Effect of plant growth regulators on direct somatic embryogenesis in camphor tree(Cinnamomum camphora L.)from immature zygotic embryos and embryogenic calli induction

A description of a successful direct somatic embryogenesis induction from immature zygotic embryos of a camphor tree （Cinnamomum camphora L.） is presented. After a subculture of 2-3 years, embryogenic calli could be derived from primary somatic embryos. Immature zygotic embryos were cultured on a Murashige and Skoog （MS） basal medium supplemented with a range of combinations of cytokinins （BA） and auxins （2,4-D or NAA） for somatic embryo induction. Primary somatic embryos could be induced directly in almost all PGR combinations. A positive effect of 2,4-D on somatic embryogenesis from immature zygotic embryos of camphor tree was obtained. BA at appropriate concentrations （〈 5 mg-L-1） had an effect similar to 2,4-D, whereas high concentrations （〉 5 mg·L^-1） of BA had the effect of restraining somatic embryo induction. NAA had a less positive effect on somatic embryogenesis than 2,4-D.

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L.‘Moldova’

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.

Effect of the thin cell layer technique in the induction of somatic embryos in Pinus patula Schl. et Cham

Pinus patula is a species commonly used for reforestation in Mexico.However,efficient methods for the mass production seedlings are required.Micropropagation particularly by somatic embryogenesis provides an option for the rapid multiplication of high-quality,genetically improved material.This study induces somatic embryogenesis in this species using the thin cell layer(TCL)technique.Two sources of explants(complete immature embryos;lTCL segments from immature embryos)were evaluated.The efficiency of TCL from longitudinal sections[lTCL]and transverse[tTCL]was evaluated.The results show using thin cell layers from immature embryos cultivated in 16 light/8 dark hours achieves induction of somatic embryos.A higher percentage of embryogenic callus was obtained when tTCL segments were used as an explant source.These results produced somatic embryos from tTCL segments of an immature embryo without germinating the seed,making the process more time efficient.In addition,this technique can be used to generate somatic embryogenesis in forest species that have low germination rates.

Morphology and ontogeny of directly differentiating shoot buds and somatic embryos in Santalum album L.

Santalum album L.is a commercially important tree that yields essential oil of high medicinal value.Regeneration research through organogenesis and embryogenesis has been documented but no report depicts comparative ontogeny of directly differentiating shoot buds(SB)and somatic embryos(SE).In the present study aseptic seedling derived hypocotyl segments(HC)and hypocotyl+root junction(HC+R)were used to induce SBs and SEs,respectively.Ontogenic differences between the structures were confirmed using scanning electron microscopy and histological analysis.MS medium containing 6-benzyladenine or BA(2.5μM)produced highest number of direct SB,while MS+BA(7.5μM)proved suitable for higher frequency of SE differentiation.The differentiating structures attained growth when transferred to MS medium containing a combination of BA and anaphthalene acetic acid or silver nitrate(AgNO3).A combination of indole-3-butyric acid and silver nitrate(AgNO3)in half-strength woody plant medium and lesser osmotic concentration(2%sucrose),induced rhizogenesis.

Optimization of Germinated Conditions for Somatic Embryos in Liriodendron Hybrids

The development of somatic embryos is a key technology in plant breeding. To gain the best cultural conditions,the factors influencing on germination and transplanting survival rate of the somatic embryos in Liriodendron hybrids were optimized,including the development period of somatic embryos,macro-element,Vitamin C,genotypes,plant hormones and transplant medium in this study. The results showed that the optimal development phase of germination was torpedo-shaped embryo,and the base cultural medium of 3/4 MS with vitamin C can keep normal form from somatic embryos. The germination rate of somatic embryo had obvious genotype difference,among which the hybrid 1 × 5002 was the most sensitive( 82. 46%),and the hybrid 1 × 4088 was the most insensitive( 13. 24%). The medium with 0. 1 mg/L of KT + 0. 1 mg/L of IBA can help promote the germination of somatic embryogenesis. In addition,the yellow soil was more suitable than mixed peat soil for transplanting Liriodendron hybrids seedlings. Therefore,the obtainment of the optimal conditions has important guidance for production practices of Liriodendron hybrids.

