安祖花胚性愈伤组织诱导及植株再生研究

为构建安祖花(Anthurium andreanum)胚性愈伤组织再生体系,以3个盆栽品种幼嫩叶片和叶柄为外植体,分析了基本培养基、植物生长调节剂组合和培养条件等因素的影响。结果表明,安祖花胚性愈伤组织诱导的最佳培养基为改良MS3+1.5 mg L–1 2,4-D+0.5 mg L–1 KT+4%蔗糖+2%葡萄糖+0.25%Phytagel,且胚性愈伤组织诱导能力差异显著,表现为?粉冠军??罗宾奴??冠军?和叶片叶柄,其中?粉冠军?叶片的胚性愈伤组织诱导率可达57.9%。胚状体分化的最佳培养基为1/2改良MSa+2%蔗糖+0.25%Phytagel,其中?粉冠军?叶片诱导的胚状体分化率可达31.6%,且在光、暗下分化率的差异不显著。分化苗移栽后的成活率可达100%。     

Plant regeneration from callus culture of a Paphiopedilum hybrid

Totipotent calli of a Paphiopedilum hybrid (Paphiopedilum callosum ‘Oakhi’ × Paph. lawrenceanum ‘Tradition’) were induced from seed-derived protocorms on a 1/2 strength Murashige–Skoog medium plus 1–10 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–1 mg l⁻¹ 1-phenyl-3-(1.2.3-thiadiazol-5-yl)urea (TDZ). These calli grew well when subcultured on the same medium, but proliferated more on 1/2 MS medium plus 5 mg l⁻¹ 2,4-D and 1 mg l⁻¹ TDZ. Calli developed further along a route of production of protocorm-like bodies and eventually formed plantlets that could be transplanted to pots and grew well. This revised version was published online in June 2006 with corrections to the Cover Date.     

High Frequency Plant Regeneration from Astragalus melilotoides hypocotyl and stem explants via somatic embryogenesis and organogenesis

An efficient and reproducible procedure is established for the plant regeneration from hypocotyl explants and hypocotyl-or stem-derived calli in Astragalus melilotoides. High frequency somatic embryo formation (98.3%) occurred direct on hypocotyls on Murashige and Skoog (MS) medium supplemented with 2.69 µM NAA and 4.44 µM BA within 5 weeks. Three types of calli were induced from the hypocotyl and stem segments on MS medium containing 9.05 µM 2,4-D and 2.22–4.44 µM BA. Both somatic embryos and adventitious buds were initiated from hypocotyl-derived calli while only adventitious buds were formed from stem-derived calli in MS medium supplemented with 2.69 µM NAA and 4.44–8.89 µM BA. Somatic embryos or adventitious buds developed into plantlets following being cultured for 3 weeks on MS medium without any growth regulators or with 14.78 µM IBA, respectively. All the regenerated plants were normal with respect to morphology and growth characters, and produced fertile seeds after planting in soil.     

Plant regeneration from protocorm-derived callus of <Emphasis Type="Italic">Cypripedium formosanum</Emphasis>

Summary Totipotent callus of Cypripedium formosanum, an endangered slipper orchid species, was induced from seed-derived protocorm segments on a quarter-strength Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and 4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thidiazuron). This callus proliferated well and was maintained by subculturing on the same medium. On average, 13 protocorm-like bodies could be obtained from a piece of 4 mm callus after being transferred to the medium with 4.44 μM N⁶-benzyladenine after 8 wk of culture. The regenerated protocorm-like bodies formed shoots and roots on medium containing 1 g l⁻¹ activated charcoal and 20 g l⁻¹ potato homogenate. After 24 wk of culture on this medium, well-developed plantlets ready for potting were established.     

