影响苹果离体叶片再生的因素分析

综述了影响苹果离体叶片再生的因素，内因主要包括基因型，外植体类型和外植体的发育状态；外因主要是添加的激素种类，浓度和配比，以胶培养条件。同时阐明了愈伤组织和不定芽诱导，产生的途径。     

秋福红树莓叶片离体再生植株研究

以红树莓品种秋福(Rubus L.cv.Autumn Bliss)离体叶片为外植体,研究适合其再生植株的叶片部位、放置方式、植物生长调节剂以及暗培养时间等条件。结果表明,叶片外植体接种于MS+TDZ2.00mg/L+IAA0.10mg/L的培养基,暗培养2～3周转移至正常光照下培养效果最好,愈伤组织形成率、不定芽再生率和外植体不定芽数分别为100.00%、95.83%和(5.57±0.27)个。将再生芽接种于1/2MS+IBA0.10mg/L的培养基中,35d后生根率达100.00%。再生植株炼苗后移栽,30d时成活率达97.30%,成功地建立了红树莓叶片的离体再生体系。     

寒富苹果叶片离体再生及四倍体诱导

为建立寒富苹果高效的离体再生体系和多倍体诱导体系,以试管苗叶片为外植体,研究了培养基中激素对寒富叶片再生芽的影响及适宜的四倍体诱导方法。结果表明,当培养基中BA质量浓度为0.5mg/L时,再生频率最高达35.7%;当培养基中BA质量浓度为2.5mg/L时,再生频率超过90%,平均再生芽数在4以上。在培养基中附加1.0mg/LTDZ,再生频率达100%,平均再生芽数达19.47。以附加15、30、60、120mg/L秋水仙素的液体再生培养基处理叶片5d,各个质量浓度处理均诱导出四倍体植株,诱变率在5.3%～22.2%之间;寒富苹果叶片在附加50mg/L秋水仙素固体再生培养基上处理5d亦获得了四倍体植株。研究结果表明寒富苹果叶片具有极强的不定芽再生能力,叶片再生过程中进行秋水仙素处理是获得其四倍体的一个有效途径。     

野生毛桃叶片愈伤组织诱导及不定芽再生

【目的】为了初步建立野生‘毛桃1号’叶片愈伤组织诱导、保存及不定芽再生技术体系,【方法】以野生‘毛桃1号’试管苗幼嫩叶片为外植体,探讨了基本培养基、激素配比等一系列因素对‘毛桃1号’叶片愈伤组织诱导和保存的影响及TDZ浓度对愈伤组织不定芽再生的影响。【结果】结果表明,适合‘毛桃1号’叶片愈伤组织诱导的培养基为LP+6-BA 1.0 mg.L-1+2,4-D 3.0 mg.L-1+AgNO30.5 mg.L-1+蔗糖30.0 g.L-1+琼脂6.0 g.L-1;保存的培养基为LP+2,4-D1.5 mg.L-1+CH 0.4 g.L-1+蔗糖30 g.L-1+CA 3.0 g.L-1或PVP 3.0 g.L-1+琼脂5.8 g.L-1,且采用持续暗培养保存,每28 d继代1次。愈伤组织在SH+TDZ 3.0 mg.L-1+NAA 0.3 mg.L-1+蔗糖30 g.L-1+琼脂6.0 g.L-1的培养基上可再生不定芽,再生率为6.83%。【结论】初步建立了‘毛桃1号’叶片愈伤组织诱导、保存及不定芽再生体系,为桃愈伤组织再生和遗传转化的研究奠定了基础。     

红将军苹果离体叶片高频再生体系研究

以中熟红富士品种红将军组培苗的叶片为试材,研究了离体叶片诱导再生不定芽过程中不同激素种类与组合、暗处理时间、取叶部位等因素对再生率的影响。结果表明,试管苗继代培养3～4代,当代培养25～30d,取顶部嫩叶3～4枚,剪除叶片边缘,将剪切的叶片中部以近轴面接触在最佳诱导分化培养基上:MS+TDZ1.0mg/L+IAA0.5mg/L+蔗糖30g/L,培养基pH值5.8,黑暗培养10d后,转入光照培养条件下,再生频率达100%,再生芽数平均4.6个。叶片再生不定芽分化后15～20d转接到苹果继代培养基MS+BA0.5mg/L+IBA0.1mg/L上,当不定芽长度约2cm时再转接到1/2MS+IBA0.5mg/L+NAA0.5mg/L+Ac0.5g/L+蔗糖2%的生根培养基上,光培养15d后,生根率可达86.0%。     

