兰州百合组培鳞茎发育研究（英文）

该研究以兰州百合商品种球鳞片为外植体材料,通过组织培养诱导丛生芽萌发及高频增殖,再以丛生芽为材料诱导其发育形成小鳞茎,调节培养基对小鳞茎进行膨大发育培养,最终形成促进兰州百合组培鳞茎膨大发育的"三步"组培培养技术路线;对发育过程中形成的丛生芽、小鳞茎及膨大鳞茎进行淀粉含量测定与生长特征参数统计,分析各步培养对鳞茎形成发育过程中淀粉含量与形态变化的影响。结果表明:所建立的"三步"培养方案培育出的组培鳞茎直径、重量与鳞片数分别为1.66 cm、2.48 g和26.33片,有效地促进了鳞茎的膨大,并能诱导鳞茎主茎杆的形成发育;在培养进程中其淀粉含量呈现逐步增加的趋势,这表明与鳞茎膨大发育正相关,同时鳞茎大小、重量及鳞片数三者也表现为正相关性;当鳞茎所含鳞片数在26片以上时,其生长点易发育形成主茎杆。该文研究了兰州百合组培鳞茎的形成与膨大发育技术,所研发的"三步"培养组成的鳞茎膨大发育组培技术有效地促进了鳞茎的膨大发育,而膨大发育的鳞茎能有效地缩短田间生长周期,从时间上提高百合生产量,同时为实现兰州百合膨大的鳞茎种球规模化生产提供技术支撑。     

百合鳞茎发育过程中碳水化合物含量及淀粉酶活性变化

以兰州百合和亚洲系"精粹"百合为试材,探讨了鳞茎发育过程中不同部位淀粉、可溶性糖含量和淀粉酶活性的变化。结果表明,母鳞茎作为百合萌发阶段的代谢源,其外部鳞片是代谢更为活跃的部位。淀粉和可溶性糖含量同时增加是百合新鳞茎开始膨大的标志。蔗糖是百合鳞茎中可溶性糖的主要形态,还原糖的变化体现了碳水化合物的供应及转化。淀粉酶在百合鳞茎发育过程中对调节和平衡碳水化合物的形态起重要作用。     

兰州百合器官离体培养外植体位置效应观察

探讨兰州百合 (Liliumdavidiivar.unicolor)鳞茎鳞片、叶片和根的不同切段的培养效应。结果表明 :其切段不定芽的分化速度和数量是下段 >中段 >上段。芽的诱导和增殖的最适外植体为鳞茎鳞片 ,兰州百合离体培养中鳞片不定芽诱导和快速繁殖的培养基为MS BA2mg L NAA 0 2mg L ,增殖培养基与诱导培养基相同 ,3周左右不定芽开始分化。叶和根不同部位中不定芽的发育能力大体与鳞茎鳞片一致 ,但低于鳞片 ,较适宜的培养基为MS BA2mg L NAA 0 4mg L ,生根培养基为 1 2MS NAA 0 3mg L ,约 15d生根 ,生根率大于95 %。月增殖率为 1∶4 ,整个繁殖周期约需 3个月。     

百合鳞茎发育过程中淀粉合成相关酶基因的克隆及表达分析

该研究通过同源克隆技术克隆腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)、颗粒结合淀粉合酶(GBSS)和可溶性淀粉合酶(SSS) 3类百合淀粉合成关键酶基因,分析这三类淀粉合成关键酶基因的表达变化,测定百合鳞茎膨大发育中淀粉含量变化。结果表明:(1) AGPase具有GlgC家族蛋白PLN02241蛋白结构特征及cl11394家族蛋白ADP_Glucose_PP与NTP_transferase结构域,获登录号KP751443; GBSS与SSS具有cl10013家族蛋白Glyco_transf_5,GT1_Glycogen_synthase_DULL1_like结构域,获登录号分别为KP751444、KP751445。(2)百合鳞茎形成与膨大发育过程中,淀粉含量呈现递增趋势,鳞茎盘开始分化茎杆时其淀粉含量最高,达到44.52%。鳞茎与叶片部位的三个淀粉合成相关酶基因表达量均逐渐增加;在鳞茎膨大后茎杆分化阶段,三个淀粉合成相关酶基因表达量达到最高,AGPase、GBSS、SSS在鳞片中的表达量分别为10.79,6.92和5.12,叶片中的表达量分别为6.79,5.22和4.41,鳞片中的表达量大幅度高于叶片;淀粉合成相关酶基因的表达量变化与淀粉含量、鳞茎的膨大发育成正相关。这为鳞茎的繁殖生产提供了可通过调节淀粉合成关键酶基因表达促进百合鳞茎膨大发育的思路。     

