Silver birch (Betula pendula) plants with aux and rol genes show consistent changes in morphology,xylem structure and chemistry

The effects of Agrobacterium pRiA4 rol and aux genes, controlled by their endogenous promoters, on tree growth and wood anatomy and chemistry were studied in 5- and 7-year-old silver birch (Betula pendula Roth) plants. Southern hybridization confirmed the following rol and aux gene combinations: control plants (no genes transferred); plants with rolC and rolD genes; plants with rolA, rolB, rolC and rolD genes; and plants with rolA, rolB, rolC, rolD, aux1 and aux2 genes. Transgene mRNA was most abundant in phloem/cambium samples and in the developing xylem, whereas no expression was detected in leaves. Plants with rolC and rolD genes or with all the rol genes were significantly shorter and had smaller leaves and a more bushy growth habit than control plants or plants with both aux and rol genes. Morphological observations and wood chemistry analyses revealed that plants with rol genes produced less xylem and broke bud later than control plants or plants with both aux and rol genes. Tension wood was detected in both control and transgenic plants irrespective of their gene combination, probably as a result of greenhouse cultivation. Xylem fibers were shorter in transgenic plants than in control plants, and plants with all the rol genes were characterized by shorter vessels compared with the control plants and a smaller proportional area of vessels compared with the other groups. In addition, silver birch plants with all the rol genes had approximately a 3.3% lower concentration of total acid soluble carbohydrates than control plants. We conclude that the rolC and rolD genes induced the typical "rol-phenotype," and that this was emphasized by concomitant expression of the rolA and rolB genes and alleviated by the presence of aux1 and aux2 genes. We observed consistent phenotypic effects of rol and aux genes on the morphology, anatomy and cell wall chemistry of the plants.     

发根农杆菌(Agrobacterium rhizogenes) Ri T-DNA遗传转化丝石竹(Gypsophila paniculata)的研究

近年来,已发展出遗传转化高等植物的一些新技术,其中有些技术如脂质体融合,微注射技术和电击导入都是基于动物细胞培养方面的工作,而另一些技术是来自于植物界独特的天然转化系统,其中包括已知能遗传转化高等植物的农杆菌(Agrobacterium)的二个种,即致瘤农杆菌(A.tumefaciens)和发根农杆菌(A.rhizogenes),这二个种都能够将其致病质粒Ti或Ri所携带的DNA序列(T-DNA)插入到双子叶植物细胞核基因组中。pTi诱发寄主产生根基肿瘤,pRi诱发寄主产生毛状根。二者的差别可能是毛状根可以从毛状根培养物获得具有完整的T-DNA序列的有生育能力的再生植株,而从致病农杆菌(A.tumefaciens)菌株所诱发的肿瘤很难获得再生植株。因此,利用发根农杆菌(A.rhizogenes)pRi作为遗传转化高等植物的基因克隆载体的研究和应用日益受到重视。     

多拷贝rol基因对转基因杨树生长及内源激素的影响

对转入多拷贝rolB、rolC基因的三倍体毛白杨进行高生长量、生根率和内源激素(IAA、ABA)含量等指标的测定,以研究多拷贝rol基因在转基因植物中的表达。结果显示:转Ri质粒三倍体毛白杨再次分别转入rolB、rolC基因后,部分株系试管苗的生长受到了抑制,但各株系生根率均有不同程度提高;rolB基因转化植株的内源IAA平均含量高于rolC基因转化植株,其IAA/ABA值亦高于rolC基因转化植株。试验结果表明rol基因的多拷贝促使转化植株快速大量生成毛状根。     

切根和施肥对银杏大苗生长的影响

为探讨切根和施肥对银杏Ginkgobiloba裸根大苗生长的影响,同时为了缩短移栽后的缓苗期,2013-2014年在浙江省长兴县选取了15年生实生银杏裸根苗24株,2015-2017年选取了5~10年生实生银杏裸根苗72株,分别进行了两次切根和施肥试验,第一次的试验结果表明:一次切掉1/2的侧根,对苗木胸径、树高生长无明显影响,切根一年后侧根上细根数量大幅增加。第二次的试验结果表明:切根处理的苗木胸径、树高增长量均低于对照CK的增长量,但差异不显著,说明切根对银杏裸根苗的生长有所抑制,但影响不大;在不切根时,施肥对苗木树高的促进作用较为显著,同时切根与开沟施肥时,对银杏树高和胸径生长均有一定的抑制作用。两因素(切根与施肥)的主效应和交互效应分析显示,对于树高生长,切根与否不是影响树高生长的主要因素,施肥才是;对于胸径生长,则恰好相反;不管是对树高生长还是胸径生长,两因素间均存在交互作用,同时也表明分期切根和开沟施肥可作为实用技术推广到生产实践中。     