欧李离体叶片再生体系的建立

【目的】以欧李试管苗叶片为外植体进行不定芽再生研究,以期为核果类果树的品种改良、基因遗传转化和功能验证等生物技术育种奠定一定的基础,【方法】使用不同的基本培养基和激素组合促进其不定芽再生并建立再生体系。【结果】结果表明,欧李幼芽增殖对基本培养基类型较敏感,改良MS培养基优于MS、F14和WPM。在MS(改良)+NAA 0.1 mg.L-1+BA 0.2 mg.L-1培养基上,增殖系数为5.6,增殖效果较好。离体叶片不定芽再生的最适植物生长调节剂组合为MS(改良)+TDZ 4.0 mg.L-1+IBA 0.2 mg.L-1,再生率达到63.1%,平均每外植体再生芽数为4.9。暗培养10 d可以提高不定芽再生率。再生不定芽1/2 MS(改良)+IBA3.0 mg.L-1培养基上生根率最高,生根率达93.3%。【结论】在改良MS培养基上TDZ与IBA组合诱导不定芽再生的效果最好。     

山杏下胚轴再生不定芽影响因子研究(英文)

山杏成熟种子在改良MS培养基上培养获得下胚轴,将其切段后培养在改良MS培养基+TDZ2.0mg/L+NAA0.5mg/L的再生培养基上,经15d的暗期培养后,其再生频率可达到37%以上。试验表明分裂素TDZ促进不定芽再生的效果优于6-BA,生长素NAA效果优于2,4-D,2-3周龄的下胚轴再生效果较好,但下胚轴的取材部位对再生无显著影响。     

Prevention of hot-water treatment damage in narcissus bulbs by pre-warming

Summary

Hot-water treatment (HWT) used to control stem nematode in narcissus bulbs can lead to yield loss through damage to flower, leaf and root initials. Warm storage of bulbs, usually at 30°C, reduces this damage. The effects of two pre-warming treatments (18°C for two weeks or 30°C for one week before HWT) were investigated in bulbs hot-water treated at a range of dates (from early-July to late-September). Experiment 1 was conducted on bulbs of cv. Carlton lifted on three dates. In the year after HWT, flower numbers were much reduced when HWT was applied after mid-August following storage at ambient temperatures, or after late-August following storage at 30°C, but numbers were only slightly reduced even with late-September HWT when given after 18°C storage. Pre-warming was somewhat more effective after early lifting. Late HWT reduced yields of bulbs harvested after two years' growth, but 18°C treatment largely prevented these losses. In Experiment 2, the beneficial effects of 18°C treatment were confirmed in cvs Carlton and Golden Harvest but not in cv. Barrett Browning. These findings are discussed in terms of growth retardation by warm temperatures.     

Adventitious shoot regeneration from in vitro leaves of adult Camellia reticulata

Experiments were conducted with Camellia reticulata cv. Captain Rawes to develop an adventitious regeneration system. Leaves were taken from axillary shoot proliferation cultures in WPM medium that had been established from mature trees. They were sectioned, and then plated in Petri dishes on media containing various combinations of cyto- kinins and auxins; the best response was induced by 2 mg I“1 BA + 1 mg I-1 IBA. The shoots obtained were multiplied by axillary branching on WPM + 2 mg I-1 BA + 2 mg I-1 Z + 2 mg I-12iP + 0.01 mg I-1 IBA. There was no significant difference in multiplication coefficient (the product of the proportion of explants forming axillary shoots and the mean number of new segments per explant) between shoots of adventitious and axillary origin, but there was between the various types of explant used in the multiplication stage: shoot tips (ST1) and nodal segments (ns) of harvested shoots longer than 14- 15 mm, and whole harvested shoots 7-10 mm long (ST2). The best results were achieved with ns and ST2 explants. Shoots of adventitious origin rooted very poorly in comparison with those of axillary origin under the same culture conditions.     

