Liter-1 NAA Research Articles

In Vitro Micropropagation of 'Yukiwariso' (Hepatica nobilis Schreber var. japonica f. magna) by Leaf Segment Culture

This study was conducted to establish a system of in vitro micropropagation of Hepatica nobilis. Leaf segments, excised from commercial plants, formed calli at high frequency in MS medium that contained 0.1-10 mg·liter-1 NAA and BA. The highest percentage of bud formation was observed to be about 20% in the medium supplemented with 0.5 or 1 mg·liter-1 NAA and 5 mg·liter-1 BA. Embryoid formation was stimulated on the medium containing 0.1 mg·liter-1 NAA and 0.5 mg·liter-1 BA, or 1 mg·liter-1 NAA and BA. When 1 to 2 mm long adventitious buds were excised from leaf segments and recultured every 8 weeks on MS medium supplemented with BA, GA3 or both for 24 weeks, they developed into shoots (referred to as the primary shoots in this text). The survival rates of the primary shoots were higher in the medium containing both BA and GA3 than in the medium with BA alone. When primary shoots were cultured on MS medium containing 5 mg·liter BA and 10 mg·liter-1 GAs, they formed the highest number of new shoots, referred to as secondary shoots in the text. When excised secondary shoots were cultured in MS medium containing 10 mg·liter-1 NAA or IBA in the dark for a week and then transplanted into MS-medium without any hormones, they developed roots at a high frequency, whereas shoots transplanted to MS-medium with NAA or IBA formed only callus at the base. Following acclimatization, rooted plants that were transplanted into pots and grown in a glasshouse, flowered within 2 years.

Planat Regeneration from Suspension Cultures in Japanese Morning Glory (Ipomoea nil (L.) Roth.)

Embryogenic suspension cultures of Japanese morning glory (Ipomoea nil (L.) Roth.) cv. Violet were established from immature embryos by using a liquid N6- based medium (N6 inorganic salts and MS organic constituents) with 3 mg·liter-1 NAA and 60 g·liter-1 sucrose. Suspension cells showed a 1.5 to 2.5-fold increase in packed cell volume in one week. When suspension cell clusters were transferred to MS media, containing 0-0.5 mg·liter-1 NAA, 60 g·liter-1 sucrose, and 3.2 g·liter-1 Gellan gum, 3.57-3.90 somatic embryos/cell clusters were produced. For shoot formation, these somatic embryos were transferred to MS medium with 0.2 mg·liter-1 IAA, 2 mg·liter-1 BA, 30 g·liter-1 sucrose and 10 g·liter-1 agar. The developing shoots were transferred to 1/2MS medium (half-strength MS inorganic salts) containing 30 g·liter-1 sucrose and 10 g·liter-1 agar for rooting. As many as 40 plantlets were obtained from a 0.1 ml packed cell volume of suspension cell clusters in 3-4 months. That the regenerated plants were successfully transferred to pots demonstrates that this plant regeneration system may facilitate micropropagation of mutant Japanese morning glory via embryo culture and transformation in I. nil.

Adventitious Bud and Embryoid Formation in Garland Chrysanthemum Leaf Culture.

Primary leaves of garland chrysanthemum seedlings were cultured on MS medium supplemented with either 0.1 mg·liter-1 BA+0.1 mg·liter-1 NAA or 0.1 mg·liter-1 BA+1 mg·liter-1 NAA. Adventitious buds were mainly formed in the presence of 0.1 mg·liter-1 BA+0.1 mg·liter-1 NAA at the cut end of the explants 6-9 days after culture, whereas embryoids were mainly formed in the presence of 0.1 mg·liter-1 BA+1 mg·liter-1 NAA at the cut ends or in various areas of the explants 9-12 days after culture. The embryoids had cotyledon-and hypocotyl-like tissues, but lacked definite root structures. Histological observations revealed that these adventitious buds and embryoids were formed directly from meristematic tissues derived from epidermal and subepidermal tissues and did not originate from callus.

