濒危植物太行菊组织培养及快繁技术研究

研究旨在建立一种珍惜濒危植物太行菊的高效再生方法,为工厂化生产提供理论基础。以MS为基本培养基,用不同激素组合对太行菊无菌苗的叶片和茎段离体培养及植株再生、炼苗移栽等过程进行研究。结果表明,太行菊的叶片分化不定芽比茎段难,茎段最适合作为外植体进行愈伤组织的诱导及不定芽分化;筛选出了最佳培养基MS+6-BA 1.0 mg/L+NAA 0.1 mg/L,在此培养基中,茎段外植体可一步成苗,不需转换培养基即可完成愈伤组织的诱导分化及不定芽增殖过程;最佳生根培养基为1/2MS+NAA 0.2 mg/L;再生苗在花园土中移栽成活率可达80%。研究简化了培养流程,建立了太行菊一步式高效再生体系。     

杨树叶片高效离体再生系统的构建

利用速生杨树（Populus.tomentosa Car）叶片为外植体建立离体培养体系,结果表明,叶脉处和叶柄处,极易发生不定芽;芽大多是从叶片上直接产生;培养基中BA浓度是影响不定芽形成的关键;BA浓度在05~2.0mg/L范围内均有不定芽发生,BA 1.0mg/L与NAA 0.05mg/L搭配有利于不定芽的形成,培养基中添加GA3 0.1~0.2mg/L可以提高不定芽的发生频率,增殖培养基采用MS+BA 0.5mg/L+NAA 0.05mg/L+GA3 0.05mg/L,不仅能够快速增殖,而且芽苗粗壮,玻化率降低。生根过程采用过渡培养效果较好,ABT3生根粉的加入,使试管苗质量大为提高,在此基础上建立起杨树叶片离体再生系统,利用该体系能够获得高的芽苗再生频率,但是不同品种间略有差异。     

Plant regeneration from in vitro cultures of cotyledon explants and anthers of Sinapis alba and its implications on breeding of crucifers

Summary Protocols for plant regeneration from cotyledon explant and anther cultures of Sinapis alba have been developed for creating doubled-haploids and somaclonal variation. Among the several cultivars tested in this study, only Arda responded well to in vitro plant regeneration both from anther-as well as cotyledoncultures. Multiple shoot formation in cotyledon explants, which always followed a brief callusing phase, was found to be the best on MS medium with ZEA (1.0mg/l) and NAA (0.1mg/l). Regeneration frequency declined sharply in the absence of auxin or presence of other cytokinins and/or auxin. The frequency of shoot regeneration also declined with reduction in the photoperiod to 16h. On MS + BAP (1.0mg/l) + NAA (1.0mg/l) medium, cotyledonary explants showed profuse callusing, which could regenerate shoots on high ZEA + low NAA/IAA medium. However, it declined with progressing time in culture. Anthers, excised from fresh as well as cold pretreated buds, cultured on 10% sucrose containing MS media with different hormonal constitution, developed calli and/or embryos. Initial culture temperature was important with embryogenesis occurring only in anthers cultured at 30°C for 3 weeks. A high temperature (35°C) treatment was lethal for both callus as well as embryo formation. While BAP + NAA and ZEA + NAA/IAA supported embryogenesis, further plant regeneration from anther-or embryo-callus could be achieved in ZEA + NAA/IAA media. Some of the regenerants flowered already in vitro and had small and sterile flowers. Cytological examination of some of the root differentiating calli indicated the presence of haploid as well as diploid cells. Shoots were rooted during prolonged incubation on the same medium or on transfer to MS (reduced)/ B5 + ZEA + NAA media.     

