Plant regeneration from leaf explants of Glycine clandestina Wendl

Fully fertile plants with the expected chromosome number 2n=40 were regenerated from excised leaf sections of Glycine clandestina. Shoots formed directly on the explants through organogenesis. Utilizing the same media and procedures fully fertile plants were also regenerated from cotyledon and hypocotyl tissue of the same G. clandestina accession. We were not successful in regenerating plants from root tissue of G. clandestina.Abbreviations 6-BA 6-Benzyladenine - FAA Formalin - NAA Naphthalenacetic acid - IAA Indoleacetic acid - Na₂EDTA Disodium salt Ethylenediamine tetraacetic acid - Fe-NaEDTA Ferric-Sodium salt Ethylenediamine tetraacetic acid     

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     

Hybrids between tobacco crown gall cells and normal somatic cells of Atropa belladonna

Protoplast fusion of Nicotiana tabacum (B6S3) crown gall cells and Atropa belladonna leaf mesophyll cells was carried out. Hybrids were selected for their capacity to grow on hormone-free media and to green in light. Shoots incapable of rhizogenesis were regenerated on the same media and grafted onto normal plants of different species. 57 hybrid cell lines differing in their genetic constitution were produced. Analysis of hybrid lines involved the determination of the lysopine dehydrogenase (LpDH) activity and the molecular forms of esterase and amylase, a restriction analysis of chloroplast DNA and a cytogenetic study.Abbreviations LS-H Linsmaier and Skoog (1965) hormone-free medium - LpDH lysopine dehydrogenase     

Plant regeneration from hypocotyl and petiole callus of Trifolium pratense L.

Red clover (Trifolium pratense L.) seedlings were screened for the ability to regenerate plantlets from hypocotyl-derived callus tissue. Media sequences described by Beach and Smith (1979) and Collins and Phillips (1982) and a variation using media from both sequences were tested. Plantlets were regenerated from three out of 642 genotypes. In all three cases, callus was initiated on B5C medium and regeneration was accomplished on SPL medium. Attempts to regenerate plants from petiole-derived callus tissue have so far been successful only with regenerants of clone F49. Petiole callus from epicotyl-derived F49 plants proved to be non-regenerative. Pollen viability varied significantly among individuals regenerated from callus cultures of clone F49. Root tip squashes from F49 regenerants revealed the normal diploid chromosome number (2n=14). The frequency of regeneration within progeny from reciprocal crosses between F49 regenerants and several non-regenerative genotypes was 29%.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - KN kinetin - NAA -naphthaleneacetic acid     

Organogenesis in cell cultures of leafy spurge (Euphorbiaceae) accessions from Europe and North America

Plants were regenerated from leafy spurge (Euphorbia esula L.) cell suspensions obtained from stem callus. A North Dakota accession was highly regenerable, but two accessions from Oregon and Austria formed only a few plantlets. Organogenesis occurred in media without growth regulators, under fluorescent lights (30 to 90 E m^–2 s^–1, 14 h photoperiod). Organogenesis was greatest in larger size clumps subcultured during maximum cell growth into media containing a reduced:oxidized nitrogen ratio of 33:67. Roots formed first and some clumps produced shoots. Organogenic suspension cultures also were initiated from hypocotyl and root segments of germinated seedlings, directly in liquid medium. Plantlets of the North Dakota accession formed in vitro adapted to greenhouse conditions. They were phenotypically similar to the parent plants.     

Regeneration by somatic embryogenesis of triploid plants from endosperm of walnut,Juglans regia L. cv Manregian

Plants were regenerated by somatic embryogenesis from endosperm tissue of open-pollinated seeds of Juglans regia L. cv Manregian. These plants were obtained by growing endosperm tissue on media similar to those used for plant regeneration from walnut cotyledons (Tulecke and McGranahan 1985). The plants appear morphologically uniform and have a triploid chromosome number of 3n=48. Nine plants have been grown to a young sapling stage in soil. This embryogenic line from endosperm has been maintained in culture for two years by the process of repetitive somatic embryogenesis.     

