白姜花胚性愈伤组织的诱导与植株再生体系的建立

将白姜花（Hedychium coronarium）未成熟花丝接种在MS + 4 mg ? L^-1 2,4-D + 4 mg ? L^-1 NAA + 1 mg ? L^-1 6-BA的培养基上,经过180 d的培养,诱导出3种愈伤组织：Ⅰ,浅黄色松散易碎;Ⅱ,白色疏松半透明水渍状;Ⅲ,黄色致密颗粒状。对愈伤组织继代培养基中的2,4-D、NAA和6-BA浓度进行优化,结果表明培养基中含有1 mg ? L^-1 2,4-D、0.25 mg ? L^-1 NAA、0.25 mg ? L^-1 6-BA时可获得胚性状态良好的愈伤组织。胚性愈伤组织在MS无机盐 + 0.25 mg ? L^-1 NAA + 0.5 mg ? L^-1 TDZ + 维生素B₅ + 100 mg ? L^-1谷氨酰胺 + 230 mg ? L^-1脯氨酸 + 100 mg ? L^-1麦芽提取物 + 45 g ? L^-1蔗糖的体胚诱导培养基中培养20 d后,转移到MS基本培养基上培养30 d,1 g胚性愈伤组织可获得50 ~ 60个体胚。成熟体胚在MS + 0.2 mg ? L^-1 IAA + 0. 5 mg ? L^-1 6-BA的萌发培养基上培养20 d时,体胚萌发率为85%。萌发的体胚转移到1/2 MS + 1 g ? L^-1活性炭的成苗培养基中可发育成正常植株,植株室外栽培成活率达90%。对100株再生植株的染色体数进行分析,发现3株三倍体（2n = 3x = 51）、6株四倍体（2n = 4x = 68）和2株六倍体（2n = 6x = 102）。     

Plantlet regeneration and stability from callus cultures of Freesia × hybrida Bailey cultivar ‘Royal’

True-to-type plantlets of Freesia × hybrida Bailey cultivar ‘Royal’ were generated from callus after 27 months of sub-culture in liquid medium. Callus was initiated from young flower pedicels cultured on semi-solid Linsmaier—Skoog (LS) medium supplemented with 5 mg/l of 2,4-D and 0.5 mg/l kinetin, and transferred to the same medium in liquid form without hormones and thereafter sub-cultured every 7–10 days. Liquid cultures with 2.4–4.3 g of callus per 25 ml medium produced largest increases in callus fresh weight. Callus generated the most shoots when cultured on LS medium supplemented with 5 mg/l of kinetin and incubated in the light, while fewer plantlets were produced when no growth regulator or GA₃ or PBA were used. Callus cultures incubated in continuous darkness did not form shoots.     

High-efficiency colony formation and whole plant regeneration from mesophyll protoplasts of Pelargonium × hortorum ‘Panaché Sud’

Summary

This study aimed to establish a plant regeneration system from protoplasts of Pelargonium hortorum, efficient enough to be used for further direct gene transfer experiments. A rapid and efficient system that allowed high efficiency colony formation (40%) and whole plant regeneration (83%), as well as rooting within 4 months, was established using mesophyll protoplasts of the cultivar ‘Panaché Sud’. Protoplast culture in liquid medium was found to be better than culture on solid medium both for cell division and colony formation. The optimum density for high colony formation (31-40%) from viable cultivated protoplasts was 3 – 5 10⁴ protoplasts ml^–1. Reducing the osmotic pressure and increasing the macronutrient and sucrose contents of the culture medium after the first week of culture facilitated the rapid development of colonies. The transfer of microcalli to mannitol-free callus-induction medium produced green calli in all cases. The highest frequency of bud and shoot regeneration from protoplast-derived calli (83%; 6.6 per callus) was obtained at a density of 3 10⁴, on medium containing 0.2 mg l^–1 indole-3-acetic acid (IAA), 1.0 mg l^–1 zeatin and 0.1 mg l^–1 thidiazuron (TDZ). The best results were obtained when the medium was gelled with Gelrite^® and cultures were maintained under low light (12 µmol s^–1 m^–2). Sixty-five percent of protoplast-derived calli underwent bud and shoot regeneration and 2.6 rootable plantlets were obtained per callus after 3 weeks on elongation medium. All acclimatised plants grew normally and gave fertile flowers. However, flow cytometry on 42 plants showed that 40 of these were tetraploids, and only two were diploids, like the mother plant. This protocol can now be used in transformation experiments and applied to other genotypes to improve regeneration.     

