TEMPORAL COMPETENCE OF EMBRYONIC PINUS PONDEROSA COTYLEDONS TO FORM MULTIPLE BUDS IN VITRO

To investigate temporal changes occurring during tissue maturation and loss of organogenic competence, embryos of Pinus ponderosa Laws, were excised from seed and placed horizontally on medium in sterile culture. On a hormone-free basal medium, cotyledons differentiated and failed to form multiple buds. When placed on a benzyladenine (BA)-containing medium, the lower cotyledons in contact with the medium formed multiple buds while those elevated above the medium did not. Cotyledons became incompetent to form buds when embryos were initially placed on basal medium for 2 days before being transferred to the BA-supplemented medium. In order to initiate buds, cotyledons of newly excised embryos had to be exposed to BA for at least 3 days. Exposure to BA for longer than 7 days did not significantly increase the number of cotyledonary buds. Cotyledons elevated above the BA-supplemented medium retained organogenic competence to form buds for up to 14 days but did not form buds unless the cotyledons were placed in contact with the medium. The presence of BA is required to retard cotyledon maturation and sustain tissue competence to initiate buds.     

The effect of carbon source on callus induction and regeneration ability in Pharbitis nil

Flower buds, cotyledons and hypocotyls of Pharbitis nil were used as plant material. Flower buds (1–2 mm long) were excised from 3-week-old plants, grown in soil. Cotyledons of 7-day-old sterile seedlings were cut into 25 mm² squares cotyledons whereas hypocotyls were cut to 1 mm long fragments. Explants were transferred into Petri dishes containing the Murashige and Skoog medium (MS), supplemented with either BA (11 μM·L⁻¹) alone or BA (22 μM·L⁻¹) and NAA (0.55 μM·L⁻¹), and different sugars: sucrose, fructose, glucose, mannose or sorbitol (autoclaved or filter-sterilized). Addition of glucose instead of sucrose to the medium stimulated the induction of callus on flower buds and cotyledonary explants, but inhibited its growth on fragments of hypocotyls. The medium supplemented with fructose (especially filter-sterilized) stimulated the development of flower elements. Organogenesis of shoots and roots on explants was also observed. Flower buds and hypocotyls were able to regenerate both organs. Addition of fructose or glucose to the medium stimulated the organogenesis of shoots, whereas root organogenesis was inhibited on all explants used. Sorbitol strongly inhibited both induction of callus and organogenesis on all explants used.     

Optimization of in vitro bud induction and plantlet formation from mature embryos of Aleppo pine (Pinus halepensis Mill.)

Summary Studies were undertaken to optimize tissue culture conditions for micropropagation of Aleppo pine (Pinus halepensis Mill.) from mature embryos and various explants of the embryo. Over 90% of the embryo explants gave rise to adventitious buds within 4 wk. Intact embryos were the most suitable explants for shoot bud induction. Both isolated cotyledons and hypocotyls produced adventitious buds, but these developed slowly and failed to elongate. N⁶-Benzyladenine (BA) alone at 5.0μM was the most effective cytokinin when added to gelled to gelled von Arnold and Eriksson’s (AE) medium containing 3% sucrose. Adventitious bud development was achieved on hormone-free AE medium, and shoot elongation was optimum on three quarter-strength Bornman’s MCM medium, with 0.1% conifer-derived activated charcoal. Shoots were multiplied on three-quarter strength MCM medium, containing 5μM BA. To induce adventitious roots on the elongated shoots, pulse treatment with 1 mM IBA for 6 h, followed by the transfer of the shoots to sterile peat:vermiculite (1:1) mixture, was beneficial. After acclimatization for 3 to 4 wk under mist, almost all the rooted shoots could be transplanted successfully to the greenhouse, where the plants exhibited normal growth habit. Histologic studies on the ontogeny of adventitious shoot formation from mature embryo explants revealed temporal structural changes in different parts of the explant. Induction of mitotic divisions on the shoot-forming medium resulted in the formation of meristemoids in the epidermal and subepidermal layers of the explant, located initially at both the tips of the cotyledons and the axils of adjacent cotyledons. Shoot buds arising in the axils of adjacent cotyledons were due to new cell division and not to any preexisting meristem.     

