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。     

‘SK4—316’胡萝卜体胚的诱导和培养

以'SK4-316'胡萝卜无菌苗的下胚轴为外植体,研究不同培养基配方和培养条件对愈伤组织诱导、体细胞胚间接发生及其同步化培养的影响,以及不同脱分化时间、脱分化培养基及外植体续存时间对体细胞胚直接发生的诱导及其培养的影响.结果表明:含3%蔗糖、0.8%琼脂的1/2MS + 2,4-D 2.5 mg/L + 6-BA(或KT)0.5 mg/L + CH 300 mg/L是诱导愈伤组织的良好培养基;1/2MS + 2,4-D 1.25 mg/L + KT 0.25 mg/L + 6-BA 0.25 mg/L(含3%蔗糖)适于愈伤组织分化并诱导体胚发生,0.02% ABA对体胚的诱导有促进作用,0.06% ABA或15% PEG能促进体胚成熟;外植体在MS + 2,4-D 1.0 mg/L固体培养基上脱分化培养48 h,再转入MS + CH 300 g/L液体培养基中可诱导体胚直接发生,但随着外植体续存于诱导培养基中时间的延长,体胚发生变异的几率也渐增.     

何首乌体细胞胚的诱导及植株再生研究

以何首乌茎尖、茎段为外植体,经体细胞胚发生途径,进行胚性愈伤组织诱导、体细胞胚的诱导、植株再生的研究.并采用临时压片法对体细胞胚的发育过程进行观察.结果表明愈伤组织诱导最适培养基为Ms＋6-BA 2.0 mg/L＋NAA 0.5 mg/L,体细胞胚诱导最适培养基为MS＋6-BA 1.0 mg/L＋NAA 0.2 mg/L.将产生的体细胞胚首先接种于MS基本培养基使其充分发育后转入MS＋6-BA 2.0 mg/L培养基中诱导出芽,出芽率高于直接采用Ms＋6-BA 2.0 mg/L培养基诱导.体细胞胚的发育过程是首先在愈伤组织表面形成许多瘤状突起即胚性细胞团,胚性细胞团继续发育成球形胚、盾形胚,球形胚、盾形胚成熟后发育成植株.     

根芹体细胞胚产量与质量的研究

对根芹的不同外植体在附加不同成分的MS培养基上进行离体培养,以诱导适合于制作人工种子用的高质量体细胞胚。下胚轴、子叶和叶片在含0.5mg/LKT,0.5mg/L2,4—D的MSO培养基上诱导与继代胚性愈伤组织,然后转移到附加有100mg/L肌醇、2g/L葡萄糖的MSO无激素培养基上悬浮培养产生体细胞胚,获得了比固体培养基及含有KT的液体培养基中产生的体细胞胚形态发育更正常的大量体细胞胚,这为人工种子制作奠定了基础。     

鱼腥草体细胞胚胎发生和植株再生

目的:利用鱼腥草的叶片和叶柄为材料,进行体细胞胚胎诱导及植株再生研究。方法:运用正交设计试验,考察在改良的MS固体培养基上添加不同种类、不同浓度的植物生长物质组合及其配比对鱼腥草愈伤组织诱导、体细胞胚胎发生及植株再生的影响。结果:鱼腥草无菌苗叶片在含有2,4-D 1.0 mg/L 6-BA 0.5 mg/L的改良MS培养基上能诱导出胚性愈伤组织;胚性愈伤组织在含有6-BA 1.0 mg/L的改良的MS培养基上诱导体细胞胚的发生;叶柄在含有6-BA 1.0 mg/L改良MS培养基上直接产生体细胞胚。体细胞胚在改良的MS NAA0.1 mg/L 6-BA 1.0 mg/L的培养基上能够快速繁殖,形成大量不定芽,在不加任何激素的MS培养基上就可以萌发出不定根,发育为成完整植株,在MS IBA 1.0 mg/L的固体培养基上能够形成大量的根。结论:建立了鱼腥草体细胞胚胎发生及植株再生的体系。     

