Repression of asexual embryogenesis in vitro by some plant growth regulators

Summary A factor that represses asexual embryogenesis has been observed in the Rutaceae, with particularly high concentrations in the naturally monoembryonic cultivars. This investigation was an initial step towards identifying the factor.Citrus reticulata Blanco Ponkan mandarin nucellus explants andDaucus carota L. ‘Queen Anne's Lace’ callus were employed to examine effects of known plant growth regulators and to determine possible identity of one or more of them with the repressive factor. The chalazal halves of ovules ofC. media L. ‘Citron of Commerce’ were used as control repressor source. Embryo initiation and growth of both test tissues were depressed markedly by 2,4-D, abscisic acid and ethephon. Slight inhibitions were obtained with IAA, kinetin and gibberellic acid. Recovery from the repressor did not occur readily inCitrus nucellus following recultures in citron-ovule-free medium; carrot callus resumed normal embryogenesis immediately upon transfer to suppressor-free medium. The repression by natural sources apparently involved the combined action of some or all natural hormones that are generically related to the above. This paper is part of B. Tisserat's Ph.D. dissertation in Botany at the University of California, Riverside. The research was supported in part by the Elvenia J. Slosson Fellowship in Ornamental Horticulture awarded to T. Murashige.     

Growth and development of Citrus pistils and fruit explants in vitro

Pistils and various fruit explants of Citrus limon L. Burm. f. and Citrus sinensis L. Osbeck were cultivated in vitro . Basal medium as well as medium supplemented with IAA, GA₃ or benzylaminopurine, supported growth of all explants for more than one year. Pistils did not enlarge considerably, but gave rise to active callus growth; callus proliferation and viability was enhanced by all hormones. Culture of fruit slice explants resulted, in addition to peel hypertrophy and callus proliferation, in a marked growth of two distinct types of juice vesicles. The growth of juice vesicle explants was promoted by all three growth hormones. – It is suggested that the successfully prolonged in vitro culture of various fruit explants, and especially of juice vesicles, may aid in studies of fruit development and physiology.     

Somatic embryos from culture ovules of polyembryonic Mangifera indica L.

Ovules were aseptically removed from 2 month old fruits of 9 naturally polyembryonic cultivars and 1 monoembryonic cultivar of mango (Mangifera indica L.). Ovules were placed into culture on solid Murashige and Skoog medium that had been modified by the addition of half strength major salts and chelated iron, 6% sucrose, 400 mg/l glutamine, 100 mg/l ascorbic acid with or without the following growth regulators: 20% (v/v) CW, 1 or 2 mg/1 BA. Somatic embryogenesis occurred from the nucellus excised from the ovules of 5 of the naturally polyembryonic cultivars after 1–2 months in culture. Somatic embryogenesis was not apparently affected by the growth regulator composition of the media; however, efficient somatic embryogenesis only occurred in liquid containing 20% CW.Abbreviations CW coconut water - BA benzyladenine     

Callus growth and somatic embryogenesis in thalamus tissue ofRanunculus asiaticus L. CultivatedIn vitro: Cytokinin effect and phenol metabolism

Summary The effect of cytokinin on growth and plant regeneration of thalamus-derived calluses ofRanunculus asiaticus L. has been investigated with various concentrations of 6-benzyladenine and 6-furfurylaminopurine (kinetin), in a medium containing 2,4-dichlorophenoxyacetic acid levels, which was decreased to 0 over three subcultures. Cytokinins, although not essential, for initiating callus production, improved subsequent callus growth and plant regeneration. No somatic embryogenesis was observed on calluses grown on media lacking cytokinins or containing only kinetin. Calluses manifested embryogenesis on media containing 6-benzyladenie plus kinetin or only 6-benzyladenine. Nondifferentiating callus was characterized by a high content of phenolic polymers and an elevated peroxidase and polyphenol oxidase activity in comparison with differentiating callus. Differences in simple phenol concentrations were observed in the two kinds of callus.     

Plantlet regeneration from a NaCl-selected salt-tolerant callus culture of Shamouti orange (Citrus sinensis L. Osbeck)

Plantlets were regenerated from a selected salt-tolerant cell line of Shamouti orange (Citrus sinensis L. Osbeck). Embryogenesis was carried out both in the presence and absence of NaCl, yielding green and white globular embryos, respectively. Greening could be induced subsequently and normal heart shape embryo development was obtained. Plantlet formation required exposure to kinetin prior to the introduction of the root-inducing hormone naphthalene acetic acid. This system differs from the designed protocol for plant regeneration from the salt-sensitive, i.e., unselected callus. It is concluded that NaCl interferes with the regeneration process, with embryogenesis and/or embryo development into plantlets. Its presence during callus growth probably changes the balance of the phytohormones which is later manifested in plant regeneration. Citrus salt-tolerant callus yields salt-tolerant embryos. Salt-tolerant calli derived from regenerated plantlets indicate acquisition of salt tolerance on the whole plant level.     