防风愈伤组织的玻璃化法超低温保存及再生

为避免连续继代造成的愈伤组织变异,探索新的种质资源保存方法,对防风愈伤组织进行了超低温冷冻保存及植株再生研究。以关防风3周龄的愈伤组织为材料,单一变量法研究适宜的玻璃化法超低温保存程序。结果显示:(1)防风愈伤组织超低温保存的最佳方案为:4℃条件下于MS+1.0mg/L 6-BA+1.0mg/L NAA+5%DMSO的继代培养基中预培养3d,60%PVS2常温装载20min,100%PVS2于2℃脱水45min后直接投入液氮。(2)防风愈伤组织经超低温保存后的相对存活率最高为79.24%,其中预培养和脱水是实现超低温冻存的关键环节,且1.0mol/L蔗糖的MS溶液洗涤、暗培养14d以上有助于冻后愈伤组织恢复生长。研究表明,玻璃化超低温冻存可以作为防风愈伤组织的保存方法,冻后愈伤可以恢复生长并再生成完整植株。     

杂交狼尾草胚性愈伤组织的诱导与植株再生

以杂交狼尾草初级愈伤为材料,运用方差分析的方法研究不同因素对杂交狼尾草胚性愈伤诱导率与植株再生率的影响。结果发现,2,4-D和TDZ对杂交狼尾草胚性愈伤诱导影响显著,最佳的胚性愈伤诱导培养基为MS+3.0 mg·L-12,4-D+0.4 mg·L-16-BA+0.2 mg·L-1 TDZ,诱导率为54%;愈伤分化培养基以附加0.1 mg·L-1 TDZ+0.2 mg·L-16-BA+3.2 mg·L-1 CuSO4的 MS培养基为最佳,再生率为68.33%,褐化率为8.33%;分化培养基中添加0.8~3.2mg·L-1的CuSO4均能促进杂交狼尾草愈伤组织分化,而添加AgNO3对杂交狼尾草愈伤分化无显著影响。该技术为离体诱变获得杂交狼尾草低温种质材料奠定了基础。     

月季愈伤组织的诱导及植株再生

以可在黑龙江地区露地越冬的5个现代月季（Rosa chinensis）品种为实验材料,分别以其无菌苗的叶片和茎段为外植体,研究了愈伤组织诱导及植株再生方法。实验结果表明：5个寒地月季品种的叶片和茎段均可诱导出愈伤组织,2,4-D诱导愈伤组织的效果较好,高浓度的细胞分裂素不适合用于月季叶片和茎段愈伤组织的诱导;TDZ在月季愈伤组织分化培养过程中具有重要作用,光照培养可促进月季愈伤组织的分化,愈伤组织的分化能力随着继代次数的增加呈下降趋势。该实验成功地从2004-8和2004-9（2个月季品种）愈伤组织中诱导出再生植株,其愈伤组织的分化率分别为45%和38%。     

Callus Induction and Plant Regeneration in Lemna Minor L.

Callus induction was obtained on Murashige and Skogg agar medium with 45 M 2,4-dichlorophenoxyacetic acid under dark at 25°C. Among the four explant types investigated, the best callus induction was obtained from two-week old fronds to which a surgical incision was applied in the basal (meristematic) region. This treatment resulted in 89.11% of fronds producing callus which continued to proliferate for another 24 months. To obtain plant regeneration pieces of calluses were transferred onto Murashige and Skoog agar medium containing 22 M indole-3-acetic acid and 4.6 M kinetin and maintained under 16-h photoperiod (irradiance of 30 mol m^–2 s^–1) at 23°C. Green fronds formed on all callus pieces. The regenerated fronds were later transferred onto Wang medium where they formed roots. The regenerated Lemna minor L. plants obtained through indirect organogenesis did not differ morphologically from individuals forming the stock collection.     

