Actinidia deliciosa in vitro II. Growth and exogenous carbohydrates utilization by explants

Changes in sugar composition (sucrose, glucose and fructose) of medium, callus, stem and leaves of in vitro proliferating explants of Actinidia deliciosa C.F. Liang, Hayward were analyzed together with explant growth at 0, 15, 30, 45 and 60 days of culturing. Autoclaving hydrolyzes a small part of the initial sucrose of the medium into glucose and fructose. In presence of Actinidia explants the initial sucrose decreased to 32% after 15 days of culturing, to 4% after 30 days and to 0.08% at the end of the culture period (60 days). Sucrose increase in the explants did not parallel with its decrease in the medium. Sucrose presence in the explants was evident only during the last month of culturing. After 15 days of culturing a large increase of glucose and fructose was found in the medium but it did not equal the hydrolyzed sucrose. The level of these two monosaccharides remained stable in the medium until the 30th day, then significantly decreased in the second month of culture; neither were completely exhausted at the end of the culture. In the whole explant the highest amount of glucose and fructose was reached after 30 days of culturing.The balance of the three sugars in the medium-explant system, as % distribution of carbon atoms, showed a utilization throughout the whole culture period.Qualitative analyses performed on medium, callus and leaves at 0, 15, and 30 days of culturing revealed the presence of glucose and fructose only and no significant amounts of other hexoses or pentoses. Starch accumulation in the leaves was also observed throughout the culturing.Paper No. 724     

Proline accumulation and sodium sulfate tolerance in callus cultures of Brassica napus L. cv. Westar

Callus cultures of Brassica napus L. cv. Westar were selected which contained 5 – 6 times more proline than unselected callus. Callus pieces from these cultures were able to survive much better after subculture to medium containing 105 mM Na₂SO₄ than unselected callus, or unselected callus cultured on exogenous proline before or during transfer to the salt. Exogenous proline was rapidly absorbed. In unselected callus there was a peak in proline accumulation ca. 2 days after transfer to Na₂SO₄, followed by a decline. In contrast proline accumulation in tolerant callus was linear with time, reaching maximum levels at 8 days. Proline levels induced by exposure to salt were maintained in the absence of stress.Abbreviations DW Dry weight - FW Fresh weight     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis> Wright,an important pharmaceutical crop

Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated from initial infected callus explants.     

Analysis of capsaicinoid biosynthesis pathway genes expression in callus cultures of Capsicum chinense Jacq. cv. ‘Umorok’

The placental tissue of the highly pungent chilli cultivar, Capsicum chinense Jacq. cv. ‘Umorok’, is used as explants for callus induction. Callus cultures were subcultured after every 32 days and growth curves for a period of six consecutive growth cycles were studied till a stable capsaicinoids producing callus cultures were obtained. The capsaicinoids content in placental tissue explants decreased gradually during the first 2 months of culture as the explants dedifferentiated to form friable callus while the biomass and capsaicinoid content did not show much change in the subsequent growth cycles. The maximum callus biomass of 7.8 g freshweight (FW) or 0.56 g dry weight (DW) per culture were obtained on the 24th day of every growth cycle and the maximum average capsaicinoids content (1.6 mg g^?1 FW capsaicin and 0.78 mg g^?1 FW dihydrocapsaicin) were obtained on the 20th day of every growth cycle. To investigate the underlying dynamics for capsaicinoid biosynthesis during callus formation, comparative gene expression analysis of the genes involved in capsaicinoid biosynthesis pathway were also studied by qRT-PCR analysis. When compared with placental tissue, all the studied genes showed reduced expression during callus formation, especially putative aminotransferase (pAMT) and pungent gene 1 (Pun1), which were extensively down regulated from the 3rd month onwards in the callus cultures. Therefore, the present study revealed that the down-regulated expression of mainly two putative genes in capsaicinoid biosynthetic pathway (pAMT and Pun1) resulted in lower accumulation of capsaicinoids in callus cultures compared to placental tissues of fruits.

