Multiple Shoot Clumps Research Articles

MICROPROPAGATION OF ONION (ALLIUM CEPA L.) THROUGH BULBLET FORMATION

Multiple shoot clumps or microclones were collected from two onion (Allium cepa L.) varieties for bulblet formation. MS medium supplemented with various concentrations of BAP, Kn, 2ip and NAA were used. The height responded shoot was observed 90% and 80% in Taherpuri and Indian variety, respectively on MS supplemented with 2.0 mg l-1 BAP+0.5 mg l-1 NAA. The best bulblet formation was obtained in MS containing 1.0 mg l-1 2ip. Finally, the rooted bulblets were hardened and established in soil under natural environment.

The effect of 2,4-dichlorophenoxyacetic acid, benzyl amino purin and cupric sulphate on in vitro propagation system from shoot apices of shoot tiller of hybrid Napier grass (Pennisetum purpureum Schum)

An efficient micropropagation method of hybrid Napier grass (Pennisetum purpureumn Schum) for in vitro plant production and material breeding was established from multiple-shoot clumps (MCS) regeneration system. This system was important for forage breeding system. Shoot apices from shoot-tillers produced MSC on Murashige-Skoog (MS) induction medium containing several combinations of BAP and 2,4-D in induction stage. The addition of 5 μM (v/v) and 50 μM (v/v) CuSO4 were added in best medium for inoculation to proliferate the clump in proliferation/multiplication stage. Plant regeneration was achieved by culturing on solid MS with several combination of medium containing NAA and BAP in regeneration stage. The best results for induction were Murashige-Skoog (MS) induction medium containing 2 mgL−1 BAP and 0.1 mgL−1 2,4-D. The proliferation stage on MS medium containing 5 μM CuSÜ4 effective for proliferation (50% multiple shoot formation). The regeneration stage using 0.1 mgL−1 NAA and 2.0 mgL−1 BAP (51.6% number of shoot can regenerate). All plantlets were successfully grown up in an acclimatization stage. Based on the results, the hybrid Napier grass regeneration via MSC was a stable tissue culture system (no albino plats), which could be applied either for further genetic transformation assay or for alternative supply of nursery plant in the future.

Pre-selection of banana somaclones resistant to Fusarium oxysporum f. sp. cubense, subtropical race 4

– Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases affecting banana crops worldwide. Therefore, the development of resistant cultivars is a promising alternative to mitigate the effects of the disease on banana plantations. The objectives of this study were to induce somaclonal variation in banana cultivars of the Silk and Cavendish types and to select somaclones resistant to subtropical race 4, thereby enabling the production of fruit in areas where this race is present. Shoot clump apexes of the Grand Naine and Maçã (Silk) cultivars were grown in MS medium. The cultures were subcultured four times. They were then challenged with fusaric acid (FA) in an experiment consisting of four treatments with different concentrations (0.1, 0.2, 0.3, and 0.4 mM) and five repetitions, each consisting of a Petri dish containing seven multiple shoot clumps in MS culture medium supplemented with 2.5 mg/L benzylamine purine. Multiple shoot clumps without the addition of FA were also used in the experiment, and were subcultured three times and maintained in a dark room. The multiple shoot clumps that survived the treatment with FA were transferred to MS medium and maintained in the growth chamber in the presence of light. The regenerated plants were later planted in tanks containing soil infested with an isolate classified as Foc subtropical race 4 (Foc STR4), and were evaluated for resistance to the pathogen at 90 days after inoculation (d.a.i.). Pathogen structures were confirmed by root clarification and root staining technique. All somaclones of the Maçã (Silk) cultivar were susceptible to Fusarium wilt and two somaclones of the Grand Naine cultivar were selected as resistant. The addition of FA as a selective agent was effective in the selection of somaclones among plants of the Grand Naine cultivar, as shown by the selection of two somaclones resistant to Foc STR4. The next step will consist of the agronomic and market potential validation of the selected somaclones, aiming to confirm their potential use by producers.

