以异源四倍体体细胞杂种为父本杂交培育三倍体柑桔植株的研究

以 3个柑桔原生质体融合而来的四倍体体细胞杂种为父本 ,与二倍体单胚性种柚子 (Citrusgrandis)以及单多胚混合型品种“华农本地早”桔 (C.reticulata)有性杂交 ,授粉后 90 d,发现种子干瘪 ,大部分种子的胚败育。将干瘪种子在 MT附加 1mg/L GA3 或 50 0 mg/L麦芽浸出物的培养基中 ,经培养抢救 ,有 2 5.6%的种子萌发成苗或继续进行胚的生长 ,后者进一步诱导能形成丛芽 ,经试管嫁接或诱导生根形成完整植株。共获得 6个组合 73棵完整植株 ,染色体数检查表明 ,2 0株为三倍体 (2 n=3x=2 7) ,32株为二倍体 (2 n=2 x=18) ,8株为非整倍体 ,其它 13株还有待于进一步检查。     

Production of aneuploid melon plants following in vitro culture of seeds from a triploid x diploid cross

Aneuploid melon plants (Cucumis melo L.) were obtained from in vitro cultured seed, which were produced by crossing triploid (3x=36) x diploid (2x=24) plants. Twenty-six fruits were obtained from pollination of 29 bisexual flowers of triploid plants. Seeds were collected from the fruits at 2, 3, 4, 5 and >7 weeks after pollination and germinated in vitro on Murashige & Skoogs (MS) medium. Embryos developed from 0.6 to 1.6% of the cultured seeds after three weeks in culture. Shoots developed from 0 to 47% of embryos after transfer to half-strength MS medium. Some (0 to 50%) of elongated shoots that rooted were subcultured on the same medium. Five rooted plantlets were obtained through culture of 5,353 seeds. Four of the plants were aneuploid, with chromosome numbers of 27, 35, 45 and 46, respectively, and the one was tetraploid (4x=48).     

Induction of Haploid Durum Wheat Plants Through Pollination of Maize Pollen

When tetraploid wheat (Triticum durum Desf. ) variety DR147 was crossed with maize (Zea mays L. ) variety suppersweet ss 7700, pollen readily germinated on the stigma and one or more pollen tubes reached the embryo sac in 83.4% of wheat florets. The frequency of fertilization and embryo formation was 44.5% and 42. 6% respectively. The hybrids were karyotypically unstable and the maize chromosomes were eliminated early in the development. Thus haploid wheat embryos were form. Although the double fertilization frequency of durum wheat X maize was high (32.7%) to form embryos and endosperms, yet the endosperms were highly abnormal. It was very difficult to produce viable mature seeds from the mother durum wheat plants. The survival of hybrid embryos produced by durum wheat X maize could be improved or prolonged by treatment with 100 ppm 2, 4-D (either by dipping inflorescences in solution or injecting 0.3 to 0.5 mL 2, 4-D solution into the uppermost internodes of the wheat stem). 9 to 13 days after pollination, caryopsis were excised from the pollinated spikes and surface sterilized for peeling of the embryos in different developing stages. The embryos were plated on MS solid medium containing 3% sucrose, 200 mg/L casein hydrolysate for embryo rescue. The experimental results revealed that the well developed embryos (larger than 0. 5 mm with scutellum structure) were easy to produce calli by callus induction or produce haploid wheat plants by embryo rescue, whereas the poorly developed embryos (globular, pear or torpedo-shaped embryos smaller than 0.3 mm) responsed very poorly. The germination frequencies of well and poorly developed embryos were 83.3 % and 12.5 %, respectively. Chromosome counts of root tip cells of the rescued plants proved their haploid nature (2n= 2x= 14).     

普通小麦与鸭茅状摩擦禾的远缘杂交：Ⅱ.未成熟胚的培养

培养了由２６个小麦品种（系）用鸭茅状摩擦禾的花粉授粉１４天后获得６４５个未成熟的胚。结果表明,未成熟胚培养的植株再生频率与胚的小麦基因型的杂交亲和性无关,胚的发育程度直接影响胚培养的结果,离体时分化完全的未成熟胚在无激素的培养基上可以迅速萌发成工轩,而分化未完全的小胚在无激素的培养基上分化进程不能继续,而且在无激素补充的情况下,萌发过程一旦起动,即使将这些胚转至补加了激素的胚分化培养基上,分化过程     

