In vitro studies on Pea (Pisum sativum L.): I. Callus formation,regeneration and rooting

Abstract

Callus production, shoot formation via organogenesis and rooting of the regenerated shoots are reported in an Egyptian variety of Pisum sativum L. Calli were initiated from hypocotyl, leaf, root and mature embryo explants when cultured on MS medium containing B5 vitamins and supplemented with 2 mg/l 2,4-D+1 mg/l kin. Among the different types of explants, hypocotyl showed best potential for callus proliferation. Hypocotyl, leaf and immature cotyledon explants were used for shoot organogenesis. The best results of shoot formation were achieved when hypocotyl explants were cultured on MS-medium supplemented with 2 mg/l BA+1 mg/l NAA. However, immature cotyledon explants showed the highest frequency of shoot formation with 1 mg/l BA. Data of in vitro rooting showed that maximum root frequency occurred on culture medium containing half strength of MS salts, 40 g/l sucrose and 2 mg/l NAA.     

Clonal propagation byin vitro culture of Corchorus (jute)

Callus was produced on cotyledon, shoot tip, hypocotyl and root explants of twoCorchorus species on several media. Cytokinin was necessary for callus production on cotyledon explants. BothC.olitorius genotypes produced most callus on media with zeatin and either NAA or IAA, and theC.capsularis genotype produced most callus on media with IAA and either zeatin or BA. High frequencies of regenerated shoots were obtained from shoot tip explants of both species, from the apical meristem and from callus. Media with 2.0 mg 1⁻¹ BA were superior for both species, and media with zeatin were equally good forC.capsularis only. More regeneration was obtained for all genotypes after subculture of callus on media with 2.0 mg 1⁻¹ zeatin. Cotyledon callus produced less regeneration, also with differences between genotypes; explants of both genotypes ofC.olitorius produced regeneration on a medium with NAA and zeatin, and theC.capsularis genotype produced regeneration on a medium with IAA and BA. Limited regeneration from root explant callus was obtained forC.capsularis only on medium with BA and IAA. Regeneration was not obtained from hypocotyl callus. Further regeneration of shoots of both species was obtained from secondary callus after subculture, and from nodal segments of regenerated shoots and of seedling shoots cultured on basic MS medium without growth hormones. Roots were produced on about 80% of all shoots after transference to medium with 0.2 mg 1⁻¹ IBA, and rooted plantlets survived and flowered normally after transference to compost.     

Efficient regeneration of sorghum, Sorghum bicolor (L.) Moench, from shoot-tip explant

Novel protocols for production of multiple shoot-tip clumps and somatic embryos of Sorghum bicolor (L.) Moench were developed with long-term goal of crop improvement through genetic transformation. Multiple shoot-tip clumps were developed in vitro from shoot-tip explant of one-week old seedling, cultured on MS medium containing only BA (0.5, 1 or 2 mg/l) or both BA (1 or 2 mg/l) and 2,4-D (0.5 mg/l) with bi-weekly subculture. Somatic embryos were directly produced on the enlarged dome shaped growing structures that developed from the shoot-tips of one-week old seedling explants (without any callus formation) when cultured on MS medium supplemented with both 2,4-D (0.5 mg/l) and BA (0.5 mg/l). However, the supplementation of MS medium with only 2,4-D (0.5 mg/l) induced compact callus without any plantlet regeneration. Each multiple shoot-clump was capable of regenerating more than 80 shoots via an intensive differentiation of both axillary and adventitious shoot buds, the somatic embryos were capable of 90% germination, plant conversion and regeneration. The regenerated shoots could be efficiently rooted on MS medium containing indole-3-butyric acid (IBA 1 mg/l). The plants were successfully transplanted to glasshouse and grown to maturity with a survival rate of 98%. Morphogenetic response of the explants was found to be genotypically independent.     

Efficient plant regeneration from cotyledon explants of bottle gourd (<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> Standl.)

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO₃ under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO₃ were diploid.     

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Summary Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l⁻¹) and indole-3-acetic acid (IAA) (0.5 mg l⁻¹). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l⁻¹) and subsequent subculture in IBA-free medium.     

