三七体细胞胚胎发生及人工种子研制

三七为五加科的重要药用植物。以三七的种胚为材料接种于MS 补加2,4-D、IAA、NAA 各1 mg/L的培养基上。接种后约二个月,在MS 补加IAA 或NAA 的培养基上可以产生体细胞胚;在MS 加2,4-D 的培养基上只产生愈伤组织而无器官分化。体细胞胚在MS+IAA 0.5 mg/L+GA_3 1 mg/L 培养基上可发育成小植株。体细胞胚用4%海藻酸钠和2%氯化钙进行人工种皮包埋后,在无菌条件下,人工种子可转换成苗,转换率达89.7%。三七的体细胞胚起源于愈伤组织内或近表层的单个胚性细胞。体细胞胚经球形、心型、鱼雷形及子叶期等诸阶段发育成植株。通过PAS 法染色发现,胚性细胞、球形胚、早心形胚阶段有明显地淀粉积累,淀粉颗粒大而密集;到晚心形胚及子叶期体胚,淀粉颗粒小而少或消失。淀粉消长变化的规律与体细胞胚的发育有着密切的关系。     

白刺花胚性愈伤组织诱导及体细胞胚发生和萌发

探讨不同因素对白刺花下胚轴、子叶2种外植体胚性愈伤组织诱导及体细胞胚发生和萌发的影响。以B5和MS为基本培养基,研究2,4-D、6-BA和TDZ对白刺花下胚轴和子叶胚性愈伤组织的诱导;在MS培养基上添加不同浓度2,4-D,研究胚性愈伤组织增殖情况;采用ABA,探究对体细胞胚发生的影响。结果表明:下胚轴比子叶更易诱导胚性愈伤组织,筛选出2种外植最佳的胚性愈伤组织诱导培养基均为MS+2.0 mg/L 2,4-D+0.5 mg/L TDZ+0.5 mg/L 6-BA,胚性愈伤组织诱导率分别为77.3%和41.0%。15.0 mg/L ABA、0.2 mg/L 2,4-D和2.0 mg/L 6-BA有利于体细胞胚发生,1/3MS+0.2 mg/L NAA+0.1 mg/L 6-BA+2.0 g/L活性炭+25 g/L蔗糖+7 g/L琼脂的培养基可使体细胞胚萌发率达80%以上,再生植株移栽成活率高达90%。白刺花外植体种类及培养基类型均会影响胚性愈伤组织的诱导,其中下胚轴诱导效果优于子叶;MS培养基较适合启动细胞脱分化形成愈伤组织,2,4-D对胚性愈伤组织的增殖保持有调控作用,ABA有利于体细胞胚的发生。     

几种匍匐翦股颖成熟胚愈伤组织诱导及植株再生

利用成熟种子作为外植体,分析了2,4-D对匍匐翦股颖胚性愈伤组织诱导与植株再生体系的影响,并对体细胞胚的发生过程进行了观察.实验结果表明,在2.0 mg/L 2,4-D 0.1 mg/L 6-BA时胚性愈伤组织的诱导频率最高.随着2,4-D浓度的增加,愈伤组织的诱导和分化能力明显下降.在再生过程中采用1.0 mg/L的6-BA达到了比较好的效果,愈伤组织的再生频率大部分在90%以上.同时发现适当提高肌醇浓度可以使苗长得较为粗壮.在实验中发现匍匐翦股颖的体细胞胚发生过程为球形胚、心形胚、鱼雷胚和子叶胚.     

石刁柏组织培养中体细胞胚发生的组织细胞学观察

以石刁柏(Asparagus officinalis)无菌苗的嫩茎切段为外植体,在含有1 mg/LNAA+0.5 mg/L BA的MS培养基上可100%地被诱导形成愈伤组织,在此条件下可长期继代,将继代的愈伤组织转入含有2 mg/L 2,4-D 0.5mg/L NAA的MS培养基上后,约有70%的愈伤组织块转变为胚性愈伤组织,这些胚性愈伤组织在3,4-D浓度进一步降低为0.5mg/L的条件下发育形成体细胞胚。切片观察表明:这些胚性愈伤组织是从愈伤组织的表层或近表层产生的。这些细胞核大,多核仁,细胞质浓、染色深的胚性细胞中的一些单个细胞处于与邻近细胞隔离状态,细胞壁也明显加厚。这些单细胞开始分裂,第一次分裂多为不均等分裂,形成一个大的基细胞和一个小的顶细胞,进一步分裂形成三细胞、四细胞、五细胞和具胚柄的多细胞原胚。原胚发育形成球形胚、梨形胚、香蕉形胚,由于在胚的一侧细胞分裂旺盛形成单子叶突起,最后形成子叶胚。其发育过程类似于单子叶植物合子胚的发育过程。     

