Shoot Induction Medium Research Articles

Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement.

Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important crop.

English

An efficient in vitro protocol has been developed for the multiplication of shoots and conservation of a rare succulent medicinal plant Caralluma stalagmifera var. longipetala growing wildly in Karnataka State. Proliferation of multiple shoots was achieved on Murashige and Skoog’s (MS) medium supplemented with various concentrations of 6-benzyladenine (BA), Kinetin (Kn), indole acetic acid (IAA), α- naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) alone or with various combinations from the nodal explants. The nodal explants cultured on medium containing BAP (2.0 mg/L) along with 0.5 mg/L Kn and 0.3 mg/L IAA produced the highest number of shoot sprouting (2.60±0.16) and maximum shoot length (3.96±0.20). The considerable frequency of callus induction and embryogenesis was noticed both in 1.0 mg/L NAA and 0.5-2 mg/L, 2, 4-D. The calli transferred to shoot induction medium containing the combination of hormones BAP (1.0 mg/L) plus IAA (0.2 mg/L) and NAA (0.1 mg/L) successfully regenerated in vitro shootlets. The in vitro rooting was achieved from both direct shoots regenerated from nodal explants and callus derived shootlets with NAA (0.2 mg/L). The in vitro rooted plantlets were successfully acclimatized (75%) in the greenhouse and gradually transferred to open field conditions. Key word: Micro propagation, medicinal plant, Caralluma stalagmifera var. longipetala.

THE ESTABLISHMENT OF THE RECIPIENT SYSTEMS OF GENETIC TRANSFORMATION FROM 'BENIHOPPE' STRAWBERRY (FRAGARIA ×ANANASSA DUCH.)

In order to research fruit-specific genetic expression, the vectors that contain a fruit-specific promoter were constructed. We wanted to prove that in strawberry, so the recipient systems of genetic transformation from 'Benihoppe' strawberry were established. Effects of the basal medium, the types and concentrations of plant hormones and plant growth regulators on shoot regeneration from leaves of strawberry were studied using orthogonal experimental designs in order to establish a shoot regeneration system from leaves. The results showed that IBA played a vital part in proliferation of shoots. The optimum medium for shoot induction was MS + TDZ 2.0 mg/L + IBA 0.2 mg/L, the regeneration rate 90.00%. The optimum medium for shoot proliferation was MS + 6-BA 0.5 mg/L + IBA 0.2 mg/L and the average number of in shoots was 7.67. The optimum medium for shoot subculture was MS + 6-BA 0.5 mg/L + 2,4-D 0.1 mg/L and the average height of shoots was 5.98 cm. The optimum medium for shoot rooting was 1/2MS + 6-BA 0.5 mg/L + 2,4-D 0.1 mg/L and average number of shoots rooting was 4.5. The calliwere fully suppressed with 20 mg/L ofkanamycin in differentiation media; the explants gradually turned white and died. The regeneration system established in this paper could be used for genetic transformation in strawberry.

희귀 수종 미선나무(Abeliophyllum distichum Nakai.)의 기내 증식 및 발근에 미치는 LED (light emitting diode) 효과

희귀 멸종위기 식물인 미선나무를 재료로 기내 증식 및 발근에 미치는 광질 및 절편체(정아지 및 액아지) 효과를 조사하였다. 광질은 냉백색 형광등(F), 100% 청색광 LED(B), 혼합광(BR; 50% blue LED+50% red LED) 및 100% 적색광(R)을 사용하였다. 줄기의 증식은 절편체 종류에 따른 뚜렷한 차이를 보이지 않았으나 광질에 따른 차이를 보였다. 전반적으로 줄기의 증식은 청색광 및 혼합광에서 촉진되는 것으로 나타났으며, 특히 청색광에서는 일부 절편에서 발근이 이루어 졌다. 줄기의 생장 역시 광질에 따른 현저한 차이가 없었다. 적색광에서 줄기 신장이 촉진된 반면 잎의 발달은 저조하였다. 발근은 전반적으로 광질에 관계없이 정아지가 액아지보다 양호하였고, 뿌리수도 증가하였으나 혼합광에서는 발근이 억제되었다. 한편 적색광에서는 뿌리의 발달이 매우 부진하여 다른 광질과 유의적인 차이를 보였다. 발근유도 시 줄기는 주로 단일 줄기로 자랐다. 이상의 결과를 볼 때 LED를 이용한 미선나무의 기내번식은 정아지를 절편으로 청색광 및 혼합광 LED (BR) 하에서 배양함이 좋은 것으로 나타났다. This study was conducted to elucidate the effect of light sources and explant types on in vitro shoot multiplication and rooting of a rare and endangered plant Abeliophyllum distichum. Both apical buds and axillary buds were used as explants under 4 different light sources, cool white florescent light (F), 100% blue light-emitting diode (LED) (B), 50% blue and 50% red LED mixture (BR), and 100% red LED (R). Clear difference was observed in terms of shoot proliferation by light sources types but not by position-dependent explant types. Multiple shoot induction rates were enhanced under both B and BR light sources. Spontaneous rooting was induced in shoot induction medium under B light source. Both the rates of rooting and numbers of roots per explant were higher in apical bud explants compared to axillary bud explants. Interestingly R light source stimulated shoot elongation but inhibited root development. Therefore, our results suggest that the use of apical bud explants under B or BR light sources is suitable for in vitro micropropagation of a rare and endangered plant species, Abeliophyllum distichum.

