Tissue culture propagation of running buffalo clover (Trifolium stoloniferum Muhl. ex A. Eaton)

Rapid propagation of running buffalo clover (Trifolium stoloniferum) was achieved on Murashige & Skoog (MS) medium. Excellent shoot proliferation and shoot growth were obtained on medium containing 0.5 or 1 mg l^-1 BA. In vitro proliferated shoots were rooted on MS or half-strength MS medium containing 0 to 0.4 mg l^-1 IAA. Both the number of roots initiated and the length of the longest root were significantly higher on MS medium than on half-strength MS medium. Rooted plantlets were successfully transferred to soil.     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

Influence of some plant growth regulators on the growth and organogenesis ofCymbidium aloifolium (L.) Sw. seed-derived rhizomesin vitro

Summary An efficient procedure is outlined forin vitro regeneration of an epiphytic orchid,Cymbidium aloifolium (L.) Sw. using rhizomes developed from seeds. Murashige and Skoog's (1962) medium (MS) containing indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or 1-naphthaleneacetic acid (NAA) stimulated growth and proliferation of rhizomes with NAA being most effective at 5.0 mg.l⁻¹ (27.0 μM). Shoot bud differentiation was induced in the apical portions of the rhizomes on MS medium containing kinetin (Kn) or N⁶-benzyladenine (BA). The highest frequency of shoot regeneration (91.5%) and the maximum number of shoot buds formed (3.5 shoots/rhizome) were recorded with BA at 1.0 mg.l⁻¹ (4.4 μM). NAA (0.1 mg.l⁻¹, 0.54 μM), whenever added to the medium in conjunction with BA (1.0 mg.l⁻¹, 4.4 μM), slightly enhanced the frequency of shoot bud regeneration (92.6%) and the number of shoot buds formed (5.2 shoots/rhizome). Moreover, an NAA-BA combination induced rooting in regenerated shoots thereby producing complete plantlets in one step. Shoots developed on cytokinin-supplemented medium were rooted on MS containing NAA at 1.0 mg.l⁻¹ (5.4 μM). Regenerated plantlets were acclimated and eventually established in a garden.     

Shoot regeneration and the relationship between organogenic capacity and endogenous hormonal contents in pumpkin

To induce multiple shoots from pumpkin (Cucurbita moschata Duch.), cotyledon explants excised from various ages of seedlings after in vitro germination were cultured on MS augmented with different concentrations of BA (0, 0.5, 1.0 or 2.0 mg l⁻¹). The highest frequency of shoot regeneration (63.7%) was observed from seven-day-old cotyledon explants cultured on MS containing 0.5 mg l⁻¹ BA. The frequency and duration of shoot formation showed close correlation with the donor seedling age. By contrast, BA supply was necessary to promote shoot formation but no differences were observed in relation to different concentrations. Multiple shoots elongated on MS supplemented with 0.1 mg l⁻¹ BA and 5–7 shoots per regenerated explant were recovered. Elongated shoots were rooted on MS, which was easier than that on 2/3MS, 1/2MS, or MS supplemented with 0.1 mg l⁻¹ NAA. The rooted shoots were then transferred to greenhouse where they grew and flowered normally. Quantitative analysis of endogenous auxin (IAA) and cytokinins (iPA and ZR) in initial cotyledon explants of different aged seedlings showed that the regeneration ability of cotyledon explants varied dependently on their endogenous iPA contents. This study therefore deduces that the various organogenic capabilities of cotyledon explants from pumpkin are the result of their endogenous hormonal contents.     

