High-frequency plant regeneration via somatic embryogenesis and organogenesis and in vitro flowering of regenerated plantlets in Panax ginseng

 The morphogenesis ability of light yellowish globular callus derived from cotyledons of mature zygotic embryos of Panax ginseng was investigated. The optimal media for somatic embryogenesis and shoot organogenesis were MS medium containing 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid, 0.1 mg l^–1 6-benzyladenine (BA), and 500 mg l^–1 lactoalbumin hydrolysate, and SH medium supplemented with 0.5 mg l^–1 α-naphthaleneacetic acid, 0.1 mg l^–1 BA, and 500 mg l^–1casein hydrolysate. The influences of glucose, mannose, fructose, and sorbose in the media on somatic embryogenesis and shoot organogenesis were revealed as differences in the numbers of somatic embryos and adventitious shoots per gram of morphogenic callus. The best regeneration of somatic embryos was obtained on medium containing glucose, with a mean of 8.7 somatic embryos per gram of callus. The best regeneration of shoots was observed on medium containing fructose, with an average of 12.2 adventitious shoots per gram of callus. Of the somatic embryos 95% were converted into regenerated plantlets, and 100% of adventitious shoots rooted to form regenerated plantlets. Regenerated plants were successfully established in soil. Flowering was observed in 5.7% of the regenerated plants derived from shoot organogenesis and in 1.4% of the regenerated plants derived from somatic embryogenesis. Received: 1 December 1998 / Revision received: 13 September 1999 / Accepted: 20 September 1999     

Plant regeneration from suspension cultures of Hosta sieboldiana

Systems for establishing suspension cultures and for inducing plant regeneration from these cultures for the Liliaceous ornamental plant, Hosta sieboldiana (Lodd.) Engl. have been developed. Pale-yellow and nodular calluses were induced from more than 20% of scape segments on MS medium containing 1 mg l^–1 picloram (PIC), 30 g l^–1 sucrose, and 2 g l^–1 gellan gum. Upon transfer of calluses to the same medium lacking gellan gum, stably-growing suspension cultures were established after 1 month. Suspension cell clusters regenerated a large number of adventitious shoots following transfer to MS media containing 0.1 mg l^–1 NAA in combination with either BA or TDZ. Over 20 shoots per 0.3 g FW of cell clusters were obtained on media containing 0.1 mg l^–1 NAA and either 1 or 5 mg l^–1 TDZ. Shoots rooted easily on plant growth regulator (PGR)-free MS medium, and plantlets were successfully transferred to soil. Plants showed no visible morphological alterations and maintained the diploid level as indicated by flow cytometric analysis.     

In vitro adventitious shoot formation from leaf cultures of Clerodendrum inerme (L) Gaertn.

In vitro adventitious shoots (about 28) of Clerodendrum inerme were regenerated from leaf segments on MS medium containing BA (4 mg/L). These shoots developed directly from the leaf explants without callusing after 5 weeks. Leaf explant when cultured in MS medium containing BA (2 mg/L) and NAA (0.5 mg/L) developed compact callus that became nodular and regenerated shoots (about 50) after 5 weeks. The in vitro developed shoots were rooted in MS medium supplemented with IAA (2 mg/L). The hardened plantlets were successfully established in the field with 90% survival.     

A simple and efficient plant regeneration system for leaf protoplasts ofNierembergia repens by inducing single shoots on the microcolonies

