Regeneration of garlic plants (allium sativum L., CV. “Chonan”) via cell culture in liquid medium

Summary Aiming at the genetic improvement of garlic cultivars, a cell suspension protocol was established which includes the induction of friable callus, establishment of cells in liquid medium, plating, regeneration, and bulb formation. Calluses of various textures from compact to friable and from green to yellowish were obtained by culturing explants excised from inner leaves of garlic bulbs on Marashig-Shoog (MS) medium with 2,4 dichlorophenoxy acetic acid (2,4-D), (1.1 mg/liter [5.0 μM]), picloram (1.2 mg/liter [5.0 μM]), and kinetin (2.1 mg/liter [10 μM]). Friable callus occurred on MS-A contained 2,4-D alone (1.0 mg/liter [4.52 μM]) and this callus was used to develop cell suspension cultures, which were maintained in liquid MS-B medium with a 2,4-D/benzyl adenine (BA) (0.5 mg/liter [2.25 μM]: 0.5 mg/liter [2.22 μM]) ratio. High plating efficiency was obtained on MS-C medium with different naphthalene acetic acid/BA combinations. Regeneration occurred after transfer of the caulogenic mass to MS-C medium containing 10 mg/liter (74.02 μM) and 20 mg/liter (148.04 μM) adenine for 60 days, followed by transfer to adenine-free medium. Plantlets transplanted to soil showed normal phenology. Shoots grown on modified MS medium supplemented with indolylbutryic acid (3.0 mg/liter [14.7 μM]) stimulated bulb formation by 30 days in culture.     

Tissue Culture of Maize I. Callus Induction and Growth

Callus induction and subculture was successful with mature embryos and stem sections of seedlings of Zea mays L. on Linsmaier and Skoog's medium modified to contain 4 mg/I of 2,4-D and 1 g/I of casamino acids. — 2,4-D was superior to NAA and IAA for both callus induction and growth. Callus subcultured on NAA formed abundant roots on agar-solidified media and numerous root-like primordia in liquid cultures. — Kinetin had no effect on callus induction in the presence of 2,4-D and neither kinetin nor gibberellic acid stimulated callus growth during subculture. — Callus grew equally well on the medium of Linsmaier and Skoog, that of Schenk and Hildebrandt, and the B-5 medium of Gamborg and Eveleigh containing 2% sucrose, 4 mg/I of 2,4-D and 1 g/I of casamino acids. — The callus grew more rapidly at 25°C than at 30°C or 35°C. Little difference was noted at any temperature in callus growth in alternating light (16 h) and dark (8 h) or continuous dark. — Sucrose was superior to glucose and maltose in both liquid and agar-solidified cultures. Lactose and galactose failed to support callus growth.     

High efficiency plant regeneration by somatic embryogenesis from callus of mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor)

Summary Tissue culture methods were developed for reproducible induction and maintenance of embryogenic (E) callus established from developmentally mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor). Embryogenic callus was obtained by culturing seeds and mature embryos of wheat on Linsmaier and Skoog’s (LS) medium containing 5 or 2 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D), respectively, and for sorghum mature embryos on LS medium containing 2 mg/1 2,4-D plus 0.5 mg/liter kinetin. Plant regeneration from E callus was achieved for several months and quantified on a fresh-weight basis of E callus. Phenotypically normal plants were regenerated from E callus cultured on LS medium supplemented with 0.1 mg/liter IAA plus 0.5 mg/liter benzyladenine (BA) for wheat and 1.0 mg/liter IAA plus 0.5 mg/1BA for sorghum. Wheat research was funded by the United States Agency for International Development, Washington, DC, cooperative agreement DNA-4137-A-00-4-53-00. Sorghum research was supported by the Gas Research Institute, Chicago, IL, contract 5084-260-0973. Expert technical asistance was provided by Nitschka S. ter Kuile, Barbara J. Ashton, Laurie Osborne, Erin Scott, and Kathleen M. Petersen.     