     

Regeneration of garlic plants (allium sativum L., CV. “Chonan”) via cell culture in liquid medium

Summary Aiming at the genetic improvement of garlic cultivars, a cell suspension protocol was established which includes the induction of friable callus, establishment of cells in liquid medium, plating, regeneration, and bulb formation. Calluses of various textures from compact to friable and from green to yellowish were obtained by culturing explants excised from inner leaves of garlic bulbs on Marashig-Shoog (MS) medium with 2,4 dichlorophenoxy acetic acid (2,4-D), (1.1 mg/liter [5.0 μM]), picloram (1.2 mg/liter [5.0 μM]), and kinetin (2.1 mg/liter [10 μM]). Friable callus occurred on MS-A contained 2,4-D alone (1.0 mg/liter [4.52 μM]) and this callus was used to develop cell suspension cultures, which were maintained in liquid MS-B medium with a 2,4-D/benzyl adenine (BA) (0.5 mg/liter [2.25 μM]: 0.5 mg/liter [2.22 μM]) ratio. High plating efficiency was obtained on MS-C medium with different naphthalene acetic acid/BA combinations. Regeneration occurred after transfer of the caulogenic mass to MS-C medium containing 10 mg/liter (74.02 μM) and 20 mg/liter (148.04 μM) adenine for 60 days, followed by transfer to adenine-free medium. Plantlets transplanted to soil showed normal phenology. Shoots grown on modified MS medium supplemented with indolylbutryic acid (3.0 mg/liter [14.7 μM]) stimulated bulb formation by 30 days in culture.     

Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-dichlorophenoxyacetic acid

Cucumber (Cucumis sativus L.) leaf explants were cultured either continuously on standard medium containing 4.5 µM 2,4- dichlorophenoxyacetic acid (2,4-d) and 4.4 µM benzylaminopurine, or first cultured for various periods at different levels of 2,4-d, picloram or naphthaleneacetic acid (NAA), and then transferred to standard medium. When cultured continuously on standard medium, less than 10% of the explants formed embryogenic callus. Initial culture on picloram or NAA, or on 2,4-d at a low concentration (1.4 µM) did not result in any embryogenic callus formation. Embryogenic callus formation increased to 40% if during the initial phase of the culture (10 days), the 2,4-d concentration was raised to 14 µM. Prolonged culture on 14 µM 2,4-d resulted in less embryogenic callus formation.Abbreviations BA benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Somatic embryogenesis in Canary Island date palm

Shoot regeneration was obtained from leaves of in vitro cultures of wild pear genotypes. The highest regeneration rates, ranging from 40% to 64%, depending on the genotype, were obtained using leaves wounded by three cuts transversely to the mid-rib, a Quoirin and Lepoivre macro-salt composition, 250 mg l^-1 cefotaxime and maintaining the explants in darkness for the first 30 days (induction phase), then transferring them to an auxin-free medium in light (expression phase). A concentration of 8.8 μM BA induced the highest number of explants to produce adventitious shoots. TDZ was less effective than BA and induced hyperhydricity in regenerated shoots. The histological studies revealed that the regenerated shoots originated mainly from callus formed by epidermal and sub-epidermal cells and by cells of the vascular tissue. The regenerated shoots were micropropagated, rooted and transplanted to the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Adventitious shoot regeneration from Sinningia speciosa leaf discs in vitro and stability of ploidy level in subcultures

Summary Leaf explants of Sinningia speciosa were cultured in vitro on Murashige and Skoog (MS) basal medium with various growth substances in order to regenerate shoots. On MS medium supplemented with indoleacetic acid (IAA) and kinetin, 80% of the explants produced green callus and 25 to 30 shoots with roots per explant. On MS supplemented with IAA and N⁶ benzyladenine (BA), 80% of the explants produced green callus and 40 to 50 shoots per explant but lacked roots. After 3–4 mo., these shoots were removed from the initial explants and transferred separately onto MS supplemented with indolebutyric acid for their elongation and successive rooting (3 mo.). Histological studies showed that the callus was associated with mesophyll cell layers, primarily with the spongy parenchyma. The shoots regenerated at the callus surface and were associated with newly differentiated vascular areas. Recurrent regenerations were obtained from leaf explants or apical meristems excised from shoots of the previous subcultures. These explants, as compared to initial cultures, had a high frequency of regeneration and also produced more shoots per explant. Chromosome numbers of root tip cells of the mother plant and of all in vitro-regenerated plants remained constant: 2n=26.     

Embryogenesis and plant regeneration from tissue culture of Canada wildrye,Elymus canadensis L.

