Induction of lincomycin and streptomycin resistance by nitrosomethylurea and ethyl methanesulphonate in Capsicum annuum L.

A high frequency of plastid-encoded antibiotic-resistant variants of Capsicum annuum were isolated on selective media following treatment by ethyl methanesulphonate (EMS) and nitrosomethylurea (NMU). Seeds and explants were mutagenized with 0.1% EMS and 5 mm NMU separately. Non-mutagenized cotyledons (controls), mutagenized cotyledons from EMS-treated seedlings and NMU-treated cotyledons were placed on regeneration medium supplemented with the antibiotics streptomycin or lincomycin. Resistant shoots appeared at a high frequency in mutagenized cotyledons, whereas in controls morphogenesis was suppressed, accompanied by bleaching. The stability of streptomycin and lincomycin resistance was confirmed by leaf assay. NMU-treated cotyledons gave a higher frequency of variants than cotyledons from EMS-treated seedlings. The mutagenic effect of EMS was more pronounced using whole seeds rather than cotyledons; in contrast, NMU was more effective in inducing variations in cotyledons than in seeds. Received: 1 October 1996 / Revision received: 12 May 1997 / Accepted: 3 June 1997     

Prolific shoot regeneration from immature embryo explants of sainfoin (Onobrychis viciifolia Scop.)

Summary Immature cotyledons and embryo axes of sainfoin were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid (NAA) to induce adventitious shoot regeneration. The highest frequency of shoot regeneration occurred following an initial callus growth on a MS medium containing 0.5 mg/l BAP and 2 mg/l NAA. Immature embryo axes showed higher regeneration capacity than immature cotyledons, however, shoot elongation was best achieved on immature cotyledons. Regenerated shoots were excised and rooted in half strength MS medium with 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA. The rooted plantlets were finally transferred to compost.     

Influence of growth regulators on plant regeneration from epicotyl and hypocotyl cultures of two groundnut (Arachis hypogaea L.) cultivars

This study was designed to evaluate the effect of phytohormones on plant regeneration from epicotyl and hypocotyl explants of two groundnut (Arachis hypogaea) cultivars. Explants cultured on media with auxins and in combination with cytokinin produced high frequency of callus. After four weeks, callus from these cultures was transferred to medium with cytokinin and reduced auxin, shoot buds regenerated from the cultures. A high rate of shoot bud regeneration was observed on medium supplemented with 2.0 mg/L BAP and 0.5 mg/L NAA. Among the different auxins tested, NAA was found to be most effective, producing the highest frequency of shoot buds per responding cultures. Of the two explants tested, epicotyl was found to be best for high frequency shoot bud regeneration. Multiple shoots arose on MS medium supplemented with BAP or kinetin (1.0–5.0 mg/L) plus IBA (1.0 mg/L), with maximum production occurring at 5.0 mg/L. The elongated shoots developed rootsin vitro upon transfer to MS medium supplemented with NAA or IBA (0.5–2.0 mg/L) and kinetin (0.5 mg/L) for 15 days.In vitro produced plantlets, were transferred to soil and placed in a glasshouse developed successfully, matured, and set seeds.     

High frequency plant regeneration from cotyledon and hypocotyl explants of ornamental kale

A high frequency shoot regeneration system for ornamental kale [Brassica oleracea L. var. acephala (D.C.) Alef.] was firstly established from seedling cotyledon and hypocotyl explants. The ability of cotyledon and hypocotyl to produce adventitious shoots varied depending upon genotype, seedling age and culture medium. The maximum shoot regeneration frequency was obtained when the explants from cv. Nagoya 4-d-old seedlings were cultured on Murashige and Skoog (MS) medium supplemented with 3 mg dm⁻³ 6-benzylaminopurine (BA) and 0.1 mg dm⁻³ naphthaleneacetic acid (NAA). The frequency of shoot regeneration was 65.0 % for cotyledons, 76.1 % for hypocotyls; and the number of shoots per explant was 4.3 for cotyledons, 8.2 for hypocotyls. Hypocotyl explants were found to be more responsive for regeneration when compared with cotyledons. Among the 4 cultivars tested, Nagoya showed the best shoot regeneration response. The addition of 3.0 mg dm⁻³ AgNO₃ was beneficial to shoot regeneration. Roots were formed on the base of the shoots when cultured on half-strength MS medium.     

