In vitro regeneration from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit

Methods to regenerate whole plants from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit were established. To optimize shoot induction, leaf explants were cultured on media containing different types and combinations of plant growth regulators. Growth was initiated within 17–24 days culture, and included callus formation, and root or shoot organogenesis ranging from 20 to 100% regeneration. Shoots were induced only when explants were cultivated on MS medium containing a combination of NAA and kinetin, NAA and BAP, IAA and Kinetin, or IAA and BAP. On media containing NAA and BAP, dark incubation was critical for efficient direct shoot regeneration from explants. Direct shoot formation and the highest number of shoots per explant (17.6) were obtained from leaf explants cultured in the dark for 30 days on MS medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ BAP. Shoots cultured on MS medium containing 0.1 mg l⁻¹ NAA formed tuberous roots with microtubers within 42 days. Healthy regenerated plants were acclimated and transferred to a greenhouse.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segments was studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine (BA) and α-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shoot regeneration. High rates of bud initiation and shoot development were obtained both with BA supplemented medium, in the range from 1 mg L⁻¹ to 3 mg L⁻¹, and with 0.1 mg L⁻¹ NAA supplemented medium. NAA concentrations above 1 mg L⁻¹ significantly reduced bud initiation and shoot elongation. The results obtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were compared. The highest adventitious bud induction was observed in Petri dishes for internodes cultured in 2 mg L⁻¹ BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds. The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further development, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm in height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed, with basal ones exhibiting higher regeneration frequency.     

Direct plant regeneration from mature leaf explants of pistachio, Pistacia vera L.

A protocol was developed for direct shoot and plantlet regeneration from in vitro regenerated leaf explants of male Pistacia vera L. cv. ‘Atl?’. Leaves excised from axenic shoot cultures of pistachio were used to induce organogenesis on a Murashige and Skoog (MS) medium with Gamborg vitamins supplemented with combinations of different concentrations of BAP and IAA. The highest adventitious shoot regeneration in 35% of the explants, with the number of shoots ranging from 2 to 3 per explant, occurred in the explants cultured during the establishment phase in the medium with 1 mg l⁻¹ IAA and 2 mg l⁻¹ BAP. For shoot multiplication, the highest number of new microshoot/explants (5.76) was obtained in a culture medium supplemented with 1 mg l⁻¹ BAP, but it was not significantly different from the number obtained at 2 mg l⁻¹ BAP. A high rooting frequency (84%) for microshoots was recorded on a medium supplemented with 2 mg l⁻¹ IBA. In vitro rooted plantlets were transferred to pots filled with a mixture of soil, sand and peat (1:1:1). They were weaned in a growth room and finally moved to a greenhouse. This protocol could be utilized for in vitro clonal propagation of this economically important plant.     

Callus induction and plant regeneration from embryonic axes of Kosteletzkya virginica

Kosteletzkya virginica, a perennial dicot halophytic species of the Malvaceae, is native to American salt marsh. It was introduced into China as a potential species to improve coastal wetlands and to develop ecologically sound saline agriculture. K. virginica adapts excellently to the tidal-flat habitats in China's east coast, with multiple eco-benefits; in particular, its seed oil could be used to produce biodiesel. The purpose of this study was thus to develop a standardized protocol to induce a high frequency of callus and subsequent plantlet regeneration system for a K. virginica breeding program with the final objective of applying transgenic techniques to improve seed oil yield. The embryonic axes of K. virginica were used as explants for callus induction, shoot induction from the callus and then adventitious root induction from the shoots on nine culture media with different hormone combinations. The best results were achieved on the following media: (1) 93.94% callus induction on MS medium supplemented with 1.0 mg L⁻¹ indole-3-acetic acid (IAA), 0.3 mg L⁻¹ kinetin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (2) 65.83% shoot induction on 1/2MS medium supplemented with 0.1 mg L⁻¹ IAA, 0.5 mg L⁻¹ zeatin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (3) 96.67% rooting on MS medium containing 30 g L⁻¹ sucrose and 8 g L⁻¹ agar. The survival rate of plantlets by organogenic regeneration was 85% after being transplanted into potting soil in flowerpots and placed in the greenhouse. This experiment indicates that we established successful callus induction and plant regeneration protocols for K. virginica.     

