Adaptation of in vitro regeneration protocol for Brazilian wheat genotypes

ABSTRACT: The availability of an efficient protocol for in vitro regeneration is imperative for genetic transformation. Using genotypes adapted to the target region as a transgenic platform accelerates the development of cultivars. Therefore, this study aimed to adapt an in vitro regeneration protocol for Brazilian wheat genotypes. For this purpose, the in vitro regeneration capacity of immature embryos from six Brazilian wheat genotypes using two protocols of regeneration of somatic embryos was analysed. Furthermore, combinations of 2,4-D and picloram in the callus induction medium were tested in order to improve regeneration efficiency. Genotypes with higher regeneration efficiency were BR18-Terena and PF020037, yielding 0.42 and 1.13 shoots per explant using the Hu and the Wu protocol, respectively. Adding 1mgL-1 2,4-D in the callus induction medium was the most favourable, producing 3.73 and 3.07 shoots per explant for PF020037 and BR18-Terena, respectively. In conclusion, a protocol for regeneration for two Brazilian wheat genotypes recommended and developed to be cultivated at the Cerrado region has been adapted.

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In vitro Regeneration of Dalbergia sissoo Roxb. and the Potential for Genetic Transformation

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha4028248 ◽

2012 ◽

Vol 40 (2) ◽

pp. 140 ◽

Cited By ~ 2

Author(s):

Hafiz Mamoon REHMAN ◽

Iqrar Ahmad RANA ◽

Siddra IJAZ ◽

Ghulam MUSTAFA ◽

Faiz Ahmad JOYIA ◽

...

Keyword(s):

Acetic Acid ◽

Genetic Transformation ◽

Callus Induction ◽

In Vitro Regeneration ◽

Induction Medium ◽

Native Forest ◽

Ms Medium ◽

Dalbergia Sissoo ◽

Callus Induction Medium

Dalbergia sissoo Roxb. ex DC. (Sissoo) is a native forest tree species in Pakistan. Many ecological and economical uses are associated with this premier timber species, but dieback disease is of major concern. The objective of this study was to develop a protocol for in vitro regeneration of Sissoo that could serve as target material for genetic transformation, in order to improve this species. Callus formation and plantlet regeneration was achieved by culturing cotyledons, immature seeds, and mature embryos on a modified Murashige and Skoog (1962) (MS) medium supplemented with plant growth regulators. Callus induction medium containing 2.71 ?M 2, 4-dichlorophenoxyacetic acid (2,4-D) and 0.93 ?M kinetin produced better callus on all explants tested compared to other treatments, such as 8.88 ?M 6-benzylaminopurine (BA) and 2.69 ?M ?-naphthalene acetic acid (NAA), or 2.71 ?M 2, 4-D and 2.69 ?M NAA. Shoot regeneration was best on MS medium containing 1.4 ?M NAA and 8.88 ?M BA compared to other treatments, such as 1.4 ?M NAA and 9.9 ?M kinetin, or 2.86 ?M indole-3-acetic acid and 8.88 ?M BA. Murashige and Skoog medium containing 1.4 NAA ?M and 8.88 ?M BA was better in general for regeneration regardless of callus induction medium and the type of explant used. Rooting was best on half-strength MS medium with 7.35 ?M indole-3-butyric acid. Regenerated plantlets were acclimatized for plantation in the field. Preliminary genetic transformation potential of D. sissoo was evaluated by particle bombardment of callus explants with a pUbiGus vector. The bombarded tissue showed transient Gus activity 1week after bombardment. Transformation of this woody tree is possible provided excellent regeneration protocols. The best combination for regeneration explained in this study is one of such protocols.

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Mature Embryo-Based in vitro Regeneration of Indica Rice Cultivars for High Frequency Plantlets Production

Bangladesh Rice Journal ◽

10.3329/brj.v20i2.34132 ◽

2017 ◽

Vol 20 (2) ◽

pp. 81-87

Author(s):

HN Barman ◽

ME Hoque ◽

RK Roy ◽

PL Biswas ◽

MAI Khan ◽

...