Isolation,Culture and Plant Regeneration of Protoplasts from an Embryogenic Callus Tissue of Gossypium hirsutum

Protoplasts were isolated and cultured from hypocotyl embryogenic callus tissue of Gossypium hirsutum L. cv. "Lumian 6". The highest yields of viable protoplasts were obtained from a vigorous embryogenic callus 7 to 9 d old subcultured on MS medium supplemented with 2 mg/L IAA and 1 mg/L KT using a solution of 1% cellulase Onozuka R-10, 1% pectinase, 0.7 mmol/L KH2PO4, 2.5 mmol/L Ca2+ , and 0.5 mol/L osmoticum (mannitol), at pH 5.8 and at a temperature of 30 ℃. After separation and purification (in 21% sucrose floatation medium), the protoplasts were laid up in a quiet liquid protoplast culture medium containing K3 salts, NT vitamins with 0.1 mg/L 2,4-D, 0.2 mg/L KT and 0.45 mol/L glucose for 10 to 15 min. The protoplasts were fractioned into an upper and a lower layer in the centrifugal tube. Most of the protoplasts in the lower layer were smaller, round and rich in cytoplasts in which contain many granular substances. When this kind of protoplasts were cultured in the thin liquid protoplast culture medium with a density of 1 x l0s to 5 x los protoplasts/mL, the division and the callus formation of the regenerated cells were easily observed. The first divisions occurred in 3 days and small cell clusters could be seen after 2 to 3 weeks in the culture. At this moment, the addition of the protoplast culture medium with decreased osmoticum once or twice is needed for the continuous protoplasts division to form calli. Regenerated calli, 3 to 5 mm in diameter, were transferred in succession on MS medium with 2 mg/L IAA and 1 mg/L KT for the initiation of embryogenesis. The embryoids germinated on the hormonefree MS medium and a number of plantlets were obtained. It seems that using vigorous embryogenic callus and decreasing osmoticum are the two critical factors for plant regeneration of cotton protoplasts.     

地皮消愈伤组织诱导及植株高效再生体系的建立

以地皮消(Pararuellia delavayana)无菌实生苗带叶茎尖和带节茎段为外植体, 探讨不同植物激素种类及组合对愈伤组织诱导、芽丛发生及植株再生的影响。结果表明, 地皮消组织培养和植株再生的适宜外植体为带节茎段, 其在MS+1.0 mg·L^-1 6-BA+0.5 mg·L^-1 NAA+0.1 mg·L^-1 KT培养基中培养17天后, 约85.38%的外植体产生出分化能力较强的愈伤组织; 25天后约97.55%的愈伤组织开始分化出绿色芽丛; 30天后不定芽分化系数可达15.38。不定芽增殖继代6次后出现玻璃化现象, 且随着继代次数的增加, 玻璃化现象加重, 增殖率明显下降; 采用MS和B₅培养基交替使用可改善试管苗玻璃化现象并保持较高的增殖率。不定芽生根的适宜培养基为MS+0.5 mg·L^-1 NAA, 生根率可达100%。再生苗移栽成活率达95%以上。该研究建立了地皮消无性快速繁殖体系, 为保护地皮消野生资源及种苗繁育提供了有效途径, 也为其遗传转化研究奠定了基础。     

Plant Regeneration from Immature Embryo Cultures of Vigna unguiculata

Micropropagated plants of two annual haloxerophytic Asiatic Salsola species (S. pestifer and S. lanata) were obtained from zygotic embryos cultured on Murashige and Skoog (MS) agar medium supplemented with 0.5 μM benzylamino-purine (BAP) and 0.3 μM indole-3-acetic acid (IAA) or with 0.5 μM 6 γ, γ-dimethylallylaminopurine and 0.3 μM IAA. The callus induction from shoot and leaf explants derived from plants propagated in vitro were obtained on MS agar medium with various concentration of auxins and cytokinins. The best medium for growth and proliferation of calluses of both studied species was MS medium containing 9.0 μM 2,4-dichlorophenoxyacetic acid. It was also determined that beginning of plant regeneration from callus of S. lanata was induced by 8.8 μM BAP. This revised version was published online in July 2006 with corrections to the Cover Date.     