In vitro vegetative propagation of Chinese cabbage

Explants from cotyledons, axillary buds, inflorescence stems and flower buds of Brassica campestris ssp. pekinensis (Lour.) Olsson (Chinese cabbage, cv. Wongbok) were cultured on MS medium with growth regulators. Multiple shoots were obtained from cotyledons, axillary buds and flower buds but not from inflorescence stems. Propagation of shoots from cotyledons was more successful than from axillary buds and flower buds. The vegetative propagation rates varied amongst clones derived from cotyledons of the same cultivar and seed lot. The propagation rates of the cotyledon-derived material followed a normal distribution with an average propagation rate of 2.6 shoots per two weeks subculture when cultured on MS media plus 44.4 m benzyladenine (BA) and 14.8 m -indolebutyric acid (IBA). Shoots from three clones were cultured on MS medium with nine different concentrations of BA. The concentration of BA which promoted the highest rate of shoot propagation varied for the three clones and was in the range 44.4 to 177.6 m.     

In vitro shoot organogenesis from excised immature cotyledons and microcuttings production in Stone Pine

Adventitious buds were induced on isolated immature cotyledons of Pinus pinea L. in the presence of benzyladenine (BA). The response to different BA concentrations also depended upon the culture medium used (modified MS, SH and GD). A wide range of BA concentrations (5, 25 or 50 M) can be applied to the GD and SH media, which are the media with the lower nitrogen content, without damaging effects. In the MS medium, which has the highest nitrogen concentration, the range of BA that can be applied was narrower and the highest BA concentration was lethal. The addition of indolebutyric acid (0.05, 0.25 or 0.5 M) to the induction medium, decreased the response of cotyledons. The increase in the concentration of sucrose from 3% to 5% did not increase the number of responding cotyledons. The addition of activated charcoal (0.5 and 3 g l^-1) or indolebutyric acid (1.5 or 3 M) did not speed up the elongation of explants. Elongation of the buds produced shoots with two different phenotypes, each phenotype having a different multiplication rate.Abbreviations BA benzyladenine - GD Gresshoff & Doy medium - IBA indolebutyric acid - MS Murashige & Skoog medium - SH Schenk & Hildebrandt medium     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.     

In vitro flowering and pod formation from cotyledons of groundnut (Arachis hypogaea L.)

Summary The response of groundnut cotyledons to the presence of various growth regulators in concentrations from 0.1 to 5 mg/l has been studied in detail using several genotypes of groundnut on two different media. Cotyledons with embryo axis, cultured on Blaydes' medium with cytokinins, produced shoots, in the axils of which 2–7 flower buds could be seen. The frequency of flower bud induction in general increased with increasing concentrations of cytokinins, the optimal levels being 3 mg/l of KN or 4 mg/l of BAP. Cotyledons without embryo axis, cultured on Blaydes' medium with BAP (0.5 mg/l), produced a cluster of flower buds directly, ranging in number from 8–28, without any vegetative growth. Excised embryo axes cultured on the same medium gave plantlets without flower buds. The growth regulators IAA, NAA, GA₃ and ABA failed to induce flower buds in independent treatments. However, lower concentrations of IAA and NAA in combination with cytokinins exerted a positive influence on flowering. The blooming of the flower buds was facilitated on media supplemented with low concentrations of cytokinins. Six percent of the induced flowers resulted in gynophore development and ultimately formed pods when cultured under complete dark conditions in modified MS medium supplemented with kinetin.     

Direct shoot organogenesis and plant regeneration in safflower

Summary  Adventitious shoot buds were induced directly on the adaxial surface of the cotyledons of eight safflower cultivars after 14 d of culture initiation on Murashige and Skoog's (MS) medium supplemented with various levels of 6-benzylaminopurine (BA). Maximum shoot organogenesis of 54.4% with 10.2 shoots per responding cotyledon was obtained with 8.87 μM BA in the cv. S-144. Regenerated shoots were classified into three groups on the basis of their morphological features and were found to be correlated with the levels of BA. The highest number of normal shoots was obtained from 2.2 μM BA treatment. The regenerated shoots of group I (normal shoots) were rooted on half-strength MS medium supplemented with 5.3 μM α-naphthaleneacetic acid, 3% sucrose and 0.8% bacto-agar. Rooted plantlets were successfully transferred to soil and appeared morphologically normal. Histological studies revealed that shoot buds originated adventitiously from subepidermal cells.     

