Efficient embryogenic callus induction and plant regeneration from embryonic axis explants inQuercus acutissima

Embryogenic callus ofQuercus acutissima was successfully induced from embryogenic cultures, and plants were regenerated from the callus. The development of the techniques involved will allow mass propagation and gene transformation in this species. Embryogenic cultures were formed from embryonic axis explants (i.e., embryos without cotyledons) excised from immature embryos, after culture on Murashige and Skoog (MS) medium containing indolebutyric acid and benzyladenine. Attempts to induce embryogenic cultures from cotyledon explants were unsuccessful. Embryogenic calli were induced at high frequency from embryogenic cultures on MS medium containing 2,4-dichlorophenoxyacetic acid. However, benzyladenine inhibited embryogenic callus formation. Somatic embryo development from embryogenic calli occurred on MS medium in all of the seven cell lines tested. Germination of somatic embryos was induced on half strength MS medium without plant growth regulators. Finally, acclimated plants growing in soil were obtained.     

Influences of antibiotics on plantlet regeneration via organogenesis in Ioblolly pine （Pinus taeda L.）

Three antibiotics ampicillin, carbenicillin, and cefotaxime were evaluated for their effects on induction, growth, and differentiation of organogenic calli, as well as rooting of regenerated shoots of three Ioblolly pine (Pinus taeda L.) genotypes. Of the antibiotics administered, cefotaxime maximally increased the frequency of callus formation and growth rate of organogeni ccalli, carbenicillin maximally increased the frequency of shoot regeneration and the average number of adventitious shoots per piece of organogenic callus, ampicUlin maximally decreased the rooting frequency of regenerated shoots and mean number of roots per regenerated shoot, in comparison with antibiotic-free media. Compared with the control, ampicillin minimally increased the frequency of callus formation, cefotaxime minimally increased the frequency of shoot regeneration, and carbenicillin minimally decreased the rooting frequency of regenerated shoots in three Ioblolly pine genotypes tested. All three antibiotics increased the frequencies of callus formation and shoot regeneration, and reduced the rooting frequency ot regenerated shoots suggested that the establishment of an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into Ioblolly pine need to select a suitable antibiotic. This investigation could be useful for optimizing genetic transformation of conifers.     

草地早熟禾耐盐愈伤组织筛选的适宜NaCl浓度和继代次数研究

草地早熟禾(Poa pratensis L.)是禾本科早熟禾属多年生草本植物,是我国北方地区应用最广泛的冷季型草坪草.近年来,由于土壤的盐化问题日益严重,培育耐盐的草地早熟禾品种显得尤为重要.体细胞无性系变异是来源于已经存在的细胞变异和培养过程中诱导的变异[1].变异的诱导与选择为提高植物的耐盐性提供了一条有效的途径.     

Somatic embryogenesis and plant regeneration inAcanthopanax sciadophylloides

Somatic embryos ofAcanthopanax sciadophylloides Franch. et Sav. were differentiated from both zygotic and somatic embryos and calli, and plants were regenerated from these somatic embryos. A zygotic embryo, enclosed within a small portion of the endosperm, was incubated on Murashige and Skoog (MS) media supplemented with various combinations (range 0–10.0 mg/l) of 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D). After 4 months, swelling of the zygotic embryos and callus formation was observed. When the swollen embryos were transferred to MS medium supplemented with 0.5 mg/l of 2,4-D, somatic embryos were formed in one to two months. After subculture on the same medium, new embryos were differentiated from various parts of the older somatic embryos. The calli were cultured on medium supplemented with 2.0 mg/l of 2,4-D and BAP for three weeks. Proliferated calli were transferred to medium supplemented with 1.0 mg/l of 2,4-D and BAP. Somatic embryos were differentiated from the calli within one to two months. Somatic embryos were germinated on half-strength MS medium without plant growth regulators and the plantlets were grown in soil. A part of this paper was presented at the 106th Annual Meeting of the Japanese Forestry Society (1995) & First Asia-Pacific Symposium on Forest Tree Genetic Improvement (Beijing).     

Caulogenic callus induction and adventitious bud formation from embryos of long-term stored seeds ofPicea jezoensis

Caulogenic calli with a high differentiation potency were induced from mature embryos ofPicea jezoensis seeds stored over a long time, for 29 years, resulting in the active formation of adventitious buds. Embryos began to induce calli within 3 weeks of cultivating on LP medium containing 3 μM BAP and 1 μM 2,4-D. Then, the calli proliferated and transformed into caulogenic calli with bud primordia in 8 weeks. The caulogenic calli increased actively with the addition of 500 mg/l ofl-glutamine in the medium. Furthermore, caulogenic calli, induced on LP medium containingl-glutamine, resulted in the formation of adventitious buds, which elongated after transferring the calli into LP medium with 0.1 μM BAP, but withoutl-glutamine. It appears that the number of adventitious buds and the process of shoot elongation are influenced by the kind of nitrogen contained in the medium for callus induction. A part of this study was presented at the 107th Annual Meeting of the Japanese Forestry Society (1996).     