三叶青毛状根的诱导及其液体培养体系的研究

为建立三叶青毛状根的诱导和液体培养体系,采用发根农杆菌株系C58C1和ATCC15834进行毛状根的诱导,通过无菌三叶青叶片预培养、浸染、共培养、筛选、形态和PCR鉴定等过程获得三叶青毛状根根系,并对三叶青毛状根和两年生块根总黄酮进行比较分析。试验结果表明,发根农杆菌C58C1能从三叶青叶片诱导出毛状根,三叶青毛状根在不添加生长素的1/2MS液体培养基中生长状态良好,其总黄酮含量约是2年生三叶青块根的1/5。     

In vitro plant regeneration from cotyledon-derived callus cultures of leguminous tree Gleditsia caspica Desf.

An in vitro regeneration system was developed using organogenic callus derived from in vitro grown cotyledonary explants of Gleditsia caspica Desf., an important leguminous tree. Murashige and Skoog (MS) basal medium augmented with 0.2 g L^?1 myo-inositol and various concentrations of either 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid, or indole-3-butyric acid (IBA) alone as well as combined with cytokinins was used for callus induction. The highest frequency of organogenic yellowish-white and nodular callus (93 %) was obtained from explants grown on medium supplemented with 13.5 μM 2,4-D and 4.4 μM benzyladenine (BA). The yellowish-white and nodular callus when transferred to MS medium supplemented with BA (2.2–17.7 μM) or kinetin (KT; 2.3–18.8 μM) solely or in combination with 2.3 μM 2,4-D produced several microshoots after 5 weeks culture. The calli cultured on MS medium with 4.4 μM BA singly showed superior growth response and produced both maximum shoot regeneration (94 %) and the highest mean number (4.3) of microshoots per callus. Transfer of regenerated microshoots onto modified MS basal medium fortified with 5.8 μM gibberellic acid and 4.4 μM BA resulted in the maximum number of internodes per shoot and the highest shoot elongation after a period of 6 weeks. Optimum rooting of 90 %, an average 6.1 roots per shoot, and a mean root length of 3.6 cm was observed when half-strength MS medium was supplemented with 9.8 μM IBA and 0.92 μM KT. The regenerated healthy plants with well-developed shoots and roots showed a survival rate of 77 % after acclimatization and transplanting to garden soil for a 10-week hardening period under ex vitro conditions.     

Mevalonate kinase activity during different stages of plant regeneration from nodular callus cultures in white pine (Pinus strobus)

Mevalonate kinase (MK) catalyzes a step in the isoprenoid biosynthetic pathway, which leads to a huge number of compounds that play important roles in plant growth and development. Here, we report on changes in MK activity in white pine (Pinus strobus L.) during plant regeneration by adventitious shoot organogenesis from cotyledons of mature embryos, including nodular callus induction, shoot formation and rooting. Nodular calli were induced from Pinus strobus (PS) embryos by culture in nodular callus induction medium in a 0-, 8- or 16-h photoperiod. Mevalonate kinase activity peaked in nodular calli after three weeks of culture on nodular callus induction medium in a 16-h photoperiod, whereas frequency of nodular callus formation peaked after 4 weeks of culture on nodular callus induction medium in darkness. During adventitious shoot formation, MK activity peaked in shoots derived from dark-grown nodular calli after 3 weeks on bud formation medium, and frequency of shoot formation was highest in dark-grown nodular calli cultured on bud formation medium for 4 weeks. During rooting, MK activity peaked 2 weeks after transfer of adventitious shoots to rooting medium and rooting frequency was highest in adventitious shoots after 3 weeks on rooting medium. Although during nodular callus induction in darkness MK activity was inversely related to frequency of nodular callus formation, MK activity was highly correlated with frequency of shoot formation and with rooting frequency. The observed increase in MK activity preceding rooting suggests that MK could serve as a marker for rooting of white pine shoots in vitro.     