Genotypic response of cowpea Vigna unguiculata (L.) to in vitro regeneration from cotyledon explants

Summary A plant regeneration system applicable to 17 cowpea genotypes was developed. Cotyledons were initiated on 1/3 MS medium containing 15 to 35 mg N⁶-benzyladenine (BA) per 1 (66.6 to 155.3 μM) for 5 to 15 d. For shoot regeneration, the explants were transferred to a medium containing 1 mg BA per 1 (4.4 μM). Within 1 wk, shoot formation was visible at the proximal end of the cotyledons. Regeneration percentages (1% to 11%) and the numbers of shoots (4 to 12 per explant) were significantly influenced by genotype. Culture duration and BA concentration in the initiation stage significantly affected regeneration capacity. Explants initiated on media containing 15 mg BA per 1 for 5 d resulted in the highest percentage of explants capable of regeneration. Conversely, the highest number of shoots was obtained from explants initiated on media supplemented with 35 mg BA per 1. Whole plants were obtained on a plant growth regulator-free medium. To our knowledge, this is the first report of plant regeneration from U.S. commercial cowpea cultivars and breeding lines. This system is adaptable to diverse cowpea genotypes and will facilitate cowpea genetic transformation. Published with the approval of the Director of the Arkansas Agricultural Experiment Station.     

In vitro micropropagation of the macarronesian evergreen treePersea indica (L.) K. Spreng

Summary Shoot propagation ofPersea indica (L.) K. Spreng was achieved using seedling axillary buds cultured on MS (Murashige and Skoog, 1962) medium with 1 mg/l (2.8 μM) N⁶-benzyladenine (BA). Forty percent of the obtained shoots did not elongate, but showed bud proliferation, which was maximal (three axillary buds per shoot) at the end of the seventh subculture. Sixty percent of the shoots elongated, did not show bud proliferation, and formed calluses at their base. Successful rooting (84.6%) was achieved dipping the base of each elongated shoot in 3 g/l (16.11 mM) indolebutyric acid (IBA) for 1–2 s, and transferring to half strength MS medium without growth regulators. These shoots presented an acclimatization success of 100%. Results suggest that micropropagated elongated shoots ofP. indica can be adequately used in reforestation programs.     

提高西瓜离体培养植株再生效率的研究

本文以“京欣1号”母本和“伊选”西瓜4天苗龄子叶为外植体,研究离体培养植株高频率再生体系。结果表明：“伊选”子叶远轴端外植体的再生频率仅为10％,子叶近轴端外植体在5mg／LBA 0．1mg／L IAA的激素组合下植株再生频率为100％,平均每个外植体的丛生芽数在所有组合中最多,为10.3个;“京欣1号”母本子叶近轴端外植体在2mg／LBA 0．5mg／L IAA激素组合下植株再生频率为100％,平均每个外植体的丛生芽数在所有组合中最多,达6．9个。本试验条件下,子叶近轴端外植体接种4天即分化出不定芽,至再生苗的移栽仅需40天,在MS 0．1mg／L NAA的生根培养基上的生根率为97．3％,移栽成活率达98．5％。     

Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Capsicum annuum L.

 A highly efficient three-stage protocol for the regeneration of chilli pepper (Capsicum annuum L.) from cotyledon explants was developed. This protocol used PAA in both the shoot-bud induction medium and the medium for elongation of the shoot buds. A superior medium for the induction of buds from the cotyledons was MS medium supplemented with BA (5 or 7 mg/l) + PAA (2 mg/l). Buds were elongated during the second stage on medium containing BA (2 or 5 mg/l) + PAA (2 mg/l). On this medium most of the buds elongated, and their number also increased due to the formation of new buds; bud elongation was achieved in 100% of the cultures provided the buds were induced in the primary stage on a medium supplemented with BA+PAA. The shoots that elongated in the second-stage rooted at 100% frequency on a medium supplemented with NAA (1 mg/l). The complete plantlets with well-developed root and shoot systems were transferred to field conditions where they grew to maturity, flowered and fruited normally. While shoot-bud induction from the cultured cotyledons was also observed on media supplemented with BA (5 or 7 mg/l) alone or in combination with IAA (0.2–2 mg/l), buds induced on these media were often distorted, with most not developing into normal shoots in the second-stage subculturing; a rosette of buds was seen in the second stage subculturing. On the other hand, PAA in combination with BA in the primary induction medium and second-stage medium promoted normal development and the elongation of shoot buds. Received: 28 July 1998 / Revision received: 22 December 1998 / Accepted: 19 February 1999     