Somatic embryogenesis and plant regeneration of Canada wildrye (Elymus canadensis L.) from tissue culture was investigated by culturing immature embryos and inflorescences on MS medium containing 2 mg/l 2,4-D. The optimum size of explants for maximum embryogenic callus formation was 1.0 to 1.5 mm for embryos and 4 to 6 cm for inflorescences. Plant regeneration from the subcultured embryogenic callus was attempted monthly using hormone-free MS medium or MS medium with 0.5 mg/1 2,4-D and 0.3 mg/l GA3. Three hundred and fifty seven plantlets were regenerated from the callus cultures of both explant sources during a six month period. Ten chlorophyll deficient plants accounting for 2.8% of the total regenerants were observed. One plant with white striped leaves survived and was found to be an octoploid.Abbreviations GA3 gibberellic acid - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Induction of callus from axillary buds of taro (Colocasia esculenta var. esculenta,Araceae) and subsequent plantlet regeneration

Summary Axillary buds of taro (Colocasia esculenta var. esculenta, Araceae) cultured on half strength Murashige-Skoog medium (HMS) containing taro extract (HMSTE) and 2, 4, 5-trichlorophenoxyacetic acid produce a compact, hard, slow growing callus which is not very active morphogenetically and produces only a few plantlets. When cultured on HMSTE plus 5 mg 1^–1 each of naphthaleneacetic acid and benzyl adenine (HMSNB) the buds produce a fast growing, friable and morphogenetically active callus. Meristematic regions form on the friable callus after 30 days on HMSNB. If transferred to HMSTE at this point the callus gives rise to plantlets. Addition of taro extract to the media is required for the culture of buds, induction of callus and plantlet regeneration.Abbreviations BA benzyl adenine - BNA b-naphthoxyacetic acid - CW coconut water (liquid endosperm) - DW dry weight - FW fresh weight - HMS half strength Murashige-Skoog medium - HMSCW HMSTE plus 100 ml CW 1^–1 - HMSNB HMSTE plus 5 mg 1^–1 each NAA and BA - HMSTE HMS plus 25 ml taro extract 1^–1 - HMSTR HMSTE plus 2 mg 2,4,5-T 1^–1 - MNA methyl-1-naphthaleneacetate - NAA naphthaleneacetic acid - OCPAA ortho-chlorophenoxyacetic acid - TE taro extract - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

连钱草愈伤组织诱导及增殖研究

对连钱草Glechoma longituba (Nakai) Kupr.愈伤组织培养作了初步的探索,以不同的培养条件,利用连钱草的顶芽、叶、叶柄为外植体,研究了连钱草愈伤组织的培养。结果表明：在以MS培养基和LS培养基为基本培养基附加不同外源激素2,4-D、NAA、KT、BA条件下,连钱草在一个较宽的生长范围内,均可诱导产生连钱草的愈伤组织,但不同外植体、不同类型植物激素及其不同浓度对愈伤组织发生均有一定影响：作为外植体,连钱草叶柄和叶都可顺利诱导出愈伤组织;生长素2,4-D对连钱草外植体的脱分化起促进作用,但NAA却抑制愈伤组织的形成;细胞分裂素KT和BA均能与2,4-D组合促进愈伤组织的诱导。MS+2,4-D在光暗交替条件下和LS+2,4-D在黑暗条件下有利于连钱草愈伤组织的诱导,最佳诱导和增殖条件是MS+2,4-D（1.5 mg·L^-1）+BA（1.0 mg·L^-1）光、暗交替（光照14 h·d^-1）。在此条件下, 30 d后,叶的诱导率达91.38%,叶柄的诱导率达100%;愈伤组织继代培养14 d后,平均增殖率达202.2%。     

Comparison of two methods for callus culture and plant regeneration in wheat (Triticum aestivum)

Callus growth and development involve a complex relationship between the explants used to initiate callus, the constituents of the medium and the environmental conditions during culturing. Use of high molecular weight osmotica such as polyethylene glycol (PEG-4000) results in non-solidification of agar medium used for culturing and selection. Thus, a new filter paper bridge technique was compared with the existing agar medium for callus initiation, multiplication, and plant regeneration of wheat. The yield of both total and embryogenic callus was doubled and significantly higher number of regenerants was obtained on filter paper bridges compared to agar medium.     