Micropropagation ofCereus peruvianus mill. (cactaceae) by areole activation

Summary Currently,Cereus peruvianus plants can be rapidly clonedin vitro via adventitious organogenesis using callus cultures; however, somaclonal variation is a problem. A method is described herein using lateral bud explants to produce multiple shoots for clonal propagation. Apical and lateral explants were cultured on MS (Murashige and Skoog, 1962) media with factorial combinations of the auxins indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), and cytokinins 6-ben-zyladenine (BA) and N-(2-furanyl-methyl)-1-purine-6 amine (kinetin) at the concentrations 0.0, 0.01, 0.1, 1.0 mg“l⁻¹. Positive results were obtained from the lateral explants in all conditions tested, but apical explants did not respond toin vitro multiplication ofC. peruvianus cactus at all growth regulator combinations tested. Formation of axillary shoots inC. peruvianus seems most frequent in medium containing BA at 1.0 mg·l⁻¹ (4.44 μM) and IAA or NAA at 1.0 mg·l⁻¹ (5.71 μM or 5.37 μM respectively), but the frequency of shoot formation in the BA or kinetin and NAA or IAA combinations indicated that any of the combinations tested can be used for multiplication ofC. peruvianus plants regenerated from callus tissue culture. Root formation occurred in all (100%) of the cactus shoots after 9 wk in the same culture medium. All the cacti that developed at the different auxin and cytokin combinations continued growth after transfer to a potting mix of red earth (Paleudult) and ground river sand (1∶1).     

Growth and steroidal saponin production in hairy root cultures of Solanum aculeatissimum

Summary Hairy root cultures of Solanum aculeatissimum were established by trans-formation using Agrobacterium rhizogenes strain 15834. Root growth and production of steroidal saponin were investigated under various culture conditions. Transformed roots grew better in Gamborg's B5 medium containing 3 % sucrose under continuous light than in the dark. Also, the roots turned light green when cultured under continuous light. Green hairy roots produced aculeatiside A (6.71mg ·) L^–1 and aculeatiside B (6.39mg · L^–1) after 8 weeks of culture, while no steroidal saponin was detected in hairy roots cultured in the dark. Of the three culture media tested, Gamborg's B5 medium was superior for growth and steroidal saponin production. Growth and steroidal saponin production were enhanced when 100g · L^–1 auxin except for 2,4-D was added to the medium. The addition of 2,4-D inhibited growth. Production of steroidal saponin was highest with NAA. Transformed roots used in this experiment were confirmed that hairy roots examined contain both TL-DNA and TR-DNA region of Ri plasmid by PCR amplification analysis of DNA.Abbreviations MS medium Murashige and Skoog's medium (1962) - B5 medium Gamborg's B5 medium (1968) - LS medium Linsmaier and Skoog's medium(1965) - HPLC High performance liquid chromatography - NAA -Naphthaleneacetic acid - IAA Indole-3-acetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - PCR polymerase chain reaction     

Cryopreservation of embryogenic cell suspensions of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> L. (G) Don.

The present study describes the potential of in vitro grown adventitious roots of Hypericum perforatum L. commonly known as St. John’s wort at low nutrient and auxin levels in the liquid medium for micropropagation. Roots were regenerated from shoot-derived callus on MS medium containing 4.0 mg l⁻¹ Indole-3 acetic acid (IAA). IAA and Indole-3 butyric acid (IBA) were equally effective for the induction of roots from shoot cultures. Half strength MS medium containing 1.0 mg l⁻¹ IAA was most found suitable for culturing roots in liquid medium. A total biomass of 4.13 ± 0.67 g comprising 226 ± 34.4 shoots and shoot buds along with roots was obtained per culture starting with 200 mg roots inoculum. Pretreatment with kinetin (2.0 mg l⁻¹) enhanced the shoot multiplication. Shoots proliferated profusely from excised roots in static liquid medium supported with glass bead matrix. Growtek^™ vessel was found suitable and cost effective system for high throughput plantlet production. In vitro grown roots regardless of their source of origin were an excellent and easy to handle source of explant for aseptic production of plantlets without loosing the morphogenetic potential over the generations. The plants exhibited 84–99% similarity among themselves through RAPD. The in vitro shoots produced can either be multiplied or rooted perpetually, and alternatively they can also be explored for the in vitro production of hypericin and hyperforin.     