Cocoa (<i>Theobroma cacao</i>L.) Somatic Embryos Tolerate Some Ice Crystallization during Cryopreservation

Secondary somatic embryos (SSEs) of cocoa, a recalcitrant tropical, seed-producing species, were cryopreserved using a vitrification approach and Differential Scanning Calorimetry (DSC) was employed to optimise sucrose preculture and Plant Vitrification Solution 2 (PVS2) incubation. The objective of the study was to evaluate the influence of sucrose preculture and PVS2 dehydration on water content of SSE that will enable it to survive cryostorage. SSEs were precultured for 3 or 5 days on media containing 0.5 M or 0.75 M sucrose and cryoprotected in loading solution (2 M glycerol and 0.4 M sucrose in medium) for 20 min before they were dehydrated with cold PVS2 for 0 - 90 min. Thermal analysis revealed the occurrence of ice crystallization in the SSEs with the extent declining with increasing PVS2 exposure. Maximal survival of SSEs was promoted by preculture on 0.5 M sucrose medium and dehydration with PVS2 for 45 - 60 min, which was characterised by small ice crystallization. Exposure of SSEs beyond 60 min leads to excessive dehydration as characterized by no change in the thermograms. Based on these findings, preculture of SSEs on 0.5 M sucrose medium and dehydration with cold PVS2 for 60 min has been adopted for the successful cryopreservation of cocoa germplasm.

橡胶树体胚苗质量调查与分析

天然橡胶是我国重要的战略物资和工业原料,主要来源于橡胶树。橡胶树生长周期长,其苗木质量直接影响天然橡胶产业的发展,橡胶树体胚苗在前期研究中表现一致性好,且具有速生、高产的优势。海南省天然橡胶新型种植材料创新基地是当前规模生产的橡胶树体胚苗的基地,现生产品种有热研73397和热研917。本研究分析比较了热研73397体胚试管苗在2018—2020年间的株高、茎粗、叶片数、主根长等指标的差异性,比较了2020年2个品种各指标的差异性,并对不同年份不同品种进行分级比较,以期阐明橡胶树体胚苗规模生产的情况。结果表明:(1)2018—2020年,热研73397的株高、茎粗、叶片数、主根长度在各区间的比例相当;株高、叶片数、主根长度在3 a间的差异不显著;茎粗在2018和2019年之间差异显著,且3 a均值均在2.00 mm以上,最大值和最小值的差值仅为0.09 mm。(2)2020年,热研73397和热研917体胚苗的株高一致,均值均为8.0 cm,且大部分集中在5.0~10.0 cm,分别占66.13%、66.91%;热研73397和热研917的茎粗范围分别为0.80~4.23 mm、1.13~3.73 mm,均值分别为2.11 mm和2.18 mm,2个品种的茎粗在1.50~3.00 mm之间高度集中,分别占85.94%、83.82%;热研73397和热研917叶片数范围分别为0~6片和1~4片,2个品种叶片数绝大多数为1~3片,分别占88.82%、96.32%,其中热研917叶片数为2片的占比高达52.94%;热研73397的主根长度均值为9.5 cm,而热研917的为9.1 cm,2个品种的主根长度多数集中在8.0~12.0 cm之间,分别占74.12%、79.41%;方差分析结果表明,2020年2个品种的株高、茎粗、叶片数差异不显著,主根长度差异显著(P<0.05)。(3)2018—2020年调查的橡胶树体胚苗中,不同年份不同品种的橡胶树体胚苗,一级苗比例为19%~22%,二级苗比例为56%~62%。据此,规模化生产橡胶树体胚试管苗质量稳定,为橡胶树体胚苗生产工艺的推广应用提供参考。