Genotypic differences in organogenesis from callus of ten triticale lines

Summary Callus was obtained from immature excised embryos of triticale using MS medium supplemented with 3 mg/l 2,4-D and 1 mg/l kinetin. The presence of 2,4-D was essential for continued callus proliferation. Plantlets were induced from the calli by sub-culturing on medium either devoid of auxin or containing 0.1 mg/l 2,4-D. The capacity to produce callus and to form organs and plantlets differed markedly among the genotypes used. Lines also had distinct response to presence and absence of 2,4-D in the regeneration media. The callus of most triticale lines used differentiated into organs more readily on MS medium supplemented with 0.1 mg/l 2,4-D than on medium without growth regulators. Very high frequencies (up to 75%) of plantlet regeneration were observed in several of the triticale lines studied.     

‘黑珍珠’番茄植株再生体系的研究

为了研究‘黑珍珠’番茄植株再生,以‘黑珍珠’番茄幼嫩叶片为外植体诱导愈伤组织,通过愈伤组织诱导培养、愈伤组织分化培养、不定芽增殖培养、生根培养和试管苗移栽,建立高效快速的‘黑珍珠’番茄再生体系。结果表明：最适宜的诱导叶片愈伤组织的培养基为MS+ 1.0 mg/L 6-BA+ 0.1 mg/L NAA,叶片外植体愈伤组织诱导率最高可达98.2%;诱导出的愈伤组织在MS+ 1.5 mg/L 6-BA+ 0.2 mg/L IBA培养基上能很好的分化出不定芽;MS+4.0 mg/L KT+ 0.01 mg/L IBA 培养基可实现不定芽芽增殖;最适宜的生根培养基为1/2MS+ (0.05~0.08) mg/L NAA,试管苗移栽成活率达92%。     

薄荷离体培养及植株再生的研究

以薄荷茎段为外植体,研究了不同植物激素对薄荷愈伤组织的诱导及其增殖、不定芽分化和诱导生根的影响。结果表明,愈伤诱导最适培养基为MS 6-BA1.5mg/L 2,4-D0.5mg/L NAA0.5mg/L,其愈伤组织生长良好;最佳愈伤增殖培养基为MS NAA1.0mg/L或MS IAA2.0mg/L,其褐化率均较低;最佳诱导分化培养基为MS 6-BA2.0mg/L NAA0.1mg/L,分化率达77%;生根培养基为1/2MS NAA2.0mg/L,且根较为粗壮。     

Plant regeneration from immature inflorescence callus cultures of wheat,rye and triticale

Summary Callus cultures were initiated from inflorescence explants of wheat, rye and triticale on MS medium supplemented with 2 mgl^-1 2,4-D+5% CW or 2 mgl^-1 2,4-D+0.5 mgl^-1 BA. On transfer of the cultures to medium supplemented with 15% CW+0.2 mgl^-1 NAA or 1 mgl^-1 BA+0.1 mgl^-1 IAA, shoot buds and embryoids were produced. Full fledged plantlets obtained on MS medium supplemented with NAA were transferred to the field. Cytological analysis showed the plants to be diploid. However, the regenerated plantlets were shorter, produced fewer tillers and had lower fertility compared to the control.Abbreviations BA Benzyladenine - CW coconut water - IAA indoleacetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Intergeneric hybridization between Brassica species and Crambe abyssinica

A protocol for high frequency callus induction and plant regeneration from sunflower (Helianthus annuus L.) anthers is described. Different variables using Murashige & Skoog (MS) basal medium supplemented with 2.0 mg/l α-naphthaleneacetic acid (NAA) and 1.0 mg/l N₆-benzyladenine (BA) were tested for their ability to enhance the frequency of anther callusing and subsequent embryogenesis. Of these, agar concentration, sucrose concentration, carbohydrate source had significant effect on callusing, while differences due to incubation under dark vs light conditions, cold pretreatment of capitula for 1 to 6 days prior to anther inoculation and genotype on callusing were non-significant. However, all these factors exerted highly significant influence on embryogenesis when calli from the various media were transferred to medium supplemented with 0.1 mg/l NAA and 0.5 mg/l BA. With the procedure developed, callusing as high as 100% and embryo formation at a frequency of 44% was achieved. Although complete embryos were formed the frequency of their conversion to whole plantlets was low (14.3%). Hence, the embryogenic pathway was bypassed to obtain multiple shoots by transferring embryogenic calli with developing embryos to MS medium supplemented with 0.5 mg/l BA. Elongated shoots rooted on half-strength MS medium supplemented with 0.5 mg/l NAA. Cytological analysis of embryogenic callus and somatic embryos revealed haploids at a frequency of 30% while that of rooted plants showed haploid regenerants at a frequency of 8.3%. Nevertheless, the frequency of putative haploid plants could be enhanced through mass multiplication using nodal explants of the regenerants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation of a pure thornless loganberry by meristem tip culture