Keyword(s):

Callus Induction ◽

High Frequency ◽

Indica Rice ◽

In Vitro Regeneration ◽

Mature Embryo ◽

Ms Medium ◽

Rice Varieties ◽

Growing Medium ◽

Regeneration Efficiency

The study was conducted at Biotechnology Division of Bangladesh Rice Research Institute (BRRI) to investigate the effects of plant growing medium and plant growth regulator (PGR) for the callus induction and high frequency plantlets regeneration of indica rice. Ten indica rice varieties viz. BR5, BR11, BRRI dhan28, BRRI dhan29, BRRI dhan33, BRRI dhan41, BRRI dhan47, BRRI dhan48, BRRI dhan49 and BRRI dhan50 were cultured on MS, N6 and LS media. The MS medium was found better for callus induction as compared to N6 and LS media. Among the tested varieties BRRI dhan48 induced the highest percent and best quality callus. Interaction effects of BRRI dhan48 to MS medium yielded 71.55% callus induction. The regeneration efficiency of BRRI dhan48 was tested on MS medium supplemented with different combination of NAA plus BAP and NAA plus kinetin. MS medium supplemented with 2.0 mg L-1 NAA and 2.0 mg L-1 Kn was found the best in respect of percent regenerated (76.67%) plantlet as well as for the growth of plantlets in vitro.Bangladesh Rice j. 2016, 20(2): 81-87

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Development of a protocol for the micropropagation of mature Eucalyptus grandis clones through somatic embryogenesis

10.51415/10321/1806 ◽

2001 ◽

Author(s):

◽

Andiswa Tsewana

Keyword(s):

Somatic Embryogenesis ◽

Embryo Development ◽

Callus Induction ◽

Embryogenic Callus ◽

Somatic Embryos ◽

Casein Hydrolysate ◽

Induction Medium ◽

Mature Trees ◽

The Media ◽

Callus Induction Medium

Biotechnology techniques such as micropropagation VIa somatic embryogenesis offer potential significant advances in the improvement of forest species, which could sustain forest production in South Africa, as well as globally, without increased use of land. In order to apply such techniques to commercial breeding and clonal programmes of E. grandis species, it is necessary to develop reliable and efficient protocols applicable to explants of proven superior genotypes. Most of the research on E. grandis somatic embryogenesis has used the genetically variable embryos or seedlings as explant sources, which results in the propagation of material of unproven genetic value. In order to exploit somatic embryogenesis maximally for cloning of superior trees, somatic embryos have to be induced from highly selected and, hence, mature trees. The aim of this investigation was to develop such a protocol for E. grandis and to test its applicability to various E. grandis hybrids. Somatic embryos were induced from buds, stems, leaves and petioles, with petioles and buds giving the best results. Thus, these were selected for further studies which involved testing the effect of medium composition on embryogenic callus induction. Media used for this purpose contained MS or B5 nutrients, 1 mg.l' 2,4-D, 0.5 g.r! glutamine, 0.5 g.r! casein hydrolysate, 4 g.r! Gelrite and 30 or 50 g.rl sucrose. All the media tested were able to support induction of embryogenic callus, although the number of explants producing embryogenic calli was affected significantly by the media composition (10-91 %). Callus induction media with B5 nutrients seemed to have a significant effect onn the developmental stage of embryos in the callus induction medium. Presence of 50 g.r! sucrose in the callus induction medium reduced the embryo yield, but the progress of embryo development was enhanced. The callus induction medium containing B5, 1 mg.l' 2,4-D, 0.5 g.rl glutamine, 0.5 g.r! casein hydrolysate, 4 g.r! Gelrite and 30 g.l' sucrose was chosen for subsequent studies. Of all the media tested for embryo development, the medium with B5, 2.5 mg.l' 2iP, 0.5 g.r! glutamine, 0.5 g.r! casein hydrolysate, 4 g.r! Gelrite and 50 g.r! sucrose was found to be the most suitable for embryo development to the cotyledonary stage. Experiments involving incorporation of both ABA and 2iP aiming at maturation of E. grandis somatic embryos led to an increase in size of the cotyledonary embryos formed but not to germination.

