Clonal propagation of kurrat (Allium ampeloprasum var. kurrat)

Summary Two protocols for clonal propagation of kurrat (Allium ampeloprasum var.kurrat) using explants from the basal plates of mature plants are described. In direct formation, explants were cultured in Murashige and Skoog (MS) medium and supplemented with either benzyladenine at 0.0 or 4.4 μM, or supplemented with 7.0 μM benzyladenine and 0.1 μM naphthaleneacetic acid. Shoots appeared after 4 wk of culture. In the two-step procedure, explants were cultured first on MS medium supplemented with 1.4 μM 2,4-dichlorophenoxyacetic acid and 1.4 μM kinetin, and incubated in the dark for 4 wk. They were then transferred to MS medium supplemented with 4.4 μM benzyladenine for shoot formation. All shoots were rooted on MS medium containing 5 g·liter⁻¹ activated charcoal. Normal viable plants were successfully established in soil.     

Antifungal cinnamic acid derivatives from Persian leek (Allium ampeloprasum Subsp. Persicum)

A cinnamic imidate, (1Z,2E)-methyl 3-(-p-hydroxy-m-methoxyphenyl)-N-(-p-hydroxyphenethyl) acrylimidate, named persicoimidate (1), has been isolated and characterized from bulbs and seeds of Persian leek, Allium ampeloprasum Subsp. Persicum. Two cinnamic acid derivatives, N-feruloyl tyramine (2) and N-caffeoyl tyramine (3) were isolated from bulbs and seeds. Compound 2 has been previously reported from garlic and leek, while compound 3 is described in Allium plants for the first time. The chemical structures of the isolated compounds have been elucidated unambiguously by spectroscopic methods, including 2D NMR and MS. The isolated compounds were evaluated for their antifungal activity against four fungal pathogens, the soil-borne pathogen Penicillium italicum, the air-borne pathogens Aspergillus niger and Botrytis cinerea, and the antagonistic fungi Trichoderma harzianum to evaluate the possible involvement of such compounds in resistance to pathogen attack.     

In vitro plant regeneration from callus of shoot apices in apple shoot culture

A new reliable protocol for the induction of adventitious shoot formation and plant regeneration from apple callus has been developed. High regeneration frequency was obtained with this method in four different genotypes (Jork9, M26, Gala and McIntosh) and callus maintained regeneration ability for several months. The procedure consists of inducing vegetative shoot apices, excised from in vitro shoots, for 20 days in darkness on an MS medium without glycine, supplied with 17.8 μM BA, 2.7 μM NAA and 250 mg l⁻¹ cefotaxime. The explants are then transferred to a fresh auxin-free medium and given light. Histological studies revealed that all the regenerated shoots originated from callus. Regenerated shoots were multiplied, rooted and successfully established in soil. Received: 2 April 1999 / Revision received: 10 November 1999 / Accepted: 15 November 1999     

In vitro shoot regeneration from cotyledons of immature ovules ofImpatiens platypetala Lindl.

Summary An efficient and reproducible protocol has been developed for in vitro shoot regeneration from cotyledonary explants derived by germinating immature ovules ofImpatiens platypetala Lindl. ‘TR6-27-2’. Cotyledonary explants were cultured on a modified Murashige and Skoog (MS) agar-solidified medium containing 7.5g · liter^−1 sucrose, 22.2µ M N⁶-benzyladenine (BA), and 0.54µM α-naphthaleneacetic acid (NAA). The induction of organogenic tissues occurred after 6 to 8 wk in culture. Exogenous auxin and cytokinin were essential for the induction of organogenic tissues and survival of explants, and BA was most effective for the induction of organogenic tissues, compared with other cytokinins tested. The addition of glutamine (500 mg · liter^−1) was also important for growth of organogenic tissues after induction and for reducing explant death during culture. The induction of organogenic tissue was also influenced by the type of cotyledon cultured and the age of the donor seedlings. On average, eight shoots per explant were induced from organogenic tissues larger than 0.5 cm in diameter 6 to 8 wk after transfer to a modified MS agar-solidified medium without NAA and BA reduced to 4.44µM. Shoots longer than 0.5 cm in length were successfully rooted 2 to 4 wk after transfer to a basal MS medium containing 30g · liter^−1 sucrose.     

Utilization of Leek (Allium ampeloprasum var. porrum) for Inulinase Production

Inulinase production by Rhodotorula glutinis was carried out in this study, using leek (Allium ampeloprasum var. porrum) as an alternative carbon source due to its high inulin content and easy availability. Taguchi orthogonal array (OA) design of experiment (DOE) was used to optimize fermentation conditions. For this purpose, five influential factors (leek concentration, pH, incubation temperature, agitation speed, and fermentation time) related to inulinase production were selected at four convenient levels. The results showed that maximum inulinase activity was obtained as 30.89 U/mL, which was close to the predicted result (30.24 U/mL). To validate the obtained results, analysis of variance (ANOVA) was employed. Consequently, leek has a great potential as an effective and economical carbon source for inulinase production, and the use of Taguchi DOE enhanced enzyme activity about 2.87-fold when compared with the unoptimized condition.     