百合鳞片离体培养诱导小鳞茎发生的研究

百合鳞片离体培养诱导愈伤组织和小鳞茎可在MS+NAA0.2mg/l+6BA2mg/l(pH5.8——6.0)同一培养基上一次完成。接种后9天左右即可获得小鳞茎,一个月左右即可诱导形成完整植株。通过外部形态观察和细胞组织学研究表明小鳞茎的发生与不定芽的发生过程相类似,并讨论了小鳞茎发生过程中的极性现象,细胞起动形成愈伤组织的部位,诱导小鳞茎发生过程中淀粉消化的生理意义以及光对小鳞茎发生的作用等问题。     

温度对兰州百合鳞片埋培繁殖的影响

为了筛选出兰州百合鳞片埋培繁殖的最适温度和鳞片层次,解决兰州百合种源不足、繁育周期长的问题,该文以兰州百合鳞片为材料,采用温度(20、25、30℃)和鳞片层次(外层、中层、内层)二因素完全随机区组设计,研究了二因素对兰州百合鳞片埋培繁殖效果的影响。通过对鳞片疑似发病率、分化率及小鳞茎分化数进行统计与分析,结果发现不同温度处理及各鳞片层次对鳞片疑似发病率、分化率及小鳞茎分化数的影响存在显著或极显著差异。结果表明:(1)温度越高,鳞片的疑似发病率越低,在埋培2周时,20℃处理下疑似发病率最高(38.67%),30℃处理下最低(10%);各层次鳞片的疑似发病率由高到低依次为外层中层内层。(2)在25、30℃处理下,小鳞茎分化率最高,埋培结束(6周)时分别为91.33%、90.89%;中层及内层鳞片小鳞茎分化率极显著高于外层鳞片。(3) 30℃处理下鳞片形成小鳞茎数最多,在埋培6周时达到每片2.00粒;同时中层及内层鳞片小鳞茎分化数显著高于外层鳞片。综上结果表明,兰州百合鳞片埋培繁殖以选用中层(3~4层)、内层(5~7层)鳞片在25~30℃条件下繁殖效果最好。     

应用细胞工程技术选育四倍体龙牙百合的研究

用二倍体龙牙百合鳞片为外植体,培养在添加２,４Ｄ,６-ＢＡ的ＭＳ培养基上,诱导愈伤组织、不定牙与小鳞茎分化,建立了二倍体的体细胞无性系。用浓度０.０２％～０.０５％的秋水仙碱或再加２％～４％的二甲基亚砜溶液处理二倍体小鳞茎、鳞片与愈伤组织均诱导出变异试管苗,通过多代筛选与增殖培养首次获得了变异苗无性系,选育出同源四倍体龙牙百合,其染色体数目为４８（２ｎ＝４ｘ＝４８）。同源四倍体苗粗壮,叶片宽而厚,小鳞茎增殖速度快,小鳞茎所含核酸、蛋白质、氨基酸、淀粉、脂肪、ＡＴＰ及维生素Ｂ２的量均高于二倍体     

兰州百合的组织培养(简报)

以兰州百合植株地下部幼嫩鳞片作为外植体,在MS 6—BA0．5mg/L NAA0．5mg/L的培养基上诱导产生不定芽效果最佳。靠近鳞茎盘部位的外植体诱导出的芽数量较多、质量较好。在添加NAA0．15mg/L的1／2MS生根培养基中可正常发根。     

淡黄花百合的组织培养与快速繁殖

以淡黄花百合的鳞片为外植体进行试管培养,筛选出各培养阶段适宜的培养基分别为:(1)丛生芽诱导,MS 6-BA 2.0 mg/L NAA 0.2 mg/L 蔗糖3%;(2)继代增殖,MS 6-BA 1.5 mg/L NAA 0.1 mg/L 蔗糖3%;(3)生根及小鳞茎生长,1/2 MS NAA 0.5～1.0 mg/L 蔗糖3% 活性碳0.1%,1/2 MS NAA 0.5 mg/L 蔗糖6% 活性碳0.1%.     

