渗透胁迫条件下小麦成熟胚愈伤组织的诱导及植株再生的研究

用不同浓度的PEG6000及NaCl对5个小麦品种的成熟胚组织培养物进行处理,研究在渗透胁迫条件下基因型和激素对成熟胚愈伤组织的诱导及分化的影响。结果表明,小麦整株水平与细胞水平的抗性存在一定相关,不同基因型对干旱与盐胁迫的敏感程度不同,成熟胚愈伤组织的诱导率和植株再生率表现出明显的差异。初步得到了晋麦47、长武134、红芒麦的耐旱愈伤组织以及晋麦47、长武134的耐盐愈伤组织,并获得了晋麦47和长武134具有一定抗性的再生芽。     

Plasticity in Sex Expression of Spinach (Spinacia oleracea) Regenerated from Root Tissues

The sex expression of spinach (Spinacia oleracea L.) was modified by a procedure where plants were regenerated from root callus of spinach over three successive generations. Somatic embryos obtained from the root callus of female or male plants were grown in vitro under long day conditions until flowering. Although the regenerants derived from somatic embryos derived from female plants were all female at the first generation, few progenies produced male flower organs and expressed gynomonoecy in subsequent generations. Once the female plants regenerated into gynomonoecy, they never expressed complete femaleness at post generation. However, the male plants easily altered into andromonoecy at subsequent generations, and several andromonoecious plants showed the sex reversion at post generation, although this phenomenon did not occur in the female progenies. No absolute sex conversion was found in both female and male progenies. The plasticity of the sex expressions of spinach indicates that female and male individuals might essentially be capable of generating both flower organs.     

Regeneration of Immature Inflorescences of Barley In Vitro

Two spring barley cultivars, Golden Promise and Galan, were screened for callus induction and shoot regeneration from cultured immature inflorescences. Genotype Galan have better regeneration capacity in in vitro conditions than Golden Promise.     

Control of direct and indirect somatic embryogenesis by exogenous growth regulators in immature zygotic embryo cultures of rose

Immature zygotic embryos of rose (Rosa hybrida L.; cv. Sumpath) did not form somatic embryos or embryogenic calluses when cultured on half-strength Murashige and Skoog's medium supplemented with various con-centrations of 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole growth regulator. However, the zygotic embryos produced somatic embryos without an intervening callus phase at a frequency of 27.3% on medium with 4.44 M 6-benzyladenine (BA) alone. Immature zygotic embryos formed embryogenic calluses at a frequency of 25% on medium with a combination of 1.36 M 2,4-D and 4.44 M BA. Upon transfer to medium without growth regulators, embryogenic calluses produced numerous somatic embryos that subsequently developed into plantlets. Somatic embryos were induced directly from immature zygotic embryos, or indirectly via an intervening callus phase, by manipulating the exogenous growth regulators. Plantlets were successfully transplanted to potting soil and grown to maturity in a greenhouse.     

An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.)

An efficient method for in vitro micro propagation and genetic transformation of plants are crucial for both basic and applied research. Maize is one of the most important cereal crops around the world. Regeneration from immature embryo is hampered due to its unavailability round the year. On the contrary mature embryo especially tropical maize is recalcitrant toward tissue culture. Here we report a highly efficient regeneration (90%) system for maize by using 2 different approaches i.e., embryogenic and organogenic callus cultures. Seeds were germinated on MS medium supplemented with 5 mg/l 2,4-D and 3 mg/l BAP. Nodal regions of 2 wks old seedlings were longitudinally split upon isolation and subsequently placed on callus initiation medium. The maximum frequency of embryogenic callus formation (90%) was obtained on MS medium supplemented with 2 mg/l 2,4-D and 1 mg/l BAP in the dark conditions. The compact granular organogenic callus formation (85% frequency) was obtained on MS medium supplemented with 2.5 mg/l 2,4-D and 1.5 mg/l BAP at light conditions. MS medium supplemented with 2 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA promoted the highest frequency of shoot induction. The highest frequency of root formation was observed when shoots were grown on MS medium. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for obtaining more stable transformants from mature seeds of Indian tropical maize cultivar (HQPM-1).     

