Shoot Tip Culture Research Articles

Phenolic Compositions of Litchi Shoot Tips and Zygotic Embryos Collected in Different Months and Their Effects on the Explant Browning and Its Control

In the research it was examined the changes in total phenolic contents and seven major phenolic compounds (gallic acids, +(–) catechin, catechol, Chlorogenic acid, o-coumaric acid, rutin and quercetin) of two litchi cultivars (Purbi and Bedana) shoot tips and fruits (for zygotic embryos) collected in different months, in order to determine their effects on the explants browning during establishment stage of shoot tip culture. The concentrations of phenolic compounds varied depending on the cultivars and the months. Phenolic compounds showed various correlation coefficients with the explants browning. Total phenolic content and some individual phenolic compounds including +(–) catechin, catechol, gallic acid, chlorogenic acid and rutin quantified in this study showed significant positive correlations with the explants browning, while o-coumaric acid and quercetin did not exhibit any significant one. According to our results, explants browning are affected by the phenolic compounds at different ranges. In both litchi cultivars, shoot tips and fruits (for zygotic embryos) collected in March exhibited the lowest explants browning during the establishment stage as compared to those collected in the other months. So it may be possible to increase the success of shoot tip and zygotic embryo culture with the selection of the most suitable terms of explants collection. Browning of explants could be controlled by the use of antioxidants both in semi-solid and liquid culture.

Efficient Micropropagation of Pear Germplasm Using Soot Tips and Nodal Explants

We micropropagated pear (Pyrus species) using shoot tips and nodal explants from three pear genotypes. The ability to establish shoot tip cultures, proliferate shoots, induce rooting, and acclimatize the resulting plantlets are all elements of in vitro micropropagation. Shoots were induced from shoot tips on Murashige and Skoog medium (MS) with five different plant growth regulator combinations. The highest shoot formation rates were achieved for the three genotypes using MS supplemented with 1.0 ㎎/L N 6 -benzyladenine (BA) and 0.1 ㎎/L gibberellic acid (GA₃). The maximum shoot number and shoot length for the three cultivars were recorded with 2.0 ㎎/L BA and 0.2 ㎎/L indole-3-butyric acid (IBA) in multiplication medium using nodal explants produced from microshoots. Nodal explants with one or two axillary buds cultured for three weeks initiated roots on medium supplemented with various concentrations of 1-naphthaleneacetic acid (NAA) or/and IBA in half-strength MS medium for adventitious rooting. The highest rooting response was with the combination of 0.2 ㎎/L NAA and 0.2 ㎎/L IBA. A combination of NAA and IBA resulted in a significant increase in the rooting ratio over NAA or IBA alone. In this medium, the root formation rate according to ranged from 68.9% for the BaeYun No. 3 genotype to 51.8% for the Hwanggeum genotype. We also investigated the influence of the concentration the polyamine phloroglucinol in rooting medium. For all three genotypes, the highest rooting ratio, longest root length, and greatest root number were observed in the treatments with 75-150 ㎎/L phloroglucinol. Most rooted plants were acclimatized successfully.

English

Sugarcane (Saccharum officinarum L.) is one of the most important crops in Kenya and has wide range of economic importance. The sugar industry contributes up to 15% to the country’s agricultural gross domestic product and an estimated 25% of the population depends on the industry for their livelihood. However, the industry has been facing several challenges including declining yields due to use of poor quality planting materials. There is an increasing pressure to enhance the productivity of sugarcane in order to sustain profitable sugar industries in Kenya, while there are several diseases attacking sugarcane and reducing its quality. Seed multiplication of newly released varieties of sugarcane is one of the major constraints in Kenya as it takes 6-7 years to produce sufficient quantity of improved seed material. In vitro culture offers a practical and fast method for mass propagation of disease-free clonal materials. Successful protocols for shoot tip culture, callus culture, embryo culture, virus free plant production and somatic embryogenesis have already been established. Thus, in vitro technology can be used to enhance productivity of sugarcane in Kenya. Despite several advantages of applying micro-propagation technique in sugarcane such as quick multiplication of newly released varieties, rejuvenation of old deteriorated varieties; production of disease free seed; easy transportation of seed material; elimination of viruses; high cane productivity and sugar yield etc., this technique is not gaining popularity up to the desired extent. There are several constraints like the high cost of production and appearance of some variants in micropropagated population among others. The present article describes the status, challenges and opportunities of in vitro technology for the sugar industry in Kenya. Though, some problems have now been resolved to considerable extents which have been described in this review however, some constraints still require intensive research work to be resolved so that a safe and efficient exploitation of this technique can be ensured in sugarcane seed production programmes for enhanced yields and quality. Key words: Sugarcane, somaclonal variation, in vitro culture, meristems, micro-propagation, callus.

