1 DM in the parental population. The selection response exhibited a quadratic relationship, with selection for increased CT concentration more effective than for reduced CT concentration. Compared with the parental population, acid detergent lignin (ADL) was higher, and crude protein (CP) and in vitro digestible dry matter (IVDDM) concentrations were lower for the high-tannin population and the converse was true for the low-tannin population. Lignin concentration was positively correlated with CT concentration (r_g=0.66 P0.01).Abbreviations ADF acid detergent fiber - ADL acid detergent lignin - CP crude protein - CT condensed tannins - IVDDM in vitro digestible dry matter - NDF neutral detergent fiber - NIRS near infrared reflectance spectroscopy     

Plant recovery of cryopreserved apical meristem-tips of Melia azedarach L. using encapsulation/dehydration and assessment of their genetic stability

A cryopreservation process usingencapsulation/dehydration technique was setup for apical meristem-tips of invitro plantlets of `paradise tree' (Melia azedarach L. var. gigantea,clone `El dorado'). Apical meristem-tipswere cultured for one day on MS basalmedium with 2 M BA and 0.5 M IBAand encapsulated with 3% sodium alginate.The highest shoot proliferation rate aftercryopreservation was obtained whenencapsulated apical meristem-tips werepregrown for 3 days in liquid medium with0.5, 0.75 and 1 M of sucrose for 24 hoursprogressively, desiccated for 5 hours withsilicagel followed by rapid or slowcooling. Survival after freezing in liquidnitrogen ranged between 67–83% andshoot proliferation ranged between 43–60%. This cryopreservation treatmentpreserved genetic stability, when it wasevaluated using the electrophoreticpatterns of nine isozyme systems and RAPDprofiles.     

Identification of somatic hybrids of dihaploid Solanum tuberosum lines and S. brevidens by species specific RAPD patterns and assessment of disease resistance of the hybrids

Summary Symmetric somatic hybrids were produced by electrofusion of protoplasts of two dihaploid tuber-bearing potato (Solanum tuberosum L.) lines and Solanum brevidens Phil., a diploid non-tuber-bearing wild potato species. A total of 985 plants was obtained. Verification of nuclear hybridity of putative hybrids was based on additive RAPD patterns, general morphological characteristics and chromosome counts. 53 (90%) calli regenerated into plants which were identified as somatic hybrids. Most of the hybrids were aneuploids at the tetraploid (4×) or hexaploid (6×) level. The 20 hybrids tested expressed a high level of resistance to potato virus Y (PVY ^N ) characteristic of the S. brevidens parent. Resistance to late blight (Phytophthora infestans (Mont.) de Bary) varied between hybrids, but was on average better than that of the fusion parents. Resistance of hybrids to bacterial stem rot (Erwinia carotovora subsp. atroseptica (van Hall) Dye) was not superior to that of commercial potato cultivars.     

Pollen grain size in four ploidy levels of genusAvena

Summary Pollen grain size in many genera positively correlated with chromosome number. In this study, oat (Avena) pollen grain size was examined in diploids (2×=14, one species, for accessions), tetraploids (4×=28, five species, 20 accessions), hexaploids (6×=42, one species, eight cultivars), and in 10 octoploid (8×=56) accessions. Mature anthers ready to dehisce pollen were sampled from one to six plants per accession, and pollen grains were squeezed out of the anther with tweezers. Oat pollen grains are slightly elliptical, and the length of the major axis was found to be highly correlated with the minor axis width (r=0.94^**). Pollen grain length, 39.3 m for diploids, 41.3 m for tetraploids, 47.0 m for hexaploids, and 48.8 m for octoploids, was positively correlated (r=0.86^**) with ploidy level. No genomic or species effects appeared to influence this trait. Only tetraploidAvena vaviloviana accession PI 412767 produced two distinct class sizes of pollen grains, 99% normal (43.0 m) and 1% large (52.7 m).     