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Induksi Kalus dan Regenerasi Beberapa Genotipe Gandum (Triticum aestivum L.) secara In Vitro

Jurnal AgroBiogen ◽

10.21082/jbio.v6n2.2010.p57-62 ◽

2016 ◽

Vol 6 (2) ◽

pp. 57

Author(s):

Atmitri Sisharmini ◽

Aniversari Apriana ◽

Sustiprijatno Sustiprijatno

Keyword(s):

Triticum Aestivum ◽

Plant Regeneration ◽

Callus Induction ◽

Basal Medium ◽

Triticum Aestivum L ◽

Induction Medium ◽

In Vitro Plant Regeneration ◽

Regeneration Medium ◽

Wheat Genotypes

Callus Induction and In Vitro Plant Regeneration of Wheat Genotypes (Triticum aestivum L.). Atmitri Sisharmini, Aniversari Apriana, and Sustiprijatno. Development of a reliable in vitro plant regeneration procedure for wheat is a prerequisite for its improvement by genetic transformation. The purpose of this study was to obtain methods of callus induction and regeneration of wheat genotypes. This experiment was conducted at ICABIOGRAD. Immature embryos from four wheat genotypes, ie Perdix, Naxos Wew, Combi and Fasan were used to induce callus formation and regeneration rate of callus. For the preparation of callus induction medium, MS-L7 basal medium was supplemented with combination of growth regulators 2,4 dichlorophenoxy acetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram). While, plant regeneration medium was prepared using MS basal medium supplemented with combination of three growth regulators i.e. IAA, BAP and kinetin. The results showed that genotype, in vitro culture medium and growth regulators played a dominant role in callus induction and plantlet regeneration. All the 4 genotypes responded positively to callus induction, however, variability was observed not only among the genotypes but also within callus induction medium used. The best induction medium was the MS-L7 basal medium supplemented with combination of phytohormon 4 mg/l 2,4-D + 2 mg/l picloram (GIK-3) which showed 100% callus induction frequency. Whereas, the best regeneration medium was shown by MS basal medium with combination of phytohormon 1.5 mg/l BAP dan 0.5 mg/l kinetin (RG3). Regarding plant regeneration, Perdix was the most responsive genotype to be regenerated with regeneration frequency of 57.33%. The successfully acclimatized planlets in greenhouse were obtained from Perdix and Naxos Wew genotypes. These results will potentially facilitate genetic transformation research of wheat in Indonesia.

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Induction of Morphogenic Callus and In Vitro Regeneration of Sugarcane (Saccharum Officinarum L.)

Agriprima Journal of Applied Agricultural Sciences ◽

10.25047/agriprima.v5i1.380 ◽

2021 ◽

Vol 5 (1) ◽

pp. 61-67

Author(s):

Sitti Inderiati ◽

FNU Yanti ◽

Eka Ria Mentari

Keyword(s):

Callus Induction ◽

Callus Formation ◽

In Vitro Regeneration ◽

Shoot Proliferation ◽

Saccharum Officinarum ◽

Induction Medium ◽

Ms Medium ◽

Shoot Initiation ◽

Morphogenic Callus

In vitro propagation is a method to produce massive healthy new planting materials quickly. An experiment was carried out for morphogenic callus induction and regeneration of a domestic sugarcane variety. The Explants used was an inert folded leaf and incubated on modified MS medium augmented with 1 mg/l, 2.5 mg/l, and 5 mg/l of 2,4-D for callus induction. The leaf calluses were subcultured on MS medium enriched with different growth regulators for shoot initiation and multiplication. The highest percentage of callus formation was achieved in the medium containing 2.5 mg/l of 2,4-D, while the fastest callus initiation was noticed in MS medium supplemented with 5 mg/l 2,4-D, and maximum proliferation and the morphogenic response of callus were obtained in 3rd subculture. Two types of callus observed on the induction medium were dry nodular friable and smooth compact. This highly morphogenic callus was white to white creamy in color and easy to separate. The highest shoot proliferation rate was found on the medium containing 2 mg/l Kinetin + 1 mg/l IAA and no growth was noticed on the medium containing Kinetin alone. Therefore, the study suggests that the growth hormone of cytokinin in combination with auxin is necessary for in vitro regeneration of sugarcane callus culture.