提高榨菜离体培养植株再生频率

采用榨菜“浙桐1号”品种为材料,以MS为基本培养基,通过对不同植物生长调节剂的组合和不同外植体等主要因素的筛选,大幅度提高了榨菜离体培养植株再生频率。结果表明,2mg／L6．BA 0．2mg／L2,4-D的组合较为适宜,其不定芽再生频率可达50％,且外植体以下胚轴为好：而CPPU和2,4-D的适宜组合为1．5mg／L 0．2mg／L,其不定芽再生频率高达66．67％,最适外植体为带柄子叶。同时,研究结果显示,添加0．25～1mg／L的GA,对榨菜已分化的不定芽的伸长有抑制作用;子叶柄和下胚轴外植体的分化具有极性现象。     

Direct shoot regeneration from mature embryo as a rapid and genotype-independent pathway in tissue culture of heterogeneous diverse sets of cumin (Cuminum cyminum L.) genotypes

Summary A rapid and one-step protocol for direct regeneration of shoots from cumin embryo explants has been developed. Embryo explants with shoot meristems were cultured on shoot regeneration medium for 15&#x2013;22 d. After embryo culture, shoots were regenerated from the area adjacent to the region between the cotyledons and embryo axis within 2 wk, without any intermediate callus phase. Shoot proliferation and elongation were achieved on shoot regeneration medium without subculture. Among the different combinations of 6-benzylaminopurine, &#x03B1;-naphthaleneacetic acid (NAA), and indole-3-acetic acid (IAA) tested, 0.8 mgl^&#x2212;1 (4.3 &#x03BC;M) NAA in combination with 0.3 mgl^&#x2212;1 (1.71 &#x03BC;M) IAA in the B5 medium resulted in the most efficient direct shoot regeneration. No significant difference was detected for the number of regenerated explants when different heterogeneous endemic varieties were compared. This plant regeneration procedure was applicable to different cumin genotypes and regenerated plants were phenotypically normal.     

Shoot regeneration and the relationship between organogenic capacity and endogenous hormonal contents in pumpkin

To induce multiple shoots from pumpkin (Cucurbita moschata Duch.), cotyledon explants excised from various ages of seedlings after in vitro germination were cultured on MS augmented with different concentrations of BA (0, 0.5, 1.0 or 2.0 mg l⁻¹). The highest frequency of shoot regeneration (63.7%) was observed from seven-day-old cotyledon explants cultured on MS containing 0.5 mg l⁻¹ BA. The frequency and duration of shoot formation showed close correlation with the donor seedling age. By contrast, BA supply was necessary to promote shoot formation but no differences were observed in relation to different concentrations. Multiple shoots elongated on MS supplemented with 0.1 mg l⁻¹ BA and 5–7 shoots per regenerated explant were recovered. Elongated shoots were rooted on MS, which was easier than that on 2/3MS, 1/2MS, or MS supplemented with 0.1 mg l⁻¹ NAA. The rooted shoots were then transferred to greenhouse where they grew and flowered normally. Quantitative analysis of endogenous auxin (IAA) and cytokinins (iPA and ZR) in initial cotyledon explants of different aged seedlings showed that the regeneration ability of cotyledon explants varied dependently on their endogenous iPA contents. This study therefore deduces that the various organogenic capabilities of cotyledon explants from pumpkin are the result of their endogenous hormonal contents.     

High frequency in vitro propagation of Phyllanthus amarus Schum. & Thom. by shoot tip culture

With the aim of micropropagation of Phyllanthus amarus, an important medicinal herb, shoot tips were cultured in Murashige and Skoog's medium supplemented with kinetin/ BAP singly or in combination with IAA. Growth regulators at lower range (0.1-1.0 mg L(-1)) stimulated direct regeneration of shoots. Kinetin was superior to BAP and kinetin-IAA combination was more suitable than kinetin alone. About 15 shoots were yielded per explant after 30 days of culture in the medium containing kinetin and IAA both at 0.1mg L(-1). The cluster of proliferated shoots elongated and rooted simultaneously under the same treatment following another subculture, thus shortening the total time schedule of micropropagation. Shoot tips of regenerated shoots were continuously used to regenerate new shoots with periodic transfer to fresh medium resulting in a steady supply of normal, healthy plants without any deviation in the production rate during a continuous one year culture. Micropropagated plants were successfully established in soil with high survivality (80%).     

High-frequency plant regeneration from cultured cotyledons of Arabidopsis thaliana

Wild-type plants of Arabidopsis thaliana strain Columbia regenerated at a high frequency from immature cotyledons cultured on a shoot-inducing medium containing 1.0 mg/l 6-benzylaminopurine and 0.1 mg/l 1-naphthaleneacetic acid. Cotyledon segments expanded rapidly and produced numerous shoots after 2–3 weeks in culture. Regeneration occurred in the absence of the original shoot apex. Hypocotyl segments from immature embryos produced root hairs and callus in culture but only rarely developed shoots. Hygromycin, kanamycin and G-418 inhibited cotyledon expansion and shoot formation in culture. Vancomycin was much less toxic to cotyledon segments than either carbenicillin or cefotaxime. Immature cotyledons therefore yield numerous regenerated plants that may be useful in future transformation studies.     