Efficient soybean regeneration and Agrobacterium-mediated transformation using a whole cotyledonary node as an explant.

An optimized regeneration and Agrobacterium-mediated transformation protocol based on whole cotyledonary node explants was developed in soybean (Glycine max) cultivar Zhong Huang 13. Adding 6-benzylaminopurine (BAP) in a germinating medium could significantly increase regeneration efficiency; the optimal BAP concentration for shoot formation was 0.5 mg/L. The concentrations of plant growth regulators in a shoot induction medium were optimized by the orthogonal test [L9 (3(3))]. The best combination for shoot regeneration was a medium of Murashige & Skoog salts with B5 vitamins (MSB) supplemented with 3.5 mg/L BAP, 0.2 mg/L indole-3-butyric acid (IBA), and 0.2 mg/L kinetin (KT). Under this favorable condition, one node could regenerate 28-30 shoots. Soybean whole cotyledonary nodes were transformed by inoculation with A. tumefaciens strain EHA105 harboring a vector pBI121 containing a β-glucuronidase gene (gus). GUS assay, polymerase chain reaction, and Southern blot analysis indicated that the gus gene was transformed into soybean plants with 23.1% transformation efficiency. Transgenic plants could be obtained within 5-6 weeks, which was about 4 weeks less than that of a traditional single cotyledonary node method.

Abscisic acid promotes shoot regeneration in Arabidopsis zygotic embryo explants

An efficient shoot organogenesis protocol for Arabidopsis zygotic embryo explants of Landsberg erecta ecotype was established. This de novo shoot organogenesis protocol has three different steps, i.e., induction of callus in an auxin-rich callus induction medium, the formation of green-organogenic callus in the shoot induction medium (SIM), and the final morphological differentiation of shoot in the hormone-free shoot development medium (SDM). Abscisic acid (ABA), auxin, and cytokinin (CK) were used in the SIM. Individual plant growth regulators as well as their combination were studied to understand their importance in the shoot induction treatment. We found that a combination of ABA + CK and ABA + CK + auxin induced higher shoot organogenic ability in the callus than ABA, CK, and auxin alone. Optimum ABA concentration on shoot organogenesis was determined to be 10−5 M. Morphological characterization of callus induction and shoot organogenesis events indicated that calli were derived from the cotyledons of zygotic embryo explants and the formation of green organogenic calli was specific to the exogenous inclusion of ABA + CK in the SIM. During the time of shoot development, the green organogenic callus became darker green due to the formation of anthocyanins. Shoot organogenic calli in the SIM and the SDM were easily identified by the green-colored calli and anthocyanin pigments, respectively. Furthermore, we demonstrated the significance of exogenous and endogenous ABA in shoot organogenesis by fluridone treatments. The inclusion of ABA in SIM has a significant effect on shoot formation.

Efficient plant regeneration from shoot apex explants of maize (Zea mays) and analysis of genetic fidelity of regenerated plants by ISSR markers