Characterization of alfalfa (Medicago sativa L.) plants regenerated from selected NaCl tolerant cell lines

Selection of stable, NaCl tolerant alfalfa (Medicago sativa L.) cell lines was accomplished by a step-up selection procedure, whereby cell lines originally selected for tolerance at 0.5% NaCl were subsequently selected at 1.0% NaCl. Sodium chloride tolerant cell lines retained tolerance following four subcultures (16 weeks) on control media (0% NaCl). Plants were regenerated from selected NaCl tolerant cell lines of three initial genotypes, one diploid (2n=2x=16) and two tetraploids (2n=4x=32). In addition, plants were regenerated from control cell lines maintained on 0% NaCl media for the same duration. Plants regenerated from NaCl tolerant cell lines were characterized by extensive somaclonal variation compared to plants regenerated from control lines. Morphologically, all plants regenerated from NaCl tolerant cell lines are abnormal and many (44.7%) were extreme dwarfs (maximum height of 5 cm). The grossly aberrant phenotypes prevented an in-depth characterization of many of the plants regenerated from NaCl tolerant cell lines. Most plants regenerated from NaCl tolerant cell lines had unbalanced polyploid chromosome sets with the most extreme cytogenetic variant having 106 chromosomes. In contrast, 98.5% of the plants regenerated from control cell lines were euploid (85% were tetraploid, 15% were octoploid). Isozyme phenotypes of the plants from NaCl tolerant cell lines were also extensively altered, compared to plants from control cell lines. In vitro NaCl tolerance was maintained following plant regeneration for nine of the 12 regenerants tested. Importantly, whole plant NaCl tolerance was expressed in two of the seven regenerated plants tested at the whole plant level; however, only one of these plants has flowered and is both male and female sterile; the other plant has never flowered. Although NaCl tolerant alfalfa cell lines are efficiently selected, the extensive somaclonal variation that accompanied the selection was a deterrent to successful recovery of heritable NaCl tolerance.     

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     

Clonal propagation of Cephaelis ipecacuanha

Shoot cultures of Cephaelis ipecacuanha A. Richard were established by using shoot tips as initial explants. Multiple shoots were obtained from node segments upon culture on B5 medium supplemented with NAA-BA (0.01–3, 5 mg/l). These shoots were rooted on B5 and 1/2 MS media containing IAA or NAA, and the regenerated plants were transferred to soil and grown in a greenhouse. The emetic alkaloids of the regenerated plants, mother plants and leaves of shoot cultures were analyzed by TLC and HPLC. Seven months of growth under greenhouse condition, the contents of the emetic alkaloids in the regenerated plants were comparable to those of the mother plants.Abbreviations B5 Gamborg B5 (1968) medium - MS Murashige-Skoog (1962) medium - 1/2 MS a half strength MS medium - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - Kin kinetin - BA 6-benzylaminopurine - TLC thin layer chromatography - HPLC high performance liquid chromatography     

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.     

Plant regeneration from immature embryos of peanut

Plant regeneration from immature embryos of peanut (Arachis hypogaea L.) can be accomplished through somatic embryogenesis. The highest frequency of somatic embryo formation occurred on B5 medium plus 0.5–1.0 mg/l picloram. Shoots and plants developed from the somatic embryos only after extended culture on basal medium. Shoots were excised from thick embryonic roots and rerooted on Murashige and Skoog medium containing half the normal concentration of inorganic salts. This technique should be useful for the production of interspecific hybrid plants from immatureArachis embryos.     

Regeneration and rapid multiplication of Bowiea volubilis Harv. in tissue culture

Callus cultures were raised from segments of inflorescence axis of Bowies volubilis Harv. (Liliaceae) on a modified basal medium of Murashige and Skoog (1962) (MS) supplemented with 1 mg 1^–1 2,4-dichlorophenoxy acetic acid (2,4-D) + 15% v/v coconut milk. Shoot primordia developed after 2–3 subcultures when auxin concentration was lowered. Rooted bulbous plants were obtained in MS medium without any hormone.Shoots were induced directly on scales of regenerated bulbs used as secondary explants on modified MS medium supplemented with 2 mg 1^–1 6-benzylaminopurine (BAP) + 0.05 mg 1^–1 2,4-D. These shoots grew and multiplied rapidly in shake culture using liquid MS medium. From each scale, 400–600 bulblets could be produced in 16–20 weeks period. Eighty percent of plants have survived on transferring to potted soil.     

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     

Plant regeneration from shoot callus of rosewood (Dalbergia latifolia Roxb.)

In vitro propagation of trees using cell, tissue and organ culture is a fast emerging area. We report here the clonal propagation of Indian rosewood (Dalbergia latifolia Roxb.) from shoot callus cultures of 5 year old trees. Bud regeneration was obtained on MS media supplemented with BA and NAA. About 35% of the cultures showed organogenesis. Shoots measuring about 3–5 cm can be excised and rooted in White's medium supplemented with 1–2 mg/L IAA. Rooted plants were successfully established in soil.Abbreviations BA Benzyladenine - CM Coconut milk - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - NAA Naphthaleneacetic acid - PVP-360 Polyvinyl pyrrolidone     

Haploid plants regenerated via anther culture in wild barley (Hordeum spontaneum C. Kock)