南瓜高效再生体系的建立

南瓜(Cucurbita moschata)再生率较低, 为建立高效的南瓜再生体系, 以南瓜子叶为外植体, 进行35组不同激素浓度的不定芽诱导研究。结果表明, 南瓜再生受培养基中激素浓度和配比的影响, 适宜浓度6-苄氨基腺嘌呤(6-BA)能有效促进不定芽形成; 单独使用脱落酸(ABA)诱导使南瓜子叶发黄, 但与6-BA组合使用可显著提高外植体的再生能力, 1.0 mg?L ^-16-BA与0.5 mg?L ^-1ABA组合南瓜芽再生率高达90.26%。将不定芽置于MS培养基中进行生根培养, 再生苗移栽易成活。从子叶接种到苗再生约需70天。     

Plant Regeneration and Transformation of Sweet Papper (Capsicum frutescens)

Three strains of sweet pepper, (Capsicum frutescens) “Shuang Feng”, “Zhong Jiao No. 2” and “Zhong Jiao No.3” were screened out of six Chinese cuhivars for their high capacity of regeneration. The normal flowering and fertile regenerated plants have been obtained from cotyledons of seedlings from 10 to 16 days old by a four-step culture procedure; short induction, shoot elongation, rooting of excised shoot and transplanting into soil. MS was the basal medium in all steps, supplemented with different kind and different concentration Of phytohomores. Optimal shoot ioduction medium is MS +4–6mg/LBA+0.5mg/LIAA which gives rise a shoot regeneration frequency of 100%. 35% of the induced shoots elongated on the medium of MS+2mg/L Zeatin or 2mg/L. BA+l–3mg/L GA, and subsequently rooted on MS medium or in addition of 0.1–0.5mg/LNAA. The regenerants were transplanted into soil and developed into normal plants. In the transformation of sweet pepper using the leaf disc method, two kinds of wild type Agrobacterium tumefaciens, C58 and GV3111, have been screened out in regard to their high infection capacity. The transient expression of GUS gene was detected and Kanamycin-resistant shoots from infected cotyledons have been obtained. Further assay and transfering the TMV-resistant and CMV-resistant genes into sweet pepper are in progress.     

南瓜高效再生体系的建立

南瓜(Cucurbita moschata)再生率较低, 为建立高效的南瓜再生体系, 以南瓜子叶为外植体, 进行35组不同激素浓度的不定芽诱导研究。结果表明, 南瓜再生受培养基中激素浓度和配比的影响, 适宜浓度6-苄氨基腺嘌呤(6-BA)能有效促进不定芽形成; 单独使用脱落酸(ABA)诱导使南瓜子叶发黄, 但与6-BA组合使用可显著提高外植体的再生能力, 1.0 mg∙L ^-16-BA与0.5 mg∙L ^-1ABA组合南瓜芽再生率高达90.26%。将不定芽置于MS培养基中进行生根培养, 再生苗移栽易成活。从子叶接种到苗再生约需70天。     

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     

白花蛇舌草叶片离体培养及试管无性系的建立

以白花蛇舌草叶片为材料,建立了白花蛇舌草叶片高效不定芽发生、植株再生体系。研究了不同激素及其组合对外植体不定芽发生的效应。结果显示,在MS基本培养基中单纯添加6BA,当6BA浓度为0.1mg/L和0.5mg/L时,不能诱导离体叶片发生不定芽,6BA浓度在1.0～5.0mg/L范围内,诱导率随BA浓度升高而增加,适宜浓度为3.0mg/L;当6BA与NAA配合使用时,其诱导率随着培养基中6BA与NAA的相对比值的提高而提高;以MS+BA3mg/L+NAA0.01mg/L作为继代培养基,建立起白花蛇舌草高效、稳定的试管无性系,为白花蛇舌草遗传转化研究奠定了基础。     