High frequency plantlet regeneration from callus and artificial seed production of rock plant Pogonatherum paniceum (Lam.) Hack. (Poaceae)

Pogonatherum paniceum (Lam.) Hack. is a rock plant with good potential for vegetative recovery on naked lands. A high frequency in vitro regeneration system was developed for P. paniceum. Calli were induced from explants of mature seeds, seedlings, young leaves, and stem segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 2.0 mg L⁻¹ α-naphthalene acetic acid (NAA) and 0.2 mg L⁻¹ 6-benzylaminopurine (BAP). High induction rates (59.57%) and regeneration rates (100%) were obtained from mature seed explants; calli were sub-cultured for over 2 years and still retained a high regenerative capacity. One seed explant resulted in 69,997 plants in 1 year. Shoot buds derived from calli were used for encapsulation in liquid MS medium containing 3% sucrose and two different alginate matrices (3% sodium alginate (w/v) + MS medium containing 3% sucrose and 3% sodium alginate + 1% activated carbon (w/v) + MS medium containing 3% sucrose) with a 20-min exposure to 2% CaCl₂ and 0.3% bavistin (w/v). The capsule with 3.0% sodium alginate (w/v) and 1% activated carbon (w/v) showed a higher conversion rate (61.58%) and stronger plantlets under non-aseptic conditions. These systems are useful for the rapid clonal propagation and dissemination of artificial seed material of P. paniceum for eco-recovery.     

Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl.

Efficient protocols were established for in vitro seed germination, neo-formation of secondary (2°) protocorms from primary (1°) protocorms and multiple shoot buds and protocorm-like body (PLB) induction from pseudo-stem segments of in vitro-raised seedlings of Cymbidium giganteum. Four nutrient media, namely Murashige and Skoog (MS), Phytamax (PM), Mitra et al. (M), and Knudson ‘C’ (KC) were evaluated for seed germination and early protocorm development. In addition, the effects of peptone, activated charcoal (AC) and two plant growth regulators [6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D)] were also studied. Both M and PM supplemented with 2.0 g l⁻¹ peptone or 1.0 mg l⁻¹ BAP resulted in ∼100% seed germination. Media supplemented with 2.0 g l⁻¹ AC could effectively induce large protocorms (1.6 ± 0.1 mm in diameter). Neo-formation of 2° protocorms from 1° protocorms was achieved in liquid and agar-solidified PM medium fortified with different concentrations and combinations of auxins (α-naphthalene acetic acid (NAA) and 2,4-D) and cytokinins [BAP and kinetin (KN)]. The highest number of 2° protocorms was obtained in liquid medium (10.7 ± 0.9/1° protocorm) supplemented with 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ NAA. Although protocorms proliferated profusely in liquid medium, these did not develop further unless transferred to agar-solidified medium within 6–8 weeks. Multiple shoot buds and PLBs were induced from pseudo-stem segments on agar-solidified PM medium fortified with different concentrations and combinations of BAP and NAA and the maximum number of PLBs (6.00 ± 0.20) was recorded when BAP and NAA were applied at 2.0 mg l⁻¹ each. A solid root system was induced from PLBs and shoot buds when these were transferred to half-strength PM or M media fortified with 0.5 mg l⁻¹ indole-3-acetic acid. Well-rooted plants were transferred to the greenhouse with 95% survival.     

High-frequency embryogenesis,regeneration of broccoli (Brassica oleracea var. italica) and analysis of genetic stability by RAPD

High-frequency somatic embryogenesis and shoot regeneration of broccoli (Brassica oleracea var. italica) were achieved. Cotyledon and hypocotyl explants from four varieties of broccoli were cultured on MS and modified MS media (mMS, supplemented with PG-96 organic components) with different combinations of growth regulator. The effects of genotypes, different explants, growth regulator combinations, organic components and AgNO₃ on induction of calli and shoots were evaluated. The optimal media for inducting calli/shoots and roots were mMS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar supplemented with NAA at 0.5 mg l⁻¹, 6-BA at 3.0 mg l⁻¹, AgNO₃ at 4.0 mg l⁻¹ and MS medium containing 3% sucrose and 0.8% (w/v) agar supplemented with NAA at 0.2 mg l⁻¹, respectively. The callus induction percentages were over 90% in all four varieties; shoot induction percentage was 92.5% and the average number of shoot per explant was 4.1 from cotyledon explant in variety Bishan. In this study, we established high-efficient embryogenesis and shoot regeneration system of broccoli and analyzed genetic stability of regenerants at DNA level using RAPD molecular marker. Out of 62 arbitrary primers screened using PCR amplification, 79 polymorphic bands were amplified from 20 primers. The results demonstrated the genetic stability of regenerants from the same variety.     

