黄瓜离体器官再生体系的优化

优化通过器官直接再生方式的黄瓜离体再生体系,为遗传转化奠定基础。以‘长春密刺’黄瓜的子叶节为外植体,探讨在黄瓜再生过程中,适宜的无菌苗获得培养基、适宜的外植体类型、无菌苗的适宜苗态、不定芽诱导培养基和芽伸长培养基中适宜的激素组合与比例。结果表明,最适的无菌苗获得培养基为1/2MS+30 g/L蔗糖+7 g/L琼脂(pH=5.8);不同外植体的再生率为子叶节>下胚轴>子叶;子叶完全出壳但未展平的无菌苗比其他苗态的再生率高,子叶展平后,再生率迅速下降;当6-BA（6-苄基氨基嘌呤）和ABA（脱落酸）浓度一定时,最适的AgNO₃为2 mg/L;当AgNO₃浓度一定时,6-BA和ABA浓度的最佳组合为1.5 mg/L 6-BA+0.5 mg/L ABA;不定芽诱导的最适培养基为MS+1.5 mg/L 6-BA+0.5 mg/L ABA+2 mg/L AgNO₃,出芽率为90%,每外植体出芽数为3.5;芽伸长培养基中加入0.10 mg/L 6-BA,能够促进再生芽的伸长。本研究成功优化了黄瓜的再生体系,得到了健壮的黄瓜成株。     

大豆子叶节植株再生体系的优化及转EPSPS基因的研究

为提高农杆菌介导转化大豆子叶节再生体系的遗传转化效率,优化了激素水平、基因型、抗生素及筛选压力等影响植株再生的多个因素,并用EPSPS基因转化大豆子叶节。结果表明,不定芽诱导培养基中6-苄氨基嘌呤(6-BA)浓度为1.6mg/L时,不定芽诱导率最高,黑农37和合丰35的不定芽诱导率较吉育91高,吲哚丁酸(IBA)诱导生根的适宜浓度为0.5~1.0mg/L。头孢霉素的最适抑菌浓度为500mg/L,草甘膦有效筛选压力为8mg/L,并获得转EPSPS基因的抗性植株。     

白果强丰番茄高效再生体系的建立

以番茄白果强丰的子叶和下胚轴为外植体,通过不同激素处理,诱导愈伤组织、不定芽及不定根的形成,以期建立白果强丰高效再生体系,为其遗传转化奠定良好的基础。结果表明,各处理对愈伤组织的诱导没有显著差别,出愈率均达到了93%以上。出芽率方面,以外植体为子叶的,以处理Ⅱ(MS+6-BA1.0mg/L+IAA0.2mg/L)的出芽率最高,为93.33%;外植体为下胚轴的,以处理Ⅳ(MS+6-BA2.0mg/L+IAA0.2mg/L)出芽率最高,为80.00%。两者相比,子叶作为外植体的出芽率高。诱导不定芽生根时,两种处理生根率均为100%,但处理Ⅵ(MS+IAA0.5mg/L)不定根数多,根系较发达,为较优不定根诱导培养基。     

小豆子叶节遗传转化体系建立

以小豆品种‘京农6号’子叶节为外植体进行离体再生,旨在建立‘京农6号’子叶节再生遗传转化体系。本研究对农杆菌菌株EHA105侵染子叶节的时间、诱导愈伤生成的适宜6-BA浓度、诱导不定芽的激素浓度及配比及诱导不定根生长的IBA浓度等影响子叶节再生遗传转化体系的因素,结果发现农杆菌EHA105侵染的最佳时间为50 min,GUS阳性率为67.5%;诱导愈伤的适宜6-BA浓度为2.0 mg/L,诱导率为91.4%;诱导不定芽的最适激素是5.0 mg/L ZT,出芽率为62.0%;而0.1 mg/L IBA诱导不定根的效果最好,生根率可达100.0%。本研究建立了小豆‘京农6号’子叶节外植体的遗传转化体系,获得了再生植株,为小豆的CRISPR/Cas9基因编辑技术在小豆功能基因组学和育种中的应用提供了技术参考。     