Regeneration and Genetic Fidelity Analysis of <i>Chlorophytum borivilianum</i> Using Flower Stalk as Explant Source

Chlorophytum borivilianum is a critically endangered plant well known for its medicinal properties for diabetes mellitus, diarrhea, arthritis, sterility, and erectile dysfunction, etc. Due to low viability and long dormancy of seeds, in vitro regeneration is required for large scale cultivation of this plant. In the present study, direct plant regeneration was optimized using flower stalk as explant. Nodal segments of flower stalk were sterilized and kept for direct regeneration on different combinations of BAP and KIN supplemented media. The highest, 15.27 ± 1.14 number of shoots were produced on medium containing BAP (2 mg/L) per nodal segment. The multiple shoot clumps regenerated from flower stalk were separated carefully and kept on rooting media. A maximum of 16.87 ± 1.53 roots per plant was observed in MS media having 0.5 mg/L of NAA. The rooted plantlets were shifted into the pot containing soilrite for hardening and acclimatization. The genetic stability of hardened plants was confirmed by start codon targeted, and inter simple sequence repeats molecular markers. All the 18 randomly selected plantlets showed similar genetic homogeneity to the mother plant. It is the first report on in vitro regeneration along with the genetic fidelity analysis of the regenerated plantlets from flower Stalk of C. borivilianum. As the standardized method of regeneration and mass multiplication is quite efficient and genetically stable, the protocol will be useful for the large-scale production of C. borivilianum to meet the market demand.

Effect of silver nitrate and growth regulators on direct shoot organogenesis and in vitro flowering from internodal segment explants of Alternanthera sessilis L.

Abstract The present report deciphers a rapid method for efficient shoot regeneration via direct organogenesis from internodal segment explants of Alternanthera sessilis. Internodal segment explants were placed on MS medium containing various doses of BAP and/or KIN (0.5–10.0 mg L 1) for shoot bud initiation. High frequency shoot bud induction (89%) was recorded on MS medium containing 2.5 mg L 1 BAP. Of the two cytokinin combinations tested, highest percent of plant regeneration (100%) with 81.1 shoots/culture was obtained on medium fortified with 2.5 mg L 1 BAP along with 50 mg L 1 adenine sulphate (AdS) and 0.5 mg L 1 silver nitrate (AgNO3) combination. Interestingly, BAP at 2.5 mg L 1 exhibited in vitro flowering from elongated shoots. Elongated shoot buds were carefully excised from the multiple shoot clumps and transferred onto half-strength MS medium without and/or fortified with different doses of NAA/IBA (0.1–1.0 mg L 1) for root initiation. IBA at 0.5 mg L 1 was found to be the best concentration for maximum percent of rooting (95.7%) over NAA. Well rooted plantlets were transferred into field conditions where they were successfully survived with 95%. It is noteworthy to mention that the random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) marker analysis between mother (control) and in vitro derived plantlets was found to be identical (monomorphic banding pattern) and no variability was observed with ISSR marker while RAPD marker showed 1.19% polymorphism. Hence, molecular results confirm that the in vitro regenerated plantlets were found to be the true-to-type in nature. In conclusion, the suitable growth hormonal combinations were identified for large scale production of genetically identical plantlets from internodal segment explants of Alternanthera sessilis.

In vitro Propagation of Red Lotus (Nelumbo nucifera Gaertn) - An Aquatic Edible Plant in Vietnam

Background: Lotus is an important aquatic plant with great economic value, not only as an ornamental flower but also as a source of herbal medicine. In general, lotus is usually propagated through the rhizome or tuber but the normal propagation rate is very low and it often depends on the quality of water environment and the weather conditions where it is grown. Lotus propagation by tissue culture has many predominant advantages compared with the traditional propagation methods. The current study aims to study the in vitro propagation of Hue’s Red Lotus, a famous local lotus in Hue city, Vietnam. Methods: Fresh mature seeds of Red lotus (23 to 25 days old) were used as initial materials for in vitro propagation. Experiments are carried out to assess sterilization ability by HgCl2, 0.1% and determine the appropriate formula for shoot regeneration, multiple shoot clumps induction, root induction of multiple shoot clumps and acclimatization. Result: The optimum formula for seeds of Vinh Thanh Red Lotus is to sterilize surface of seeds by 70% ethanol for 30 seconds and then HgCl2 0.1% for 16 minutes. The samples were cultured on MS (Murashige Skoog) medium supplemented without plant growth regulators for in vitro shoot regeneration. MS medium added with 0.5 mg/L BAP gave the highest multiplication rate (5.60 shoots per explant) and good quality shoots. Adding 0.5 mg/L NAA in rooting medium provided the highest rooting effect and good root quality (18.17 roots/shoot clumps). The survival rate of transplanting plants was 40% on mud medium which were soaked for one week in 2g/L KMnO4 after two weeks in the greenhouse.

