Production,maintenance and plant regeneration from cell suspension cultures of Stevia rebaudiana (Bert.) Bertoni

A method is described for producing and maintaining Stevia rebaudiana suspensions and regeneration of plants from calli derived from cell suspensions. Suspension cultures composed of isolated cells (ca. 10%) and cellular aggregates (5–100 cells) were obtained in 20–30 days by using friable callus as the initial inoculum in liquid medi with BA (0.5 mg/l)+2,4-D (1.0 mg/l), and periodic filtering (100–500 m sieves) with 6–7 days interval between subcultures. Cultures derived from actively growing calli are mainly diploid (2n=22) whereas those derived from senescent calli showed a wide variation in chromosome number (55–200). Stock cell suspensions which had been maintained for 3 years were plated on basal LS agar medium with BA (0.5 mg/l)+2,4D (0.5 mg/l) to form callus. Calli originating from predominantly 2n cell suspensions when transferred to medium with K (2.0 mg/l)+NAA (0.02 mg/l) were able to form buds. Shoot elongation and further rooting of isolated shoots was better on LS medium devoid of growth regulators. Variation in rooting capacity, plant vigour, morphological characters and chromosome number was found amongst regenerated plants.Abbreviations BA Benzylaminopurine - 2,4-D 2,4 - Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - LS Linsmaier & Skoog     

小冰麦异附加系的体细胞无性系建立及其变异的研究

从7种小冰麦异附加系的幼叶和成熟胚诱导出愈伤组织,建立了体细胞无性系,获得大量试管苗,并移栽成活。实验设计了适于小冰麦异附加系组织培养的 WG 培养基。愈伤组织诱导采用二次诱导方法。第一诱导培养基为 WG_2附加4mg/1 2,4-D、1mg/1 NAA。第二诱导培养基为 WG_2附加2m//1 2,4-D、0.5mg/1 NAA,和0.25mg/1KT。分化培养基为 WG_3附加0.5mg/1 KT、1mg/1 NAA 和100mg/1 Ad。再生植株的染色体检查表明,异附加系无性系的染色体数变异明显。保持2n=44的再生植株只有34.4%,而且变异植株中回复到2n=42的植株较多。再生植株中约有1/2发生了形态变异。在变异植株的花粉母细胞中观察到染色体的交换、易位等结构变化。特别在愈伤组织细胞中观察到多条染色体融合成多着丝点染色体和体细胞的染色体交叉,说明无性系中发生了染色体的交换和易位。     

Study on the Establishment and Variation of Somaclones from Wheat-Wheatgrass Alien Additional Lines

Calli were induced from the young leaves and mature embryos of seven wheat-wheatgrass alien additional lines, and somaclones were established from the calli. A large number of plantlets were obtained and survived after transplantation. The medium WG used for tissue culture of wheat-wheatgrass alien addition lines was set up in the experiment. The methods induced callus with two kinds of media were used. The first medium WG2 supplemented with 4 mg/l 2, 4-D and l mg/l NAA was used for callus formation. The second medium was WG2 with 2 mg/l 2, 4-D, 0.5 mg/l NAA and 0.25 mg/l KT. Differentiation medium used was WG3 with 0.5 mg/l KT, 1mg/l NAA and 100 mg/l adenylic acid. The examination of the chromosomes in regenerated plants indicated that the chromosomal number variation was remarkable. The plants maintaining 2n=44 were only 34.4% of the total regenerated plants, and the plants with the chromosomal number returned to 2n=42 made a relatively greater part of the variational plants regenerated. About half of the regenerated plants showed morphological variations. The crossover, translocation and other chromosomal structural changes were found in PMCs of the variational plants regenerated. Meanwhile, the polycentric chromosome derived from the fusion of several chromosomes and chiasmata of somatic chromosomes were clearly observed in callus cells, indicating that crossover and translocation occurred in somacloaes.     

Tissue,cell culture and micropropagation of Mandevilla velutina,a natural source of a bradykinin antagonist

Leaf, stem and root explants of Mandevilla velutina were cultured in vitro and produced vigorous callus in LS basal medium containing one auxin (2,4-D or NAA) plus BAP. Calli can be subcultured indefinitely with vigorous growth. Subculture of calli to NAA (1.0 mg/l) plus BAP (5.0 mg/l) caused profuse regeneration of shoots. Isolated shoots were rooted in basal medium plus NAA (5.0 mg/l) or IBA (8.0 mg/l). Rapidly growing cell suspensions can be easily obtained from friable callus cultured in liquid medium.Abbreviations LS Linsmaier & Skoog - 2,4-D 2,4 dichlorophenoxi-acetic acid - NAA -naphthalene-acetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine - IBA indole-3-butyric acid     

