Agrobacterium infection of hemp (Cannabis sativa L.): establishment of hairy root cultures

Abstract

Experimental conditions were optimized for hemp, a difficult to transform plant, to be effectively infected with either Ri or Ti plasmid-bearing agrobacteria and to establish stably transformed tissues. Hypocotyl of intact seedlings was the most responsive material and the response depended on both bacterial strain and plant variety. Transformed tissues, hairy roots and tumors, were cultured and stabilized in vitro and showed the characteristic traits of fast and phytohormone-independent growth as well as high incidence of lateral branching and abundance of root hairs in the case of roots. They all contained T-DNA of the corresponding Ri or Ti plasmid as revealed by PCR analysis with specific primers and further hairy roots induced by AR10GUS strain showed normal pattern of β-glucuronidase positive staining. To our knowledge, this represents the first reported protocol for the establishment of Cannabis sativa hairy root cultures.     

Nitrogen fertilization and sex expression affect size variability of fibre hemp (Cannabis sativa L.)

Mechanical harvesting and industrial processing of fibre hemp (Cannabis sativa L.) require uniformity of stem length and weight. In 1991 and 1992 we carried out field experiments to investigate the effects of soil nitrogen level (80 and 200 kg ha^-1N) and row width (12.5, 25 and 50 cm) on the variability of weight and height in hemp plants. The crops were sampled 5 times between early June and early September. Row width did not affect size variability. At final harvest coefficients of variation (CV) of both weight and height were about 1.5 times higher at 200 than at 80 kg ha^-1N. Distributions of dry weight were positively skewed at all sampling dates except the first, with skewness larger at 200 than at 80 kg ha^-1N. Distributions of height were negatively skewed at all sampling dates except the first at 80 kg ha^-1N. At 200 kg ha^-1N they changed from negative skewness during the first part of the growing season to negative kurtosis in the second part of the growing season. More suppressed plants were present at 200 than at 80 kg ha^-1N. Contrary to most published results, we did not find a reduction of CV of weight nor of CV of height at the onset of self-thinning. Suppressed hemp plants can survive relatively well in the low-light environment under the canopy. Sexual dimorphism contributed to variability of height and weight, but the effects were smaller than those of nitrogen fertilization. The ratio of female to male plants was higher at 200 than at 80 kg ha^-1N, as a result of a shift in sex-ratio within the population of suppressed plants. As suppressed plants were much more slender than dominating plants, self-thinning eliminated the most slender individuals in a hemp crop. However, the presence of many more heavy individuals of low slenderness at 200 than at 80 kg ha^-1 N was probably the major cause of the difference in slenderness between 200 and 80 kg ha^-1 N.     

Flavonoid glycosides and cannabinoids from the pollen of Cannabis sativa L

Chemical investigation of the pollen grain collected from male plants of Cannabis sativa L. resulted in the isolation for the first time of two flavonol glycosides from the methanol extract, and the identification of 16 cannabinoids in the hexane extract. The two glycosides were identified as kaempferol 3-O-sophoroside and quercetin 3-O-sophoroside by spectroscopic methods including high-field two-dimensional NMR experiments. The characterisation of each cannabinoid was performed by GC-FID and GC-MS analyses and by comparison with both available reference cannabinoids and reported data. The identified cannabinoids were delta9-tetrahydrocannabiorcol, cannabidivarin, cannabicitran, delta9-tetrahydrocannabivarin, cannabicyclol, cannabidiol, cannabichromene, delta9-tetrahydrocannabinol, cannabigerol, cannabinol, dihydrocannabinol, cannabielsoin, 6a, 7, 10a-trihydroxytetrahydrocannabinol, 9, 10-epoxycannabitriol, 10-O-ethylcannabitriol, and 7, 8-dehydro-10-O-ethylcannabitriol.     

