Plant Regeneration In Tissue Culture Research Articles

Forever young: stem cell and plant regeneration one century after Haberlandt 1921.

Plants are characterized by a post-embryonic mode of organ development, which results in a need for these photoautotrophic organisms to regenerate lost parts in the course of their life cycle. This capacity depends on the presence of "pluripotent stem cells," which are part of the meristems within the plant body. One hundred years ago, the botanist Gottlieb Haberlandt (1854-1945) published experiments showing wounding-induced callus formation, which led ultimately to plant regeneration in tissue culture and thence to the techniques of "plant biotechnology," with practical applications for mankind. Here, we recount Haberlandt's discovery within the context of his long research life and his most influential book Physiologische Pflanzenanatomie. In the second part, we describe and analyze a plant tissue-culture regeneration system using sterile, dark-grown sunflower (Helianthus annuus) seedlings as experimental material. We document that excised hook segments, which contain a "stem cell niche," can regenerate entire miniature H. annuus-plantlets that, raised in a light/dark regime, develop flowers. Finally, we discuss molecular data relevant to plant regeneration with reference to phytohormones and conclude that, one century after Haberlandt, 1921, the exact biochemical/genetic mechanisms responsible for the capability of stem cells to remain "forever young" are, although already complex, really just beginning to become known.

A tissue culture system for callus formation and plant regeneration using tuber discs of Dioscorea tokoro Makino, a pharmaceutical yam species

Yam (Dioscorea sp.) is a recalcitrant plant species with respect to plant regeneration in tissue culture. We report here on callus formation and plantlet regeneration in the pharmaceutical yam species D. tokoro Makino. Explants of leaves, nodes, internodes, roots, and tuber discs of three lines of D. tokoro were cultured on media containing the synthetic auxins 2,4-dichlorophenoxyacetic acid or picloram at 10 µM. Explants of nodes, roots, and tuber discs formed calluses at high frequency in all lines we examined, regardless of type of auxin added to the media. However, explants of leaves and internodes rarely formed calluses. Some calluses derived from tuber discs differentiated shoots through the formation of bud-like structures by culture on media with or without 10 µM 6-benzyladenine. The T-1110 line showed the highest shoot formation efficiency. The regenerants from the calluses derived from tuber discs successfully acclimatized to a greenhouse, grew normally and ultimately formed tubers in soil.

Use an Organic Biostimulant (Vermicompost Tea) For Enhancement In Vıtro Callus Growth in Sainfoin (Onobrychis viciifolia Scop.)

The health and vitality of callus growth is one of the prerequisites for the success of further in vitro studies. This study investigated the efficiency of different percentage (0%, 10%, 20%, 30%, and 40%) of vermicompost tea as an organic substance on in vitro callus growth in sainfoin. Morpho-physiological responses of calli to vermicompost tea measured under in vitro conditions. As a result of this investigation, a combination of plant growth regulators (4 mg/l BAP and 0.5 mg/l NAA) with 20% of vermicompost tea causing significant callus initiation and growth in sainfoin stem explants. Under the light of these scientific findings, vermicompost tea might be used as an organic bio stimulant for efficient callus growth and complementary to commercial chemical hormones in sainfoin. This research is important due to it can contribute positively to the plant species that are difficult in terms of callus growth and plant regeneration in tissue culture.

Enhancing plant regeneration in tissue culture

Micropropagation is used for commercial purposes worldwide, but the capacity to undergo somatic organogenesis and plant regeneration varies greatly among species. The plant hormones auxin and cytokinin are critical for plant regeneration in tissue culture, with cytokinin playing an instrumental role in shoot organogenesis. Type-B response regulators govern the transcriptional output in response to cytokinin and are required for plant regeneration. In our paper published in Plant Physiology, we explored the functional redundancy among the 11 type-B Arabidopsis response regulators (ARRs). Interestingly, we discovered that the enhanced expression of one family member, ARR10, induced hypersensitivity to cytokinin in multiple assays, including callus greening and shoot induction of explants. Here we 1) discuss the hormone dependence for in vitro plant regeneration, 2) how manipulation of the cytokinin response has been used to enhance plant regeneration, and 3) the potential of the ARR10 transgene as a tool to increase the regeneration capacity of agriculturally important crop plants. The efficacy of ARR10 for enhancing plant regeneration likely arises from its ability to transcriptionally regulate key cytokinin responsive genes combined with an enhanced protein stability of ARR10 compared with other type-B ARRs. By increasing the capacity of key tissues and cell types to respond to cytokinin, ARR10, or other type-B response regulators with similar properties, could be used as a tool to combat the recalcitrance of some crop species to tissue culture techniques.