Organogenesis and somatic embryogenesis in Codiaeum variegatum (L.) Blume cv. “Corazón de Oro”

Summary Adventive organogenesis and somatic embryogenesis were induced from leaf explants taken from in vitro or in vivo plants of Codiaeum variegatum cv. “Corazón de Oro.” Shoot multiplication occurred with N⁶-benzyladenine (BA) alone, where the simultaneous production of adventitious buds and somatic embryos occurred at the fourth subculture, and on leaves not in contact with the medium. A medium with BA and 2,4 dichlorophenoxy acetic acid (2,4-D) produced the largest organogenic response, for both in vivo- and in vitro-produced explants. Somatic embryogenesis was only induced when such explants were transferred to a medium lacking 2,4-D. Thus, a medium with BA only produced the largest percentage of explants with shoots and embryos. Replacing BA with thidiazuron induced up to 100% bud regeneration on in vitro-produced explants by 60 d, but was slower for in vitro-grown explants. Both types of embryos exhibited growth arrest that was partially overcome by transfer to hormone-free basal medium with activated charcoal. Rooted plants from all explants were successfully obtained on a medium with indole-butyric acid (IBA).     

High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad)

Cotyledons of various ages from seedlings of eight watermelon (Citrullus vulgaris) cultivars were cultured on MS medium supplemented with different combinations of phytohormones. High frequency shoot regeneration (60.0–92.0%) was induced from 5-day-old cotyledons of cultivars cultured on MS medium containing 5.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l indole-3-acetic acid (IAA). Multiple shoot buds elongated on MS medium containing 0.2 mg/l kinetin (KT) and 5–10 shoots per expiant could be recovered depending on the cultivars. Elongated shoots rooted on MS medium with 0.1 mg/l -naphthalene acetic acid (NAA). Zeatin riboside (ZT) had a similar efficiency as BA in shoot induction, and it was significantly more functional than 2-isopentenyladenine (2iP) or kinetin (KT). Cotyledons from 5-day-old seedlings were the most responsive to shoot induction.Abbreviation BA 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - KT kinetin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - ZT zeatin riboside     

Organogenesis and embryogenesis inHelianthus tuberosus and in the interspecific hybridHelianthus annuus ×Helianthus tuberosus

A method of plant regeneration from cotyledons ofHelianthus tuberosus, Helianthus annuus ×Helianthus tuberosus and for the backcross of the interspecific hybrids onH. annuus was developed. Induction of somatic embryogenesis and plantlet regeneration from anther culture of the interspecific hybridsH. annuus ×H. tuberosus is reported.Cotyledons were cultured on Murashige and Skoog basal medium (MS) supplemented with indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin) or N⁶-benzylaminopurine (BAP). Shoot regeneration occurred on most of the media tested, but the best results were obtained on media with a high concentration of cytokinins (BAP or kinetin: 4 mg l^–1) and lower concentration of auxin (IAA: 0.5–1 mg l^–1).Embryogenic callus and adventitious buds were initiated from only two anthers of the hybridH. annuus ×H. tuberosus cultured on the MS medium containing BAP (0.2 mg l^–1) and 1-naphtalenacetic acid (NAA: 0.1 mg l^–1). Prolonged culture of these embryogenic calli and buds on the original medium with successive subculture on MS basal medium without growth regulators resulted in embryo formation and shoot differentiation. The plantlets, after rooting, were established in soil.     

In vitro plant regeneration via organogenesis of cowpea [Vigna unguiculata (L.) Walp.]