Induction of callus and plant regeneration in Vicoa indica

Callus cultures were initiated from the stem and leaf explants of aseptically grown Vicoa indica. A simple method is described for plant regeneration from callus and the rapid multiplication of the plants thus obtained. Callus initiation was optimum in Gamborg B5 (B5) basal medium containing either 2.0 mg l^-1 naphthaleneacetic acid (NAA) with 0.2 mg l^-1 kinetin (Kn) or 2.0 mg l^-1 6-benzylaminopurine (BAP) with 0.2 mg l^-1 NAA. The calli initiated on B5 medium were able to proliferate on both Murashige and Skoog (MS) and B5 basal medium. Shoot primordia were obtained from greenish callus on passage to B5 basal medium containing 3.0 mg l^-1 BAP and 1.0 mg l^-1 Kn. On further subculture onto B5 medium containing 0.2 mg l^-1 Kn the shoot primordia developed into plantlets.     

Production of multiple shoots from thidiazuron-treated mature embryos and leaf-base/apical meristems of barley (Hordeum vulgare)

The in vitro plant regeneration frequencies for immature scutella, leaf-bases/apical meristems (LB/AM) and mature embryos of four commercially important barley genotypes were compared. Production of shoots from mature embryos or calluses of LB/AM incubated on media containing 1.0 or 2.0 mg l^–1 6-benzylaminopurine (BA) were comparable to regeneration frequencies obtained for scutella-derived calluses of the same genotypes. Incubation of excised mature embryos and LB/AM on media containing the plant growth regulator, thidiazuron (TDZ), resulted in an increased shoot production. However, TDZ treatment did not stimulate plant regeneration from calluses derived from scutella or LB/AM. Shoots formed from TDZ-treated mature embryos and LB/AM were induced without a callus interphase and the in vitro culture system gave a three- to eight-fold higher regeneration frequency than recorded for scutella-derived calluses on BA medium. The simplicity and rapid development of shoots using the mature embryo system could potentially be used for the regeneration and genetic transformation of barley over alternative regeneration systems.     

In vitro propagation of Sandersonia and Gloriosa

The in vitro reactions of two closely related endangered species Sandersonia and Gloriosa were studied. Both species responded similarly in vitro. Callus production was initiated on a basal medium containing 2, 4-dichlorophenoxyacetic acid. Roots were formed on removal of this hormone. Tuber explants from particular parts of the tuber produced plantlets on a range of hormone concentrations. Repeated longitudinal sectioning of the bud also resulted in multiple plantlet formation.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of the heavy metal tolerant and hyperaccumulator <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> (Brassicaceae)

Plants were regenerated from root explants of Arabidopsis halleri (L.) O’Kane and Al-Shehbaz via a three-step procedure callus induction, induction of somatic embryos and shoot development. Callus was induced from root segments, leaflets and petiole segments after incubation for 2 weeks in Murashige and Skoog medium (MS) supplemented with 0.5 mg/l⁻¹ (2.26 μM) 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.05 mg/l⁻¹ (0.23 μM) kinetin. Only calli developed from root segments continued to grow when transferred to a regeneration medium containing 2.0 mg/l⁻¹ (9.8 μM) 6-γ-γ-(dimethylallylamino)-purine (2ip) and 0.05 mg/l⁻¹ (2.68 μM) α-naphthalenacetic acid (NAA) and eventually 40 of them developed embryogenic structures. On the same medium 38 of these calli regenerated shoots. Rooting was achieved for 50 of the shoots subcultured in MS medium without hormones. The regeneration ability of callus derived from root cuttings, observed in this study, makes this technique useful for genetic transformation experiments and in vitro culture studies.     