苹果原生质体分离培养及植株再生

以悬浮培养细胞及叶片作为分离原生质体的起始材料 ,对影响苹果原生质体分离和培养的因素进行了系统研究。优化的原生质体培养基为 :改良MT 维生素C 5mg/L BA0 .5～ 1mg/L 2 ,4 D 0 .2mg/L 蔗糖 0 .6 5mol/L 谷氨酰胺 50 0mg/L CH 10 0mg/L ME50 0mg/L ;以葡萄糖代替蔗糖作渗透压调节物质效果好 ,且在培养过程中不需降压 ;适宜的低密度培养 (0 .5× 10 5/mL)可减少褐变。悬浮系原生质体培养 5～ 6d出现第 1次细胞分裂 ,15～ 2 0d形成多细胞团 ,4 0～ 50d形成肉眼可见的小愈伤组织。经培养诱导出不定芽 ,并进一步诱导生根长成完整植株。获得了平邑甜茶、M2 6 、嘎啦 3种基因型的原生质体再生植株。     

朱顶红‘Red Lion’胚性愈伤组织诱导及体细胞胚发生

以‘Red Lion’朱顶红(Hippeastrum vittatum)的鳞片为外植体进行体细胞胚诱导和形态学与组织学的胚性鉴定。结果表明,鳞片在MS+2 mg·L~(-1) BA+1 mg·L~(-1) NAA+2 mg·L~(-1) TDZ+30 g·L~(-1)蔗糖培养基上胚性愈伤组织诱导率可达90.63%,而不添加TDZ的培养基上胚性愈伤组织诱导率为0。胚性愈伤组织可呈白色松散、透明膨松、透明致密、微棕色松脆和翠绿色松脆等类型;非胚性愈伤组织呈微棕色致密状。胚性愈伤组织中可明显观察到球形胚、心形胚和棒状胚3个发育时期。体细胞胚在不添加植物生长调节剂的MS培养基上可发育形成芽和根,长成完整小植株。     

‘过山香’香蕉原生质体培养及植株再生

 以'过山香'香蕉的胚性悬浮细胞（Embryogenic cell suspensions,ECS）为起始材料分离原生质体,酶解液的组成为：3.5%纤维素酶R-10、1%离析酶R-10、0.15%果胶酶Y-23、204 mmol/L KCl、67 mmol/L CaCl2和0.41 mol/L甘露醇,原生质体产量为3.1×107个/mL PCV ECS （PCV：packed cell volume,细胞密实体积）。分别以培养基‘A’和‘B’为培养成分在液体浅层培养和看护培养两种培养系统中进行原生质体培养。结果表明：在液体浅层培养系统中,采用培养基‘B’比采用培养基‘A’效果好,原生质体的细胞分裂频率和细胞团形成频率分别约是采用培养基‘A'的3倍和10倍;所获得的培养物为只能增殖而不能进一步分化的非胚性细胞团。在看护培养系统中,采用培养基‘A’与采用培养基‘B’时,细胞分裂频率和细胞团形成频率没有显著地差异;所获得的细胞团具有典型的胚性细胞特征。将从看护培养中获得的细胞团转移到体胚诱导培养基上,培养45 d后,从105个原生质体获得1550个体胚。继续在体胚诱导培养基上培养30 d,7.8%的体胚能萌发。萌发的体胚在MS+0.1%活性炭的培养基中发育成健壮植株,移栽后成活良好。     