Plant regeneration of pansy (Viola wittrockiana) ‘Caidie’ via petiole-derived callus

An in vitro plant regeneration protocol for pansy (Viola wittrockiana) cultivar ‘Caidie’ from petioles was established as following: callus induction on a half-strength MS medium supplemented with 0.45 μmol l⁻¹ 2,4-d plus 8.9 μmol l⁻¹ BA, callus subculture on medium F (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 0.44 μmol l⁻¹ BA) and then on medium T (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 2.2 μmol l⁻¹ BA), shoot regeneration on medium D3 (MS media supplemented with 2.9 μmol l⁻¹GA₃, 23.6 μmol l⁻¹ AgNO₃, 0.02% active charcoal and 4.5 μmol l⁻¹ TDZ), shoot multiplication on medium M (half-strength MS medium containing NAA 1.1 μmol l⁻¹, TDZ 9.1 μmol l⁻¹ and GA₃ 8.7 μmol l⁻¹), and then shoot elongation and rooting on medium R (MS medium supplemented with 1.1 μmol l⁻¹ NAA and 1.1 μmol l⁻¹ BA). Subculture on appropriate medium was found to be important for successful shoot regeneration.     

Micropropagation of the bromeliad Guzmania ‘Hilda’ via organogenesis and the effect of α-naphthaleneacetic acid on plantlet elongation

This study established a highly effective micropropagation system to obtain good plantlet proliferation from floral organs via callus induction and bud differentiation in Guzmania ‘Hilda’ bromeliad. The best frequencies of organogenic callus formation (20% in petal and 35% in ovary explants) were obtained on media containing a combination of 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) with 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 1.5 mg l⁻¹ 2,4-D with 0.5 mg l⁻¹ NAA, respectively. Organogenic calli were cultured on medium with 1.0 mg l⁻¹ NAA and 0.5 mg l⁻¹ 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) induce the differentiation and regeneration of adventitious buds into plantlets. When the plantlets were cultured in a medium with optimum NAA concentration (0.5–1.0 mg l⁻¹) significant improvement in regeneration and elongation was achieved within one month. This overcame the difficulty of delayed elongation in Guzmania plantlets. More than 99% of the regenerated and acclimatized plantlets developed to the flowering stage.     

In vitro high frequency direct plant regeneration from whole leaves of blackberry

High frequency and direct (without callus) plant regeneration was achieved from whole leaf explants of thornless blackberry (Rubus hybrid) cv. Black Satin (EC No. 381258; PI No. 553272) in vitro. Leaf blade explants from 1-, 3- and 5-month-old mother cultures were cultured on Murashige and Skoog (MS) medium with thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), indol-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA), alone or in combination. Three-month explants cultured on 0.02 mg l⁻¹ TDZ produced a high regeneration frequency (91.7%) and the most shoots/leaf explant (17.3). The shoot primordia developed within 3 weeks from the point of detachment of the petiole from the leaf blade. The age of the explant source significantly affected the shoot regeneration potential of the leaf explants. Leaves excised from 3-month-old in vitro-cultured shoots performed better than those from 1- and 5-month-old shoots. Shoots rooted best on half-strength MS basal medium with 0.5 mg l⁻¹ IBA and 90% of the plantlets survived acclimatization. The regenerated plantlets were morphologically similar to the mother plants.     

Efficient in vitro multiplication in Ornithogalum ulophyllum Hand.-Mazz. from twin scale explants

Ornithogalum ulophyllum Hand.-Mazz. with beautiful white flowers is an important medicinal and ornamental plant of the Middle Eastern countries and need exploitation for commercial propagation. The study reports in vitro mass proliferation of bulblets achieved from twin scales and “in vitro regenerated bulblet” explants on MS medium supplemented with various concentrations of BAP–NAA. The best regeneration on twin scales and “in vitro regenerated bulblets” was obtained on MS medium containing 2 mg l⁻¹ BAP–0.5 mg l⁻¹ NAA and 2 mg l⁻¹BAP–1 mg l⁻¹ NAA, respectively. However, bulb scales seemed to be more potent for bulblet regeneration. A large number of the developing bulblets rooted on the regeneration medium. Remaining non-rooting bulblets were rooted on MS medium containing 1 mg l⁻¹ NAA. All plants were acclimatized in the environmental chamber for 4 weeks and were transferred to the greenhouse for flowering. Regenerated bulblets developed into morphologically normal plants.     