Stomatal and leaf growth responses to partial drying of root tips in willow

Root tips of intact willow (Salix dasyclados Wimm., Clone 81-090) plants were partially dried by exposure to ambient greenhouse air and then kept in water-vapor-saturated air for up to 3 days. The drying treatment increased abscisic acid (ABA) concentrations in both the root tips subjected to drying and in the xylem sap, while it reduced leaf stomatal conductance and leaf extension rate. Despite the decrease in stomatal conductance, leaf water potentials were unaffected by the root drying treatment, indicating that the treatment reduced hydraulic conductivity between roots and foliage. After roots subjected to drying were returned to a nutrient solution or excised, ABA concentrations in the remaining roots and in the xylem sap, stomatal conductance of mature leaves and extension rate of unfolding leaves all returned to values observed in control plants. The 4-fold increase in xylem sap ABA concentration following the root drying treatment was not solely the result of reduced sap flow, and thus may be considered a potential cause, not merely a consequence, of the observed reduction in stomatal conductance.     

Frost hardiness gradients in shoots and roots of picea mariana seedlings

Frost hardiness of tissues along the length of the stem and the root was investigated in first‐year black spruce (Picea mariana (Mill.) B.S.P.) seedlings. Frost hardiness of 1 cm long stem and root segments was evaluated based on Index of Injury, calculated from post‐freezing electrolyte leakage. Frost hardiness was tested approximately weekly beginning seven weeks after seedlings were transferred from an 18 to a 10 h photoperiod, both at day/night temperatures of 26°C/16°C. Trees were transferred to temperatures of 10°C day and 5°C night at a 10 h photoperiod after a further 18 days. Frost hardiness was greater at the terminal bud and least at the root tips. Although shoots were generally more frost hardy than roots, differences in hardiness along the stem and root axes were gradual, rather than abruptly differing at the shoot‐root interface. All tissues, including root tips, increased in frost hardiness after conditioning for 18 days under short photoperiods (10 h) and warm temperatures (26?C/16°C, day/night). Under cold temperatures (10°C/5°C, day/night) all tissues, excepting the root tips, tolerated — 16°C with little subsequent electrolyte leakage.     

不同培养条件对金铁锁毛状根生长的影响

利用发根农杆菌ATCC15834诱导金铁锁获得毛状根,研究金铁锁毛状根的最优培养条件。结果表明,在5种培养基中,以在1/2MS培养基中皂苷的总产量最大;添加3%蔗糖、水解乳蛋白(LH)及IBA利于毛状根的生长及总皂苷的积累;而添加IAA、NAA、6-BA及蛋白胨抑制其生长。     

Factors influencing axillary shoot proliferation and adventitious budding in cedar

We developed procedures for in vitro cloning of Cedrus atlantica Manetti and C. libani A. Rich explants from juvenile and mature plants. Explant size was one determinant of the frequency of axillary bud break in both species. Shoot tips and nodal explants mainly developed calli, whereas bud sprouting occurred in defoliated microcuttings cultured on a modified Murashige and Skoog medium without growth regulators. Isolation and continuous subculture of sprouted buds on the same medium allowed cloning of microcuttings from C. atlantica and C. libani seedlings and bicentennial C. libani trees, thus providing a desirable alternative for multiplying mature trees that have demonstrated superior characteristics. We also report adventitious bud differentiation from isolated embryos of C. atlantica. Neither auxin treatments nor other methods tested, including infection with Agrobacterium rhizogenes, were effective in inducing root initiation.     