黄山栾树体细胞胚的发生和组织学观察

黄山栾树无菌苗的节间和叶柄离体培养后,其体细胞胚发生的结果表明：节间愈伤组织可诱导产生体细胞胚,而叶柄愈伤组织则生根：节间愈伤组织诱导培养基为MS＋3．0mg．L～2,4．D＋0．5～3．0mg．L-1NAA;节间胚性愈伤组织诱导培养基为MS＋2．0nag．L-2,4-D;胚性愈伤组织转移到无植物生长调节剂的MS培养基上可发育成正常植株。组织学观察表明,体细胞胚在胚性愈伤组织中有的发生于愈伤组织表层细胞,有的发生在愈伤组织内部。黄山栾树体细胞胚的形成经历球形胚、心形胚、鱼雷胚和子叶胚几个阶段,这与合子胚的发育途径相似。     

秦艽(Gentiana macrophylla Pall.)离体培养再生过程中体细胞胚的发生(简报)

本文以秦艽叶片和茎段作为外植体,通过离体培养对秦艽植株再生途径进行研究。愈伤组织在添加2mg/L 2,4-D和0.5mg/L BA的MS培养基上诱导,两周内可出现愈伤组织。愈伤组织在相同激素配比并附加500mg/L LH的MS培养基上继代。愈伤组织的分化在添加有0.1mg/L 2,4-D和0.5mg/L BA的MB培养基上进行。通过显微观测,疑似体细胞胚可以在叶片和茎段的愈伤组织上产生。形态学和组织学的分析进一步证实了秦艽离体再生过程中体细胞胚发生的现象。体细胞胚和合子胚一样,也经历球形、心形、鱼雷和子叶胚等发育时期。相对独立的结构说明秦艽的体细胞胚可能是单细胞来源。体细胞胚在愈伤组织的表面和内部都有出现。在本实验中,体细胞胚发生途径是在秦艽愈伤组织形成后观察到的唯一再生途径。     

2.4-D、6-BA对人参体细胞胚胎发生过程的影响研究

本实验以人参芽胞、二年生人参根、实生苗的茎、叶为外植体研究了体细胞胚的发生条件,并对其发生过程中可溶性蛋白、相关酶活性及内源激素的变化等进行了研究。结果表明,诱导愈伤组织的培养基为MS+2,4-D 4.0mg/L + BA 0.2mg/L;在MS+2,4-D 1.0mg/L + KT 0.2 mg/L培养基上继代培养,可获得胚性愈伤组织;在无2,4-D的培养基上可诱导出胚状体。将胚状体转入无任何激素的MS培养基上继续培养,之后转入1/2MS培养基上获得再生植株。组织细胞学观察表明人参胚状体的起源方式为单细胞起源。在体细胞胚胎发生过程中,多糖和淀粉含量在早期胚时较低,可溶性蛋白含量、POD及PPO活性在早期胚时最高;IAA在早期胚时期含量最高,在成熟胚时期ABA含量最高,而ABA/IAA比值在成熟胚时较高,利于体细胞胚的发育成熟。cDNA-AFLP 分析表明胚状体发育不同时期的人参培养物基因表达不同,从而导致了分化和发育。培养物HPLC分析表明胚胎发生试管苗总皂苷含量比子叶胚时期高4倍多。单体皂苷差异较大。     

小麦遗传转化受体系统建立的研究

选用‘小偃22’和‘宁春16’小麦品种的成熟胚和幼胚进行培养,研究不同种类的胚和培养因子对愈伤组织诱导和分化的影响。结果表明,幼胚和成熟胚的愈伤组织诱导率无明显差异,但较高浓度的2,4-D有利于成熟胚的诱导,而幼胚培养时2,4-D浓度的影响效果因品种而异;两种外植体分化率的高低与KT/IAA的配比均有密切关系,但高浓度的激素水平不利于成熟胚的分化;诱导培养基中低浓度的2,4-D有利于所诱导的愈伤组织的分化。同时,在诱导培养基中添加低浓度的KT能显著提高两品种成熟胚愈伤组织的分化率;各种培养基处理与品种间都存在显著的互作效应,‘小偃22’成熟胚培养的最佳培养基组合为MSD 3.0 mg/L 2,4-D和MSD 0.5 mg/LIAA 1.0 mg/L KT,幼胚培养为MSD 4.0 mg/L 2,4-D和MSD 0.5 mg/L IAA 1.0 mg/L KT;‘宁春16’成熟胚培养为MSD 4.0 mg/L 2,4-D和MSD 1.0 mg/L IAA 1.0 mg/L KT,幼胚培养时为MSD 1.0 mg/L 2,4-D和MSD 2.0 mg/L IAA 2.0 mg/L KT。     