An efficacious regeneration system was developed from shoot apex explants of Zea mays using a two-step culture procedure. Seventeen Indian genotypes were assessed for their regeneration potential. The maximum response of shoot induction was obtained from explants cultured on Murashige and Skoog medium supplemented with 4.5 µM thidiazuron and 26.7 µM glycine. Maximum mean number of shoots (17.2) was observed in genotype COH (m)-5 while NPK was the least responsive (6.7). Shoot clumps transferred from shoot induction medium to multiplication (second) medium amended with 1.1 µM thidiazuron and 0.88 µM N 6 -benzylaminopurine showed increased number of shoots in COH (m)-5 (36.1 shoots); NPK was the least responsive with an average of 9.5 shoots. The best response in root induction, with a larger number of roots (10.5) and longer roots (6.6 cm), was observed in Murashige and Skoog medium supplemented with 7.3 µM indole-3-butyric acid and 7.9 µM phloroglucinol. Analysis of variance indicated that plant regeneration response varied greatly among the genotypes. In vitro raised plants were successfully transferred to the field after hardening, with a 99 % survival rate. Inter simple sequence repeats analysis revealed that the similarity matrix pair-wise value was 1, the Mantel test value was p 1.0; Analysis of molecular variance genetic variances were 93 % within the population and 7 % between populations; Principal component jolliffe cut off was 0.15, Principal component and Principle coordinate analysis % variance was 13.19. These values were congruent for both the mother and the in vitro-raised plants, confirming genetic integrity.

Shoot regeneration and cryopreservation of shoot tips of apple (Malus) by encapsulation–dehydration

Here, we report an efficient and widely applicable method for cryopreservation of Malus shoot tips by encapsulation–dehydration using adventitious shoots. Shoots were induced from leaf segments cultured on a shoot induction medium containing 2–3 mg L−1 thidiazuron, depending on genotype, and 0.5 mg L−1 indole-3-butyric acid. Shoot tips (3 mm in length) containing six leaf primordia excised from 11-wk-old adventitious shoots were encapsulated and precultured with 0.5 M sucrose for 5 d, followed by air-drying for 6 h prior to direct immersion in liquid nitrogen. With our protocol, we obtained a mean organogenesis rate of 100%, a mean of 4.5 adventitious shoots per explant (leaf segment), and a mean shoot recovery of 57.0% from cryopreserved shoot tips in four Malus species. Inter-simple sequence repeat (ISSR) analysis did not reveal any polymorphic bands in regenerants recovered from either leaf segments or cryopreserved shoot tips of ‘Gala’. To the best of our knowledge, this is the first report on cryopreservation of Malus shoot tips using adventitious shoots derived from leaf segments and is the most widely applicable protocol so far reported for cryopreservation of Malus. Establishment of this protocol provides an alternative means for cryopreservation of Malus.

Effect of Different Auxin Concentration on Rooting in Pigeonpea (Cajanus cajan [L.] Millsp.) cv. Manak (H77216) ViaCotyledonary Node Explants

Pigeonpea ( Cajanus cajan [L] Millsp.) is an important grain legume of the semi-arid tropics. It provides protein rich food. Seeds of pigeonpea were collected and surface sterilized, these sterilized seeds were germinated aseptically in shoot induction media (SIM) fortified with MS media supplemented with 2mg/l BAP. Cotyledonary node explants (excised from 15 day old seedlings) were transferred to shoot elongation media (SEM) for elongation fortified with MS media + 1.0 mg/l GA 3. Elongated shoots were transferred to root induction media (RIM) using different concentration of IAA and IBA. Highest percentage (93) of rooting was observed on modified MS media + 1.0 mg/l IBA.

Optimized shoot regeneration for Indian soybean: the influence of exogenous polyamines

A simple and efficient regeneration protocol was established for soybean [Glycine max (L.) Merrill]. Cotyledonary node explants from 7-day-old in vitro seedlings were used as explants. The effect of different plant growth regulators [N 6 –benzyladenine (BA), kinetin (KT), thidiazuron (TDZ), gibberellic acid (GA3), zeatin riboside (ZTR), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA)] along with polyamines (Spermidine, spermine, and putrescine) were investigated at different stages of regeneration using direct organogenesis system. Exogenous spermidine (137.69 μM) in shoot induction medium containing optimal BA concentration (2.22 μM) induced maximum number of shoots (39.02 shoots/explant) compared to BA (2.22 μM) alone. Regenerated shoots elongated well in shoot elongation medium containing GA3 (1.45 μM) and spermine (74.13 μM), and developed profuse roots in root induction medium containing putrescine (62.08 μM). Rooted plantlets were successfully hardened and acclimatized with a survival rate of 92 %. The amenability of the standardized protocol using cultivar PK 416 was tested on four more Indian soybean cultivars JS 90–41, Hara soy, Co1, and Co2 of which PK 416 was found to be the best responding cultivar, with a maximum of 96.94 % shoot induction.