余甘子下胚轴离体培养中的器官形成与植株再生(英文)

以余甘子(phyllanthusemblicaL.)下胚轴为外植体,通过器官发生途径诱导形成不定芽,探讨不同激素组合对下胚轴器官发生和植株再生的影响,以期建立有效的再生体系,为以后的遗传转化研究奠定基础。结果表明,在附加0.1μmol/LNAA、5μmol/LBA和30g/L蔗糖MS培养基上培养,5周后不定芽再生频率达87.5%,平均每个外植体再生芽数为9.7个,为最佳组合。将分化的不定芽转移至含有10μmol/LIBA的MS培养基上,5周后生根,发育成健康的植株,生根率在29.2%。     

普通狗牙根种子组培技术优化及植株再生

以普通狗牙根的成熟种子作外植体,通过胚性愈伤组织的发生进行植株再生。结果表明:诱导狗牙根种子产生愈伤组织的最佳培养基为:MS+2,4-D 3 mg/L+壳聚糖4 g/L;暗培养优于光培养,最高出愈率为87.0%;所诱导愈伤组织在1/2原激素浓度下继代,以MS+6-BA0.1 mg/L为分化培养基,获得了再生植株;半透明粘稠状愈伤组织的分化率最高,为63.1%。     

Production of virus-free plantlets by anther culture of LiliumבEnchantment’

Anthers of the LiliumבEnchantment’, excised at the uninucleate microspore stage, were cultured on MS media containing 6% sucrose with auxin and cytokinin. When anthers were cultured on the medium with 2 mg l⁻¹ picloram and zeatin, 12–86% of them formed nodular calli. Anthers excised from greenhouse- and field-grown plants showed different responses: anthers of greenhouse-grown plants had a significantly higher capability to form callus and regenerate bulblets than those of field-grown plants. In anthers from greenhouse-grown plants, bulblet formation was dependent on the time at which anthers were excised from donor plants: anthers collected from early forced mother-plants had a higher capability of forming bulblet than others. All regenerated plantlets were diploid, which was substantiated by histological observation showing that the anther-derived calli originated from anther wall tissues. Virus tests by ELISA were made for 49 plantlets selected randomly at transplanting: 20 plantlets (41%) were virus-free, and the rest showed positive reactions for lily symptomless virus, cucumber mosaic virus and/or tulip breaking virus.     

酿酒葡萄原生质体再生植株

将酿酒葡萄品种白诗南、梅郁的花丝接种在含６ＢＡ２．０ｍｇ·１－１、２,４－Ｄ０．５ｍｇ·１－１的Ｂ５诱导培养基上,诱导产生胚性愈伤组织。胚性愈伤组织经液体悬浮培养形成含大量胚性细胞团的悬浮培养物。用Ｃｅｌｌｕｌａｓｅ　　Ｒｓ２％、Ｐｅｃｔｏｌｙａｓｅ　Ｙ２３０．３％,５ｍｍｏｌ·１－１ＣａＣｌ２·２Ｈ２Ｏ、０．６ｍｏｌ·１－１甘露醇、０．３％葡聚糖硫酸钾、ｐＨ５．６～５．８的酶混合液从胚性细胞团分离得到原生质体。原生质体培养到第５天出现第一次分裂,４０ｄ形成上百个细胞的大细胞团,５０ｄ形成０．５～１．０ｍｍ大小的小愈伤组织。这些小愈伤组织在含６ＢＡ０．５ｍｇ·１－１的Ｂ５分化培养基上分化出胚状体进而形成幼苗,在生根培养基上生根形成再生植株。     

君子兰种子离体培养的研究

试验对君子兰品种'油匠'、'胜利'、'和尚'的种子离体培养进行了研究,结果表明,直接从果实中剥离而采用70%酒精10 s 0.1%升汞8 min的消毒效果最好,污染率仅5.68%.基因型对种子组织培养的影响较大,'油匠'的种子较易于诱导出愈伤组织,诱导率可达72.97%.'胜利'的种子最适诱导培养基为MS 2,4-D 2 mg/L BA 2 mg/L,其诱导率为52.94%,最适分化培养基为MS NAA 1 mg/L BA 1 mg/L,分化率达72.22%.君子兰种子在培养基上诱导产生愈伤组织并能大量分化成苗,对君子兰组培工厂化生产有较大意义.     