Colchicine, trifluralin, and oryzalin promoted development of somatic embryos in Ilex paraguariensis (Aquifoliaceae)

A protocol for somatic embryogenesis and plant regeneration of Ilexparaguariensis St. Hil. from embryos cultures was developed. Heart stage zygotic embryos were removed from seeds of immature, light green fruit and treated with antimicrotubule agents (0.1; 0.2, and 0.5% colchicine for 24 and 48 h; 1; 10, and 20 M of either trifluralin, - trifluoro- 2,6-dinitro-N,N- dipropyl-p-toluidine, or oryzalin, 3,5-dinitro-N₄, N-dipropylsulphate during 48 h). The embryos were cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar (1/4MS), and 0.46 M zeatin. Cultures were incubated in darkness at 27 ± 2 °C. All thetreatments provoked a diminution of the number of germinated embryos and in some of the treated embryos somatic embryogenesis was induced. Somatic embryo maturation and conversion into whole plants could be achieved by culturing the embryos on 1/4MS lacking hormones and incubated at 27 ± 2 °C, 14 h photoperiod (116 mol m^-2s^-1). Mostof the plants regenerated from somatic embryos appeared morphologically normaland grew under greenhouse conditions. Only 2 plants out of 152 studied contained the tetraploid number of the chromosomes (2n = 4x = 80), meanwhile the rest of the plants had the normal diploid number of chromosomes (2n =2x = 40). Somatic embryos with abnormal morphology were also observed.     

Potential of a Fusarium eumartii culture filtrate on the screening for wilting resistance in potato

Summary Fusarium solani f.sp. eumartii Carp. Snyder and Hansen (Fusarium eumartii) is a soil inhabitant that induces the so-called Potato Wilt and Stem End Rot disease. Prior to wilting, the pathogen induces peculiar small bronze spots on the leaflets. Failure to isolate F. eumartii from infected leaflets suggests the involvement of a toxin in the disease. The fungus was grown in liquid Richard's medium and thereafter a filtrate was obtained dialyzing (MW cutoff 12,000–14,000) and sterilizing the culture by filtration (0.22 m). Potato leaves treated with both the pathogen or the filtrate showed symptoms of bronze spots and significantly higher electrolyte leakage when compared to controls. Tomato leaves showed neither bronze spots nor electrolyte leakage after plant inoculation with the pathogen or with the filtrate treatment. Both, the absence of visible symptoms and the lack of electrolyte leakage in tomato could be associated to a certain degree of host specificity of the F. eumartii filtrate towards potato. The filtrate also induced symptoms similar to infections by F. eumartii in adult plants and in vitro plantlets of cultivars Huinkul MAG and Kennebec. Callus responses to the filtrate were related to responses of the cultivars to the pathogen in greenhouse. These results show the potential of the culture filtrate of F. eumartii for use in screening for wilting resistance.     

Production of intergeneric hybrid plants between <Emphasis Type="Italic">Sandersonia aurantiaca</Emphasis> and <Emphasis Type="Italic">Gloriosa rothschildiana</Emphasis> via ovule culture (Colchicaceae)

Intergeneric hybrid plants between Colchicaceous ornamental plants, Sandersonia aurantiaca and Gloriosa rothschildiana, have successfully been produced via ovule culture. After 5 days of reciprocal cross-pollination, a few pollen tubes were observed in the ovary. Although seeds were obtained in both reciprocal cross-combinations, they did not germinate under ex vitro conditions. Ovules with placental tissues isolated 14 days after cross-pollination of S. aurantiaca × G. rothschildiana were cultured on a medium containing 0.01 mg l^–1 each of -naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), on which 41.5% of ovules swollen and produced callus-like structures within 10 weeks. When such swollen ovules were transferred to a medium containing 0.1 mg l^–1 each of NAA and BA, 7.5% of the initially cultured ovules produced rhizome-like structures within 6 weeks. Among the rhizome-like structures, those derived from two independent ovules (3.7% of the initially cultured ovules) produced multiple shoots following transfer to a medium containing 0.25 mg l^–1 NAA and 2.5 mg l^–1 BA. Multiple shoot-derived plantlets were established on a plant growth regulator-free medium, and they were successfully transplanted to pots. Early verification of their hybridity was accomplished by flow cytometry (FCM) analysis, chromosome observation and rDNA analysis.     

Oil content, tocopherol composition and fatty acid patterns of the seeds of 51 Cannabis sativa L. genotypes