A simple and efficient protocol for plant regeneration from mesophyll protoplasts ofNierembergia repens is described. The protoplasts divided in modified half-strength MS (/12 MS) medium containing benzylaminopurine (BA) and a-naphthaleneacetic acid (NAA) and formed visible colonies after 2 weeks which produced single adventitious shoots 4 weeks later. Plating efficiency (11.2%), percent colony formation (0.84%), and the number of shoot-forming colonies (368/dish) were highest in /12 MS containing 0.1 mg/l BA and 0.05 mg/l NAA. However, the percentage of colonies with shoot formation was highest (31.8%) in /12 containing 0.05 mg/l BA and 0.01 mg/l NAA. Almost all of the remaining colonies (97.5%) also regenerated shoots upon transfer onto MS medium containing 0.05 mg/l BA. The shoots with 2–3 leaves readily rooted 3–5 days after insertion in /12MS lacking plant growth regulators. Regenerated plantlets were easily established in soil 50 days after protoplast isolation. All the regenerants were normal and possessed diploid chromosome numbers.Abbreviations BA Benzylaminopurine - FDA fluorescein diacetate - MES 2-(Morpholino)ethanesulfonic acid - MS Murashige and Skoog (1962) medium - /12MS half strength MS - NAA -naphthaleneacetic acid - PGR plant growth regulator     

Rapid micropropagation system in vitro and antioxidant activity of Scabiosa tschiliensis Grunning

A rapid micropropagation system for Scabiosa tschiliensis Grunning, an ethnic medicinal plant, has been developed. Calluses were induced from leaf and petiole explants on Murashige and Skoog (MS) medium supplemented with 2.0 mg l^?1 thidiazuron and 0.5 mg l^?1 2,4-dicholorophenoxyacetic acid. In this medium, callus induction rate was about 94.05 %. Adventitious shoots developed from leaf (86.30 %) and petiole (83.33 %) calluses when cultured on MS medium containing 4.0 or 2.0 mg l^?1 N⁶-benzyladenine (BA), respectively. Up to 73.85 % of the regenerated shoots formed complete plantlets on MS medium supplemented with 2.0 mg l^?1 indole-3-butyric acid, with an average of 3.25 roots per shoot. Quantitative analysis of flavonoids showed that the phytochemical profiles of calluses and regenerated plants were similar to that of wild-type plants. The 2, 2-diphenyl-1-picrylhydrazyl assay revealed that the flavonoid extracts of calluses, adventitious shoots and wild-type plants had stronger antioxidant activities, the inhibitory concentrations being 23.944, 31.329 and 26.502 μg ml^?1, respectively, where 50 % of DPPH was scavenged (IC₅₀). Results showed that this perennial herb could be used as a potential source of new natural antioxidants.     

Plant regeneration of rose (Rosa hybridia) from embryogenic cell-derived protoplasts

Culture conditions are described for sustained cell division and plant regeneration from protoplasts of rose (Rosa hybrida L. `Sumpath'). Protoplasts were enzymatically isolated from 2-week-old embryogenic cell suspension cultures. Freshly isolated protoplasts were plated as a thin layer onto protoplast culture medium (half-strength 21 Murashige and Skoog's medium containing 60 g l^–1 myo-inositol, 4.4 M BA, and 1.4 M 2,4-D) at a density of 5×10⁴ protoplasts ml^–1. The plating efficiency reached 3.9% after 2 weeks of culture. However, few protoplasts underwent cell division when cultured in protoplast culture medium in which 60 g l^–1 myo-inositol was replaced with the same osmolarity of 90 g l^–1 mannitol, indicating that myo-inositol is essential for sustained cell division of protoplasts. Colonies were formed after 8 weeks of culture at a frequency of 0.2%. Colonies were then transferred to colony culture medium (0.4% Gelrite-solidified protoplast culture medium) and maintained by subculturing at 4-week intervals to form embryogenic calluses. Upon transfer to half-strength MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Somatic embryos were transferred to half-strength MS basal medium containing 48 mg l^–1 ferric ethylenediamine di-(o-hydroxyphenylacetate), where they subsequently developed into plantlets at a frequency of 30.9%. The plantlets had the same chromosome number of 2n=3x=21 as the source plant. They were successfully transplanted to potting soil and grown to maturity in a greenhouse.     

Callus induction and high-frequency plant regeneration from hypocotyl and cotyledon explants of Arctium lappa L.