Arachis hypogaea L. Direct Organogenesis and SDS - PAGE Analysis in Groundnut

The present research work was carried out to observe the effect of various concentrations of Benzyl amino purine (BAP) in terms of multiple shoot induction with special emphasis on qualitative and quantitative changes of proteins during multiple shoot formation. From whole embryonated cotyledons of groundnut. The shoot induction medium containing different concentrations of Benzyle amino purine ranged from 5.0 to 25.0mg/l along with lower concentration of Indole acetic acid (IAA).The multiple shoots were observed in all the BAP concentrations at a varying levels. Among the various levels of BAP, 25.0mg/l of BAP plus 0.50mg/l of IAA was found to be the most effective in terms of multiple shoot induction. The growth parameters like plantlet height, fresh and dry weight also highly influenced by the concentration of the BAP. The root induction was achieved in micropropagated shoots by using Indole butyric acid (IBA) at a concentrations of 1.0 to 5.0mg/l. The mean number of roots, root length was highly influenced by IBA. In all the concentrations of IBA the rooting was associated with moderate and heavily associated basal callusing. The quantitative estimation of protein content in regenerated shoots also increased with higher concentrations of BAP. The SDS-PAGE analysis showed that there was a nine visible bands with 82.2, 76.4, 57.5, 27.6, 23.7, 21.4, 18.2, 16.5 and 12.8 kDa were observed in multiple shoots derived from all the concentrations of BAP. Whereas in whole embryonated cotyledonary explants there was a entirely different protein banding pattern with 82.2, 76.4, 57.5, 27.6, 23.7, 21.4, 18.2, 16.5 and 12.8 kDa were recorded. Original Research Article British Biotechnology Journal, 4(6): 720-732, 2014 721

Cytokinin, Carbon Source, and Acclimatization Requirements for <i>in Vitro</i> Propagation of <i>Scutellaria barbata</i> D. Don and <i>Scutellaria racemosa</i> Pers.

Micropropagation protocols to minimize hyperhydricity were optimized for medicinal Scutellaria barbata and Scutellaria racemosa. Six cytokinins and eight different carbon sources at two different incubation periods of 14 and 21 days were studied for adventitious shoot bud induction using nodal explants. In S. barbata, 5 μM meta-Topolin and 0.1 μM NAA supplemented shoot induction medium produced four shoots each after 14 and 21 day incubation. Observation of S. racemosa nodal explants recorded four and five shoots after 14 and 21 day incubation. In both species, control explants (no plant growth regulators in the medium) consistently resulted in the bud break with two shoots in both 14 and 21 day incubation. The effect of carbon source on shoot regeneration was studied by supplementing eight different sugars at 0.1 M concentration to the optimized shoot induction medium (5 μM meta-Topolin and 0.1 μM NAA). S. barbata nodal explants cultured on shoot induction medium supplemented with fructose and glucose for 14 days produced 10 and nine adventitious shoots respectively; and after 21 day incubation adventitious shoot count reached 19 in glucose supplemented medium. S. racemosa explants in the same experiment produced five shoots in maltose and four shoots in sorbitol supplemented medium after 14 day incubation; whereas after 21 day incubation, sucrose and maltose produced five shoots; fructose, glucose, and sorbitol produced four shoots. Regenerated plants were successfully acclimatized and Scanning Electron Microscopy of the leaf surface revealed differences in stomatal behavior and cuticle deposition between in vitro and acclimatized plants. The antioxidant assay conducted on both Scutellaria species showed considerable total polyphenol content, TEAC activity and flavonoid content in fresh and dried leaf samples attributing to their medicinal potential.

Silicon Promotes Adventitious Shoot Regeneration and Enhances Salinity Tolerance ofAjuga multifloraBunge by Altering Activity of Antioxidant Enzyme

We investigated the effect of Si concentration on shoot regeneration and salinity tolerance of Ajuga multiflora. Addition of Si to the shoot induction medium significantly increased the frequency of shoot induction. The average number of shoots regenerated per explant decreased on the medium containing NaCl alone, while there was less decrease when the shoot induction medium was supplemented with both NaCl and Si. The shoot induction percentage increased linearly with increasing concentration of Si in the NaCl containing medium. Addition of Si to the shoot induction medium significantly increased SOD, POD, APX, and CAT activity in regenerated shoot buds as compared with the control. The inclusion of Si to the NaCl containing medium significantly increased the SOD activity in leaves and roots, while it decreased POD, APX, and CAT activity in both organs. Scanning electron microscopic analysis showed that there are no distinct differences in the structure of stomata between the control and Si-treated plants. However, NaCl treatment significantly affected the structure and number of stomata as compared to the control. Wavelength dispersive X-ray analysis confirmed the high Si deposition in trichomes of plants grown in the Si containing medium but not in plants grown in the medium without Si.