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。     

根芹体细胞胚产量与质量的研究

对根芹的不同外植体在附加不同成分的MS培养基上进行离体培养,以诱导适合于制作人工种子用的高质量体细胞胚。下胚轴、子叶和叶片在含0.5mg／LKT,0.5mg／L2,4 一 D的MSO培养基上诱导与继代胚性愈伤组织,然后转移到附加有100mg／L肌醇、2g／L葡萄糖的MSO无激素培养基上悬浮培养产生体细胞胚,获得了比固体培养基及含有KT的液体培养基中产生的体细胞胚形态发育更正常的大量体细胞胚,这为人工种子制作奠定了基础。     

甜菜原生质体培养直接产生体细胞胚

甜菜(Beta vulgaris L.)是重要的经济植物,是制糖的重要原料。近10年来已相继有人开展了甜菜原生质体的培养,但到目前为止,在国内外还未有关于甜菜原生质体培养中体细胞胚发生的报道,尤其是从原生质体直接的发生。本研究从甜菜未授粉的胚珠诱导产生的愈伤组织制备原生质体,分裂后直接产生体细胞胚,现报道于下。用于分离原生质体的愈伤组织是从甜菜(Beta vulgaris L.)品系1804/6未授粉胚珠诱导产生的。将该愈伤组织在 MS 培养基附加1.0mg/L 2,4-D 0.5mg/L 6-BA 中     

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。     

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 immature and mature embryos of a minor millet Paspalum scrobiculatum L.

Immature and mature zygotic embryos of Paspalum scrobiculatum L. cv. PSC 1 cultured on MS or N₆ nutrient medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), formed embryogenic callus. Induction of embryogenic callus and subsequent somatic embryogenesis was possible at a lower concentration of 2,4-D on N₆ than MS medium. Immature embryos were highly totipotent, forming somatic embryos at a higher frequency than mature embryos. Addition of amino acids (L-proline or L-tryptophan) to 2,4-D medium resulted in significant enhancement of embryogenesis on culture of mature embryos. Silver nitrate also supported an increased frequency of embryogenesis. Thus it is possible to have high frequency of somatic embryogenesis on culture of mature embryos, which are available in abundance and with ease than immature embryos. The somatic embryos readily germinated and formed plantlets on hormone-free regeneration medium. The regenerated plantlets were successful on transfer to soil and set seed.     

领春木体细胞胚胎发生及植株再生

以领春木（Eupteleapleiospermum Hook．f.etThoms．）种子幼胚为试验材料,对领春木体细胞胚胎发生进行了研究。结果表明：在附加1．0mg·L-1 2,4．D＋0．5mg·L-1 6-BA＋3％蔗糖＋0．8％琼脂的Ms培养基上可诱导出愈伤组织。愈伤组织在附加0．5mg·L-1 NAA＋0．5mg·L-1 6．BA的培养基上可形成体细胞胚;体胚在附加0．5mg·L-1 6-BA＋0．05mg·L-1 NAA＋0．1％PVP的1／2MS培养基上能大量增殖;将成熟体胚转移到不添加任何植物生长调节剂的MS培养基上,培养60d,形成正常植株。     

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.     

百合体细胞胚胎发生和植株再生

以切花百合(Lilium)品种‘黄天霸’(‘Manissa’)花器官为外植体诱导体细胞胚胎发生与植株再生。结果表明,不同花器官、不同激素配比对愈伤组织形成均具有显著影响。花丝为最佳外植体,激素对愈伤组织诱导的影响效应为NAA>6-BA>2,4-D,最适培养基为MS+1.0 mg.L-1NAA+0.2 mg.L-16-BA;激素诱导体细胞胚胎发生的影响效应为2,4-D>KT>6-BA,最佳培养基配方为MS+1.0 mg.L-12,4-D+0.2 mg.L-1KT+1.0 mg.L-16-BA;MS培养基添加IBA可促进体细胞胚萌发成苗,体细胞胚芽成苗的最佳培养基为MS+0.2 mg.L-16-BA+1.0 mg.L-1IBA。     

Direct somatic embryogenesis and plant regeneration from single cell suspension cultures of Elymus giganteus Vahl

