Micropropagation of fastigiate bird cherry (Prunus padus L.) and adventitious shoot formation from leaves

A shoot tip of a mature clone of fastigiate bird cherry (Prunus padus L.) was successfully established in vitro. Culture of shoot tip explants on a Murashige and Skoog (MS) based medium with phloroglucinol (PG) resulted in micropropagation, but the clonal line gradually became hyperhydric on this medium. This problem was overcome using PG- free medium based on either MS or Driver and Kuniyuki Walnut medium (DKW). Heavier cultures with more shoots were obtained on DKW medium with fructose or glucose rather than sucrose or sorbitol. Leaf explants placed on DKW basal medium with benzyladenine (BA) produced adventitious shoots. Addition of 1-naphthaleneacetic acid to media with BA increased regeneration. More leaves produced shoots on medium with sucrose or sorbitol rather than glucose or fructose. Adventitious shoots were excised and micropropagated. Shoots were rooted by insertion into DKW medium with indol-3yl- butyric acid, followed by transfer to hormone-free DKW. PG increased the proportion of shoots that produced adventitious roots.     

Rapid plant regeneration protocol for cluster bean (Cyamopsis tetragonoloba L. Taub.)

Summary

An efficient in vitro regeneration procedure using thidiazuron (TDZ) has been developed to allow high frequency, multiple shoot induction from cotyledonary node explants of cluster bean (Cyamopsis tetragonoloba). Shoot bud induction occurred on Murashige and Skoog (MS) medium after 4 weeks in the presence of TDZ, followed by transfer onto shoot multiplication and elongation media containing MS salts, B5 vitamins, and different combinations of auxins and cytokinins. Multiple shoots were induced at all levels of TDZ in the medium, but the best proliferation capacity occurred at 5 µM TDZ. Combinations of auxins and cytokinins showed a stimulatory effect on shoot multiplication and also on the length of the newly formed shoots. Maximum shoot induction [i.e., the highest number of shoots (16.0 ± 0.94) per explant] was obtained on agar-solidified medium containing 5 µM benzyladenine (BA) with 0.5 µM indole-3-acetic acid (IAA). Rooting of in vitro-regenerated shoots was achieved in ex vitro conditions by a pulse treatment with 300 µM indole-3-butyric acid (IBA) for 15 min. Rooted plantlets were transferred to soil where 70 – 75% attained sexual maturity and produced viable seeds under greenhouse conditions. The present regeneration system is efficient and can be used in various in vitro manipulation studies.     

Effect of leaf excision time and age,BA concentration and dark treatments on in vitro shoot regeneration of M.26 apple rootstock

SUMMARY

The effect of benzyladenine (BA) concentrations both during the last proliferating subculture before regeneration (10-222 and, 444 µM) and during organogenesis (11.1 and 22.2 µM), leaf excision time (15 and 30 d from the beginning of the subculture), leafage and dark treatments, on adventitious shoot regeneration of M.26 apple roostock were evaluated. Leaves excised 30 d after the beginning of the last proliferating subculture and grown wkhout BA in thè medium gave the highest percentages of organogenesis, while the number of "regenerated shoots per leaf did not differ significantly among the different BA x leaf excision time combinations. The highest BA concentration (22.2 µM) in the organogeneticmedium produced thehighest percentage of regenerating leaves, with no differences between the lengths and numbers of shoots per regenerating leaf. The first twMjnfurled apical leaves showed a greater regenerative ability than the third and fourth ones, whereas the lengths and numbers of regenerated shoots per leaf were similar. The highest leaf organogenetic ratejyas observed when darkness was imposed at the begirP-ning of the last proliferating subculture and/or at the beginning of the organogenetic phase, but more regenerated shoots per leaf were obtained with darkness provided at the beginning or at the end of the lastproliferating subculture; shoot lengths were similar in all the dark treatments. The great influence onorganogenesis of all the treatments applied in the last proliferating subculture indicates the importance of this stage inpreparing explants for shoot regeneration and thus the possibility of using inductive factors in this phase.     

Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons

An adventitious shoot regeneration and rooting protocol was developed for green ash (Fraxinus pennsylvanica) seedling explants. The best regeneration medium for freshly isolated hypocotyls and cotyledons was Murashige and Skoog (MS) supplemented with 13.3 μM 6-benzylaminopurine (BA) plus 4.5 μM thidiazuron (TDZ), and 22.2 μM BA plus 4.5 μM TDZ, respectively. Seventy-six percent of hypocotyl segments and 24% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 2.7 ± 0.5 and 2.3 ± 1.3, respectively. The effect of in vitro-germinated seedling age on adventitious shoot regeneration from hypocotyl and cotyledon explants was also studied. Results showed that hypocotyl and cotyledon explants from freshly isolated embryos exhibited a higher organogenesis potential than 4–15-day-old explants. Adventitious shoots from hypocotyls and cotyledons were established as proliferating shoot cultures following transfer to MS basal medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. A high rooting percentage (73–90%) was achieved when in vitro shoots were rooted on woody plant medium (WPM) containing 4.9 μM indole-3-butyric acid (IBA) and IAA (0, 2.9, 5.7, or 8.6 μM) with a combination of 10-day dark culture period followed by a 16-h photoperiod. The highest rooting (90%) of adventitious shoots or the number of roots per shoot (3.0 ± 1.0) was obtained on WPM with 4.9 μM IBA plus 5.7 μM IAA. Rooted plants were successfully acclimatized to the greenhouse and 100% survived after overwintering in cold storage. This regeneration system using hypocotyls and cotyledons provides a foundation for Agrobacterium-mediated genetic transformation of F. pennsylvanica for resistance to the emerald ash borer.     

悬铃木下胚轴及叶片再生不定芽的影响因素分析

 以悬铃木的下胚轴和离体叶片为材料, 研究了基因型、植物生长调节剂、光照条件、外植体部位等因素对其不定芽分化的影响。结果表明: 1) 6-BA与IBA的浓度比对下胚轴不定芽的分化影响较大,以6-BA ∶IBA小于20 ∶1较为合适; 而叶片的不定芽再生受基因型的影响显著, 在4种供试材料中, PH2的再生能力最强, 但0.1～1.0 mg·L^-1 TDZ不利于其不定芽再生。2) 黑暗培养对下胚轴的不定芽分化有一定的促进作用, 但不利于叶片的不定芽再生, 50～100 lx的弱光有利于叶片的不定芽分化。3) 不同部位的下胚轴切段及叶片的再生能力差异显著, 其中靠近子叶的下胚轴切段和试管苗顶端的2枚叶片再生能力较强。此外, 刻伤叶片的再生效果明显优于叶片切块, 说明悬铃木的叶片与下胚轴的离体培养存在明显的生物全息现象。     

The effects of explant and cytokinin type on regeneration of Prunus microcarpa

We investigated in vitro regeneration ability of Prunus microcarpa subsp. tortusa using various explants (root, cotyledon and hypocotyl pieces) and cytokinins [benzyladenine (BA), meta-Topolin (mT) and thidiazuron (TDZ)]. Sectioned cotyledon, root and hypocotyl pieces of in vitro grown seedlings were cultured on Nas and Read Medium (NRM) containing BA (7.5, 10, 12.5, 15 or 17.5 μM), mT (2.5, 5.0, 7.5, 10 or 12.5 μM) or TDZ (2.5, 5.0, 7.5, 10 or 12.5 μM). As a measurement of morphogenetic reaction, the ratios of regenerating explants and the numbers of primary adventitious shoots per regenerating explant were analyzed. Cotyledon explants exhibited higher regeneration ratios than hypocotyl explants, and the root explants were inappropriate for regeneration. Both BA and mT were effective on shoot regeneration but higher regenerating explant ratios were obtained when BA was used. In comparison with BA and mT, the effect of TDZ on enhancing explant regeneration ability was insignificant. Mean number of adventitious shoot per regenerating explant was between 1 and 4, and regenerating explant ratios were between 0% and 77%. The practical appliacations of the results are discussed.     

