Somatic Embryogenesis and Plantlet Regeneration in Callus Cultures Derived from Mature Zygotic Embryos of Slash Pine

1TheworkwassupportedbyChineseNationalHighTechnologyDevelopmentProgram"863project"IntroductionSomaticembryogenesishasagreatpotentiaIforrapidProPaationofsuPCriorconifcrspccics.ThefirstreportonsomaticembryogenesisandplantlctrcgencrationinconifersPeCieswasach…     

Somatic embryogenesis from immature and mature zygotic embryos ofCryptomeria japonica I: Embryogenic cell induction and its morphological characteristics

Embryogenic cells (ECs) of sugi (Cryptomeria japonica D. Don) were induced from immature and mature zygotic embryos cultured on different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D)-containing modified Campbell and Durzan medium. The rate of induction of ECs varied depending on the stage of embryos collected. The highest percentage of induction (35%) was obtained with immature zygotic embryos collected July 18 and July 30, 1997, when 1 M of 2,4-D was added to the induction medium. The ECs easily proliferated when subcultured in a medium of the same composition as the induction medium within 3 weeks. Morphological characteristics of nonembryogenic cells and embryogenic cells of different developmental stages were studied under an inverted fluorescence microscope.Part of this work was presented at the 109th annual Japanese Forestry Society meeting at Tochigi, April 1998     

Mechanical Properties of Picea sitchensis

This study establishes the basis for the assignment of Danish - grown Sitka spruce (Picea sitchensis) to the European strength class system (EN 338). In total, 712 boards were chosen to be representative of the normal Danish commodity. These were visually graded for strength according to Nordic rules (INSTA 142), and subsequently tested to failure in either bending or tension. The results show that Danish - grown Sitka spruce graded to levels T1 and T2 satisfy the requirements of EN 338 at the C18 and C24 levels, respectively. Results for Danish - grown Sitka spruce concur with those for Danish - grown Norway spruce (Picea abies). This study shows that Sitka spruce and Norway spruce of similar origins exhibit highly comparable mechanical properties.     

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L. ''Moldova''

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape 'Moldova' (Vitis vinifera L. 'Moldova'). Primary calli were initiated on Nitsch and Nitsch (NN) medium supplemented with 1.0mg'L-1 2,4-D and 0.5 mg'L-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg-L-1 6-BA and 2 mg.L-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg.L-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dy-namic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic em-bryos, including typical epiderma, cotyledon primordium and vascular tissue.     

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L. ＇Moldova＇

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape 'Moldova' (Vitis vinifera L. 'Moldova'). Primary calli were initiated on Nitsch and Nitsch (NN) medium supplemented with 1.0mg'L-1 2,4-D and 0.5 mg'L-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg-L-1 6-BA and 2 mg.L-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg.L-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dy-namic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic em-bryos, including typical epiderma, cotyledon primordium and vascular tissue.     

Dynamics of swaying of Picea sitchensis

Six 26-year-old Sitka spruce (Picea sitchensis (Bong.) Carr.) trees growing in a Scottish plantation were swayed manually to determine their mechanical dynamics. The natural frequency of sway of the intact trees (mean height 14.2 m and mean stem diameter 14.5 cm at 1.3 m) was on average 0.35 Hz. The variation of this frequency with tree size was found to be well described by engineering mechanics theory. In particular, shape parameters could be defined for both intact and branchless trees, which, along with stem size, density and elasticity, could predict the natural sway frequency using a simple formula. The damping of sway was found to consist of three components, (1) interference of branches with those of neighbors, (2) aerodynamic drag on foliage, and (3) damping in the stem. For the sample of six trees, which spanned the diameter range at the experimental site, the importance of these three components to overall damping was in the ratio 5/4/1 for the median sized tree. Interference between neighbors depended on the distance to neighbors, as well as on the sizes of the chosen tree and its neighbors. Aerodynamic damping was larger for larger trees and the energy lost to this force was similar in magnitude to that calculated to be lost using drag coefficients from published wind tunnel and other studies. The amount of damping from the stem was linearly related to stem diameter.     

