Zinc deficiency and the formation of white structures in immature embryo cultures of wheat (Triticum aestivum L.)

The effect of microelements on the induction of embryogenic callus from epiblast and scutellum of wheat (Triticum aestivum L.) embryos was studied by the sequential omission of each of the microelements from Murashige & Skoog medium. Omission of iron caused a marked decrease in yield and poor shoot formation from embryogenic callus. The yield of embryogenic callus on medium without added manganese was also reduced. Omission of boron, copper-cobalt, iodine, and molybdenum had little effect on the induction of embryogenic epiblast callus. By contrast there was a marked increase in the formation of white structures on the medium without any microelements or, specifically without addition of zinc. Since the formation of typical embryoids of wheat is associated with the formation of white structures, our result highlights the importance of certain microelements on somatic embryogenesis of wheat.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium Murashige & Skoog medium     

植物激素对棉花体细胞胚胎发生的诱导及调节作用

选用11种激素研究了外源激素对棉花胚性愈伤组织增殖、胚胎发生和发育的调控作用。结果表明不同激素对棉花胚性愈伤组织增殖、胚胎发生与发育的影响不同。除2,4-D和BA对棉花胚性愈伤组织的增殖影响不大外,其他激素对棉花胚性愈伤组织的增殖均具有抑制作用,且具有一定的时间效应,同时还受基因型的影响。激素对棉花体细胞胚的形成和发育的影响极大,2,4-D既抑制了体细胞胚的形成,又抑制了体细胞胚的发育;TDZ的作用与2,4-D相似,显抑制了体细胞胚的形成,且诱导获得的体细胞胚均停留在球形胚阶段;GA也抑制了体细胞胚的形成,且不利于体细胞的成熟与萌发;BU-30对棉花体细胞胚形成与发育的影响不大。其他7类生长素类物质和细胞分裂素类物质对棉花体细胞胚的形成均具有促进作用,且依IBA、ABA、IAA、BA、KT、ZT、2iP序增强,其总胚数为对照的1．193—3．852倍;其中2iP的促进作用最大,可使产生的体细胞胚数提高2．852倍。     

Somatic embryogenesis in Asparagus officinalis can be an in vitro selection process leading to habituated and 2,4-D dependent embryogenic lines

Long-term embryogenic lines were repeatedly obtained from nine asparagus (Asparagus officinalis L.) genotypes by the selection of rare events, which consisted of the emergence of either a few somatic embryos or an embryogenic callus from a restricted area of a primary callus. In the first case, somatic embryos emerged from 1 % of calli induced with naphtaleneacetic acid and transferred to a medium without auxin. Isolated and subcultured on hormone free medium, these embryos developed habituated embryogenic lines (H lines) growing by adventive embryogenesis. In the second case, 3 % of primary calli developed then subcultured on 2,4-dichlorophenoxyacetic acid (2,4-D) produced a new type of friable and yellowish-white callus, constituted of clusters of globular somatic embryos which can be continuously maintained on 2,4-D (2,4-D lines). Among 2,4-D lines, two types were identified by subculturing them on hormone–free medium. Half of the 2,4-D lines were habituated and half were 2,4-D dependent. Most plants regenerated from H lines exhibited a strong increase in embryogenic capacity compared to control plants, unlike plants regenerated from the 2,4-D dependent lines. This increased embryogenic capacity was transmitted to the progeny as a monogenic dominant trait. H lines would therefore be issued from mutation(s) occurring in vitro, conferring both the embryogenic and habituated phenotypes. On the contrary, in the 2,4-D dependent lines, the embryogenic processes appeared to remain under exogenous auxin control and no evidence of a mutational origin could be inferred from the behaviour of regenerated plants.     

High-frequency somatic embryogenesis from germinated zygotic embryos of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and evaluation of the effects of medium strength,sucrose, GA<Subscript>3</Subscript>, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l⁻¹ 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA₃), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA₃ negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l⁻¹ BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.     