The response of ectomycorrhizal fungi on Pinus densiflora seedling roots to liquid culture

Japanese red pine (Pinus densiflora) seedlings taken from a nursery were grown in liquid culture with all roots entirely submerged in water. The aims of the present study were to observe the reactions of preformed ectomycorrhizae to liquid culture and to examine new infections by ectomycorrhizal fungi on newly formed root tips in liquid culture. Inoculation levels were controlled by trimming the ectomycorrhizal roots to one of three selected root lengths. The results showed that the mantles of pre-formed ectomycorrhizae were lost during the 8 weeks of the liquid culture and that these preformed root tips became blackish and wrinkled, but Hartig nets remained in these blackish root tips. On the newly formed lateral root tips, no ectomycorrhizal mantles were formed, although Hartig nets were found. In addition, the level of inoculum did not affect the frequency of Hartig nets in newly formed root tips, suggesting that the density of inoculum was more important than the amount of inoculum for new infection. In conclusion, ectomycorrhizal fungi endured and infected new roots while submerged; however, only Hartig nets were formed and not mantles.     

Lignan formation in hairy root cultures of Edelweiss (Leontopodium nivale ssp. alpinum (Cass.) Greuter)

A hairy root line of Edelweiss (Leontopodium nivale ssp. alpinum (Cass.) Greuter) was obtained upon transformation with Agrobacterium rhizogenes strain ATCC15834. Elicitation of this line with silver nitrate, sucrose, methyl jasmonate and yeast extract at various concentrations in most cases resulted in a stimulation of lignan biosynthesis. Through elicitation with 6% sucrose the roots accumulated the pharmacologically active lignans leoligin and 5-methoxy-leoligin at levels of 0.0678% and 0.0372%, respectively, without significant growth inhibition. These lignan levels were comparable to those found in intact roots of cultivated Edelweiss. The biotechnological production of leoligin could be an attractive option for the continuous, field culture-independent production of the valuable secondary metabolites leoligin and 5-methoxy-leoligin.     

Life cycles of individual roots in fine root system of Chamaecyparis obtusa Sieb. et Zucc.

Recent studies have remarked on differences in the life cycles of individual fine roots. However, the dynamics of individual roots with different life cycles, such as ephemeral and perennial, during root system development are still unknown. We examined individual roots during fine root system development in a mature stand of Chamaecyparis obtusa Sieb. et Zucc. (Cupressaceae) using the sequential ingrowth core method and an anatomical method. The visual classification, i.e., orange, red, brown, intact dead, and fragmented dead, of fine roots corresponded well with the anatomical classification. Orange and red roots contained passage cells, and brown roots contained cork cambium. The proportions of protoxylem groups differed among visual classes. Brown secondary roots were mainly triarch (43%) and tetrarch (40%) and rarely diarch (12%), whereas fragmented dead roots, which constituted more than 95% of the dead roots, were mainly diarch (67%). These results imply that triarch and tetrarch roots tend to form secondary roots, whereas diarch roots tend to become dead roots without secondary growth. Using the numbers of root tips and clusters, root system development could be classified into three stages: colonization, branching within the root system, and maintenance. During the colonization stage, mainly triarch and tetrarch roots, which tend to be secondary growth, invaded ingrowth cores. During the branching stage, primarily diarch roots, which tend to be ephemeral, emerged. Fine root system development involved the recruitment of different individual roots during the life cycle depending on the growth stage.     

Ri质粒诱导药用植物毛状根技术及应用

毛状根是产生植物次生代谢产物的重要生物反应器。本文简述了发根农杆菌Ri质粒诱导植物产生毛状根的基本原理与方法及影响因子,对毛状根培养次生代谢物在药用植物上的应用作了概述,并提出其产业化前景、存在的问题。     

银杏非根尖细胞的染色体观察

由于雌雄异株植物银杏的种子及幼苗无法区分性别 ,因而在研究其性别时不能用传统的种子萌发取根的方法来进行染色体制片 ,而从雌、雄成株上取根也不切实可行。为解决该问题 ,本文以银杏的幼小花药、叶、胚珠以及小孢子、雌配子体游离核为材料 ,主要采用放线菌酮 (Cycloheximide,1 0 0mg/kg)作预处理药物 ,常规制片均得到了优良的染色体标本。     

Agrobacterium rhizogenes-mediated transformation to improve rooting ability of eucalypts

In an attempt to improve rooting in in vitro propagated Eucalyptus clones, root-inducing genes on the Ri plasmids of three Agrobacterium rhizogenes strains were inserted into the Eucalyptus genome. Root development was monitored in vitro and after the plantlets had hardened off. Only transformed roots grew as root cultures in hormone-free liquid medium. The potential use of this procedure for improving rooting of clonal material is discussed.     

