中林美荷杨不定芽分化的因素分析与植株再生

对中林美荷杨进行组织培养,采用不同外植体为组织培养材料,以6-BA和NAA或IBA不同激素组合,比较1/2MS与MS不同盐浓度培养基诱导不定芽产生的情况。结果表明叶柄不定芽诱导优于茎段和叶片,茎段形成层不定芽诱导效果也较好。叶柄诱导不定芽的最佳培养基及激素组合是：1/2MS + 6-BA0.5 mg·L^-1+ NAA0.05 mg·L^-1,较MS基本培养基不定芽生长正常,芽诱导率高(76%),增殖倍数达4.7个/叶柄。通过不定芽的继代培养及壮苗、生根,形成完整植株,炼苗移栽成活率高。     

Direct shoot organogenesis and plant regeneration in safflower

Summary  Adventitious shoot buds were induced directly on the adaxial surface of the cotyledons of eight safflower cultivars after 14 d of culture initiation on Murashige and Skoog's (MS) medium supplemented with various levels of 6-benzylaminopurine (BA). Maximum shoot organogenesis of 54.4% with 10.2 shoots per responding cotyledon was obtained with 8.87 μM BA in the cv. S-144. Regenerated shoots were classified into three groups on the basis of their morphological features and were found to be correlated with the levels of BA. The highest number of normal shoots was obtained from 2.2 μM BA treatment. The regenerated shoots of group I (normal shoots) were rooted on half-strength MS medium supplemented with 5.3 μM α-naphthaleneacetic acid, 3% sucrose and 0.8% bacto-agar. Rooted plantlets were successfully transferred to soil and appeared morphologically normal. Histological studies revealed that shoot buds originated adventitiously from subepidermal cells.     

Induction of adventitious shoots in vitro in Campanula carpatica

Efficient protocols have been developed to induce adventitious shoots in different types of explants of Campanula carpatica Jacq. More than five shoots per explant developed on hypocotyls of 5-week-old seedlings after 2 weeks of culture. Hypocotyls produced twice as many shoots as the cotyledons. TDZ proved to be about 6 times more efficient than BA. NAA had to be added to the regeneration medium to obtain the optimal balance of auxin and cytokinin to induce shoot regeneration. Significant differences were noted between different growth regulator concentrations in their effects on shoot organogenesis. BA induced double the number of callus clumps as TDZ. Incubation of explants in the dark produced about 6 shoots per explant while those in the light produced about 2 shoots per explant. Explants derived from 5-week-old seedlings were five times more regenerative compared to those derived from 15-week-old seedlings. Explants from cv. White Uniform were more organogenic than those from cv. Blue Clip. Root segments were also found to form shoots when treated with CPPU.     

Comparative adventitious shoot induction in kentucky coffeetree root and petiole explants treated with thidiazuron and benzylaminopurine

Summary Adventitious shoot induction and elongation was compared between root and petiole explants of Kentucky coffeetree (Gymnocladus dioicus L.) explants treated with a factorial combination of benzylaminopurine (BA) and thidiazuron (TDZ). Petiole explants initiated more adventitious shoots compared to root explants. Up to 83% of petiole explants initiated shoots compared to 67% of root explants. Maximal shoot induction was approximately 12 or five shoots per responding explant for petiole and root explants, respectively. For both explant types, TDZ was more effective than BA for shoot induction. There was an interaction between BA and TDZ on shoot induction in petiole explants, with the greatest percentage of explants forming shoots and the highest number of shoots initiated on the combination of 0.5 μM TDZ plus 10μM BA and 1.0μM TDZ plus 5 or 10 μM BA. In contrast, increasing concentrations of BA inhibited shoot initiation in root explants with and without TDZ. While BA inhibited shoot initiation in root explants, it promoted shoot initiation in petiole explants. In contrast, TDZ was equally effective at inducing shoots in root and petiole explants. This suggests that root and petiole explants of Kentucky coffeetree could be a useful model system for studying the differences, in apparent mode of action between TDZ and BA on adventitious shoot initiation.     