大麻性别的RAPD和SCAR分子标记

利用随机扩增多态性DNA(randomamplifiedpolymorphicDNA,RAPD)技术获得与大麻性别连锁的分子标记.将10株雄性大麻或10株雌性大麻的单个DNA样品等量混合分别组成雄性或雌性DNA池(DNApool),以提供具有相同遗传背景的雌、雄性DNA样品.每个随机引物分别用三个不同的循环程序进行PCR扩增.在30个随机引物中,用引物401扩增得到一条约2.5kb雄性多态性片段.对该片段进行了克隆和序列分析,并根据序列分析结果将上述RAPD分子标记转化为重复性和特异性更好的SCAR(sequencecharacterizedamplifiedregions)分子标记.     

Establishment of a highly efficient regeneration system from leaf base segments of oat (Avena sativa L.)

A reliable and efficient protocol for the regeneration of fertile plants derived from leaf base segments of young in-vitro-grown oat seedlings has been developed successfully. Callus induction and shoot regeneration were achieved when the basal region of young seedlings was cultured on auxin-containing medium. Callus induction efficiencies as well as regeneration frequencies were correlated with the developmental stage and the genotype of the explants. In five different genotypes of oat, an average of 25 plants per explant could be produced and for the most responsive genotype more than 50 regenerants per explant could be regenerated reproducibly. This high regeneration potential makes oat leaf bases a very attractive target for transformation. Received: 6 May 1997 / Revision received: 10 August 1997 / Accepted: 15 September 1997     

The influence of temperature on leaf development and growth in potatoes in controlled environments

Three controlled environment experiments were conducted at different temperatures to determine the relation between temperature and leaf development and growth in the potato (cv. Maris Piper). Developmental stages are defined for the appearance and duration of leaf extension in the potato and comparisons made with other temperate zone crops. The rate of leaf appearance was linear over the temperature range (9–25°C) and above 25°C there was no further increase in the rate. The temperature coefficient for the rate of appearance of leaves was 0.032 leaves (degree days)^-1 using a base temperature of 0°C. The duration of extension of an individual leaf decreased with increase in temperature up to 25°C such that the thermal duration was constant at 170 degree days using a base temperature of 0°C for leaf positions 4–10 on the main stem. At higher leaf positions the thermal duration was similar or greater. The advantages and limitations of controlled environment work as a parallel to field experimentation are discussed.     

A controlled-environment chamber for measurement of canopy photosynthesis by small stands of lettuce (Lactuca sativa L.)

Abstract. A controlled-environment chamber constructed from a standard chest freezer was used to grow and measure the CO₂ exchange of small stands of lettuce ( Lactuca sativa L. ev. Ambassador). The chamber, with horizontal air flow, provided good control of air temperature ( c. 6 to 16°C), irradiance (0–300 μmol PAR m ²S¹ and CO₂ (350–1000 μmol mol⁻¹). The photosynthetic response to changes in these variables was measured using an inexpensive CO₂ dosing system which recorded the input rate required to maintain a constant concentration of CO₂ (to ± 2.5%). Characteristis of the growth environment and the changes in response to temperature and irradiance are described.     

Leaf area index, canopy structure and photosynthesis of red clover (Trifolium pratense L.)

Abstract. The influence of leaf age, total leaf area and its dispersion in space on canopy photosynthesis were studied using microswards of red clover ( Trifolium pratense L.) which were established in the greenhouse. Two varieties, Renova (flowering) and Molstad (non-flowering), were sown in separate plastic boxes at densities of 225, 400 and 625 plants per m².
Vertical distribution of photosynthetically active radiation (PAR), leaf area, leaf age and ¹⁴CO₂-fixation were determined periodically. Net photosynthesis and dark respiration of canopies were measured. Maximum photosynthetic capacity of individual leaves was measured on plants taken from the intact canopy or from plants where shading of the growing leaves had been prevented.
Net photosynthetic rate of canopies increased linearly with leaf area index (LAI) up to an LAI of 3.5 and then declined at higher LAI, independent of variety and sowing density. Below the optimum LAI, net photosynthesis depended mainly on interception of PAR. Decrease in canopy photosynthesis above the optimum LAI was due to a higher proportion of old leaves with decreased photosynthetic capacity, and not to an increase in respiring plant parts. It is concluded that LAI and position of leaf age categories in the canopy are more important than vertical distribution of leaf area in determining canopy photosynthesis of red clover.     