Plant Regeneration Through Somatic Embryogenesis in Leaf Derived Callus of Plumbago Rosea

Regeneration of Plumbago rosea L., a rare medicinal plant, via somatic embryogenesis in callus cultures derived from leaf explants was described. Optimum callus formation was achieved on semi-solid Murashige and Skoog (MS) medium supplemented with 0.25 mg dm^–3 kinetin and 2.0 mg dm^–3 1-naphthaleneacetic acid (NAA). Somatic embryogenesis was achieved upon transferring the 4-week-old callus to a medium containing 1.0 mg dm^–3 kinetic (Kn), 0.5 mg dm^–3 gibberellic acid (GA₃) and 0.1 mg dm^–3 NAA. Embryo maturation and germination was achieved on the half-strength MS basal salts supplemented with 0.01 – 0.25 mg dm^–3 Kn and 2 % (m/v) saccharose. An average of 50 – 60 plantlets were obtained from 150 mg of embryogenic callus within 4 week of subculture. Out of the 50 plantlets about 28 survived in the greenhouse.     

宜昌百合胚性愈伤组织诱导及植株再生体系的研究

采用L₉（3⁴）正交试验等方法,以野生宜昌百合（Lilium leucanthum）鳞片为外植体,分别在光照和黑暗培养条件下,研究了TDZ、NAA、2,4-D不同浓度配比对宜昌百合的胚性愈伤组织诱导效果的影响,并采用石蜡切片技术对愈伤组织进行了的组织学观察。在此基础上,探讨了NAA对体胚苗壮苗生根的影响。结果表明：在光照条件下诱导胚性愈伤组织的最佳培养基为MS+TDZ 0.5 mg·L^-1+NAA 1.0 mg·L^-1+2,4-D 0.05 mg·L^-1,诱导率为62.690%;黑暗条件下诱导胚性愈伤组织的最佳培养基为MS+TDZ 0.5 mg·L^-1+NAA 1.5 mg·L^-1+2,4-D 0.1 mg·L^-1,诱导率为59.423%。胚性愈伤组织黄绿色或深绿色,颗粒状明显;非胚性愈伤组织松散易碎、呈水渍状,经石蜡切片观察可观察到胚性愈伤组织细胞核大、胞质浓,且细胞内含丰富淀粉粒,细胞直径为50~80 μm。非胚性愈伤组织细胞较大,未见明显细胞核,直径约为100~180 μm。体胚苗壮苗生根的最适宜培养基为1/2MS+NAA 0.2 mg·L^-1+AC 1 g·L^-1,培养60 d后,幼苗移至草炭：蛭石：珍珠岩=1：1：1基质中,成活率为90%。本试验成功建立了宜昌百合鳞片胚性愈伤组织的再生体系,为宜昌百合利用胚状体进行无性育种以及种质资源的创新奠定了基础。     

A reliable protocol for plant regeneration from callus culture of <Emphasis Type="Italic">Phalaenopsis</Emphasis>

Summary Callus of Phalaenopsis Nebula was induced from seed-derived protocorms on 1/2 Murashige and Skoog (MS) basal medium plus 0–1.0 mg l⁻¹ (0–4.52 μM) N-phenyl-N′-1,2,3,-thiadiazol-5-yl urea (TDZ) and/or 0–10 mg l⁻¹ (0–45.24 μ M) 2,4-dichlorophenoxyacetic acid (2,4-D). Protocorms 2 mo. old performed better than 1-mo.-old protocorms for callus induction. More calluses formed on 1/2 MS basal medium supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. These calluses could be maintained by subculturing every month with basal medium supplemented with 0.5 mg l⁻¹ (2.27 μM) TDZ and 0.5 mg l⁻¹ (2.26 μM) 2,4-D. Protocorm-like bodies were formed, and plants regenerated from these calluses on 1/2 MS basal medium alone or supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. Plantlets were then potted on sphagnum moss in the greenhouse and grew well. No chromosomal abnormalities were found among the root-tip samples of 21 of the regenerated plantlets that were successfully acclimatized.     