High Frequency of Shoot Regeneration from Hypocotyls and Stem Segments of Antirrhinum majus (Snapdragon)

A broadly applicable direct shoot regeneration method from hypocotyls and stem explants has been developed for six cultivars of Antirrhinum majus L. In order to establish a stable and high frequency of shoot regeneration system, leaves, hypocotyls and stem explants of six cultivars were tested with 72 combinations of auxin (naphthaleneacetic acid (NAA) or 3-indoleacetic acid (IAA)) and cytokinin (6-benzylaminopurine (BA) or zeatin (Z)). A few adventitious shoots were directly regenerated from hypocotyl segments of cv. Orchid on MS medium with NAA + BA, IAA + BA, NAA + Z and IAA + Z. High frequency of direct shoot regeneration was obtained from hypocotyl segments on MS medium with 0.05, 0.1 or 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z and 0.5 mg l⁻¹ IAA + 2 mg l⁻¹ Z. Finally, stable and high frequency (92–100%) of shoot regeneration with more than 10 adventitious shoots per explant was achieved from the hypocotyls and stem explants of all six cultivars on MS medium with 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z. The shoots emerged directly from the hypocotyls and stem segments 4 weeks after culture initiation.     

Micropropagation of three berry fruit species using nodal segments from field-grown plants

Plant micropropagation of blueberry (Vaccinium corymbosum L. cv. Berkeley), blackberry (Rubus sp. cv. Smoothstem) and raspberry (Rubus idaeus L. cv. Gradina) was carried out from nodal segments of adult field‐grown plants. Hardwood and softwood cuttings were studied as explant sources. The cultures successfully established were softwood from all three species, and hardwood only from blueberry. Shoot‐bud establishment from blueberry was achieved by culturing explants in WPM salts with MS vitamins for 15 days, and then 30 days in the same medium with 18 mM Zeatin. The best results of multiplication were obtained in the same medium with 25 mM 2iP. For blackberry, shoot‐bud establishment was achieved by culturing explants in MS medium for 15 days, and then in the same medium with 4 mM BA and 0.25 mM IBA. This medium was also the best for blackberry multiplication. Raspberry explants (cvs Gradina and Willamette) were cultured in MS medium for 15 days and then transferred to MS medium supplemented with 4 μm BA and 0.25 mM IBA. After 30 days of culture, only ‘Gradina’ explants survived, from which shoot‐bud establishment was obtained in a modified MS medium (Anderson's macronutrients except calcium, with Sequestrene as the iron source) with the same growth regulators. Multiplication was achieved by subculturing explants in the same medium either with 4 mM BA plus 0.25 mM IBA or with 8 mM BA plus 0.25 mM IBA. Shoots of at least 1 cm in length from all species were rooted ex vitro in a mixture of peat and Perlite (1:1, v/v) in a mist chamber, and 100% of rooting plants were acclimated. Bacterial, fungal and viral diseases were detected in stock plants, while tests carried out in both shoots and regenerated plants revealed the absence of any kind of disease.     

Micropropagation of <Emphasis Type="Italic">Scabiosa caucasica</Emphasis> Bieb. Cv. Caucasica blue

Summary Micropropagation of Scabiosa caucasica cv. Caucasica Blue was achieved by culturing, separating axillary and adventitious shoots, or node sectioning on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA). The highest frequency of adventitious shoots regenerated from nodal or internodal explants and leaf blade (with or without petiole) appeared to occur on MS medium with 4.4 and 18 μM BA, respectively. Addition of 0.19 or 1.9 μM α-naphthaleneacetic acid to the BA-containing medium promoted callus formation and reduced shoot organogenesis. During micropropagation, shoot nodal explants derived from in vitro shoots cultured on MS medium supplemented with 4.4 μM BA yielded 8.9 shoots per explant within 40 d after culture initiation.     

In vitro regeneration of Alstroemeria cv. ‘Yellow King’ by direct organogenesis

The common techniques for the in vitro production of Alstroemeria plants are based on rhizomes as explants, which have low multiplication rates and a high risk of carrying viral diseases. To overcome these problems, we developed a protocol for the in vitro regeneration of Alstroemeria cv.‘Yellow King’, by testing for shoot induction several explant sources (leaf, stem apices, rhizomes and immature inflorescence apices), temperature and light/dark regimes, hormone and salt concentrations. For shoot multiplication and rooting, several hormone concentrations were tested. We found that only the young floral apices produced adventitious shoots by direct organogenesis. The highest shoot induction rate (10.4 shoots per explant) was obtained by incubation in the dark for 15 days at 8 °C followed by 15 days at 25 °C and a 16-h/8-h light/dark regime, on a Murashige and Skoog (1962) liquid medium at 50% of the salt concentration, supplemented with 2.5 mg l⁻¹ KIN, 1.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, using a piece filter paper to support the explant. The highest shoot multiplication rate (9 shoots per explant) was obtained on a liquid MS medium at full strength supplemented only with BA at 1.0 mg l⁻¹. In vitro rooting of shoots was induced also on a liquid MS medium, either with or without plant hormones.     