An efficient protocol for the production of triploid plants from endosperm callus of neem,Azadirachta indica A. Juss

Triploid plants of neem were obtained by immature endosperm culture. Immature seeds, at the early dicotyledonous stage of embryo development, is the best explant to raise endosperm callus on MS + NAA (5 mumol/L) + BAP (2 mumol/L) + CH (500 mg L-1). Maximum shoot bud differentiation from the endosperm callus occurred on MS + 5 mumol/L BAP. Shoots were multiplied by forced axillary branching and rooted in vitro. The plants were established in soil. Over 66% of the plants were triploid with chromosome number 2n = 3x = 36. A characteristic feature of the shoots of endosperm origin is the presence of a large number of multi-cellular glands.     

Micropropagation of an elite Darjeeling tea clone

Shoot cultures of Camellia sinensis (L.) O. Kuntz var. T-78, an elite Darjeeling tea clone, were established from cotyledonary nodes and shoot tips of germinated seedlings as well as from nodal explants of field grown plants. Shoot multiplication rate ranged from 4x in nodal explants to 35x in cotyledonary nodes after 18 weeks of culture. Rooting was achieved in 80–90% micro-shoots by either placing them on an inductive medium for 10 d and then transferring shoots to hormone-free medium, or by treating micro-shoots with a chronic dose of IBA (500 mg/l) for 30–40 min. Rooted plants were established in soil under glasshouse condition at 60% frequency after hardening phase of 4–6 weeks. The regenerated plants show a constant chromosome number of 2n=30 and are morphologically true to type. This procedure can be applied for conservation and utilisation of an elite clone of Darjeeling tea.     

Agrobacterium-mediated transformation of seedling-derived maize callus

Efficient production of seedling-derived Type I callus was demonstrated for several corn genotypes including commercial inbred lines. Seeds were germinated on MS-based medium containing 10 mg l(-1) picloram and 3 mg l(-1) 6-benzylaminopurine, which induced the development of axillary buds in the area of coleoptilar node. Nodal sections of 7-10-day old seedlings were isolated, split longitudinally, and placed on callus induction medium supplemented with 2.2 mg l(-1) picloram and 0.5 mg l(-1) 2,4-dichlorophenoxyacetic acid. For lines L4 and L9 the frequency of embryogenic callus induction was 38-42% based on calli per split nodal section. Frequency of callus induction from split nodal sections of seeds germinated on media without growth regulators was 0-3%. Seedling-derived callus of five genotypes was used for Agrobacterium-mediated transformation. Two constructs containing the green fluorescence protein gene and genes for either neomycin phosphotransferase II or glyphosate selection were used in transformation experiments. Transformation frequency varied from 2 to 11% and about 60% of the T(0) plants had 1-2 copies of transgenes.     

转HAL1基因番茄的耐盐性

利用农杆菌介导的叶盘法,把HAL1 基因转入番茄,Southern杂交检测得到转基因植株.耐盐实验表明, T1代转基因番茄在150 mmol/L的NaCl胁迫下仍有43%的发芽率,200 mmol/L的NaCl胁迫下发芽率为6%,而对照种子在100和150 mmol/L的NaCl胁迫下发芽率分别为11.0%和0.转基因番茄的电解质相对外渗率小于对照,而根冠比和叶绿素含量大于对照,转HAL1基因显著提高了番茄的耐盐性.盐胁迫下Na 、K 的累积状况表明,转基因番茄根、茎、叶的K /Na 均有所提高,根系的SK/Na增大,茎、叶的RSK/Na和RLK/Na减小,说明根系对K /Na 离子的选择吸收和运输能力加强.不但选择吸收K /Na ,而且表现出整株水平上的有利于耐盐的K /Na 区域化分配.     

Production and characterization of amphiploids of Aegilops kotschyi and Ae. biuncialis with Secale cereale, and of backcross hybrids of Ae. biuncialis x S. cereale amphiploids with 2x and 4x S. cereale

Amphiploids (2n = 6x = 42) of Ae. kotschyi and Ae. biuncialis with self-compatible S. cereale were produced from F1 sterile hybrids (2n = 3x = 21) through colchicine treatment and callus tissue regeneration. The amphiploids resembled the F1 plants in overall morphology, but were larger in all respects and self-fertile. The spikelets consisted mostly of 3 well-developed florets. Selfed seeds were obtained from some colchicine-doubled sectors and callus regenerates. Most of the produced seeds were well developed. Backcrosses between amphiploids and rye (2x and 4x) resulted in obtaining (Ae. biuncialis x S. cereale amphiploid) x S. cereale hybrids via embryo culture. The BC1 plants (2n = 4x = 28 and 2n = 5x = 35, respectively) were phenotypically intermediate between the parents and vigorous in vegetative growth. Some seeds were obtained only from the 35-chromosome BC1 hybrids.     