SHOOT AND TREE PRODUCTION FROM ASPEN TISSUE CULTURES

Trees were produced from firm white callus tissue of triploid quaking aspen (Populus tremuloides Michx.), initiated on a modified Wolter and Skoog defined medium and subcultured monthly for two years. When subcultured to medium without auxin, kinetin or supplements, but containing 0.15 mg/liter BAP (6-benzylaminopurine), multiple stunted shoots appeared on most inocula. However, at 0.05 mg/liter BAP, only a few vigorous shoots per piece were initiated, but seven rooted on the callus: two in the dark with BAP and five in 200 ft-c of light with 0.04 mg/liter 2,4-D. After proliferation of the roots on the medium surface, four shoots elongated and were planted in semi-sterilized soil, then were given 3100 ft-c of light for rapid growth into trees. Both light sources were on for 16 hr/day. Two trees were also grown from stunted shoots excised from the callus and rooted in soil.     

The Effects of Agitated Liquid Medium on in vitro Cultures of Hevea brasiliensis

The initiation and prolonged growth of callus, from stem explants of young plants of Hevea brasilienies on solid medium yielded a heterogeneous callus, with areas which are the result of compact growth interspersed with brown necrotic tissue and soft white tissue formations. Subculturing this callus (O callus) to agitated liquid medium and returning it to solid medium resulted in the production of a homogeneous friable and rapidly growing callus (Rl callus) The two established lines O and Rl have remained stable over one year in culture and differ in gross morphology, anatomy, growth and auxin content. Both were maintained on Murashige and Skoog's medium, with 2 mg/1 2,4-D and 0.5 mg/I kinetin. R 1 but not O showed enhanced growth at the lower 2,4-D level of 0.2 mg/l: both lines failed to continue growing when 2,4-D was omitted. It is suggested that the changes resulting from subculture in agitated liquid medium are related to those undergone by callus cultures which become habituated. Thus the Rl callus line is regarded as partially habituated. Subculture in agitated liquid medium also resulted in the production of large numberr of polyploid cells but these did not persist over the long periods of subsequent growth on agar medium, Enhanced auxin production by the establihed Rl callus line was thus observed in the absence of a detectable level of polyploidy.     

Growth and tropane alkaloid production inAgrobacterium transformed roots and derived callus ofDatura

Small callus pieces excised from theAgrobacterium transformed root line D₂ ofDatura stramonium, were cultured onto solidified MS medium supplemented with a 1.0 μM kinetin and three different concentrations (0.1, 0.5 and 1.0 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D), and were examined for their alkaloid productivity in relation to organization level and growth rate. Growth of transformed roots (in a MS liquid medium without plant growth regulators) was greater than that of transformed calli excised from them and cultured separately. The addition of 1.0 μM 2,4-D to the culture medium had a positive effect on callus biomass production, while it inhibited root formation by this tissue (the lower the 2,4-D concentration in the medium the greater the number of roots which emerged from the calli). Hyoscyamine production was also higher in the transformed roots than in the transformed calli, and in these tissues the production of hyoscyamine was positively correlated with organogenesis index (i.e. its ability for rooting). At the same time, the epoxidation of hyoscyamine to scopolamine only took place in the transformed calli. This occurred to a greater extent at the lower concentrations of 2,4-D in the culture medium. The mode through which the 2,4-D could control the alkaloid production of transformed callus is discussed.     