Micropropagation of Dianthus caryophyllus L. — Control of Vitrification

Vitrification is a morphological and physiological disorder affecting in vitro regenerated plants. Vitrified shoots of carnation (Dianthus caryophyllus L.) regenerated from cultured cotyledons were abnormally glassy, thick and bushy with wider translucent leaves. Such vitrified shoots were recovered by culturing them on a medium supplemented with GA₃. Differentiation of shoot buds from the cultured cotyledons of D. caryophyllus occurred on MS medium supplemented with BAP (2.0 mg/l). Shoot buds subcultured on the same medium resulted in further prolific development of shoot buds and bushy shoot growths. Key words: carnation, shoot morphogenesis, micropropagation, cotyledons vitrification.     

Silver nitrate promotes shoot development and plant regeneration of chile pepper (Capsicum annuum L.) via organogenesis

Summary Chile pepper (Capsicum annuum L.) plants were regenerated from cotyledon explantsin vitro in four major stages: bud induction, bud enlargement, shoot elongation, and root development. Bud induction medium contained 0.5 mg/L (2.9μM) indole-3-acetic acid and 2 mg/L (8.9 μM) N⁶-benzyladenine. Bud enlargement occurred, and an occasional shoot appeared when medium with 2 mg/L (6μM) gibberellic acid, 2 mg/L (8.9 μM) N⁶-benzyladenine, and 5 mg/L (29.4 μM) silver nitrate was used. Most shoots elongated after placement on a third medium without plant growth regulators or on fresh plates of bud enlargement medium. Incubations were for 2, 2, and 4 weeks, respectively, at 28.5°C and continuous light. Treatment with silver nitrate was necessary for multiple shoot production and elongation to occur in the third culture stage and was most effective when present in the second-stage medium but not in the bud induction medium. Sixteen to 26% of the shoots rooted in medium with 1 mg/L (5.4 μM) 1-naphthaleneacetic acid after 1 month. Additional shoots transferred to a second rooting medium with 0.1 or 1.0 mg/L (0.54 or 5.4 μM) 1-naphthaleneacetic acid developed roots, increasing the overall rooting efficiency to 70–72%. Most rooted shoots grew well and produced viable seeds when grown in the greenhouse. Other cytokinins tested for plant regeneration were zeatin and thidiazuron. Zeatin induced few shoots and fewer well-developed plants. Thidiazuron induced multiple shoots 4 months after culture began, but many were small and did not elongate further. Phytagar tissue culture grade proved superior to other agars tested, increasing bud induction frequency from 0-33% to 80–93% and eliminating explant hyperhydricity.     

Regeneration of whole plants from protoplasts isolated from tissue-cultured shoot primordia of garlic (Allium sativum L.)

Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x10⁴ protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants.     

High efficiency <Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from cotyledon explants of <Emphasis Type="Italic">Incarvillea sinensis</Emphasis>

Summary A simple and effective procedure has been developed for plantlet regeneration from cotyledon-derived callus of the medicinally important herb and ornamental species, Incarvillea sinensis. An average of 18.4 adventitious shoots per explant were obtained from 100% cotyledon explants cultured on half-strength Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ 6-benzylaminopurine for 3 wk, followed by another 4 wk on hormone-free 1/2×MS medium. The cotyledon explants continued to expand and regenerate new shoots upon repeated subculturing onto fresh medium. Most regenerated shoots (66.9%) were rooted on 1/4×MS mediumcontaining 1.0 mg l⁻¹ indole-3-acetic acid, with an average of about 3.8 roots per shoot. Regenerated plants with well developed shoots and roots were successfully acclimatized in soil and were normal phenotypically.     

Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Capsicum annuum L.

 A highly efficient three-stage protocol for the regeneration of chilli pepper (Capsicum annuum L.) from cotyledon explants was developed. This protocol used PAA in both the shoot-bud induction medium and the medium for elongation of the shoot buds. A superior medium for the induction of buds from the cotyledons was MS medium supplemented with BA (5 or 7 mg/l) + PAA (2 mg/l). Buds were elongated during the second stage on medium containing BA (2 or 5 mg/l) + PAA (2 mg/l). On this medium most of the buds elongated, and their number also increased due to the formation of new buds; bud elongation was achieved in 100% of the cultures provided the buds were induced in the primary stage on a medium supplemented with BA+PAA. The shoots that elongated in the second-stage rooted at 100% frequency on a medium supplemented with NAA (1 mg/l). The complete plantlets with well-developed root and shoot systems were transferred to field conditions where they grew to maturity, flowered and fruited normally. While shoot-bud induction from the cultured cotyledons was also observed on media supplemented with BA (5 or 7 mg/l) alone or in combination with IAA (0.2–2 mg/l), buds induced on these media were often distorted, with most not developing into normal shoots in the second-stage subculturing; a rosette of buds was seen in the second stage subculturing. On the other hand, PAA in combination with BA in the primary induction medium and second-stage medium promoted normal development and the elongation of shoot buds. Received: 28 July 1998 / Revision received: 22 December 1998 / Accepted: 19 February 1999     

Effect of TDZ on plant regeneration from mature seeds in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