平陆百合鳞片组织培养中小鳞茎发生的条件及生物全息现象初探

本文研究了山西平陆百合在组织培养中鳞片分化形成小鳞茎的条件,其小鳞茎的发生符合生物全息理论。试验结果表明:百合鳞片小鳞茎的分化能力与外加植物激素的种类和浓度关系很大。在生长素类物质中,IRA 对鳞片分化小鳞茎效果最好,不仅数量多,而且小鳞茎的体积大,生活力强;NAA 从次之;而2,4-D 的诱导效果最差。细胞分裂素类(KT 或6-BA)与生长素类(IBA、     

Effect of low temperature on dormancy breaking and growth after planting in lily bulblets regenerated in vitro

Lilies regenerating on scale segments may develop dormancy in vitro depending on the culture conditions. The dormancy is broken by storage for several weeks at a low temperature (5 °C). The effect of the low temperature on sprouting, time of leaf emergence and further bulb growth was studied. Dormant and non-dormant bulblets were regenerated in vitro on bulb scale segments cultured at 20 °C or 15 °C, respectively. The low temperature not only affected the number of sprouted bulblets but also the time of emergence. The longer the cold storage, the faster and more uniform leaf emergence occurred. Both dormant and non-dormant bulblets grew faster after a low temperature treatment of six weeks. Thus, during dormancy breaking the tissue is prepared not only for sprouting but also for subsequent bulb growth. These processes are rather independent as low temperature stimulates growth in non-dormant bulblets whereas these bulblets sprout also without treatment at low temperature. Moreover, the hormone gibberellin induces rapid sprouting but has no influence on further bulb growth. Good growth in bulblets exposed to the low temperature coincided with production of an increased leaf weight. However, the relationship is not absolute as bulblets that were cold-treated for six weeks grew larger than bulblets cold-treated for four weeks but the formation of leaf biomass was similar. During storage at low temperature starch was hydrolyzed in the bulb scales and sugars accumulated. This indicates that during this period, preparation for later bulb growth involves mobilization of carbohydrate reserves which play a role in leaf growth and development of the photosynthetic apparatus. Starch hydrolysis proceeded in the outer scales after planting. Approximately six weeks later, the switch from source to sink took place in the bulblet, which became visible as a deposition of starch in the middle scales.     

Lilium candidum bulblet and meristem development

Lilium candidum L., commonly known as the Madonna lily, is a wild Lilium species with medicinal properties and excellent potential as an ornamental crop, but one that has been scarcely investigated. The aim of this research was to study (1) tissue culture propagation of L. candidum bulblets, (2) early bulblet development, and (3) the effect of temperature and bulblet weight on bulblet and plant growth and meristem development. An investigation of the effect of explant type and temperature on in vitro bulblet propagation showed that scales were the most efficient explants for in vitro propagation and that exposing the regenerating bulblets to 15°C for 4 wk increased bulblet weight but reduced the number of bulblets produced. For bulblets planted in soil after 12 wk of exposure to 15°C or 25°C, the fastest growth was observed in the bulblets that had been exposed to 15°C and that had a larger initial size. Histological examination showed that young in vitro-grown bulblets had a rudimentary meristem comprising few cells with no layer organization. After 12 wk of growth, all bulblets showed a layered meristem, regardless of bulblet size or exposure to 15°C. However, an increased amount of leaf primordia was detected in larger bulblets. Furthermore, the histological examination revealed that in L. candidum, as opposed to other lily species, there had been no real "phase change" in the meristem and that the phase change from juvenile to vegetative adult occurred at a much later stage in L. candidum than in other species.     

Phase change in lily bulblets regenerated in vitro

During the development of the lily ( Lilium ), three phases can be distinguished: the juvenile, the vegetative adult and the flowering phase. Juvenile bulblets sprout with one or a few leaves whereas vegetative adult bulblets sprout with a stem with elongated internodes. The transition to the vegetative adult phase was studied in lily ( Lilium  × cv. Star Gazer) bulblets regenerating on bulb scale segments in vitro. The phase change was marked by the development of a tunica-corpus structure in the apical meristem which leads to the formation of an actively growing stem primordium. This structure is absent in juvenile bulblets. Juvenile bulblets first developed competence for phase change during a culture period of at least 6 weeks at 25°C. Subsequent induction of the phase change occurred during a period of 2 weeks at lower temperature (15°C). A major factor influencing phase transition was bulblet weight. Small bulblets never formed a stem whereas large bulblets always formed a stem under inducing conditions. Large bulblets more often formed a stem than small ones but the relation between bulb growth and phase transition was not absolute. A high sucrose concentration, a large explant and a prolonged period for competence development stimulated bulb growth but also phase transition independently of growth. Lowering the concentration of MS-minerals reduced bulb growth but did not affect phase transition. Under these conditions, phase change was correlated with a low phosphorus content.     