Performance and Frost Hardiness of Picea sitchensis x Picea glauca Hybrids in Scotland

The growth of seven Picea sitchensis x Picea glauca hybridswas compared with the growth of two P. sitchensis provenancesand Picea glauca var.albertiana at two sites in northern Scotland.The sites were at Aultmore (an exposed, dry site with a mineralsoil) and Shin (a frosty, wet site with deep peat). They wereof the type considered more suited to Pinus contorta than P.sitchensis. At age 10, in 1984, most of the hybrids, at both sites, wereabout 10 and 20 per cent taller than P. sitchensis of Masset(Q.C.I.) and Ketchikan (Alaska) provenance, respectively. P.glauca var. albertiana grew very poorly, especially at Aultmore. At Aultmore, the frost hardiness of three of the tallest hybrids,the two P. sitchensis provenances, and P. glauca var.albertiana,was tested at about 3-weekly intervals throughout 1982 and 1983.Detached shoots were subjected to artificial frosts in a programmablechamber. P. glauca var. albertiana was frost susceptible atbudburst, but at all other times it was relatively very frosthardy (eg. to^–10°C in mid-August). Also, the hybridswere consistently more hardy than P. sitchensis of even Ketchikan(Alaska) provenance from July onwards. However, the hybridswere less frost hardy than P. sitchensis of Masset (Q.C.I.)provenance in early spring (they dehardened a week earlier inMarch-April) and their buds were equally as frost susceptibleat the time of budburst. In 1983, trees of P. glauca var. albertianaburst their buds about a week sooner than P. sitchensis. It was concluded that P. sitchensis x P. glauca hybrids canperform better than P. sitchensis at sites considered ‘marginal’for P. sitchensis, and that their good performance may be partlyattributed to, or associated with, their greater summer andautumn frost hardiness. A programme of inter-specific hybridizationis being pursued.     

Effects of gibberellin A(4 + 7) and bark-ringing on cone initiation in mature Picea sitchensis grafts

Large numbers of cones (strobili) were induced in a 10-year-old plot of mature grafts of Picea sitchensis (Bong.) Carr. All trees injected with 20 mg GA(4 + 7) in June initiated female and male cones in the same year. This treatment increased the number of female cones per plant 12-fold above the controls, more than doubling the percentage of cones that were female. Complete bark-ringing (done in May of the previous year) showed an additive effect with GA on the number of female cones formed, but a negative interaction on the number of male cones induced. Ringing promoted male cone production most when used alone. Treatments, singly or combined, also increased the proportion of cones that were lateral, compared with the preponderance of terminal male cones in the controls. The effects were apparently not directly associated with alterations in vegetative vigor, although these occurred causing a reduction in the proportion of buds containing vegetative shoots the following year. The clones differed in most characteristics, but both sparse and prolific clones were induced to reproductive activity. Viable seed yields per tree, and notional production from seed-orchards were enhanced almost 10-fold by GA injection, and about 4-fold by bark-ringing and GA + bark-ringing.     

西加云杉木材中单宁的提取、分布及组分

研究西加云杉木材中植物单宁的提取工艺、分布和组分构成,为木材单宁变色的防治提供参考.采用有机溶剂萃取法,通过单因素试验并结合L9(34)正交试验,优化西加云杉木材中植物单宁的提取工艺;采用香草醛-紫外法测定西加云杉木材内植物单宁在径向(由髓心至树皮方向)和轴向的含量分布;通过定性鉴定试验确定单宁类型,并采用高效液相色谱...     

火炬松体细胞胚胎发生和干化体细胞的氧化物酶活性(英文)

培养于附加2,4-D、BA和KT的愈伤组织诱导培养基上的火炬松成熟合子胚在培养3-9周后形成白色、半透明、有光泽的粘性愈伤组织。这类愈伤组织形成于成熟合子胚的子叶,但当用NAA或者IBA代替愈伤组织诱导培养基中的2,4-D时,它的诱导频率明显降低。这种粘性愈伤组织在分化培养基上形成体细胞胚。体细胞胚经过去50μm ABA和8.5%PEG600处理后成为耐干化胚。扫描电镜观察表明,萌发处理36小时后,耐干化胚恢复到干化处理之前的状态且大小和形态正常,而不耐干化胚不能恢复到干化处理之前的状态且表面撕裂。过氧化物酶活性的分析结果表明,耐干化胚有更高的过氧化物酶活性。耐干化胚的高过氧化物酶活性可能与催化H2O2的分解和保护体细胞胚免受氧化的伤害有关。     

美国西加云杉出国考察报告

简要介绍了西加云杉的生物生态学特性及其生境条件 ,并提出了该树种引种的研究方向。     

Initiation of somatic embryogenesis from immature zygotic embryos of oocarpa pine (Pinus oocarpa Schiede ex Schlectendal)