Summary Thornless Loganberry (TL) is a periclinal chimeral blackberry in which a layer of mutant (thornless) epidermis surrounds a core of wild-type (thorny) tiusse. Due to its chimeral arrangement, TL produces thorny adventitious root cuttings and thorny offspring. To separate the chimera into its components parts, meristems of TL were grow in vitro on modified Murashige and Skoog medium to yield callus and adventitious shoots. One of these shoots has survived, flowered, and produced thornless offspring from seed. The importance of this non-chimeral TL is discussed.List of terms BA 6-benzylaminopurine - GA₃ gibberellic acid - NAA naphthalene acetic acid - hybridberry polyploid bramble interspecific hybrids - MS Murashige & Skoog (1962) high mineral salt medium - TL Thornless Loganberry - TL_tc tissue culture-derived (non-chimeral) Thornless Loganberry     

安祖花愈伤组织再生体系的建立及染色体检变

试验以安祖花幼嫩叶片和无菌苗叶柄为外植体,通过愈伤组织诱导途径,建立快速高效的安祖花再生体系。结果表明：最适宜的诱导叶片和叶柄愈伤组织的培养基分别为1/2MS+1.0 mg/L 6-BA+0.2 mg/L 2,4-D和1/2MS+1.0 mg/L 6-BA+0.1 mg/L 2,4-D;诱导出愈伤组织在1/2MS+2.0 mg/L KT+0.1 mg/L NAA培养基上能很好的分化不定芽苗;1/2MS+2.0 mg/L 6-BA+0.2 mg/L NAA培养基可对再生芽实现增殖与复壮;最适宜的生根培养基为1/2MS+0.05 mg/L NAA。试验通过观察安祖花继代过程中愈伤组织细胞染色体变异的情况,发现随着继代次数的增多染色体变异细胞的频率也随之上升,并且染色体变异多为非整倍体变异。     

Genotypic and exogenous factors affecting shoot regeneration from anther callus of linseed (Linum usitatissimum L.)

Summary The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. Hella. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l^-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l^-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.Abbreviations B5 Gamborg's (1975) medium - BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - N6 Chu's (1978) medium - NAA -naphthaleneacetic acid - MS Murashige & Skoog's (1962) medium - ZEA zeatin     

豌豆愈伤组织及不定芽诱导影响因素的研究

以豌豆品种陇豌1号为试验材料,对愈伤组织和不定芽诱导过程中种子处理、外植体选择、最适培养基、不定芽诱导及生根等因素进行研究。确定了最适的种子消毒处理为75%乙醇消毒30s,0.1%升汞消毒10min。以下胚轴为外植体诱导愈伤组织,最适激素配比为0.2mg/L 2,4-D和1.0mg/L TDZ,愈伤组织诱导率为91.11%。具有分化能力的愈伤组织持续培养在只添加TDZ的培养基上即可诱导出不定芽,但这些不定芽生根率仅为8.89%,为此分析了影响生根的因素,为后续的研究提供依据。     

Somatic cell genetic studies inBrassica species. II. Production of androgenetic haploid plants inBrassica nigra (L.)Koch

Summary Callus growth and its subsequent regeneration into complete plantlets was achieved from in vitro cultured anthers ofBrassica nigra (L.)Koch. Callus was induced on a modified N6 medium containing trace elements, organics of B5 medium and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Morphogenesis of callus in the form of shoots on MS medium containing indole-3-acetic acid (IAA) and N⁶-benzyladenine (BA) 0.5 mg/l each and embryoids on MS medium containing 0.5–1.0 mg/l IAA and 3.0–5.0 mg/l BA could be accomplished. Chromosomal analysis revealed presence of 41% haploids (n=8) amongst the regenerated plants.     