黄花补血草愈伤组织的诱导和植株再生

以黄花补血草(Limonium aureum(L.)Hill.)无菌苗为材料,研究了不同激素配比条件下不同外植体愈伤组织的诱导及植株再生.结果表明,黄花补血草无菌苗叶片、叶柄和幼根均可作为离体培养的外植体,但叶片和叶柄的诱导率明显高于幼根.将外植体接种于分别添加0.5 mg/L NAA或1.0 mg/L 2,4-D或0.3-0.5 mg/L 6-BA 0.5-2.0 mg/L NAA的MS培养基上,经过14-28 d培养后,可脱分化产生乳白色、红色或浅绿色颗粒状或致密愈伤组织,频率达到70%以上.在MS 0.3 mg/L 6-BA 1.0 mg/L NAA培养基上,红色和绿色颗粒状愈伤组织经过1-2次继代后,均可分化产生不定芽,进而形成丛生芽,分化率达到100%.将高约3 cm的丛生芽切下,接种于分别添加0.5 mg/L IAA或0.5 mg/L IBA的1/2 MS培养基上可产生不定根,获得完整的再生植株.     

罗布麻愈伤组织诱导及植株再生

以罗布麻(Apocynum venetum L.)当年的成熟种子和5周龄的幼苗叶片为外植体,研究了不同激素组合、暗培养对愈伤组织及植株再生的影响.结果表明,幼苗作外植体诱导愈伤的最佳培养基为添加1.0 mg/L 6-BA 0.2 mg/L IBA的MS培养基;继代培养中1.0 mg/L 6-BA与0.2 mg/L IBA组合愈伤致密而生长迅速,长时间培养硬化的愈伤组织可用添加0.5 mg/L 6-BA和0.1 mg/L IBA培养基和初期暗培养获得大量质地疏松、增殖迅速的愈伤组织;再生苗诱导以0.5 mg/L 6-BA 0.2 mg/L IBA组合为佳;1/2MS附加NAA 0.6 mg/L为适宜的生根培养基,初步建立了罗布麻离体再生体系.     

矮丛蓝莓叶片的愈伤组织诱导及植株再生

以矮丛蓝莓'Blomidon'品种的试管苗叶片为外植体,研究不同培养基、激素、转接时间以及转接方式对愈伤组织诱导及植株再生的影响.结果表明:WPM1/2为适宜的培养基;添加0.5 mg·L~(-1) CPPU+1.0 mg·L~(-1) ZT+0.5 mg·L~(-1) TDZ和0.5 mg·L~(-1) CPPU+0.5 mg·L~(-1) ZT+1.0 mg·L~(-1) TDZ诱导愈伤率较高达100%;ZT、CPPU与生长素IAA、IBA的配比诱导愈伤率效果不佳;添加ZT诱导的愈伤组织适合20 d后转接,CPPU诱导的愈伤组织适合50 d后转接;转接方式均以固体培养为宜.矮丛蓝莓'Blomidon'最适宜培养基为WPM1/2+0.5 mg·L~(-1) CPPU+1.0 mg·L~(-1) ZT+0.5 mg·L~(-1) TDZ,其诱导愈伤组织50 d后转接平均分化芽数最多达10.26个.     