Haploid Plant Regeneration from Protoplast Cultures of Durum Wheat (Triticum durum Desf.)

Haploid suspension callus cultures from embryos of durum wheat (Triticum durum Desf. ) × maize (Zea mays L. ) crosses were used for protoplast isolation. Experimental results from enzyme digestion showed large numbers of viable protoplasts released from both suspension culture and solid culture of callus cut into small pieces of 1 mm in size prior to incubation in an enzyme solution containing 2.0% cellulase RS and 0.5 % pectolyase Y-23. Division frequency of protoplasts isolated from suspension cultured callus was quite different from that of solid cultured callus, however, the former being 5.20%, and the latter less than 1.0% when cultured on KM8p medium containing 1.0 mg/L 2, 4-D using LMP (low melting point) agarose embedding method. Embryogenic ealli could be selected out from protoplast-derived microcalli after 2 to 3 subcultures. Plants could be regenerated from protoplast-derived embryogenic calli after 20 days of culture on differentiation medium I (MS basal medium supplemented with 0.2 mg/L 2, 4-D, 1.0 mg/L BAP, 0. I mg/L NAA, 3 %/4 su- crose, 200 mg/L casein hydrolysate, 146 mg/L glutamine, 300 mg/L aspartic acid) and (components were the same as I without 2, 4-D) respectively. The plant regeneration frequency was about 20%. Chromosome count of root tip cells of 4 plants of the 22 protoplast- derived plants sampled at random revealed haploid in nature (2n= 2x= 14).     

Introgression mapping of genes for winter hardiness and frost tolerance transferred from Festuca arundinacea into Lolium multiflorum

Genes for winter hardiness and frost tolerance were introgressed from Festuca arundinacea into winter-sensitive Lolium multiflorum. Two partly fertile, pentaploid (2n = 5x = 35) F(1) hybrids F. arundinacea (2n = 6x = 42) x L. multiflorum (2n = 4x = 28) were generated and backcrossed twice onto L. multiflorum (2x). The backcross 1 (BC(1)) and backcross 2 (BC(2)) plants were preselected for high vigor and good fertility, and subsequently, a total of 83 BC(2) plants were selected for winter hardiness after 2 Polish winters and by simulated freezing tests. Genomic in situ hybridization (GISH) was performed on 6 winter-hardy plants selected after the first winter and shown to be significantly (P < 0.05) more frost tolerant than the L. multiflorum control. Among the analyzed BC(2) winter survivors, only diploid (2n = 2x = 14) plants were found. Five plants carried 13 intact L. multiflorum chromosomes and 1 L. multiflorum chromosome with a single introgressed F. arundinacea terminal chromosome segment. The sixth BC(2) winter survivor appeared to be Lolium without any Festuca introgression capable of detection by GISH. A combined GISH and fluorescence in situ hybridization analysis with rDNA probes of the most winter-hardy (after 2 winters) and frost-tolerant BC(2) plant revealed the location of an F. arundinacea introgression on the nonsatellite arm of L. multiflorum chromosome 2, the same chromosome location reported previously as a site for frost tolerance genes in the diploid and winter-hardy species Festuca pratensis.     

The use of mutagens to increase the efficiency of the androgenic progeny production in <Emphasis Type="Italic">Solanum nigrum</Emphasis>

Pollen embryogenesis was successfully induced in Solanum nigrum L. (2n=6×=72). Stimulation of androgenesis expressed as the frequency of androgenic responsive anthers was observed after 10 and 20 mM ethyl methanesulphonate (EMS), 10 and 20 mM sodium azide (NaN₃) and 0.2 mM N-nitroso-N-methylurea (MNU) treatment applied on seeds for 24 h. The frequency of androgenesis on the medium with sucrose was higher than on the medium with maltose. Androgenic regenerants originated also in the anthers collected from donor plants where survival after mutagenic treatment was lower than 50 %. Green haploid (3x), aneuploid (to 8x) and dihaploid (6x) plants were obtained. The high frequency of aneuploids among androgenic plants is explained by cell division irregularities in microsporial calli.     