金花梨子叶不定梢再生研究

以金花梨花后65 d的成熟胚子叶为外植体,研究了不同培养基、BA浓度、暗培养条件及AgNO3浓度对子叶不定梢再生的影响。结果表明:梨成熟胚子叶在适宜培养条件下能发生大量不定梢,培养15 d为初发生期,25~30d为高峰期,40 d后结束发生。暗培养预处理有利于提高子叶再生频率、再生不定梢数和不定梢质量。在NN69+BA3.0mg/L+NAA 0.2mg／L培养基上,暗培养3周后转光下培养获得了85.2％不定梢再生率,平均再生不定梢4.4个。培养基中加入0~4mg／LAgNO3对梨子叶不定梢再生有促进作用,但浓度大于4mg/L时抑制子叶不定梢再生。     

Regeneration and transformation of the premier UK apple (Malus × pumila Mill.) cultivar Queen Cox

Summary

A number of factors were assessed for their effects on in vitro shoot proliferation and adventitious shoot regeneration. More in vitro leaves of a quality suitable for use in regeneration and transformation experiments were obtained from shoots on DKW proliferation medium compared with MS medium, and also on MS and DKW media containing phloroglucinol. Compared with MS medium, shoot proliferation was greater on MS with halved levels of NH₄NO₃ and KNO₃. Adventitious shoots were hyperhydric on MS-based but not on DKW-based regeneration medium. More adventitious shoots regenerated on media solidified with Sigma Agargel than on media with Sigma Phytagel or Gelcarin (FMC). Viable transformed shoots were recovered on Sigma Agar or Agargel but not Phytagel. Wounding of leaf explants by stabbing with needles, and stabbing combined with scoring with a scalpel, increased the number of calli regenerating, and these methods, as well as solely scoring with a scalpel, increased the number of calli regenerating shoots compared with the control. Combined stabbing and scoring resulted in more calli producing shoots than solely scoring or stabbing. Vortexing leaf explants with silicon carbide whiskers increased the percentage of subsequently formed calli that regenerated shoots compared with the control. Transformed shoots were regenerated following co-cultivation with Agrobacterium tumefaciens EHA101 harbouring the binary vector pSCV1.6 (with selectable marker gene npt II and GUS reporter gene uid A). The number of transformed shoots as a percentage of explants varied from 0.5% to 2.2%. Molecular analysis of the four extant transformed lines confirmed integration of the transgene and indicated that in three lines there was one integration site, and in one line there were four sites of integration.     

Clonal propagation of Poncirus trifoliate through culture of shoot primordia

Summary

In Poncirus trifoliate, a highly efficient clonal propagation system for the culture of shoot primordia was devised. Shoot primordia were induced at the base of hypocotyl tissue cultured on MS medium supplemented with 44.4 µM BA, 3% sucrose and 0.8% agar. In MS liquid medium (44.4 µM BA, 3% sucrose) on a rotary shaker at two revolutions per minute, shoot primordia of Poncirus grew in size and number. Plant regeneration occurred on MS solid medium. Frequency of regeneration was highest on MS basal medium containing 3% sucrose and 0.8% agar. About 75 shoot buds regenerated from one shoot primordium. Histological observations showed that shoot buds arose from cells in the hypodermal layers of the shoot primordium. The shoot bud developed a vascular system, which became connected to the shoot primordium tissue. Regenerated shoots rooted on 1/2 MS basal medium or 1/2 MS medium supplemented with 0.5 or 5.0 µM IBA. These rooted shoots were acclimatized easily under intermittent mist.     