日本落叶松胚状体干化处理对萌发的影响

[目的]提高日本落叶松胚状体的萌发能力,优化日本落叶松良种繁育技术。[方法]本研究根据子叶数量和胚轴形态,对胚状体进行分类,并分别统计其萌发率;利用“滤纸容器法”对类型Ⅰ胚状体进行干化处理,统计其萌发情况。[结果]胚状体分为10种类型。类型Ⅰ胚状体萌发率为4.48%,其余类型胚状体难以萌发。干化处理14 d后,类型Ⅰ胚状体萌发率提高到69.43%,且具有干化响应的胚状体更易萌发。[结论]本研究表明日本落叶松胚状体干化处理后萌发率明显提高,为该物种良种繁育提供依据。

转录因子CmHAT1调控板栗体细胞胚胎发育的功能研究

【目的】体细胞胚胎发生是板栗有效的离体再生方法。然而,目前板栗体细胞胚胎由于发育慢、再生效率低等问题,制约了板栗体细胞胚胎的再生进程,难以在后续基因功能验证和新种质创制过程中发挥作用。本文旨在通过研究HDZIPⅡ基因家族成员CmHAT1在板栗体细胞胚胎发育过程中的作用,进一步加速板栗体细胞胚胎的发育进程。【方法】利用生物信息学的方法鉴定了板栗HD-ZIPⅡ基因家族成员,并分析了该家族成员在板栗体细胞胚胎不同发育时期的表达情况,筛选调控体细胞胚胎发育的关键候选基因CmHAT。进一步通过烟草亚细胞定位实验、荧光定量和遗传转化方法,研究关键基因CmHAT的亚细胞定位和基因功能。【结果】(1)板栗HD-ZIPⅡ基因家族有7个成员,大部分CmHD-ZIPⅡ成员在板栗体细胞胚胎发育时期均有表达,其中CmHAT1在愈伤组织时期表达较低,在球形胚时期表达上调,心形胚时期表达最高,而在子叶形胚时期表达下调,因此筛选出CmHAT1作为调控体细胞胚胎发育的关键候选基因。(2)CmHAT1定位在细胞核;与转化空载体相比,过表达CmHAT1使体细胞胚胎数量显著减少,体细胞胚胎体积显著增大,而沉默CmHAT1后体细胞胚胎的表型没有显著性差异。【结论】在同等质量的愈伤诱导体细胞胚胎的情况下,过表达CmHAT1后可以调控板栗体细胞胚胎的数量和体积,影响体细胞胚胎的发育。本研究为进一步优化板栗再生体系和遗传转化体系奠定了基础,为利用体细胞胚胎系统创制板栗新种质提供了科学依据。

‘西林3号’核桃体细胞胚胎诱导与增殖的影响因素

【目的】筛选最佳的‘西林3号’核桃体细胞胚胎诱导和增殖培养条件。【方法】通过进行不同消毒方法、不同外植体、不同采样时期、不同激素浓度配比、不同蔗糖与谷氨酰胺浓度处理,对核桃胚性愈伤组织的诱导情况进行研究,筛选最佳诱导条件;同时对比不同激素浓度对核桃体细胞胚胎增殖率的影响,筛选最佳增殖培养基。【结果】雄花(花药)、种子、幼胚的最佳消毒方法分别为70%乙醇15 s+10%NaClO浸泡4 min、70%乙醇30 s、70%乙醇5 min,培养4 d后的存活率高,污染率低。幼胚是诱导胚性愈伤组织的最佳材料,5月31日是最佳的采样时间,诱导率分别为(65.00±7.07)%和(85.83±1.17)%。以愈伤组织为材料时,诱导胚性愈伤组织的最佳激素浓度配比为改良DKW培养基添加1.0 mg/L 6-BA、2.0 mg/L KT、0.01 mg/L IBA,诱导率为(37.94±1.33)%。以愈伤组织为材料时,诱导胚性愈伤组织的最佳蔗糖质量浓度为30 g/L,诱导率为(82.67±0.94)%,谷氨酰胺不是诱导胚性愈伤组织的影响因素。体胚增殖的最佳激素浓度配比为改良DKW培养基添加0.5 mg/L 6-BA、1.0 mg/L KT、0.005 mg/L IBA,增殖率为98%。【结论】外植体和采样时间的选择直接影响体胚诱导的结果,对于幼胚这类果实内部材料来说,仅进行简便快捷消毒即可。激素和碳源是胚性愈伤组织诱导与发生的关键因素,谷氨酰胺不是胚性愈伤组织诱导的影响因素。在进行体胚继代增殖培养时,适当添加激素可以抑制体胚成熟,提高增殖率。