Summary A yellowish, nodular callus was induced from mature embryos of Elymus giganteus Vahl on MS medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l kinetin, from which a cell suspension culture was initiated in liquid MS medium supplemented with 0.5 mg/l 2,4-D, 1.0 mg/l kinetin and 0.2 mg/1 naphthaleneacetic acid (NAA). By filtering through a series of sieves with decreasing mesh sizes and collecting the resultant filtrate, a suspension culture composed mainly of single embryogenic cells was established. In a medium containing 0.3 mg/l 2,4-D, 1.0 mg/l 6-benzylaminopurine (6-BAP) and 500 mg/l casein hydrolysate (CH), the single cells underwent direct somatic embryogenesis resulting in the formation of proembryos. These proembryos developed into mature embryos when placed in a double-layer liquid overlay culture. Intact plants were developed from somatic embryos when they were transferred onto solidified MS medium without added growth regulators.     

A rapid protocol for somatic embryogenesis from immature leaflets of groundnut (Arachis hypogaea L.)

Summary Plant regeneration via somatic embryogenesis was developed in two groundnut varieties. Somatic embryogenesis was induced from immature leaflets on MS medium with different concentrations of the auxins 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) in combination with 0.5 mg/l of the cytokinin BA. The highest frequency of somatic embryo formation occurred on MS medium fortified with 20 mg 2,4-D per l. Of the two auxins tested individually 2,4-D was more effective for induction of embryogenesis as well as production of embryos. Embryo development and maturation was achieved on MS medium supplemented with N⁶-benzyladenine (BA) (0.5–2.0 mg/l) and 2,4-D (0.5 mg/l). Plant conversion frequency from somatic embryos was highest in presence of 2.0 mg BA per l and 0.5 mg NAA per l. The frequency of embryogenesis and plant regeneration was higher in the VRI-2 cultivar than in the other cultivar tested. Regenerated plants were transferred to soil, grown to maturity, and produced viable seeds.     

Effects of ABA on secondary embryogenesis from somatic embryos induced from inflorescence culture ofAralia cordata Thunb

In order to investigate the effect of ABA on secondary embryogenesis from somatic embryos inAralia cordata Thunb., embryogenic callus and somatic embryos were induced from inflorescence on solid MS basal medium supplemented with 1.5 mg/L 2,4-D after eight weeks without subculture. For mass production of somatic embryos, embryogenic cell clumps were maintained in liquid MS medium supplemented with 1.0 mg/L 2,4-D, and then transferred to 2, 4-D-free medium. When developing embryos at various stages were cultured separately in liquid medium with ABA (0 to 2.0 mg/L) for three weeks, and then cultured in ABA-free liquid medium for two weeks, torpedo-shaped embryos exhibited secondary embryogenesis of 65.9% in only 0.2 mg/L ABA pretreatment. Cotyledonary embryos in cultures by 0.2, 0.5 and 1.0 mg/L ABA pretreatment also exhibited secondary embryogenesis (73%, 9.4% and 6.0%, respectively). However, globular and heart-shaped somatic embryos treated with ABA did not form secondary embryos on their hypocotyl surfaces. When cotyledonary embryos were cultured in ABA-free medium or 0.2 mg/L ABA treated medium for three weeks, and then in ABA-free liquid medium for 6 weeks, the germination frequency was lower in medium with 0.2 mg/L ABA (45.9%) than in hormone-free medium (56.8%). This result seems to be related to the high frequency of secondary embryogenesis. It is suggested that secondary embryogenesis by ABA application depends upon the stage of embryo cultured and the ABA concentration.     

Direct somatic embroygenesis from immature embryos of rosewood (Dalbergia latifolia Roxb.)

Direct regeneration of somatic embryos was obtained from immature zygotic embryos of Dalbergia latifolia. Immature embryos dissected from green pods 90 d after flowering gave the highest frequency of somatic embryo formation. Preculture on high 2,4-D medium for 4 weeks induced direct somatic embryogenesis, which was expressed during the second culture phase in the presence of low 2,4-D along with a high sucrose concentration. Embryos were separated and transferred to the maturation medium containing MS + 0.5–1.0 mg/L BAP, where embryos developed into plantlets. Somatic embryos failed to convert into complete plants without BAP treatment. This method of direct regeneration of somatic embryos without a callus phase has direct application for genetic manipulation studies.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - KIN Kinetin