魔芋茎尖玻璃化冻存研究

研究了魔芋茎尖玻璃化冻存的主要技术环节，建立了较适宜的魔芋种质资源超低温保存体系：在实体显微镜下，从继代培养2周的不定芽上切取1～1.33mm大小的茎尖，经0.7mol/L蔗糖的MS-NH+4培养液室内预培养2d，玻璃化溶液PVS2室温下处理10～20min，直接投入液氮中保存。保存后，茎尖经40～77℃水浴快速化冻，1.2mol/L蔗糖的培养液     

树莓试管苗茎尖包埋玻璃化法超低温保存

摘要：本试验对树莓茎尖包埋玻璃化法种质资源保存进行了研究,以期建立简单、高效的树莓种质资源超低温保存体系。试验以树莓试管苗茎尖为材料,探讨了不同因素对包埋玻璃化法超低温保存后成活率的影响。结果表明：长约1mm的树莓试管苗茎尖用海藻酸钙包埋后,采用分步预培养的方法,最后终止蔗糖浓度为0.9mol/L的MS培养基中进行预培养3d,再用含2M甘油和0.9M蔗糖的装载液处理90min, 在0℃下用PVS2处理180min后投入液氮中保存1h,在40℃下化冻3min,用含1M蔗糖的MS液体培养基洗涤20min,最后转到恢复培养基上,30d后在没有形成愈伤组织的情况下形成新的植株。以上的保存程序应用于6个树莓品种,成活率达87%。该结果为树莓种质资源的长期保存提供了理论依据。     

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     

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.     

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 in vitro-grown meristems of strawberry (Fragaria × ananassa Duch.) by encapsulation-vitrification

Alginate-coated meristems from in vitro-grown strawberry (Fragaria × ananassa Duch.) were successfully cryopreserved following dehydration by a vitrification solution. Excised meristems from cold-hardened plantlets at 4 °C for two weeks in the dark were encapsulated into alginate-gel beads containing a mixture of 2 M glycerol plus 0.4 M sucrose. These encapsulated and cryoprotected meristems were dehydrated with a highly concentrated vitrification solution (designated PVS2) for 2 hr at 0 °C prior to a plunge into liquid nitrogen. Successfully encapsulated vitrified meristems remained green and then developed shoots within one week after plating without intermediary callus formation. The average rate of shoot formation of encapsulated vitrified meristems amounted to nearly 90%. The cryogenic protocol was successfully applied to four cultivars of strawberry. It was also confirmed that encapsulated vitrified meristems cooled to - 196 °C produced higher shoot formation than encapsulated dried meristems. Besides, the recovery growth was much earlier than the latter. The encapsulation-vitrification method is easy to handle and produces high levels of shoot formation. Thus, the protocol promises for cryopreservation of strawberry germplasm. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Diversity of heat-stable genotype specific resistance to Meloidogyne in Maranon races of Lycopersicon peruvianum complex

Accessions of Lycopersicon peruvianum complex and their F₁progenies were screened for genotype specific resistance to Mi-1-avirulent M. incognita and M. javanica biotypes at25 ^°C and at 32 ^°C (a temperature at which Mi-1resistance is not expressed), and to Mi-1-virulent M.incognita at 25 ^°C. All entries of the L. peruvianumChotano-humifusum race accessions LA2157 and LA2334 were resistantto Mi-1-avirulent biotype at 25 ^°C and at 32 ^°C,indicating that the accessions are homozygous for the heat-stableresistance. The L. peruvianum Maranon race accessions LA1626,LA1708, LA2172, LA2185, LA2326 and LA2328 segregated for heat-stableresistance to Mi-1-avirulent biotype. The F₁ progeny tested ofLA392 × LA2157, LA2334 × LA2157, LA2328 × LA2326,LA2328 × LA2185, LA1708 × LA2328 andLA1626 × LA2172 were resistant to Mi-1-avirulent biotype at32 ^°C. There were differences in the segregating accessions andF₁ hybrids for expression of heat-unstable and heat-stable resistanceto Mi-1-avirulent Meloidogyne spp. The L. peruvianumLA392 and LA2163 and L. chilense LA1968, LA1972, LA2404, LA2405,LA2406, LA2748, LA2930, and the L. peruvianum × L.chilense hybrids were homozygous susceptible with all entries testedsusceptible at 32 ^°C. Cuttings of these L. peruvianumaccessions and their F₁ progenies were susceptible to Mi-1-virulent M. incognita biotype at 25 ^°C.     

香石竹茎尖试管苗继代培养玻璃化现象的研究

香石竹红色品种茎尖试管苗继代培养玻璃化现象是香石竹脱毒试管苗生产的一个主要障碍。采用改善光照,在培养中提高糖和琼脂的浓度,降低细胞分裂素的用量,对克服香石竹茎尖试管苗继代培养玻璃化有明显效果,但对一些红色系品种效果并不理想,继代培养玻璃苗率仍达61%以上。     

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.     