High frequency shoot regeneration from cotyledon explants of cucumber via organogenesis

Organogenic callus induction and high frequency shoot regeneration were achieved from cotyledon explants of cucumber. About 86.2% of cotyledon explants derived from 5-day-old in vitro raised seedlings produced green, compact nodular organogenic callus in MS medium containing NAA (2.69 μM) and BA (4.44 μM) after two successive transfers at 20 days interval. Adventitious shoots were produced from the organogenic callus when it was transferred to MS medium supplemented with NAA (1.34 μM), BA (8.88 μM), zeatin (0.91 μM) and l-glutamine (136.85 μM) with shoot induction frequency of 75.6%. Shoot proliferation occurred when callus with emerging shoots was transferred in the same medium at an interval of 20 days. Shoots (1.0 cm length) were excised from callus and were elongated in MS medium fortified with GA₃ (1.44 μM) and BA (4.44 μM). The elongated shoots were rooted in MS medium supplemented with IBA (3.42 μM) and BA (4.44 μM). Rooted plants were acclimatized in green-house and subsequently established in soil with a survival rate of 80%. This protocol yielded an average of 35 shoots per cotyledon explant in a culture duration of 120–140 days.     

Morphogenic gradients of adventitious bud and shoot regeneration in epicotyl explants of Citrus

The in vitro responses of epicotyl explants from ‘Cravo’ rangpur lime (Citrus limonia Osb.), ‘Foster’ grapefruit (C. paradisi Macf.), and ‘Pera’ sweet orange (C. sinensis (L.) Osb.) were characterized for the first time. Further analysis was performed in ‘Cravo’ rangpur lime and ‘Foster’ grapefruit aiming to verify the in vitro morphogenesis of five distinct regions of the epicotyl under different treatments. It was observed the same general pattern of morphogenic gradient along the epicotyl axis in both citrus cultivars, with greater organogenic response as the distance of the explants from the cotyledonary node increased. This morphogenic gradient was influenced by factors related to plant material, composition of the culture medium, and conditions of incubation. The regions of the epicotyl farthest from cotyledons could be used as a source of explants in experiments of genetic transformation of the genotypes evaluated aiming to improve the efficiency of production of transgenic Citrus plants.     

甜樱桃砧木离体叶片愈伤组织诱导及不定芽再生

叶片再生效率的高低直接影响目的基因转化的成功率,为建立稳定、高效的樱桃不定芽离体再生体系,以30～40d苗龄的甜樱桃砧木ZY-1的组培继代苗为试材,取上部幼嫩叶片或不含腋芽的茎段,分别从培养基生长调节剂配比、接种材料类型、叶片接种部位、接种方式以及培养条件等方面进行了不定芽再生技术研究。结果表明,培养基中添加7.0mg/L的6-BA与0.5～1.0mg/L的IBA配比时不定芽再生率和出芽数均较高,TDZ和NAA不适于诱导ZY-1叶片再生不定芽;接种继代苗茎段比叶片再生率高;接种叶片以选择嫩叶横切2刀、远轴面向下接触培养基的方式为好;叶片不同部位处理以带叶柄的基部叶块最易再生;叶片接种后在25℃室温条件下,先暗培养3周再照光,利于不定芽的再生。     

香玲核桃离体叶片再生体系的建立

为解决核桃种质资源的实验室保存及转基因研究等问题,研究了暗培养时间、叶片放置方式及不同浓度激素组合(BA、NAA、TDZ)对香玲核桃叶片再生的影响。试验结果表明,最适宜的暗培养时间为14d;近轴面放置效果最好;不同组合的激素配比对叶片再生不定芽能力不同,最适宜香玲核桃叶片再生的培养基为:MS+BA0.5mg·L-1+NAA0.5mg·L-1+TDZ1.0mg·L-1+sucrose30g·L-1+agar6g·L-1,在该培养基上,离体叶片不定芽再生率达60%,每叶片平均再生不定芽数超过1.1个。筛选得到了香玲核桃离体叶片的最适分化及生根培养基,对离体再生培养条件进行优化,为香玲核桃的种质资源保存及遗传转化研究奠定了基础。     

In vitro morphogenesis and adventitious shoot mass regeneration of Vriesea reitzii from nodular cultures