Quantitative trait loci (QTLs) detection for plant regeneration ability from seed culture in rice (Oryza sativa L.)

Quantitative trait loci (QTLs), which were related to the ability of callus induction and plant regeneration in seed culture of rice, were analyzed using a mapping population from a cross between the rice cultivars 'Samgang' (tongil type) and 'Nagdong' (japonica). A tongil type rice cultivar, 'Samgang' showed lower frequency (20%) of plant regeneration than that (35%) of japonica rice, 'Nagdong'. Transgressive segregations were observed for the ability of callus induction and plant regeneration from the seed-derived calli of 58 doubled haploid (DH) lines. The ability of plant regeneration of 58 doubled haploid lines showed a continuous distribution with comparatively wide range (10.0 to 66.7%) of variation. Composite interval mapping analysis was used to identify the QTLs controlling callus induction and plant regeneration ability. Four significant QTLs, qCWS6, qCWS8, qCWS9 and qCWS11, associated with callus weight per seed were detected on chromosomes 6, 8, 9, and 11 with LOD values of 3.30, 2.60, 2.70 and 2.43, explaining 36% of the total phenotypic variation. Three significant QTLs, qPR1, qPR6, and qPR11, for the ability of plant regeneration were located on chromosome 1, 6, and 11 at LOD score of 2.25, 2.15 and 2.55, accounting for 24 % of the total phenotypic variation. The present study should be useful for improving the efficiency of plant regeneration in tissue culture of indica rice by means of marker-assisted selection.

Inheritance of somatic embryogenesis using leaf petioles as explants in upland cotton

Somatic embryogenesis (SE) is a critical step leading to plant regeneration in tissue culture of many plant species. The objective of the present study was to analyze the inheritance of SE in cotton (Gossypium hirsutum L.) using leaf petioles as explants. A high embryogenic callus (HEC)—producing line, W10, was selected by petiole callus culture from a commercial Chinese cotton cultivar CRI24 and crossed with a non embryogenic line, TM-1 and a low embryogenic (LEC) commercial Chinese cotton cultivar, CRI12, respectively. The parental lines, F1 and F2 were grown in field conditions for sources of leaf petioles as explants. The F1 plants were similar to the HEC parent in embryogenic callus (EC) induction, indicating that high EC ability is dominant. The classical Mendelian analysis showed that the high EC ability in the HEC line W10 is controlled by two independent dominant genes with complementary effect, designated Ec 1 and Ec 2 , while the LEC line CRI12 contains one dominant gene Ec 2 . A joint segregation analysis confirmed that SE ability in cotton is controlled by two major genes with epistatic effects along with other polygenes. A SSR marker analysis identified three quantitative trait loci (QTLs) on two linkage groups, one of which harbored a major QTL (qEc1) which is assigned to the major gene Ec 1 . This qualitative and quantitative genetic study has provided an incentive to fine map the genes responsible for SE towards the isolation of the SE genes in cotton.

Maize tissue culture plant regeneration ability can be improved by polyethylene glycol treatment

Type II maize callus of the HiII genotype can be separated into regenerable and non-regenerable types based on the visible morphology of the callus. When the non-regenerable morphotype of callus initiated 6 mo or a yr earlier was treated with from 2% to 20% polyethylene glycol (PEG; 3,350 molecular weight) for three subculture periods of 21 d each, the morphotype changed to regenerable, and the callus did become highly regenerable. The PEG treatments did not improve the plant regeneration ability of the regenerable morphotype or of an old culture initiated 4 yr earlier.

Factors for high frequency plant regeneration in tissue cultures of Indian mustard (Brassica juncea L.)

