Micropropagation and induction of autotetraploid plants of <Emphasis Type="Italic">Chrysanthemum cinerariifolium</Emphasis> (Trev.) Vis

Rapid propagation technology was established and optimized in vitro for Chrysanthemum cinerariifolium (Trev.) Vis., an important botanical insecticide plant with a huge international market. A large number of buds could be induced directly from epicotyl and hypocotyl explants on Murashige T; Skoog F. J. Plant. Physiol. 15: 473–479; (1962) medium [Murashige and Skoog (MS) medium] supplemented with 0.3 mg l⁻¹ benzyladenine (BA) and 0.3 mg l⁻¹ α-naphthaleneacetic acid (NAA). Root induction and development could be observed within 15 d after inoculation on 1/2 MS medium supplemented with 0.2 mg l⁻¹ indole-3-acetic acid (IAA) and 0.1 mg l⁻¹ rooting powder (ABT). Furthermore, a polyploid breeding study in vitro was reported to obtain superior breeding lines with high yield and good quality. Autotetraploid lines of C. cinerariifolium were obtained by colchicine treatments and identified by root-tip chromosome determination and stoma observation. The chromosome number of the autotetraploid plantlet was 2N = 4x = 36. Obtained autotetraploid lines will be of important genetic and breeding value and be used for further selection and plant breeding.     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

Direct shoot regeneration from nodal explants of <Emphasis Type="Italic">Sida cordifolia</Emphasis> Linn

A method was developed to initiate multiple shoots from mature nodal explants of Sida cordifolia Linn. High frequency of regeneration was achieved on Murashige and Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 6-benzylaminopurine, 0.5 mg l⁻¹ α-naphthalene acidic acid, 1.0 mg l⁻¹ adenine sulfate, and 10% (v/v) coconut milk. Multiple shoots were initiated within 21 d and the above media was capable of inducing the formation of more than 20 shoots from each explant. Regenerated shoots were successfully rooted on half-strength MS medium supplemented with 2.0 mg l⁻¹ indole-3-butyric acid and 3% (w/v) sucrose. Rooted plantlets were established in soil. The regenerated plantlets showed no morphological differences from the parent material. This protocol could be useful for germplasm conservation, cultivation, and genetic improvement of S. cordifolia.     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

Effects of salinity on growth and compatible solutes of callus induced from <Emphasis Type="Italic">Populus euphratica</Emphasis>

Summary The present study aimed to evaluate the response to salinity of Populus euphratica, which is more salt-resistant than other poplar cultivars, at the cellular level. To this purpose, callus was induced from shoot segments of P. euphratica on Murashige and Skoog (MS) medium supplemented with 0.5 mg l⁻¹ (2.2 μM) 6-benzyladenine (BA) and 0.5 mg l⁻¹ (2.7 μM 1-naphthaleneacetic acid (NAA). Callus was transferred to MS medium supplemented with 0.25 mg l⁻¹ (1.1 μM) BA and 0.5 mg l⁻¹ NAA. The relative growth rate of callus reached a maximum in the presence of 50 mmol l⁻¹ NaCl and growth was inhibited with increasing NaCl concentrations. Examination of the changes of osmotic substances under salt stress showed that accumulation of proline, glycine betaine, and total soluble sugars increased with increasing salt concentrations. The results indicate that the response of the callus of P. euphratica to salt stress is similar to that of the whole plant.     

In vitro micropropagation of Gymnema sylvestre – A multipurpose medicinal plant

The nature of the explant, seedling age, medium type, plant growth regulators, complex extracts (casein hydrolysate, coconut milk, malt extract and yeast extract) and antioxidants (activated charcoal, ascorbic acid, citric acid and polyvinylpyrrolidone) markedly influenced in vitro propagation of Gymnema sylvestre. A maximum number of shoots (57.2) were induced from 30 day old seedling axillary node explants on Murashige and Skoog (MS) medium containing 6-benzyladenine (1 mg l⁻¹), kinetin (0.5 mg l⁻¹), 1-napthalene acetic acid (0.1 mg l⁻¹), malt extract (100 mg l⁻¹) and citric acid (100 mg l⁻¹). High frequency of rooting was obtained in axillary node explant derived shoots (50%) on half strength MS medium supplemented with IBA (3 mg l⁻¹). The plantlets, thus developed, were hardened and successfully established in natural soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