Shoot regeneration via organogenesis was achieved from axenic cowpea [Vigna unguiculata subsp. unguiculata L. (Walp.) Verde.] hypocotyls and cotyledons of advanced breeding lines and varieties. Cotyledons and embryos were excised from green immature pods. The apical parts of the embryos were removed and the hypocotyls were transferred to regeneration media. Cotyledons and hypocotyls were tested on media with gradients of several hormonal and putrescine combinations. Cowpea cotyledons and hypocotyls exhibited a pattern of shoot formation that occurred in three distinct phases. Multiple shoots developed within 45 days from the wounded region of the primary hypocotyl and cotyledons in different media containing a high cytokinin concentration. The induced plant explants were then grown for 20 days in low-intensity light (10 μmol m^–2 s^–1) on the same medium and numerous shoot buds emerged de novo from the upper part of the hypocotyl and the wounded part of the cotyledons. These buds had no apparent vascular connection with the parent tissues. The plant regeneration capability of this procedure was tested with several cowpea genotypes, five of which (83D-442, 86D-1010, 93K-624, Vita 3 and Ife Brown) responded positively with shoot development and were able to form roots and whole plants. Some somaclonal variation was observed. Received: 14 June 1996 / Revision received: 14 December 1996 / Accepted: 25 January 1997     

Shoot Organogenesis from Immature Zygotic Embryo Cultures of Ginkgo biloba

Immature zygotic embryos were cultured on Murashige and Skoog's medium (MS) supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), benzyladenine (BA) and zeatin or with various concentrations of 2,4-D alone. The maximum number (8 per embryo) of adventitious buds formed from cotyledons of heart stage embryos cultured on MS medium with 1 mg dm⁻³ BA and 0.01 mg dm⁻³ NAA. The adventitious buds originated from procambial strands of immature embryo cotyledons and then developed into adventitious bud primordia within 20 d. Adventitious buds transferred to hormone free MS medium grew into shoots, but did not produce plantlets because the shoots failed to root. This revised version was published online in July 2006 with corrections to the Cover Date.     

A liquid cytokinin pulse induces adventitious shoot formation from Douglas-fir cotyledons

Summary The effects of high-concentration, 2-h liquid pulses of N⁶-benzylaminopurine (BA) and thidiazuron (TD) on adventitious bud and shoot formation were tested in cotyledons of Douglas-fir (Pseudotsuga menziesii). Seedling age proved important; on average, cotyledons from the youngest seedlings formed 10-fold more buds than cotyledons from the oldest seedlings. Optimal cytokinin concentrations for the youngest cotyledons were 400 and 800 M BA, and 100 and 200 M TD. Shoots developed best from buds induced with 300, 400, and 800 M BA. Four gelling agents were tested; BRL agarose yielded more than three times the number of buds, and Gelrite nearly twice the number of buds, as either Sigma agar or Difco Bacto-Agar. One of the best treatments (400 M BA, agarose) yielded more cotyledons with buds, and more buds per cotyledon, than when cytokinins were incorporated into the growth medium.Abbreviations BA N⁶-benzylaminopurine - TD thidiazuron - SH Schenk and Hildebrandt (1972) - NAA 1-naphthaleneacetic acid Paper 2691 of the Forest Research Laboratory, Oregon State University, Corvallis. Mention of trade names or commercial products does not constitute endorsement or recommendation for use by the authors or Oregon State University.     

Micropropagation of Camptotheca acuminata decaisne from axillary buds, shoot tips, and seed embryos in a tissue culture system

Summary Micropropagation of the anti-cancer plant Camptotheca acuminata Decaisne from axillary buds and seed embryos was investigated. Axillary buds from greenhouse seedlings required a period of culture in media free of N⁶-benzyladenine (BA) before multiple shoot induction began. Direct induction of multiple shoots on BA-containing medium resulted in high mortality of the axillary buds. Multiple shoot induction from the greenhouse axillary buds was best achieved on B5 with 4.4 μM BA+0.5μM α-naphthaleneacetic acid, forming an average of three 2-mm tall shoots per bud in 8 wk. Elongation of these multiple shoots was successful at a lower BA level (0.22 μM) on B5 medium. Both in vitro and ex vitro rooting of the microcuttings was feasible with indole-3-butyric acid in the culture media, but ex vitro rooting led to high plantlet survival. Seed embryos were not ideal explants for multiple shoot induction. Shoot tips and axillary buds of in vitro-germinated seedlings showed an optimal multiple shoot formation on B5 with 8.9 μM BA, double the optimal BA level for greenhouse axillary buds. Using axillary buds to propagate C. acuminata plants in vitro is feasible for mass propagation of desired clonal lines high in camptothecin concentrations.