An efficient system for high frequency plant regeneration was established through investigating various factors such as plant growth regulator combinations, explant types and ages, and addition of <TEX>$AgNO_3$</TEX> influenced on shoot regeneration in Brassica juncea L. cv. BARI sarisha-10. Murashige and Skoog (MS) medium supplemented with 0.1 mg/L NAA (1-naphthaleneacetic acid) and 1 mg/L BA (6-benzyladenine) showed the maximum shoot regeneration frequency (56.67%) among the different combinations of NAA and BA. Explant type, explant age, and addition of <TEX>$AgNO_3$</TEX> also significantly affected shoot regeneration. Of the four type of explants (cotyledon, hypocotyl, root, and leaf explants)- cotyledon explants produced the highest shoot regeneration frequency and hypocotyls explants produced the highest number of shoots per explant, whereas root explants did not produce any shoot. The cotyledonary explants from Four-day-old seedlings showed the maximum shoot regeneration frequency and number of shoots per explant. Shoot regeneration frequency increased significantly by adding <TEX>$AgNO_3$</TEX> to the medium. Two mg/L <TEX>$AgNO_3$</TEX> appeared to be the best for shoot regeneration with the highest shoot regeneration frequency (86.67%) and number of shoots per explant (7.5 shoots). Considerable variation in shoot regeneration from cotyledonay explants was observed within the B. juncea L. genotypes. The shoot regeneration frequency ranged from 47.78% for cv. Shambol to 91.11% for cv. Rai-5. In terms of the number of shoots produced per explant, B. juncea L. cv. Daulot showed the maximum efficiency. MS medium supplemented with 0.1 mg/L NAA showed the highest frequency of rooting. The regenerated plantlets were transferred to pot soil and grown to maturity in the greenhouse. All plants were fertile and morphologically identical with the source plants.

Somatic Embryogenesis and Plant Regeneration in Tissue Cultures of Artemisia annua L.

Mature seeds of Artemisia annua L. were placed onto Murashige and Skoog`s (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). After 6 weeks of culture, off-white, compact calluses were formed on the plumule of seedlings at a frequency of 5.9%. Calluses were subcultured on the same medium. After an additional 2 weeks of subculture, calluses produced a few somatic embryos at a frequency of 28.8%. Upon transfer to MS basal medium, calluses producing a few somatic embryos gave rise to numerous somatic embryos, which subsequently developed into plantlets. Plantlets were successfully transplanted to potting soil and grown to maturity in a greenhouse.

Over-expression of Chinese Cabbage Calreticulin 1, BrCRT1, Enhances Shoot and Root Regeneration, But Retards Plant Growth in Transgenic Tobacco

Calreticulin (CRT) is a ubiquitously expressed, high capacity Ca(2+)-binding protein that is involved in intracellular Ca(2+) homeostasis and molecular chaperoning in the endoplasmic reticulum (ER). A cDNA encoding a calreticulin, BrCRT1 (Brassica rapa Calreticulin 1), has been isolated from Chinese cabbage (B. rapa subsp. pekinensis) flower bud. Constitutive over-expression of the BrCRT1 gene promotes robust shoot production and root formation at sub-optimal concentrations of BA/NAA, which are important factors controlling plant regeneration in tissue culture. In contrast, the suppressed BrCRT1 line exhibited a slight reduction of shoot and root regeneration. In spite of enhanced regeneration in tissue culture, the seedling and plant growth rate was inhibited in soil. The steady state level of BrCRT1 transcripts was sensitive to exogenous auxins and cytokinins, and rapidly accumulated within 30 min, and this induction required de novo protein synthesis. Together with the results of transgenic tobacco plants and mRNA analysis in Chinese cabbage, our data suggest that BrCRT1 genes may up-regulate the competency of vegetative tissue to respond to hormonal signals involved in shoot and root regeneration processes.

A differentially expressed bZIP gene is associated with adventitious shoot regeneration in leaf cultures of Paulownia kawakamii

Plant regeneration in tissue culture is important for the successful application of transgenic technology in crop improvement. We isolated a bZIP motif containing cDNA (PKSF1) from excised leaf cultures of the fast-growing tree Paulownia kawakamii by a novel subtractive hybridization strategy. PKSF1 codes for a leucine zipper transcription factor and is preferentially expressed during adventitious shoot regeneration. It showed about a sixfold higher level of expression in the shoot-forming tissues compared to the callus-forming cultures of Paulownia. In situ hybridization analysis showed that PKSF1 expression was localized in the developing shoot primordia of the shoot-forming cultures. It was also expressed in the apical meristem and provascular tissues in the vegetative shoot apex. Quantitative polymerase chain reaction results showed that only basal levels of transcripts were present in callus-forming tissues at day 0 and day 10, whereas maximal expression was seen in shoot-forming tissues at day 10, suggesting that PKSF1 is associated with adventitious shoot bud development in Paulownia.

Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.)

Hypocotyl segments of 2- to 3-week-old radish (Raphanus sativus L. cv. F1 Handsome Fall) seedlings produced yellowish compact calli when cultured on Murashige and Skoog's (MS) medium supplemented with 1 mgl(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and α-naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. When subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced white, compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on medium containing 0.1 to 3 mgl(-1) 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto medium containing 0.1 mgl(-1) 2,4-D and 1 mgl(-1) abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron.