In Vitro Studies of Sugarcane Variety Co-91017 through Micropopagation of Shoot Tip Culture

Saccharum officinarum is an important sugarcane variety in Tamil Nadu. In our present study to optimization and standardization of protocol for induction of callus and regeneration of whole plantlets was established through in vitro culture of Sugarcane (Saccharum officinarum L.) by using shoot tip as an explant. The multiple shoot regeneration at various frequencies was observed by using different concentration and combination of growth regulators containing MS medium. The highest percentage of callus induction was observed in MS medium supplemented with 2.5 mg/L, 2-4 D. The best response in terms of multiple shoot induction was observed on MS medium supplemented with BAP 2.0 mg/L + IBA 0.5 mg/L. When in vitro shoot lets were inoculated on to the half-strength semi solid MS medium supplemented with 2.5 mg/L NAA, rooting was more profuse. Rooted shoots were transplanted in the green house for hardening and their survival rate was 85% in the field condition.

A novel approach for propagation of recalcitrant pistachio cultivars that sidesteps rooting by ex vitro grafting of tissue cultured shoot tips

Pistachio (Pistacia vera L.) cultivars need to be grafted on suitable rootstocks for their propagation, but grafting success is uncertain, preventing producers to meet plant demand. Thus, there is a need for a better grafting method that in addition to increase success rate, allows us an early detection of success. Here, we have developed an alternative grafting technique that achieves these goals using young potted seedlings of terebinth rootstock and reduced size micropropagated scions from commercial cultivars. During micropropagation of pistachio cultivars we studied the effects of the application of antioxidants together with the growth regulators meta-topolin and paclobutrazol on in vitro cultures to overcome the issues identified in the different phases of micropropagation: culture initiation, multiplication and rooting. Grafting succeeded using scions derived from in vitro cultured micro-shoots both from acclimated and non-acclimated plants. With this protocol, up to 75.7 % successful grafts were obtained, and grafted plants resumed growth from day 3, being ready for transplant a few months later. Sustained on our results, we present a simple and reliable system to reduce production time by ex-vitro grafting of shoot-tips from in vitro cultures onto 4 weeks old terebinth (Pistacia terebinthus L.) rootstock seedlings grown in pots.

Direct Regeneration of Plants Derived from in vitro Cultured Shoot Tips and Leaves of Poplar (Populus × euramericana ‘Neva’)

Direct Regeneration of Plants Derived from in vitro Cultured Shoot Tips and Leaves of Poplar (Populus × euramericana ‘Neva’)

TDZ and 4-CPPU Induce Embryogenic Response on Scalps of Recalcitrant East African Highland Banana