不同杂种优势类群玉米幼胚愈伤组织的诱导及植株再生特性的研究

以Reid、唐四平头和其它种质等3个杂种优势类群共19份玉米自交系为试验材料,以玉米幼胚作为外植体,研究了基因型、培养基和激素对玉米幼胚愈伤组织的诱导及植株再生的影响,结果表明供试材料均能进行愈伤组织的诱导,但是仅有12个自交系能再生植株。N6和改良N6培养基有助于提高愈伤组织的质量及其生长速度,2,4-D在愈伤组织的诱导中起着关键性作用。在诱导培养基中添加0.2mg／L的6-BA或KT会使胚性愈伤组织的诱导频率下降以及降低愈伤组织的质量。在胚状体诱导培养基中添加1mg／L的KT能促进绿苗的分化,但是浓度过高会使丛生苗分化过多。此外,通过对不同杂种优势类群自交系玉米幼胚培养特性的分析,发现在唐四平头类群的4个自交系中,黄早四的绿苗分化率仅为0.5％,其它3个自交系不能再生植株。但是,从Reid和其它种质类群的供试自交系中筛选出了胚性愈伤组织的诱导频率和绿苗分化率均较高的、适合于遗传转化的受体材料,如3189／4380、4380／陕综5、8103、先早17、18-599红、18-599白、501、178和冀53。     

Plant regeneration from callus and protoplasts in Medicago polymorpha

Seventeen ecotypes of the wild species Medicago polymorpha adapted to a Sardinian (Italy) environment have been evaluated for their response to tissue culture. The accession Samughero-Albi was the more respondent for callus induction and, together with Usassai, showed the highest regeneration capacity on media containing 1 mg l^-1 2iP and 0.1 mg l^-1 IAA. The morphogenetic response was also affected by the explant source. The hypocotyl-derived-calli were the best regenerating tissues. Regenerated plantlets were difficult to root and it was possible to obtain plants with a well developed root system only after 5–7 weeks of culture on media containing 2iP and IAA both at 0.2 mg l^-1. Mesophyll cells were the best protoplast yielding source but only those isolated from roots were able to divide and to regenerate plants. Results are discussed in relation to the genotype specificity for the morphogenetic response and the feasibility of using M. polymorpha in the somatic hybridization with M. sativa.Abbreviations NAA -naphthaleneacetic acid - 6-BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - 2iP N⁶-²-isopentenyl-adenine - IAA indole-3-acetic acid - GA₃ gibberellic acid - GFMS growth regulator free MS medium - Prol proline - Malt maltose     

The Effect of Different Plant Growth Regulators on Adventitious Shoot Formation from Virginia Pine (Pinus virginiana) Zygotic Embryo Explants

The effects of different plant growth regulators on in vitro adventitious shoot formation in Virginia pine (Pinus virginiana Mill.) zygotic embryo explants were quantitatively evaluated. Using Tang and Ouyang (1999) (TE) basal medium supplemented with 11.4 μM indole-3-acetic acid (IAA) and 2.2 μM N⁶-benzyladenine (BA), callus was observed after 3–6 weeks of culture. Calluses were transferred to TE basal medium supplemented with 0.49 μM indole-3-butyric acid (IBA) and 8.8 μM BA for 6–9 weeks, where they produced numerous small shoot primordia. They were then transferred to TE basal medium supplemented with 0.49 μM IBA and 4.4 μM BA to promote growth and elongation of adventitious shoots. After elongated shoots were transferred to TE medium containing 0.05 μM α-naphthaleneacetic acid (NAA) for 6 weeks, adventitious roots were formed. Regenerated plantlets were established in soil in greenhouse.     

Nitrate accumulation by wheat (Triticum aestivum) in relation to growth and tissue N concentrations

The accumulation of nitrate in relation to total N concentrations ([N]_i) in tissues of wheat (Triticum aestivum L., cv Sicco) grown in solution culture was investigated. Root, shoot and leaf tissues showed qualitatively similar relationships between internal nitrate concentrations and [N]_i, both expressed on a tissue water basis. At low [N]_i, no nitrate was detectable but once a particular [N]_i was exceeded, nitrate accumulated as a linear function of [N]_i. The threshold [N]_i values for nitrate accumulation were 110, 450, and 550 mM for roots, total shoot and leaf 4, respectively. The slope of the relationship between nitrate and [N]_i indicated that in all tissues nitrate accounted for 50–55% of the extra N accumulated above the threshold [N]_i. All growth requirements for N were satisfied before nitrate accumulated.     