In vitro axillary shoot proliferation and somatic embryogenesis of yellow pitaya Mediocactus coccineus (Salm-Dyck)

Yellow pitaya (Mediocactus coccineus) seeds were sown on Murashige and Skoog (1962) mineral salt medium. After germination, epicotyls were placed on media enriched with a combination of naphthaleneacetic acid (NAA) (0.05, 0.27 or 0.54 M) and benzyladenine (BA) (2.2 or 4.4 M). The apical tip was excised from half of the shoots and the other half were kept intact. Different values for proliferation rate, shoot length and thickness were observed on each medium. The cotyledons and roots were placed on MS medium supplemented with NAA (2.7 or 5.4 M) and embryogenic calluses were formed. Somatic embryos were induced on these media and then they normally developed on a growth regulator-free medium.Abbreviations BA benzyladenine - MS Murashige and Skoog - NAA -naphthalenacetic acid     

Callus induction and plant regeneration from explants of commercial cultivars of leek (Allium ampeloprasum var. porrum L.)

Summary Plant regeneration capacity was studied for 8 cultivars and 4 accessions of leek (A. ampeloprasum var. porrum L.). Compact callus was induced on embryo and leaf explants on three different media. The highest frequency of compact callus formation (up to 90%) was obtained when mature, zygotic embryos were cultured on MS medium, containing 30 g/l sucrose and 1 mg/l 2,4-D. Regeneration occurred through somatic embryogenesis on MS medium, supplemented with 1 mg/l kinetin. Plants could be regenerated from all cultivars and accessions tested. These cultivars and accessions could be classified into three groups with respect to shoot formation frequency. The results suggest a distinct influence of the genotype on the morphogenic response of leek embryo explants in vitro.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium - N₆ medium from Chu et al. (1975) - B₅ medium from Gamborg et al. (1968) - BDS Dunstan and Short medium (1977)     

Long-term multiplication of onion (Allium cepa L.) by cyclic shoot regeneration in vitro

We succeeded in cultivating onion plants in vitro with a high potential for shoot regeneration. The apex must be destroyed or injured to obtain axillary buds. This capacity was restricted to the abaxial base of the youngest sheaths. It was shown necessary to restore plant individuality before further proliferation; this process constituted one cycle. For successive regeneration each cycle was composed of three steps: shoot proliferation in the presence of a cytokinin, shoot individualization and plant development in the absence of growth regulators. Effect of growth regulators on the physiological status of onion plants cultured in vitro is discussed.Abbreviations BA 6-benzyladenine - NAA naphthaleneacetic acid     

Development of a cyclic somatic embryogenesis regeneration system for leek (Allium ampeloprasum L.) using zygotic embryos

Summary In leek (Allium ampeloprasum L.) a cyclic system of somatic embryogenesis was developed. Somatic embryos used for cyclic embryogenesis were able to develop the same type of embryogenic callus as zygotic embryos in the primary cycle. For the first time a comparison of the efficiencies of both expiants was made. Ten families were investigated for somatic embryogenesis. There was a genetic relationship with respect to somatic embryo production between the reciprocal crosses. From each family one genotype was selected for investigating cyclic somatic embryogenesis. Different levels of somatic embryo production were found between the expiants of zygotic and somatic embryos. The two best genotypes, 92.001-03 and 92.002-33 produced twice as many somatic embryos as the overall average. On average, 56% of the somatic embryos finally developed into greenhouse plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - MS medium Murashige and skoog (1962) basal medium     

In vitro plant regeneration from callus derived from root explants of Lathyrus sativus

Protocols have been developed for the in vitro production of plants from callus derived from root explants of Lathyrus sativus cv. P-24. Callus and shoot regeneration were achieved only in MS medium supplemented with 10.7 M naphthaleneacetic acid and an increased concentration of kinetin (0.9 M for 14 days to 1.4 M for 18 days) during callusing. The shoots obtained rooted in 1/2 MS supplemented with 0.5 M indolebutyric acid. During the year plants have been regenerated several times. The requirement for growth regulators is very specific and narrow.Abbreviations IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - BA benzyladenine     

In vitro multiple shoot regeneration and plant production in Alysicarpus rugosus DC. var. heyneanus Baker

A protocol for in vitro multiple shoot regeneration and plant production through seedling (shoot tip) culture was established for Alysicarpus rugosus DC. var. heyneanus Baker. Maximum number of adventitious shoots (14.4) per shoot tip explant were initiated after two subcultures on MS solid medium supplemented with IAA (2.85 microM) plus BAP (2.22 microM) after 4 weeks. Shoot elongation (3.0-3.5 cm) was achieved on MS medium without any hormones. Stunted shoots elongated on half MS medium without growth hormones. Rooting occurred in MS medium containing IAA (1.14 - 2.85 microM) alone or in combination with IBA (0.89 - 2.46 microM) and or NAA (1.07 - 2.69 microM). Maximum rooting was established in MS medium supplemented with IAA (2.85 microM). The plants were acclimatized successfully with 55% survival in pot containing cocoa peat and sand (1:1). After a month, hardened plants were transferred to pots with manure, garden soil and sand (1:2:1) for further growth and finally planted in field.     