High frequency of shoot multiplication and bulblet formation of garlic in liquid cultures

In vitro shoot proliferation and bulblet production of garlic (Allium sativum L.) was studied in liquid cultures. Shoots grown in vitro were used as explants and were cultured in MS medium supplemented with 2% (w/v) sucrose and 0.5 mg l^–1 2-iP. Three culture methods (semi-solid, liquid-immersion and raft) were compared for shoot proliferation. Explants in liquid (immersion) culture exhibited an increased multiplication rate and fresh weight of shoots after 3 weeks of culture as compared with the other treatments. Bulblet formation and growth were studied in liquid medium with different concentrations of sucrose (2–13%). MS medium containing 11% (w/v) sucrose was optimal for bulblet development and bulblets developed in this medium within 9 weeks in culture. The highest multiplication rate was (135 bulblets/explant) found when explants were cultured in bulbing medium (MS medium containing 0.1 mg l^–1 NAA+11% (w/v) sucrose) supplemented with 10 M JA. Growth retardants CCC, B-9, ABA also promoted induction and growth of bulblets. Darkness promoted the bulblet induction and growth compared to light conditions (16-h photoperiod of 50 mol m^–2 s^–1). The dormancy of bulblets was broken by cold treatment at 4 °C for 8 weeks.     

Stimulating effect of spermine on bulblet formation in bulb-scale segments of Lilium longiflorum

When bulb-scale segments of Lilium longiflorum were cultured on a medium containing auxin and cytokinin, the proportion of the expiants with newly-formed bulblets was significantly increased by the application of different polyamines. The most effective polyamine was spermine, where more than 90% of segments formed an average of 5 bulblets as compared to controls where less than 50% explants formed an average of 1.5 bulblets. Application of arginine one of the precursors putrescine biosynthesis, slightly promoted bulblet formation. The putrescine-stimulated bulblet formation was strongly inhibited by simultaneous addition of an inhibitor of the spermidine synthase, cyclohexylamine. The spermidine-promoted bulblet formation, however, could not be suppressed by this inhibitor. The promotive effect of spermidine on bulblet formation was reversed by an inhibitor of the spermine synthase, N-(3-aminopropyl)cyclohexylamine, but application of this inhibitor with spermine did not show any apparent effect on the bulblet formation. Endogenous level of spermine increased in common during bulblet formation that were stimulated by exogenous polyamines. Thus, spermine seemed to be the main stimulating chemical on bulblet formation in lily bulb-scale segments.Abbreviations APCHA N-(3-aminopropyl)cyclohexylamine - Arg arginine - BA benzyladenine - CHA cyclohexylamine - MS Murashige and Skoog's - NAA naphthaleneacetic acid - Orn ornithine - Put putrescine - Spd spermidine - Spm spermine     