High frequency in vitro embryogenic callus induction and plant regeneration from indiangrass mature caryposis

Indiangrass [Sorghastrum nutans (L.) Nash.] is native to the North America and is an important component of the original tall grass prairie. It is also an important ornamental and forage grass. Recently, it has been proposed as an ideal biomass producer for cellulosic ethanol production. Genetic transformation is an important tool for introducing important agronomic traits into plants, but an efficient and reproducible in vitro regeneration protocol is a prerequisite for successful genetic transformation. In this report, we used mature caryopses as explants and tested the effect of various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (1–5) and kinetin (KT) (0, 0.1, and 0.2) on embryogenic callus induction using LS basal medium. Caryopses cultured on media supplemented with 2,4-D alone generally outperformed those cultured on media supplemented with both 2,4-D and kinetin for embryogenic callus induction. The best treatment is LS basal medium supplemented with 3 mg l⁻¹ 2,4-D. LS basal medium supplemented with KT of 0, 0.5, 1, 2 or 5 mg l⁻¹ were tested for regeneration efficiency which was shown to increase as the KT concentration increased. The quality of the shoots produced on the medium containing KT at 5 mg l⁻¹, which produced the highest regeneration frequency appeared to be lower as leaves become vitrified. Shoots were moved to a rooting medium containing either 0 or 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA). Rooted plantlets were then transferred to soil-containing pots and were placed in a mist room for 1 week before they are transferred to a normal greenhouse where they all survived. The reported regeneration protocol is very efficient and highly reproducible in spite of the heterogeneous nature of the tested cultivar; thus it should be suitable for genetic transformation.     

Effect of position and orientation of leaflet explants with respect to plant growth regulators on micropropagation of Zamioculcas zamiifolia Engl. (ZZ)

Micropropagation studies on Zamioculcas zamiifolia Engl. (ZZ) as to the position and orientation of leaflet explants and plant growth regulators were carried out. Explants consisted of leaflet lamina from the basal or apical part of the leaflet with or without petiolule or midrib that were placed vertically into the medium except for the explants with midrib from the basal part of the leaflet that were placed horizontally as well. The explants were cultured on solid Murashige and Skoog medium (MS) with 30 g l⁻¹ sucrose, supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2 or 4 mg l⁻¹ and 6-benzyladenine (BA) at 0 or 4.44 μM in all (four) possible combinations, or with 1-naphteleneacetic acid (NAA) at 0 or 5.38 μM and BA at 0 or 4.44 μM in all (four) possible combinations (establishment medium). The morphogenic response was direct from all types of leaflet explants and varied only with respect to different plant growth regulators of the medium: 2,4-D combined or not with BA formed somatic embryo-like structures; NAA alone produced tubers and roots; BA alone resulted mainly in leaves; NAA combined with BA produced mainly roots. The intensity of the response varied accordingly to the explant type and orientation. Explants with petiolule or midrib from the basal part of the leaflet showed the highest morphogenic response compared to explants without petiolule or midrib or to explants from the apical part of the leaflet, in all the plant growth regulator combinations used. Explants with midrib from the basal part of the leaflet placed vertically into the media showed higher morphogenic response compared to those placed horizontally on the medium surface. With the objective to regenerate plantlets, explants were subcultured on MS with NAA and BA at various concentrations based on the explant response in the establishment medium, taking into consideration the initial explant type. The initial explant type did not affect the response in the subculture. Most plantlets (a tuber with roots and one leaf with one pair of leaflets) were produced by explants with embryo-like structures induced in a medium with only 2,4-D. Explants with tubers induced in a medium with NAA gave plantlets at 65–85% when subcultured in a medium with 4.44 μM BA alone or in combination with 2.69 μM NAA. Explants with leaves induced in a medium with BA and explants with roots induced in a medium with NAA and BA gave plantlets at low percentages (20–40%). The best response was produced by explants with embryo like structures induced in a medium with only 2,4-D which gave plantlets at 100% when subcultured in the medium with 2.69 μM NAA and 2.22 μM BA. Plantlets raised in different treatments were transplanted ex vitro after 22 weeks and exhibited 80–100% survival.     