Micropropagation systems based on nodular cultures (NCs), are considered as an intermediary in vitro morphogenetic route, diverging from regenerative systems based on organogenesis and somatic embryogenesis. The aim of this study was to establish a regenerative protocol based on the induction and development of NCs in Vriesea reitzii, an endangered bromeliad from the Atlantic forest which also has ornamental value. Additionally structural analyses were performed in order to better understand this in vitro morphogenetic route. NCs were regenerated in MSB culture medium free of PGR or supplemented with different levels of NAA alone or in combination with in combination with 2-iP. The subculture of these NCs on MSB medium supplemented with 10 μM of GA₃ promoted the synchronized shoot elongation. A regenerative efficiency of 12.4 g g⁻¹ of NCs was obtained, and this results in 5300 microshoots after 10 weeks in culture. The structural analyses of the NCs revealed that the regenerative process occurs from the proliferation of meristematic cell groups resulting in the development of multiple shoot meristems and buds. The development of NCs leads to the formation of monopolar structures called microshoots, which evolve to elongated shoots. Intermediary features shown in NCs are consistent with their classification as an intermediary system among organogenesis and somatic embryogenesis.     

Globe artichoke plants obtained from shoot apices through rapid in vitro micropropagation

A rapid method of in vitro propagation for globe artichoke (Cynara scolimus L.) is described. Shoot apices and subterminal stem segments were cultured on modified Murashige and Skoog (1962) medium (MS) with NaH₂PO₄ (50 mg/l), m-inositol (100 mg/l), L-tyrosine (100 mg/l), adenine (40 mg/l), indoleacetic acid (IAA, 0.5 mg/l), kinetin (K, 10 mg/l). sucrose (4%) and agar (0.7%) at pH 5.5. On this medium, a high number of proliferating shoots was obtained. The number of these shoots was increased and their overall development improved by sub-culturing on a MS medium with a reduced concentration of K (5 mg/l) and without the additional amount of sodium dihydrogen phosphate. In these conditions, a 4.5 rate of shoot proliferation was reached after 3 weeks.To induce rooting, the proliferated shoots were transferred to a medium containing half MS, thiamine-HCl (1 mg/l), m-inositol (100 mg/l), ascorbic acid (10 mg/l), naphthaleneacetic acid (NAA, 2 mg/l), sucrose (2%), agar (0.7%) at pH 5.5. In these culture conditions about 84% of plantlets rooted.Cytological analyses performed on root tips of 20 randomly chosen plantlets showed that all the analysed plants contained the diploid number of chromosomes. The plantlets were successfully transferred to soil and the method described seems to be suitable for rapid propagation of globe artichoke.     

Effect of explants source on shoot proliferation and adventitious regeneration in 10 Buddleia cultivars

Viable shoot cultures and weaned plants were obtained from cultured apical meristems with 10 Buddleia cultivars giving viabilities of 32–72%. The number of shoots produced, the micropropagation rate and the root number produced in vitro was higher in meristem derived shoots compared to those derived from shoot-tips. The subsequent growth rate of meristem derived plants, in the greenhouse, was also greater. The number of roots produced by conventional cuttings collected from meristem derived plants was significantly higher than in cuttings which were collected from plants derived from shoot-tips or from the original stock plants.Endogenous bacteria were not detected in either shoot cultures derived from meristems or in 10-week-old weaned plants derived from meristems whereas those derived from shoot-tips showed the presence of endogenous bacteria when sterilized explants were cultured on nutrient agar or on tryptic soy broth.Factors affecting adventitious bud and shoot production in leaf and internode explants was determined for ‘Lochinch’, ‘Border Beauty’, ‘Ile de France’ and ‘Pink Delight’ using meristem derived shoot cultures. Adventitious shoots appeared after 4 weeks of culture, in both types of explant when cultured on MS supplemented with 0.5–5.0 μM TDZ. The highest percentage regeneration was achieved from bisected internode explants cultured on 0.5 μM TDZ, with 93–100% regeneration among the cultivars whereas BA was less effective. The best response was obtained using 5.0 μM TDZ which gave over 10–11 shoots per explant in all bisected explants for all cultivars.     