Adventitious shoot regeneration from in vitro leaves of adult Camellia reticulata

Experiments were conducted with Camellia reticulata cv. Captain Rawes to develop an adventitious regeneration system. Leaves were taken from axillary shoot proliferation cultures in WPM medium that had been established from mature trees. They were sectioned, and then plated in Petri dishes on media containing various combinations of cyto- kinins and auxins; the best response was induced by 2 mg I“1 BA + 1 mg I-1 IBA. The shoots obtained were multiplied by axillary branching on WPM + 2 mg I-1 BA + 2 mg I-1 Z + 2 mg I-12iP + 0.01 mg I-1 IBA. There was no significant difference in multiplication coefficient (the product of the proportion of explants forming axillary shoots and the mean number of new segments per explant) between shoots of adventitious and axillary origin, but there was between the various types of explant used in the multiplication stage: shoot tips (ST1) and nodal segments (ns) of harvested shoots longer than 14- 15 mm, and whole harvested shoots 7-10 mm long (ST2). The best results were achieved with ns and ST2 explants. Shoots of adventitious origin rooted very poorly in comparison with those of axillary origin under the same culture conditions.     

In vitro propagation of three endangered cactus species

We tested the feasibility of in vitro culture techniques for the propagation of the three endangered cacti species Escobaria minima (Baird) D. Hunt, Mammillaria pectinifera (Ruempler) F.A.C. Weber and Pelecyphora aselliformis Ehrenberg. Twenty-five MS-based proliferation media were tested in preliminary experiments, with different combinations of the auxin NAA and either of the cytokinins BA, kinetin or TDZ. TDZ induced a good proliferation rate, albeit associated with abundant callus formation and hyperhydricity of axillary shoots. A high multiplication rate combined with good quality proliferated shoots and little or no callus induction was observed on media containing BA for E. minima and M. pectinifera and on a medium containing kinetin for P. aselliformis. These results were also confirmed in subsequent experiments in which different explants (shoot tips, bases and longitudinal sections) were used. Micropropagated plantlets were successfully restored to the field, where they reached the flowering stage. Plantlet regeneration of M. pectinifera and P. aselliformis from callus induced on media containing TDZ, but not 2,4-D, was also achieved.     

‘艾西丝’南瓜子叶的离体培养

用‘艾西丝’南瓜子叶作外植体离体培养,成功地建立了一套子叶高频率诱导再生芽的程序。在ＭＳ＋ＢＡ ４．０ｍｇ／Ｌ＋ＩＡＡ０．４ｍｇ／Ｌ的培养基上,以４ｄ苗龄子叶基部切块为外植体,其不定芽的诱导率最高,达５０％,在ＭＳ＋ＢＡ０．２５－０．５ｍｇ／Ｌ的培养基上继代,增殖培养效果较好,在无激素的１／２ＭＳ培养基上最易生根。     

欧洲甜樱桃幼胚子叶离体培养再生植株研究

 以欧洲甜樱桃（Prunus avium L.）幼胚子叶为试材,研究了子叶不同发育时期、BA和NAA配比、品种基因型和培养条件等对幼胚子叶离体再生不定芽的影响。结果表明,以MS为基本培养基,附加BA 2.0 mg·L^-1＋NAA 0.2 mg·L^-1,用PF（子叶的长度/胚的长度×100）﹦50～80发育阶段的子叶,那翁幼胚子叶再生不定芽效果最好,再生率最高可达79.2﹪。暗培养2周后再转入光照下培养,对欧洲甜樱桃子叶再生不定芽具有一定的促进作用。欧洲甜樱桃其他品种的子叶再生率,雷尼尔为72.2﹪,先锋为61.1﹪,拉宾斯为33.3﹪。子叶再生不定芽全部发生于子叶的正面近胚芽端切口处,表现出明显的极性效应。     

Plant regeneration of an endangered medicinal plant Hydrastis canadensis L.

Goldenseal (Hydrastis canadensis L.) is an endangered medicinal plant used to treat sore eyes and mouths, cold and flu and also as a dye. The objective of this study was to develop an efficient in vitro propagation protocol for goldenseal. Significantly more shoots (26 shoots per leaf explants) were induced on a medium containing 2.5 μM thidiazuron (TDZ) and 5.0 μM 1-naphthaleneacetic acid (NAA) than any other treatment. Sub-culturing regenerated shoots on a medium with 5.0 μM 6-benzylaminopurine (BA) induced the maximum rate of shoot multiplication. Growth of the regenerated shoots in a temporary immersion bioreactor resulted in significant increases in biomass, shoot height and shoot multiplication. The regenerated shoots from the temporary immersion bioreactor formed roots when transferred onto a medium with 1.0–2.0 μM indole-3-butyric acid (IBA). Regenerated whole plantlets were acclimatized and maintained in standard greenhouse conditions for further growth. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of this rare, medicinally important species.     