马蹄金(Dichondra repens Forst.)组织培养和快速繁殖

为了探究马蹄金组织培养及快繁技术,本研究以马蹄金为材料,使用75%的酒精和10%NaClO对不同外植体(胚根,下胚轴,子叶)进行消毒处理,在不同激素浓度配比的MS培养基中进行丛生芽诱导、愈伤组织诱导及分化,成功建立了马蹄金的组培快繁体系。结果表明:利用马蹄金下胚轴为外植体直接诱导不定芽效果较好,其最适程序为:流水冲洗30 min+75%酒精30 s+10%NaClO 9 min,污染率为5%,成活率为75%;下胚轴不定芽诱导的最适培养基为MS+1 mg/L 6-BA+0.3 mg/L NAA,诱导率为93.55%,出芽指数为8.86;胚根愈伤组织诱导的最适培养基为MS+1 mg/L 6-BA+0.5 mg/L NAA,诱导率为100%,下胚轴愈伤组织诱导的最适培养基为MS+0.5 mg/L 6-BA+1 mg/L NAA,诱导率为95.24%,子叶愈伤组织诱导的最适培养基为MS+0.3 mg/L 6-BA+0.5 mg/L NAA,诱导率为100%;愈伤组织不定芽分化的最适培养基为1/2MS+1.5 mg/L 6-BA+0.5 mg/L NAA,芽诱导率为20%,平均芽数为12.83;组培苗诱导生根的最适培养基为不加激素的1/2MS培养基;生根苗以瓶盖全开的炼苗方式移栽到以河沙为基质的育苗盒中成活率为100%,且添加蒸馏水与自来水对植株生长并无明显影响。本研究成功建立了马蹄金组织培养与快繁体系,为马蹄金种质资源改良及优质种苗生产提供了技术参考。     

杜梨子叶离体再生体系的建立

为梨树品种改良、遗传转化及功能验证奠定基础,以杜梨(Pyrus betulaefolia)子叶外植体为试验材料,研究了基本培养基、植物生长调节剂、暗培养时间、碳源、外植体等因素对其再生能力的影响,筛选出适合杜梨不定芽分化的培养及生根培养条件,建立杜梨子叶的再生体系。结果表明,子叶诱导不定芽生长最佳培养基为NN69+6-BA5mg/L+IBA0.05mg/L+蔗糖35g/L,再生频率为100%;出芽后最佳继代培养基为MS+6-BA1.0mg/L+NAA0.1mg/L,平均再生芽数4.84;诱导不定芽生根最佳培养基为1/2MS+IBA1.0mg/L+蔗糖30g/L,生根率为80%。通过上述条件的优化,建立了杜梨子叶高效的再生体系。     

辣椒6421子叶外植体离体培养

以辣椒自交系6421子叶外植体为实验材料,通过筛选6-BA浸泡预处理浓度、优化不定芽诱导培养基、芽伸长培养基和生根培养基配方来提高辣椒子叶离体培养效率。结果表明:6-BA浸泡预处理子叶外植体最佳浓度为50 mg/L,最佳不定芽诱导培养基为MS+4.0 mg/L 6-BA,外植体浸泡处理1 min后接种至该培养基中,培养15 d后不定芽诱导率达93.21%;最佳不定芽伸长培养基为MS+6.0 mg/L 6-BA+0.1 mg/L IAA,培养20 d后芽伸长率可达23.21%;最佳生根培养基为1/2 MS+0.25 mg/L IAA+0.25 mg/L IBA,培养30 d后生根率可达90.12%。     

黄瓜子叶节不定芽的诱导及对甘露糖耐性的研究

为了进一步优化黄瓜组织培养体系,将非杭生素筛选标记用于黄瓜遗传转化中,以黄瓜新津研四号、绿地黄瓜和新津优1号为材料,研究黄瓜3个基因型在两种培养基中诱导不定芽的能力以及3个基因型子叶节对甘露糖的耐性。结果表明,黄瓜3个基因型不定芽诱导能力相似,L4(MS+lmg/L6-BA+0.5mg/L ABA+4mg/LKT)培养基诱导黄瓜不定芽的效果好于LS(MS+2mg/L6-BA +0.025mg/L IAA+4mg/L KT)培养基;当用甘露糖作为黄瓜子叶节遗传转化杭性筛选剂时,适宜筛选浓度为5mg/L。     

