红姜花胚性细胞悬浮体系的建立和原生质体培养

以红姜花(Hedychium coccineum)未成熟花丝和花药为试材,研究了红姜花胚性愈伤组织的诱导、细胞悬浮体系的建立以及原生质体的培养。结果表明:在MS+4mg/L 2,4-D+4mg/L NAA+1mg/L 6-BA+30g/L蔗糖+7g/L琼脂上经过120d培养诱导出了愈伤组织,愈伤组织在增殖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。胚性愈伤组织通过3个月的悬浮培养,得到均质稳定的胚性细胞悬浮系。以胚性悬浮细胞(ECS)为起始材料分离原生质体,酶解试验表明,在原生质体分离过程中,用于原生质体分离的酶液需要保持一定的渗透压以保护原生质体不被破坏。当甘露醇的浓度为0.14mol/L时,原生质体产量最高,达到2.25×105个/mL PCV ECS(packed cell volume,PCV)。在看护培养系统中,原生质体经过7d的培养,细胞第一次分裂,经过14d培养,细胞分裂频率达到12.3%,28d时,细胞团形成频率达到4.2%。所形成的细胞团具有典型的胚性细胞特征。     

枇杷原生质体植株再生

以普通批粑（价iobotlya joponica Lindl．）‘解放钟’等品种的不同发育期胚为材料诱导愈伤组织。起源于鱼雷形胚的胚性愈伤组织是游离原生质体的合适材料。最合适的游离原生质体的酶混合液为：l％纤继素酶＋2％果胶酶干！2％甘露醇，其原生质体得率高于L 07／g愈伤组织，原生质体成活率在 95％以上。原生质体经液体培养 4天后，出现第一次细胞分裂，并继续分裂，形成大量肉眼可见的小愈伤组织。愈伤组织在含3％山梨醇的培养基土培养3周后，其表面出现茎原基，并进一步发育成芽苗。芽苗移入生根培养基后生成完整植株。已获得解放钟和‘白梨’移栽成活的原生质体再生植株。     

番木瓜胚性细胞悬浮系高效遗传转化体系的建立

【目的】建立番木瓜高效遗传转化体系，为番木瓜基因功能研究和重要农艺性状改良提供新的技术支撑。【方法】以紫晖番木瓜胚性细胞悬浮系（embryogenic cell suspensions,ECS）为遗传转化受体，利用植物表达载体pCAMBIA1301和农杆菌介导法进行遗传转化，对抗生素浓度筛选、侵染时间、继代培养、抗性胚的诱导与萌发以及植株再生整个过程进行探索，最后获得抗性再生植株。【结果】通过设置不同浓度的头孢霉素和潮霉素处理，观察ECS细胞状态，筛选、确定头孢霉素和潮霉素最适处理质量浓度分别为200 mg·L-1和5 mg·L-1。工程菌和ECS共培养侵染2 d后转到含有头孢霉素和潮霉素的液体筛选培养基上进行继代培养，继代周期为14 d。经GUS染色验证，表明继代3次后的ECS几乎全部为转化细胞。将以上ECS转移到液体胚诱导培养基中进行培养，2个月后可获得大量球形体细胞胚，且GUS组织染色为蓝色。将球形体细胞胚转到半固体成熟培养基上培养，2个月后可获得成熟子叶期体细胞胚。子叶期体细胞胚在萌发培养基上光培养30 d后，可获得再生芽。任意选取再生芽进行GUS染色，均可染成蓝色。抗性再生芽...     