Micropropagation and accumulation of essential oils in wild sage (Salvia fruticosa Mill.)

A protocol for in vitro propagation of the wild three-lobed sage (Salvia fruticosa Mill.) (Synonym, Salvia triloba L.) was developed. Shoot tips were excised from in vitro seedlings and established on MS, Nitch and Nitch (NN), or B5 medium. For shoot proliferation, in vitro nodal and apical explants were cultured on MS medium containing 0.25–2 μM 6-benzylaminopurine (BA), 6-furfurylaminopurine (kinetin), or thidiazuron (TDZ). Proliferated microshoots were rooted on MS medium supplemented with 2.7–11.4 μM indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), or α-naphthaleneacetic acid (NAA). Results indicated that shoots established at 100% regardless of media type, however, shoot height, nodes per shoot, and leaf number were highest for explants established on MS medium compared to NN or B5. Number and height of proliferated shoots, nodes per shoot, and leaf number were highest for nodal explants cultured on a medium containing 0.75 μM BA. Microshoots cultured on a medium supplemented with 2.7 μM IBA exhibited the highest rooting percentage compared to those cultured with IAA or NAA. Essential oil composition in microshoots and shoots of greenhouse-grown plants was determined by gas chromatography/mass spectrometry. The major essential oils detected in both plant materials were α-pinene, 1,8-cineole, camphor, and borneol. No α-thujone or β-thujone was detected. The content of essential oils, camphor, and borneol were higher in the microshoots than in shoots of greenhouse-grown plants.     

不同培养条件对‘丰香’草莓离体叶片再生的影响

 以草莓品种‘丰香’离体叶片为外植体, 探讨了基本培养基、不同细胞分裂素、暗培养、硝酸银浓度以及不同植物生长调节剂组合对不定芽再生的影响。结果表明, 基本培养基中以MS 最为适合,WPM、QL 、AS 培养基均不利于不定芽的再生, 而TDZ 的诱导效果好于BA。以MS 基本培养基附加TDZ2.0 mg·L - 1和IBA 0.8 mg·L ^-1可以使‘丰香’叶片不定芽的再生率高达72.33 % , 平均每叶再生芽5.59个。暗培养14 d 可以将‘丰香’叶片的不定芽再生率提高到90.09 %。硝酸银对于提高‘丰香’叶片的不定芽再生没有明显效果, 但在一定程度上改变了细胞分化的方向。     

Silver nitrate and adenine sulphate induced high regeneration frequency in the recalcitrant plant Cosmos bipinnatus using cotyledon explants

Plant regeneration ability was studied in the medicinal-ornamental plant, Cosmos bipinnatus ‘Sonata white’, which is a dicotyledonous recalcitrant plant to shoot induction. Cotyledons were used as sources of explants to investigate plant regeneration. High frequency of direct shoot induction was obtained when BA (5 mg/l) and AgNO₃ (5 mg/l) were used in combination with 20 mg/l adenine sulphate (73.8%) in Murashige and Skoog (MS) medium. The highest shoot number per explant (5.7) was induced on MS medium supplemented with 5 mg/l BA, 5 mg/l AgNO₃, and 40 mg/l adenine. Eight week-old shoots were transferred to root induction media containing MS and half-strength MS medium with different concentration of IBA. The highest rate of root induction (70.8%) was obtained on half-strength MS medium with 1.5 mg/l IBA within four weeks. The plantlets were transferred to pot and kept in the greenhouse condition. Seventy percent of the plantlets successfully acclimatised.