黄花矶松组织培养及培养基筛选研究

试验选用种子培养的黄花矶松无菌苗的子叶、茎段、下胚轴作为外植体材料,研究不同外植体的离体培养技术及其适宜的培养基。结果表明,生长素2,4-D对不定芽诱导具有明显的促进作用,在其浓度为1.5 mg/L时诱导率最高,子叶是诱导不定芽的良好外植体,最适培养基为MS+ 2,4-D 1.5 mg/L+ 6-BA 2.0 mg/L+ NAA 1.0 mg/L。黄花矶松的最适增殖培养基为MS+6-BA 1.0 mg/L+ NAA 1.0 mg/L,而且是以丛生芽的方式进行增殖的;最适生根培养基是1/2 MS+ KT 1.0 mg/L+ IBA 1.0 mg/L。     

植物激素直接诱导非洲菊花蕾不定芽的研究

以非洲菊幼嫩花蕾为外植体,研究了细胞分裂素类和生长素类对直接诱导非洲菊不定芽的影响。结果表明,6-BA诱导不定芽的效果略优于KT,当6-BA与KT分别是5.0 mg/L 和6.0 mg/L 时,诱导的不定芽数和不定芽长度最大;NAA诱导不定芽的效果最好,IBA次之,2,4-D最差,NAA的最适浓度是0.2 mg/L,较高浓度的生长素类虽然不利于不定芽的分化,但是有利于芽的伸长生长。     

甘蓝型油菜花茎高效再生体系研究

研究了甘蓝型油菜花茎外植体高效诱导不定芽的再生体系,结果表明：以含有2,4-D（0.5~1.0mg/L）的培养基对外植体进行预培养,以及在分化培养基中加入AgNO3（2~6mg/L）,可显著降低外植体褐化坏死的频率,提高了不定芽的发生频率及其再生能力;6BA（2.5 mg/L）与NAA（0.1mg/L）配合使用有利于提高不定芽发生频率;再生的不定芽90%可长根     

新疆海岛棉的丛生芽诱导和茎尖遗传转化的研究

 以新疆海岛棉新海17号、新海14号、85H为材料,对海岛棉器官发生再生体系和遗传转化进行研究。结果表明：茎尖系统再生能力强,茎尖组培成苗率可达90.5%。在MSB5培养基中,6-BA浓度为0.5 mg ·L^-1时,芽诱导率最高;但每个外植体的出芽数所需的最佳浓度是1.5 mg·L^-1;当浓度为2.0 mg ·L^-1时,开始有抑制出芽的现象;每个外植体最多能诱导4个芽。用农杆菌介导法转化棉花的茎尖,对侵染的损伤和Kan的耐受能力强,对Kan棉花的茎尖选择压达100 mg· L^-1;抗性绿苗率可达88.9%。     

Efficient Micropropagation Protocol for <Emphasis Type="Italic">Jatropha</Emphasis> Curcas Using Liquid Culture Medium