Regeneration from long-term embryogenic callus of the <Emphasis Type="Italic">Rosa hybrida</Emphasis> cultivar kardinal

Summary Media components used for three stages of development: (1) callus maintenance, (2) maturation of embryos, and (3) conversion of embryos to plants were shown to affect regeneration of plants for the commercially important red rose cultivar Kardinal. Embryogenic callus was maintained for 5yr on either Schenk and Hildebrandt’s basal salts medium (SH) supplemented with 13.6 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or Murashige and Skoog’s basal salts medium (MS) supplemented with 18.1 μM dicamba and 0.46 μM kinetin. Maturation of embryos was three times higher using callus maintained on the SH medium supplemented with 2,4-D while conversion of cotyledonary-stage embryos to plants was significantly higher (10 times) using callus that had been maintained on MS medium with dicamba and kinetin. Maximum maturation (13.5%), and conversion (15.2%), occurred when callus was cultured on MS maturation medium without hormones. Cotyledonary-stage embryos cultured on MS conversion medium supplemented with abscisic acid (5–20 μM) produced plants that survived at a significantly higher rate (two times) in the greenhouse than when embryos were cultured without abscisic acid. The highest rate of plant regeneration occurred when embryogenic callus of ‘Kardinal’ was maintained on MS medium supplemented with dicamba and kinetin, maturation of embryos occurred on MS maturation medium without hormones, and conversion of cotyledonary-stage embryos occurred on MS conversion medium supplemented with abscisic acid.     

Relationship between auxin-induced cell proliferation and somatic embryogenesis in culture of carrot cotyledons

We elucidated the relationship between cell proliferation and somatic embryogenesis in the culture of carrot cotyledons. Fresh weights of the cotyledon expiants were determined every five days while being cultured on a medium containing 2,4-D. Callus production increased exponentially from Day 20 to Day 25, showing a two-fold rate of proliferation. To examine the embryogenic potential of the callus, we pre-cultured cotyledon explants on an MS medium with 2,4-D, then transferred them to an MS basal medium at five-day intervals. Somatic embryos formed most frequently when the cotyledons were pre-cultured for 20 days on an MS medium that contained 5 μ2,4-D. The frequency of somatic embryo formation was 81%, while that of normal embryos with two cotyledons was 51% among those formed on a hormone-free medium. We used FACScan analysis to relate the embryogenic potential of the callus to the S phase in the cell cycle of cultured cells. The S phase was high after 25 days of culture on the medium with 5 μM 2,4-D. In contrast, the frequency of normal embryogenesis was higher at Day 20 of the pre-culture period. Culturing embryogenic calli on a medium with 5 μM 2,4-D was most favorable for producing somatic embryos with two cotyledons. We verified that active somatic embryogenesis was apparently related to cell division activity; somatic embryos induced from actively dividing cells were apt to accompany cotyledonary abnormality.     

High Frequency of Plant Production via Somatic Embryogenesis from Callus or Cell Suspension Cultures inEleutherococcus senticosus

Hypocotyl segments ofEleutherococcus senticosuscultured on Murashigeand Skoog's (MS) medium with 4.5 µM2,4-D produced somaticembryos directly from the surface of explants without interveningcallus formation. When these somatic embryos were subculturedto the same MS medium with 4.5 µM2,4-D, friable embryogeniccalli were formed mainly from radicle tips of somatic embryos,but at a low frequency (5%). Selected embryogenic calli weremaintained on MS agar or liquid medium with 4.5 µM2,4-D.To induce somatic embryo development, embryogenic calli andcell clumps were transferred to MS medium lacking 2,4-D. Thefrequency of somatic embryo formation differed between culturetypes with 1570 embryos formed per Petri dish from callus cultureand 5514 embryos formed per flask from cell suspension cultures.Somatic embryos formed on agar medium had larger cotyledonsthan those of embryos formed in liquid medium. GA₃treatmentwas necessary to induce germination from somatic embryos. Therate of plant conversion was 97% in somatic embryos from callusculture and 76% in embryos from liquid culture. Regeneratedplantlets were successfully acclimatized in the glasshouse.Copyright1999 Annals of Botany Company Eleutherococcus senticosus, micro propagation, somatic embryogenesis.     