Effect of sucrose concentrations on somatic embryogenesis in carnation (Dianthus caryophyllus L.)

The effect of sucrose concentration on callus induction followed by differentiation of embryogenic callus derived from petal explants of four carnation cultivars (Nelson, Sagres, Spirit and Impulse) was investigated. Embryogenic calli were produced on Murashige and Skoog [Murashige, T., Skoog, F.A., 1962. Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 154, 73–479] basal medium (MS) culture medium containing six concentrations of sucrose (3, 6, 9, 12, 15 and 18%, w/v) all supplemented with 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 μM 6-benzyladenine (BA). Maximum frequency of embryogenic callus was obtained from the media containing 9 and 12% sucrose. Somatic embryos were induced on a hormone-free MS media containing the seven concentrations of sucrose. Development of somatic embryos was enhanced by increasing sucrose concentration from 1.5 to 12%, while it was reduced in higher concentrations of 15 and 18%. However, normal embryos were not developed in the media containing 1.5 and 3% sucrose. Ninety-five percent of somatic embryos were regenerated to form the entire plantlets when they transferred onto the half-strength hormone-free MS culture medium containing 3% sucrose. Plantlets were also continued to grow normally under greenhouse condition.     

Somatic embryogenesis and plant recovery from callus of ‘Nabali’ Olive (Olea europea L.)

Regeneration via somatic embryogenesis from callus was studied in ‘Nabali’ olive (Olea europea L.). Among different explant sources (leaf blades, leaf petioles, hypocotyls of germinated seeds and roots of germinated seeds), roots gave the highest (46%) callus induction. Somatic embryogenesis was induced from root callus on embryogenesis induction medium (EIM) containing 5.0 μM 2,4-D, 0.5 μM kinetin and 5.0 μM NAA in darkness. Embryo regeneration was studied by transferring the callus from EIM to embryogenesis expression medium (EEM) containing different concentrations (0.0, 5.0, 10.0 and 15.0 μM) of 2-isopentenyladenine (2iP), BA, thiadiazuron (TDZ), zeatin or kinetin. Among the tested concentrations, 2iP at 10.0 μM outperformed the other growth regulators. 2,4-D at 5.0 μM in the EIM was satisfactory for embryogenesis induction. Sucrose at 0.2 M evoked higher embryogenesis than any other concentration of fructose and glucose in EIM, while sorbitol and mannitol at 0.1, 0.2 or 0.3 M reduced embryogenesis significantly and inhibited it totally at 0.4 M. Somatic embryos were rooted by transferring them to hormone-free medium (HFM). About 85% of embryos converted to rooted plantlets, 5% showed secondary embryogenesis and 10% were not developed and died. Rooted plantlets gave 95% survival when acclimatized ex vitro. Acclimatized plantlets developed into whole plants in the greenhouse and they were phenotypically similar.     