柑桔原生质体分离及再生植株的研究

用0.3％的果胶酶和0.3％的纤维素酶处理锦橙(Citrus sinensis Osbeck)胚性愈伤组织16小时,得到91.2％具有活性的原生质体。培养4天时,出现第一次细胞分裂,二个月时,再生出胚状体。胚状体转入加有赤霉素和硫酸腺嘌呤的培养基中一个月,再生成完整的植株。原生质体再生出的胚状体中,没有发现有染色体数目的变异。锦橙非胚性愈伤组织的原生质体能分裂形成细胞团,但没有再生出植株。对这种原生质体的再生和利用作了讨论。     

阿拉伯半乳糖蛋白对香蕉悬浮细胞胚性状态的影响

采用火箭琼脂糖凝胶电泳法测定了培养15 d后的香蕉悬浮细胞液体培养基中的阿拉伯半乳糖蛋白(ara-binogalactan-proteins,AGPs)的含量,发现具有体胚发生能力的贡蕉、大蕉和龙牙蕉过山香品种的胚性悬浮细胞(em-bryogenic suspension cells,ECS)培养基中的AGPs含量分别达到36、48、56 mg.L-1,而在不能进行体胚诱导的香芽蕉威廉斯品种的非胚性悬浮细胞(non-embryogenic suspension cells,NECS)的培养基中检测不到AGPs,说明悬浮细胞分泌到培养基中的AGPs含量与香蕉悬浮细胞的胚性状态有一定的关系。在香蕉ECS的悬浮培养过程中,添加不同浓度的βGlcY试剂(β-glucosyl Yariv)到悬浮培养基中可引起胚性细胞与非胚性细胞(ECS/NECS)的比值下降;在香蕉的ECS体胚诱导过程中,添加不同浓度βGlcY试剂到体胚诱导培养基中,引起体胚发生频率下降。这些结果表明,AGPs在香蕉悬浮细胞的胚性保持过程中起到重要的作用,并且具有剂量效应。     

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%。     

香榧体细胞胚发生、发育的形态与细胞学观察

将香榧(Torreya grandis‘Merrillii’)的未成熟合子胚置于SH+0.1 mg·L-1 NAA+500 mg·L-1AC+3%蔗糖+0.5 g·L-1 Gln培养基上暗培养45 d,诱导产生半透明颗粒状胚性愈伤组织;将胚性愈伤组织转入SH+20 g·L-1 PEG+10 mg·L-1 ABA培养基中暗培养3个月诱导体细胞胚。采用碘—碘化钾染色和石蜡切片技术对体胚起源、形态发育与细胞组织学进行了观察。结果表明:胚性愈伤组织起源于合子胚胚轴表皮或皮层细胞的对称分裂。胚性愈伤组织含有两类细胞,一种是细胞质浓厚、细胞核大、体积小的圆形胚性细胞,另一种是高度液泡化拉长的细胞。胚性愈伤组织包含由这两种细胞构成的原胚团Ⅰ、原胚团Ⅱ和原胚团Ⅲ,以及一些游离细胞。原胚团Ⅲ在无植物生长调节剂的SH基本培养基上形成原胚,原胚接入成熟培养基,历经球形、棒状、心形、鱼雷形胚后发育成子叶胚。将子叶胚转入萌发培养基后胚根伸长,胚芽发育长出针叶,形成完整的再生植株。同时在离体培养中合子胚胚柄处退化的裂生多胚也能重新发育形成胚体。在初生体胚发育过程中表面常伴有次生体细胞胚的形成。     

芹菜胚性细胞悬浮系原生质体分离及再生植株

利用芹菜胚性细胞悬浮系成功分离得到大量原生质体, 获得芹菜大量原生质体的最佳反应体系为: 酶液组成为3.0%纤维素酶Onozuka R210、1.0%离析酶R210、11%甘露醇、0.5% CaCl₂ ·2H₂O和0.1% MES; 摇床转速为80 r/min, 温度(25 ±2) ℃, 酶解时间5～6 h; 原生质体产量为25.00 ×10⁶ /g, 原生质体活力83.41%。原生质体浅层培养, 培养基为1 /2 MS + 1 mg/L 2, 4-D + 0.5 mg/L KT + 11%甘露醇+ 500 mg/L水解络蛋白, 两天后, 重新再生细胞壁之后进行第1次分裂, 逐步降低渗透压至甘露醇3% ,大约30 d形成小细胞团。小愈伤组织经增殖培养后在1 /2MS + 500 mg/L CH + 0.25 mg/L KT固体分化培养基诱导出不定芽, 30 d后再转入MS基本培养基, 获得完整的再生植株。     