青藏高原5种石竹科垫状植物的核型研究

垫状植物是高山冰缘带植物区系的重要组成成分,是植物适应高寒环境趋同进化的特殊生活型。该研究以采自西藏和云南高山冰缘带的5种石竹科垫状植物种子为材料,采用滤纸萌发法、常规压片法镜检观察分析其核型,为青藏高原垫状植物积累核型资料,并为探讨极端环境下特殊生活型植物的核型进化机制提供依据。结果表明:5种植物均为二倍体,染色体核型分别为:密生福禄草(Arenaria densissima),2n=2x=20m+2sm,2A;山生福禄草(A.oreophila),2n=2x=22m,1A;团状福禄草(A.polytrichoides),2n=2x=20m+2sm,2A;大花福禄草(A.smithiana),2n=2x=20m+2sm,1A;囊种草(Thylacospermum caespitosum),2n=2x=18m+4sm,2A。囊种草与福禄草亚属植物的染色体数目一致,为二者在分子系统发育框架下形成的独立分支提供核型证据。并讨论了5种垫状植物古多倍体物种形成的核型进化与中新世青藏高原及周边地区地质抬升的关联。     

Selection of somaclonal variants with low-temperature germinability in melon (Cucumis melo L.)

Summary Plants were regenerated from adventitious buds and somatic embryos (R₀) of melon (Cucumis melo L.), the cultivar Andes. Somaclonal variants of melon with low temperature germinability were selected from the progenies (R₁) of R₀ plants. Among 5,618 R₁ seeds harvested from 23 R₀ plants that were regenerated from adventitious buds 4 seeds germinated after 5 days of culture at 15 °C (selection rate; 0.07%). However, among 374 R₂ seeds harvested from 2 R₁ plants no seed germinated after 7 days of culture at 14 °C. Among 9,181 R₁ seeds harvested from 50 R₀ plants regenerated from somatic embryos 110 seeds germinated after 5 days of culture at 15 °C (selection rate; 1.20%). Among 3,717 R₂ seeds harvested from 17 R₁ plants 113 seeds germinated after 7 days of culture at 14 °C (selection rate; 3.04%). R₃ seeds were collected from these R₂ plants following self-pollination. Forty-five of the 47 lines (R₃) originated from 10 R₀ plants showed higher germination rates than that of the original cultivar. Selected lines with low-temperature germinability showed greater fruit growth rate than the original cultivar during the middle stage when they were cultivated in a greenhouse under low-temperature conditions. Of fruits harvested from 31 lines, 15 lines showed greater fruit volume than the original cultivar.     

The effects of level of 2,4-D and time in culture on regeneration rate and chromosome numbers of regenerants from calli of the hybrid Triticum aestivum cv. Chinese Spring ph1b x Thinopyrum ponticum (2n = 10x = 70).

Thinopyrum ponticum (Podp.) Barkworth &D. R. Dewey (2n = 10x = 70) has excellent resistance for both leaf and stem rusts. Long-term callus cultures were established from the immature embryos of a hybrid between Triticum aestivum L. (2n = 6x = 42) x Th. ponticum. They were maintained in culture for over 2 years and continued to grow and have organogenetic capacity. With increasing time on a maintenance medium, the plant regeneration rates of the hybrid calli decreased when transferred to regeneration media containing 0.1, 0.2, or 0.5 mg/L 2,4-D, but the rate of decrease was much higher at 0.5 mg/L than at either 0.1 or 0.2 mg/L 2,4-D. After 3 months of subculture, the highest plant regeneration rate was obtained on the medium containing 0.5 mg/L 2,4-D (1.11 plantlets/callus), while on the 24th month of subculture the highest plant regeneration rate was obtained on the medium containing 0.1 mg/L 2,4-D (0.20 plantlets/callus). Thus, it was shown that as the calli aged it was important to reduce the level of 2,4-D in the regeneration medium. Over 2 years, a total of 667 regenerants were successfully transferred and grown to maturity. Chromosome numbers in root-tip cells were determined for 539 regenerants and ranged from 36 to 70. Telocentric chromosomes were frequent. A fertile plant was found among the regenerants after 15 months of subculture. It had 56 chromosomes with 2.15 (1-6) univalents, 22.76 (17-26) closed bivalents, 3.55 (1-9) open bivalents, and 0.41 (0-3) trivalents and was highly resistant to stem rust race 15B-1. Callus culture of wide hybrids can be used to introgress characters from alien species into wheat.     