In vitro regeneration of Camellia reticulata by somatic embryogenesis

Camellia reticulata L. plantlets were regenerated by direct and indirect somatic embryogenesis from immature zygotic embryos. Initial explants (cotyledon sections and embryonic axes) produced somatic embryos without intermediate callus tissue when grown on Murashige and Skoog’s basal medium with 30 gl^-1 sucrose and no growth regulators; the somatic embryos completed their development in 4-6 weeks in the same medium. Embryogénie competence was increased by 0.5 and 1 mg l^-1 IBA. Histological observation showed the embryos to originate from epidermal and subepidermal cells of the cotyledon and hypocotyl explants. Secondary somatic embryos developed directly from the cotyledons and hypocotyl region of primary somatic embryos by a process that was morphologically very similar to that occurring on zygotic explants. Direct repetitive embryogenesis was maintained by this system. Up to 40% germination occurred when mature somatic embryos were isolated and incubated in medium supplemented with 1 mgl^-1 GA₃ + 1 mgl^-1 IAA. Indirect somatic embryogenesis was induced in callus differentiated on cotyledon explants after three months’ culture in media containing IBA or NAA and/or BAP, embryogenic capacity being retained by callus subcultured on 0.5 mg l^-1 IBA + 1 mg l^-1 BAP.     

分蘖洋葱茎尖愈伤组织诱导及植株再生

 阿城紫皮分蘖洋葱鳞茎茎尖愈伤组织诱导培养基以MS + 2 ,4-D 2. 0 mg·L^-1 + KT0. 5 mg·L^-1最佳, 出愈率为100 %; 分化培养基以MS + NAA 0. 1 mg·L^-1 + BA 0. 4 mg·L^-1最佳,分化率为88. 3 % , 平均成苗数为10. 2 ; 生根壮苗最佳培养基为1/ 2 MS + PP3330. 1 mg·L^-1+NAA 0. 01 mg·L^-1 + IBA 1. 5 mg·L^-1, 生根率100 % , 移栽成活率达100 %。     

苹果体细胞悬浮培养及植株再生研究

以苹果试管苗叶片的再生不定芽嫩叶为试材,对苹果体细胞悬浮系的建立及植株再生的影响因素进行了研究.结果表明:在MS+NAA 0.5 mg/L+BA 2.0 mg/L培养基上可诱导获得高活力的愈伤组织.将该愈伤组织转入MS+2,4-D 2.0 mg/L+BA 1.0 mg/L液体培养基中培养,采用无菌筛网分离获得含单个细胞和少于8～10个细胞的小细胞团进行继代培养,建立体悬浮细胞培养系.将悬浮细胞转到MS+2,4-D 2.0 mg/L+BA 1.0 mg/L的固体增殖培养基上暗培养25 d后,可形成微型愈伤组织,将该愈伤组织转到MS+BA 5.0 mg/L+IAA 0.5 mg/L的植株再生培养基上暗培养30 d后转入光下,光照培养30 d后53％的愈伤组织可再生植株.该研究建立的苹果体细胞悬浮培养技术,不仅可用于细胞融合及遗传转化过程中杂种细胞和转化细胞的分离及植株再生研究,而且对于利用组织培养技术筛选变异株系也具有重要意义.     

苦苣菜再生体系建立研究

以苦苣菜的嫩茎为材料,进行愈伤组织的诱导、分化、试管苗生根、移栽及移植所需要条件的研究,并建立起苦苣菜嫩茎的再生体系。结果证明：MS＋BA0．6mg／L＋2,4-D1．6mg／L是嫩茎颗粒状愈伤组织诱导的理想培养基;MS＋AgNO3 2．0mg／L＋BA1．0mg／L＋NAA0．1mg／L是诱导愈伤组织颗粒和不定茅分化培养的理想培养基;1／2MS＋IAA0．2mg／L＋NAA0．1mg／L是试管苗生根培养的理想培养基;河沙和炉灰渣是试管苗移栽的理想基质,移栽成活率为96％和98．5％,移植到山坡上的试管苗生长旺盛,根系发达。     