In vitro regeneration of plantlets from shoot callus of mature trees ofDalbergia latifolia

A successful procedure was established for in vitro mass multiplication of Indian rosewood (Dalbergia latifolia Roxb.). In vitro regeneration of plantlets was achieved from callus of shoot tips and shoot segments of over 50-year-old elite trees on Murashige & Skoog's medium containing naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). For rooting, regenerated shoots from the calli were excised and first treated with White's liquid medium or half-strength Murashige & Skoog's medium, supplemented with indole-3-acetic acid, indole-3-butyric acid and naphthaleneacetic acid for 48 h to 72 h. Following this treatment, plantlets were transferred to hormone-free half-strength MS medium. Rooted plantlets were then transferred to pots and grown in the greenhouse.Abbreviations BAP 6-benzylamino pruine - CH casein hydrolysate - CM coconut milk - 2, 4-D dichlorophenoxyacetic acid - GA gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - PVP-10 polyvinyl pyrrolidone - YE yeast extract     

In vitro plant regeneration of spinach from mature seed-derived callus

Summary A system for the regeneration of spinach (Spinacia oleracea L.) from mature dry seed explants has been established. The response of two commercial spinach cultivars, ‘Grandstand’ and ‘Baker’, was examined. Callus proliferation was most prominent on MS medium supplemented with 9.3 μM of 6-furfurylaminopurine (kinetin) and 3.39 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious shoot formation was observed within 8 wk after callus was transferred onto regeneration medium. Shoot regeneration was best from callus induced on 9.3 μM kinetin and 4.56 μM 2,4-D. The regeneration medium contained 9.3 μM kinetin, 0.045 μM 2,4-D, and 2.89 μM gibberellic acid (GA₃). Shoots were rooted on hormone-free medium, and plants grown in a greenhouse showed normal phenotype. This system is beneficial in rapid propagation of spinach plants, particularly when only a limited number of seeds are available.     

枸杞髓组织离体培养及高频率植株再生的研究

枸杞髓组织在４种ＭＳ培养基上都能诱导出愈伤组织,诱导率５３．７％～１００％。在培养基ＭＳ＋６－ＢＡ０．１ｍｇ／Ｌ＋ＮＡＡ０．５ｍｇ／Ｌ获得的愈伤组织,呈颗粒状,分散性能好,胚性细胞多．将其转移到ＭＳ＋６－ＢＡ０．５ｍｍ／Ｌ＋ＮＡＡ０．０１ｍｇ／Ｌ的分化培养基上获得大量绿色小芽,小芽在ＭＳ＋６－ＢＡ０．２ｍｇ／Ｌ的培养基上得到快速繁殖,繁殖系数５０～１５０株／芽·月。丛生芽在ＭＳ＋ＮＡＡ０．２ｔｍｇ／Ｌ的培养基上形成完整植株     

Influence of silver nitrate (ethylene inhibitor) on cucumber in vitro shoot regeneration

The effect of addition of silver nitrate (AgNO₃) on organogenesis of proximal and distal cotyledon and hypocotyl explants of five cucumber (Cucumis sativus L.) cultivars was investigated. Distal cotyledon and hypocotyl were unresponsive while only poor shoot regeneration was observed in proximal cotyledon and hypocotyl explants of all cucumber cultivars. The addition of different concentrations of AgNO₃ (10, 30 and 50 μM) to the medium, however, induced shoot regeneration in distal cotyledon except Suyo Long cultivar and effectively increased shoot regeneration response as well as the number of shoots per explant in proximal cotyledon and hypocotyl of all cucumber cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of plant growth regulators and basal media onin vitro shoot proliferation and rooting ofMyrtus communis L.