グラジオラス木子茎頂からの不定はい形成と花色変異

グラジオラス木子茎頂からの不定胚培養系の確立と得られた再分化個体に生じた花色変異について検討し, 以下の結果を得た.1. 木子茎頂からの不定胚形成率は, MS培地に5mg・liter-1NAAを添加した区で最も高く51.1%であった.不定胚形成率には品種間差異が認められ, 'トパーズ', 'トラベラー'が高く'ヘクター'は低くかった.2. 不定胚由来個体からは花色変異体が得られた.花色変異はいずれも個体全体におよんでいた.花色変異の出現には品種間差異があり, 'トパーズ'を除く'トラベラー', 'ヘクター'で発生した.また花色は淡色化および濃色化の両方に変異していた.3. 'トラベラー'から得られた花色変異系統は, 原品種に比べて草丈, 花穂長, 小花, 小花数がやや小型化または減少の傾向にあった.花色変異系統を分球と木子により増殖して得られた一部の開花個体では, 原品種と同様の花色に戻る花色復帰が認められた.4. 不定胚由来個体に花色変異が認められたことから, 不定胚培養系はグラジオラスの育種に有効であることが示唆された.

Ｉｎ ｖｉｔｒｏ培養により再生したササユリ子球の維管束形成

本実験は, ササユリの再生した子球を用いて植物生長調節物質と維管束組織の発達との関係を明らかにする目的で行った.培養8週間後の再生子球のりん片では, 1本の大維管束と2本の小維管束が認められた.維管束の配列ではりん片と短縮茎は並立維管束, 根は放射維管束であった.りん片の大維管束は2節下位のりん片の維管束と連絡し, 小維管束は1節下位のりん片の維管束と連絡していることが明らかになった.また, 根原基は短縮茎内部に形成された輪状の組織から発生していた.植物生長調節物質無添加区で培養した再生子球では維管束の走向に規則性が認められたが, NAA 0.01mg・liter-1とBA 2.0mg・liter-1の添加区では規則性が認められなかった.また, NAA 0.01mg・liter-1とBA 2.0mg・liter-1を添加した培地で培養した場合, 子球の中央部に多くの維管束が認められた.以上の結果から, 高濃度のBAは子球の維管束の配列を不規則にすることが明らかとなった.

クロユリのｉｎ ｖｉｔｒｏ培養による増殖に関する研究 （第１報） 母球からの出葉，着葉，開花および形成された新球などの特徴と小球状りん片からの子球形成

Wild black lily bulbs were planted in pots and grown for 8 months in a glasshouse. The bulbs produced scapes with 1a2 terminal flowers and 5a6 leaves with a verticillate phyllotaxis. Tow new bulbs were formed on the mother-bulb as it was consumed and eventually rotted. Morphological characteristics matched those of Fritillaria camtschatcensis Ker-Gawl. Small globule-scales of the bulbs were excised and cultured on MS-media without plant hormones in the dark or under light of 40μmol·m-2·sec-1 at 15, 20, and 25°C for 8 weeks. The optimum condition for bulblet formation was a MS-medium containing a combination of 0.1 mg·liter-1 NAA and 0.1 mg·liter-1 24-epibrassinolide (EB) at 20°C in the dark.

培養増殖中の温度と継代がスターチス・シヌアータの開花に及ぼす影響

1. スターチス・シヌアータ(Limonium sinuatum Mill.) 'Early Blue'の花穂切片を外植体とし, 4週間の初代培養を20℃ならびに27℃下で行った後, 20℃4週間の発根培養に移し, 器外に植え出して20℃で栽培した.20℃区では67%が抽だいしたのに対し, 27℃区では13%にとどまった.2. 初代培養後, 20℃での4週間の継代培養を繰り返し, 最後に発根培養を行って植え出したところ, 初代培養後ただちに発根培養に移した場合, 抽だい率は67%であったのに対し, 1回継代培養を行うと抽だい率は43%と低くなり, 2回以上継代培養を行うと全く抽だいが観察されなくなった.3. 継代培養中に2℃4週間の低温処理を行い発根培養に移した場合, また発根培養中に同様の低温処理を行った場合, いずれも植え出し後の抽だい率が著しく高くなった.4. 継代培養中に2℃4週間の低温処理を行っても, その後20℃で増殖のための継代培養を繰り返すと, 植え出し後の抽だい率が低下した.5. 以上より, スターチス・シヌアータの培養苗生産においては, 高い培養温度や継代培養の繰り返しが培養苗の開花能力の消失を引き起こす原因となること, 培養中に低温処理を行うことにより培養苗に開花能力を再付与しうることが示された.