Organogenesis and embryogenesis inHelianthus tuberosus and in the interspecific hybridHelianthus annuus ×Helianthus tuberosus

A method of plant regeneration from cotyledons ofHelianthus tuberosus, Helianthus annuus ×Helianthus tuberosus and for the backcross of the interspecific hybrids onH. annuus was developed. Induction of somatic embryogenesis and plantlet regeneration from anther culture of the interspecific hybridsH. annuus ×H. tuberosus is reported.Cotyledons were cultured on Murashige and Skoog basal medium (MS) supplemented with indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin) or N⁶-benzylaminopurine (BAP). Shoot regeneration occurred on most of the media tested, but the best results were obtained on media with a high concentration of cytokinins (BAP or kinetin: 4 mg l^–1) and lower concentration of auxin (IAA: 0.5–1 mg l^–1).Embryogenic callus and adventitious buds were initiated from only two anthers of the hybridH. annuus ×H. tuberosus cultured on the MS medium containing BAP (0.2 mg l^–1) and 1-naphtalenacetic acid (NAA: 0.1 mg l^–1). Prolonged culture of these embryogenic calli and buds on the original medium with successive subculture on MS basal medium without growth regulators resulted in embryo formation and shoot differentiation. The plantlets, after rooting, were established in soil.     

Regeneration of transformed plants from hairy roots of <Emphasis Type="Italic">Plumbago indica</Emphasis>

Spontaneous shoot regeneration was observed from Agrobacterium rhizogenes-induced hairy roots of Plumbago indica when these were incubated in liquid MS medium for a period of 3 weeks under continuous light. Insertion of the rolB gene in putative transformed plants was confirmed by PCR and sequencing. Transformed plants grown for a period of 1 week on solid MS medium containing 0.5 mg l⁻¹ 6-benzyladenine and then transplanted to growth regulator-free medium showed better overall growth than control plants. Transformed plants had a higher root bio-biomass and an increased plumbagin content relative to non-transformed plants.     

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.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

Effect of genotype,explant type and growth regulators on organogenesis in Morus alba

Plantlets of the mulberry (Morus alba L. vars. Chinese White, and Kokuso-27) were produced from callus cultures. For callus induction, leaf, internodal segments, and petiole explants of Chinese White, Kokuso-27 and Ichinose varieties were grown on MS basal medium fortified with 2,4-D and 6-benzylaminopurine (BA). Callogenesis was dependent on the nature of explant used, the genotype and growth regulators supplemented in the medium. Leaves were the best explant type used for callus induction. Best callogenesis was obtained on MS medium containing a combination of 1 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ BA (95-100%). Calluses formed shoots on MS medium supplemented with 1 mg l⁻¹ BA. Supplementation with 0.1 mg l⁻¹ 2,3,5-triiodobenzoic acid (TIBA) in this medium enhanced shooting response. Presence of TIBA in the medium also improved the long-term organogenic potential of the callus. Regenerated shoots produced roots on Murashige & Skoog (MS) medium containing either 0.5 mg l⁻¹ indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Seventy percent of the rooted plants were established in the field where they are performing well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration from petioles of Parthenocissus tricuspidata planch

Summary A protocol of somatic embryogenesis and plant regeneration from petiole segments of Parthenocissus tricuspidata Planch. has been developed. Embryogenic tissue was induced on B5 (Gamborg) basal medium supplemented with 2.25–9.0 μM 2,4-dichlorophenoxyacetic acid, 500 mg l⁻¹ casein hydrolysate (CH), and 0.1 gl⁻¹ activated charcoal. Somatic embryos were induced on B5 medium containing various concentrations of benzyladenine (BA) (4.44, 6.66, and 8.88 μM) and α-naphthaleneacetic acid (NAA) (0, 0.54, and 1.61 μM) plus 500 mg l⁻¹ CH. Ninety percent of normal somatic embryos were converted into plantlets directly on Murashige and Skoog (MS) medium free of plant growth regulators. Shoots could be induced from abnormal somatic embryos on MS medium containing 4.44 μM BA, 0.05 μM NAA, and 500 mg l⁻¹ CH. Genotypic differences were found in the process of somatic embryogenesis and plant regeneration. Histological analysis confirmed the process of somatic embryogenesis. Regenerated plantlets with well-developed roots were successfully acclimatized in greenhouse and all plants showed normal morphological characteristics.     