The influence of macronutrients and growth regulators on in vitro shoot proliferation and rooting of an East Spanish population ofMyrtus communis L. were studied. Preincubation of field material on a medium without mineral salts prevented the browning from phenolic exudates. For multiplication, nodal segments of 5 mm fromin vitro produced shoots were cultured on Murashige and Skoog (MS), Schenk and Hildebrandt (SH) and Heller (H) media (full strength or diluted to 1/2 or 1/4), with 6-benzylaminopurine (BAP) at concentrations 4.4, 13.3 and 22.2 ΜM or kinetin (K) at concentrations 4.7, 14.0 and 23.2 ΜM. The optimum shoot proliferation was on quarter-strength MS medium with 4.4 ΜM BAP, whereas the maximum number of nodal segments was produced on half-strength MS medium with 4.4 ΜM BAP. Rooting of shoots was obtained by adding 2.5 – 24.6 ΜM indole-3-butyric acid (IBA) and broad range of macronutrients; Lloyd and McCown (WPM) and Gresshoff and Doy (GD) media both full strength or diluted to 1/2 were optimum. No rooting was obtained in the presence of α-naphthaleneacetic acid (NAA). Acknowledgements: This study was supported by a grant from Conselleria de Cultura, Educació i Ciència de Ia Generalitat Valenciana. The authors are grateful to Man Cannen Perea for her helpful comments.     

In vitro regeneration of cereals based on multiple shoot induction from mature embryos in response to thidiazuron

The in vitro competency of mature cereal embryos (winter, spring and durum wheats, oat, barley and triticale) was assessed for direct multiple shoot production on culture media containing the plant growth regulators, thidiazuron (TDZ) and/or 6–benzylaminopurine (BAP). Mature embryos of CDC Dancer oat showed the best response, with 69 shoots per explant on culture medium containing a combination of 4.5 μM TDZ and 4.4 μM BAP. TDZ alone induced about 16 shoots per explant from the oat. Among the wheat genotypes, durum wheat showed the most number of shoots (35) per explant on culture medium containing 4.5 μM of TDZ and 4.4 μM of BAP. With TDZ alone, shoot regeneration for durum wheat ranged from 27–32 shoots per explant. The regeneration frequency from the three winter wheat genotypes ranged from 11–25 shoots per explant and was highest on culture medium containing 9.1 μM TDZ and 4.4 μM BAP. The latter culture medium was also effective for a triticale genotype, inducing 34 shoots per explant. The regeneration from mature embryos of barley genotypes ranged from 5–9 shoots per explant. The mature embryos of all the cereals tested could be used for in vitro regeneration with TDZ and TDZ+BAP combinations.     

In vitro plant regeneration of Aster scaber via somatic embryogenesis

We established an in vitro plant regeneration system via somatic embryogenesis of Aster scaber, an important source of various biologically active phytochemicals. We examined the callus induction and embryogenic capacities of three explants, including leaves, petioles, and roots, on 25 different media containing different combinations of α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The optimum concentrations of NAA and BA for the production of embryogenic calli were 5.0 μM and 0.05 μM, respectively. Media containing higher concentrations of auxin and cytokinin (such as 25 μM NAA and 25 μM BA) were suitable for shoot regeneration, especially for leaf-derived calli, which are the most readily available calli and are highly competent. For root induction from regenerated shoots, supplemental auxin and/or cytokinin did not improve rooting, but instead caused unwanted callus induction or retarded growth of regenerated plants. Therefore, plant growth regulator-free medium was preferable for root induction. Normal plants were successfully obtained from calli under the optimized conditions described above. This is the first report of the complete process of in vitro plant regeneration of A. scaber via somatic embryogenesis.     

大叶种胡椒实生苗茎尖培养和合子胚培养研究

利用各种表面消毒方法对采自海南岛三个地区的胡椒大田植株的外植体进行消毒试验,由于内源性污染,除胡椒成熟种子外,其它各种大田外植体的表面消毒均未能成功。以胡椒成熟种子无菌萌发的实生苗茎尖作外植体,在1/2MS(MS或B5)+1.5mg/LBA+0～0.2mg/LIAA(或NAA)上可实现丛生芽增殖。茎尖水平或竖直接种方法显著影响茎尖的增殖;水平接种茎尖的生长和增殖效果优于竖直茎尖接种方式。茎尖增殖率随BA浓度的增加而提高,但BA浓度大于2.0mg/L时会使苗芽的质量降低,愈伤组织产生严重,苗芽细小,抽出不明显,颜色发黄甚至变白。附加或不附加100mg/LAdSO4对丛生芽增殖没有明显影响。生根培养基以1/2MS+1.0mg/LIBA+0.5～1.0mg/LIAA为最优,生根率可达100%;在细沙∶土∶椰糠(1∶1∶1)的基质中常规炼苗,成活率可达98%以上。液体纸桥法对胡椒种胚进行培养,在不附加任何生长调节物质的培养基(MS、B5或SH)上只产生单苗,而在附加不同种类和不同浓度的生长调节物质的培养基上则诱导形成愈伤组织,但未能实现分化;以胡椒无菌萌发的实生苗胚轴和叶片切段作外植体进行培养,较易诱导产生愈伤组织,但难以实现分化。