The focus of the current project was to establish somatic embryogenesis protocols for the tropical pine species Pinus oocarpa using immature zygotic embryos (ZEs) as explants. Somatic embryogenesis is best supported by mimicking natural seed-embryo developmental conditions, through a tissue culture medium formulation based on the mineral content of the seed nutritive tissue [megagametophyte (MG)]. A novel culture medium (P. oocarpa medium, PO) was tested in combination with different plant growth regulator (PGR) concentrations and compared with standard Pinus taeda media for the initiation of somatic embryogenesis from immature ZEs of P. oocarpa. Immature MGs containing immature ZEs of two mother trees were used with 12 and 8% extrusion rates for mother tree genotypes 3 and 5, respectively. In both mother trees the percentage capture was 2%. Multiplication of two captured cell lines (T5C2S01 and T5C1S12) was improved by lowering the concentrations of PGRs to 2.5 μM each 2,4-dichlorophenoxyacetic acid and abscisic acid (ABA) plus 1.0 μM each 6-benzylaminopurine and kinetin. Mature somatic embryos formed on 40 μM ABA, 6% (w/v) maltose, 12% (w/v) PEG 8000 and 0.6% (w/v) Phytagel. While PO medium appeared suboptimal for somatic embryo induction, it did exhibit potential for enhanced culture proliferation and subsequent improved maturation with cell line T5C2S01, where microscopic analysis revealed better embryo morphology on PO medium than on 1250 medium. However, this enhancement was not observed with cell line T5C1S12. Germination was preceded by partial desiccation for a period of 2-3 weeks before transferring the embryos to germination medium. Germination was observed after 7 days under low light, and apical primordia slowly expanded after transfer to ex vitro conditions. To our knowledge, this is the first report on the production of somatic seedlings in P. oocarpa.     

Storage protein changes during zygotic embryogenesis in interior spruce

The major storage proteins isolated from protein bodies of embryo tissues of interior spruce Picea glauca (Moench) Voss/Picea engelmanii Parry had apparent molecular weights of 41, 35, 33, 24 and 22 kD. Minor proteins of 30 and 27.5 kD were also observed. Based on their solubility characteristics, the 41 kD protein was identified as a water and buffer-soluble albumin, and the 35, 33, 24 and 22 kD proteins were characterized as buffer-insoluble, high salt-soluble globulins. Two-dimensional electrophoresis revealed each protein was composed of several isoelectric variants. Developmentally specific accumulation of storage proteins was observed during embryogenesis. The 41 kD protein only accumulated during the later stages of cotyledon maturation, whereas the other storage proteins began to accumulate during the early stages of embryo development. All storage proteins showed major accumulations during cotyledon maturation.     

水曲柳体细胞胚与合子胚发生的细胞学研究

水曲柳(Fraxinus mandshurica)属木犀科(Oleaceae)白蜡树属,是我国东北重要珍贵硬阔树种之一,主要分布于小兴安岭、长白山、辽宁东部山地等地区, 以材质优良而著称.由于长期不合理的采伐利用,目前可利用的资源急剧减少,已被列为国家三级保护植物(傅立国,1992).进行水曲柳体细胞胚胎发生的研究,在资源保护、树种快繁和基因工程育种上有其重要的现实意义.     

Nutrient Concentrations in Naturally Regenerated Seedlings of Picea sitchensis in Southeast Alaska

Concentrations of N, P, K, Mg, Ca, Mn were determined in topsand roots, and S in tops, of naturally regenerated Sitka spruceseedlings growing on youthful and mature soils near Juneau,SE Alaska. The samples were taken at the end of the growingseason from sites with and without alder (Alnus sinuata) asthe principal hardwood component. P, Mg, and Mn concentrations of seedlings from mature soilswere larger than in those from youthful soils. Differences inN, K, and Ca concentrations were small. Seedlings on sites withalder tended to have more N, P, and K than on those without. Nutrient concentrations of naturally regenerated Alaskan seedlingsfrom mature soils, with alder present, resembled closely thoseof healthy 1 + 1 transplants in English forest nurseries.     