水稻对叶瘟和穗瘟部分抗性的遗传分析

在一个水稻籼籼交重组自交系群体中,选用由感病株系构成的2个亚群体和2个不同的稻瘟病菌小种,进行了水稻对叶瘟部分抗性的QTL定位,还选用由感病而且抽穗期相近的株系构成的亚群体和另一个病菌小种,进行了水稻对穗瘟部分抗性的QTL定位,将病叶面积百分比(DLA)、病斑大小(LS)和病斑数(LN)作为对叶瘟部分抗性的性状,将病斑长度(LL)和孢子量(CA)作为对穗瘟部分抗性的性状。所构建的图谱包含168个标记。应用QTLMapper 1．01b,共检测到11个表现主效应的QTL和28对双因子互作,有3个表现主效应的QTL参与对同一性状的互作。QTL的主效应对单一性状的贡献率为4．7％～38．8％,而上位性效应对单一性状的贡献率为16．0％～51．7％,QTL的主效应对大多数性状的贡献率小于互作效应,表明互作效应对于部分抗性的重要作用。对穗瘟部分抗性的两个性状LL和CA,所检测到QTL总效应的贡献率分别达到70．6％和82．6％,表明由排除了主效抗病基因的感病株系组成的亚群体适合于进行部分抗性QTL定位。     

Rice (Oryza sativa L.) cultivars tolerant to high temperature at flowering: anther characteristics

We examined the relationship between morphological characteristics of anthers and fertility in japonica rice cultivars subjected to high temperature (37.5(26 degrees C day/night) at flowering. Percentage fertility was negatively correlated with the number of cell layers that separated the anther locule from the lacuna that formed between the septum and the stomium. The cell layers consisted of the remaining septum and degraded tapetum, and serve to keep the adjacent two locules closed. Anther dehiscence therefore requires the rupture of the cell layers. We conclude that the tight closure of the locules by the cell layers delayed locule opening, and decreased fertility at high temperatures.     

The prediction of leaf canopy expansion in the leek from a simple model dependent on primordial development

In an analysis of leaf development of leek plants grown in the field in 1988, successive leaves initiated, appeared (tip and ligule) and senesced at equal intervals of accumulated temperature/thermal time. These intervals corresponded to a plastochron of 92°C days and phyllochrons of 135 (tip) and 233 (ligule) °C days. The rate of appearance of ligules was exactly equal to the rate of leaf senescence, with the result that the number of fully-expanded leaves per plant remained constant at 1.4. These data, which were compatible with results from previous seasons, were used to develop a model of the interrelationships between primordium initiation at the shoot apex and subsequent events in the development of individual leaves. Primordium initiation is considered to be the primary controlling event in the life of a leaf, and the processes of tip appearance, ligule appearance and death can be predicted from knowledge of the number of primordia which have been initiated, without reference to the environment. A model of canopy expansion, based on the central role of the shoot apex, was developed using the temperature relations of primordium initiation and additional data on leaf expansion and leaf dimensions. Leaf area indices computed in this way provided a satisfactory simulation of the thermal-time course of leaf area index observed in a previous season, 1985.     

Water-use efficiency in rice (Oryza sativa L.) in relation to resistance to salinity

Abstract The effects of sodium chloride on water-use efficiency of a number of varieties of rice have been investigated. Sensitivity to salinity in rice arises in large part from excessive rates of sodium ion transport to the shoot consequent upon high rates of transpirational water loss. Varietal differences in water-use efficiency were found: these were greater when measured for whole shoots over a period of one week, than when made as instantaneous measurements on individual leaves. Salinity had rather little effect on water use efficiency but overall resistance of seedlings to salt was, in general, greater the greater the water-use efficiency. The reasons for the differences between varieties are discussed in terms of differences in growth pattern: among the seven varieties investigated, water-use efficiency and salt-resistance were lower in dwarfed as opposed to non-dwarfed varieties.     