The oil content, the tocopherol composition, the plastochromanol-8 (P-8) content and the fatty acid composition (19 fatty acids) of the seed of 51 hemp (Cannabis sativa L.) genotypes were studied in the 2000 and 2001 seasons. The oil content of the hemp seed ranged from 26.25% (w/w) to 37.50%. Analysis of variance revealed significant effects of genotype, year and of the interaction (genotype × year) on the oil content. The oil contents of the 51 genotypes in 2000 and 2001 were correlated (r = 0.37**) and averaged 33.19 ± 1.45% in 2000 and 31.21 ± 0.96% in 2001. The -tocopherol, -tocopherol, -tocopherol, P-8- and -tocopherol contents of the 51 genotypes averaged 21.68 ± 3.19, 1.82 ± 0.49, 1.20 ± 0.40, 0.18 ± 0.07 and 0.16 ± 0.04 mg 100g^–1 of seeds, respectively (2000 and 2001 data pooled). Hierarchical clustering of the fatty acid data did not group the hemp genotypes according to their geographic origin. The -linolenic acid yield of hemp (3–30 kg ha^–1) was similar to the -linolenic acid yield of plant species that are currently used as sources of -linolenic acid (borage (19–30 kg ha^–1), evening primrose (7–30 kg ha^–1)). The linoleic acid yield of hemp (129–326 kg ha^–1) was similar to flax (102–250 kg ha^–1), but less than in sunflower (868–1320 kg ha^–1). Significant positive correlations were detected between some fatty acids and some tocopherols. Even though the average content of P-8 in hemp seeds was only 1/120^th of the average -tocopherol content, P-8 content was more closely correlated with the unsaturated fatty acid content than -tocopherol or any other tocopherol fraction. The average broad-sense heritabilities of the oil content, the antioxidants (tocopherols and P-8) and the fatty acids were 0.53, 0.14 and 0.23, respectively. The genotypes Fibrimon 56, P57, Juso 31, GB29, Beniko, P60, FxT, Félina 34, Ramo and GB18 were capable of producing the largest amounts of high quality hemp oil.     

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.     

A tissue culture technique for vegetative propagation and low temperature preservation of Beta vulgaris

Summary A tissue culture technique for clonal propagation of Beta vulgaris is described. Flower buds or flower bud clusters formed adventitious shoots on half-strength Murashige & Skoog's medium supplemented with 10 mol/l benzyladenine (BA). The shoots were multiplied by axillary bud proliferation on a medium with 1 mol/l BA and rooted on a medium with 10 mol/l indolebutyric acid. The plants were vegetative. No mutations were observed. Genotypic variation was found in shoot formation, shoot multiplication and rooting. Some genotypes showed leaf malformations which were attributed to BA. Rooted plants in culture tubes could be stored for one year at 2 or 5°C and a low light intensity.     

In vitro propagation of Japanese garden iris,Iris ensata Thunb.

Summary Explants of young scapes of Iris ensata were cultured on MS medium with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar, and this species was characterized by high variety specificity for callus, shoot and root induction. Among 23 varieties and one wild form tested, Okichidori, Miyukisudare and Meiji-l exhibited a considerable rate of shoot induction, although these induced poorly rooted shoots. In addition, two types of callus induction such as green and white calli were observed, and the induction of green-type calli was significantly correlated with that of shoots. Surprisingly, the only modification, half-strength MS inorganic salts, for the above medium proved to be very effective for shoot induction in the scape culture. For shoots obtained from the scape culture, effects of sucrose concentrations and activated charcoal on root induction were examined by using 1/2 MS with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar as the basic medium. The addition of 1% activated charcoal to the media had a marked effect for root induction independent of sucrose concentrations and varieties tested. The in vitro propagation technique of I. ensata is discussed.     

A stability analysis of time to flowering as a screen for responsiveness to temperature and photoperiod in cowpea (Vigna unguiculata)

Summary Twenty-one genotypes of cowpea (Vigna unguiculata), comprising landraces and varieties, were grown in 22 photothermal environments in Nigeria and Niger, West Africa, and a stability analysis of days from sowing to flowering (f) was carried out. Cowpeas are rarely insensitive to photoperiod; they are typically quantitative shortday plants wherein f is delayed when photoperiod (P) is longer than the critical photoperiod (P _c ). Therefore, in order to quantify genotypic variation in temperature sensitivity, genotype f was regressed against the mean trial f in circumstances where P c (i.e. approximately 13 hd^-1) and mean temperature (T) was between 19° and 28° C. Correspondingly, in order to assess genotypic variation in photoperiod sensitivity, trials where T was near optimal (25°–28° C) but where P ranged from 10–14.5 hd^-1 were used. These stability analyses detected no significant differences (P>0.05) between genotypes 9n temperature sensitivity but revealed significant differences (P<0.001) in photoperiod sensitivity. Regression coefficients from the stability analysis were strongly correlated (r=0.94, 19df) with a photoperiod sensitivity constant, c, determined from a photothermal flowering model. A stability analysis of f from field trials can therefore identify and quantify genotypic variation in response to temperature and photoperiod in cowpea.Abbreviations f days from sowing to flowering - P mean photoperiod - P _c critical photoperiod - P _ce ceiling photoperiod - T mean temperature - T _b base temperature - T _o optimum temperature - SDP short-day plant