Abbreviations: BA, 6-benzylaminopurine; IBA, Indole-3-butyric acid; MS, Murashige and Skoog; PGRs, plant growth regulators.     

秋水仙素诱导樱桃矮化砧木'吉塞拉6号'获得六倍体再生植株

 以三倍体樱桃矮化砧木'吉塞拉6号'（Prunus ceransus × P. canescens）的离体叶片为外植体,采用秋水仙素诱导处理再生出六倍体植株。将外植体首先在加有秋水仙素（50 mg. L-1）、生长素（IBA 0.5 mg. L-1）和细胞分裂素（BA 5.0 mg. L-1）的改良WPM液体培养基中培养5 d,再转移到不含秋水仙素（其它成分相同）的固体培养基上继续培养56 d,再生出形态变异明显的新梢。采用流式细胞仪鉴定染色体倍性,确定其为六倍体新梢。六倍体植株与三倍体的'吉塞拉6号'植株形态学上有明显差异。六倍体的试管苗已在大田移栽成活,并已成功高接在甜樱桃大树上。     

Direct and callus-mediated regeneration of Curcuma soloensis Valeton (Zingiberaceae) and ex vitro performance of regenerated plants

Protocols are outlined for the regeneration of Curcuma soloensis, an attractive tropical ornamental plant, from young vegetative bud explants. We used both direct and callus-mediated regeneration techniques to produce material suitable for mass propagation and the development of transgenic plants. During direct plantlet propagation, the presence of thidiazuron (TDZ) in the growing medium induced more than three times as many shoots as 6-benzylaminopurine (BA), with a mean of 18.7 shoots per explant on MS medium containing 2.5 μM TDZ compared to 5.0 shoots with 40 μM BA. Subsequently, the shoots rooted readily on MS basal medium that was free of plant growth regulators. During indirect plantlet regeneration, TDZ combined with BA and 2,4-dichlorophenoxyacetic acid (2,4-D) had significant effects on embryogenic callus induction and multiplication. The frequency of callus formation was 91.1% for explants cultured on MS basal medium supplemented with 2.5 μM TDZ, 2.0 μM BA and 1.2 μM 2,4-D. On average 7.1 shoots were produced per callus mass cultured on MS medium supplemented with 2.5 μM TDZ, 9.0 μM BA and 1.2 μM naphthaleneacetic acid (NAA). Regenerated shoots were transferred to MS medium supplemented with 2.5 μM TDZ, to produce multiple shoots. In vitro cultured plantlets readily acclimatized to greenhouse conditions, showing 100% survival rates in a sphagnum, perlite and sand (1:1:1) medium. These plants were transplanted into pots or planted in the field. The ex vitro acclimated plants grew vigorously and produced showy inflorescences 5–6 months after planting. The high-frequency of shoot multiplication and rapid flowering of tissue-cultured plants indicate that C. soloensis has great potential in the floricultural market.     

High frequency shoot regeneration from cotyledon explants of cucumber via organogenesis

Organogenic callus induction and high frequency shoot regeneration were achieved from cotyledon explants of cucumber. About 86.2% of cotyledon explants derived from 5-day-old in vitro raised seedlings produced green, compact nodular organogenic callus in MS medium containing NAA (2.69 μM) and BA (4.44 μM) after two successive transfers at 20 days interval. Adventitious shoots were produced from the organogenic callus when it was transferred to MS medium supplemented with NAA (1.34 μM), BA (8.88 μM), zeatin (0.91 μM) and l-glutamine (136.85 μM) with shoot induction frequency of 75.6%. Shoot proliferation occurred when callus with emerging shoots was transferred in the same medium at an interval of 20 days. Shoots (1.0 cm length) were excised from callus and were elongated in MS medium fortified with GA₃ (1.44 μM) and BA (4.44 μM). The elongated shoots were rooted in MS medium supplemented with IBA (3.42 μM) and BA (4.44 μM). Rooted plants were acclimatized in green-house and subsequently established in soil with a survival rate of 80%. This protocol yielded an average of 35 shoots per cotyledon explant in a culture duration of 120–140 days.