Breeding of banana conventionally is very difficult due to high levels of sterility arising from aneuploidy. Transformation of East African highland banana (EA-AAA banana) has been limited by difficulties in induction of embryogenic callus and/or embryogenic cell suspension (ECS), a requirement for efficient genetic transformation. In this study N-phenyl-N’-1,2,3-thidiazol-5-ylurea (TDZ) and N-(2-chloro-4-pyridyl)-N’-phenylurea (4-CPPU) that are highly active in regulating morphogenesis in tissue culture of many plant species were tested for enhancing embryogenic callus induction in recalcitrant EA-AAA banana through scalps. In vitro derived shoot-tips of banana cultivars Mbwazirume, Mpologoma, Nakabululu, Nakinyika and Nfuuka were subcultured on various combinations of TDZ and 4-CPPU till scalps were formed. In all the cultivars, ideal scalps for callus induction were achieved in over 50% of the shoot-tip cultures within four subculture cycles in the medium containing equal proportions of 9–13 µM of TDZ and 4-CPPU, and 26 µM of each. Embryogenic callus was formed on 2.5–20% of the scalp cultures depending on cultivar and, TDZ and 4-CPPU combination. Cultivars Nakinyika, Nakabululu, Mbwazirume and Nfuuka developed regenerable embryogenic callus. Of the plantlets regenerated 5 out of 12 and 4 out of 5 plantlets of cultivars Nakinyika and Mbwazirume, respectively, developed normal roots and were weaned onto soil. These results suggest that TDZ and 4-CPPU increases embryogenic response from scalps of EA-AAA banana which can enhance their genetic transformation.

Keynote Summary: Half a century on huanglongbing: learning about the disease, trying to control it

Keynote Summary: Half a century on huanglongbing: learning about the disease, trying to control it

CONTRIBUTION TO THE PRODUCTION SCHEME OF LOCAL CERTIFIED PROPAGATING MATERIAL OF ALMOND: IN VITRO SANITATION AND MICROPROPAGATION

Tissue culture techniques, alone or coupled with heat treatment, have largely contributed to the eradication of viruses from a wide range of infected plants and less for elimination of phytoplasma. In this study we present the elimination of both Prunus necrotic ringspot virus and Candidatus Phytoplasma phoenicium from two infected local almond accessions by using different tissue culture techniques combined with heat therapy. Shoot tip culture coupled or not with thermotherapy was the most effective to eliminate PNRSV in 100% of regenerated shootlets, while both shoot tip and stem cutting cultures associated to heat treatment were all suitable for phytoplasma elimination from regenerated shootlets. This free-pathogens material has been multiplied for successive subcultures and then rooted in vitro before being hardened in the glasshouse conditions. At this stage elimination of infectious agents has been confirmed prior to use the freed material as certified mother plants for further propagation. These results showed the important potential of traditional tissue culture techniques to be used in the certification program for local germplasm.

In vitro shoot proliferation and in vitro and ex vitro root formation of Pyrus elaeagrifolia Pallas.

Shoot-tip cultures of Pyrus elaeagrifolia Pallas, an important gene source for drought and chlorosis resistance in pear rootstock breeding, were established from a wild mature tree originated from seed. Murashige and Skoog basal medium supplemented with different concentrations of benzyladenine (BA) singly or in combination with auxin was used in the study. In the initial culture, the highest percentages (>80%) of shoot proliferation were obtained in the mediums supplemented with 9.0 μM BA and 0.5 μM indole-3-acetic acid. In the subcultures, the highest shoot proliferation rates were obtained in the medium containing 4.5 and 9.0 μM BA. The shoot proliferation rates ranged from 91.1 ± 2.4 to 96.4 ± 2.0% in the second subculture and from 76.7 ± 7.8 to 89.4 ± 3.3% in the third subculture. In the second subculture, the shoots grown on 9.0 μM BA without auxin produced the best proliferation (10.6 ± 1.6). For the in vitro rooting experiments, the highest rooting rate (54.2 ± 10.4%), root length (10.5 ± 2.4 mm), and root number (2.5 ± 0.6) were obtained from 10 days dark treatment on the medium containing half strength of macronutrients supplemented with 5 μM indole-3-butyric acid (IBA). For the ex vitro rooting experiments, shoot rooting was significantly influenced by 10 mM IBA applied as quick-dip method. The percentage of rooting was 55 ± 9.6% and root number was 1.8 ± 0.3 at this concentration.