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。     

影响沙田柚叶片离体培养的因素研究

研究了离体培养条件下影响沙田柚叶片愈伤组织诱导和分化的一些因素。结果表明 ,2 ,4 D可以诱导愈伤组织的形成 ,高浓度的蔗糖 (6% )显著提高叶片愈伤诱导率与愈伤组织重量 ,且在 2 ,4 D和蔗糖之间存在着相互作用。外源GA3处理抑制愈伤组织的诱导和生长 ,而CCC与ABA处理显著提高叶片的愈伤诱导率和愈伤组织生长量。愈伤组织转移到附加 3 .0mg/LBA的MS分化培养基上可以分化出芽 ,0 .2 5mg/LGA3的加入可以进一步提高愈伤组织的分化率和每块愈伤组织的再生芽数。     

Production of Virus-tree Nucellar Plantlets from Unfertilized Ovules of Citrus in vitro

On a series of Murashige and Tucker (MT) media supplemented with different growth regulators, the 8-week-old unfertilized ovules of Washington navel orange (Citrus sinensis) were able to regenerate perfect plantlets via somatic embryogenesis or organogenesis. The sorts and combinations of exogenous hormones had remarkable effects on the induction, growth and differentiation of its callus. It was found that the most suitable induction medium was MT medium supplemented with 1.0 mg/l BA and 0.5mg/l 1AA. The most suitable differentiation medium was MT medium supplemented with 1.0 mg/l IBA. It was proved by indicator plant examination that the nucellar plantlets free of citrus exocortis virus (CEV) and citrus tristeza virus (CTV) had been obtained from infected trees.     

Regeneration via somatic embryogenesis of the endangered wild arum (Arum palaestinum)

Somatic embryogenesis was obtained from callus of wild arum (Arum palaestinum). Callus was induced from sterilized corm bud sprouts cultured on basal medium containing 4.4???M 6-benzyladenine and 5.4???M 1-naphthaleneacetic acid. Callus was maintained under dark conditions using basal medium with 4.4 or 8.8???M 6-benzyladenine and 5.4 or 10.8???M 1-naphthaleneacetic acid. The highest callus weight and most desirable callus phenotype were achieved using basal medium containing 8.8???M 6-benzyladenine and 5.4???M 1-naphthaleneacetic acid. Friable calli were cultured in the dark on basal medium containing 4.5???M 2, 4-dichlorophenoxyacetic acid, 0.46???M 6-furfurylaminopurine, 5.4???M 1-naphthaleneacetic acid, and 1.7?mM proline to induce embryogenesis before transfer to regeneration medium. Embryos that developed on regeneration medium were transferred to medium minus plant growth regulators for germination. Ninety percent of the germinating embryos developed into rooted plantlets. Rooted plants were grown in the greenhouse and acclimatized successfully with a 95?% survival rate. This is the first report of successful somatic embryogenesis and plant regeneration in A. palaestinum.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

Probable identity of substances in citrus that repress asexual embryogenesis

Summary The embryogenetic responseof culturedDaucus carota L. ‘Queen Anne's Lace’ callus was employed to attempt fractionation and identification of a repressive factor produced byCitrus medica L. ovules. The factor was evidently synthesized and released into the medium continuously, inasmuch as citron ovules that had been autoclaved with the medium were completely infeffective. The inhibition could be attributed to volatile and nonvolatile components. A substantial part of the inhibition was prevented by continuously refereshing the atmosphere within the cultures with filtered air. Monitoring of the gases produced by citron ovule sections under conditions simulating bioassays disclosed significant evolution of carbon dioxide, ethylene and ethanol. Repression of embryogenesis was not averted by trapping the liberated ethylene. On the other hand, ethanol in concentrations equivalent to those released by citron ovules suppressed asexual embryogenesis dramatically. The adverse effect of ethanol was reversed immeditaley upon transfer to ethanol-free medium. Another investigation had disclosed anti-embryogenetic effects of auxin, abscisic acid and gibberellin. Analysis ofCitrus ovules excised from young fuits disclosed those of monoembryonic citron to contain concentrations of IAA, ABA and GA₃ several times higher than those of polyembryonic Ponkan mandrain. The nonvolatile protion might be identified with these hormonal substances. This paper is part of B. Tisserat's PhD. dissertation in Botany at the University of California, Riverside. The research was supported in part by the Elvenia J. Slosson Fellowship in Ornamental Horticulture awarded to T. Murashige.     

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree,Dalbergia sissoo Roxb

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.     