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

The Cultivation of Tissues of Cupressus lusitanica in vitro

Conditions for In vitro culture of initial explants of Cupressus lusitanica were determined as the first step of an investigation of physiological differences between tissues obtained from plants, or plant parts, of different ages. Explants proliferated well on a basal agar medium containing only Heller's mineral salts and sucrose. Addition of several vitamins, phytohormones, and coconut water to this basal medium stimulated callus growth only slightly or not at all, and transplanted callus soon died. Proliferation of initial explants was considerably stimulated by increasing the concentration of mineral salts in the medium, and the growth habit changed from compact to friable. Callus could be transplanted to, and maintained on, a high-salt medium enriched by vitamins. Whereas added auxins still stimulated proliferation of initial explants and transplanted callus only slightly, added coconut water was considerably more effective than on basal medium. Thus results confirm observations by others with tobacco tissues: Additions of phytohormones and other organic substances may not exert their potential effects if growth is limited by sub-optimal mineral nutrition.     

Plant regeneration via somatic embryogenesis from protoplasts of six explants in Coker 201 (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.     

杜仲叶片和叶柄愈伤组织的诱导和植株再生

本实验以5~6年生杜仲叶片及叶柄为外植体,研究了杜仲愈伤组织诱导及植株再生的方法。结果表明:接种于补加NAA(2.0~4.0 mg/L)或BA(1.0 mg/L)+NAA(2.0~4.0mg/L)的MS培养基上的叶片和叶柄,经21~28d培养后,脱分化形成绿色或浅绿色致密愈伤组织,频率达到70%以上。绿色致密愈伤组织在补加BA(2.25~2.75 mg/L)+NAA(0.15 mg/L)的MS培养基上经过1~2次继代之后,即出现茎芽分化,频率在15%以上,只是其中许多都是畸形苗,正常苗频率较低。此问题尚在研究之中。选择生长健壮的再生植株,切除其基部愈伤组织,然后将切口浸泡在250mg/L无菌ABT生根粉溶液中3~5sec,再插入1/4强度无激素MS培养基中, 2~3周后,在苗基部长出1~3条白色粗壮的不定根,生根频率在60%以上。     

Physiological,elemental, and stable isotope responses of the organs of mungbean to reduced atmospheric pressure

The effects of hypobaric conditions on stable isotope and mineral element concentrations during the germination of mungbean [Vigna radiata (Linn.) Wilczek] were evaluated. Mungbean seeds were cultured in lower atmospheric pressure (60 kPa) and normal air pressure (101 kPa) conditions, respectively. Oxygen and carbon dioxide partial pressures were maintained at 21 and 0.04 kPa, respectively. At 60 kPa, the fresh weight (FW) and dry weight (DW) of plants significantly increased by 5.41 and 9.62%, respectively, compared to those at 101 kPa after culturing for 7 d. Twelve mineral elements were compared among three organs (leaf, stem, and root) from seedlings grown under hypobaric and normal atmospheric conditions. This showed that lower air pressure generally improved element accumulation in the plant. A significantly lower value of δ ¹³C was observed at 60 kPa compared to that at 101 kPa. In addition, a significant increase in δ ¹⁵N value was detected in three different organs of plants grown under 60 kPa. Our survey provides a foundation for future field and laboratory studies on the influence of air pressure on plants, particularly in terms of stable isotope and mineral elements.     

Factors affecting callus and shoot formation from in vitro cultures of Lens culinaris Medik.

The influence of culture medium and explant on callus and shoot formation of lentil (Lens culinaris Medik.) has been studied. Three different explants (shoot-tip, first node and first pair of leaves) from three Spanish lentil cultivars were cultivated on two basal media: Murashige and Skoog medium (MS) and medium with mineral salts of MS medium plus vitamins of Gamborg's B5 medium (MSB), supplemented with growth regulators. Media with 2,4-D induced the formation of calli in all explants, but no organ regeneration was obtained from these calli. Multiple shoot formation was obtained from 33% to 92% of the explants in media supplemented with 2.25 mg l^–1 of BA and 0.186 mg l^–1 NAA+2.25 mg l^–1 BA; in the other media one to two shoots per explant were formed in 10 to 98% of the explants. Root formation from explants was achieved only in media with NAA or IAA. Of the explants tested, the best morphogenetic responses were obtained from nodes and the poorest from leaves.