English

Seeds of Helianthemum kahiricum have an excellent germination rate extending to about 90% within short time not more than four days after scarification (mechanical treatment) and a good protocol of disinfection. A high frequency of sprouting and shoot differentiation was observed in the primary cultures of nodal explants of H. kahiricum on Murashige and Skoog medium (MS) free growth regulators or with less concentration of kinetin (0.5 mg L -1 or 1.0 mg L -1 kin). In vitro proliferated shoot were multiplied rapidly by culture of shoot tips on MS with kinetin (0.5 mg L -1 ) which produced the greatest multiple shoot formation. The kinetin had a positive effect on the multiplication and growth, but a concentration that exceeded 2.0 mg.L -1 decreased the growth. A high frequency of rooting with development of healthy roots was observed from shoots cultured on MS/8 medium hormone-free. Key words : Helianthemum kahiricum, in vitro germination, multiplication, axillary buds. Abbreviation: Zea, Zeatin (cytokinine); IAA, indole-3-acetic acid (auxin); IBA, indole-3-butyric acid (auxin); Kin, kinetin (cytokinine); MS, Murashige and Skoog medium; NAA, 2-naphthalene acetic acid (auxin); 2iP, 2 isopentenyladenine (cytokinine).

Standardization of rapid axillary shoot multiplication throughin vitroshoot tip culture in gerbera

Rapid axillary multiplication was standardized using in vitro shoot explant of two gerbera cultivars on halfstrength MS medium supplemented with nine plant growth regulator combinations (BAP: 1.0, 1.5, 2.0 mg/l; BAP + kinetin: 1.0 + 0.25, 0.5, 1.0 mg/l and kinetin: 1.0, 2.0, 3.0 mg/l). BAP: kinetin ratio of 1: 4, 1: 2 and 1: 1 proved equally effective in inducing shoot proliferation as well as decreasing the number of days to shoot initiation. Shoot proliferation was higher on half-strength MS medium (8.12 shoots/plant) in comparison to 7.06 shoots/plant on full-strength MS medium. 100 percent rooting was achieved on MS medium supplemented with 0.75 mg/l. Root number and root length in both the cultivars improved significantly in media supplemented with IBA in comparison to NAA. Rooting response on MS solid, MS with half agar and MS liquid media was similar except that the root length in plantlets on MS liquid medium was significantly shorter. However, plantlets rooted on MS liquid and MS media with half agar exhibited a significantly improved survival (96.25%) of rooted plantlets ex vitro in comparison to those rooted on MS medium with full agar (71.25 and 86.25%, respectively).

Micropropagation of Four Coffee Cultivars (Coffea arabica L.) from Yemen through Shoot Tip Culture

The aim of this study is to optimize in vitro multiplication of Coffea arabica cvs 'Benan',' Burai',' Odayni'and 'Odayni- Bayat'. Seeds germinated on modified quarter MS medium with 1.0 g l−1 activated charcoal gave thehighest germination percentage of the four cultivars under study. BA at 8.0 mgl-1 was the most effective concentrationwith an average of 14 shoots per explant, resulting in better and morphologically superior microshoots in 'Benan' cv.However 'Burai' ,'Odayni' and 'Odayni- Bayat' cultivars, showed the highest number of shoots when they were grownon MS medium supplemented with BA at 4.0, 2.0 and 6.0 mgl-1 respectively. Root formation was 100% in response tothe application of IAA, IBA and /or NAA to the culture media for the four cultivars. Half MS supplemented with NAAat 2.0, 1.0 and 3.0 mgl-1 was found to be a suitable medium for root induction in excised micro-shoots, of 'Odayni','Burai' and 'Odayni-Bayat' cultivars, respectively. The plantlets were successfully acclimatized in the greenhouse;consequently the survived plants reached 100%.