Although several studies have been made on the micropropagation of Jatropha curcas using agar base mediums, none of them have been by using liquid medium systems. The effects of explant type and temporary immersion system (test tube, jar with filter paper boat, and growtek bioreactor) on the micropropagation of J. curcas were studied. The explant type influenced shoot quality, multiplication coefficient (MC), and rooting. Leaf explant produced more and longer shoots than nodal explant. Use of filter paper (FB) boat prevented hyperhydricity and allowed proliferation of nodal explants cultured in liquid MS (Murashige and Skoog) medium supplemented 6-benzylaminopurine (BAP) and Kinetin (KN). The best shoot bud induction (92.1±3.1%) was achieved in liquid MS medium supplemented with 2.0 mg/L KN. Leaf regeneration efficiency was compared in growtek bioreactor and in jar containing liquid MS medium supplemented with 0.5 mg/L Thidiazuron (TDZ). The best shoot bud regeneration (78.7±2.1%) was obtained in growtek bioreactor. Shoot buds achieved from nodal segment and leaf were subcultured on filter paper boats in jar and bioreactor containing liquid MS medium supplemented with BAP, Indole butyric acid (IBA), Indole-3-acetic acid (IAA), and KN. Best shoot proliferation and elongation was obtained in filter paper boats containing liquid MS medium supplemented with 1.5 mg/L BAP, 0.5 mg/L IAA, and 0.2 mg/L KN. The number of multiple shoot buds was higher in leaf explants as compared to nodal explants and the highest number of multiple shoot buds was recorded from leaf explants. Up to 76.4% rooting efficiency was obtained when the shoots were ex vitro rooted. The generated plants well established in the nursery and grew normally in outdoor conditions. The protocol has good potential for application in large-scale propagation of J. curcas using liquid medium.     

Plant regeneration from cotyledons and embryonic axes in apple: Sites of reaction and effect of pre-culture in the light

Summary Mature embryos of the cultivar Gloster were isolated from seeds of fruits stored at 1 °C. Embryonic axes and wounded cotyledons were pre-cultured in the light for 0 to 6 days on embryo growth medium (experiment 1) or for 0 to 24 hours on shoot induction medium (experiment 2). After a subsequent dark period of 3 weeks for induction of organogenesis, explants were cultured in the light on shoot elongation medium. Up to 95% of the embryos formed shoots, while the mean shoot number per embryo reached 6.4. Regeneration frequency was influenced by the type of explant: embryonic axes reacted better (75%) than the tops of the cotyledons (29%) or wounds on the cotyledons (9%). The mean total shoot number per embryo comprised means of 2.3 for embryonic axes, 1.8 for tops of cotyledons and 2.3 for wounds on cotyledons (experiment 1). Wounds on the proximal part of the cotyledon reacted much better than those on other parts. Shoot differentiation was highest when cotyledons were cultured with the adaxial side on the medium, suggesting that there exist gradations within the cotyledon for regeneration capacity (experiment 2). The number of shoots per reactive site was not normally distributed, while a high frequency of 6 or more shoots per reactive site was found (experiment 1). A pre-culture of more than 24 hours in the light seemed to have negative effect, but there was no clear evidence whether this was a light and/or a medium effect.     

花生胚轴的离体诱导与植株再生

选择不同基因型的花生品种为外植体供体,以初步建立适应河南花生品种的高效再生体系。以5天苗龄的花生无菌胚轴为外植体,将供试的4个花生品种分别接种于4种丛生芽诱导培养基上：MS+6-BA5mg/L+NAA1mg/L,MS+TDZ0.6mg/L+NAA0.4mg/L,MS+TDZ1mg/L+6-BA1mg/L+NAA0.5mg/L,MS+TDZ1mg/L+6-BA2mg/L+NAA0.5mg/L。在25℃±1℃、2000lx、16h/d光照条件下培养约30天左右,上胚轴和下胚轴均分化出愈伤组织和丛生芽点。结果发现,上胚轴的丛生芽诱导率远高于下胚轴,最高达到67%,平均每个外植体产生4.5个丛生芽,最高的可分化出30多个;上胚轴在培养基MS+6-BA5mg/L+NAA1mg/L和MS+TDZ1mg/L+6-BA1mg/L+NAA0.5mg/L的丛生芽分化较好,该研究为花生组织的离体培养和外植体遗传转化提供有效途径。     