Plant regeneration from in vitro cultured leaves of Lanzhou lily (Lilium davidii var. unicolor)

Lanzhou lily (Lilium davidii var. unicolor) is one of the best lilies which are edible in China but the efficient shoot regeneration system has not been developed. The purpose of the present study is to establish an efficient and reproducible protocol for induction of shoots in vitro from L. davidii var. unicolor leaves. Shoot regeneration from in vitro cultured leaves of L. davidii var. unicolor was tested on the 26 media based on NN [Nitsch, J.P., Nitsch, C., 1969. Haploid plants from pollen grains. Science 163, 85–87] basal medium, containing different concentrations of thidiazuron (TDZ) in combination with different concentrations of α-naphthaleneacetic acid (NAA). Shoot organogenesis occurred directly from the leaves without forming callus. Shoot regeneration mainly occurred from the cuts across the midvein and the base of the leaf explants. The highest frequency of regeneration (93.3%) and the largest number of shoots per leaf (3.83) were obtained on NN basal medium supplemented with 0.5 mg l⁻¹ TDZ and 1.0 mg l⁻¹ NAA. All the regenerated shoots formed complete plantlets on half-strength MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497] basal medium containing 0.1–0.5 mg l⁻¹ indole-3-butyric acid (IBA) with in 30 days, and 92% of the regenerated plantlets survived in the soil. This study will be useful for Agrobacterium-mediated transformation and exploitation of somaclonal variation of Lanzhou lily.     

Influence of yeast extract and casein hydrolysate on callus multiplication and somatic embryogenesis of date palm (Phoenix dactylifera L.)

Complex organic additives are known to improve growth and differentiation of in vitro plant cultures. The present investigation was conducted to determine the effect of various concentrations of yeast extract (YE) and casein hydrolysate (CH) on callus growth and somatic embryogenesis in date palm cultivar Nabout Saif. Callus induced from shoot tip explants was grown on callus multiplication medium supplemented with either YE or CH at 0.0, 0.1, 0.25, 0.5, and 1 g l⁻¹. To induce somatic embryogenesis, callus was transferred to a hormone-free medium containing the corresponding concentration of additives. The results have shown that callus weight and the number of somatic embryos were directly proportional to increases in the concentration of organic additives tested. Callus growth was best achieved when 1 g l⁻¹ of either YE or CH was added to the culture medium. At this concentration of YE, callus growth was double that of the control medium. On CH-containing media growth was 2.3 times that of the control. This indicates that CH is more effective in enhancing callus growth. However, YE was more effective in enhancing somatic embryogenesis. The data show that the best somatic embryo formation was obtained on either 1 g l⁻¹ YE or 0.5 g l⁻¹ CH which produced 45 and 30 embryos per culture, respectively, as compared to 20 embryos produced in the control treatment. Resultant somatic embryos successfully rooted and regenerated plantlets which exhibited normal growth in the greenhouse. Enhanced plant regeneration, an essential criterion for commercial micropropagation, was achieved.     

Plant regeneration of Carica papaya L. through somatic embryogenesis in response to light quality,gelling agent and phloridzin

Difficulties to develop an easy and reproducible protocol to get healthy and well formed plants from somatic embryos of papaya (Carica papaya L.) had included low germination, callus production at the base of the embryo radicle and the occurrence of hyperhydric plantlets among others, and by consequence unsuccessful transfer to the field. With the aim of improving a propagation method, the effects of light quality, gelling agent and phloridzin concentration on the germination of somatic embryos of hermaphrodite C. papaya L. var. Maradol were studied. Somatic embryos were grown on half strength MS medium, with the addition of Chen vitamins [Chen, M.H., Wang, P.J., Maeda, E., 1987. Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants. Plant Cell Rep. 6, 348–351], solidified with three distinct gelling agents: Sigma^® Agar–Agar, Difco^® Bacto agar and Phytagel^®; supplemented with phloridzin and exposed to different light qualities: blue (54 μmol m⁻² s⁻¹), red (65 μmol m⁻² s⁻¹), gro-lux (68 μmol m⁻² s⁻¹), red + blue, white (32 μmol m⁻² s⁻¹) and wide spectrum (49 μmol m⁻² s⁻¹) during a period of 4 weeks. Results show that light quality and gelling agent had important effects on germination and plant growth, while 3.0 mg L⁻¹ phloridzin had an important role on germination as well as in root development. Somatic embryos exposed to white light, culture medium solidified with 3.0 mg L⁻¹ phytagel and 3.0 mg L⁻¹ phloridzin showed longer roots. Meanwhile, germination and plant length were promoted on an improved culture medium solidified with 7.5 g L⁻¹ Difco^® Bacto agar, 3.0 mg L⁻¹ phloridzin and exposed to gro-lux lamps. Under these conditions, 70% of somatic embryos germinated and developed normal roots without hyperhydricity. The regenerated plantlets with well developed roots and shoots were successfully transferred to a greenhouse with a survival rate of 95%.     