Summary This study describes a protocol for plant regeneration from cultured seedling explants of Arctium lappa. Hypocotyls and cotyledons of A. lappa were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with 2.0mg l^−1 2,4-dichlorophenoxyacetic acid and 0.5–2.0 mg l^−1 benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0–2.0 mg l^−1 α-naphthaleneacetic acid (NAA) and 0.5–2.0 mg l^−1 BA. These regenerated shoots were rooted on MS medium with 1.0 mg l^−1 indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mgl^−1 NAA. The regenerated plants acclimatized in soil were normal morphologically and in growth characters. They flowered and set seeds in the following year after acclimatization.     

深山含笑的组织培养和快速繁殖

组织培养深山含笑的实验结果表明：利用休眠芽和种子萌发时实生苗的上胚轴及下胚轴为外植体均能诱导出愈伤组织和不定芽,其中无菌实生苗的上胚轴最易诱导出不定芽,无菌实生苗的下胚轴最易诱导出愈伤组织。外体植体在ＭＳ＋１．０－２．０ｍｇ Ｌ＾－１ ２,４－Ｄ培养基上只产生愈伤组织;在ＭＳ＋３．０ｍｇ Ｌ＾－１ ＢＡ＋０．２ｍｇ Ｌ＾－１ ＮＡＡ培养基上产生较多的不定芽和较多的愈伤组织;在ＭＳ＋２．０ｍｇ Ｌ＾     

Callus production from protoplasts of Cyclamen persicum

Cyclamen persicum (Mill.) cv. Sierra Rose protoplasts were isolated from adventitious shoots regenerated in vitro from petiole and leaf explants with yields of 1.3 × 106 protoplasts/g f.wt. of tissue. Protoplasts were embedded within agarose lenses bathed in modified KM8p medium supplemented with 1.0 mg l-1 NAA, 0.1 mg l-1 2,4-D and 0.5 mg l-1 BA. Cell division was observed after 4–5 days. After 6 weeks calli had grown out of the lenses which were transferred to modified MS medium for further callus growth. The most rapid callus growth was on medium containing 0.1 mg l-1 NAA and 10 mg l-1 TDZ. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

大果良种沙棘愈伤组织诱导及植株再生的研究

大果良种沙棘的幼嫩茎尖,茎段外植体接种在MS,1／2MS附加不同浓度配比的IAA,IBA,BA,NAA培养基上可诱导茎尖及腋芽生长,将诱导产生的无性系芽接种在MS或1／2MS附加BA0．3－0．5mg/L,NAA0．05mg/L的培养基上可形成丛生芽,同时在小叶片和嫩茎上诱导产生愈伤组织,继续培养愈伤组织表面形成大量的绿色突起,进一步分化成不定芽,在相同培养基上,不定芽上可直接产生不定芽,从而形成多达数百个的不定芽族,不定芽长至3cm时切下转至1／2MS附加IAA或IBA 0．2mg/L的培养基上可生根而形成完整 的再生植株。     

Evaluation of the in vitro methods for micropropagation of Chondracanthus acicularis (Roth) Fredericq (Gigartinales, Rhodophyta): tissue culture and production of protoplasts