High frequency somatic embryogenesis and plant regeneration in tissue cultures of Codonopsis lanceolata

Culture conditions for high frequency somatic embryogenesis and plant regeneration from cotyledonary explants of Codonopsis lanceolata are described. The maximum induction frequency of somatic embryos from cotyledonary explants was 80% on Murashige and Skoog (MS) medium containing 6% sucrose with 1 mg/l 2,4-dichlorophenoxyacetic acid and 10% coconut water. Upon transfer onto MS basal medium containing 3% sucrose, most somatic embryos developed into plantlets.

Plant regeneration in stem, petiole and leaf explant cultures of Ipomoea triloba L.

Plant regeneration in tissue cultures of Ipomoea triloba L., one of the related species of sweet potato, I. batatas (L.) LAM., was studied. Stem, petiole and leaf explants of I. triloba L. produced white friable calli on MURASHIGE and SKOOG (MS) medium supplemented with 0.5mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1.0mg/l kinetin, 3.0% sucrose and 0.8% agar, pH5.8. The induction frequency of callus reached 100.0% on all media used. When transferred onto MS medium supplemented with 0-0.4mg/l 3-indoleacetic acid (IAA), 0-10.0mg/l 6-benzylaminopurine (BAP), 3.0% sucrose and 0.8% agar, pH5.8, 65.0-100.0% of the calli formed adventitious roots. Except 0 level of IAA and BAP, the concentrations of IAA and BAP did not significantly influence the forination of adventitious roots. All calli cultured on the adventitious root formation medium were transferred onto hormone-free MS medium containing 3.0% sucrose and 0.8% agar, pH5.8, and the shoots were directly regenerated from (the green areas formed on) the stem-, petiole- and leaf-derived calli at the high frequencies. The highest frequencies of stem-, petiole- and leaf-derived calli regener-ating shoots were 68.4%, 42.1%, and 55.0%, respectively. The concentrations of IAA and BAP added to the adventitious root formation medium significantly influenced the shoot regeneration. The optimal concentrations were 0.4mg/l IAA + 2.0mg/l BAP to stem-derived calli, 0.4mg/l IAA + 5.0mg/l BAP to petiole-derived calli and 0mg/l IAA + 1.0mg/l BAP to leaf-derived calli, respectively. Some adventitious roots also produced a small number of shoots on the hormone-free MS medium. The regenerated shoots developed into whole plantlets after they were transferred onto the fresh hormone-free MS medium. No morphological variations were observed in the regenerated plants.

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl−1) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl−1 BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl−1). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.

Somatic embryogenesis and plant regeneration in tissue cultures of sweet potato (Ipomea batatas Poir.)

Leaf, shoot-tip, stem, and root explants of sweet potato (Ipomea batatas Poir.) gave rise to two kinds of callus on nutrient agar medium containing 0.5 to 2.0 mg/l 2,4-D. One callus, bright- to pale-yellow, was compact and organized, while the other was dull-yellow and friable. The former callus gave rise to numerous globular and heart-shaped embryoids. When transferred onto hormone-free medium, the embryoids readily developed into a torpedo-shape before germination. The plantlets were transplanted to soil where they flowered and formed storage roots at maturity.

Improved efficiency of somatic embryogenesis and plant regeneration in tissue cultures of maize (Zea mays L.)

Immature embryos of eleven cultivars of hybrid maize (Zea mays L.), cultured on 2,4-D-containing nutrient media, showed rapid proliferation of the scutellum and improved efficiency in the formation of embryogenic callus and somatic embryos. High concentrations of sucrose were found to be most favorable for the formation of the embryogenic callus. Embryoids obtained in cultures of all eleven cultivars germinated in vitro and produced normal green plants which were grown to maturity in soil. The effect of genotype and other factors on somatic embryogenesis in maize are discussed.

Somatic Embryogenesis and Plant Regeneration in Tissue Cultures of Panicum maximum Jacq.

Callus tissue cultures were initiated from immature embryos, mature embryos and young inflorescences of Guinea grass (Panicum maximum Jacq.) on Murashige and Skoog's (MS) medium supplemented with 2.5–10 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Calluses were transferred onto the same nutrient medium with 0.2 mg/l 2,4-D, or without 2,4-D. In callus cultures derived from immature embryos and young inflorescence segments, plantlets were produced via somatic embryogenesis after 3–5 wk. Young plants were successfully transplanted to pots and grown in the greenhouse. Plant development in callus obtained from mature embryos took place through the organization of shoot meristems. Regenerated plants were shown to have the normal tetraploid chromosome number of 2n = 4x = 32.