Plant regeneration via somatic embryogenesis in mature embryo-derived callus culture of Sinocalamus latiflora (Munro) McClure

Somatic embryogenesis and subsequent formation of plantlets was achieved from callus cultures derived from mature zygotic embryos of Sinocalamus latiflora (Munro) McClure (Bamboo). Embryogenic callus was initiated on Murashige and Skoog's medium (MS) supplemented with 6 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg/l kinetin, 250 mg/l polyvinylpyrrolidon and 5% sucrose. Prolonged culture of the embryogenic callus on the same medium resulted in embryoid formation. The embryoids developed further to yield whole plantlets when transferred to a medium containing lower concentrations of 2,4-D (3 mg/l) and kinetin (2 mg/l).     

Plant regeneration protocol of medicinally important <Emphasis Type="Italic">Andrographis paniculata</Emphasis> (Burm. F.) Wallich <Emphasis Type="Italic">ex</Emphasis> Nees via somatic embryogenesis

Summary In vitro propagation of Andrographis paniculata (Burm. f.) Wallich ex Nees through somatic embryogenesis, and influence of 2,4-dichlorophenoxyacetic acid (2,4-1) on induction, maturation, and conversion of somatic embryos were investigated. The concentration of 2,4-D in callus induction medium determined the induction, efficacy of somatic embryogenesis, embryo maturation, and conversion. Friable callus initiated from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 2.26, 4.52, 6.78, and 9.05μM 2,4-D started to form embryos at 135, 105, 150, and 185d, respectively, after explant establishment. Callus initiated at 13.56μM 2,4-D did not induce embryos even after 240 d, whereas those initiated on MS medium with 4.52μM 2,4-D was most favorable for the formation and maturation of somatic embryos. Callus subcultured on the medium with reduced concentration of 2,4-D (2.26μM) became embryogenic. This embryogenic callus gave rise to the highest number of embryos (mean of 312 embryos) after being transferred to half-strength MS basal liquid medium. The embryos were grown only up to the torpedo stage. A higher frequency of embryos developed from callus initiated on 2.26 or 4.52 μM 2,4-D underwent maturation compared to that initiated on higher concentrations of 2.4-D. The addition of 11.7μM silver nitrate to half-strength MS liquid medium resulted in 71% of embryos undergoing maturation, while 83% of embryos developed into plantlets after being transferred to agar inedium with 0.44 μMN⁶-benzyladenine and 1.44 μM gibberellic acid. Most plantlets (88%) survived under field conditions and were morphologically identical to the parent plant.     

Increased Copper Content of the Medium Improves Plant Regeneration from Immature Embryo Derived Callus of Wheat (Triticum aestivum)

The effect of various concentrations of CuSO₄ on the induction and regeneration of embryogenic callus from immature embryos of wheat was investigated. Immature embryos of wheat cvs C-306 and R-3777 were cultured on MS medium supplemented with 2,4-D (11.3 µM) and different levels of cupric sulphate, i.e. 0, 0.1 (MS level), 0.5, 1 and 5 µM. Relatively high induction frequency of callus was obtained on MS medium supplemented with 2,4-D (11.3 µM) and 0.5 µM CuSO₄. The compact, nodular, embryogenic callus was maintained on the medium having 2,4-D (11.3 µM) and proline (86.8 µM) by regular subculturing. Plant regeneration from the embryogenic callus occurred on MS medium supplemented with NAA (1.07 µM) and BAP (44.4 µM). Regenerated plantlets were rooted on MSmedium supplemented with IAA (2.85 µM). The average number of regenerated plantlets produced from primary callus induced on 2,4-D (11.3 µM) and 5x CuSO₄ was significantly higher.     

Induction of somatic embryogenesis in Pinus roxburghii Sarg.