In vitro propagation of Iris pallida

Plantlets were regenerated from callus of Iris pallida, an important perfume plant. Only the leaf base attached to the rhizome had the ability to generate yellow-colored callus on LS medium supplemented with 1 mg/l 2,4-D and 0.1 mg/l KT in the dark. Yellow calli grew with partial differentiation into white tissue, probably embryogenic, during subculture on the same medium with a 16-h photoperiod. Only yellow-colored calli with the white tissue could differentiate into plantlets after transfer to kinetin- or gibberellin- supplemented LS medium. Regenerated plantlets which grew on the medium without growth regulators were transferred to the soil. After 2 years of cultivation in soil, the regenerated plants flowered and formed rhizomes. The components of the essential oil in the rhizome of regenerated plants were essentially the same as those in natural plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - NAA alpha-naphthaleneacetic acid - LS Linsmaier and Skoog (1965) medium     

Callus induction and plant regeneration from maize mature embryos

Calli were induced from mature embryos of maize (Zea mays L.) inbred lines A632, B73 and Mol7 on MS medium supplemented with 1–2 mg/1 2,4-dichlorophenoxyacetic acid. Callus induction frequency ranged from 23–100%, with Mol7 having the highest frequency. Plants were regenerated from 4–5% of the B73 and Mol7 explants. Embryogenic and organogenic calli of B73 were maintained for more than two and one half years without losing regenerability. Of 95 regenerated plants, only one R₀ plant with abnormal pollen was detected, and no morphological variants were observed in the R₁ progeny.Abbreviations Dicamba 3,6-Dichloro-o-anisic acid - IAA 3-indoleacetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Ze zeatin     

小麦×天兰冰草杂种体细胞愈伤组织诱导及无性系建立

从普通小麦×天兰冰草杂种F_1的叶、茎、节、幼穗诱导出愈伤组织,建立了体细胞无性系,获得了大量试管苗并移栽成活。 愈伤组织诱导率及分化率以幼穗最高,茎、节、叶较低。完全展开的叶片不能形成愈伤组织,未伸展的幼叶能诱导出愈伤组织,分化程度越低的幼叶部位越容易脱分化。幼叶基部切段的愈伤组织诱导率可达90％以上。改良MS附加4mg/l 2,4-D、0.1mg/l KT、0.5mg/l NAA为最佳愈伤组织诱导培养基。最适宜的分化培养基为改良MS附加0.25mg/l KT、0.5 mg/l NAA、150 mg/l腺嘌呤核苷。杂种的愈伤组织诱导率及分化率高于两个亲本。杂种的愈伤组织长势旺盛,适应性强等都表现了小冰麦杂种在组织培养中的杂种优势,而且这种再生能力杂种优势可以通过无性系得到保持。     

Isozyme Variability in Plants Regenerated from Calli of Cereus peruvianus (Cactaceae)

Morphological and isozyme variation was observed among plants regenerated from callus cultures of Cereus peruvianus. Different morphological types of shoots (68%) were observed in 4-year-old regenerated plants, while no distinct morphological variants were observed in plants grown from germinated seeds. Isozyme patterns of 633 plants regenerated from calli and of 261 plants grown from germinated seeds showed no variation in isocitrate dehydrogenase isozyme, and the differential sorbitol dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, acid phosphatase, and peroxidase isozyme patterns observed in regenerated plants were attributed to nonallelic variation. Allelic variation was detected at three isoesterase loci. The proportion of polymorphic loci for both populations was 13.6% and the deviation from Hardy–Weinberg equilibrium for the Est-1 and Est-7 loci observed in somaclones was attributed to the manner in which the regenerant population was established. The high values for genetic identity among regenerant and seed-grown plant populations are in accordance with the low levels of interpopulation genetic divergence. In somaclones of C. peruvianus, morphological divergence was achieved within a short time but was not associated with any isozyme changes and also was not accompanied by biochemical genetic divergence.     