Micropropagation of Kebericho: An Endandered Ethiopian Medicinal Plant

Echinops kebericho, endemic to Ethiopia, is a critically endangered medicinal plant. It is among the most important medicinal plants of the country, valued primarily for its root parts. The commercial harvesting and sale of roots of E. kebericho have threatened local populations. This study aimed to develop micropropagation protocol for E. kebericho using shoot tip explants. The study started with seed germination test using seeds stored for different months. Shoot tips from in vitro germinated seedlings were cultured on shoot initiation MS media supplemented with 0, 0.1, 0.5, 1.0, 1.5 and 2.0 mg/l BAP or KN alone. Explants were cultured on shoot proliferation media fortified with Kinetin, BAP, and TDZ each at 0.0, 0.5, 1.0, 2.5, 3.0 and 5.0 mg/l either alone or in combination with 0.0, 0.1, 0.25 and 0.5 mg/l NAA. For rooting, full, half and 1/3 strength MS media supplemented with IBA and NAA alone each at 0, 0.05, 0.1, 0.5, 1.0, 1.5 and 2.5 mg/l were used. Growth regulator free MS medium was used as control. Study results showed that 100% germination was recorded in fresh seeds and dropped as low as 65.18% and 22.3% for 3 and 5 months seeds respectively. 1.0 mg/l KN and 0.5 mg/l KN + 0.1 mg/l NAA showed maximum shoot proliferation on shoot induction media and shoot multiplication media respectively. Best rooting was obtained on 1/3 MS containing 1.5 mg/l NAA with 8.23 roots and 4.82 cm root length and established under greenhouse with 83% survival.

Proteomic analysis of cotyledonary explants during shoot organogenesis in Vigna radiata

Vigna radiata or mungbean belongs to the legume family of plants. Mature mungbean seeds are rich source of dietary proteins for human nutrition. The present study was aimed to analyze the comparative protein profiles of two cotyledon types, Cot and Cot E, prior to and during early time points of shoot morphogenic induction to understand the unique differential regeneration response in these two explant types which was reported earlier. These explants were grown separately in vitro on the shoot induction medium (SIM) containing Gamborg’s B5 basal nutrient composition supplemented with 15 μM N6-benzyladenine. Isolation and characterization of the proteomes from Cot and Cot E explants at different time points, during early events of shoot differentiation, were performed using two dimensional gel electrophoresis following matrix assisted laser desorption-ionization tandem mass spectrometry. A total of 112 differentially identified proteins were classified according to their putative biological function. The differential control of protein synthesis between these explants under control condition, i.e. before in vitro culture, was also noted. In Cot E explants SIM induced prompt acquisition of competence for direct shoot morphogenesis probably through fast phytohormone signaling. Over accumulated proteins in Cot E indicated stimulation of several metabolic and associated pathways earlier than Cot explants. Abundance of stress and defense related proteins in Cot E explants was presumably to cope up with stressful cultural condition. Enhanced accumulation of folding-assisted proteins involved in organogenesis mediated cellular reprogramming in Cot E explants contributed further in rapid and efficient regeneration responsiveness.

Production of recombinant miraculin protein using transgenic citrus cell suspension culture system

Miraculin gene containing the N-terminal signal peptide was introduced into navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) callus cells by Agrobacterum-mediated transformation. Transgenic somatic embryos were screened on the shoot induction medium containing 25 mg hygromycin L−1. Citrus callus cells were reproduced from the green color somatic embryos on the callus reproduction medium. The obtained transgenic cells were cultured in Murashige and Tucker’s liquid medium containing 50 g sucrose L−1 in a shaking incubator. Similar to the native miraculin, the secreted recombinant miraculin protein formed a disulfide-linked dimer and retained taste-modifying activity. The stability of recombinant protein expression was confirmed over nine generations of callus. This production system can be an excellent alternative for producing various recombinant proteins as well as miraculin.

Regeneration System Establishment of Disease-resistant Asparagus Variety

Asparagus officinalis Geland is highly resistant to stem blight and was used to study the effect of different auxins and cytokines on callus induction and multiplication of buds and rooting.Results showed that the best culture medium for callus induction was MS + 6-BA 0.3 mg/L + 2,4-D 1.5 mg/L +Sucrose 25 g/L +Agar 7 g/L,pH 5.8.The best culture medium for callus proliferation and shoot induction was MS+NAA 0.3 mg/L +6-BA 0.5 mg/L +Sucrose 30 g/L +Agar 7 g/L,pH 5.8.The best culture medium for root induction was 1/2 MS+0.5 mg/L IBA+1 mg/L PP333+ 0.3 mg/L 6-BA+ Sucrose 30 g/L+Agar 7g/L,pH 5.8.