Embryogenic calli were initiated from embryonic explants of Pinus roxburghii using female gametophytes containing immature pre-cotyledonary embryos. Zygotic embryos were collected at different developmental stages and cultured on various media. Initiation of embryogenic calli was achieved in pre-cotyledonary zygotic embryos of a 0.1-mm to 1.2-mm embryonal head on Douglus fir cotyledon revised medium (DCR) medium supplemented with 2,4-D or NAA and BA. Embryogenic callus development was initiated from the suspensor region of immature embryos. The method of immature embryo culture was significant as rapid embryogenic callus development occurred in megagametophytes where the suspensor was stretched onto the medium from the cut micropylar end. Sixty embryogenic lines were established from 2500 explants cultured during one season. A pro-embryo with six to eight meristematic cells and suspensor of six to ten long, vacuolated cells dominated the early phase of the callus development. Cleavage polyembryony occurred in proliferating callus, constituting a method of multiplication of these somatic embryos. Somatic embryos developed to stage-I and stage-II embryos on DCR medium supplemented with 5 μM 2,4-D or 10 μM NAA. Received: 30 June 1999 / Revision received: 15 November 1999 / Accepted: 3 December 1999     

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μm kinetin or zeatin, but was highest on medium containing 4.5 μm 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 μm 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 μm 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse. Received: 14 January 1997 / Revision received: 17 June 1997 / Accepted: 5 July 1997     

Somatic embryogenesis and plant regeneration from root sections of <Emphasis Type="Italic">Allium schoenoprasum</Emphasis> L.

A protocol has been developed for somatic embryogenesis and subsequent plant regeneration in Allium schoenoprasum L. Calli were induced from root sections isolated from axenic seedlings and cultivated on media containing either Murashige and Skoog’s (MS) or Dunstan and Short’s mineral solution supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 6-benzylaminopurine (BA), 6-furfurylaminopurine (Kin) or thidiazuron (TDZ) at 1, 5 or 10 μM. The highest frequencies of callus induction were achieved on media with 5 μM 2,4-D in combination with 5 μM TDZ or 10 μM BA (78.9% and 78.4%, respectively). Calli were then transferred to 1 μM 2,4-D, where compact yellow callus turned to segmented yellowish callus with transparent globular somatic embryos at the surface. Calli that were previously grown on media with 5 μM 2,4-D in combination with 10 μM BA or 10 μM TDZ showed the highest frequencies of embryogenic callus formation (45% and 42%) as well as mean number of somatic embryos per regenerating callus. The choice of mineral solution formulation did not significantly affect callus induction or embryogenic callus formation. The embryos could complete development into whole plants on plant growth regulator (PGR)-free medium, but inclusion of Kin (0.5, 2.5 and 5 μM) in this phase improved somatic embryo development and multiplication. Subsequently transferred to 1/2 MS PGR-free medium, all embryos rooted and the survival rate of the plants in a greenhouse was 96%.     

Embryogenesis and plant regeneration from tissue culture of Canada wildrye,Elymus canadensis L.

Somatic embryogenesis and plant regeneration of Canada wildrye (Elymus canadensis L.) from tissue culture was investigated by culturing immature embryos and inflorescences on MS medium containing 2 mg/l 2,4-D. The optimum size of explants for maximum embryogenic callus formation was 1.0 to 1.5 mm for embryos and 4 to 6 cm for inflorescences. Plant regeneration from the subcultured embryogenic callus was attempted monthly using hormone-free MS medium or MS medium with 0.5 mg/1 2,4-D and 0.3 mg/l GA3. Three hundred and fifty seven plantlets were regenerated from the callus cultures of both explant sources during a six month period. Ten chlorophyll deficient plants accounting for 2.8% of the total regenerants were observed. One plant with white striped leaves survived and was found to be an octoploid.Abbreviations GA3 gibberellic acid - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant regeneration from callus and protoplasts of Brassica nigra (IC 257) through somatic embryogenesis