Summary Androgenic plants have been obtained via anther culture in four natural populations of Hordeum spontaneum. Microscopic observations revealed that androgenesis started with the formation of two vegetative-type nuclei derived from the mitotic division of the uninucleate microspores. In this species androgenesis was affected by the type and concentration of the sugars added to the culture medium: the highest response (17% of callusing anthers) was observed on media containing 80 g l^–1 maltose. The highest production of androgenic plants (per 100 anthers, 5.9 green and 4.3 albino plants) was obtained from callus grown on these same media. About half of the green plants regenerated were haploid, while the others were diploid and set seed.Abbreviations IAA indolacetic acid - BAP 6-benzylaminopurine     

大果良种沙棘愈伤组织诱导及植株再生的研究

大果良种沙棘的幼嫩茎尖,茎段外植体接种在MS,1／2MS附加不同浓度配比的IAA,IBA,BA,NAA培养基上可诱导茎尖及腋芽生长,将诱导产生的无性系芽接种在MS或1／2MS附加BA0．3－0．5mg/L,NAA0．05mg/L的培养基上可形成丛生芽,同时在小叶片和嫩茎上诱导产生愈伤组织,继续培养愈伤组织表面形成大量的绿色突起,进一步分化成不定芽,在相同培养基上,不定芽上可直接产生不定芽,从而形成多达数百个的不定芽族,不定芽长至3cm时切下转至1／2MS附加IAA或IBA 0．2mg/L的培养基上可生根而形成完整 的再生植株。     

Studies on Plant Regeneration from Mesophyll Protoplasts of Solanum tuberosum L.

Protoplasts from potato mesophyll of two strains (Solannum tuberosum L. cv. Xiao Yie Zi x Duo Zi Bia and Solanum tuberosum L. cv. Wu Meng 601) were induced to callus in culture medium of protoplasts. The callus derived from mesophyll protoplasts were transferred to MS medium with 2 mg/l ZT+0.1 mg/L IAA. Shoots regenerated from the callus were detected after 70 days of culture.The shoots which had grown to a height of 2–3 cm were transferred to MS medium with 0.05 mg/L NAA. Roots were coming out in a few days.Complete plantlets were achieved. Stern segments with 1–2 leaves were then transferred to a mixture of sterilized soil and grown, and produced tuber.     

In vitro regeneration of commercial cultivars of subterranean clover

Regeneration of subterranean clover (Trifolium subterraneum L.) was achieved by both shoot organogenesis and somatic embryogenesis. Shoots derived via organogenesis were initiated from the hypocotyls of mature imbibed seed. The hypocotyl, including the emerging radicle, was sliced longitudinally into two halves and cultured on shoot induction medium. After 30 days, adventitious shoots were formed from the hypocotyl region while the radicle showed no development. Shoots were then subcultured onto shoot multiplication medium and finally onto a root initiation medium. Histological studies revealed that shoots arose de novo and did not originate from pre-existing meristems. In the second regeneration protocol, shoot apical meristems from young seedlings were induced to form callus. Following four to six weeks culture in the dark, somatic embryos appeared spontaneously on the calli. A majority of embryos had a well-defined root pole, two cotyledonary lobes, and were capable of germination, albeit at a low frequency. Regenerated plants obtained from both protocols appeared phenotypically normal.     

Regeneration of haploid and dihaploid plants from protoplasts of supersweet (sh2sh2) corn

Summary Plants were regenerated from maize (Zea mays L.) protoplasts isolated from embryogenic cell suspensions. The donor maize suspension cultures were established from friable callus initiated from microspores of a commercial supersweet hybrid (sh2sh2). The frequency of cell colony formation was higher when protoplasts were cultured on feeder layers of maize cells as compared with a liquid thin layer method. It was demonstrated that haploid and dihaploid soil-grown plants can be regenerated from maize protoplasts isolated from haploid cell cultures.     

Obtaining a Transgenic Upland Cotton Harboring Two Insecticidal Genes

pBinLK carried two insecticidal genes, pea lectin (P-Lec)gene and soybean Kunitz trypsin inhibitor (SKTI) gene, were successfully transferred into 4 upland cotton ( Gossypium hirsutum L. ) cultivars, "Xinluzao-1 ", "Xinluzhong-2", "Jihe-321" and "Liao-9" via Agrobacterium-mediated transformation. Hypocotyl segments from aseptic seedlings were used as receipient. After co-cultivation of hypocotyl segments with A. tumefac/ens (Smith et Townsend) Conn, kanamycin-resistant calli were screened, and somatic embryos and regenerated plants were obtained through various media. Transgenic cotton plants harboring two insecticidal genes were confirmed by NPT-Ⅱ ELISA, PCR and PCR Southern. rllae results of bioassay demonstrated that the transgenic plants showed significant resistance to the larvae of cotton bollwonn (Heliothis armigera Hubner).