珙桐体细胞胚胎发生技术研究

研究探讨基本培养基类型对珙桐愈伤组织诱导的影响、植物生长调节剂对胚性愈伤组织诱导及体胚发育的影响,初步建立以未成熟合子胚为外植体,通过体细胞胚胎发生途径再生珙桐植株的培养方案。结果表明,在含有3%蔗糖、0.3%凝胶、800 mg/L水解酪蛋白、400 mg/L L-谷氨酰胺的MS、1/2MS和WPM基本培养基上均可诱导出愈伤组织,以MS+2.0 mg/L 2,4-D+0.5 mg/L 6-BA诱导率最高,可达94.0%;胚性愈伤组织在MS+0.5 mg/L NAA+1.0 mg/L 6-BA的培养基上诱导效果较好;在MS+0.5 mg/L NAA+1.0 mg/L 6-BA培养基上,体胚诱导率可达22.9%;成熟体胚在1/2MS+0.5 mg/L 6-BA+0.25 mg/L IBA的萌发培养基上能正常萌发,其后在添加1 g/L活性炭的萌发培养基上转化成完整小植株。组织细胞学观察表明,珙桐体胚起源于胚性愈伤组织,经历了球形期、心形期、鱼雷期和子叶期发育阶段,这与自然条件下的合子胚形成过程相似。

石蒜体胚发生过程的细胞学和生理特性

【目的】探究石蒜‘梦幻少女’Lycoris chinensis×radiata‘Astro Girl’体胚发生过程中的细胞学和生理生化指标的变化规律,明确‘梦幻少女’体胚发生的特征,提高石蒜属Lycoris植物种球繁殖效率,为石蒜属新品种工厂化生产提供技术支撑。【方法】采用石蜡切片、酶联免疫吸附测定技术和超高效液相色谱-串联质谱(UPLC-MS/MS)技术,测定了0~40 d‘梦幻少女’体胚发生过程中的形态和组织学特征、体胚可溶性淀粉及总蛋白质、抗氧化酶活性和多胺等生理生化指标的变化。【结果】①体胚发生过程中,从球形胚到短暂膨大约20 d,进一步分化为心形胚、棒状胚和子叶胚,膨大的球形胚后期或心形胚时期基部会形成维管束。②体胚发生过程中,总蛋白质和淀粉、抗氧化酶活性发生显著变化,淀粉变化与球形胚的形成呈正相关,与体胚形态建成和体胚成熟呈负相关。总蛋白质、过氧化物酶(POD)活性及过氧化氢酶(CAT)活性变化与子叶胚形成呈正相关,与子叶胚的成熟发育呈负相关。超氧化物歧化酶(SOD)活性变化与球形胚、子叶胚时期呈负相关,与心形胚、棒状胚时期呈正相关,表明储能作用的淀粉、蛋白质及与植物生理密切相关的抗氧化酶对体胚发生起着重要的作用。③体胚发生过程中多胺氧化酶(PAO)活性和多胺(PAs)变化显著,腐胺(Put)、精胺(Spm)和亚精胺(Spd)的变化共同决定多胺的变化趋势,多胺和多胺氧化酶活性主要在体胚分化早期发生明显积累,与球形胚分化呈正相关;在子叶胚的形成期,精胺与亚精胺呈正相关;在子叶胚成熟期,腐胺、精胺和亚精胺3个指标呈现腐胺先下降后上升,精胺、亚精胺先上升后下降的趋势。【结论】在‘梦幻少女’体胚发生的0~40 d,体胚形态经历了原胚—球形胚—心形胚—棒状胚—子叶胚的过程。其中,生理特性变化显著,淀粉的积累或消耗可能是球形胚形成、体胚后续形态发育与成熟的重要影响因素。在子叶胚形成及成熟时期,总蛋白质、过氧化酶活性和多胺的变化可能发挥着相应密切的联合作用。多氨氧化酶活性与多胺的活跃积累可能有利于体胚早期发育。