Plant regeneration from cotyledons of niger

Callus initiation from seedling explants of niger (Guizotia abyssinica Cass) cv. Ootacamund was found to be better on LS medium containing kinetin (1.4 M) plus 2,4-dichlorophenoxyacetic acid (9 M) than its analogues. Embryoids were induced directly from cotyledons on LS medium supplemented with 2,4,5-trichlorophenoxyacetic acid and 2,4,5-trichlorophenoxypropionic acid. When cotyledon-derived callus was subcultured onto medium with 10.7 M naphthalene-acetic acid and 2.3 M kinetin, embryogenesis was observed. Multiple shoots were obtained from cotyledonary explants in presence of MS medium containing 4.4 M benzyladenine and 11.4 M indoleacetic acid. Regenerated plants that were transferred to pots and grown to maturity were morphologically normal and fertile.Abbreviations NAA naphthaleneacetic acid - IAA indoleacetic acid - BA benzyladenine - GA₃ gibberellic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxypropionic acid - 2,4,5-TP 2,4,5-trichlorophenoxypropionic acid - ABA abscisic acid     

Callus induction and thallus regeneration from callus of phycocolloid yielding seaweeds from the Indian coast

The tissue culture of phycocolloid yielding seaweeds included preparation of axenic explants, callus induction, subculture of excised callus and regeneration of plantlets from pigmented callus in the laboratory. Treatment of algal material with 0.1–0.5% detergent for 10 min and 1–2% betadine for 1–5 min and 3–5% antibiotic treatment for 48–72 h successively enabled viable axenic explants to be obtained as high as 60% for Gracilaria corticata, Sargassum tenerrimum and Turbinaria conoides and 10% for Hypnea musciformis. Callus induction was more conspicuous in T. conoides than in the other three species investigated. Of the irradiances investigated, 30 μmol photons m⁻² s⁻¹ produced calluses in as many as 40% explants in G. corticata and T. conoides and 10% in H. musciformis and S. tenerrimum. The explants cultured at 5 and 70 μmol photons m⁻² s⁻¹ did not produce any callus in all the species studied except for H. musciformis in which 10% explants developed callus at 5 μmol photons m⁻² s⁻¹. Most of the species investigated showed uniseriate filamentous Type of growths and buds from cut ends and from all over the surface of explants. Nevertheless, T. conoides had three Types of callus developments, namely (1) uniseriate filamentous Type of outgrowths from the centre of the cut end of explant, (2) bubbly Type of callus and (3) club-shaped callus clumps. The subculture of T. conoides callus embedded in 0.4% agar produced two Types of filamentous growth, namely filiform (with elongated cells) and moniliform filaments (with round cells) in the 2 months period after inoculation. Further, friable callus with loose cells was also found associated with excised callus. The moniliform filaments showed prolific growth of micro-colonies resembling to somatic embryo-like growth which, in liquid cultures, differentiated and developed into propagules with deformed shoots and distinct rhizoids. The shoots of these propagules remained stunted with abnormal leaf stalks without forming triangular shaped leaves as the parental plant and rhizoids had prolific growth in the laboratory cultures. The excised callus of G. corticata continued to grow when transferred to liquid cultures and showed differentiation of new shoots within 10 days. The shoots grew to a maximum length of 5–6 cm in the 2 months period in aerated cultures in the laboratory. Dedicated to the memory of Late Dr. Rangarajan.     

In Vitro Culture of Kodo Millet: Influence of 2,4-D and Picloram in Combination with Kinetin on Callus Initiation and Regeneration

Immature inflorescences of kodo millet (Paspalum scrobiculatum L. cv. GPUK-3) were cultured on MS medium. Induction of embryogenic callus and subsequent somatic embryogenesis was possible on both 2,4-D and Picloram alone or with kinetin from spikelets as well as rachis. Immature inflorescence cultured on medium supplemented with lower levels of Picloram in combination with kinetin developed organogenic callus with shoot buds. Direct somatic embryo formation on rachis was observed at higher levels of Picloram in combination with kinetin. Plant regeneration was observed when calluses were transferred to α-napthaleneacetic acid (NAA) plus 6-benzylaminopurine (BA) supplemented MS medium. Histological observations provided a clear evidence for both somatic embryogenesis and shoot organogenesis. Profuse rooting was induced on phenylacetic acid (PAA) supplemented medium. Regenerated plants were successfully transferred to pots under field conditions where most of the plants survived and set normal seeds.