Reduced Sink Activity in Growing Shoot Tissues of Maize under Salt Stress of the First Phase may be Compensated by Increased PEP‐Carboxylase Activity

The effects of salt stress (100 mm NaCl for 6 days) on growing tissues (shoot apex, growing leaf segments, root tips) of young maize plants (Zea mays L. cv. Pioneer 3906) were investigated in comparison to an unsalinized control, focusing on assimilate supply from source leaves and the activity of sucrolytic enzymes in the sink tissues. The objectives were to test whether (i) phloem unloading in growing tissues is mainly symplastic, (ii) salinity reduces sink activity, determined either as sucrose synthase activity (indicator for the symplastic pathway) or as acid invertase activity (indicator for the apoplastic pathway), and (iii) PEP‐carboxylase activity is increased under salinity to compensate for reduced sink activity. For growing tissues of young maize shoots, it can be assumed that phloem transport of sucrose is mainly driven by symplastic unloading into the sink cells. In maize root tips, both, apoplastic and symplastic pathways, contributed to carbohydrate supply to the sink cells. The activity of acid invertase in growing shoot tissues was very low, and the alkaline invertase contributed less than 10 % to the cytoplasmic sucrolytic activity. Salt stress of the first phase (mainly osmotic stress) caused a significant inhibition of acid invertase activity in the growing leaf segments and in the root tips, which was also true for alkaline invertase activity in the root tips as well as for sucrose synthase activity in root tips and shoot apex. The decrease of sucrose synthase activity in shoot apex might be particularly detrimental for the plant growth, as this tissue with a high cell division rate relied entirely on cytoplasmic enzyme activities. Under salt stress, PEP carboxylase activity was significantly increased in growing leaves and the shoot apex of maize, whereas no significant effect was observed in the root apex. In conclusion, PEP carboxylase can have an anaplerotic function supporting the demand for metabolites in growing shoot tissues of young maize plants under salt stress. In root tips, an additional supply of organic acids to the tricarboxylic acid cycle is probably not needed, as sucrolytic sink activity, which was high even under saline conditions, can meet the demand of the sink cells.     

Medium, Explant and Genotype Factors Influencing Shoot Regeneration in Oilseed Brassica spp.

The effects of culture media, explants and genotypes on shoot regeneration in oilseed Brassica species were examined in this study. The maximum shoot regeneration frequency was obtained in Murashige and Skoog medium supplemented with 3 mg l^?1 6‐benzylaminopurine and 0.15 mg l^?1 1‐naphthaleneacetic acid. The addition of 2.5 mg l^?1 AgNO₃ was very beneficial to shoot regeneration in B. napus and Ag₂S₂O₃ (10 mg l^?1) was even superior to AgNO₃ (2.5 mg l^?1). Explant age, explant type and carbon source also significantly affected shoot regeneration. Four‐day‐old seedlings of cotyledonary explants showed the maximum shoot regeneration frequency and number of shoots per explant. Of the four explants – peduncles, hypocotyls, cotyledons and leaf petioles – cotyledons produced the highest shoot regeneration frequency (56.67 %). Four carbon sources – glucose, maltose, starch and sucrose – were compared for their respective effects on shoot regeneration from cotyledonary explants. Sucrose appeared to be the best carbon source for shoot regeneration with the highest shoot regeneration frequency (76.00 %). Considerable variation in shoot regeneration from cotyledonary explants was observed both between and within Brassica species. The shoot regeneration frequency ranged from 10.00 % for cv. R5 (B. rapa) to 83.61 % for cv. N1 (B. napus). Two B. napus, one B. carinata and one B. juncea cultivars exhibited shoot regeneration frequency higher than 70 %. In terms of the number of shoots produced per explant, B. rapa showed the highest variation, ranging from 5.64 for cv. R3 to 1.33 for cv. R5. Normal plantlets were regenerated from all induced shoots and developed normally. The regenerated plants were fertile and identical with the source plants.     

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.     

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.     