香蕉胚性细胞悬浮系玻璃化法超低温保存及再生植株遗传稳定性分析

采用玻璃化法对3个香蕉栽培品种（Musa spp. AAA）的胚性细胞悬浮系（ECS）进行超低温保存。对超低温保存程序进行优化,冻存后进行ECS重建并通过体细胞胚发生途径进行植株再生,最后对再生植株进行遗传稳定性分析。用建立的ECS玻璃化法超低温保存程序对 ‘巴西蕉’、‘北大矮蕉’和‘粤优抗1号’的ECS进行超低温保存,获得的细胞相对存活率分别为89.6%、90.9%和89.9%,并重建了‘北大矮蕉’的ECS;在冻存后体细胞胚发生途径植株再生过程中,3个品种的相对植株再生率分别为64.4%、0.9% 和14.0%。从冻存后‘巴西蕉’ECS获得的细胞胚发生途径再生植株的遗传稳定性分析结果表明：再生植株与对照在形态学方面无明显区别;简单序列重复区间（ISSR）分子标记也显示与对照相比基本无差异带出现,这初步表明超低温保存后的香蕉ECS保持了遗传稳定性。     

牡丹成熟胚离体培养的初步研究

研究了不同浓度GA3浸泡处理对解除‘凤丹'种子上胚轴休眠的影响.结果表明:浓度为500 mg/L的GA3浸泡种子48 h,胚的萌发率52.6%;经过对4种培养基的筛选,认为1/2MS+Ca2+可以作为胚培养的基本培养基;胚培养最佳培养基为1/2MS+GA30.5 mg/L.     

Regeneration of different Cyclamen species via somatic embryogenesis from callus,suspension cultures and protoplasts

The present study is the first report of the establishment of embryogenic callus cultures from seedling tissue, the regeneration of plants via somatic embryogenesis and the development of a regeneration system from protoplast to plant, using three wild species of Cyclamen, Cyclamen graecum Link, Cyclamen mirabile Hildebrand, Cyclamen trochopteranthum Schwarz (syn. Cyclamen alpinum hort. Dammann ex Sprenger). The ability to form embryogenic callus and to regenerate via somatic embryogenesis was strongly genotype-dependent for each species. From 0.5 g callus, up to 1461 somatic embryos were formed in the case of C. mirabile. Culture media with different concentrations of plant growth regulators, CaCl₂ and activated charcoal significantly influenced embryo formation in this species. Up to 1.4 × 10⁶ protoplasts were isolated from 1 g of C. graecum cell suspension. Diverse growth responses of the protoplasts in two embedding agents, agarose and alginate, were observed for the different Cyclamen species. These specific growth characteristics could be used as a selection marker for future fusion experiments. From both protoplast culture systems, somatic embryos were regenerated, grown to plantlets and acclimatised to greenhouse conditions.     

Plant regeneration from protoplasts of calamondin (Citrus madurensis Lour.)

Diploid callus cells arose from the hypocotyl region of plantlets differentiated by anther culture of Calamondin (Citrus madurensis Lour.). The callus grew vigorously on Murashige and Tucker's medium containing 10 mg l⁻¹ benzyladenine, and differentiated embryoids on a medium with 5% lactose and no plant growth regulators. This embryogenic potential was transferred to protoplasts which regenerated plantlets.

Preculture of callus in liquid medium containing 5% lactose, for 4–7 days prior to protoplast isolation, effected a high yield of protoplasts. Plating efficiency was increased in a medium solidified with agarose and/or layered with liquid medium.     

香雪兰种子胚的组织培养和植株再生

香雪兰的成熟胚和未成熟胚切段，在含有ＩＡＡ２ｍｇ／Ｌ、ＮＡＡ０３ｍｇ／Ｌ和ＢＡＰ０５ｍｇ／Ｌ的改良Ｎ６培养基上可形成半透明的瘤状愈伤组织。将这种愈伤组织培养于含有ＮＡＡ０３ｍｇ／Ｌ、ＢＡＰ０５ｍｇ／Ｌ和ＫＴ０５ｍｇ／Ｌ的ＭＮ６培养基可分化出丛生芽。生根培养基为含ＮＡＡ０５ｍｇ／Ｌ的改良Ｎ６培养基。相同的外植体可在ＭＮ６＋ＩＡＡ２０ｍｇ／Ｌ＋ＢＡＰ３０ｍｇ／Ｌ培养基上通过体细胞胚胎发生途径直接形成小植株。讨论了外源激素对香雪兰种子胚离体培养的作用。     

草莓悬浮细胞原生质体培养再生愈伤组织

以草毒品种宝交早生的花药愈伤组织建立的悬浮细胞系为试材,对原生质体分离和再生进行研究。结果表明:酶液的渗透压、浓度和配比对悬浮细胞原生质体产量和活力有重要的影响。悬浮细胞在CPW+1.0％ Cellulase R-10+0.5 Macerozyme R-10+0.05％Pectolyase Y-23+0.6mol/L甘露醇+0.5％PVP的酶液中酶解12h,原生质体产量和活力最高,分别达16.35×10~6/g和84.6％。采用液体浅层培养法对原生质体进行培养获得了再生愈伤组织。     

Somatic embryogenesis from floral tissues of feijoa (Feijoa sellowiana Berg)

The objectives of the present work were to study the embryogenic competence of floral tissues of Feijoa sellowiana and to investigate the influence of plant growth regulators on somatic embryo induction and development in order to establish a somatic embryogenesis protocol starting from somatic tissues. Petals, stamens and ovaries of floral buds were cultivated onto LPm basal medium supplemented with different levels of 2,4-D, Picloram, 2-iP, Kin and BAP. The highest embryogenic callus induction was obtained with Picloram (10 μM) and Kin (1 μM). Rates of embryogenic calluses induction in stamens and petals were significantly affected by PGRs. Embryogenic calluses were transferred to the same medium, supplemented with gradually reduced levels of PGRs-free medium. After 60 days in suspension cultures with 2,4-D (1 μM) and 2-iP (1 μM) calluses were transferred to PGR-free medium. After 30 days it was observed the development of globular somatic embryos on the surface of 18% of friable calluses previously induced with Picloram (10 μM) and Kin (1 μM). Only embryogenic calluses derived from stamens gave rise to this morphogenetic pattern.Torpedo and cotyledonary somatic embryos transferred to PGR-free culture medium were converted to complete plantlets. This is the first report of somatic embryogenesis in this species starting from somatic tissues.     

‘翠秀’黄瓜子叶原生质体的高效培养及植株再生

 分离纯化的黄瓜子叶原生质体，以5.5×10～5.5×10⁵个/mL的不同密度培养于mKMSp液体培养基中，均获得不同程度的细胞分裂。当培养密度超过1.0×10³个/mL时，原生质体可持续分裂至愈伤组织形成，最高植板率为54%。原生质体来源的愈伤组织经诱导培养产生大量体胚并再生成植株。Ca²⁺ 浓度对黄瓜原生质体的稳定和细胞分裂有重要影响。     

梅花不同外植体离体培养及体细胞胚诱导植株再生

以梅花品种‘雪梅’和‘绿萼’的叶片、叶柄、茎段及未成熟子叶为外植体进行离体培养,探讨了体胚发生途径与植株高效再生方法。结果表明,成熟组织再生能力低,不定芽再生困难。在添加1.0mg·mL-1BA,1.0mg·mL-1NAA,1.0mg·mL-1IBA的1/2MS培养基中,‘雪梅’和‘绿萼’未成熟子叶的体胚诱导率最高,分别为40.4%和25.3%;同时将初生胚转移至新鲜培养基上可产生大量次生胚。在添加0.5mg·mL-1BA,0.1mg·mL-1TDZ和1.0mg·mL-1IBA的1/2MS基本培养基中,体细胞胚发育成完整植株的频率较高,‘绿萼’达26.7%,‘雪梅’达23.8%。再生植株成功移栽至温室且生长良好。     

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。     

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.