小分子组合诱导大鼠胚胎成纤维细胞重编程为功能性神经元

目的:建立通过小分子化合物将大鼠胚胎成纤维细胞(REF)重编程为化学诱导神经元(ciNC)的方法体系,为细胞移植治疗神经退行性疾病提供安全有效的供体细胞。方法:以裴钢研究团队建立的人成纤维细胞直接重编程为神经元的方法为基础,通过更换包被液、缓解氧化应激损伤、添加神经源性保护因子、调整毛喉素浓度和小分子去除实验对诱导培养基和成熟培养基进行优化,并通过免疫荧光法和蛋白质印迹法验证不同诱导阶段细胞的相关蛋白质。结果:以原方案进行诱导时,细胞存活率仅(34.24±2.77)%。包被液更换为基质胶后,细胞存活率上升到(45.41±4.27)%;添加褪黑素后细胞存活率提高至(67.95±5.61)%,(23.43±1.42)%的细胞转变为神经样细胞;添加小分子P7C3-A20后细胞存活率进一步提升至(76.27±1.41)%,(39.72±4.75)%的细胞转变为神经样细胞;毛喉素浓度提升至30μmol/L时,神经样细胞比例达到(55.79±1.90)%;去除SP600125后,(86.96±2.15)%细胞存活,神经样细胞生成率提高至(63.43±1.60)%。以优化后的方案诱导,可经过神经前体细胞将REF诱导为ciNC。其中,神经前体细胞能够高表达神经前体细胞标志物SRY-box转录因子2、配对盒6以及神经元特异性标志物Ⅲ类β-微管蛋白,而成纤维相关蛋白波形蛋白表达量减少。神经前体细胞在成熟培养基中诱导两周后,可见大部分细胞呈神经样细胞形态,诱导后的ciNC高表达Ⅲ类β-微管蛋白和微管相关蛋白2,而成纤维相关蛋白波形蛋白表达减少。结论:优化后的小分子组合在常氧条件下能将REF重编程为ciNC。

Screening and verification of the factors influencing somatic embryo maturation of Larix olgensis

With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo.Three dominant factors emerged, and we developed a response surface model based on the Box-Behnken design.We defined the optimal conditions for the maturation of somatic embryos. The contents of abscisic acid, silver nitrate, sucrose and casein hydrolysis significantly affected the amount of maturing embryos, but inositol, maltose and glutamine had no effect. By establishing a response surface model with multiple factors, we predicted that the optimal number of L. olgensis somatic embryos was 204 ± 4 gon basal medium, containing 18.28 mg Labscisic acid,5.46 mg Lsilver nitrate and 82.67 g Lsucrose. In the verification experiments, the addition of 20 mg Labscisic acid, 5 mg Lsilver nitrate and 80 g Lsucrose to BM yielded an average of 202.06 somatic embryos per gram. These results should guide large-scale breeding of L. olgensis.

Role of hydrogen peroxide in stress-induced programmed cell death during somatic embryogenesis in Fraxinus mandshurica

We examined how reactive oxygen species, in the form of hydrogen peroxide (H2O2), affect osmotic stress–induced programmed cell death during somatic embryogenesis from cotyledon explants of Manchurian ash (Fraxinus mandshurica Rupr.). We found that substantial osmotic stress was essential for Manchurian ash somatic cells to obtain embryogenic competence. The explant cells displayed hallmarks of programmed cell death, chromatin condensation, and DNA fragmentation to oligonucleotides during somatic embryogenesis. Increasing concentrations of plant growth regulators and sucrose in the medium increased osmotic stress thereby inducing H2O2 accumulation in the explant cells. We found that H2O2 concentration was significantly decreased in explant cells when the induction medium was modified, i.e., when reducing the concentration of sucrose, which reduces the osmotic pressure of the medium, or by withdrawing plant growth regulators at mid-culture. These treatments also decreased the proportion of explant cells undergoing programmed cell death. Accordingly, a decreased rate of somatic embryo induction was observed. These results show that PCD occurred during tissue browning and death of some explant cells during somatic embryogenesis in F. mandshurica. The ROS contributed to PCD in abiotic stress stimulated F. mandshurica cells.