新疆杨组培再生体系建立

以新疆杨叶片为外植体,研究新疆杨组织培养再生体系建立的主要影响因素。结果表明:诱导愈伤组织的最佳培养基组合为MS+0.5 mg/L 6-BA+0.5 mg/L 2,4-D;愈伤组织分化的最佳培养基组合为MS+0.1 mg/L TDZ+0.3 mg/L IBA,不定芽增殖的最佳培养基组合为MS+1.0 mg/L 6-BA+0.3 mg/L IBA,诱导生根的最佳培养基组合为1/2MS+0.5 mg/L IBA,新疆杨组培苗在生根培养基上形成的根条数多,苗体健壮,生根率达到100%。本研究成功建立了新疆杨组织培养再生体系,为其转基因研究工作奠定了基础。     

Long-term callus cultures of diploid Barley (Hordeum vulgare). I. Auxin effects on culture initiation and maintenance

Summary Use of 2,4-D was superior to NAA or IAA for embryogenic callus initiation or maintenance in barley cultivar Bruce. A concentration of at least 2.0 mg/l 2,4-D was desirable for culture initiation. The developmental size of the embryo was more important than embryo age for obtaining embryogenic calli. Even brief exposures (20–40 days) of calli to concentrations of higher than 5.0 mg/l 2,4-D or 10.0 mg/l NAA resulted in inhibition of subsequent plant regeneration and therefore, concentrations above these could not be used for maintenance cultures. In the long-term maintenance cultures, the best production of embryogenic calli was with 0.1 mg/l and 1.0 mg/l 2,4-D.     

Selection of a highly-regenerative genotype of white clover (Trifolium repens L.) and plant regeneration from protoplasts derived from this genotype

Summary Callus cultures were induced from hypocotyl sections of 24 varieties of white clover (Trifolium repens L.). The calli did not show any significant difference of growth among the varieties. After the calli has been transferred to three regeneration media, green-spot formation was observed on calli derived from some seedlings. Remarkable intra- and intervarietal variations in the emergence of green spots and some trends between the origin of varieties and the frequency of green spots were observed. In most cases, the green spots turned brown without showing further differentiation, and only two genotypes formed shoots. A callus from a seedling of the Swedish variety Undrom has sustained high levels of plant regeneration throughout 24 months of culture. Protoplasts derived from this selected genotype were divided into cell colonies. 8P (Kao, 1977) medium containing 0.5 mg/1 2,4-D and 0.5 mg/1 kinetin was the most suitable medium for inducing divisions in protoplasts. When subcultured into solid B5 medium, the colonies produced calli, which when transferred to a regeneration medium, formed shoots. This genotype is expected to a useful subject for genetic engineering of white clover.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2ip 6-, -dimethylallylamino purine - NAA -naphthaleneacetic acid     

树莓品种‘Kivigold’快繁技术体系建立

以树莓（Rubus idaeus）品种‘Kivigold’带腋芽茎段为外植体,研究消毒剂HgCl2（0.1%）不同的消毒时间对外植体培养的影响,对不定芽增殖培养基和生根培养基中适宜的激素种类及浓度、生根苗移栽驯化中适宜的基质进行了筛选。结果显示,树莓品种‘Kivigold’外植体的最佳消毒时间为10 min,初代培养时只需添加6-BA即可满足外植体萌发和生长的需要;在不定芽增殖过程中,细胞分裂素主要影响不定芽的增殖,而生长素主要影响不定芽的生长,以质量浓度低于1.5 mg/L的6-BA以及质量浓度低于0.5 mg/L的NAA较为适宜,3种碳源中蔗糖更有利于不定芽的增殖和生长;经过进一步的筛选,确定适宜于‘Kivigold’不定芽增殖和生长的培养基为MS+1.00 mg/L 6-BA +0.10 mg/L NAA（含20 g/L蔗糖和5.9 g/L琼脂,pH 5.8）,适宜于‘Kivigold’不定芽生根的培养基为1/2 MS+0.10 mg/L NAA（含20 g/L蔗糖和5.9 g/L琼脂,pH 5.8）;在移栽驯化中最适宜的栽培基质为泥炭土:珍珠岩=1:1,移栽成活率可达到93.33%。     