A reliable protocol for plant regeneration from callus culture of <Emphasis Type="Italic">Phalaenopsis</Emphasis>

Summary Callus of Phalaenopsis Nebula was induced from seed-derived protocorms on 1/2 Murashige and Skoog (MS) basal medium plus 0–1.0 mg l⁻¹ (0–4.52 μM) N-phenyl-N′-1,2,3,-thiadiazol-5-yl urea (TDZ) and/or 0–10 mg l⁻¹ (0–45.24 μ M) 2,4-dichlorophenoxyacetic acid (2,4-D). Protocorms 2 mo. old performed better than 1-mo.-old protocorms for callus induction. More calluses formed on 1/2 MS basal medium supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. These calluses could be maintained by subculturing every month with basal medium supplemented with 0.5 mg l⁻¹ (2.27 μM) TDZ and 0.5 mg l⁻¹ (2.26 μM) 2,4-D. Protocorm-like bodies were formed, and plants regenerated from these calluses on 1/2 MS basal medium alone or supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. Plantlets were then potted on sphagnum moss in the greenhouse and grew well. No chromosomal abnormalities were found among the root-tip samples of 21 of the regenerated plantlets that were successfully acclimatized.     

Establishment of a highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation method for <Emphasis Type="Italic">Broussonetia papyrifera</Emphasis>

Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively. For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473, 1962) (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA) and 0.05 mg l⁻¹ indole-3-butyric acid (IBA) (CI medium) containing 5 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, and 0.5 mg l⁻¹ gibberellic acid (GA3) (SI medium), 5 mg l⁻¹ hygromycin and 250 mg l⁻¹ cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l⁻¹ hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay. Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as 44%.     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

Somatic embryo proliferation, maturation and germination in Catharanthus roseus

In the present study an efficient somatic embryogenesis method has been developed in Catharanthus roseus. Friable embryogenic callus was induced from hypocotyl of in vitro germinated seeds on Murashige and Skoog basal nutrient media supplemented with various auxins particularly 2,4-D (1.0 mg l⁻¹). However, only NAA (1.0 mg l⁻¹) produced somatic embryos in cultures. Embryo proliferation was even high on the same medium added with BAP. Cotyledonary somatic embryo germinated and converted into plantlets in BAP (0.5 mg l⁻¹) added medium following a treatment with gibberellic acid (1.0 mg l⁻¹) for maturation. Carbon sources and concentrations had a marked influence on maturation process. Plantlet conversion was better achieved when embryos were matured on 3% fructose or 3–6% maltose. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as raw material, genetic modification to embryo precursor cell may improve alkaloid yield further.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the chinese medicinal plant, <Emphasis Type="Italic">Dendrobium candidum</Emphasis> wall. ex lindl., from axenic nodal segments

Summary Dendrobium candidum Wall. Ex Lindl. is an important species used in the formulation of Shih-hu, a Chinese traditional medicine. An efficient protocol for in vitro propagation of D. candidum using the axenic nodal segments of the shoots, originated from the in vitro germinated seedlings, was developed. The seeds from 120-d-old capsules after pollination were first germinated on half-strength Murashige and Skoog (MS) basal medium supplemented with 30 g l⁻¹ sucrose. After 4 mo., the seedlings were subcultured on a similar medium supplemented with 1 ml l⁻¹ HYPONeX, 80 g l⁻¹ potato homogenate and 2 g l⁻¹ activated charcoal for further growth. Axenic nodal segments excised from 9-mo.-old seedlings were cultured on the medium in the presence of 2 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ naphthaleneacetic acid (NAA). After 75 d, 73.2% of the explants gave rise to buds/shoots. The elongated shoots were rooted on the medium containing 0.2 mg l⁻¹ NAA and the plantlets were successfully acclimatized in soil.     