Tissue was cultured and protoplasts isolated from the carrageenophyte Chondracanthus acicularis with the aim of developing micropropagation as an alternative to harvesting raw material from natural beds. Both adventitious shoots and filamentous calluses were induced by tissue culture on medium solidified with 0.4–1 % (w/v) agar. Adventitious shoots were mainly produced from discoid bases while filamentous calluses were mainly induced from basal zones and sub-apical explants. A gradient of the regeneration ability was observed from the top to the bottom of the thallus. The discoid base was the most reactive explant and produced the highest number of adventitious shoots compared to basal zones and sub-apical explants, irrespective of the concentration of agar. Protoplasts were isolated enzymatically from the whole thallus using a combination of cellulase R-10 Onozuka, macerozyme R-10, and crude extract of the gland gut of algivorous molluscs. The highest mean yield of protoplasts (1.2?×?10⁶ protoplasts g^?1 fresh weight) was obtained after 16 h of digestion with an enzyme mixture containing 2 % (w/v) cellulase R-10, 1 % (w/v) macerozyme R-10 Onozuka, 4 % (v/v) crude extract of gut gland of Haliotis, 0.8 M mannitol, 50 mM sodium citrate, 0.3 % (w/v) bovine serum albumin. Depending on the conditions, mean protoplast yields ranged from 3.14?×?10⁵ to 1.2?×?10⁶ protoplasts g^?1 fresh weight. Different factors (storage duration, mannitol, sodium citrate, crude extract of the gland gut of algivorous molluscs) were tested to improve the yield of protoplasts but none has a significantly effect.     

Plant regeneration through organogenesis from callus induced from mature zygotic embryos of loblolly pine

Mature zygotic embryos from three seed sources of loblolly pine were cultured on callus induction medium containing 10 mg l^–1 α-naphthaleneacetic acid, 4 mg l^–1 benzyladenine (BA), 400 mg l^–1 casein hydrolysate, and 400 mg l^–1 glutamine for 6 weeks. Light-yellow, loose, glossy, globular callus was formed, and the highest frequency was 35.7%. The highest differentiation frequency of callus on adventitious bud induction medium was 62.1%. After culture of calli with adventitious buds on elongation medium for 6 weeks, adventitious shoots more than 1.0 cm in height were selected for rooting. On rooting medium supplemented with 0.1 mg l^–1 indole-3-butyric acid, 1 mg l^–1 BA, and 0.5 mg l^–1 gibberellic acid, the highest rooting frequency of adventitious shoots was 46% in a culture period of 6 weeks. Established plants survived following transfer to soil at a frequency of 71%. Received: 14 May 1997 / Revision received: 25 September 1997 / Accepted: 11 October 1997     

Plant Regeneration from Mesophyll Protoplasts of Echinacea Purpurea

An efficient plant regeneration system was developed from isolated protoplasts of Echinacea purpurea L. using an alginate block/liquid culture system. Viable protoplasts could be routinely isolated from young leaves of Echinacea seedlings in an isolation mixture containing 1.0% cellulase Onozuka R-10, 0.5% pectinase and 0.3 mol l^–1 mannitol. Purified protoplasts were embedded in 0.6% Na-alginate block at a density of 1 × 10⁵/ml and cultured in a modified MS medium containing 0.3 mol l^–1 sucrose, 2.5 µmol l^–1 BA and 5.0 µmol l^–1 2,4-D. Cell colonies were observed after 4 weeks of culture, and the protoplast-derived colonies formed calluses when transferred onto 0.25% gellan gum-solidified MS medium supplemented with 1.0 µmol l^–1 BA and 2.0 µmol l^–1 IBA. Shoot organogenesis from protoplast-derived callus was induced on MS medium supplemented with 5.0 µmol l^–1 BA and 2.0 µmol l^–1 IBA. Complete plantlets were obtained from the regenerated shoots on MS basal medium. The protoplast to plant regeneration protocol developed in this study provides the prerequisite for creating novel genotypes of this valuable medicinal species through genetic manipulation.     

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam.

Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l⁻¹ IAA and 1.0–3.0 mg l⁻¹ BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l⁻¹ BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.     