Effects of cytokinin types and their concentrations on shoot proliferation and hyperhydricity in in vitro pear cultivar shoots

This study was made to clarify the effects of cytokinin type and their concentrations on shoot proliferation and hyperhydricity in in vitro Pyrus pyrifolia N. (`Hosui' and `Kosui') shoots. The shoots were subcultured in a woody plant medium supplemented with 0.5 M 3-indolyl-butyric acid, 3% (w/v) sorbitol, 0.8% (w/v) agar, and with cytokinins kinetin, 6-benzylaminopurine (BA), N-(2-chloro-4-pyridyl)-N9-phenylurea (CPPU), 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) added at concentrations 0.44, 4.40, 11.0 and 44.0 M. The highest number of shoots was induced by adding BA at concentration 11.0 M, then by 4.4 M BA, in both cultivars. TDZ and CPPU caused greater hyperhydricity in cultured explants than BA and kinetin. `Kosui' was more susceptible to hyperhydricity compared with `Hosui'.     

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

Summary A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.     

Uptake and metabolism of N6-benzyladenine and 1-naphthaleneacetic acid and dynamics of indole-3-acetic acid and cytokinins in two callus lines of Actinidia deliciosa differing in growth and shoot organogenesis

Uptake, metabolism and accumulation of N⁶‐benzyladenine (BA) and 1‐naphthaleneacetic acid (NAA) as well as changes in endogenous indole‐3‐acetic acid (IAA) and several isoprenoid‐type cytokinins (Cks) were characterized in two callus lines of Actinidia deliciosa cv. Hayward showing different growth and shoot organogenic responses to exogenously applied 2.7 µM NAA and 4.4 µM BA. Studies were carried out in callus 0, 1, 12, 24 and 48 h after the onset of their fifth subculture on a medium containing [³H]NAA or 8‐[¹⁴C]BA. Kiwifruit callus of line A presented high caulogenic response and lower growth that was positively associated with faster BA uptake, with transient accumulation of BA and isoprenoid‐type Cks, mainly zeatin, exceeding three‐ and four‐fold that of the non‐caulogenic callus, and with values of the BA/NAA ratio exceeding 1, in fact higher than the BA/NAA ratio in the culture medium. The accumulation of BA took place in both callus lines during the first 24 h of subculture and before the re‐initiating of callus proliferation. The higher growth and the low or null caulogenic response shown by line B callus were correlated with faster NAA uptake, with endogenous NAA levels two‐fold higher than in A calli, with higher IAA amounts and with values of the BA/NAA ratio below 1. Moreover, at 48 h free NAA in both kinds of callus reached levels close to those found after 35 days of subculture. Results suggest that temporal accumulation of BA and endogenous Cks is involved in the initiation of cell division leading to callus growth, whereas the maintenance of high NAA and IAA levels are related to the support of long‐term callus development. It also appears that for callus cells to become committed to shoot organogenesis, they must have a concentration of active Cks higher than a threshold value during the first 2 days of culture on fresh medium, while at the same time the concentration of auxins must not exceed a certain maximum.     

Adventitious shoot regeneration from leaf explants of Gypsophila paniculata L.