茎尖部位与激素对甘蔗‘福农39’和‘新台糖22’愈伤过程的影响

以甘蔗品种‘福农39’和‘新台糖22’的茎尖为外植体,以MS为基本培养基,研究不同激素浓度和不同茎尖部位对愈伤组织的诱导与分化的效应。结果表明:添加3.0 mg/L 2,4-二氯苯氧乙酸(2,4-D)对甘蔗品种‘福农39’愈伤组织的诱导效果最佳。在此浓度下,‘福农39’和对照‘新台糖22’愈伤组织诱导率分别达到98.9%和97.8%;茎尖生长点基部(较粗的一端)以上0?3 cm的部位愈伤组织诱导效果最好,‘福农39’和对照‘新台糖22’该部位的愈伤组织诱导率分别达到了97.8%和88.9%;添加2.0 mg/L 6-苄氨基腺嘌呤(6-BA)对甘蔗品种‘福农39’愈伤组织的分化诱导效果最好。在此浓度下,‘福农39’和对照‘新台糖22’愈伤组织分化出苗率分别达到了57.4%和74.1%;茎尖生长点基部以上0?3 cm部位诱导的愈伤组织分化出苗率最高,‘福农39’和对照‘新台糖22’在该部位的愈伤组织分化出芽率分别达到了41.5%和54.8%。本研究为后续‘福农39’转基因研究及健康种苗生产提供帮助。     

大丽花叶片离体再生体系的建立

为了建立大丽花高效遗传转化体系及解决今后通过植物基因工程选育新种质的问题,以大丽花为试材,研究光照条件、叶片生理状态、激素浓度等因素对叶片再生的影响,建立以大丽花离体叶片为外植体的高频再生体系。研究结果表明：以顶部充分展开的25天叶龄的无菌苗叶片为外植体,在含KT 7 mg/L+NAA 0.05 mg/L的MS分化培养基上,暗培养15天后转到光下培养,20天后开始有不定芽直接从叶片上分化产生,出现的高峰期在接种后30~35天,芽分化率最高可达86%,平均叶片再生芽位点数为5.0。待不定芽长至2 cm以上时,将其剪下转到生根培养基1/2MS+ NAA 0.1 mg/L上培养后得到生根的完整植株。     

In vitro induction of tetraploids in Phlox subulata L.

Tetraploid plants of Phlox subulata L. were induced successfully by treating shoot tips in vitro with colchicine. Shoot tips excised from in vitro shoots were treated with four different concentrations of colchicine (0.005, 0.01, 0.02, 0.04%) in solid MS medium supplemented with 4.54 μM TDZ and 0.49 μM IBA for 10, 20 or 30 days, respectively. The survival rates of shoots tips were affected by the concentration of colchicine and the duration of treatment. High concentration and longer duration reduced survival of the shoot tips, but the effect of duration of colchicine was more than that of concentration. Tetraploid plants were obtained in all of the treatments, but the percentages of tetraploids varied among different treatments, from 25.0% to 75.0%. The most efficient condition for inducing tetraploids was to treat shoot tips with 0.005% colchicine for 20 days, with 30.0% survival rate of shoot tips and 6 tetraploid plants out of 10 plants examined. The rooted tetraploid plants were transplanted successfully in a solar greenhouse. Under the same growing condition, significant varieties in flower bud and flower sizes were detected between 2x and 4x plants. The flower diameters of tetraploid and diploid plants were 2.91 cm and 2.24 cm, respectively.     

Trichomes and regeneration by direct organogenesis of medicinal plant Dracocephalum kotschyi L. using shoot tips (Lamiaceae)

The present study describes the procedure for micropropagation of Dracocephalum kotschyi L. using shoot tips from in vitro-germinated plants. The best response was observed for shoot tips on MS medium containing 5 mg 6-benzylaminopurine L^?1 and 0.2 mg 1-naphthaleneacetic L^?1 acid. Regeneration for other types of the explant hypocotyls and cotyledons did not show satisfactory results so that the explants did not develop into normal shoots and in turn developed into the calli after 12 days of culture. Histochemical analysis showed that only the shoot tip revealed a direct induction of more teratological protuberances that arise around the cut end of the explants. Elongation of shoot buds was obtained on MS medium containing 1 mg BAP L^?1 + 0.5 mg IBA L^?1. Regenerated shoots rooted best on the same medium of elongation. After hardening, the rooted plants were transferred to the greenhouse where they grew, matured, and flowered normally with a survival rate of 95%. We concluded that the present protocol can be efficiently used for mass propagation of Dracocephalum kotschyi.