甘蔗幼叶切段培养中的体细胞胚胎发生

本文从形态学和组织学方面研究了甘蔗幼叶胚性愈伤组织发生及体细胞胚胎的形成过程。甘蔗幼叶片切段培养于含2.4—D1.5mg/1的MS培养基上,4—6天后切段开始形成愈伤组织,约10天后愈伤组织表面出现白色颗粒状结构。将含有白色颗粒状结构的愈伤组织转移至不含激素的培养基中,7—10天后可见有小植株长出。组织学和形态学观察表明,甘蔗离体再生植株是通过体细胞胚胎发生途径。     

In vitro plant regeneration and ethylmethanesulphonate (EMS) uptake in somatic embryos of date palm (Phoenix dactylifera L.)

The effect of the auxins dicamba (3,6-dichloro-2-methoxybenzoic acid) and picloram (4-amino-3,5,6-trichloropicolinic acid) on callus growth and embryogenesis in Phoenix dactylifera L. was investigated. Maximum callus fresh weight was obtained in nutrient medium enriched with 200 µm picloram. Somatic embryogenesis and subsequent plant regeneration was achieved following transfer of such calli to hormone-free medium. Germination of the somatic embryos was influenced by treatment with the chemical mutagen ethylmethanesulphonate (EMS). Uptake of the labelled mutagen ([¹⁴C]EMS) by the somatic embryos increased with increased incubation time. Presence of dimethyl sulphoxide (DMSO) as a carrier agent during mutagenic treatment was necessary for efficient mutagen uptake.     

ADVENTIVE EMBRYOGENESIS IN CITRUS I. THE OCCURRENCE OF ADVENTIVE EMBRYOS WITHOUT POLLINATION OR FERTILIZATION

Anatomical studies of unfertilized undeveloped seeds from open- and control-pollinated fruits of ten facultative apomictic Citrus cultivars were carried out with the aid of light and epifluorescence microscopes. With or without pollination, adventive embryos autonomously developed at all positions in the nucellus in all cultivars. The adventive embryos initiated at the chalazal end of the nucellus were more vigorous than those initiated at the micropylar end. Because of the lack of endosperm and poor seed development, however, all adventive embryos within the unfertilized seeds terminated their development at the globular or early cotyledonary stages and were unable to germinate under natural conditions. The capability of unfertilized seeds to develop varied from species to species. Growth of the adventive embryos was dependent on nucellus size, but the growth rate of adventive embryos relative to nucellus size was different in different species. Neither pollination, fertilization nor subsequent zygote and endosperm development further stimulated adventive embryo initiation. Conversely, pollination and subsequent fertilization of other seeds in the same fruit slightly, but significantly, suppressed adventive embryo growth in the unfertilized seeds. These facts concerning adventive embryogenesis in unfertilized seeds indicate that neither pollination nor fertilization is essential for in vivo adventive embryogenesis and that normal endosperm is necessary for perfect development of adventive embryos initiated only in the micropylar half of the nucellus.     

青扦胚性细胞悬浮培养中影响体细胞胚发生因素的研究

试验以青扦（Ｐｉｃｅａｗｉｌｓｏｎｉ）的胚性愈伤组织为材料,以改良５９基本成分附加２４－Ｄ１ｍｇ／Ｌ及ＫＴ１ｍｇ／Ｌ为培养介质,比较了液体悬浮与半固体二种培养方式对胚性愈伤组织增殖和体细胞发生的影响,研究了液体悬浮培养过程中影响体细胞胚发生的因素。结果表明：液体悬浮培养好于半固体培养,它的胚性愈伤组织的生长率为２６８％,是半固体培养的１２４倍;体细胞胚的分化率为９３％,是半固体培养的２２倍;悬浮培养较佳的培养条件为：初始细胞密度为２％（鲜重）,蔗糖浓度为２０ｇ／Ｌ,摇床转速为１００ｒ／ｍｉｎ,ｐＨ为５８。经过两个月悬浮培养,将培养物转至１／２改良５９附加ＡＢＡ１ｍｇ／Ｌ的分化培养基上,３个月后每ｇ培养物上可获得２８５个正常的子叶期体细胞胚。     

Callus concentration regulates somatic embryo production in orchardgrass suspension cultures

Summary The effects of callus inoculation concentration and culture duration on somatic embryogenesis of orchardgrass,Dactylis glomerata L., were evaluated in suspension cultures of an embryogenic genotype Embryogen-P. Somatic embryo formation was induced in liquid SH medium containing 30 μM dicamba (SH-30 and 1.5% casein hydrolysate; embryo development was in liquid SH medium without plant growth regulators (SH-0); and embryo maturation and germination occurred on solid SH-0 medium. Callus proliferation in SH-30 suspension cultures was greatest when callus was inoculated into the liquid medium at a relatively high concentration of 4% (4 g callus/100 ml medium), but the induction of somatic embryos was highest in this medium if the callus was inoculated at a lower concentration (<2%). In a second experiment, somatic embryo yield was highest when SH-0 development medium was inoculated with suspension culture callus at 0.1% concentration and declined markedly as inoculation concentration increased. Cell concentration is a critical factor in regulating the somatic embryogenesis response in orchardgrass suspension cultures.     