NUTRITIONAL AND MORPHOGENETIC INVESTIGATIONS ON CALLUS CULTURES OF NEOMAMMILLARIA PROFILERA MILLER (CACTACEAE)

Neomammillaria prolifera (Cactaceae), when grown on Murashige and Skoog's medium supplemented with fresh coconut milk, showed very little growth. Various concentrations and combinations of growth regulators which did not cause callusing had no apparent effect on the normal growth rates of intact plants. Healthy green calli obtained on a 2,4-D and kinetin-containing medium exhibited extremely fast growth and very specific growth requirements. Relatively high amounts of 2,4-D (10–20 mg/liter), kinetin (1–2 mg/liter), and coconut milk (20–60%) were required at all times for continued proliferation of callus on subculturing. Moreover, the callus was very tolerant to extremely high concentrations of other growth regulators (IAA, NAA, IBA, and GA up to 100 mg/liter) in the presence of 2,4-D and coconut milk. These substances could not replace 2,4-D for callusing or continued growth of callus. It was not possible to establish root cultures or to induce callusing of roots. Attempts to induce differentiation in callus were unsuccessful, except for sporadic root initiation in some cultures. A comparison of these results with similar studies on other succulents demonstrates some basic physiological similarities among this group of plants.     

Callus induction and maintenance of Zea mays kernels

Summary Totipotent tissue cultures of maize (Zea mays L.) have previously been initiated from various explant tissues. In this paper, we present an alternative source of callus induction.A callus of maize (G 204 hybrid) was obtained from intact kernels grown on Linsmair and Skoog RM medium supplemented with 20 mg 2,4-dichlorphenoxyacetic acid (2,4-D) per litre. The callus growth was greatest from the first node of the seedling shoot. Occasionally, callus growth was observed from the radicle and coleopticle regions. The callus was easily transferred and maintained on a medium with 2 mg/L 2,4-D. This callus formed numerous roots and leaf-like structures when grown on a medium containing 800 mg/L yeast extract, 30 g/L sucrose and 10 g/L agar.     

葶苈愈伤组织诱导及分化研究

为保护野生资源、实现人工栽培,本研究以葶苈(Draba nemorosa)嫩茎为材料,采用组织培养方法进行愈伤组织诱导与分化、不定芽生根与试管苗生根继代增殖培养,以及移栽和定植研究。结果表明,MS+6-BA 0.4 mg/L+2,4-D 2.5 mg/L是愈伤组织诱导培养和继代增殖培养的理想培养基;MS+6-BA 0.6 mg/L+NAA 0.1 mg/L 是愈伤组织分化培养和不定芽继代增殖培养的理想培养基;1/3MS+IAA 0.6 mg/L是不定芽生根培养和生根继代增殖培养的理想培养基;试管苗移栽成活率为86.8%,定植成活率为96.4%;定植苗保持了野生葶苈的植物学性状。     

Toward the production of artemisinin through tissue culture: Determining nutrient-hormone combinations suitable for cell suspension cultures

Summary Artemisia annua L. is the source of a potent antimalarial, artemisinin. As part of a program to produce artemisinin through tissue culture, a series of 14 multifactorial experiments were conducted to determine suitable conditions for initiating and maintaining friable callus fromA. annua. In the first six experiments, three different nutrient formulations [Gamborg B5 (B5), Murashige and Skoog (MS), and Whetmore and Rier (WR)], each with 32 combinations of auxins and cytokinins [2,4-dichlorophenoxyacetic acid (2,4-D) with benzyladenine (BA), or 1-naphthaleneacetic acid (NAA) with 6-furfurylaminopurine (kinetin)], were tested. Both B5 and WR nutrients supported friable callus formation from leaf explants with some combinations of auxin and cytokinin. Inasmuch as friable callus seemed to be produced over a wider range of auxin and cytokinin concentrations in combination with B5, the remaining experiments were conducted solely with this nutrient formulation. In the remaining eight experiments, it was determined that friable callus formed when combinations of NAA with kinetin or 2,4-D and BA were used with B5 medium. Lighter colored, more friable callus formed in response to 2,4-D and BA than with NAA and kinetin. No single combination of concentrations of auxin and cytokinin seemed to be “ideal” for producing friable callus. Ranges of 2,4-D from 0.5 to 2.0 with BA between 0.025 and 0.1, or NAA between 0.5 and 2.0 with kinetin between 0.5 and 1.0 mg/liter, produced acceptable results.     