グラジオラス花らい子房からのカルス形成と植物体再生

Callus formation and plant regeneration from flower bud organs in Gladiolus were studied. The results were as follows: 1. Callus was not inducible from upper parts of the perianth, but was obtained from their lower parts and from ovaries at frequencies of 56.0% and 94.0%, respectively; about 64.0% of these calli regenerated shoots. 2. Among ovaries at different developmental stages, those samples 2∼3 days before anthesis were most efficient in callus formation and shoot regeneration. 3. Calli were induced from all explants of ovaries on MS medium containing 5mg·liter-1 NAA and 5mg·liter-1 BAP. 4. Shoots were regenerated most efficiently from the calli on MS medium containing 2mg·liter-1 BAP. 5. Callus formation from ovaries varied little among varieties, but shoot regeneration from the calli varied a great deal, e.g. regenerating frequency of cv. Traveler was much higher than that of cv. Hector. 6. When regenerated shoots were transferred to the MS medium, shoots and roots developed and subsequently corms formed at the base of the shoots. After corms were released from their dormancy by a low temperature treatment and transplanted into the field, they developed shoots and roots and then flowered. Flower shape and color of the regenerated plant had the same characteristics as the mother plant.

トルコギキョウプロトプラストからの植物体再生

Optimal conditions to regenerate plants from Eustoma grandiflorum 'Bicolor Purple' protoplasts investigated in this study led to an efficient plant regeneration methood.Protoplasts were isolated enzymatically from young leaves of Eustoma grandiflorum seedlings cultured aseptically on MS medium for 50 days. The enzyme solution contains 2.0% Cellulase Onozuka R10, 0.2% Macerozyme R10, 0.1% CaCl2•2H2O and 9.0% mannitol (pH 5.5). Isolated protoplasts were cultured in a modified MS (200 mg•liter-1 NH4NO3) medium containing 1.0% sucrose, 9.0% mannitol, 1.0 mg•liter-1 NAA and 0.1mg•liter-1 BA at the density of 12×104•ml-1.Cell division occurred after 45 days in culture. A fresh medium was added weekly to promote colony formation. Colonies (0.51.0 mm in diameter) were transferred to a modified MS agar medium containing 0.1 mg•liter-1 BA and 1.0 mg•liter-1 NAA for callus proliferation. Shoots regenerated rapidly upon transferring the calli to MS mediumcontaining 0.3 mg•liter-1 BA.The regenerated shoots were propagated on MS agar medium containing 0.1 mg•liter-1 BA and 1.0 mg•liter-1 GA3. Hyperhydration of shoots was prevented by ventilating the culture bottles through PTFE membrane (18 mm diam.) attached to the cap. Shoots which elongated more than 1 cm were treated with Oxyberon powder (0.5% IBA) and then transplanted to rock wool cubes. The IBA-treated shoots formed the roots and grew into normal plants. But the percentage of bicolor flowers in protoplast-derived Eustoma grandiflorum plants was significantly lower than the usual means of obtaining plants fromseeds.

組織培養によるフキ（Ｐｅｔａｓｉｔｅｓ ｊａｐｏｎｉｃｕｓ Ｆｒ．Ｓｃｈｍｉｄｔ）ウイルスフリー株の大量増殖

The vegetative propagation rate of Japanese butterbur plants is low, it often permits four viruses to be transmitted to the next generation through the rhizomes as in other vegetatively propagated crops. All of the butterbur plants in the field are eventually inflicted with viruses. The purpose of this study is to establish an efficient masspropagation system of virus-free plants by tissue culture.Shoots from flower-heads were induced on Murashige and Skoog's (MS) solid medium containing 0.1 mg•liter-1 NAA and 1.0mg•liter-1 BA. After 12 weeks of culture, 5.2 shoots per explant were obtained from 80% of the flower-heads.Rapid clonal propagules were obtained when 2-cm shoots were transferred to MS liquid medium supplemented with 0.01mg•liter-1 NAA and 3.0mg•liter-1 BA and placed on a gyrating, rotary shaker set at 100 rpm. After 8 weeks of culture, 80.9 shoots from axillary buds were obtained. These shoots rooted remarkably well in half-strength B 5 liquid medium without phytohormones while being gyrated.All regenerated plants became acclimated readily when grown in a sandy loam at 25°C.Although no cucumber mosaic virus (CMV) or butterbur mosaic virus (BuMV) was detected in the plants, 8.1% were infected with arabis mosaic virus (ArMV). Thus, 91.9% of the regenerated plants were virus-free.These results indicate that this micropropagation system of using flower-heads and axillary buds seems adaptable for a large-scale system of supplying virus-free Japanese butterbur plants.