不同盐浓度对木麻黄无性系愈伤组织的影响

以短枝木麻黄4个无性系幼枝茎段为外植体，在不同NaCl浓度的培养基上进行愈伤组织诱导，观察不同盐浓度对木麻黄外植体愈伤组织分化的影响。试验结果表明：培养基中NaCl浓度达到0．064mol／L(0．374％)，比正常浓度高出320倍的情况下木麻黄外植体表现出很强的耐盐性，仍然能诱导出正常的愈伤组织；不同的NaCl浓度的各个处理之间愈伤组织的诱导率、大小、出芽率、继代愈伤组织的大小均没有显著差异；但无性系之间的愈伤组织的诱导率及愈伤组织的大小均有极显著差异；不同继代间愈伤组织的大小有极显著差异。     

抗生素对火炬松的器官发生植株再生的影响(英文)

本文研究了氨苄青霉素、羧苄青霉素和头孢霉素等3种抗生素对火炬松愈伤组织的生长和分化及不定芽生根的影响。结果表明,头孢霉素最有利于愈伤组织的诱导和生长,羧苄青霉素最有利于芽的分化,氨苄青霉素降低了不定芽的生根频率。所有试验的3种抗生素提高愈伤组织的形成和芽再生,但降低了芽的生根频率。这些结果表明,选择合适的抗生素对优化火炬松遗传转化体系有重要作用。图3表4参25。     

Plantlet regeneration from leaf protoplasts ofPopulus euphratica

Protoplasts were isolated from the leaves of sterile plants ofPopulus euphratica Oliv. by using 1% Cellulase “Onozuka” RS and 0.25% Pectolyase Y-23 in 0.6m of mannitol solution. Protoplasts were cultured in modified Murashige and Skoog's (MS) medium which contained no ammonium ions but was supplemented with BAP (6-benzylaminopurine), 2,4-D (2,4- dichlorophenoxy-acetic acid), and 1% sucrose at the cell density of 9×10⁴/ml. Cell divisions occurred in every culture medium, especially in the medium containing 0.5 mg/l of BAP and 0.1 mg/l of 2,4-D, in which callus was successfully induced by successive culture through cell cluster formation. Shoots were regenerated from the callus, and their growth was enhanced on 1/2 MS medium containing 0.8 mg/l of BAP. Finally, shoots were rooted and plantlets were regenerated on 1/2 MS medium without a hormone. A part of this paper was presented at the 106th Annual Meeting of the Jpn. For. Soc. (1995).     

Selection for salt and drought tolerance in protoplast- and explant-derived tissue cultures of Colt cherry (Prunus avium x pseudocerasus)

Colt cherry (Prunus avium x pseudocerasus) callus cultures were derived from leaf protoplasts, protoplasts of root cell suspension cultures, or by direct culture of leaf and root tissues. Survival of calli cultured on basal proliferation medium containing 25, 50, 100 or 200 mN (millinormal) NaCl, Na(2)SO(4) or KCl, or iso-osmotic (with NaCl) concentrations of mannitol ranged from 1 to 15%. After six transfers on the same medium, surviving cell lines were subjected to three cycles of direct recurrent selection; i.e., in each cycle, they were cultured alternately on basal proliferation medium, and on basal proliferation medium supplemented with NaCl, KCl, Na(2)SO(4) or mannitol. Salt- or mannitol-tolerant cell lines selected in this way had smaller cells than unselected cell lines, and they grew more rapidly and had higher callus and cell survival rates than unselected cell lines when cultured in the presence of salt or mannitol. Cells lines selected for tolerance to one agent (sodium salt, potassium salt or mannitol) showed minimal tolerance to another agent. However, when plants were regenerated from salt- or mannitol-tolerant callus and new cultures derived from them, the new cultures showed tolerance to all of the salts and mannitol. Plant regeneration from the new cultures was not achieved under the conditions that led to the regeneration of the parent plants from callus.     