A method to obtain plants from embryogenic callus of Brassica nigra and protoplasts of hypocotyl expiants is described. Callus was initiated on Murashige and Skoog medium containing kinetin (kn) and 2,4-dichlorophenoxy acetic acid (2,4-D). Lowering of auxin induced embryo formation. Supplementation with gibberellic acid (GA₃) enhanced embryogenic response tenfold. Passage through liquid medium devoid of growth regulators was essential for the growth of embryos. Secondary embryos were produced on transfer to solid basal medium. Embryogenic callus retained its morphogenic ability even after 12 subcultures. Both primary and secondary embryos produced fertile plants. Hypocotyl-derived protoplasts were also regenerated to plants following the same protocol. The survival of plants on transfer to soil was about 80%. The seeds from plants derived from callus and protoplasts were viable.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - IAA indole acetic acid - kn kinetin - GA₃ gibberellic acid     

High frequency plant regeneration from immature ovule-derived embryogenic cell suspension cultures of Chelidonium majus var. asiaticum

Culture conditions for high frequency plant regeneration via somatic embryogenesis in cell suspension cultures of Chelidonium majus var. asiaticum are described. Immature ovules formed embryogenic calluses at a frequency of 40% when cultured on Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The optimum ovule size for embryogenic callus formation ranged from 1 to 1.5 mm in length. Cell suspension cultures were established from embryogenic calluses using MS liquid medium containing 4.52 μM 2,4-D. Upon plating onto MS basal medium, cell aggregates from cell suspension cultures produced somatic embryos which then developed into plantlets. Regenerated plantlets were transplanted to potting soil and grown to maturity in a growth chamber. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from embryogenic suspension cultures of Chinese yam (Dioscorea opposita thunb.)

A competent, embryogenic suspension culture of Chinese yam (Dioscorea opposita Thunb. cv. ‘Nagaimo’) has been obtained. Embryogenic callus was induced from stem segments cultured on an agar-solidified MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). One month following placement of the embryogenic callus in a liquid medium containing 2,4-D, the embryogenic tissue began to proliferate rapidly. Established suspension cultures consisted almost entirely of early-stage pro-embryos with very little contamination from non-embryogenic tissues. Under optimum conditions, suspension culture packed cell volume increased 2.5-fold per week. Following transfer of the tissue to a hormone-free medium, the embryogenic tissue developed. Globular embryos were formed within 4 weeks and addition of benzyl adenine further enhanced development and germination. Plantlets were regenerated by culturing embryos on a hormone-free agar-solidified medium.     

Somatic embryogenesis from immature and mature embryos of a minor millet Paspalum scrobiculatum L.

Immature and mature zygotic embryos of Paspalum scrobiculatum L. cv. PSC 1 cultured on MS or N₆ nutrient medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), formed embryogenic callus. Induction of embryogenic callus and subsequent somatic embryogenesis was possible at a lower concentration of 2,4-D on N₆ than MS medium. Immature embryos were highly totipotent, forming somatic embryos at a higher frequency than mature embryos. Addition of amino acids (L-proline or L-tryptophan) to 2,4-D medium resulted in significant enhancement of embryogenesis on culture of mature embryos. Silver nitrate also supported an increased frequency of embryogenesis. Thus it is possible to have high frequency of somatic embryogenesis on culture of mature embryos, which are available in abundance and with ease than immature embryos. The somatic embryos readily germinated and formed plantlets on hormone-free regeneration medium. The regenerated plantlets were successful on transfer to soil and set seed.     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

Comparative study of somatic embryogenesis from immature and mature embryos and organogenesis from leaf-base of Triticale

Immature and mature zygotic embryos of hexaploid, Triticale var. DT-46 formed an embryogenic callus, with subsequent somatic embryo formation upon subculture to MS (Murashige and Skoog, 1962) or N₆ (Chu et al., 1975) nutrient medium supplemented with various concentrations (9.0–22.5 M) of 2,4-dichlorophenoxyacetic acid (2,4-D). Of the two types of explants, embryogenic tissue from immature embryos responded at a higher frequency, to form somatic embryos over the callus surface. Leaf-base segment cultured on to 2,4-D-containing medium formed a tissue which did not form somatic embryos and instead differentiated into shoot-buds. N₆ medium proved to be more effective than MS in support of somatic embryogenesis or shoot-bud formation. Regeneration of plantlets occurred on 2,4-D-free basal medium. These in vitro-formed plantlets were successfully transferred to soil and set seed.