Somatic embryogenesis and shoot organogenesis from leaf explants of <Emphasis Type="Italic">Primulina tabacum</Emphasis>

Primulina tabacum is a rare and endangered species that is endemic to China. Establishing an efficient regeneration system is necessary for its conservation and reintroduction. In this study, when leaf explants collected from plants grown in four ecotypes in China are incubated on Murashige and Skoog (MS) medium containing 5.0 μM thidiazuron (TDZ) for 30 days, then transferred to medium containing 5.0 μM 6-benzyladenine (BA), adventitious shoots are then observed. Conversely, when leaf explants are incubated on medium containing 5.0 μM BA for 30 days, then transferred to medium containing 5.0 μM TDZ, somatic embryogenesis is induced. This indicates that somatic embryogenesis and shoot organogenesis could be switched simply by changing the order of two cytokinins supplemented in the culture medium. Histological investigation has revealed that embryogenic cells are induced within 30 days following incubation of explants in medium containing TDZ. Only if embryogenic cells were induced, TDZ could enhance somatic embryogenesis and BA could stimulate shoot organogenesis. When comparing explants from different ecotypes, leaf explants from Zixiadong in Hunan Province could induce low numbers (1–2) of either somatic embryos or adventitious shoots on medium containing either 5.0 μM TDZ or 5.0 μM BA, respectively. Whereas, leaf explants from plants collected from the other three ecological habitats could induce 50–70 somatic embryos/adventitious shoots per explant. Moreover, somatic embryos could induce secondary somatic embryogenesis and adventitious shoots on different media. All regenerated shoots developed adventitious roots when these are transferred to rooting medium, and over 95% of plantlets have survived following acclimatization and transfer to a potting mixture (1:1, sand:vermiculite).     

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.     

禿杉组织培养研究

本文详细报道了从秃杉(Taiwania flousiana Gaussen)离体胚诱导不定芽、不定根及从无菌苗茎端培养再生植株的过程。诱导不定芽要求较低的蔗糖浓度(以3％最好);同时BA是必须的,在附加0.1—3 mg/1 BA的White培养基上,从离体胚的子叶或胚轴上诱导了不定芽的发生(以1 mg/1最好);NAA与BA结合使用,对不定芽诱导无促进作用;适当提高光照有利于不定芽的诱导。在诱导不定芽的同时,在子叶表面还观察到有许多无结构的“不定突起”。不定芽起源于子叶表皮下1—2层细胞。IBA对诱导离体胚上产生不定根效果较好。在有或无生长素的培养基上,从生长1月龄的无菌苗茎端培养获得了不定根的产生,在加有细胞分裂索的培养基上,从无菌苗上产生了腋芽。     

Cauliflower inflorescence protoplast culture and plant regeneration

A yield of 2–4×10⁶ protoplasts/g F.W. could be obtained when fresh cauliflower inflorescence segments were digested with 2% cellulase Onozuka R-10, 1% cellulase RS and 0.4% Macerozyme R-10 in CPW18S for 7 to 10 h. Purified protoplasts were cultured in K8p liquid and agarose medium. Although protoplasts in liquid medium divided earlier than in agarose, protoplast-derived cells in liquid culture could not avoid browning. With agarose culture, sustained division and callus formation could be achieved. After 20 days, calli were transferred onto B5 agar medium with ZT 1.5 mg l^-1, BA 0.5 mg l^-1 and IAA 0.1 mg l^-1 for shoot formation. The frequency of bud formation varied from 56.7% for calli of 1mm in size to 5.6% for 5mm calli. The shoots formed were rooted in B5 medium containing 0.5 mg l^-1 IBA, and the regenerated plants were transplanted to pots and grew normally. It took about two months from protoplasts to the regenerated plants.Abbreviations Ade adenine - BA 6-benzyl aminopurine - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4,-dichlorophenoxyacetic acid - GA₃ gibberellic acid - Gln glutamine - NAA -naphthylacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - ZT zeatin     

Plants regenerated from mesophyll protoplasts of white mulberry

INTRODUCTIONProtoplastcultureis0neofthen1ostrapidlydevel0pingareasinp1anttissueculture,becauseofitsimportancei11plantgeneticmanipulation.However,sofar,thereareonlyafewforesttreespeciesinwhichplantregenerationfr0mprotoplastshaJsbeensuccessful,namelyLiriode…