In vitro plant regeneration of Ilex paraguariensis (aquifoliaceae)

Summary Nodal segments as well as shoot tips and apical meristems of 2-yr-old “maté” plants (Ilex paraguariensis St. Hil.) were cultured in vitro to establish micropropagation systems. Maximum shoot regeneration was achieved when nodal segments were cultured with 1/4 Murashige and Skoog (MS) medium with 3% sucrose. We induced roots to differentiate by transferring the regenerated shoots onto the same medium, solidified with 2.5 g Phytagel per 1 and supplemented with indole-3-butyric acid (7.4 μM) and finally transferring shoots to 1/4 MS medium with 3% sucrose and lacking growth regulators. Plants were successfully established in soil.     

Callus induction and <Emphasis Type="Italic">de novo</Emphasis> regeneration from callus in Guar (<Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>)

Guar (Cyamopsis tetragonoloba L. Taub) is a drought tolerant and multipurpose grain legume cash crop grown primarily under rainfed conditions in several countries. The effect of various growth regulators and their combinations on a variety of explants, namely the embryo, cotyledons, cotyledonary nodes, shoot tip and hypocotyle, has been studied and an efficient system for callus induction and regeneration from callus has been developed. It was established that Murashige and Skoogs culture medium containing 2,4-dichlorophenoxyacetic acid (10.0M) in combination with 6-benzylaminopurine (5.0M) with embryo or cotyledon explants is most suitable for induction of green and friable morphogenic callus, with a range of 82.5–95% of cultured explants responding to callus induction. Efficient de novo shoot regeneration was achieved by culturing the callus obtained on this medium on Murashige and Skoogs medium containing 1-naphthlenacetic acid (13.0M) in combination with 6-benzylaminopurine (5.0M) with a range of 82.1–88.4% of callus clumps producing 20–25 shoots. In vitro rooting of cultured shoots was obtained on half-salt concentration of Murashige and Skoogs culture medium supplied with indole-3-butyric acid (5.0M) on which 82–90% of cultured shoots produced healthy roots. The in vitro regenerated plants were grown to pod setting and subsequent maturity under greenhouse conditions.     

Effect of air desiccation and salt stress factors on in vitro regeneration of rice (Oryza sativa L.)

Enhancement of callus induction and its regeneration efficiency through in vitro techniques has been optimized for 2 abiotic stresses (salt and air desiccation) using 3 rice genotypes viz. BR10, BRRI dhan32 and BRRI dhan47. The highest frequency of callus induction was obtained for BRRI dhan32 (64.44%) in MS medium supplemented with 2, 4-D (2.5 mgL⁻¹) and Kin (1.0 mgL⁻¹). Different concentrations of NaCl (2.9, 5.9, 8.8 and 11.7 gL⁻¹) were used and its effect was recorded on the basis of viability of calli (VC), relative growth rate (RGR), tolerance index (TI) and relative water content (RWC). It was observed that in all cases BRRI dhan47 showed highest performance on tolerance to VC (45.33%), RGR (1.03%), TI (0.20%) and RWC (10.23%) with 11.7 gL⁻¹ NaCl. Plant regeneration capability was recorded after partial air desiccation pretreatment to calli for 15, 30, 45 and 60 h. In this case BRRI dhan32 gave maximum number of regeneration (76.19%) when 4 weeks old calli were desiccated for 45 h. It was observed that air desiccation was 2-3 folds more effective for enhancing green plantlet regeneration compared to controls. Furthermore, desiccated calli also showed the better capability to survive in NaCl induced abiotic stress; and gave 1.9 fold (88.80%) increased regeneration in 11.7 gL⁻¹ salt level for BRRI dhan47. Analysis of variance (ANOVA) showed that the genotypes, air desiccation and NaCl had significant effect on plant regeneration at P < 0.01.     