Adventitious shoots were successfully regenerated from leaf explants of Gypsophila paniculata L. The efficiency of shoot regeneration for cv. Arbel was tested on 18 media based on Murashige and Skoog basal medium containing different concentrations of thidiazuron or 6-benzylaminopurine in combination with naphthaleneacetic acid. Both explant age and that of the cuttings used as leaf donors affected the regeneration efficiency. The highest efficiency of adventitious shoot regeneration was obtained with the oldest leaves originating from the youngest cutting analyzed; on thidiazuron-containing medium, shoots regenerated on average from 67% of the leaves, with an average of seven shoots per explant. This regeneration procedure was suitable for all six commercial cultivars studied. Regenerated shoots elongated, rooted and successfully acclimatized to the greenhouse where they were grown to flowering. Received: 25 July 1998 / Revision received: 11 November 1996 / Accepted: 30 November 1996     

The influence of FeEDDHA in red raspberry cultures during shoot multiplication and adventitious regeneration from leaf explants

We examined the effect of FeEDDHA on multiplication via axillary branching and adventitious shoot regeneration from leaf explants in five red raspberry cultivars. When applied during multiplication, FeEDDHA reduced chlorosis, increased content of chlorophyll and iron but had no effect on the number of side shoots in four of the five cultivars. Addition of FeEDDHA to regeneration medium increased the percentage of regenerating leaves in some cultivars and the number of adventitious shoots in all five cultivars.     

Isoperoxidase and isopolyphenol oxidase spectra in male and female tissues ofActinidia deliciosa in vitro

The isoperoxidase and isopolyphenol oxidase patterns were studied in undifferentiated calli and regenerated shoots and roots of two cultivars ofActinidia deliciosa: one female cv. Hayward and one male cv. Matua. One characteristic, very stable isoperoxidase band (0.85<Rf<0.90) was found only in the male callus, shoots and roots cultured on media supplemented with zeatin (ZEA) or indole-3-butyric acid (IBA). In contrary, one isopolyphenol oxidase band (0.35<Rf<0.40) was typical for male callus and shoots cultured on the medium enriched with ZEA and another one (0.85<Rf<0.90) for male shoots and roots cultured on the medium with IBA. These specific bands had never been found in female cultivar.     

Adventitious shoot formation from leaf explants of Rhododendron

A protocol for high frequency adventitious shoot regeneration adventitious shoot regeneration from leaf explants of Rhododendron spp. has been developed. The highest percentage of regeneration and the greatest number of shoots were obtained when leaf explants were cultured on Anderson's medium containing 4.9 M IBA and 73.8 M 2iP. Genotypic variation was observed for adventitious shoot regeneration potential among the seven cultivars tested. Regeneration frequencies ranged from 0 to 96%. Lodestar had the highest rate of regeneration after 3 months of culture with 96% shoot regeneration and an average of 14 shoots per explant. Regenerated shoots were rooted in soil in about 2 months. This protocol should be useful in applying gene transfer techniques to Rhododendron improvement.Abbreviations IAA 1-H-indole-3-acetic acid - NAA 1-naphthaleneacetic acid - IBA 1-H-indole-3-butyric acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - 2iP N-(3-methyl-2-butenyl)-1-H-purine-6-amine     

Shoot apical meristem: In vitro regeneration and morphogenesis in wheat (Triticum aestivum L.)

Summary We report a less genotype-dependent in vitro regeneration system capable of producing multiple shoot clumps and whole plants in four different wheat genotypes. Shool apical meristems from 7-d-old-seedlings produced axillary and adventitious shoots and somatic embryos on media containing N⁶-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). All four genotypes responded positively to shoot multiplication depending upon media composition. Scanning electron microscopies of cultures showed a proliferating budding state that gave rise to adventitious shoots and somatic embryos on further multiplication. The percentage of relative shoot apical meristem multiplication was 80–90%, and the average number of shoot meristems per multiplied shoot was 40–50 in all genotypes. Among different concentrations of phytohormones, 2 and 4 mgl⁻¹ BA (8.8 and 17.7 μM) in combination with 0.5 mg l⁻¹ 2,4-D (2.26 μM) gave the best results. Actively multiplying shoot clumps were recovered with high frequency among 3-mo.-old cultures. These shoot clumps regenerated normally and produced fertile plants containing viable seeds. This in vitro system might prove useful for the production of transgenic plants of wheat in a relatively genotype-independent manner.     

High frequency adventitious shoot induction and plant regeneration from leaves of statice

An efficient regeneration system for large-scale propagation of statice (Limonium altaica cv. Emille) was developed using leaves from mature plants. Leaf segments (5×5 mm sections) were cultured on Murashige and Skoog's medium supplemented with N⁶-benzyladenine (BA) and thidiazuron (TDZ) individually and in combination with indole-3-acetic acid (IAA) and α-naphthaleneacetic acid (NAA). Prolific direct adventitious shoot regeneration occurred on most of the media. The best response in terms of frequency of shoot regeneration (99.5%) and number of shoots per explant (112 shoots per explant) was observed on medium supplemented with 2.85 μM IAA and 1.14 μM TDZ. The shoots rooted easily on half strength MS medium and MS medium with indole-3-butyric acid. In vitro propagated plants could be transferred to soil with survival rates of more than 95%. This revised version was published online in June 2006 with corrections to the Cover Date.     