影响非洲菊叶柄离体培养再生的因素

以3个非洲菊品种的叶柄为材料,研究了不同激素及其浓度对非洲菊离体培养再生的影响。结果表明:3个品种的叶柄在含单一细胞分裂素6-BA的培养基上培养时都不能被诱导出愈伤组织;而在仅含生长素NAA的培养基上培养时均可被诱导出愈伤组织,并且在其愈伤组织发生部位都有不定根发生,但只有品种6267能从不定根发生部位直接分化出不定芽;当在含NAA的培养基上再附加6-BA时也可被诱导出愈伤组织,但无不定根发生。所产生的愈伤组织在分化培养基上培养时只有由品种Ⅱ叶柄在同时含有NAA和6-BA的诱导培养基上产生的愈伤组织才可以分化出不定芽。表明愈伤组织的诱导与分化、不定芽和不定根的发生与品种及培养基中的激素种类有关。     

甜椒抗菌蛋白基因(harp)转化桉树的研究

用不同浓度的2,4-D与IAA对桉树叶盘进行了愈伤组织的诱导及植株再生的试验,建立了新的桉树再生体系.进一步用含甜椒抗菌基因(hrap)的农杆菌侵染桉树叶盘,发现侵染后对愈伤组织诱导率无明显影响,却抑制了桉树的植株再生.对再生苗的PCR和Sou thern杂交检测证实已将hrap基因已转入桉树植株的基因组中.     

Monitoring genetic stability inQuercus serrata Thunb. somatic embryogenesis using RAPD markers

Genetic stability of propagules regeneratedvia somatic embryogenesis is of paramount importance for its application to clonal forestry. Random amplified polymorphic DNA (RAPD) markers were used to determine the genetic stability in somatic embryogenesis ofQuercus serrata Thunb. (Japanese white oak). Forty samples from an embryogenic line, consisting of regenerated plantlets, somatic embryos, and embryogenic calli, were examined using 54 decanucleotide primers. A total of 6520 clear reproducible bands obtained from these studies exhibited no aberration in RAPD banding pattern among the tested samples. Our results show that somaclonal variation is absent in our plant propagation system. The genetic stability is discussed in terms of the origin of somatic embryos.     

榉树叶器官再生植株技术

以榉树（Zelkova schneideriana）叶片为外植体,先诱导愈伤组织的产生,再诱导愈伤组织分化出芽,研究榉树叶器官再生植株技术,结果表明：榉树叶片诱导出愈伤组织很容易,幼嫩叶片的诱导率可达到90％以上,而由愈伤组织诱导芽分化则相对较难,仅有10％左右的诱导率,老叶片诱导的愈伤无法再分化出芽。     

Establishment of an in vitro plant regeneration protocol for Casuarina cunninghamiana Miq. via indirect organogenesis

An in vitro plant regeneration protocol via indirect organogenesis from morphogenetic callus was established for Casuarina cunninghamiana Miq. Effects of plant growth regulator NAA (naphthaleneacetic acid) and BAP (6-benzylaminopurine), sucrose and AgNO₃ on callus induction, adventitious bud differentiation and shoot development were examined. Explants used were epicotyl fragments from 45-day-old seedlings. The largest callus (4.29 mm in diameter) was obtained after 1 month on a basic culture medium consisting of Murashige and Skoog ? macro- and full strength micro- elements, Nitsch and Nitsch vitamins, supplemented with 0.54 μM NAA, 3.30 μM BAP, and 30 g L⁻¹ sucrose. The calli were subcultured in the same medium above for 2 months. They were then cultured for another 2 months for adventitious bud differentiation and shoot development. The highest mean adventitious bud differentiation, number of shoots formed per callus and number of shoots ≥2 cm long per callus (47.50%, 27.38 and 4.75, respectively) were achieved on the above medium modified with NAA at 0.27 μM and supplemented with AgNO₃ 1 mg L⁻¹. Shoots were successfully rooted without plant growth regulator and the rooted plantlets survived and grew normally. This protocol for in vitro plant regeneration provides a tool not only for vegetative propagation but also for plant genetic transformation and gene function studies of C. cunninghamiana.     

盾叶薯蓣愈伤组织培养及其高产系的筛选

以盾叶薯蓣的块茎、茎、叶和茎尖为材料,对其进行了愈伤组织培养,并根据愈伤组织个体间生长速度和皂甙含量差异,进行了高产系的筛选．结果表明：盾叶薯蓣不同外植体（块茎、茎、叶、茎尖）均能诱导出愈伤组织,其中以茎尖形成愈伤组织最快,皂甙元含量最高;不同培养基、ｐＨ 值、接种量、温度、激素等因子对愈伤组织的形成、生长及皂甙元含量有很大的影响;改良 Ｍ Ｓ培养基、２,４－Ｄ 浓度为２．０～４．０ ｍ ｇ／Ｌ、ＢＡ 为 ０．５ ｍ ｇ／Ｌ 的激素配比使愈伤组织的生长量、产物含量最高;对无性系进行筛选和 ＨＰＬＣ 检测,以 １２号无性系生长速率和皂甙元含量最高,其皂甙含量达 ３．７％ ,生长量比普通系高２１．５％ ．     