盐肤木体细胞胚胎发生及植株再生

以盐肤木(Rhus chinensis Mill.)幼胚为外植体,研究不同植物生长调节剂组合对其愈伤组织诱导及体细胞胚胎发生的影响,以建立盐肤木体细胞胚胎发生及植株再生体系。结果表明,最适愈伤组织诱导培养基为MS+6-BA 0.2 mg/L+2,4-D 1.0 mg/L,诱导率为84.57%,诱导出的初代愈伤组织白色或淡黄色,质地疏松,表面光滑,为非胚性愈伤。初代愈伤组织转移到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L培养基上培养1个月后,长出淡黄色质地紧密的胚性愈伤组织,诱导率高达100%,在此培养基上胚性愈伤组织增殖倍数为854.73%。所获得的胚性愈伤组织转接到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L+蔗糖4%的培养基上培养1个月后可诱导体细胞胚胎发生,诱导率可达32.67%。诱导得到的体细胞胚胎经历球形胚、心形胚、鱼雷胚、子叶胚进一步分化发育成苗。无菌苗炼苗后栽种到泥炭土∶蛭石∶珍珠岩为2∶1∶1的生长基质上,能100%稳定成活。经过细胞学观察分析,体细胞胚的发育与合子胚相似。     

白鹤芋体细胞胚胎发生及组织学研究

本研究以白鹤芋品种‘Mojo’的根茎为外植体,研究不同外源激素配比对体细胞胚胎发生的影响,筛选出最佳胚性愈伤及体胚的诱导配方。其后采用体式显微镜和石蜡切片技术对体胚发生途径中的典型时期进行形态学和组织学观察。结果表明,4 mg/L 2,4-D与0.5 mg/L KT作用时,白鹤芋胚性愈伤组织诱导效果最佳,诱导率达87.44%;体细胞胚胎诱导的最佳配方为1.0 mg/L6-BA+0.2 mg/L TDZ+0.2 mg/L NAA。白鹤芋是单子叶植物,其体细胞胚胎的形态形成过程与单子叶植物胚的形态形成过程类似。研究结果表明,白鹤芋体细胞胚胎的形态形成主要分为以下几个阶段:胚性细胞、胚胎、球形胚、椭圆形胚、盾形胚、子叶型胚、成熟体细胞胚。     

Somatic embryogenesis and plantlet regeneration in Cornus florida

Somatic embryos were initiated from 12 to 15 weeks postanthesis (WPA) zygotic embryos of Cornus florida L. (flowering dogwood) cultured on Murashige-Skoog (MS) or Schenk and Hildebrandt (SH) medium amended with either 3 mg/L 2,4-D or 5 mg/L 2,4-D and 1 mg/L kinetin. White, opaque globular and early cotyledonary stage embryos were formed directly on detached cotyledons from 2 of the 5 trees sampled after 7 weeks of culture. Morphologically mature embryos developed after an additional 4 weeks incubation on medium without growth regulators; however, many of the embryos were fused in pairs along the entire length of the hypocotyl-radicle axis. Indirect embryogenesis was observed from callus cultures initiated from 9 to 15 WPA zygotic embryos. These cultures have continued to produce embryos for 16 months. Many of the embryos formed roots on germination medium, but only 12% formed plantlets and none developed past the first true leaf stage.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - FPA Formalin-propionic acid-ethanol (50%) - WPA weeks post-anthesis     

云南大叶茶体细胞胚发生及体细胞胚苗形成体系的建立

利用云南大叶茶(Camellia sinensis var.assamica Kitamura)胚性细胞系(CL_1)中悬浮培养物,建立了高频率同步化体细胞胚发生及体胚苗形成体系。以改良的MS为基本培养基,将CL_1中培养物由液体保持培养基(0.1mg/L 2,4-D 0.5mg/L 6-BA)继代转入液体诱导培养基(0.05mg/L 2,4-D 0.50mg/L6-BA),暗培养诱导28d,转入不含任何激素的液体分化培养基中再培养28d,获得了不同发育时期的体细胞胚,其发生频率为81.5％。不同发育时期的体细胞胚用不同目的细胞筛收集,在液体生长培养基(1/2 MS 1.0mg/L GA_3 0.5mg/L 6-BA)中培养发育成熟。ABA有利于高质量体细胞胚的形成。20～70月大小的体细胞胚在固体生长培养基中成苗转换率为75％。在液体悬浮培养条件下观察记录了体细胞胚发育过程,证实其过程与合子胚的形态发生过程相似。     