Pea (Pisum sativum L. cv. Espace) seeds directly cultured on thidiazuron (TDZ)-containing medium formed high numbers of shoots. The number of shoots per seedling depended on the concentration and duration of the TDZ treatment. The best treatment was 12-wk incubation on MS medium supplemented with 4 mg/l TDZ followed by 4-wk culture on MS medium supplemented with 0.5 mg/l benzylaminopurine (BA) and produced more than 400 shoots/seedling. Isolated shoots rooted at a high frequency on MS medium containing 2–3 mg/l indole-3-butyric acid and 2 mg/l α-naphthalene acetic acid. In addition to the formation of shoots, bud-containing tissues (BCT) were formed at the cotyledonary nodes, shoot nodes, tendrils, stipules, and internodes. The BCT from the cotyledonary nodes and the shoot nodes was maintained in its pure state on MS medium supplemented with 4 mg/l TDZ by repeated culture. Shoot development was accomplished when the BCT were left on MS medium supplemented with 4 mg/l TDZ without subculture prior to transfer onto MS medium supplemented with 0.5 mg/l BA.     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997     

裂叶悬钩子组织培养研究 Ⅰ.裂叶悬钩子叶外植体培养直接再生不定芽及植物激素对它的影响

本文首次报道裂叶悬钩子(Rubus laciniatus Wild)叶外植体培养在改良的NN~(69)培养基上附加2—4mg/1 6-BA和0.1mg/1 NAA或1—3mg/1 2,4-D和0.1mg/1 NAA,两者都可直接从完整叶片、叶片下切段或叶柄诱导出不定芽。诱导频率达20—48%。而不定芽绝大部分发生在叶轴处或叶柄基部。完整叶片的不定芽诱导率与叶片下切段无差别,但比叶柄基部诱导率要高。6-BA对叶轴处不定芽诱导率比2,4-D的要高。此外,不需继代培养,不定芽数可达10—20个,继代培养一个月左右,每个不定芽能形成丛生芽数可达40一60个。另外,本文还讨论了细胞分化过程中的极性现象。     

Plant Regeneration and Transformation of Sweet Papper (Capsicum frutescens)

Three strains of sweet pepper, (Capsicum frutescens) “Shuang Feng”, “Zhong Jiao No. 2” and “Zhong Jiao No.3” were screened out of six Chinese cuhivars for their high capacity of regeneration. The normal flowering and fertile regenerated plants have been obtained from cotyledons of seedlings from 10 to 16 days old by a four-step culture procedure; short induction, shoot elongation, rooting of excised shoot and transplanting into soil. MS was the basal medium in all steps, supplemented with different kind and different concentration Of phytohomores. Optimal shoot ioduction medium is MS +4–6mg/LBA+0.5mg/LIAA which gives rise a shoot regeneration frequency of 100%. 35% of the induced shoots elongated on the medium of MS+2mg/L Zeatin or 2mg/L. BA+l–3mg/L GA, and subsequently rooted on MS medium or in addition of 0.1–0.5mg/LNAA. The regenerants were transplanted into soil and developed into normal plants. In the transformation of sweet pepper using the leaf disc method, two kinds of wild type Agrobacterium tumefaciens, C58 and GV3111, have been screened out in regard to their high infection capacity. The transient expression of GUS gene was detected and Kanamycin-resistant shoots from infected cotyledons have been obtained. Further assay and transfering the TMV-resistant and CMV-resistant genes into sweet pepper are in progress.     

In vitro morphogenesis from cotyledon and epicotyl explants and flow cytometry distinction between landraces of Bambara groundnut [<Emphasis Type="Italic">Vigna subterranea</Emphasis> (L.) Verdc], an under-utilised grain legume

The morphogenetic competence of Bambara groundnut was assessed for different landraces, explant sources and media compositions. With cotyledon explants, the best callusing occurred on a medium containing 3 mg/l BAP + 0.5 mg/l NAA, while roots were produced with 3–5 mg/l BAP + 0.5 mg/l NAA. Shoots regenerated (∼6%) from cotyledons on media with BAP alone (3–5 mg/l) or combined with 0.01–0.1 mg/l NAA. Flowers were regenerated on 5 mg/l BAP + 0.5 mg/l NAA, without any intervening callus phase. With epicotyls, the highest callusing was on 3 mg/l BAP + 0.5 mg/l NAA, and shoots regenerated (15–20%) on 3 mg/l BAP alone or with NAA at concentrations that depended on the landrace studied. Regenerated shoots rooted on hormone-free medium, and plants transferred to the greenhouse were all morphologically normal and fertile. Flow cytometry showed that most regenerants were diploid and in addition permitted to distinguish between landraces according to their relative nuclear DNA content. This is the first report on de novo regeneration in vitro of Bambara groundnut, an important yet neglected legume crop.