BAP and IBA pulsing for in vitro multiplication of banana cultivars through shoot-tip culture

BAP and IBA pulsing for in vitro multiplication of banana cultivars through shoot-tip culture

VIRUS COMPOSITION INFLUENCES VIRUS ELIMINATION SUCCESS AND IN VITRO GROWTH CHARACTERISTICS OF THE GRAPEVINE cv. PLAVAC MALI

Shoot tip cultures coupled with thermotherapy was used for the production of virus- free candidate clones of the Croatian autochthonous and most important red-berried grapevine cultivar Plavac mali. The procedure was successful for the elimination of Grapevine leafroll- associated virus 3 (GLRaV-3) and Grapevine fleck virus (GFkV), but not Grapevine leafroll- associated virus 1 (GLRaV-1). Results showed that a selective virus eradication occurred depending on the initial composition of the viral population of treated samples. When the in vitro growth of cv. Plavac mali explants harbouring different viruses was compared, it was found that, similarly to virus elimination, tissue proliferation was virus composition- dependent. This is a first report on virus elimination in cv. Plavac mali and, by and large, from grapevines in Croatia.

Eliminating Potato Virus Y (PVY) and Potato Leaf Roll Virus (PLRV) Using Cryotherapy of in vitro-grown Potato Shoot Tips

Potato virus Y (PVY) and potato leafroll virus (PLRV) are among the most damaging potato viruses and prevalent in most potato growing areas. In this study, cryopreservation was used to eradicate PVY and PLRV using two cryogenic methods. Potato shoot tips proliferated in vitro were cryopreserved through droplet-vitrification and encapsulation-vitrification using plant vitrification solution 2 (PVS2; 30% glycerol + 15% dimethyl sulfoxide + 15.0% ethylene glycol + 13.7% sucrose) and modified PVS2. Both cryogenic procedures produced similar rates of survival and regrowth, which were lower than those from shoot tip culture alone. The health status of plantlets regenerated from shoot tip culture alone and cryopreservation was checked by reverse transcription-polymerase chain reaction. The frequency of virus-free plants regenerated directly from highly proliferating shoot tips reached 42.3% and 48.6% for PVY and PLRV, respectively. In comparison, the frequency of PVY and PLRV eradication after cryopreservation was 91.3~99.7% following shoot-tip culture. The highest cryopreserved shoot tip regeneration rate was observed when shoot tips were 1.0~1.5 mm in length, but virus eradication rates were very similar (96.4~99.7%), regardless of shoot tip size. This efficient cryotherapy protocol developed to eliminate viruses can also be used to prepare potato material for safe long-term preservation and the production of virus-free plants.

Blueberry red ringspot virus eliminated from highbush blueberry by shoot tip culture

In order to find an effective elimination method of Blueberry red ringspot virus (BRRV), shoot tips of 2–3 mm size originating from buds of highbush blueberry cv. Darrow naturally infected by BRRV and collected in winter months were regenerated and multiplied on woody-plant medium (WPM) with 1.5 mg/l zeatin and 20 g/l sucrose. Developed shoots tested negative by polymerase chain reaction (PCR) for BRRV were transferred onto a rooting medium consisting of WPM supplemented with 1 mg/l indole-3-butyric acid. Rooting plants were transferred into soil and tested twice by PCR 6 months apart. Ninety per cent eradication of BRRV was achieved while isolating shoot tips, multiplying them in one vegetative generation, and regenerating whole 50–60 cm high plants within 30 months. All plants developed rich fruits after their overwintering in a cold greenhouse and were tested BRRV negative in summer 2013.  

Effect of temperature on shoot tip culture of Paris polyphylla var. chinensis (Franch.) Hara

Bioactive compounds from rhizome of Paris polyphylla var. chinensis (PPC) have been reported to have many pharmaceutical activities such as antitumor, anticancer and hemostatic [1]. However, the propagation of PPC had become a severe problem due to deforestation and overharvesting. Hence, appropriate methods are required for propagation [2]. The study of PPC rhizome shoot tip culture was limited by bacterial contamination in normal tissue culture laboratory. Therefore, environmental factors such as temperature were studied on the propagation of shoot tip explants of PPC in MS free hormone medium. They were cultured in a gradient incubator at 5 to 18 °C for 45 days. It was found that temperature is a crucial factor controlling in vitro culture of PPC, and the temperature between 14 to 16 °C were better than other temperature treatments in controlling endophytic bacteria and PPC growth. The shoot tip explants cultured at this range of temperature grow up to a stem of 7.5 cm length. At a temperature lower than 14 °C, the shoot tip explants could not grow. At the temperature higher than 18 °C, the shoot tip explants did not grow because the explants were contaminated by endophytic bacteria. It could be concluded that the temperature between 14 to 16 °C is the suitable culturing condition for shoot tip culture of PPC.