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     

模拟大气温度和CO_2浓度升高对双季稻氮素利用的影响

未来气候主要表现为大气温度和CO2浓度升高的变化趋势,升温2℃和CO2浓度达到450μL L–1(同比增加60μL L–1)情景是哥本哈根共识下的安全阈值。本研究采用自主研制的开顶式气室(open-top chamber,OTC)进行双季稻大田原位模拟试验,以早稻两优287和晚稻湘丰优9号为试验材料,设置了大田(UC)、对照(CK)、增温2℃(CT)、增CO2 60μL L–1(CC)和同时增温2℃增CO2 60μL L–1(CTC)5个处理,研究温度和CO2浓度升高对双季稻产量和氮素利用的影响。结果表明,早稻CT的籽粒产量和氮素积累量均低于CK,CC和CTC比CK提高籽粒产量19.7%和2.0%,提高氮素积累量15.7%和5.1%;晚稻CT、CC和CTC籽粒产量和氮素积累量比CK分别提高9.2%、14.4%和18.8%,及7.3%、10.2%和15%。茎叶氮素转运率和贡献率早稻CC和CTC略低于CK,晚稻CC、CTC均高于CK。氮素吸收利用率早稻以CC最高(45.7%),晚稻以CTC最高(48.5%),分别比CK提高了35.5%和33.1%。氮素农学利用率与之一致,早稻和晚稻的CC和CTC均最高(23.1 kg kg–1和26.9 kg kg–1),比CK提高了56.3%和46.2%。氮素生理利用率早稻和晚稻均以CC最高,相比CK提高了12.7%和10.5%,但差异不显著。CK与UC之间各项指标差异不大,这表明OTC覆盖对水稻生长造成的影响在可接受误差之内。综上所述,本研究认为温度升高2℃对早稻产量和氮素利用倾向于不利影响,对晚稻则相反;CO2浓度增加60μL L–1对早稻和晚稻产量和氮素利用倾向于有利影响;同时增温和增CO2对早稻表现抵消作用,对晚稻表现协同作用。     

Variation of PGM and IDH isozymes for identification of alfalfa varieties

Growth habit, heading date and Vrn genotypewere examined for wheat landraces cultivated in China,Korea and Japan, to study their ecogeographicaldifferentiation in east Asia. Spring type landracesaccounted for 43.6% of the whole, and the frequencyvaried between the localities, being closely relatedto the degree of winter coldness. Spring typelandraces mainly adapted to north and south Chinawhere average January temperature is under –7 ^°Cand over 4 ^°C, respectively. On the contrary,winter type adapted to areas of average Januarytemperature from –7 ^°C to 4 ^°C. As toheading date, significant difference was not observedbetween spring and winter type landraces but betweenlocalities, and those cultivated in north China weresignificantly later in heading. It is thereforeindicated that spring type mainly adapts to areaswhere wheat is sown in spring to avoid frost injury,and where winter temperature is not low enough tovernalize winter type wheat. Genetic analysis forspring type landraces showed that the relativefrequency of four Vrn genes was different witheach other. Vrn3 was most widely and frequentlyfound among the four genes, followed by Vrn1 andVrn2. Only seven landraces proved to be thecarrier of Vrn4. The frequency was alsodifferent between localities. Genotype with Vrn1plus other dominant gene(s) adapted to spring sowingto avoid severely cold winter in north China, whilegenotype with only Vrn3 adapted to winter sowingin south China and southwest Japan. It is thereforeconcluded that at least three ecotypes, differing ingrowth habit and Vrn genotype, areallopatrically distributed in east Asia, as a resultof adaptation to winter coldness in each locality.     

In vitro induction of tetraploid plants from callus cultures of diploid bananas (<Emphasis Type="Italic">Musa acuminata</Emphasis>, AA group) ‘Kluai Leb Mu Nang’ and ‘Kluai Sa’

Tetraploid plants were induced successfully from diploid bananas Musa acuminata (AA genome) ‘Kluai Leb Mu Nang’ and ‘Kluai Sa’ (2n = 2x = 22) with in vitro oryzalin treatment. Calluses from in vitro-grown shoot tips of both cultivars were treated with oryzalin at concentrations of 1.5 or 3 mg l⁻¹ for 24, 48 and 72 h, respectively. The oryzalin treatments produced tetraploids at a frequency of 15.6% in ‘Kluai Leb Mu Nang’ and 16.7% in ‘Kluai Sa’ as detected by flow cytometry. Chromosome counting showed that the tetraploid plant chromosome number was (2n = 4x = 44). The selected tetraploid plants were transplanted in the field and variations in the morphological characteristic of leaf shape and fruit bunch compared to normal diploid plants were found under the same growing condition even after 3 years of cultivation.     

Plant Regeneration from in vitro Cultured Anthers of Black Mustard (Brassica nigra Koch)

Anthers of Brassica nigra, excised from fresh as well as cold-pretreated (3 days at 3 ± 2°C) buds cultivated on modified B₅ medium (Gamborg et al. 1968) containing sucrose level varying from 2 % to 10 %, along with 1O^?6M BAP (benzylaminopurine) and 9 × 10^?6M 2,4-D (2,4-dichlorophenoxyacetic acid), developed calli and/or embryos. The latter response was observed only in anthers reared on media containing 6 % or higher levels of sucrose. On media containing two or four per cent sucrose, the anthers produced calli, exclusively. The growth of embryos was inhibited or else they started callusing if left on the media containing higher levels of sucrose. However, on transfer to MS medium (Murashige and Skoog 1962), containing 2 % sucrose, embryos started callusing and subsequently a few secondary embryos differentiated. Such embryos were sub-cultured on MS + 5 × 10^?6M BAP + 2 % sucrose, wherein numerous shoots developed from embryos. The shoots were rooted by transferring to a medium containing 5 × 10^?6M NAA (naphthalene acetic acid). Within two months of culture, some of these plants started flowering in vitro.     

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.