An efficient in vitro method for mass propagation of Potentilia potanii wolf

Summary This study reports a method for high-frequency shoot organogenesis and plant establishment of Potentilla potaninii Wolf. Hypocotyl and cotyledon explants of P. potaninii were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of benzyladenine (BA) and &#x03B1;-naphthaleneacetic acid (NAA) to induce adventitious shoot formation for micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants grown on MS medium supplemented with 5.0 mgl^&#x2212;1 BA and 1.0 mgl^&#x2212;1 NAA. The regenerated shoots rooted most efficiently on half-strength MS medium supplemented with 1.0 mgl^&#x2212;1 NAA and 0.5 mgl^&#x2212;1 indole-3-acetic acid or indole-3-butyric acid. The acclimatized plants with normal morphology and growth characters flowered and set seeds in the following year.     

Plant regeneration from protocorm-derived callus of <Emphasis Type="Italic">Cypripedium formosanum</Emphasis>

Summary Totipotent callus of Cypripedium formosanum, an endangered slipper orchid species, was induced from seed-derived protocorm segments on a quarter-strength Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and 4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thidiazuron). This callus proliferated well and was maintained by subculturing on the same medium. On average, 13 protocorm-like bodies could be obtained from a piece of 4 mm callus after being transferred to the medium with 4.44 μM N⁶-benzyladenine after 8 wk of culture. The regenerated protocorm-like bodies formed shoots and roots on medium containing 1 g l⁻¹ activated charcoal and 20 g l⁻¹ potato homogenate. After 24 wk of culture on this medium, well-developed plantlets ready for potting were established.     

Somatic embryogenesis and plant regeneration from protoplasts isolated from embryogenic cell suspensions of wheat (Triticum aestivum L.)

We describe the early formation of somatic embryos followed by plant regeneration from protoplasts isolated from an embryogenic wheat cell suspension, which was initiated from small granular (0.2 to 1 mm in size) embryogenic calli. These granular calli formed embryogenic cell suspensions within 20 days in liquid culture, and were selected gradually from young inflorescence-derived nodular embryogenic calli of the winter wheat cv. Kehong 1041. The division frequency of protoplasts was 11 to 16%, and the frequency of differentiation into plants was about 0.001% (number of plants formed divided by the total number of protoplasts plated). About 20% of somatic embryos present in the culture formed directly from protoplast-derived cells within 15 days of cultures.     

Enhancing rice callus regeneration by extracellular products of Tolypothrix tenuis (Cyanobacteria)

Cyanobacteria produce a wide array of substances with activity in many biological systems. The aim of the present research was to compare the effect of differently treated extracellular products (EP) from Tolypothrix tenuis (Cyanobacteria) on rice plantlet regeneration as well as on the pigments, protein, total free porphyrin contents, and 5-aminolevulinate dehydratase (ALA-D) activity in rice callus during differentiation. Rice embryo calli were regenerated in Murashige & Skoog medium supplemented with EP with protein (EPp) or without protein (EPnp) or autoclaved (EPa), as well as with benzyladenine (BA) and benzyladenine + naphthaleneacetic acid (BA + NAA). At day 75, calli percentage regeneration were: EPnp (84%), EPp (58%), BA + NAA (45%), BA (44%), EPa (40%). The same trend was found for chlorophyll a, b, total and carotenoid contents. Protein content in BA, BA + NAA and EPnp treatments was 35% higher than in EPp and EPa. Total free porphyrin was similar in all treatments. ALA-D activity in BA, EPp and EPa treatments was 28% higher than in BA + NAA and EPnp. The extracellular bioactive substance(s) from T. tenuis would contain a mixture of thermolabile plant growth regulators that replaced and improved the effects of synthetic plant growth regulators on rice callus organogenesis. Calli were rhizogenic in all the regeneration media tested. The pigment content of the calli was related to percentage regeneration but not to the total free porphyrin and ALA-D activity.