Selection and characterization of a rice mutant resistant to 5-methyltryptophan

Summary A rice plant resistant to 5-methyltryptophan (5MT) was selected from mutagenized M3 seeds (Oryza sativa L. var. Sasanishiki) originating from panicles treated with ethylene imine (0.2%) 2 h after flowering. When germinated on 5MT-containing medium, the seeds (M4) from selfed plants segregated with a 3 resistant:1 sensitive ratio, indicating that the plant was heterozygous for a resistance gene and that the resistance was dominant. The resistance was also expressed in callus derived from seeds. Analysis of the free amino acids in seeds, seedlings, and calli showed that homozygous resistant plants (TR1) contained higher levels of total free amino acids than sensitive plants. In particular the levels of tryptophan, phenylalanine, and histidine were, respectively, 8.5, 5.4, and 4.9 times higher than those in the sensitive plants.     

Chromosome doubling and mode of reproduction of induced tetraploids of eastern gamagrass (Tripsacum dactyloides L.)

Eastern gamagrass, (Tripsacum dactyloides L.) is a perennial, warm-season grass that is being developed as a forage plant. Shoots were derived from callus initiated from immature embryos and immature inflorescences of diploid (2n=2x=36) gynomonoecious eastern gamagrass. These shoots were induced to microtiller in the presence of 3 mg/l benzyladenine. Amiprophosmethyl (10, 15, or 20 μm) was applied to 27 microtillers for 3–5 days to induce chromosome doubling. All 14 surviving plants were tetraploid, (2n=4x=72), as determined by flow cytometry or chromosome counts. These plants were morphologically normal and produced seed. Test crosses were made with a known diploid. Flow cytometry and chromosome counts showed that the progeny were triploid, proving that the induced tetraploids reproduce sexually. Received: 12 February 1997 / Revision received: 18 February 1998 / Accepted: 13 March 1998     

Effects of germination on chemical composition and functional properties of sesame (Sesamum indicum L.) seeds

The changes of chemical composition and functional properties of derooted sesame (Sesamum indicum L.) seeds (DSS) before, during, and after germination were investigated. Sesame seeds germinated in dark chambers maintained near 100% relative humidity at 35 degrees C without presoaking reached >99% germination rate in 4 days with the final moisture content stayed ca. 2% (w/w), characterizing sesame seeds as orthodox seeds that are suitable for long term storage at low temperature and humidity under defined environment. With noticeable reduction in fat content (23%), germinated DSS were found rich in linolenic acid, P, and Na, increasing from 0.38% (w/w), 445 mg/100 g, and 7.6 mg/100 g before germination to 0.81% (w/w), 472 mg/100 g, and 8.4 mg/100 g after germination, respectively. DSS after germination contained considerable amount of Ca (462 mg/100 g), higher than that of soybean. Germinated DSS presents an excellent source of sesamol (475 mg/100 g), a potent natural antioxidant, and alpha-tocopherol (32 mg/100 g), the most active form of vitamin E.     

Induction of haploid and diploid plants though in vitro anther culture of haploid wheat (n=3x=21)

Summary Five haploid plants of wheat were used for anther culture. Embryos were formed and six plants were regenerated. Of these, two were haploid (n=3x=21) and two diploid (2n=6x=42). The two diploids derived from the anthers of the same haploid wheat plant gave seeds, but the fertility was reduced in one of them showing, abnormalities at meiosis.     

Somatic embryogenesis in Vigna radiata (L.) Wilczek.

Somatic embryogenesis was achieved from immature cotyledon derived callus of mungbean, V.radiata (L.) Wilczek in MS liquid medium. Embryogenic callus was induced on MS medium with NAA (5 mg/L). Differentiation of somatic embryos was observed when embryogenic callus was transferred to MS liquid medium containing 2,4-D (1.5 mg/L) and L-proline (50 mg/L). The torpedo shaped embryos were transferred to MS liquid medium with BAP and ABA (1 mg/L each) for maturation and germination. Fifty per cent of torpedo shaped embryos were converted into tiny plants (8-9 plants out of 17) after one week of culture. The germinated embryos were isolated and transferred to MS half strength basal (solid) medium for further development.