Influence of Sucrose on Growth and [6]-Gingerol Production of In vitro-Grown Ginger (Zingiber officinale Roscoe)

The effect of sucrose on growth and [6]-gingerol production of in vitro- grown Zingiber officinale was investigated. Individual shoots from multiple shoot clumps were cultured in Murashige and Skoog (MS) basal medium supplemented with varying amounts of sucrose, i.e. 30, 60, 90 and 120 g/L. After three months of incubation, the growth and [6]-gingerol production of the cultures were compared. Results showed an increasing number of microrhizomes formed in response to increasing concentration of sucrose from 30-90 g/L except at a higher concentration of 120 g/L which was found to be already inhibitory. Likewise, the highest [6]-gingerol production was observed in medium supplemented with 90 g/L sucrose suggesting a positive correlation of [6]-gingerol production with the number of microrhizomes. These results suggest that sucrose concentration can be manipulated to improve [6]-gingerol production in ginger tissue culture.

In vitro plant regeneration of lotus (Nelumbo nucifera)

Abstract In this study, an efficient and reproducible plant regeneration system for lotus (Nelumbo nucifera Gaertn.) was established using shoot apical meristems from the buds and one-week-old aseptically germinated embryos as explants. Multiple shoot clumps were induced on Murashige and Skoog (MS) basal medium supplemented with various combinations of N6-Benzylaminopurine (6-BA) and α-Naphthalene acetic acid (NAA). The maximum response was obtained with 2.22 μM 6-BA, and produced 21.33 shoots per explant after four weeks. After five subcultures, multiple shoot clumps were transferred to MS basal medium supplemented with various combinations of 3-Indolebutyric acid (IBA), NAA and sucrose for root induction. After four weeks, plantlets with well-developed roots were achieved on MS basal medium supplemented with 0.54 μM NAA and 30 g/L sucrose with 100% rooting rate. The successfully acclimated plantlets were transferred to pots with the addition of 2 g/L KMnO4 into the soils, and finally fertile plants with much bigger leaves were obtained in the greenhouse. The survival rate was 97.33%.

Efficient nursery production and multiple-shoot clumps formation from shoot tiller-derived shoot apices of dwarf napiergrass (Pennisetum purpureum Schumach.)

The objective of this study was to improve an in vitro tissue culture system for the dwarf napiergrass l.Pennisetum purpureum Schumach.). Additionally, it was needed special treatments for acclimatization, and carried the risks of somaclonal variation and physiological aberations. Shoot apices as initial explants were isolated aseptically from shoottillers, and cultured on in vitro MS (Murashige and Skoog, 1962) medium containing 3.002 sucrose and 0.3o/o phytagel. The most effective phltohormone treatment for multiple-shoot clumps (MSCs) induction was 0.1 mg L-l 2,4-D plus 2.0 mg L-1 BAP. The addition of 50 p M CuSO+ could increase the percentage of MSCs proliferation. Plant regeneration frequency was achieved up to 84%o by culturing the MSCs on solid MS medium containing 0.1 mg L-1 NAA and2.0 mg L-l BAP. A11 regenerants were successfully grown up in the soil. Compared to control plants, in vitro regefierated plants did not reveal any significant difference (P > 0.05) on morphological characteristics and DNA content. The results of this study suggest that improved protocols for in vitro propagation would provide high quality nursery plant production of dwarf napiergrass in grassland field. Key words: dwarf napiergrass; flow cytometry; multiple-shoot clumps formation; plant regeneration.