濒危植物盘龙参种子的非共生萌发及种苗的快繁研究

以盘龙参种子为材料,筛选离体条件下适宜种子非共生萌发、种苗快繁的培养基。结果表明:盘龙参种子在无植物生长物质的培养基中能够萌发,但不能发育成苗;在含有1.0mg·L-1 KT、0.1mg·L-1 IAA和0.1mg·L-1 GA3的培养基中萌发,并能进一步发育形成种苗;在较高浓度生长素(1.2mg·L-1 NAA)的培养基中不能萌发。种苗在较高浓度细胞分裂素和较低浓度生长素配比的培养基中能够增殖,最高增殖系数可达到2.8,转入壮苗生长培养基中培养80d后可以移栽温室。最适增殖培养基为1/2MS+12.0mg·L-1 6-BA+0.1mg·L-1 NAA+10.0mg·L-1腺嘌呤;最适壮苗生根培养基为1/2MS+1.0mg·L-1 KT+0.1mg·L-1 IAA+10.0mg·L-1腺嘌呤。     

Eugenol from normal and transformed root cultures of Coluria geoides

Transformed root cultures of Coluria geoides Ledeb. were established with the use of Agrobacterium rhizogenes LBA 9402. Both normal and transformed root cultures were investigated for their growth and yield of eugenol. Normal roots were grown in B5 medium-supplemented with 0.2 mg l^-1 of kinetin and 0.2 mg l^-1 of 1-naphthaleneacetic acid (NAA). Hairy roots grew well in hormone-free B5 medium. Both hairy roots and normal roots produced glycosidic bound eugenol. as with the roots of intact plants, eugenol was the main component of the total essential oils obtained from hairy root and normal root cultures. The yield of eugenol from normal roots was 0.1–0.25% of the dry wt. and depended on the development stage of the culture. Yield of eugenol from hairy roots was 0.08–0.1% of the dry wt. NAA modified the hairy root morphology and influenced the yield of eugenol.Abbreviations NAA 1-naphthaleneacetic acid     

Plant regeneration from protoplasts ofCapsicum annuum L. cv. California Wonder

Plant regeneration from mesophyll protoplasts of pepper,Capsicum annuum L. cv. California Wonder has been demonstrated via shoot organogenesis. Protoplasts isolated from fully expanded leaves of 3-week-old axenic shoots when cultured in TM medium supplemented with 1 mg l ⁻¹ NAA, 1 mg l ⁻¹ 2,4-D, 0 5 mg l ⁻¹ BAP (CM 1) resulted in divisions with a frequency ranging from 20–25 %. Antioxidant ascorbic acid and polyvinylpyrrolidone (PVP) in the medium and incubation in the dark helped overcome browning of protoplasts. Microcalli and macrocalli were formed in TM medium containing 2 mg l ⁻¹ NAA and 0.5 mg l ⁻¹ BAP (CM II) and MS gelled medium containing 2 mg 1 ⁻¹ NAA and 0 5 mg 1 ⁻¹ BAP (CM III), respectively. Regeneration of plantlets was possible via caulogenesis. Microshoots, 2–5 percallus appeared on MS gelled medium enriched with 0.5 mg l ⁻¹ IAA, 2mg l ⁻¹ GA and l0mg l ⁻¹ BAP (CM IVc). Rooting of microshoots was obtained on half strength gelled medium containing 1 mg l ⁻¹ NAA and 0.5mg l ⁻¹ BAP. Protoplasts isolated from cotyledons failed to divide and degenerated eventually.     

High-efficiency plant regeneration from leaf explants of male himalayan poplar (Populus ciliata wall.)