Shoot Formation on Floral Organs of Japanese Iris in Vitro.

For the purpose of achieving micropropagation in Japanese iris (Iris ensata Thunb.), various explants, namely pistils, perianths, stamens, anthers, filaments, perianth-ovary junctions, ovaries and pedicels, were cultured in vitro on MS media with or without BA and NAA.Shoot formation was observed from explants of perianth-ovary junctions and upper portions of ovaries on media containing BA and NAA. Explants of lower portions of ovaries formed shoots only on the medium containing 5 mg•liter-1 BA and 5 mg•liter-1 NAA. A higher frequency of shoot formation was observed in explants of perianth-ovary junctions than those of ovaries.No roots were differentiated from any explants except those from ovaries and pedicels. Most explants of lower portions of ovaries showed root formation on media with BA and NAA, but those from upper portions showed root formation only on a medium with 1 mg•liter-1 each of BA and NAA. Those of pedicels showed root formation with a frequency of 33 percent on a medium without plant growth regulators.Callus formation occurred from ovary and stamen explants. The frequency of callus formation from ovary explants was 100 percent on media with BA and NAA. Stamen explants showed 11 percent callus formation on a 5 mg•liter-1 BA plus 5 mg•liter-1 NAA medium.

カラジウムの葉色別外植体の培養により得られた再生植物体の葉色の変異

Plantlets were regenerated from three variegated cultivars of caladium (Caladium × hortulanum Birdsey) by in vitro propagation of leaf segments. Leaf segments of 'Freida Hemple' were taken from green and red areas of the leaf blades; those of 'C. R. 5' were obtained from green, white, and red areas; whereas those of 'Pink Cloud' were taken from green, white, and pink areas. The segments were then cultured on modified MS medium supplemented with 1.0 mg • liter-1 BA and 1.0 mg • liter-1 NAA.1. All green and red leaf segments of 'Freida Hemple' formed multiple shoot primordia. In 'C. R. 5' and 'Pink Cloud', the green segments formed the highest number of multiple shoot primordia, whereas the red and pink leaf segments yielded the lowest percentage of primordia.2. The variegation pattern of leaf blades in 'Freida 'Hemple' and 'C. R. 5' were stable, whereas in 'Pink Cloud' the patterns were unstable and variants were observed. In 'Pink Cloud' the percentage of variants in which the original white color of the leaf vein changed to green was high in the pink leaf segments but low in the green leaf segments.

Varietal Differences in Leaf Color Variation of Plants Regenerated from in vitro Culture of Leaf Blade in Caladium Cultivars

Expanding leaves of nine cultivars of caladium (Caladium Xhortulanum Birdsey) were cultured on modified MS medium supplemented with 1.0 mg•liter-1 BA and 1.0 mg•liter-1 NAA from which multiple shoot primordia were obtained. The multiple shoot primordia were in turn, cultured on modified MS medium without any growth regulators to regenerate plantlets.Leaf color variations in the plantlets regenerated from the multiple shoot primordia were very different among the nine cultivars. Leaf color variants were not observed in the plantlets regenerated from their multiple shoot primordia of 'Aaron', 'Frieda Hemple', 'John Peat', 'Candidum Junior', and 'C. R. 5', but they were observed with high frequency in those of 'Pink Cloud', 'White Wing', 'Rosary', and 'Ginger Land'.All leaf color variants in the regenerated plantlets changed from the white or red veins to the green. Variants regenerated from the multiple shoot primordia of 'Pink Cloud' and 'Ginger Land' had green veins and the laminas retained the color of the original parent plants. On the other hand, the variants regenerated from those of 'White Wing' and 'Ro•sa•ry' had both green laminas and veins.When the green vein variants of 'Pink Cloud', and green leaf ones of 'White Wing' and 'Rosary' were cultured, the regenerated plantlets appeared similar to the parental stock.