Growth and enzyme production by Bacillus subtilis and Flavobacterium pectinovorum in Picea sitchensis

Summary When Sitka spruce (Picea sitchensis) is stored in water, an increase takes place in the permeability of the wood. This increase is due to bacterial degradation of the tori and bordered pit membranes and partial degradation of the crossfield pits. It has been demonstrated that two bacteria—Bacillus subtilis and Flavobacterium pectinovorum—isolated from this water stored wood, grow and produce amylase, xylanase and pectinase in sapwood blocks suspended in sterile pond water. Since these organisms have also been shown to increase the permeability of sapwood blocks, it has been concluded that they are primarily responsible for the permeability changes that occur when spruce poles are stored in lake water.When the two organisms are incubated together in sapwood blocks, growth of F. pectinovorum is synergistically affected, while growth of B. subtilis is antagonised. However, no antagonism of enzyme production was evident. The levels of biological activity in the sapwood in these laboratory experiments were much higher than the values observed in the natural lake system. The concentration effect obtained in the laboratory system, in contrast to the dilution effect occurring in the lake environment, is undoubtedly responsible for this variance. Results indicate that the rate at which permeability changes occur could possibly be accelerated by replacing the natural lake system with an enclosed tank system.     

The elasticity and vertical distribution of stress within stems of Picea sitchensis

Eight 22-year-old Picea sitchensis (Bong.) Carr. trees growing in a Scottish forest were bent using a winch and cable. The shapes of the bent stems were modeled using the structural theory of a cantilever beam, with non-uniform stem taper and large deflections taken into account. The Young's modulus of elasticity for the stems was estimated to be 2.0-6.4 GPa, which is lower than that reported for sawn green timber of this species, but similar to that for intact green stem sections. The longitudinal distribution of stress along the stems had a maximum at a height that was dependent on taper. Maximum stress occurred higher up in the more tapered stems.     

Slash pine (Pinus elliottii Engelm.) somatic embryogenesis I. Initiation of embryogenic cultures from immature zygotic embryos

Immature female gametophytes and enclosed zygotic embryos of slash pine (Pinus elliottii Engelm.) collected at various developmental stages were cultured either on eight initiation media supplemented with different plant growth regulator (PGR) combinations or on LPM medium solidified with two brands of agar. Embryonal-suspensor cell masses extruded from gametophyte explants infrequently produced white or translucent mucilaginous embryogenic cultures. Variation in extrusion frequency was observed across seed sources (families), explant collection dates, agar types and culture media. Increased explant maturity (as related to collection dates) reduced cell extrusion rates. Four types of extrusions were grouped according to their morphology. One type of extrusion produced the greatest number of established and long-surviving embryogenic cultures. This extrusion type (type I) was associated with an extrusion condition termed empty in which the entirety of the embryonal-suspensor mass was extruded out of the gametophyte. Three other extrusion types (types II, III and IV) showed less culture establishment and survival.     

Seasonal Changes in the Frost Hardiness of Provenances of Picea sitchensis in Scotland

Changes in the natural level of frost hardiness of shoots offour provenances of Picea sitchensis were monitored over twogrowing seasons by detaching shoots from 7 to 10-year-old treesgrowing in a nursery in Scotland, and subjecting them to freezingtemperatures under conditions which simulated night frosts. Six seasonal phases of frost hardiness were identified (Fig.3).

1. During each autumn, killing temperatures (the level of hardiness)decreased from –5°C to below –20°C, beginningseveral weeks after shoot elongation ceased. Alaskan provenanceshardened in September, apparently in response to shorteningday lengths alone, whereas an Oregon provenance did not hardenuntil November, after repeated frosts. Queen Charlotte Islandsprovenances were intermediate.
2. From November to March allprovenances were hardy to below –20°C,which is adequateto prevent direct freezing injury at mostplantation sites.
3. In March-April, several weeks before bud-burst, old shootsdehardenedto killing temperatures of about –10°Cin responseto warm temperatures, and southerly provenancesdid so beforenortherly ones.
4. During bud-burst the newly-emergingshoots were hardy to only–3°C to –5°C untilthey were about 3.5 cmlong. All provenances burst bud at thesame time and were equallyfrost susceptible at this time.
5. DuringMay-July the elongating shoots fluctuated in hardinessbetween–5°C and –10°C apparently in responsetofluctuating ambient temperatures.
6. In August 1980 there wasa period of late summer dehardeningto killing temperaturesof about –3°C.

Seasonal changes in hardiness are discussed in relation to changesin shoot growth and environmental factors. The main opportunitiesfor selecting frost hardy genotypes seem to be in the rate ofautumn hardening, the time of pre-bud burst dehardening, andthe time of bud-burst.