Combination of thidiazuron and 2-isopentenyladenine promotes highly efficient adventitious shoot regeneration from cotyledons of mature sunflower (Helianthus annuus L.) seeds

Genetic improvement of sunflower (Helianthus annuus L.) through the use of biotechnological tools requires a reliable in vitro shoot regeneration system. Tissue culture protocols reported to date for sunflower suffer from low efficiency, poor reproducibility, genotype dependence and a tendency for flowering in vitro. The present study describes an efficient protocol system for shoot regeneration via direct adventitious shoot organogenesis from cotyledons of mature seeds of sunflower. About 169 media combinations comprising 12 different growth regulator combinations in various concentrations were assessed for induction of shoots from cotyledons derived from mature seeds and also from seedling tissues of 2?C20-day-old seedlings. Appearance of shoots from seedling tissues was sporadic and the frequency of shoot regeneration was low. Cotyledon explants from mature seeds were consistent with regard to frequency of adventitious shoot regeneration and number of shoots per explant. A high frequency (93.86?%) of adventitious shoot regeneration was obtained within 2?weeks of culture initiation on Murashige and Skoog (MS) medium supplemented with 9.84???M 2-isopentenyladenine (2-iP), 2.85???M indole-3-acetic acid (IAA) and 0.45???M thidiazuron (TDZ). Use of 2-iP in the shoot induction and elongation media prevented precocious flowering. Statistical analysis revealed significant effects of explant orientation, age of seedlings, and genotype on adventitious organogenesis. Maximum shoot regeneration was obtained when cotyledons from 0 and 1-day-old seedlings were placed with their adaxial surface in contact with the medium surface. The protocol developed was tested on 42 genotypes and found to be applicable to a wide range of genotypes. Histological studies indicated that the shoots originated predominantly through adventive organogenesis from the sub-epidermal and cortical regions.     

Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.)

A protocol is presented for direct adventitous shoot organogenesis and complete plant regeneration from seedling-derived explants of pomegranate (Punica granatum L.), a tropical fruit tree. Murashige and Skoog (1962) (MS) medium enriched with 8.9 mumol/L benzyladenine (BA), 5.4 mumol/L naphthaleneacetic acid (NAA) and 10% coconut water (CW) induced adventitious shoot bud differentiation in axenic seedling-derived cotyledons as well as hypocotyl segments. The cotyledons were more responsive than the hypocotyls. Addition of ethylene inhibitors such as AgNO3 (10-40 mumol/L) and aminoethoxyvinylglycine (AVG) (5-15 mumol/L) to the medium markedly enhanced regeneration frequency as well as number of shoots obtained per explant. The promotive effect of AVG and AgNO3 on shoot organogenesis was observed only in cotyledon explants. The regeneration medium containing AgNO3 (20 mumol/L) or AVG (10 mumol/L) induced adventitious shoot buds from 57% or 53% of the cotyledon explants respectively. These shoot buds developed into shoots upon transfer to a regeneration medium without AgNO3 and AVG. The promotive effect of AVG on shoot regeneration was reversed by exogenous application of 20 mumol/L 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound. On the other hand, shoot regeneration stimulated by AgNO3 was relatively less affected by CEPA. Regenerated shoots were rooted in half-strength MS medium (1/2 MS) containing 0.54 mumol/L NAA. The well rooted plantlets were acclimatized and eventually established in soil.     

Influence of growth regulators and explant type on in vitro shoot propagation and rooting of red sandal wood (Pterocarpus santalinus L.).

In vitro shoot regeneration in Pterocarpus santalinus L. was achieved when detached cotyledons from in vitro germinated seedlings were cultured on MS medium containing NAA (0.1 mg/L), BA (1 mg/L) and kinetin (1 mg/L). The regenerated shoots rooted on 1/4 strength MS medium with IAA (1 mg/L) and the fully developed plantlets were successfully established in the soil.     

Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Helianthus annuus L.