Efficient nursery plant production of dwarf cogongrass (Imperata cylindrica L.) through mass propagation in liquid culture

AbstractDwarf cogongrass (Imperata cylindrica L.) was developed as a dwarf mutant through heavy‐ion beam irradiation 7 years ago. The dwarf mutant could be expected to use as a new variety for a cover plant with low maintenance, although it has poor seed fertility. To establish an efficient nursery production system for dwarf cogongrass, we attempted mass propagation of it in liquid culture and investigated the genetic stability of its regenerants. Multiple‐shoot clumps (MSCs) were initiated from apical meristems of dwarf cogongrass on Murashige and Skoog (MS) solid medium supplemented with 0.1 mg L−1 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 2.0 mg L−1 benzylaminopurine (BAP). Mass propagation conditions were established from MSCs cultured in MS liquid medium containing 0.05 mg L−1 2,4‐D and 0.5 mg L−1 BAP. The fresh weight of the MSCs per flask increased by more than 16 times in 14 days of liquid culture. Two different sizes of MSCs were produced in liquid culture. When smaller MSCs (<2 mm in diameter) were transferred to half‐strength hormone‐free MS solid medium, plant regeneration occurred at high frequency (93.3%). These tissues showed high regenerative potential with approximately 350 green shoots recovered within 50 days from 60 regenerating clumps. Furthermore, root elongation was vigorous in the regenerants growing in the same medium. Regenerated plants were acclimatized in hydrated Jiffy‐7 pellets for 30 days and then grown in soil as nursery plants. The plant height of regenerants was almost the same as original dwarf cogongrass and significantly lower than the wild type plants (P < 0.01). Analysis of amplified fragment length polymorphism (AFLP) banding patterns generated using 10 primer combinations showed no major genetic variations among the regenerated plants and original dwarf cogongrass.

Tissue Culture and Plant Regeneration of Rumex gmelini Turcz.

With the hypocotyls and seedling rhizomes of Rumex gmelini Turcz.as explants,the tissue culture of R.gmelini was studied by adding different concentrations of 6-BA and 2,4-D into MS basic mediums.The results showed that the better materials for multiple shoot clumps induction were seedling rhizomes;the best medium for multiple shoot clumps induction was MS+6-BA 3.0 mg·L-1+2,4-D 0.1 mg·L-1,the induction rate was 81.2%;the rooting medium was MS basic mediums without hormones,the rooting rate could get 92%,and under this conditions the plant regeneration could be obtained rapidly.After transplantation,about 80% of these seedlings could survive.

Fertile transgenic Brachiaria ruziziensis (ruzigrass) plants by particle bombardment of tetraploidized callus

We have produced transgenic plants of the tropical forage crop Brachiaria ruziziensis (ruzigrass) by particle bombardment-mediated transformation of multiple-shoot clumps and embryogenic calli. Cultures of multiple-shoot clumps and embryogenic calli were induced on solidified MS medium supplemented with 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/L 6-benzylaminopurine (BAP) or 4mg/L 2,4-D and 0.2mg/L BAP, respectively. Both cultures were bombarded with a vector containing an herbicide resistance gene (bar) as a selectable marker and the β-glucuronidase (GUS) reporter gene. Sixteen hours after bombardment, embryogenic calli showed a significantly higher number of transient GUS expression spots per plate and callus than multiple-shoot clumps, suggesting that embryogenic callus is the more suitable target tissue. Following bombardment and selection with 10mg/L bialaphos, herbicide-resistant embryogenic calli regenerated shoots and roots in vitro, and mature transgenic plants have been raised in the greenhouse. Polymerase chain reaction (PCR) and DNA gel blot analysis verified that the GUS gene was integrated into the genome of the two regenerated lines. In SacI digests, the two transgenic lines showed two or five copies of GUS gene fragments, respectively, and integration at different sites. Histochemical analysis revealed stable expression in roots, shoots and inflorescences. Transgenic plants derived from diploid target callus turned out to be sterile, while transgenics from colchicine-tetraploidized callus were fertile.

Role of ethylene and jasmonic acid on rhizome induction and growth in rhubarb (Rheum rhabarbarum L.)