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 embryogenic callus induction and plant regeneration from mature caryposis of big bluestem and little bluestem

Big bluestem (Andropogon gerardii Vitman) and little bluestem [Schizachyrium scoparium (Michaux) Nash.] are native to the North America and are important forage grasses and ornamental grasses. Both grasses are proposed as ideal biomass producers for cellulosic ethanol production. To apply genetic transformation, which is an important tool for incorporating desirable agronomic traits into plants to both species, however requires an efficient and reproducible regeneration protocol. We used mature caryopses from big and little bluestem as explants and tested the effect of various combinations of 2, 4-dichlorophenoxyacetic acid (2, 4-D) (1, 2, 3, 4 or 5 mg l⁻¹) and kinetin (KT) (0, 0.1 or 0.2 mg l⁻¹) on embryogenic callus induction with LS as the basal medium. The highest percentage of embryogenic calli induction occurred on medium containing 2, 4-D alone at 2 mg l⁻¹ for ‘Bison’ and on medium containing 4 mg l⁻¹ 2, 4-D alone for ‘Bonilla’ big bluestem. For little bluestem, the highest percentage of embryogenic callus induction occurred on medium containing 3 mg l⁻¹ 2, 4-D plus 0.1 mg l⁻¹ kinetin, suggesting that addition of KT is beneficial. Shoot regeneration took place on LS basal medium without any plant growth regulator for both species, although the addition of KT increased both regeneration frequency and the number of shoots produced per callus. Rooting of shoots reaching about 2 cm long occurred readily with or without α-naphthaleneacetic acid (NAA). Rooted plantlets were all successfully established in the soil.     

Effect of nitrogen and sulfur interaction on growth and pungency of different pseudostem types of Chinese spring onion (Allium fistulosum L.)

Effects of N and S supply on the growth and pungency (estimated as pyruvic acid levels) of Chinese spring onion (Allium fistulosum L. var. giganteum Makino) were investigated in two pot experiments using soilless growing media. In the first experiment the effects of S supply (0.01 and 4.00 mmol L⁻¹ SO₄²⁻) on the growth and pungency of Chinese spring onion were investigated among four cultivars with fleshy root type or long pseudostem type. In the second experiment the effects of different S (0.01 and 4.00 mmol L⁻¹ SO₄²⁻) and N (1.5, 3.0, 6.0, 12.0 and 24.0 mmol L⁻¹ N) supply levels on the growth and pungency of Chinese spring onion were studied. Fleshy root spring onion had stronger pungency and larger pseudostem diameter than long pseudostem spring onion, and the pungency of fleshy root spring onion was regulated to a greater extent by N and S supply compared with long pseudostem spring onion. Increasing S supply level significantly increased the biomass, N and S uptake and pungency of all cultivars tested. The biomass of Chinese spring onion of fleshy root type (cv Longyao) and long root type (cv Zhangqiu) was more influenced by N supply than it was by cultivar or S supply. Low S supply decreased the pungency of the two cultivars with increasing N supply. No significant differences in N or S uptake or pungency were observed in the two cultivars with different S supply at the N supply level of 1.5 mmol L⁻¹ N, however, cultivar differences in N and S uptake and pungency were investigated at high N supply (12.0 mmol L⁻¹ N) and S supply (4.0 mmol L⁻¹ SO₄²⁻). Excessive N supply (24.0 mmol L⁻¹) significantly inhibited plant growth, retarded S assimilation, and decreased pungency. It is therefore essential to apply the optimum recommended rate of N fertilizer in Chinese spring onion production.