小盐芥下胚轴再生体系的建立

以小盐芥下胚轴为外植体,研究了不同激素与不同浓度组合对愈伤诱导和不定芽分化的影响。结果表明,MS 6-BA 2,4-D组合能诱导出愈伤,诱导率为100%。MS 6-BA NAA组合既能诱导出愈伤又能分化不定芽,愈伤诱导率为100%,激素不同浓度不定芽分化率不同,最佳分化培养基为MS 2.5mg/L 6-BA 0.1mg/L NAA,不定芽分化率为43%。生根培养基为1/2MS,生根率为74%。     

Production of intergeneric hybrid plants between <Emphasis Type="Italic">Sandersonia aurantiaca</Emphasis> and <Emphasis Type="Italic">Gloriosa rothschildiana</Emphasis> via ovule culture (Colchicaceae)

Intergeneric hybrid plants between Colchicaceous ornamental plants, Sandersonia aurantiaca and Gloriosa rothschildiana, have successfully been produced via ovule culture. After 5 days of reciprocal cross-pollination, a few pollen tubes were observed in the ovary. Although seeds were obtained in both reciprocal cross-combinations, they did not germinate under ex vitro conditions. Ovules with placental tissues isolated 14 days after cross-pollination of S. aurantiaca × G. rothschildiana were cultured on a medium containing 0.01 mg l^–1 each of -naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), on which 41.5% of ovules swollen and produced callus-like structures within 10 weeks. When such swollen ovules were transferred to a medium containing 0.1 mg l^–1 each of NAA and BA, 7.5% of the initially cultured ovules produced rhizome-like structures within 6 weeks. Among the rhizome-like structures, those derived from two independent ovules (3.7% of the initially cultured ovules) produced multiple shoots following transfer to a medium containing 0.25 mg l^–1 NAA and 2.5 mg l^–1 BA. Multiple shoot-derived plantlets were established on a plant growth regulator-free medium, and they were successfully transplanted to pots. Early verification of their hybridity was accomplished by flow cytometry (FCM) analysis, chromosome observation and rDNA analysis.     

In vitro tetraploid induction via colchicine treatment from diploid somatic embryos in grapevine (Vitis vinifera L.)

A protocol for in vitro induction of tetraploids via colchicine-treated somatic embryos from immature zygotic embryos of diploid grapevine (Vitis vinifera L.) is reported. Embryogenic callus was initiated from immature zygotic embryos cultured on Nitsch and Nitsch (NN) medium supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The callus was transferred to NN medium containing 1.0 mg/l α-naphthalene acetic acid (NAA) and 0.5 mg/l benzyladenine (BA) to establish somatic embryogenesis. The vigorously growing globular embryos were selected and treated by 0, 10 or 20 mg/l colchicine for 1, 2 or 3 days, and then immediately transferred to NN medium supplemented with 0.03 mg/l NAA and 0.5 mg/l BA, for somatic embryo conversion and plant regeneration. The number of surviving embryos and regenerated plantlets following colchicine treatment decreased with increasing colchicine concentration and treatment time. Among 29 randomly investigated plantlets regenerated from colchicine-treated somatic embryos, five solid tetraploids (2n = 4× = 76) were identified by chromosome counting analysis; all others were diploid (2n = 2× = 38). Ploidy level of plant regenerated was also determined from leaves using flow cytometry. No chimeras with both 2C and 4C nuclei was produced from colchicine-treated somatic embryos. Significant differences in leaf stomata parameters were observed between diploid and induced tetraploid plantlets.