Plant regeneration of Fraxinus angustifolia by in vitro shoot organogenesis

The in vitro regeneration of Fraxinus angustifolia through bud/shoot organogenesis on embryonic explants was investigated. Embryo axes and cotyledons from mature seeds underwent adventitious regeneration after exposure to growth regulators, the embryo axes showing a greater regenerative potential than cotyledons. While the MS medium proved effective for the induction and initiation phases, it was inadequate for bud development, and was replaced by DKW medium. A constant supply of benzyladenine (BA) appeared essential for all phases. The gelling agents used had an important role in morphogenesis: Duchefa Gelrite positively affected the number of new buds per explant. The rooting of developed shoots was easily achieved on auxin-free WPM medium and most of the rooted plantlets “hardened” after a period of acclimation in a mist greenhouse.     

Somatic embryo,adventitious root and shoot regeneration in in vitro grown quince leaves as influenced by treatments of different length with growth regulators

The purpose of this work was to acquire more information on the capacity of in vitro grown quince (Cydonia oblonga Mill.) leaves to simultaneously regenerate somatic embryos, adventitious roots and shoots, and to evaluate the variations induced on regeneration response by treatments of different length with growth regulators. After 2 days of liquid treatment with 2,4-dichlorophenoxyacetic acid, the leaves were cultured for 0, 3, 6, 9, 12, 15, 18 and 21 days on gelled growth medium containing the basal components of Murashige and Skoog and kinetin (Kin) 4.5 μM + naphthaleneacetic acid (NAA) 0.5 μM. At the end of each treatment period, the leaves were cultured on a transfer medium in the absence or in the presence of a growth regulator combination represented by N⁶-benzylaminopurine (BA) 2.66 μM + gibberellic acid 0.58 μM + indole-3-butyric acid 0.3 μM. The culture period for all the treatments was fixed to 52 days.     

High efficiency shoot regeneration from calluses of strawberry (Fragaria X ananassa Duch.) stipules of in vitro shoot cultures

A highly efficient method of shoot regeneration was developed from calluses of four culti- vars of strawberries (Fragaria x ananassa Duch.), ‘Addie’, ‘Dana’, ‘Gea’ and ‘Santana’, grown in vitro. Optimum shoot regeneration (84-96% of the explants), with four to eight shoots was obtained from calluses developed from stipules near the internal zone between petiole and stipule, on Murashige and Skoog (1962) or Gamborg et al. (1968) media, supplemented with 3% (w/v) glucose, 10 [iM BAP and 2.5 IBA and 0.8% agar. The calluses continued to produce shoots for at least six subsequent subcultures. This has been reported, up to now, only in juvenile explants. Microscopic observation showed no preformed buds or meristematic groups of cells in the connecting zone of petiole and stipule prior to culture. However, there were several layers of cells in this area containing higher amounts of starch, which were not observed in the cells of the bottom or in the external side of the stipule. Regeneration from stipules occurred five to six days earlier in whole leaves (stipule + petiole + lamina) than in leaves without laminae, but the final percentage was the same in the cases of all explants. The percentage of regenerating calli from the other explant sources (leaf lamina, petiole and root) was low and dependent on cultivar. Cv. Gea, which showed the highest regeneration capacity, regenerated 32% from leaf laminae, 16% from petiole and root calluses, followed by cv. Addie with 12% from leaf laminae only; the others failed to regenerate from calluses derived from these tissues. The regenerated shoots were successfully rooted and hardened for further observations on eventual somaclonal variation.     

An improved protocol for adventitious shoot regeneration and plant formation in Psoralea corylifolia L.

An effective protocol was developed for in vitro regeneration of Psoralea corylifolia through enriched cotton moistened-liquid (CML) and solid culture systems. Prolific adventitious shoot buds were achieved from hypocotyl explants of 2-week-old cultures on enriched CML Phillips and Collins (L2) medium supplemented with different concentrations and combinations of thidiazuron (TDZ), benzyladenine (BA), kinetin (KIN), naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and bavistin (BVN). Combination of 2 μM TDZ, 0.5 μM BA, 100 mg l⁻¹ BVN and 2 μM NAA produced a greater number of adventitious shoots per explant (93.5) when transferred to half-strength enriched solid L2 medium. Regenerated shoots (40–50 mm in length) were exposed simultaneously for rooting as well as hardening in moistened (1/8-L2 basal salt solution with 5 μM IBA and 100 mg l⁻¹ BVN) soil mixture and vermiculite (3:1, v/v). The plants were subsequently established in the field. The survival percentage differed with seasonal variations.