Summary This study reports a protocol for high-efficiency plant regeneration from leaf explants of male Himalayan poplar (Populus ciliata Wall.). Shoots were regenerated at high frequencies from explants grown on Murashige and Skoog (MS) medium supplemented with 0.5 mg l⁻¹ kinetin and 0.2 mg l⁻¹ indole-3-acetic acid (IAA). Regenerated shoots developed roots in MS medium supplemented with 0.1 mg l⁻¹ IAA. Himalayan poplar plantlets could be produced within 2 mo. after acclimatization in a sterile mixture of sand and soil.     

Direct plant regeneration of curry leaf tree (<Emphasis Type="Italic">Murraya koenigii</Emphasis> koenig.), an aromatic plant

Summary An efficient protocol for plant regeneration from stem segments of Murraya koenigii was developed by culturing on Murashige and Skoog (MS) medium supplemented with 2.5 mg l⁻¹ benzyladenine (BA), 25 mgl⁻¹ adenine sulfate, 0.25 mgl⁻¹ indole-3-acetic acid (IAA), and 3% sucrose. The frequency of shoot bud regeneration was higher on similar medium in subsequent subcultures. The regenerated shoots were rooted on half-strength basal MS medium supplemented with 0.25–0.5 mgl⁻¹ IAA or 1-naphthaleneacetic acid (NAA) within 8–12 d of culture. The maximum percentage of rooting was obtained on MS medium supplemented with IAA and NAA, each at 0.25 mgl⁻¹. During acclimatization, 95% of rooted plantlets survived were grown normally under greenhouse conditions.     

Genotypic and media effects on plant regeneration from cotyledon explant cultures of someBrassica species

Conditions were established for efficient plant regeneration from cotyledon explant calli in different cultivars ofBrassica juncea, B. campestris andB. carinata on Murashige & Skoog's (MS) medium supplemented with various combinations of cytokinins and auxins. Regeneration frequency, however, varied with genotype and the different growth hormone combinations in media. Almost in all species, MS medium with zeatin (1.0 mg 1^-1) and IAA (0.1 mg l^-1) was found to be best for shoot organogenesis followed by the ones containing high kinetin (2.0 mg l^-1) and low IAA (0.02 or 0.2 mg l^-1) concentrations. On these media, the cotyledonary explants invariably underwent callusing followed by multiple shoot formation, which could be separated and subcultured for further propagation. Number of shoots per cotyledon explant cultured varied from 0 to as many as 50. InB. juncea andB. campestris, the regeneration frequency declined sharply in the absence of auxin in medium. BAP in combination with NAA yielded no or a reduced number of shoots. Shoot organogenesis also declined with the reduction in photoperiod from continuous light to 16 hours. Shoots were easily rooted during prolonged incubation on the same medium and whole plants were transferred to pots in the greenhouse and grown to maturity.Abbreviations BAP 6-benzylaminopurine - KIN kinetin - IAA indole-3-acetic acid - MS medium after Murashige & Skoog [8] - NAA -napthaleneacetic acid - ZEA Zeatin     

Improvement of ginsenoside production by jasmonic acid and some other elicitors in hairy root culture of ginseng (Panax ginseng C. A. Meyer)

Summary Hairy root cultures of Panax ginseng, established after the infection of root sections with Agrobacterium rhizogenes KCTC 2703, were cultured in phytohormone-free Murashige and Skoog (MS) liquid medium containing different concentrations of jasmonic acid and some other elicitors, in order to promote ginsenoside accumulation. Jasmonic acid in the range 1.0−5.0 mg l⁻¹ (4.8–23.8 μM) strongly improved total ginsenoside production in ginseng hairy roots. Peptone (300 mg l⁻¹) also showed some effect on ginsenoside improvement; however its effect was much weaker than that of jasmonic acid. Ginsenoside content and productivity were 58.65 and 504.39 mg g⁻¹, respectively. The Rb group of ginsenoside content was increased remarkably by jasmonic acid, while Rg group ginsenoside content changed only slightly compared to controls. However, jasmonic acid also strongly inhibited ginseng hairy root growth.