Plant regeneration from excised hypocotyl explants of <Emphasis Type="Italic">Platanus acerifolia</Emphasis> willd

Summary A method of plant regeneration from hypocotyl segments of Platanus acerifolia Willd, has been developed. Hypocotyl slices were cultured on Murashige and Skoog (MS) basal medium supplemented with a range of combinations of cytokinins [6-benzyladenine (BA) or kinetin] and auxins [indole-3-butyric acid (IBA), indole-3-acetic acid, α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid] for adventitious shoot induetion. The highest regeneration frequency was obtained with MS medium containing 2.0 mg l⁻¹ (8.88 μM) BA and 0.5 mg l⁻¹ (2.46 μM) IBA. Adventitious buds and shoots were differentiated from hypocotyl-derived cellus or directly from the wounded sites within 4–8 wk. The regenerated shoots were elongated and proliferated efficiently on multiplication medium. Complete plantlets were transplanted to the soil and grew normally in the greenhouse after root formation on rooting medium for 4–6 wk.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

Regeneration of sorghum from shoot tip cultures and field performance of the progeny1

A system for rapid plant regeneration through somatic embryogenesis from shoot tip explants of sorghum [Sorghum bicolor (L.) Moench] is described. Somatic embryogenesis was observed after incubation of explants in dark for 6–7 weeks through a friable embryogenic callus phase. Linsmaier and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2 mg l⁻¹) and kinetin (0.1 mg l ⁻¹) was used for induction of friable embryogenic calli and somatic embryos. Germination of somatic embryos was achieved about 5 weeks after transfer onto Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (2 mg l⁻¹) and indole-3-acetic acid (0.5 mg l ⁻¹) under light. Seeds from in vitro-regenerated plants produced a normal crop in a field trial, and were comparable to the crop grown with the seeds of the mother plant used to initiate tissue culture. The simplicity of the protocol and possible advantages of the system for transformation over other protocols using different explants are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Chimonanthus praecox</Emphasis> (L.), a winter flowering ornamental shrub

A procedure for the micropropagation of Chimonanthus praecox (L) Link, wintersweet, has been developed using buds from adult trees excised in spring. Shoot cultures established on Murashige and Skoog (1962) medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BAP) and 0.1 mg l⁻¹ indole-3-butyric acid (IBA) were difficult to maintain in vitro through extended periods of time due to browning of the medium, shoot and leaf necrosis, and hyperhydricity. A treatment combining the use of 0.1% w/v activated charcoal and addition of a double phase agar-solidified/liquid medium improved propagation, enabling a successful in vitro propagation scheme to be developed. Optimal shoot multiplication occurred on medium containing 0.5 mg l⁻¹ BAP, and rooting on medium with 2.0 mg l⁻¹ IBA for 7 d, followed by transfer to hormone-free medium. Rooted plantlets were easily acclimated in a glasshouse and replanted and cultured outdoors.     

Micropropagation of <Emphasis Type="Italic">eclipta alba</Emphasis> (L.) hassk—An important medicinal plant

Summary An efficient and reproducible protocol for mass propagation of Eclipta alba (L.) Hassk, an important medicinal plant, was standardized by culturing shoot tips and nodal segments taken from in vitro raised plants. Maximum shoot proliferation occurred when the explants were cultured on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ benzylaminopurine (BAP). The shoot buds formed were further multiplied and maintained on medium containing BAP (0.5 mgl⁻¹) and gibberellic acid (0.5 mgl⁻¹). Rooting was best achieved on MS medium supplement with 1 mg⁻¹ indole-3-butyric acid. Rooted plantlets attained maturity and flowered normally in the field.     

Establishment of an <Emphasis Type="Italic">Agrobacteriuim</Emphasis>-mediated cotyledon disc transformation method for <Emphasis Type="Italic">Jatropha curcas</Emphasis>

Jatropha curcas contains high amounts of oil in its seed and has been considered for bio-diesel production. A transformation procedure for J. curcas has been established for the first time via Agrobacterium tumefaciens infection of cotyledon disc explants. The results indicated that the efficiency of transformation using the strain LBA4404 and phosphinothricin for selection was an improvement over that with the strain EHA105 and hygromycin. About 55% of the cotyledon explants produced phosphinothricin-resistant calluses on Murashige and Skoog (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA), 0.05 mg l⁻¹ 3–indolebutyric acid (IBA), 1 mg l⁻¹ phosphinothricin and 500 mg l⁻¹ cefotaxime after 4 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, 0.5 mg l⁻¹ gibberellic acid (GA3), 1 mg l⁻¹ phosphinothricin and 250 mg l⁻¹ cefotaxime, and about 33% of the resistant calli differentiated into shoots. Finally, the resistant shoots were rooted on 1/2 MS media supplemented with 0.3 mg l⁻¹ IBA at a rate of 78%. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. 13% of the total inoculated explants produced transgenic plants after approximately 4 months. The procedure described will be useful for both, the introduction of desired genes into J. curcas and the molecular analysis of gene function.     

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.     

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.