Intergeneric somatic hybridization of rice (Oryza sativa L.) and barley (Hordeum vulgare L.) by protoplast fusion

An intergeneric somatic hybrid was obtained upon fusion of protoplasts of rice and barley. Protoplasts isolated from suspension cultures of rice cells were fused by electrofusion with protoplasts that had been isolated from young barley leaves. Some of the resultant calli formed green spots and shoots. Only one shoot formed roots, and it was subsequently successfully transferred to soil in a greenhouse. Its morphology closely resembled that of the parental rice plant. Cytological analysis indicated that the plant had both small chromosomes from rice and large chromosomes from barley. Southern hybridization analysis with a fragment of the tryptophan B (trpB) gene revealed both a rice-specific band and a barley-specific band. Mitochondrial (mt) and chloroplast (cp) DNAs were also analyzed using the same method. The plant was shown to contain novel mitochondrial and chloroplast sequence rearrangements that were not detected in either of the parents. Received: 5 March 1997 / Revision received: 4 September 1997 / Accepted: 13 September 1997     

亚麻荠种植和利用的研究现状

介绍了亚麻荠(Camelina sativa(L.)Crantz)的种植历史、栽培特性、种子成分、生物学特性、栽培措施、抗病虫害能力以及对其产物的应用.亚麻荠耐旱和抗病虫草能力强的特性符合农业可持续发展的方向.作为一种低投入的经济作物,亚麻荠的种植在我国有着广阔的发展前景.     

水稻籼粳亚种间杂种低温花粉不育的QTL分析

为探明籼粳杂种低温花粉不育的遗传基础,以籼稻品种3037和粳型广亲和品种02428的F2分离群体进行了低温花粉不育的遗传分析。推迟播种后,F2群体各单株孕穗期的日平均温度为21～23℃,调查了F2群体各单株的花粉育性。利用108对SSR引物构建了包含157个F2单株,覆盖12条染色体的分子标记连锁图谱。该连锁图的总长度为1857．8cM,标记间平均距离为16．26cM,标记较均匀地分布在12条染色体上。采用区间作图法对F2群体花粉不育进行QTL分析,共检测到2个低温花粉不育QTLS,即qLTSPS2和qLTSPS5,分别位于第2、5染色体,其加性效应分别为0．021、0．045,显性效应分别为-0．246、-0．251,显性度分别为11．7和4．8,具有超显性效应．超显性是QTL作用的主要方式,这2个位点杂合基因型在低温环境下具有降低花粉育性的作用,分别解释表型变异的15．6％、11．9％。另外,两因素的方差分析表明这两个QTL之间不存在互作。     

莴苣大孢子发生过程中钙的分布动态

莴苣胚囊发育为蓼型,减数分裂形成的4个大孢子中只有合点端的一个大孢子可继续发育,其余3个大孢子从珠孔端依次退化.大孢子母细胞中的钙沉淀颗粒很少,减数分裂后的四分体中的钙沉淀颗粒稍有增加.以后,4个大孢子中的钙沉淀颗粒在数量上有明显差异:即将退化的大孢子中钙明显减少,而未退化大孢子细胞质中则保持有较多的细小钙沉淀颗粒.大孢子的退化是一种细胞程序死亡现象,细胞中的钙浓度降低时可能启动了大孢子细胞的程序性死亡过程,而细胞中的钙浓度高时则保持大孢子细胞的继续发育.文章首次揭示了大孢子发生过程中钙的分布特征.     