Somatic Embryogenesis in Lily Bulb Scale Cultures

Abstract： Somatic embryogenesis from lily bulb scales has not been studied in details, although tissue culture methods have been applied to the propagation for decades. The effects of different kinds and concentration of auxins for oriental lily somatic embryogenesis were investigated （Lilium hybrida var. Sorbonne）. 2, 4-dichlorophenoxyacetic acid （2, 4-D）, thidiazuron （TDZ） and α-naphthaleneacetic acid （NAA） media with benzyladenine（6-BA） and lactalbumin hydrolysate （LH） were used for embryogenic callus in the darkness. The best response on embryogenic callus formation was obtained on MS media supplemented 2, 4-D 2.0 mg·L^-1, 6-BA 0.5 mg·L^-1 and LH 300 mg·L^-1. Transfer embryogenic callus to the media with TDZ, 6-BA, kinetin （KT） supplemented 2, 4-D. The highest number of somatic embryos has been produced on medium with 0.5 mg·L^-1 2, 4-D and 0.3 mg·L^-1 KT. Germinated embryos with shoot axes were changed to MS media with 6-BA 0.5 mg·L^-1. The results suggest that in vitro culture of somatic embryogenesis from lily bulb scales can be used for plant regeneration.

Effect of Rare Earth Elements on the Induction Frequency of the Somatic Cell Embryo in The Fruit of Chinese Wolfoerry

A part of lanthanides could raise the induction fraquency(IF) of the somatic cell embryo(SCE) in the fruit of Chinese wolfbeny. The effect of thorium and yttrium used for this purpose is not obvious and even plays a role of inhibition when they are used with a concentration of more than 4 ppm.When the combinations of different rare earth elements (RE) are used, the diversity of the effects amongthem is large. Some of them help to raise the iF of the SCE while the others inhibit the generation of SCE.The mischmetal results in the best effect, giving a relative IF of 295.4%.

Breeding and Simple-rapid Regeneration Protocol for Jisheng 1 with Glandless Trait and High-frequency Somatic Embryo Production Ability

Most of model cotton varieties used in tissue culture have glands on both the reproductive and vegetative parts of the plant.These glands contain compounds that are toxic to human and non-ruminant animals.The presence of these compounds limits their usage as food and feed.To obtain a glandless cotton variety with high-frequency somatic embryo production ability,27 glandless varieties

Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model

This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

Morphology of somatic embryogenesis and plantlet formation in tissue cultures of lantern tree (Koelreuteria bipinnata var.integrifoliola)

Somatic embryogenesis ofKoelreuteria bipinnata var. integrifoliola was observed, plantlet formation in different types of somatic embryos was studied and the effect of abnormal embryos on plantlet formation was identified. Results show that somatic embryos of K. bipinnata var. integrifoliola include normal embryos, embryos with abnormal cotyledons, vitrified embryos, albino embryos, secondary embryos, linked embryos, embryos with abnormal growing points and embryos with expanding hypocotyl. After 40 d of callus culture, the response of normal somatic embryos from K. bipinnata var. integrifoliola was 26.7%, embryos with abnormal cotyledon 30.3% while other types of somatic embryos were below the 20% level. Most of normal embryos developed into plantlets and plantlet formation reached 94.9%. But the percentage of plantlet formation decreased apparently in abnormal embryos： the number of embryos with abnormal cotyledon declined to 76.1%, that of linked embryos to 47.4% and other types of abnormal embryos to below the 20% level. Albino embryos and embryos with abnormal growing point did not develop at all into plantlets. Embryos with abnormal cotyledons, linked embryos and embryos with abnormal growing points were observed during early stages of somatic embryogenesis, but vitrified, secondary and albino embryos and calli of embryos were observed at later stages. Increasing sucrose concentrations can decrease the occurrence of vitrified embryos, but the number of albino embryos decreased with an in- creasing in sucrose concentration.