Elimination of mixed ‘Odontoglossum ringspot’ and ‘Cymbidium mosaic’ viruses from Phalaenopsis hybrid ‘V3’ through shoot-tip culture and protocorm-like body selection

Phalaenopsis is one of the most popular orchid plants in the global market. Several viruses have been reported to negatively impact its growth, yield and quality. A mixed infection of Odontoglossum ringspot virus and Cymbidium mosaic virus was detected in Phalaenopsis hybrid “V3” (Phal. Yukimai × Phal. Taisuco Kochdian) plants in Taiwan. In the present communication, we report a relatively simple protocol for elimination of these two viruses using shoot-tip culture, and early isolation and selection of virus free protocorm-like body (PLB) through subcultures. The indexing of viruses in the field grown plants, PLBs and tissue culture plants was carried out by the Indirect enzyme-linked immunosorbent assay (ELISA), and one-step multiplex reverse transcription polymerase chain reaction (RT-PCR). The induction of PLBs in shoot-tips was achieved on 1/2× Murashige and Skoog's basal medium supplemented with 1% sucrose and 0.9% Bacto-agar. PLBs could be maintained, proliferated and converted to plantlets on 1 g/L Hyponex medium supplemented with 1% sucrose, 6% potato pulp, 0.5 g/L tryptone, 0.25% activated charcoal and 0.6% Bacto-agar. The regenerated plantlets from virus-free PLBs acclimatized easily in a greenhouse and showed 100% survival rate. All the tissue culture-raised Phalaenopsis plants in the greenhouse tested negative for the two viruses. Our study demonstrates that some PLB lines selected at the first subculture as virus-free were found to be infected with virus at second subculture, however, re-occurrence of virus was never found in PLB lines at third subculture onwards. Hence, at least 3 subcultures are necessary to authenticate that the cultures are free of viruses. Simple culture media without plant growth regulators used in the present study minimizes the chances of somaclonal variations and ensures genetic uniformity of cultured plantlets, a highly desirable trait in the orchid industry. The method developed in the present study has potential of virus elimination, mass propagation of genetically uniform and virus-free Phalaenopsis plants.

Rapid in vitro propagation system through shoot tip cultures of Vitex trifolia L.-an important multipurpose plant of the Pacific traditional Medicine.

A rapid and efficient plant propagation system through shoot tip explants was established in Vitex trifolia L., a medicinally important plant belonging to the family Verbenaceae. Multiple shoots were induced directly on Murashige and Skoog (MS) medium consisting of different cytokinins, 6-benzyladenine (BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP), BA at an optimal concentration of 5.0μM was most effective in inducing multiple shoots where 90% explants responded with an average shoot number (4.4±0.1) and shoot length (2.0±0.1cm) after 6weeks of culture. Inclusion of NAA in the culture medium along with the optimum concentration of BA promoted a higher rate of shoot multiplication and length of the shoot, where 19.2±0.3 well-grown healthy shoots with an average shoot length of 4.4±0.1cm were obtained on completion of 12weeks culture period. Ex vitro rooting was achieved best directly in soilrite when basal portion of the shoots were treated with 500μM indole-3-butyric acid for 15min which was the most effective in inducing roots, as 95% of the microshoots produced roots. Plantlets went through a hardening phase in a controlled plant growth chamber, prior to ex-vitro transfer. Micropropagated plants grew well, attained maturity and flowered with 92% survival rate. The results of this study provide the first report on in vitro plant regeneration of Vitex trifolia L. using shoot tip explants.