Experiments were conducted to elucidate the hormonal induction and regulation of rhizome growth in rhubarb (Rheum rhabarbarum L.). It was found that ethylene is the key regulator of rhizome induction and development. The role of jasmonic acid (JA) and its interaction with ethylene in rhizome induction and growth were also examined. Both ethylene and JA have a significant effect on promoting rhizome formation in vitro. Conversely, the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) (1.5 μM) inhibited rhizome induction in multiple-shoot clumps in vitro, and suppressed the stimulatory effects of exogenously applied ethephon (1 mg l−1) and JA (10 ng l−1) in promoting mini-rhizome formation, further confirming the role of endogenous ethylene in the process. In addition, rhizome growth was significantly enhanced in the presence of both ethylene and JA (ethephon 1 mg l−1 and JA 10 ng l−1) compared to JA alone. These results suggest that endogenous ethylene is the key regulator of rhizome growth in rhubarb and JA promotes rhizome formation, possibly through inducing endogenous ethylene synthesis.

Production of polyploids from cultured shoot tips of Eucalyptus globulus Labill by treatment with colchicine

Polyploids from cultured shoot tips of Eucalyptus globulus were produced by treatment with colchicine. Results showed that the combination of 0.5% colchicine and treating multiple shoot clumps for 4 days was the most appropriate conditions for E. globulus polyploidy induction and the effect of the use of multiple shoot clumps for colchicine polyploidy-induced was better than single buds. By comparing the polyploidy plants with normal diploid ones in morphology, leaves of polyploid plants were thicker, larger, and darker green. The chromosome number of polyploidy plants that had been identified in morphology was 2n = 4x = 44, while that of diploids was 2n = 2x = 22.

Particle inflow gun-mediated transformation of multiple-shoot clumps in rhodes grass ( Chloris gayana)

Rhodes grass (Chloris gayana) is one of the most important warm-season forage grasses. It is cultivated in tropical and subtropical parts of the world and is mostly used for grazing and hay production. We have established a particle-bombardment transformation protocol for rhodes grass using multiple-shoot clumps (MSCs) as the target tissue. A vector pAHC25 containing a herbicide-resistance gene (bar) together with the beta-glucuronidase (GUS) gene was used in transformation experiments. The most efficient recovery of bialaphos-resistant tissue was achieved when the bombarded MSCs were first cultured for 15 d on bialaphos-free medium before being subjected to selection pressure. The resistant tissues regenerated transgenic plants that displayed GUS gene expression. Under optimized conditions, 251 target pieces yielded 46 transgenic plants from 4 independent transgenic lines.

Efficient transformation of beet (Beta vulgaris) and production of plants with improved salt-tolerance

Small bud tips of 1–3 mm in length were taken from multiple shoot clumps that derived from immature inflorescence cultures of beet as recipient for the Agrobacterium-mediated transformation and transgenic plants were obtained from eight genotypes. The optimal genetic transformation protocol was established as followed: the buds were immersed in Agrobacterium suspension of OD600 =0.3–0.5 for 5–10 min, with vacuum infiltration (0.3–0.5 × 105 Pa) or supplemented with 0.01% Silwet L-77, co-cultured for 2–4 days and followed by 10-day culture on medium containing 100 mg l−1 cefotaxime, then the buds were selected on medium containing 10 mg l−1 hygromycin B for three consecutive generations. The percentage of hygromycin-resistant buds after three selections varied from 13.3 to 30.6% with genotypes. The results of PCR and further Southern blotting of genomic DNA of hygromycin-resistant buds or plants showed that the exogenous hpt and AtNHX1 gene had been integrated into the genomes of some transformed buds or plants. The transgenic buds or plants with AtNHX1 gene encoding Na+/H+ antiport on the vacuole membrane of Arabidopsis showed improved salt-tolerance than the controls. AtNHX1gene inherited in some transgenic lines as Mendelian segregation. This result revealed that it was feasible to improve salt-tolerance of beets by the introduction of AtNHX1 gene into cultured buds.