Effects of IBA on the rooting of branch cuttings of Chinese jujube (<Emphasis Type="Italic">Zizyphus jujuba</Emphasis> Mill.) and changes to nutrients and endogenous hormones

‘Zhongqiusucui’ jujube secondary shoots were treated with 3-indolebutyric acid (IBA) at three concentrations, 500, 1000 and 1500 mg/L. Results show that IBA could significantly enhance rooting and root characteristics of cuttings and were best with IBA at 1500 mg/L. In the rooting process, the formation of adventitious roots was related to the consumption and accumulation of nutrients (soluble sugars and proteins) and the changes in endogenous hormones in phloem, leaf tips and leaf bases. The rooting of cuttings had a positive correlation with the consumption of soluble sugars during the period of callus formation and with the accumulation of soluble sugars during adventitious root formation and growth. Rooting was positively related to the breakdown of soluble proteins in the phloem when the callus formed, and had a positive correlation with its accumulation during adventitious root formation and growth. Leaf tips and leaf bases showed a reverse trend in changes of soluble protein. However, together with the phloem, leaf tips and leaf bases regulated and controlled the formation and development of adventitious roots. The main activities of soluble proteins exist in the leaf tips as this was the main source of soluble proteins. The relation between rooting and IAA (indole-3-acetic acid) content in phloem was positive and thus a high concentration of IAA could benefit the induction and formation of adventitious roots. However, rooting was negatively related with ABA (abscisic acid) and GA (gibberellic acid) and a high concentration of both could inhibit the induction and formation of adventitious roots. Rooting had a positive correlation with phloem IAA/ABA ratios, and higher ratios could improve rooting. Low concentrations of ZR (zeatin riboside) triggered the induction of adventitious roots, while higher concentrations promoted root growth. Endogenous hormones in leaf tips and bases had an impact on rooting. The activities of endogenous hormones mainly existed in leaf tips because they play a major role in the production and consumption of IAA and its ABA content increased during rooting. The ZR in leaf tips influenced the rooting of cuttings, especially in the callus formation and rooting stage. Leaf tips were the main source of GA.     

沙棘主要蛀干害虫危害特性及种群动态变化

沙棘木蠹蛾主要危害沙棘栽植树的根部和干部，红缘天牛同时危害沙棘栽植树和根蘖苗的干部，已成为宁夏沙棘林中两大主要蛀干害虫。通过对沙棘树不同高度范围内沙棘木蠹蛾幼虫和红缘天牛幼虫的调查发现，沙棘木蠹蛾幼虫主要分布在地下根部和干部40～120cm的范围内，而红缘天牛主要分布在树干40～120cm的高度内。红缘天牛幼虫数量随时间变化大致呈现向树干上部增加的变化趋势。沙棘木蠹蛾幼虫数量随时间变化呈现明显的向根部转移的趋势，7月末，有近98％的幼虫在根部危害。之后，由于初孵幼虫的大量出现，使干部幼虫数量急剧增加，越冬前，虽然有部分转移至根部危害，但到9月末仍有近43％的幼虫停留在树干40～120cm处越冬，直到次年越冬结束。沙棘木蠹蛾和红缘天牛在沙棘树干部40cm以上危害位置的较大重叠，必然造成它们在时间、空间和营养上的激烈竞争。     

Ginkgolide B produced endophytic fungus (Fusarium oxysporum) isolated from Ginkgo biloba

To screen the presence of ginkgolide B-producing endophytic fungi from the root bark of Ginkgo biloba, a total of 27 fungal isolates, belonging to 6 different genus, were isolated from the internal root bark of the plant Ginkgo biloba. The fungal isolates were fermented on solid media and their metabolites were analyzed by TLC. The obtained potential ginkgolides-producing fungus, the isolate SYP0056 which was identified as Fusarium oxysporum, was successively cultured in the liquid fermentation media, and its metabolite was analyzed by HPLC. The ginkgolide B was successfully isolated from the metabolite and identified by HPLC/ESI-MS and (13)C-NMR. The current research provides a new method to produce ginkgolide B by fungal fermentation, which could overcome the natural resource limitation of isolating from the leaves and barks of the plant Ginkgo biloba.