野葛愈伤组织诱导与不定芽分化

采用MS为基本培养基，附加NAA、6-BA和IAA3种激素．诱导野葛不同外植体愈伤组织形成；再将愈伤组织接种在不同浓度6-BA的MS培养基上．分别在光照和黑暗条件下进行不定芽的诱导。结果表明．野葛幼叶、茎段和顶芽在适宜激素的诱导下均能形成愈伤组织，但不同激素组合其愈伤组织诱导率不同。幼叶愈伤组织诱导的最适培养基为MS NAA1．0mg／L 6-BA1．0mg/L．其诱导率为75％．茎段愈伤组织诱导的最适培养基为MS NAA1．0mg／L 6-BA3．0mg／L IAA0．2mg/L，其诱导率为70％。不同外植体形成的愈伤组织其不定芽的分化诱导率不同．茎段愈伤组织在不同浓度6-BA下均能诱导出不定芽；光照有利于芽的分化．在光培养条件下，茎段愈伤组织不定芽平均诱导率为66．7％。     

Agrobacterium tumefaciens-mediated GUS gene transfer to Sophora japonica L.

Agrobacterium-mediated genetic transformation of Sophora japonica was standardized using the Agrobacterium tumefaciens strain LBA4404 that harbored the binary vector pBI121 containing genes forβ-glucuronidase (GUS) and neomycin phos-photransterase (nptⅡ). S. japonica transformants were selected by the ability of the leaf explants to produce kanamycin-resistant calli that regenerated into kanamycin-resistant plantlets. Successful transformation was confirmed by histochemical assay for GUS activity, PCR analysis and Southern blot. The period of nearly two months was required for the regeneration of transgenic plantlets from the explants. The transformed plants resembled their parents in morphology.     

尾巨桉愈伤组织诱导与植株再生研究

分别以尾巨桉幼苗的茎段和叶片作外植体进行愈伤组织诱导、不定芽分化及植株再生培养研究。结果表明：在改良H培养基上茎段和叶片两种外植体均能诱导出愈伤组织,7-10d产生愈伤组织,愈伤组织诱导率达90%以上。在改良H＋6-BA1.0mg·L-1＋NAA0.5mg·L-1＋蔗糖5%培养基上,茎段愈伤组织的分化率达22.2%,每块愈伤组织有效芽数达10-20个,并且诱导出了根芽齐全的单生型小苗。     

In vitro flowering of green and albino Dendrocalamus latiflorus

To propagate Dendrocalamus latiflorus, we used in vivo inflorescences to produce calli on Murashige and Skoog basal (MS) medium supplemented with 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg/l kinetin, 250 mg/l polyvinyl pyrrolidone (PVP), and 1% coconut milk. Multiple shoots were generated on MS medium supplemented with 0.1 mg/l thidiazuron (TDZ). The green plantlets were successfully transferred to soil. Multiple albino shoots also regenerated and were able to proliferate on medium containing cytokinins, especially TDZ. Albino multiple shoots rooted in medium containing α-naphthaleneacetic acid (NAA), and callus formation was observed in the presence of 2,4-D and picloram. Green and albino regenerates flowered after 8 months of subculture. The flowering ratio increased to 44% after three treatments in medium containing 1 mg/l TDZ. Morphological observations revealed that the in vitro green and albino flower organs were normal. However, pollen derived from the in vitro flowers of both the green and albino plants were sterile.     

雷公藤胚性愈伤组织再生植株的增殖及其稳定性

以雷公藤胚性愈伤组织为材料,研究继代培养时间对植株再生和基本培养基、生长素浓度及其与细胞分裂素组合对再生苗增殖的影响.结果表明:在继代培养过程中,愈伤组织再生率和再生苗数经历了一个由低到高再到低的过程,以8个月到1年之间的胚性愈伤组织的再生能力最强,达100%,每块愈伤组织平均形成的芽数也比较多,雷公藤胚性愈伤组织在继代20个月后已经丧失再分化能力.愈伤组织再分化过程易发生变异,从叶形上看,再生苗出现6种类型的变化.再生苗茎段增殖以1/2MS为基本培养基附加2.0 mg·L-1IBA为好,以1/2MS+2.0mg·L-1IBA+O.5 g·L-1活性炭的培养基有利于芽苗生根培养,生根率达100%.移栽至珍珠岩与腐殖质土(1:1)的混合基质中成活率达96%.