Optimization of in vitro multiple shoot clump induction and plantlet regeneration of Kentucky bluegrass (Poa pratensis)

An efficient protocol for Kentucky bluegrass (Poa pratensis L.) in vitro culture was established using shoot apices of seedlings as explants. The optimal procedure of this protocol for majority of the genotypes was that meristematic cell clumps and small calluses were firstly induced from the bases of explants on initial culture medium supplemented with 0.9 μM 2,4-d and 8.9 μM 6-BA for 20 d, then were separated and transferred to shoot clumps induction medium containing 8.9 μM 6-BA for the formation of multiple shoot clumps. The percentage of multiple shoot clumps and numbers of shoots per clump were deeply related with the combinations of different plant growth regulators, duration of initial culture, the intensity of illumination and genotypes. Histological observation of the induced explants revealed that the meristematic cell clumps were produced from repeated division of the cortical cells and original meristematic primodium cells of explants, and the multiple shoots were formed via organogenesis pathway in the meristematic cell regions of cultures on shoot clumps induction medium. In this study, plantlets were efficiently regenerated on large scale from seven cultivars of Kentucky bluegrass. Hence the meristematic cell clumps and small calluses in this protocol could be considered good targets for genetic transformation of Kentucky bluegrass.

INVESTIGATIONS INTO IN VITRO MANIPULATION OF SOLANUM CENTRALE (BUSH TOMATO)

Solanum centrale J.M. Black (Solanaceae) commonly called bush raisin or bush tomato, or known locally as kampurarpa, is a small clonally spreading undershrub with yellow fruit that dry on the plant to resemble a raisin. The fruit provides excellent nourishment and is recognised as possibly the most important of all Central Australian native plant foods, and is one of the key commercially significant bushfood species. One of the main obstacles to the horticultural cultivation of S. centrale is the low germination rate of the seed. In this research, a medium was developed that stimulated multiple shoot initiation from different type of explants, resulting in multiple shoot clumps forming on various media within 6 weeks. Aseptic cultures were initiated from mature plants, using apical shoots of 5 mm in length and placed on basic MS medium supplement with 2 pM BAP. Three types of explants were investigated: apical buds, nodal cuttings with one axillary bud per explant, and leaf segments of 10 mm2 including the mid vein. The formation of adventitious shoots was achieved using half MS medium with B 5 vitamins and BAP, kinetin, and zeatin at concentrations of 1, 5, 10 and 25 pM with IAA at 1 pM. Roots formed when clumps of shootlets were transplanted to the glasshouse environment and planted in perlite:sand:peat potting mixture.

Shoot apical meristem: In vitro regeneration and morphogenesis in wheat (Triticum aestivum L.)

We report a less genotype-dependent in vitro regeneration system capable of producing multiple shoot clumps and whole plants in four different wheat genotypes. Shool apical meristems from 7-d-old-seedlings produced axillary and adventitious shoots and somatic embryos on media containing N6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). All four genotypes responded positively to shoot multiplication depending upon media composition. Scanning electron microscopies of cultures showed a proliferating budding state that gave rise to adventitious shoots and somatic embryos on further multiplication. The percentage of relative shoot apical meristem multiplication was 80–90%, and the average number of shoot meristems per multiplied shoot was 40–50 in all genotypes. Among different concentrations of phytohormones, 2 and 4 mgl−1 BA (8.8 and 17.7 μM) in combination with 0.5 mg l−1 2,4-D (2.26 μM) gave the best results. Actively multiplying shoot clumps were recovered with high frequency among 3-mo.-old cultures. These shoot clumps regenerated normally and produced fertile plants containing viable seeds. This in vitro system might prove useful for the production of transgenic plants of wheat in a relatively genotype-independent manner.

Establishment of multiple shoot clumps from maize (Zea mays L.) and regeneration of herbicide-resistant transgenic plantlets

A kind of quick, efficient and season-free inducing embryoid and multiple shoot clumps system from shoot tip meristems that derived from elite inbreds of maize was established. The herbicide-resistant gene als (coding Acetolactate synthase) isolated from a mutant of Arabidopsis thaliana was transferred to tissue pieces of maize multiple shoot clumps by microprojectile bombardment. Herbicide-resistant tissue and regenerants were obtained through selections with herbicide chlorsulfuron. PCR analysis and Southern blot hybridization indicated that gene als has been transferred to some regenerants. The test of spraying chlorsulfuron displayed that the transgenic plantlets and R(1) plants had favorable herbicide-resistant trait. We have established a new genotype-free system of maize which could rapidly and efficiently produce large quantities of transgenic plantlets.