We have developed a highly efficient three-stage protocol for plant regeneration in sunflower (Helianthus annuus L.) from embryonal cotyledons. This protocol uses phenylacetic acid (PAA) for both shoot-bud induction and the elongation of smaller buds. The medium used for inducing bud formation from the cotyledons was modified MS medium supplemented with 3 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l PAA. Buds were elongated on MS medium supplemented either with only 0.2 mg/l gibberellic acid (GA₃) or with 0.2 mg/l GA₃ + 0.1 mg/l PAA + 0.3 mg/l BAP. The elongated shoots were then transferred onto rooting medium containing 1 mg/l PAA. The complete plantlets with well-developed roots were transferred to field conditions where they survived and set normal seeds. The induction of shoot buds from embryonal cotyledons was also observed on modified MS medium supplemented with 0.5-5 mg/l BAP in combination with 0.5-5 mg/l !-naphthaleneacetic acid (NAA). In this case, the formation of callus took place along with shoot-bud formation, which hindered further development of the latter. The presence of PAA with BAP in the primary bud induction medium promoted normal development and elongation of shoot buds.     

中国木薯栽培种通过器官发生再生植株研究

本文研究了中国木薯栽培种四种外植体通过器官发生再生植株的条件。结果表明:在MS附加0.05mg/L TIBA,1mg/L BA的培养基上“NZ 188”初步的萌发胚状体“切头”后切口处可直接产生丛芽,出芽率为43%。“SC201”胚状体子叶块在MS附加0.5 mg/L NAA,0.5mg/L BA的培养基上可直接出芽,出芽率为42%,在MS附加0.5mg/L IBA,1.5mg/L BA培养基上·出芽率为31%,AgNO_3和ABA单独使用或配合使用均不利于芽的再生。“NZ188”胚状体下胚轴在MS附加0.5mg/LNAA,0.5mg/L BA的培养基上形成的愈伤组织转入MS附加1mg/L NAA,2mg/L BA的培养基上,3周后大多数愈伤组织有绿点出现、仅4.4%外植体分化出芽。“HZ188”无菌苗茎段接种在MS附加0.05mg/L TIBA,2mg/LBA的固体培养基上,2周后形成大量愈伤组织,4周后仅见一块愈伤组织分化出芽。     

In Vitro Regeneration of Achillea millefolium L from Shoot-tips and Root Segments of Seedlings

This report describes, for the first time, an efficient plant regeneration system for Achillea millefolium L (yarrow), a medicinal plant, via shoot multiplication from shoot-tips and adventitious shoot regeneration from root segments. Higher numbers of shoots were obtained when shoot-tips were cultured on MSMO medium supplemented with 3.0 mg l^?1 BA and 0.5 mg l^?1 IAA, or 5.0 mg l^?1 KIN and 1.0 mg l^?1 IBA, producing 17.3 and 17.0 shoots per explant at 100% frequency, respectively. For adventitous shoot regeneration, only root segments developed shoots when cultured on medium containing a combination of 1 mg l^?1 TDZ, 0.5 mg l^?1 IAA and 0.5 mg l^?1 GA3 (18.9 shoots per explant at 100% frequency), while other types of explants (i.e., cotyledons, leaf lamina and petiole segments) or hormonal combinations tested were found ineffective. Regenerated shoots rooted readily on MSMO medium containing different concentrations of IAA, IBA, NAA or 2,4-D, however, NAA at 0.5 mg l^?1, or IBA at 0.5 or 1.0 mg l^?1 were found to be the most productive. Nearly all of the regenerated plants (98%) survived through the hardening process when the rooted plantlets were kept at 55–65% relative humidity for 2 weeks, which were then planted in pots containing potting soil and kept at 25–35% humidity.     