Effects of Auxin, Cytokinin and Nitrogen Concentration on Morphogenesis of Tissue-cultured Shoot Apex of Chinese Yam (Dioscorea opposita Thunb.).

Most shoot apices of Chinese yam (Dioscorea opposita Thunb. cv. Nagaimo) developed into shoots in modified Murashige-Skoog (MS) medium in which the nitrogen level was reduced to one tenth of the recommended concentration and containing 0 to 0.1 mg•liter-1 NAA and 0.1 mg•liter-1 BA. Bases of shoot apices developed friable calli in a medium supplemented with 1.0 mg•liter-1 NAA. The composition of the basal medium had little effect on shoot formation, but morphogenic calli were produced by excised apices on a medium having 1 to 2 mg•liter-1 BA and low concentration of NAA. Morphogenic callus resembled shoot primordium in that adventitious buds were formed on its surface. When morphogenic calli were subcultured on MS medium containing 0 to 0.1 mg•liter-1 BA and 0.01 to 0.1 mg•liter-1 NAA, a large number of plantlets were regenerated.

Embryoid and Callus Formation from Pollen Grains of Eggplant and Pepper by Anther Culture.

Effective methods for the production of haploid plants by culturing anthers of eggplant (Sola-num melongena L.) and pepper (Capsicum annuum L.) were studied. Anthers containing pollen grains at the uninucleate stage were plated on MS medium supplied with various concen-trations of 2, 4-D and kinetin and then exposed to high and low temperatures. Embryoids from pollen grain and adventitious shoots from calli were obtained, and they grew to plantlets upon transferring to a MS medium.Results obtained for eggplant were as follows:1. Anthers cultured on MS medium supplied with 0.1 mg•liter-1 of both 2, 4-D and kine-tin and treated with 35°C for 24 hr formed calli and embryoids at the highest frequency, but low temperature treatment (5°C for 24 or 48 hr) was ineffective or rather inhibitory.2. Regeneration of embryoid and adventitious buds from calli was induced by transferring calli to MS medium without phytohormones or with 1 mg•liter-1 kinetin plus 0 or 0.05 mg•liter-1 NAA.3. Plantlets on MS media supplied with 0.8% agar and 58% sucrose grew normally and vigorously.Results for pepper were as follows:4. Calli were formed at high frequency on MS media supplied with 0.2 mg•liter-1 2, 4-D plus 0.1 mg•liter-1 kinetin or 0.1 mg•liter-1 both of 2, 4-D and kinetin. On the other hand, embryoids were directly formed at high frequency on MS media supplied with 0.02 mg•liter-1 kinetin or 0.004 mg•liter-1 2, 4-D plus 0.1 mg•liter-1 kinetin.

Callus Formation and Plant Regeneration from Protoplast Derived from Cotyledons and Hypocotyls of Radish(Raphanus sativus L.)and Other Cruciferous Plants.

Protoplasts were isolated from radish hypocotyls and cotyledons of 4- to 7-day-old seed-ling with three enzyme mixtures, three concentrations of mannitol and two incubation peri-ods. The most effective conditions for protoplast isolation were to shake sliced 5-day-old cotyledons at 27°C for 2 hr in the enzyme solution containing 2% Cellulase Y-C, 0.2% Pec-tolyase Y-23, CPW salts and 0.3 M mannitol. Protoplasts isolated from 16 cruciferous cul-tivars under the most effective condition were compared with other two enzyme mixtures and two materials; cotyledon and hypocotyl. Although the isolation efficiency differed depend-ing on cultivars, the best condition was as same as radish. As the initial plating medium for cell division of protoplasts, Pelletier's B medium (1983) was used, which supplemented 1 mg. liter -1 both of 2- (1-naphthyl) acetic acid (NAA) and N- (phenylmethyl) -1H-purine-6-amine (BA), 0.25 mg•liter-1 2, 4-dichlorophenoxy acetic acid (2, 4-D), 0.5 M mannitol and 20 g•liter-1 glucose. Then C medium supplemented with 0.2 mg•liter-1 NAA, 1 mg•liter-1 BA, 0.1 mg•liter-1 2, 4-D, 0.02 mg•liter-1 gibberellic acid3 (GA3) and 2 mg•liter-1 zeatin was added every 710 days. Colonies of about 2 mm in diameter were formed after 4 weeks, and transferred to E medium supplmented with 1 mgliter-1 NAA and BA, 0.1 mg•liter-1 2, 4-D, 1 mg•liter-1 3- (3-indolyl propanoic acid (IPA), 0.02 mg•liter-1 GA3 and 2 mgliter -1 zeatin. Colonies developed into calli after more 1 month at 25°C, 16 hr daylength, and they were transferred to E medium supplemented with same phytohormones or 1 mg•liter-1 BA. Regenerated shoots were excised and transferred to Murashige and Shoog (MS) medium, and most of shoots rooted. Callus was formed from protoplasts of all cultivars, but plating efficiency differed depending on the cultivars. Shoot regeneration was not achieved in the turnip cultivar 'Shogoin Ookabu' and in all radish cultivars except 'Moriguchi'.