Accumulation of Glutamine by Suspension Cultures of Symphytum officinale

A callus was induced from the veins of a leaf of Symphytum officinale, comfrey, on a medium containing the inorganic elements reported by Murashige and Skoog with addition of 3% sucrose, 0.5 mg/liter 2,4-D and 0.3~3.0 mg/liter kinetin.

Suspension cultures of this cell line obtained from the callus were shown to accumulate a large amount of L-glutamine intracellularly, The effect of growth hormones and nutrients on accumulation of the amino acid has been examined in suspension cultures. The most suitable concentrations of 2,4-D and kinetin for glutamine accumulation were 0.3 mg/liter each. The presence of potassium nitrate as a nitrogen source was beneficial for growth and ammonium nitrate stimulated the accumulation of glutamine. High levels of these nitrogen sources in the medium were required for obtaining a high level of glutamine. The concentration of glutamine accumulated reached to approximately 20% of dry cell weight when S. officinale was incubated in the medium containing 0.495 % of ammonium nitrate and 0.570% of potassium nitrate which corresponded to three times higher levels than those in a Murashige and Skoog’s medium.

Most of the amino acid was found intracellularly but a small amount was excreted into the medium in the later stages of the incubation. Addition of a cationic surfactant, cetyltrimethylammonium bromide, to the cultures caused to increase the amount of the amino acid in the culture filtrate.

The contents of free amino acids in leaves of S. officinale were compared with those in the callus. The level of glutamine in the callus was 260 times higher than that in the intact plant.     

In vitro shoot tip multiplication of cowpea Vigna unguiculata (L.) walp

Summary Shoot multiplication was induced in cowpea, cv. Georgia-21, from shoot tip explants. Shoot tips, 5 mm long, were isolated from in vitro-grown seedlings and cultured on MS medium containing N⁶-benzyladenine (BA) at 1, 2.5, or 5 mg/liter (4.4, 11.1, or 22.2 μM) or 6-furfurylaminopurine (kinetin) at 1, 2.5, or 5 mg/liter (4.6, 11.6, or 23.2 μM) combined with 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.3 μM) or naphthaleneacetic acid (NAA) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.7 μM). Cultures were maintained at a 12-h photoperiod (40 μmol·m⁻²·s⁻¹) and 23 ± 2° C. Treatments with BA induced greater shoot proliferation than those with kinetin. The highest number of shoots was produced on 5 mg (22.2 μM) BA per liter in combination with NAA or 2,4-D at 0.01 mg/liter (0.05 μM). Callus proliferated from the basal ends of shoot pieces in all treatments. The cultures also formed roots in the presence of kinetin, but not on BA-containing medium. To produce whole plants, the shoots were separated and rooted on 0.1 mg (0.5 μM) NAA per liter. Resulting plants grew normally under greenhouse conditions. Shoot tips provide an excellent explant source for cowpea micropropagation and can be used for callus induction.     

催吐萝芙木离体培养和植株再生体系的建立

为建立催吐萝芙木(Rauvolfia vomitoria Afzel.)的快繁再生体系,以茎段为外植体,比较了植物生长调节剂对其愈伤组织诱导、分化及生根的影响。结果表明,诱导愈伤组织的适宜培养基为MS+2,4-D 1.0 mg L~(–1)+TDZ 0.5 mg L~(–1)或MS+2,4-D2.0 mg L~(–1)+TDZ 0.5 mg L~(–1),出愈率达100%且生长状况良好;诱导丛生芽的最佳培养基为MS+6-BA 3.0 mg L~(–1)+NAA 0.1 mg L~(–1),出芽率为46.6%,平均出芽数为3.04。这为催吐萝芙木的快速繁殖和遗传转化研究奠定了基础。     