Co-ordination between leaf initiation and leaf appearance in field-grown maize (Zea mays): genotypic differences in response of rates to temperature

BACKGROUND AND AIMS: In maize (Zea mays), early flowering date, which is a valuable trait for several cropping systems, is associated with the number of leaves per plant and the leaf appearance rate. Final leaf number depends upon the rate and duration of leaf initiation. The aims of this study were to analyse the genotypic variation in the response to temperature of leaf appearance rate and leaf initiation rate, and to investigate the co-ordination between these processes under field conditions. METHODS: Sixteen hybrids of different origins were grown under six contrasting environmental conditions. The number of appeared leaves was measured twice a week to estimate leaf appearance rate (leaves d(-1)). Plants were dissected at four sampling dates to determine the number of initiated leaves and estimate leaf initiation rate (leaves d(-1)). A co-ordination model was fitted between the number of initiated leaves and the number of appeared leaves. This model was validated using two independent data sets. KEY RESULTS: Significant (P < 0.05) differences were found among hybrids in the response to temperature of leaf initiation rate (plastochron) and leaf appearance rate (phyllochron). Plastochron ranged between 24.3 and 36.4 degree days (degrees Cd), with a base temperature (Tb) between 4.0 and 8.2 degrees C. Phyllochron ranged between 48.6 and 65.5 degrees Cd, with a Tb between 2.9 and 5.0 degrees C. A single co-ordination model was fitted between the two processes for all hybrids and environments (r2= 0.96, P < 0.0001), and was successfully validated (coefficient of variation < 9 %). CONCLUSIONS: This work has established the existence of genotypic variability in leaf initiation rate and leaf appearance rate in response to temperature, which is a promising result for maize breeding; and the interdependence between these processes from seedling emergence up to floral initiation.     

Effects of co-cropping Bidens pilosa (L.) and Tagetes minuta (L.) on bioaccumulation of Pb in Lactuca sativa (L.) growing in polluted agricultural soils

Polluted agricultural soils are a serious problem for food safety, with phytoremediation being the most favorable alternative from the environmental perspective. However, this methodology is generally time-consuming and requires the cessation of agriculture. Therefore, the purpose of this study was to evaluate two potential phytoextractor plants (the native species Bidens pilosa and Tagetes minuta) co-cropped with lettuce growing on agricultural lead-polluted soils. The concentrations of Pb, as well as of other metals, were investigated in the phytoextractors, crop species, and in soils, with the potential risk to the health of consumers being estimated. The soil parameters pH, EC, organic matter percentage and bioavailable lead showed a direct relationship with the accumulation of Pb in roots. In addition, the concentration of Pb in roots of native species was closely related to Fe (B. pilosa, r = 0.81; T. minuta r = 0.75), Cu (T. minuta, r = 0.93), Mn (B. pilosa, r = 0.89) and Zn (B. pilosa, r = 0.91; T. minuta, r = 0.91). Our results indicate that the interaction between rhizospheres increased the phytoextraction of lead, which was accompanied by an increase in the biomass of the phytoextractor species. However, the consumption of lettuce still revealed a toxicological risk from Pb in all treatments.     

The effect of temperature differences between porometer head and leaf surface on stomatal conductance measurements

It is generally known that instantaneous values of leaf conductance as measured with a dynamic porometer need to be corrected for the temperature difference, ΔT, between the porometer cup and the sampled leaf. Leaf conductances, obtained with a Delta-T AP4 dynamic porometer, with and without correction for ΔT are compared for a bush species (Guiera senegalensis) and two forb species (Jacquemontia tamnifolia and Mitracarpus scaber). With temperature differences predominantly varying within the ±2.5 °C recommended by the manufacturer, it appears that the differences between uncorrected and corrected conductances are very large, up to 100% on average, especially for the two forbs. Furthermore, it is shown that, using the Mitracarpus data, a relatively small error of ±0.5 °C in ΔT can cause a difference of 25–50% in the final conductance value, in particular for the high conductance range. An error of ±0.5 °C may easily occur: the accuracy of ΔT as measured by the thermistors in the porometer is 0.2 °C and the temperature variation within a leaf can be much larger. This result will have implications for upscaling of leaf conductances to canopy values or may explain why upscaled values appear not to correspond with down-scaled values, obtained from eddy correlation measurements and an inverted canopy transpiration model.