Water status of callus from Hevea brasiliensis during induction of somatic embryogenesis

To improve somatic embryogenesis in Hevea brasiliensis , the water and plant growth regulator status of the culture medium was studied. Induction of embryogenic tissue from the internal integument of immature seeds was clearly favored by stabilizing the water potential of the culture medium at –0.7 MPa, by using low and decreasing concentrations of 3,4-dichlorophenoxyacetic acid and benzyladenine or by incorporating 10^-7 M abscisic acid in the medium. Each of these changes in the medium favored a specific water status in the callus, namely a high relative water content (93 to 95%) and an elevated water potential (–0.9 MPa). These characteristics were apparently important for initiating somatic embryogenesis, and their decrease corresponded to the loss of embryogenic potential in the callus. Thus, the relative water content and water potential of callus appear to be good markers of its embryogenic state.     

Effect of physical desiccation on plant regeneration efficiency in rice (Oryza sativa L.) variety super basmati

This experiment assessed the effect of partial physical desiccation on plant regeneration efficiency in scutellum-derived embryogenic calluses of rice (Oryza sativa L.) variety Super basmati. A number of callusing cultures were developed, and efficient callus induction was observed on MS (Murashige and Skoog) basal medium supplemented with 2.0 mg/L 2,4-dichlorophenoxy acetic acid. The calluses were proliferated on the same medium for 3 weeks and then shifted to dehydration desiccation treatment for 72 h. The desiccated calluses were cultured on different media for somatic embryogenesis and plant regeneration. A medium with 2.0 mg/L α-napthaleneacetic acid, 10.0 mg/L abscisic acid , 2.0 mg/L kinetin was best for somatic embryogenesis only, but not for further plant development. After 10 d, differentiated calluses were sub-cultured on medium with various concentrations and types of carbohydrates (carbon source) in ¹MS_2j medium. A large number of plantlets (14.51±2.81 and 8.56±2.90 plants/callus) were regenerated via chemical desiccation, on MS with 3% maltose+3% sorbitol and 6% sucrose, respectively. Under dehydration on only simple MS (3% sucrose), 11.23±3.22 plants/callus were developed. Under conditions of dehydration and chemical desiccation, plant regeneration rates were higher than the calluses cultured on simple MS medium in the presence of plant growth regulator. After somatic embryogenesis, >25% plants were sterile. The protocol used here may allow maximum regeneration of normal and fertile plantlets of super basmati rice within 3 months.     

IN VITRO SOMATIC EMBRYOGENESIS AND REGENERATION OF SOMATIC EMBRYOS FROM PIGMENTED CALLUS OF KAPPAPHYCUS ALVAREZII (DOTY) DOTY (RHODOPHYTA,GIGARTINALES)1

In vitro somatic embryogenesis and regeneration of somatic embryos to whole plants through micropropagules was successfully demonstrated from pigmented uniseriate filamentous callus of Kappaphycus alvarezii (Doty) Doty in axenic cultures. More than 80% of the explants cultured on 1.5% (w/v) agar‐solidified Provasoli enriched seawater (PES) medium showed callus development. The callus induction rate was consistently higher for laboratory‐adapted plants. The excised callus grew well in subcultures and maintained its growth for prolonged periods if transferred to fresh medium in regular intervals. Some subcultured calli (<10%) did undergo transformation and produced densely pigmented spherical or oval‐shaped micropropagules (1–5 mm in diameter) that subsequently developed into young plantlets in liquid PES medium. The micropropagule production was further improved through somatic embryogenesis by a novel method of culturing thin slices of pigmented callus with naphthaleneacetic acid (NAA) or a mixture of NAA and 6‐benzylaminopurine. Transfer of embryogenic callus along with tiny somatic embryos to liquid medium and swirling on orbital shaker facilitated rapid growth and morphogenesis of somatic embryos into micropropagules that grew into whole plants in subsequent cultivation in the sea. The daily growth rate of one tissue cultured plant was monitored for seven generations in field and found to be as high as 1.5–1.8 times over farmed plants. The prolific somatic embryogenesis together with high germination potential of somatic embryos observed in this study offers a promising tool for rapid and mass clonal production of seed stock of Kappaphycus for commercial farming.