Application of bioreactors for large-scale micropropagation systems of plants

Summary The application of bioreactor culture techniques for plant micropropagation is regarded as one of the ways to reduce production cost by scaling-up and automation. Recent experiments are restricted to a small number of species that, however, demonstrate the feasibility of this technology. Periodic immersion liquid culture using ebb and flood system and column-type bubble bioreactors equipped with a raft support system to maintain plant tissues at the air and liquid interface were found to be suitable for micropropagation of plants via the organogenic pathway. Balloon-type bubble bioreactors proved to be fit for micropropagation via somatic embryogenesis with less shear stress on cultured cells. Several cultivars of Lilium were successfully propagated using a two-stage culture method in one bioreactor. A large number of small-scale segments were cultured for 4 wk with periodic immersion liquid culture to induce multiple bulblets from each segment, then the bulblet induction medium was changed into bulblet growth medium by employing a submerged liquid bioreactor system. This culture method resulted in a nearly 10-fold increase in bulblet growth compared to conventional culture with solid medium. About 20 000 cuttings of virus-free potato could be obtained from 120 singlenode explants in a 20-liter balloon-type bubble bioreactor after 8 wk of culture. The percentage of ex vitro survival and root induction of the cuttings was more than 95%. Other successful results were obtained from the micropropagation and transplant production of chrysanthemum, sweetpotato, Chinese foxglove. Propagation systems via somatic embryogenesis in Acanthopanax koreanum and thornless Aralia elata were established using a liquid suspension of embryogenic determined cells. More than 500 000 somatic embryos in different stages were harvested from a 10-liter balloon-type bubble bioreactor after a 6-wk culture. Further development of these embryos in solid medium and eventually in the field was successful. The bioreactor system could reduce initial and operational cost for micropropagation, but further development of sophisticated technology might be needed to apply this system to plant micropropagation industries.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration and bulblet growth from lily bulbscale explants as affected by retardants,sucrose and irradiance

Bulbscales of oriental lily hybrid Star Gazer were used as the explants. Bulblets were formed on the basal portion of the excised bulbscales on MS medium supplemented with growth retardants, different sucrose concentrations and exposed to continuous light or dark. ^{Alar, Cycocel} and Paclobutrazol in concentration 1 mg dm⁻³ produced higher number of bulblets as compared to the control. The number of bulblets, however, decreased with the increase in concentration of the growth retardants. The number of bulblets was higher at 90 than at 60 g dm⁻³ sucrose and when the bulbscales were exposed to continuous light than to darkness. The growth retardants, higher sucrose concentration and continuous dark stimulated fresh mass of bulblets. The number of bulblets having roots and leaves decreased in medium with Alar, Cycocel and Paclobutrazol as compared to the control. A few bulblets produced roots and leaves in medium with 90 g dm⁻³ sucrose and none of the regenerated bulblets produced leaves under continuous dark.     

Effect of Methyl Jasmonate on Morphology and Dormancy Development in Lily Bulblets Regenerated In Vitro

Scales of lily bulbs are swollen petioles. Lily scale fragments cultured in vitro regenerate bulblets consisting of scales that may or may not carry a leaf blade. The bulblets are dormant and require a cold treatment to sprout. We added the gaseous plant growth regulator methyl jasmonic acid (MeJA) in the headspace of the tissue-culture container and studied the effect on plantlet morphology (scale/leaf-blade formation) and dormancy development in three lilies, Lilium speciosum “Rubrum No. 10,” L. longiflorum “Snow Queen,” and the Asiatic hybrid “Connecticut King.” Methyl jasmonic acid strongly reduced leaf-blade formation in Lilium longiflorum and Connecticut King. This was a specific effect as scale formation was affected much less. The specific inhibition of leaf-blade formation was not observed in Lilium speciosum. In this lily, high concentrations of methyl jasmonic acid (MeJA) inhibited leaf-blade and scale formation to similar extents. Methyl jasmonic acid reduced dormancy development in all three lilies, with the largest effect observed in Connecticut King. In this Asiatic hybrid, almost all bulblets that had regenerated at 300 or 1000 μl l⁻¹ MeJA in the headspace, did not require a dormancy-breaking treatment to achieve sprouting after planting in soil. Previously, it has been found in lily that treatments that reduce leaf-blade formation promote dormancy development. The present findings with MeJA do not agree with this. In the three lilies, the various parameters that were studied—regeneration, scale weight, leaf-blade weight, and dormancy development—were very differently affected by MeJA.     

Effects of storage temperature and sucrose on bulblet growth, starch and protein contents in in vitro cultures of Hyacinthus orientalis

The scale segments of the bulblets of Hyacinthus orientalis L. cv. Anna Marie were examined to improve their growth and development with cold-pretreatment and sucrose. The cold-pretreated (4 °C for 4 months) segments showed higher growth and better development of the bulblets on medium without sucrose than ones stored at 20 °C. A rapid decrease in starch content of bulb pieces was found during the first 2 weeks in all cultures and thereafter the content decreased gradually. A scanning electron microscopic observation during the bulblet growth and development showed a gradual decreasing trend of the starch granules from 2 to 16 weeks of the cultures. SDS-PAGE electrophoresis revealed the presence of a characteristic polypeptide of approximately 45 kD, which is assumed to be a major storage protein in the bulblets.