红姜花体细胞胚胎发生及植株再生的研究

以红姜花（Hedychium coccineum）的花丝和花药为外植体,通过体细胞胚胎发生途径建立了植株再生体系。结果表明：外植体在MS + 4 mg ? L-1 2,4-D + 4 mg ? L-1 NAA + 1 mg ? L-1 6-BA + 30 g ? L-1 蔗糖 + 7 g ? L-1 琼脂的培养基上经过120 d诱导出愈伤组织,愈伤组织在MS + 1 mg ? L-1 2,4-D + 0.25 mg ? L-1 NAA + 0.25 mg ? L-1 6-BA + 30 g ? L-1 蔗糖 + 7 g ? L-1琼脂的培养基上经过继代筛选,获得浅黄色松散易碎的胚性愈伤组织。胚性愈伤组织在MS无机盐 + B5维生素+ 100 mg ? L-1谷氨酰胺 + 230 mg ? L-1 脯氨酸 + 100 mg ? L-1麦芽提取物 + 0.02 mg ? L-1 NAA + 0.02 mg ? L-1 TDZ + 0.5 ~ 1.0 mg ? L-1 2,4-D + 45 g ? L-1蔗糖的培养基中通过3个月的悬浮培养,可得到均质稳定的胚性细胞悬浮系（ECS）。胚性悬浮细胞在SH无机盐 + B5维生素 + 100 mg ? L-1谷氨酰胺 + 230 mg ? L-1脯氨酸 + 100 mg ? L-1麦芽提取物 + 0.25 mg ? L-1 NAA + 0 ~ 0.20 mg ? L-1 TDZ + 45 g ? L-1蔗糖 + 7 g ? L-1琼脂的体胚诱导培养基中培养10 d,可见到白色半透明体胚发生,20 d后体胚发育成熟。当TDZ浓度为0.15 mg ? L-1时,体胚诱导率高达4 500个 ? mL-1 PCV ECS（PCV：细胞密实体积）。在SH无机盐 + B5维生素 + 0.20 mg ? L-1 IAA + 0.25 ~ 1.0 mg ? L-1 6-BA + 30 g ? L-1蔗糖 + 7 g ? L-1琼脂的体胚萌发培养基上,体胚萌发率高达100%。将萌发的体胚转移到1/2MS + 1 g ? L-1活性炭成苗培养基中,进一步发育成正常的再生植株,植株室外栽培成活率达90%。     

Micropropagation of Cryptanthus with leaf explants with attached intercalary meristems excised from greenhouse stock plants

By using the leaves with attached intercalary meristems from greenhouse grown stock plants, five cultivars of Cryptanthus were cultured on modified MS media with 4.5 μM NAA and IBA and 3 μM BA to induce adventitious shoot formation from callus tissue. Contamination was 17–21% for explants taken from stock plants which were sprayed weekly with Agribrom and 27–75% for those taken from stock plants which were not treated. More than 99% true to type plantlets were obtained from non-chimeric plants. Green and albino plantlets were obtained from chimeric plants. The chimeric C. ‘Coster's Favorite’ DeCoster also produced a few chimeric plantlets with intermarginal pink stripes in addition to the green and albino plantlets. Most of the non-chimeric plants took a shorter time to produce plantlets of transplantable size (8–12 mm) than the chimeric ones. Except for albino plantlets, survival rate of plantlets exceeded 95%. A minimum average of 500 rooted plantlets can be obtained in a year from a single well-callused leaf explant. The protocol in this report should speed up the mass production and introduction of desirable new cultivars and hybrids of non-chimeric Cryptanthus.     

Direct somatic embryogenesis and plant regeneration from leaf cultures of ornamental species of Dianthus

Protocol for direct somatic embryogenesis from leaf explants of economically important species of Dianthus, viz. D. caryophyllus, D. barbatus and D. chinensis has been developed. Murashige and Skoog’s (MS) liquid medium supplemented with 2,4-D (1 mg/l) was used for direct induction of somatic embryogenesis without an intervening callus phase. Initially globular structures were observed after 21 days of culture of leaf explants in liquid medium. Development of embryos to heart and torpedo stages was achieved in the liquid medium incorporated with polyethylene glycol (PEG 6000) at a concentration of 2.5%. Embryo maturation was further promoted by addition of casein hydrolysate (CH) (200 mg/l) in MS liquid medium. Embryos germinated to form plantlets on solid MS medium supplemented with GA₃ (1 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions.