Soybean somatic embryogenesis: Effects of hormones and culture manipulations

Somatic embryos were induced in cultures of immature soybean (Glycine max (L.) Merr) embryos, or isolated cotyledons on MS modified medium supplemented with NAA and 2,4-D, BAP and ABA. When NAA and 2,4-D were compared at similar concentrations (25 and 23 M), 2,4-D produced larger number of somatic embryos, however, embryogenesis efficiency was improved in media containing NAA by using higher levels (100–150 M) of the auxin. Somatic embryo morphology varied with auxin type: NAA-induced embryos more closely resembled zygotic embryos than did 2,4-D-induced embryos. Additions of BAP or ABA to auxin-containing media had either no effect or reduced embryo production, although ABA altered the morphology of 2,4-D-induced embryos. In media containing both NAA and 2,4-D, the latter was dominant in terms of embryo morphology. The effects of subculture frequency and of transfers between 2,4-D and NAA media were investigated: Subculture frequency influenced mainly the frequency of normal embryos, while preculture on 2,4-D increased subsequent embryogenesis efficiency on NAA medium but reduced the frequency of normal embryos.Abbreviations Em^-2 s^-1 microEinsteins per square meter per second - NAA -naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - ABA abscisic acid - BAP benzylamino purine This paper (No. 86-3-96), is published with the approval of the director of the Kentucky Agricultural Experiment Station.     

一品红体细胞胚胎发生与植株再生

一品红不同部位愈伤组织诱导能力存在差异,嫩茎>幼花序>嫩叶。愈伤组织的长势主要受生长素的影响,细胞分裂素对愈伤组织生长有促进作用;但在含6-BA和NAA的培养基中诱导出的愈伤组织,其胚性明显强于单独用NAA诱导出的愈伤组织。液体悬浮培养是一品红体细胞胚胎高频发生的中间步骤。不同浓度BA对一品红体细胞胚的萌发率影响不大,萌发培养基中KNO₃含量加倍可提高萌发率。     

In vitro plant regeneration of Aster scaber via somatic embryogenesis

We established an in vitro plant regeneration system via somatic embryogenesis of Aster scaber, an important source of various biologically active phytochemicals. We examined the callus induction and embryogenic capacities of three explants, including leaves, petioles, and roots, on 25 different media containing different combinations of α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The optimum concentrations of NAA and BA for the production of embryogenic calli were 5.0 μM and 0.05 μM, respectively. Media containing higher concentrations of auxin and cytokinin (such as 25 μM NAA and 25 μM BA) were suitable for shoot regeneration, especially for leaf-derived calli, which are the most readily available calli and are highly competent. For root induction from regenerated shoots, supplemental auxin and/or cytokinin did not improve rooting, but instead caused unwanted callus induction or retarded growth of regenerated plants. Therefore, plant growth regulator-free medium was preferable for root induction. Normal plants were successfully obtained from calli under the optimized conditions described above. This is the first report of the complete process of in vitro plant regeneration of A. scaber via somatic embryogenesis.     

Induction,germination and shoot development of somatic embryos in cassava

Four Indonesian and two Latin-American cassava genotypes (Manihot esculenta Crantz), were evaluated for their ability to develop somatic embryos from young leaf lobes. All genotypes formed somatic embryos but they differed in the frequency of embryos induced. The best genotypes, M. Col 22 and Tjurug, produced germinating embryos (GE) on 81% (22.1 GE/initial leaf lobe) and 46% (4.3 GE/initial leaf lobe) of the cultured leaf lobes, respectively. Up to 57% of the germinating embryos of M. Col 22 and 12% of Tjurug produced either normal or malformed shoots. Most malformed shoots developed into shoots with normal morphology after prolonged culture. All shoots formed roots after transfer to medium without BAP. Roots of all normal and most malformed regenerants had the original ploidy level (2n=36). Regardless of whether the plants were multipliedin vitro (150 plants) or in the greenhouse (30 plants) there were no morphological differences compared to parent plants.     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Micropropagation of 21 species of Mexican cacti by axillary proliferation

Summary We have developed micropropagation systems for 21 species of Mexican cacti using explants from seedlings germinatedin vitro or shoot segments of juvenile 2–3-yr-old greenhouse plants. The species propagated belong to the generaAstrophytum, Cephalocereus, Coryphantha, Echinocactus, Echinocereus, Echinofossulocactus, Ferocactus, Mammillaria, Nyctocereus, andStenocactus. Multiple shoot formation from areoles was achieved in Murashige and Skoog (MS) medium supplemented with either 1 or 2 mg N⁶-benzyladenine (BA) per 1 (4.44 or 8.87 μM) or BA at 1 or 2 mg/l plus naphthaleneacetic acid at 0.1 or 1 mg/l (0.54 or 5.37 μM). The requirements of growth regulators for optimal shoot proliferation, the velocity of the response, and the number of buds produced by explant were different among the genera and species studied. Rooting of the shoots generatedin vitro was achieved in MS medium supplemented with indoleacetic acid at 0.5–1 mg/l (2.85–5.71 μM) or indolebutyric acid at 0.5–1 mg/l (2.46–4.90 μM). Finally, 70–95% of the rooted plants transferred to potting medium survived.     

Long-term multiplication of onion (Allium cepa L.) by cyclic shoot regeneration in vitro

We succeeded in cultivating onion plants in vitro with a high potential for shoot regeneration. The apex must be destroyed or injured to obtain axillary buds. This capacity was restricted to the abaxial base of the youngest sheaths. It was shown necessary to restore plant individuality before further proliferation; this process constituted one cycle. For successive regeneration each cycle was composed of three steps: shoot proliferation in the presence of a cytokinin, shoot individualization and plant development in the absence of growth regulators. Effect of growth regulators on the physiological status of onion plants cultured in vitro is discussed.Abbreviations BA 6-benzyladenine - NAA naphthaleneacetic acid     

极东锦鸡儿幼胚子叶的体细胞胚胎发生和植株再生

以极东锦鸡儿未成熟合子胚子叶为外植体进行其体细胞胚胎发生和植株再生研究。在添加不同BA与NAA或2,4-D,外加500mg·L~（-1）水解酪蛋白、30g·L~（-1）蔗糖和8g·L~（-1）琼脂的MS培养基上诱导产生了体细胞胚。在5mg·L~（-1）NAA＋5mg·L~（-1）BA和5mg·L~（-1）2,4-D＋1mg·L~（-1）BA处理中体胚诱导率分别为14%和10%;NAA处理每外植体上诱导出的体胚数量最多为4.3个,而2,4-D为10.5个。体细胞胚经成熟培养后,在添加0.01mg·L~（-1）NAA、20g·L~（-1）蔗糖和6g·L~（-1）琼脂的MS培养基上萌发率达到58.94%。萌发的体胚在MS培养基上长成正常小植株,再生率为87%。经炼苗后的体胚苗移植到草炭土：蛭石：珍珠岩=5：4：1（V/V/V）的栽培基质中,可以正常生长,移栽成活率为40%。     

稀有植物裸果木的组织培养及植株再生

对稀有植物裸果木进行组织培养研究,在MS培养基上裸果木的下胚轴脱分化形成愈伤组织,并进一步分化形成再生植株。激素种类及其浓度是器官脱分化与植株再生的决定因素。诱导下胚轴形成愈伤组织的最适培养基为MS 1mg／L 6-BA 0．5mg／L NAA;芽分化诱导的最适培养基为MS 1 mg／L6-BA;生根的最适培养基为不含任何激素的1／2MS培养基。     

珍稀濒危植物蒙古扁桃的组织培养及植株再生

对珍稀濒危植物蒙古扁桃进行组织培养获得再生植株。实验结果表明,在MS培养基上蒙古扁桃幼苗茎尖,茎切段和叶片等外植体均可以脱分化形成愈伤组织,并进一步分化形成再生植株。器官的脱分化与再分化决定于培养基中的激素种类及其浓度。诱导愈伤组织形成的最适培养基为MS＋6－BA0.8mg/L NAA0.1mg/L,芽分化诱导最适培养基为MS＋6－BA0.8mg/L,诱导生根的最适培养基是MS＋IBA0.5mg/L。     

Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production

Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as “Indian Ginseng”, is a medicinally and industrially important plant of the Indian subcontinent and other warmer parts of the world. The plant has multi-use medicinal potential and has been listed among 36 important cultivated medicinal plants of India that are in high demand for trade due to its pharmaceutical uses. The medicinal importance of this plant is mainly due to the presence of different types of steroidal lactones- withanolides in the roots and leaves. Owing to low seed viability and poor germination, the conventional propagation of W. somnifera falls short to cater its commercial demands particularly for secondary metabolite production. Therefore, there is a great need to develop different biotechnological approaches through tissue and organ culture for seasonal independent production of plants in large scale which will provide sufficient raw materials of uniform quality for pharmaceutical purposes. During past years, a number of in vitro plant regeneration protocols via organogenesis and somatic embryogenesis and in vitro conservation through synthetic seed based encapsulation technology have been developed for W. somnifera. Several attempts have also been made to standardize the protocol of secondary metabolite production via tissue/organ cultures, cell suspension cultures, and Agrobacterium rhizogenes-mediated transformed hairy root cultures. Employment of plant tissue culture based techniques would provide means for rapid propagation and conservation of this plant species and also provide scope for enhanced production of different bioactive secondary metabolites. The present review provides a comprehensive report on research activities conducted in the area of tissue culture and secondary metabolite production in W. somnifera during the past years. It also discusses the unexplored areas which might be taken into consideration for future research so that the medicinal properties and the secondary metabolites produced by this plant can be exploited further for the benefit of human health in a sustainable way.     

Identification of a novel elite genotype for in vitro culture and genetic transformation of cotton

Hypocotyls of cotton (Gossypium hirsutum L.) cultivars cv. YZ-1, Coker 312 and Coker 201 were inoculated on Murashige and Skoog callus induction medium. YZ-1 exhibited a very high regeneration potential, with 81.9 % of the explants inoculated differentiated into embryogenic callus within 8–10 weeks. During the process of callus maintenance (subculture for 1 to 3 years), the total embryos number in Coker 312 and Coker 201 calli dropped sharply, and the percentage of embryo germination decreased. On the contrary, the callus of YZ-1 consistently maintains a high frequency of plant regeneration after long-time subculture. Transgenic kanamycin-resistant calli of Coker 201 partially lost the ability of somatic embryogenesis and plant regeneration. The stress produced by the transformation procedure slightly affected somatic embryogenesis and plant regeneration of YZ-1, which showed minimum loss of plant regeneration ability.     

温度对枳椇次生胚发生和植株再生的影响

在含有2,4-D的培养基上所诱导和增殖的枳棋体细胞胚只能形成无再生能力的畸形胚。通过次生胚发生途径可以在不加植物生长调节物质的培养基诱导出发育正常的体细胞胚,子叶期胚的次生胚诱导能力高于早期体胚。相对高的温度(30℃)有利于次生胚的诱导,相对低的温度(20℃)更有利于次生胚的发育、植株再生和移栽成活。     

Adventitious shoot regeneration from vegetative shoot apices in pear and putative role of cytokinin accumulation in the morphogenetic process

Adventitious shoot regeneration was obtained from callus produced from main vegetative apices of pear of in vitrogrown shoots of Italian cultivars Spadona and Precoce di Fiorano and wild pear genotypes ISF54 and ISF61. The highest morphogenetic response was obtained on a medium containing 8.8 M 6-benzyladenine, 1.0 M -naphthaleneacetic acid and 250 mg l^–1cefotaxime. The explants were maintained for 30 days in darkness and then transferred to an auxin-free medium and to the light. Histological studies revealed that the new vegetative buds originated from callus that completely altered the morphology of the explant tissues by the 30th day of culture. The in situ localisation of cytokinins, performed using antibodies with marked specificity against zeatin (Z) and isopentenyladenine, revealed an accumulation of Z in the cambiform cells of the leaf primordia and in the shell zone of the new forming buds showing a primary role of this cytokinin in cell differentiation of in vitro pear organogenesis.     

组织培养中畸形胚状体及超度含水态苗的研究

畸形胚状和超度含水态苗是组织培养中常见的2种形态、生理异常现象,由于其难以发育成苗或很难移栽成活而严重影响组织培养的成功率。概述了这2种畸形现象的的形态特征及防止方法等,并讨论了应进一步研究的问题。     

Somatic embryogenesis and plant regeneration in Cyphomandra betacea (Cav.) Sendt

Callus cultures with globular proembryogenic structures were induced from zygotic embryos and hypocotyl segments of Cyphomandra betacea on MS medium supplemented with 2,4-D. Proembryogenic structures produced somatic embryos and plantlets on regulator-free basal medium. Pieces of embryogenic callus subcultured on medium with the same original composition gave rise to new globular structures and the potential for plantlet regeneration has been maintained for over a year. The histological examination of these proembryogenic structures suggested that somatic embryos arise from single cells. Regenerated plants are phenotypically normal, having diploid chromosome numbers (2n = 24).     

Influence of external factors on secondary embryogenesis and germination in somatic embryos from leaves of Quercus suber

Somatic embryogenesis was obtained in cultures of leaves from young seedlings of Quercus suber L. A two-stage process, in which benzyladenine and naphthaleneacetic acid were added first at high and then at low concentrations, was required to initiate the process. Somatic embryos arose when the explants were subsequently placed on medium lacking plant growth regulators. The embryogenic lines remained productive, by means of secondary embryogenesis, on medium without growth regulators. However, this repetitive induction was influenced by the macronutrient composition of the culture medium. Both low total nitrogen content and high reduced nitrogen concentration decreased the percentage of somatic embryos that showed secondary embryogenesis. Our results suggest that alternate culture on medium that increases embryo proliferation and a low salt medium prohibiting embryo formation will partially synchronize embryo development. Chilling slightly reduced secondary embryogenesis but gave a modest increase in germination. Maturation under light followed by storage at 4 °C for at least 30 days gave the best results in switching embryos from an embryogenic pathway to a germinative one. Under these conditions 15% of embryos showed coordinated root and shoot growth and 35% formed either shoots or mostly roots. These percentages were higher than those of embryos matured in darkness. This result indicates that a specific treatment is required after maturation and before chilling to activate the switch from secondary embryo formation to germination.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BA indolebutyric acid - MS Murashige & Skoog (1962) medium - SH Schenk & Hildebrandt (1972) medium - G Gamborg (1966, PRL-4-C) medium (macronutrients in mg l^–1: NaH₂PO₄·H₂O, 90; Na₂HPO₄, 30; KCl, 300; (NH₄)₂SO₄, 200; MgSO₄·7H₂O, 250; KNO₃, 1000, CaCl₂·2H₂O, 150) - PGR plant growth regulator     

Genetic Control of Totipotency of Plant Cells in an in vitro Culture

The main approaches have been considered to studying the genetic control of plant cell totipotency in an in vitro culture. The capacity of cultured plants for callusogenesis, organ formation, and somatic embryogenesis depends on the activity of genes that determine and maintain the meristematic state of cells, level of hormones in the cells, and sensitivity to hormones, as well as on the activity other genes that control different stages of plant morphogenesis.     

两个苏丹草品系成熟种子的组织培养和植株再生研究

该研究以苏丹草品系S722和Sa的成熟种子为外植体、MS培养基为基础培养基,2,4-D和NAA各3个浓度共6个处理对这两个苏丹草品系成熟种子进行愈伤诱导,探讨不同品系在不同植物生长物质浓度及植物生长物质组合中诱导愈伤组织和继代培养以及分化的能力。结果表明：苏丹草S722和Sa成熟种子的愈伤诱导率差异不显著,平均诱导率为17.19％。诱导培养基中2,4-D浓度为0.5或1 mg?L-1时,诱导效果最佳,而添加NAA不能提高愈伤诱导率。在继代培养中,设定2,4-D和6-BA各两个浓度共4个处理组合,处理1(2,4-D 1 mg?L-1＋6-BA 0 mg?L-1)的继代培养效果最佳。为了解不同植物生长物质对愈伤分化的影响,设定6-BA、NAA 各两个不同浓度、KT 3个不同浓度共5个处理组合对继代培养的愈伤进行分化培养。在5个处理中,处理1(6-BA 2 mg?L-1＋NAA 0 mg?L-1＋KT 0 mg?L-1)对 S722成熟种子诱导的愈伤分化率最高,达33.3％。在这两个苏丹草品系中,S722更容易分化培养。综上结果表明,2,4-D浓度为1 mg?L-1时诱导愈伤和继代培养效果较好,6-BA浓度为2 mg?L-1时分化效果较好。另外,针对不同苏丹草品系进行组织培养和植株再生时,适当调整植物生长物质浓度能提高植株再生的成功率。     

Effects of medium composition and culture duration on in vitro morphogenesis of sweet potato

In vitro morphogenesis of sweet potato (Ipomoea batatas) shoot explants after cultures in callus initiation medium (CIM) with two sucrose contents and plant regeneration medium (PRM) with three growth regulator combinations for different durations was studied. After 4 weeks, explants on 5 % sucrose CIM had significantly more shoots but similar or lower root fresh mass and callus fresh mass than those on 3 % sucrose CIM subsequent to transfer for 6 weeks on all three PRM. Cultures transferred to growth regulator-free PRM after 4 and 12 weeks on 5 % sucrose CIM formed plants through organogenesis and embryogenesis, respectively. Embryogenic cultures from 4 weeks on CIM + 10 weeks on callus proliferation medium when transferred to PRM without growth regulator for 4 and 8 weeks produced multiple embryos in the prior and both embryos and shoot buds in the later.