小叶龙竹（Dendrocalamus barbatus）愈伤组织诱导及植株再生

以小叶龙竹种子为外植体,通过研究MS培养基中不同植物生长调节剂浓度组合对外植体愈伤组织诱导和不定芽分化的影响以及不同配比对生根的作用,建立了稳定的繁殖再生体系。试验结果表明愈伤组织诱导的最适培养基为MS＋2,4-D5．0mg·L-1;不定芽分化的最适培养基为MS＋2,4-D0．5mg·L-1＋6．BA1．0mg·L-1＋KT0．25mg·L-1;小苗的最适生根培养基为1／2MS＋6-BA0．5mg·L-1＋NAA1．0mg·L-1＋IBA0．4mg·L-1,建立的繁殖再生体系将为进一步利用分子生物学技术对竹类植物进行遗传改良奠定基础。     

Plant regeneration through somatic embryogenesis on Holostemma ada-kodien,a rare medicinal plant

Plant regeneration through indirect somatic embryogenesis has been established on Holostemma ada-kodien Schult. Type of auxin significantly influenced somatic embryogenesis. Friable callus, developed from leaf, internode and root explants on Murashige and Skoog (MS) medium supplemented with 2,4-D (1.0 mg l^–1), was most effective for the induction of somatic embryos. Subculture of the friable callus developed on 2,4-D (1.0 mg l^–1) onto solid or liquid 1/2 MS medium with 0.1 or 0.5 mg l 2,4-D turned the callus embryogenic. Suspension cultures were superior to static cultures (solid medium) for the induction of somatic embryos. Transfer of embryogenic callus to liquid 1/2 or 1/4 MS medium with lower levels of 2,4-D (0.05–0.1 mg l^–1) induced the highest number of somatic embryos. An average of 40 embryos were obtained from 10 mg callus. Fifty per cent embryos exhibited maturation and conversion upon transfer to 1/10 MS basal solid medium. Plantlets were established in field conditions and 90 per cent survived.     

Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat (Triticum aestivum L.)

 Anthers from a doubled-haploid line of spring wheat (Triticum aestivum L.) cv. Pavon 76 were plated in liquid P-4 medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at four concentrations (0.5, 1.0, 2.0, 4.0 mg/l) for 5, 10, 15, and 25 days before being transferred to another medium with the same or reduced 2,4-D concentrations for the remainder of the induction phase for a total of 45 days. Incubation with 0.5 mg/l 2,4-D for 45 days produced lower callus yield and plant regeneration, indicative of insufficient auxin for callus induction. Callus yield and regeneration frequencies were higher with 1.0 mg/l 2,4-D. With 2.0 or 4.0 mg/l 2,4-D, an induction period of 10 or 15 days was sufficient for initiation of callus development. The extended presence of 2–4 mg/l 2,4-D in the medium beyond the initiation phase was detrimental to plant regeneration. Thus optimal callus induction and plant regeneration could be obtained through manipulating the 2,4-D concentration and the duration of its presence in the induction medium. Received: 1 December 1997 / Revision received: 15 February 1999 / Accepted: 26 February 1999     

Somatic embryogenesis inGnetum ula Brongn. (Gnetum edule) (Willd) Blume

Summary Somatic embryos ofGnetum ula (Gnetum edule) an endangered gymnosperm closely related to the angiosperms have been induced in vitro. Megagametophyte tissue with immature embryos was cultured on Murashige and Skoog medium. A mucilaginous, translucent embryogenic callus was obtained with 5 mg/l BA. Callus induced with 2,4-D was non-embryogenic. The embryogenic callus in liquid half strength Murashige and Skoog medium without inorganic nitrates supplemented with 2.5 g/l casein hydrolysate and 0.5 g/l L-glutamine gave rise to immature embryos. The embryos matured when treated with 60 g/l sucrose and 10 mg/l abscisic acid.Abbreviations MS Murashige and Skoog - BA 6-benzylaminopurine - 2,4-D 2,4 - dichlorophenoxyacetic acid - ABA abscisic acid     