New approaches for shoot production and establishment of in vitro root cultures of Cleome rosea Vahl.

Alternative methods for in vitro shoot culture of Cleome rosea, a Brazilian herbaceous species with ornamental value and medicinal potential, were evaluated. A protocol for rapid in vitro multiplication of roots, a valuable source of medicinal compounds, was also developed. Stem explants were cultured in liquid media (continuous immersion and paper bridge), while root explants were cultivated in continuous immersion and on solidified media. The highest numbers of shoots, 20 ± 4.6 shoots/explant, were obtained from stem explants incubated in a continuous immersion system in a liquid medium supplemented with 2.2 μM BA. Root explants cultivated in liquid media produced only hyperhydrous adventitious shoots. However, these explants generated 5.8 ± 0.8 shoots/explant by indirect organogenesis when cultivated on solidified medium supplemented with 2.2 μM BA. In addition, root multiplication was achieved in liquid medium in the presence of α-naphthaleneacetic acid. Adventitious shoots developed on newly formed roots when inoculated on solidified medium supplemented with 2.2 μM BA. Shoot microcuttings developed roots when transferred onto solidified MS medium without growth regulators. Rooted microcuttings were efficiently acclimatized when transferred ex vitro.     

Adventitious shoot regeneration from leaf explants of almond (Prunus dulcis mill.)

Summary A method has been developed to facilitate shoot formation from leaf explants of almond. Leaves were dissected from micropropagated shoot cultures of the commercial cultivars Nonpareil and Ne Plus Ultra, and sections incubated on Almehdi and Parfitt's (1986) basal medium (AP) with varied plant growth-regulator conditions. Three auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA), in combination with two cytokinins, benzylaminopurine (BA) and thidiazuron (TDZ), were tested at various concentrations along with casein hydrolysate (CH) to determine, the conditions most conducive to adventitious shoot regeneration. Response to the tested plant growth-regulator conditions varied with genotype. Of the three auxins tested, NAA and IBA induced adventitious shoots from Ne Plus Ultra explants, but only IBA was effective for Nonpareil. For the cytokinins, shoot development from Ne Plus Ultra occurred in the presence of either BA or TDZ, whereas for Nonpareil only TDZ was effective unless CH was incorporated in the basal medium. The inclusion of CH (0.1% w/v) improved callus morphology, and increased regeneration frequencies for both cultivars. Maximum regeneration frequencies for Ne Plus Ultra (44.4%) and Nonpareil (5.5%) were achieved on AP basal salts supplemented with CH, IBA (9.8 μM), and TDZ at 22.7 and 6.8 μM, respectively.     

Shoot organogenesis and somatic embryogenesis from leaf and shoot explants of <Emphasis Type="Italic">Ochna integerrima</Emphasis> (Lour)

Ochna integerrima is a medicinal and ornamental plant in Southeastern Asia. It has been listed as a rare and endangered species in China. Here we studied the effects of plant growth regulators and their concentrations on the induction of somatic embryogenesis and shoot organogenesis from leaf and shoot explants of O. integerrima for the first time. Cytokinins played a crucial role in somatic embryogenesis and shoot organogenesis. Among them, a higher concentration of thidiazuron (10.0–15.0 μM TDZ) could induce both somatic embryogenesis and adventitious shoot formation whereas low concentrations of TDZ (5.0 μM) could only induce adventitious shoots. However, 6-benzyladenine (BA at 5–15 μM) could only induce adventitious shoots. Shoot explants induced more adventitious shoots and somatic embryos than leaf explants when cultured on medium with the same concentration (5–15 μM) of TDZ or 15 μM BA. Medium containing 0.5 μM α-naphthaleneacetic acid and 8 μM indole-3-butyric acid and 0.1% activated charcoal could induce adventitious roots within 1 month. An efficient mass propagation and regeneration system has been established.