Quantitative studies of embryogenesis in normal and 5-methyltryptophan-resistant cell lines of wild carrot

The frequency of embryo formation was determined in normal and 5-methyltryptophan-resistant (5-MT^r) cell lines of wild carrot (Daucus carota L.) grown in the presence or absence of 2-isopentenyladenine (2-ip) and 2,4-dichlorophenoxyacetic acid (2,4-D). 2-ip stimulated the intitation of embryo formation and also accelerated embryo development. 2.4-D inhibited embryo differentiation at several stages: at 0.1 mg/l, it stopped regeneration at the earliest stage, resulting in callus growth instead of embryo formation; at 0.04 mg/l 2,4-D, some globular embryos were produced, but they did not develop into more advanced embryos. Variant cell lines with higher levels of auxin (indole-3-acetic acid, IAA) were used to study the effect of an elevated endogenous concentration of auxin on embryogenesis. IAA at these concentrations suppressed regeneration in the same manner as the exogenous auxin, 2,4-D, did. This result confirms the hypothesis that high levels of IAA are responsible for the suppression of regeneration in the 5-MT^r cell lines.     

Studies on Somatic Embryogenesis and Histological Observation in Gossypium hirsutum L. cv. Coker 312

A reproducible 3-step procedure of somatic embryogenesis of Gossypium hirsutum L. cv. Coker 312 has been developed. Calli were initiated on LSC medium containing 0.1mg/L 2,4-D plus 0.1 mg/L KT from cotyledon tissue of 5-day-old-seedlings, and subcultured on the same medium with 4 mg/L NAA and 1 mg/L KT. Embryogenic calli and few globular embryos developed at a frequency of 67.5% after 55 days’ culture in the latter medium. When the embryogenic calli were transferred to growth regulator-free medium, embryogenesis occured and all stages of normal zygotic embryos, globular-, heart- and arrowhead- or torpedo-shaped embryo, ,were developed. Cyto-histological study showed that embryogenic calli were very easily distinguished from non-embryogenic calli. Embryoids were mainly initiated from the cells in the peripheral area of embryogenic calli. At the early stage the development of embryoid was limited in a boundary of thicken cell wail. There were 2 peaks of starch accumulation in the process of embryogenesis, one was at the early globular stage, and the other at the later torpedo-shaped stage.     

三叶半夏悬浮培养下的体细胞胚胎发生及植株再生

用三叶半夏幼嫩叶片诱导产生的胚性愈伤组织建立了胚性细胞悬浮系,研究了悬浮培养下体细胞胚胎的发生及植株的再生。结果表明,胚性悬浮细胞在附加1．0mg／L2,4-D、0．2 mg／LBA和300mg／L LH的MS液体培养基中产生大量的球形胚,转入液体分化培养基(MS 0．1 mg／LNAA 0．2 mg／L BA 300 mg／L LH)中进一步发育成心形胚、鱼雷形胚和子叶形胚。收集成熟胚转移到MS固体分化培养基上培养获得了再生植株。另外还观察到某些成熟胚上产生了许多次生胚。     

Establishment of a System with High Synchronous Frequency of Somatic Embryogenesis and Embryo Seedling Formation in Camellia sinensis var. assamica

The system of high synchronous frequency of somatic embryogenesis and somatic embryo seedling formation was established by means of embryonic cell hne 1 ( CL1 ) of Camellia sinensis var. assamica Kitamura. Modified MS was used as the basic medium. Cultures of CL1 was transferred to the aqueous induced medium (0.05 mg/L 2,4-D + 0.50 mg/L 6-BA) from the maintenance medium (0.1 mg/L 2,4-D + 0.5 mg/L 6-BA) for somatic embryos induction under dark condition. 28 days later, they were cultured in the liquid differentiation medium. Various kinds of somatic embryos were obtained after another 28 days. The frequency of somatic embryos was 81.5 %. Various mesh sizes of sieves were applied to collect the somatic embryos in different developmental stages which could develop to mature stage in the aqueous growth medium ( 1/2 MS + 1.0 mg/L GA3 + 0.5 mg/L 6-BA). ABA was effective to promote the formation of highly qualified somatic embryo. The mature somatic embryos sized 20 to 70 mesh had the conversion frequency 75 %. The development of somatic embryogenesis studied under a cell suspension culture system was similar to the zygotic embryogenesis.     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.