Factors influencing shoot regeneration from cotyledons of tetraploid <Emphasis Type="Italic">Isatis indigotica</Emphasis> fort

Summary An efficient in vitro plant regeneration system from cotyledons was established in tetraploid Isatis indigotica Fort. Factors influencing shoot regeneration from cotyledons, including culture medium type, combinations of plant growth regulators, and sucrose concentrations in the medium, as well as illumination were investigated. Murashige and Skoog's (MS) medium was found to be best for promoting shoot regeneration, followed by Gamborg's B₅ and White's medium. The highest shoot regeneration frequency was achieved from cotyledons cultured on MS medium supplemented with 2.0 mgl⁻¹ (8.9 μM) 6-benzyladenine and 1.0 mgl⁻¹ (5.4 μM) α-naphthaleneacetic acid (NAA), with 97.9% regeneration, associated with a high number of multiple shoots developed per explant (8.6 shoots per explant). A sucrose concentration of 3% present in the medium and light conditions were beneficial for shoot regeneration. The shoots developed were rooted in a half-strength MS medium supplemented with 1.0 mgl⁻¹ (5.4 μM) NAA and successfully transplanted in soil in pots with over 85% survival. The establishment of an efficient plant regeneration procedure from cotyledons provides a basis for the rapid in vitro multiplication of tetraploid Isatis indigotica Fort., one of the most extensively used medicinal plants in China currently under great shortage.     

杉木再生系统的比较研究

通过对杉木(Cunninghamia lanceolata Hook)再生系统的比较研究,建立了子叶、下胚轴、茎段和针叶等四个再生体系。子叶在附含1mg/L BA和0.1mg/L NAA的DCR培养基中再生频率为93.7%±0.45%,平均芽数为3.76±0.25;下胚轴在附含2mg/L BA和0.2mg/L NAA的DCR培养基中再生频率为96.2%±0.35%,平均芽数为17.4±0.18;茎段在附含1mg/L BA和0.1mg/L NAA的DCR培养基中再生频率达100%,平均芽数为3.28±0.11;针叶在附含1.5mg/L BA和0.1mg/L NAA的DCR培养基中再生频率达84.5%±0.45%,平均芽数仅为1.42±0.08。不定芽在附含0.2mg/L BA和0.02mg/L NAA的DCR培养基中有效伸长。芽苗经预处理后在附含0.3mg/L IBA的1/2 DCR培养基中生根效果最佳。     

李砧木Marianna离体叶片再生体系优化研究

以李砧木‘Marianna’试管苗新梢顶端第1片叶为外植体,研究激素组合、基本培养基种类及外植体类型等对不定芽再生的影响。结果表明:1/2 MS基本培养基和WPM培养基再生率显著高于MS和SH培养基;叶片附带叶柄的外植体再生率和再生不定芽数显著高于叶柄和切除叶柄的叶片外植体;最佳再生培养基为1/2MS+2.0mg/L TDZ+0.1 mg/L IBA+0.25%琼脂+3.0%蔗糖,最高再生率和再生不定芽数分别为81.7%和7.46±1.38个;最佳生根培养基为1/2MS+0.5~1.0 mg/L IBA,能获得96.7%生根率、较高的生根数和根长。     

ORGANOGENESIS FROM HELIANTHUS ANNUUS INBREDS AND HYBRIDS FROM THE COTYLEDONS OF ZYGOTIC EMBRYOS

Dormant mature and immature zygotic embryos of sunflower (Helianthus annuus L.) inbreds and hybrids were dissected and cultured on modified Murashige and Skoog's medium supplemented with 0.1 mg/l benzyl adenine (BA), 0.5 mg/l naphthalene acetic acid (NAA), adenine sulfate, and casamino acids. For certain inbreds and hybrids, adventitious shoot formation occurred from callused cotyledonary tissue, particularly along the cut edges. The developmental stage of the zygotic embryo was critical. Eighty percent of immature H. annuus ‘Mammoth Russian’ embryos produced adventitious shoots from cotyledons, while mature embryos did not. The organogenic potential of mature cotyledons diminished as soon as one day after germination was initiated. Different forms of regeneration were observed in two inbred lines, and when these were crossed, both forms of regeneration were observed at a lower frequency from the immature embryos of the hybrid. Rooted shoots were successfully grown to maturity in a greenhouse.