In vitro propagation through axillary bud multiplication in different mulberry genotypes

Axillary buds from 5 genotypes of mulberry belonging to 4 species were cultured on modified MS basal medium. A total of 30 media combinations were tried for all the genotypes. The response of axillary buds and the requirement for growth regulators varied with genotype. In Morus indica BAP (0.25–0.5 mg/l), and in M. alba and M. rotondifolia GA₃ (0.5–1.0 mg/l)were found to induce sprouting. Two genotypes of M. bombycis, namely Schimanochi and Mizusawa, developed healthy shoots on the incorporation of 2,4-D (0.5–1.0 mg/l) and BAP (0.5–2.0 mg/l), respectively. IBA (0.5 mg/l), along with cytokinin/auxin/gibberellin, had no effect on bud growth but helped root induction. Shoots developed from the axillary buds were further multiplied as nodal explants. MS basal medium supplemented with 0.5 mg/l IBA and LS vitamins was found best to produce healthy plantlets in all the genotypes. An average 89% survival was observed on transferring the plantlets to soil.Abbreviations MS Murashige and Skoog (1962) - LS Linsmaier and Skoog (1965) - IBA 3-indole-butyric acid - GA₃ Gibberellic acid - BAP 6-Benzylaminopurine - Kn Kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid     

Plant regeneration from callus cultures of Valeriana wallichii DC.

Petiole expiants of Valeriana wallichii. DC., a threatened medicinal plant, were used for inducing callus. Optimum callus formation was observed on Murashige and Skoogs' (1962) medium supplemented with 3.0 mg/l NAA and 0.25 mg/l Kn. Shoot regeneration was achieved upon transferring the callus to medium containing 1.0 mg/l Kn and 0.25 mg/l NAA. Complete plantlets were obtained on the same medium or upon transfer of the regenerated shoot buds to medium containing 5.0 mg/l Kn and 1.0 mg/l IAA. Nearly a thousand callus regenerated plants were successfully transferred to the field following previously standardized hardening procedures.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichloro phenoxyaceticacid - 2iP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's medium (1962) - NAA -napthalene aceticacid - Z Zeatin     

High frequency plant regeneration of Cymbopogon martinii (Roxb.) Wats by somatic embryogenesis and organogenesis

Embryogenic callus cultures were obtained upon repeated sub-culture of non-embryogenic callus from nodal segments of Cymbopogon martinii (Roxb.) Wats. Murashige and Skoog's medium supplemented with 1mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l kinetin and Linsmaier and Skoog's medium supplemented with 2mg/l 2,4-dichlorophenoxyacetic acid and 0.4 mg/l kinetin were used as maintenance media for non-embryogenic and embryogenic cultures, respectively. Plant regeneration occurred through organogenesis in MS basal media containing 2 mg/l kinetin, 1 mg/l 6-benzylaminopurine, 0.2 mg/l biotin, 0.2 mg/l Ca-pantothonate and 0.1 mg/l napthalene acetic acid. Embryogenesis was induced in LS medium supplemented with 1 mg/l kinetin, 0.5 mg/l 6-benzylaminopurine and 0.1 mg/l 3-indole acetic acid. Plant regeneration at high frequency was recorded both through organogenesis and embryogenesis in different passages of long term callus cultures.Abbreviation MS Murashige and Skoog medium - LS Linsmair and Skoog medium - BAP 6-benzylaminopurine - kin kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - CH Casein hydrolysate - CaP calcium pantothonate - NAA napthalene acetic acid     

Plant regeneration from hordeum spontaneum and hordeum bulbosum immature embryo derived calli

Callus cultures were initiated from isolated immature embryos of Hordeum spontaneum and Hordeum bulbosum on MS or B5 basal medium supplemented with 2 mg/1 2,4-D. Shoot regeneration occurred on transfer of tissue to media containing 1 mg/1 IAA and 1 mg/1 zeatin. The regenerated shoot buds were rooted on basal medium without hormones. The in vitro regenerated plants were transferred to soil and were grown to fertile mature plants. A low percentage of albino plants was observed among the regenerated plants. No major differences were detected between the two species in respect to their potency to form callus or to the regeneration capacity. The regeneration capacity of calli decreased gradually and ended after 6 months in culture.Abbreviations IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - MS Murashige and Skoog medium     

Regeneration of plantlets from the callus of stem segments of adult plants of Ficus religiosa L.

Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS Murashige and Skoog (1962) - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Callus induction and plant regeneration in Panicum bisulcatum and Panicum milioides

Surface sterilized seeds and mesocotyls from sterile seedlings from Panicum bisulcatum Thumb., as well as basal parts of leaves and mesocotyls from sterile seedlings, and seeds from Panicum milioides Nees ex. Trin were used as explants to induce callus on a Murashige and Skoog medium supplemented with 2.5 to 10 mg/l of 2,4-D. Subculturing of the white callus from P. milioides and of the brown callus from P. bisulcatum on a medium containing 0.1 mg/l 2,4-D and 10 g/l sucrose led in both species to the appearance of green structures from which plants could be regenerated. Plants were regenerated by an organogenetic process in P. milioides, while P. bisulcatum plants were regenerated both via organogenesis and somatic embryogenesis. 1032 and 94 plants, from P. bisulcatum and P. milioides, respectively, were transferred into soil, and about 90% of them were grown to maturity and set seeds.Abbreviations MS Murashige and Skoog medium (15) - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid     

Plant regeneration from callus cultures of Durum and emmer wheat

Callus cultures were initiated from isolated mature embryos of Triticum turgidum L. Thell ssps durum and dicoccum on a basal medium supplemented with 2,4-D, 2,4,5-Cl₃POP or 2,4-D+CM. Shoot bud regeneration was observed on 2,4,5-Cl₃POP medium. In both the cultivars of durum, further development of shoot buds occurred on transfer of tissues to basal medium whereas in dicoccum basal medium supplemented with coconut milk or coconut milk with NAA (0.2 mg/l) was necessary. The regenerated shoot buds were induced to root on basal medium supplemented with NAA. The in vitro obtained plants were transferred to soil and successfully grown to maturity. Chlorophyll variants were observed among the regenerated plants of dicoccum.Abbreviations BA benzyladenine - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,iP 6---dimethylallylamine purine - IAA indoleacetic acid - NAA -naphthalene acetic acid - Kn kinetin - 2,4,5-Cl₃POP 2,4,5-trichlorophenoxypropionic acid - MS modified Murashige and Skoog's medium - RH relative humidity - Z zeatin     

Plant regeneration and initiation of cell suspensions from root-tip derived callus of Oryza sativa L. (rice)

Root-tip derived suspended callus of Oryza sativa cv. Thaipei showed the capacity for plant regeneration via organogenesis. Cell cultures were induced in liquid Murashige-Skoog medium containing 2 mg/l 2.4-dichlorophenoxyacetic acid. Dicamba or Picloram were effective for induction of organogenesis. Shoots and roots differentiated following subculture on medium lacking auxins but containing kinetin. At 1 and 4 mg/l Dicamba and 1 mg/l Picloram normal green plants were regenerated whereas with 7 mg/l Dicamba in the medium only albino plantlets were obtained. Regenerated plantlets were grown to maturity and set seed. Cell suspension cultures, initiated from the root-tip derived calli, provided suitable material for protoplast isolation.Abbreviations BM Basic medium - 2.4 -D 2,4-dichlorophenoxyacetic acid - Dicamba 3,6-dichloro-2-methoxy benzoic acid - Picloram 4-amino-3,5,6-trichloropicolinic acid     

Interspecific somatic hybridization between lettuce (Lactuca sativa) and wild species L. virosa

Somatic hybrids between cultivated lettuce (Lactuca sativa) and a wild species L. virosa were produced by protoplast electrofusion. Hybrid selection was based on inactivation of L. sativa with 20mM iodoacetamide for 15 min, and the inability of L. virosa protoplasts to divide in the culture conditions used. Protoplasts were cultured in agarose beads in a revised MS media. In all 71 calli were formed and 21 of them differentiated shoots on LS medium containing 0.1mg/l NAA and 0.2mg/l BA. Most regenerated plants exhibited intermediate morphology. These plants were confirmed as hybrids by isoenzyme analysis. The majority of somatic hybrids had 2n=4x=36 chromosomes, and had more vigorous growth than either parent. Hybrids had normal flower morphology, but all were sterile.     

Micropropagation of an endangered plant species: Coronopus navasii (Brassicaceae)

Plantlets of Coronopus navasii, an endangered species from SE Spain, were successfully regenerated from shoot and root segments excised from young seedlings. Initiation of multiple buds and development of leaves were obtained in MS modified medium plus l mg/l BAP and 0.1 mg/l NAA. Rooting was achieved by transfer of the isolated shoots to fresh MS medium without plant growth regulators. Plant survival of 47% was obtained six weeks after removal from in vitro culture conditions.Abbreviations BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - MS medium see Murashige and Skoog 1962     

Somatic embryogenesis and plantlet regeneration in the soybean Glycine max

A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. TGM119 were immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form calli. Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was filtered to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging drop cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the calli. Calli containing somatic embryos were transferred into liquid medium and incubated under low light conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask.Abbreviations KT kinetin - CM coconut milk - BA benzyladenine - NAA napthalene acetic acid - IAA indole acetic acid - 2,4-D 2,4 dichlorophenoxy acetic acid - MS Murashige and Skoog medium