メロン子葉プロトプラストからの不定芽再分化

Protoplasts, isolated from cotyledons of aseptically germinated seeds of the melon 'Charen-tais', were used to investigate the effects of hormones and osmotic conditions on cell divi-sion and plantlet regeneration.1. Division of protoplasts occurred at a high frequency during the initial incubation peri-od on the modified MS medium (200 mg•liter-1 NH4NO3 and 1% sucrose) containing a wide range of 2, 4-D concentrations. Protoplast division was continuous in a medium containing 0.05 mg•liter-1 2, 4-D and 0.5 mg•liter-1 BA even as the protoplasts mature and formed cell walls.2. Three osmotic conditions; mannitol only and an equimolor conc. of mannitol and sucrose and that of mannitol and glucose were used to clarify the role of the osmoticum on the shoot regeneration from calli. No obvious difference among the three osmotic conditions was ob-served in the frequency of cell division at initial culture and in callus formation. The callus grown in a medium containing mannitol and glucose differentiated shoots at the highest fre-quency among the media tested.3. More shoots were regenerated in a MS medium containing 0.5 mg•liter-1 GA3 and 2 mg•liter-1 BA than were formed with a combination of GA3 and zeatin. The GA3 com-bined with cytokinins promoted more shoots formation from calli than did the IAA-cytokinin combinations.The following protocol for shoot regeneration of melon was quite efficient: a) Culture the protoplasts in the modified MS medium supplemented with 0.05 mg•liter-1 2, 4-D, 0.5 mg•liter-1 BA, 0.2 M mannitol and 0.2 M glucose. b) Transfer the callus grown in the above medium to regeneration medium supplemented with 2 mg•liter-1 BA and 0.5 mg•liter-1GA3. On the average, three shoots per callus were regenerated.Many regenerated shoots may appear abnormal and will not elongate normally, in which case, transplant the explant intact onto a MS medium containing 0.1 mg•liter-1 BA and 0.2 mg-liter-1 GA3. Subculture the explant several times to promote elongation of the shoots. Finally, subculture the elongated shoot on MS agar medium with 1 mg•liter-1 NAA for 24 hr and then transfer it to one without any hormone. The treated shoot will regener-ate roots and grow into a normal plant.

In Vitro Propagation of Prayer Plants

Abstract Lateral buds excised from unrooted cuttings of Maranta leuconeura E. Moor. ‘Kerchoviana’, prayer plant, were cultured on Linsmaier and Skoog (LS) medium supplemented with various combinations of N6-benzylaminopurine (BA), kinetin, α-anaphthaleneacetic acid (NAA), and 3-indoleacetic acid (IAA) for the production of multiple shoots and complete plants. Of 48 combinations of growth regulators, 4 produced the most vigorous, well-formed shoots after a culture period of 6 weeks: 2.0 mg·liter-1 kinetin, 0.2 mg·liter-1 BA, 0.2 mg·liter-1 BA plus 0.1 mg·liter-1 NAA, and 2.0 mg·liter-1 kinetin plus 1.0 mg·liter-1 IAA. Enhanced shoot production occurred when shoots were transferred at 12-week intervals and when they were maintained on 0.2 mg liter-1 BA. Complete plants with strong, fibrous root systems were produced after the shoots were transferred to basal medium lacking growth regulators for 3-4 weeks.