Effect of 2,4-Dichlorophenoxyacetic Acid and NaCl on the Establishment of Callus and Plant Regeneration in Durum and Bread Wheat

Optimal callus induction and plant regeneration were obtained in bread and durum wheat by manipulating the NaCl concentration in the induction medium. Immature embryos from a high regeneration line of spring wheat (Triticum aestivum L.), 'MPB-Bobwhite 26', and an elite durum wheat (Triticum turgidum var. durum L.), 'Mexicali', were cultured in E3 induction medium consisting of Murashige and Skoog (MS) medium, 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 2% sucrose and 0.9% Bacto agar. The treated embryos were transferred to E3 liquid medium supplemented with various levels of 2,4-D and NaCl. Incubation on medium containing 2.5 mg l^–1 2,4-D for 45 days produced callus and plant regeneration in 'MPB-Bobwhite 26', but lower callus yield and plant regeneration in 'Mexicali', indicating that 2,4-D alone was not sufficient for callus induction and plant regeneration in this durum variety. Callus yield and regeneration frequencies were higher in 'Mexicali' embryos that were incubated in media containing 2 mg l^–1 2,4-D and 2 mg l^–1 NaCl. The presence of NaCl in the medium beyond the initiation phase was detrimental to plant regeneration. The use of NaCl in the callus formation could form the basis for improved transformation of durum wheat varieties.     

Plant regeneration from protoplasts of Japanese lawngrass

Embryogenic callus of Japanese lawngrass (Zoysia japonica Steud.) was induced from sterile mature seeds on LS medium with 5 mg / l of 2,4-D. Embryogenic callus selected visually under microscope was proliferated in liquid N6 medium with amino acids (N6-AA medium). Protoplasts were isolated from suspension cells by the treatment of enzyme mixture containing pectolyase Y-23 and cultured in K8p medium with 2 mg / l of 2,4-D at the density of 10⁶ / ml. Plants were regenerated by transferring the protoplasts derived callus to MS medium and incubating at 28 °C under light for two months. Plantlets were successfully transplanted in the soil.Abbreviations 2,4-D 2,4-dichrolophenoxyacetic acid - MES 2-(N-Morpholino) ethanesulfonic acid     

Plant regeneration using immature zygotic embryos of <Emphasis Type="Italic">Tribulus terrestris</Emphasis>

The genus Tribulus is the source of a number of steroidal saponins and other bioactive compounds which are of medicinal and pharmaceutical importance and plant regeneration of Tribulus terrestris has been reported. The objective of this study was to evaluate the potential of immature zygotic embryos of Tribulus terrestris as an explant for plant regeneration. Embryos were cultured on MS medium supplemented with 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), alone or in combination and callus and shoot or embryo formation evaluated. With 2.5 mg/l NAA or 2,4-D, callus formation frequency was 100% but 57% with 2.5 mg/l TDZ. The combination of 2.5 mg/l TDZ and NAA or 2,4-D also elicited callus formation frequency of 100%. The callus formation frequency was lower with lower levels of these growth regulators. On a medium with 0.5 mg/l TDZ, 17.4% of the 2,4-D-derived callus (2.5 mg/l), developed embryo-like structures and this increased to 37.3 and 41.4% respectively, when TDZ was combined with 0.5 mg/l indole-3-butyric acid (IBA) or 2,4-D. Both shoot formation and embryo-like structures developed in cultures with 2.5 mg/l TDZ, alone or in combination with 0.5 mg/l IBA or 2,4-D. The optimum sucrose level for morphogenetic response of embryo-derived callus was between 5.0 and 7.5%. Embryo-like structures were also observed when the 2,4-D-derived callus was cultured in a liquid containing benzyladenine (BA) and IBA. Plants were regenerated from both embryo-like structures and shoot buds on solid MS medium containing 0.2 mg/l IBA and rooted plantlets were transferred to soil.