Protocol optimization and histological analysis of in vitro plant regeneration of RB92579 and RB93509 sugarcane cultivars

The objectives of this study were to establish appropriate conditions for obtaining plant regeneration and acclimatization of the 'RB92579' and 'RB93509' sugarcane cultivars and to elucidate the shoots origin through histological analysis. For both cultivars, obtaining shoots showed better results with the culture of explants on a callus induction medium containing 2.0mg L-1 2,4-dichlorophenoxyacetic acid, followed by cultivation on a shoot induction medium containing 0.1mg L-1 kinetin and 0.2mg L-1 benzilaminopurine. The MS medium without growth regulators proved to be appropriate for elongation and rooting of shoots and the use of the composed substrate of vermiculite + MS salts was effective for acclimatization. Histological analysis revealed that the origin of the shoots in both cultivars occurred through indirect organogenesis.

In Vitro Adventitious Shoot Regeneration via Indirect Organogenesis from Inflorescence Explants and Peroxidase Involvement in Morphogenesis of Populus euphratica Olivier

The inflorescences as explants for rapid propagation in vitro remained unknown in Populus euphratica Olivier. Here, we reported that multiple shoots were initiation from calli of both male and female inflorescences. The optimum medium for shoot induction from male inflorescences was lactose sulfite medium containing 1.0mg L(-1) 6-benzylaminopurine (BA) and 0.5mg L(-1) α-naphthalene acetic acid (NAA) or Murashige and Skoog (MS) medium containing 0.5mg L(-1) BA and 0.2mg L(-1) NAA. The optimum medium of shoot induction from female inflorescence calli was the MS medium containing 0.5mg L(-1) BA and 0.2mg L(-1) NAA. Rooting of regenerated shoots was obtained on 1/2 MS medium supplemented with 0.5∼1.0mg L(-1) indole-3-butyric acid (IBA) and the highest frequency rooting was on medium containing 0.5mg L(-1) IBA. No shoots were obtained on medium without BA and NAA. Peroxidase (POD) activity was measured by polyacrylamide gel electrophoresis during shoot induction and differentiation stages. The results showed that two bands of POD (2a and 2b) activity appeared lowest during the early 8days at the dedifferentiation phase of leaves inducing calli, whereas POD 2a, 2b activity appeared to be increasing at the homeochronous dedifferentiation phase of inflorescence. Five most intensive bands, POD 1a, 1b, 1c, 2a, and ab, appeared in 8th and 28th days at the redifferentiation phase during shoot morphogenesis. These results demonstrated that the POD was involved in shoot morphogenesis from both leaf and inflorescence explants of Populus euphratica.

Development of an efficient plant regeneration protocol for sweet potato ( Ipomoea batatas L.) cv. Blesbok

An efficient and reproducible plant regeneration protocol for the South African sweet potato (Ipomoea batatas Lam.) cultivar Blesbok was developed in this study. The effect of different hormone combinations and type of explant on shoot regeneration was evaluated in order to optimize the regeneration protocol. Explants in the form of stem sections, leaf discs, apical shoots and axillary buds derived from in vitro stock plant cultures were cultured on Murashige and Skoog (MS) media supplemented with 36 combinations of naphthalene acetic acid (NAA) (0, 0.01, 0.1, 0.2, 0.5, and 1 mg/L) and 6-benzylaminopurine (BAP) (0, 0.01, 0.1, 0.2, 0.5 and 1 mg/L). The highest percentage of shoot regeneration was obtained when apical shoot explants (31%) and axillary bud explants (22%) were cultivated on MS supplemented with 0.01 mg/L NAA + 1 mg/L BAP. Leaf discs and stem section explants produced highly recalcitrant callus that did not regenerate into shoots in shoot induction medium (SIM). Callus from apical shoots explants cultured on SIM developed into shoots. The shoots rooted readily on root induction medium (RIM) and then in hormone free MS medium. Regenerated plants appeared normal and showed a 100% survival rate when transferred to soil. The regeneration protocol described in this study will be used in a plant transformation protocol to produce transgenic sweet potato with broad virus resistance.