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In vitro regeneration of Persian poppy (Papaver bracteatum)

Biologia ◽

10.2478/s11756-010-0079-6 ◽

2010 ◽

Vol 65 (4) ◽

Cited By ~ 4

Author(s):

Sara Rostampour ◽

Haleh Sohi ◽

Ali Dehestani

Keyword(s):

Ascorbic Acid ◽

Callus Induction ◽

High Performance ◽

High Efficiency ◽

In Vitro Regeneration ◽

Naphthalene Acetic Acid ◽

Dichlorophenoxyacetic Acid ◽

Papaver Bracteatum ◽

Callus Proliferation

AbstractPersian poppy (Papaver bracteatum Lindl.) is an important commercial source of medicinal opiates and related compounds. In this research, calli were induced from seeds, roots, cotyledons and hypocotyls of P. bracteatum at a high efficiency. The optimized callus induction media consisted of the Murashige and Skoog (MS) basic media supplemented with 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L kinetin and 15 mg/L ascorbic acid. The concentrations of 2,4-D and ascorbic acid were found critical to callus induction and proliferation. Subsequent subcultures resulted in excellent callus proliferation. Ascorbic acid at concentration 15 mg/L increased the callus proliferation significantly. Maximum callus growth was achieved when the explants were incubated at 25°C. MS salts at full strength were found inhibitory for callus induction, while ľ MS salts were found to favor callus induction. Shoot regeneration of calli in vitro was achieved on ľ MS medium containing 0.5 mg/L benzylamine purine and 1.0 mg/L naphthalene acetic acid. Analysis of alkaloid extracts from Persian poppy tissues by high-performance liquid chromatography showed that thebaine accumulated in the tissues of plants. The thebaine alkaloid profile of the Persian poppy is a well-defined model to evaluate the potential for metabolic engineering of thebaine production in P. bracteatum.

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IN VITRO REGENERATION AND MASS PROPAGATION OF AZIMA TETRACANTHA [LAM.] FROM THE LEAF EXPLANTS THROUGH CALLUS CULTURE

Kongunadu Research Journal ◽

10.26524/krj202 ◽

2017 ◽

Vol 4 (2) ◽

pp. 52-56

Author(s):

Mallika Devi T

Keyword(s):

Callus Induction ◽

In Vitro Regeneration ◽

Leaf Explants ◽

Shoot Formation ◽

Ms Medium ◽

Apical Leaf ◽

Half Strength ◽

Regenerated Plantlets ◽

Azima Tetracantha

In the present study the protocol for callus induction and regeneration in Azima tetracantha has been developed in culture medium. The young apical leaf explants were used for callus induction on MS medium containing BAP and NAA at 1.0 and 0.4mgl-1 respectively showed maximum callus induction (73%). The amount of callus responded for shoot formation (74%) was obtained in the MS medium containing BAP (1.5 mgl-1) and NAA (0.3mgl-1).The elongated shoots were rooted on half strength medium supplemented with IBA (1.5 mgl-1) and Kn (0.4 mgl-1) for shoots rooted. Regenerated plantlets were successfully acclimatized and hardened off inside the culture and then transferred to green house with better survival rate.

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The Effect of Daminozide, Dark/Light Schedule and Copper Sulphate in Tissue Culture of Triticum timopheevii

Plants ◽

10.3390/plants10122620 ◽

2021 ◽

Vol 10 (12) ◽

pp. 2620

Author(s):

Dmitry Miroshnichenko ◽

Anna Klementyeva ◽

Sergey Dolgov

Keyword(s):

Tissue Culture ◽

Somatic Embryogenesis ◽

Callus Induction ◽

Copper Ions ◽

Induction Medium ◽

Tissue Cultures ◽

Triticum Timopheevii ◽

Dark Light ◽

Green Plants ◽

Callus Induction Medium

Triticum timopheevii Zhuk. is a tetraploid wheat that is utilized worldwide as a valuable breeding source for wheat improvement. Gene-based biotechnologies can contribute to this field; however, T. timopheevii exhibits recalcitrance and albinism in tissue cultures, making this species of little use for manipulation through genetic engineering and genome editing. This study tested various approaches to increasing in vitro somatic embryogenesis and plant regeneration, while reducing the portion of albinos in cultures derived from immature embryos (IEs) of T. timopheevii. They included (i) adjusting the balance between 2,4-D and daminozide in callus induction medium; (ii) cultivation using various darkness/illumination schedules; and (iii) inclusion of additional concentrations of copper ions in the tissue culture medium. We achieved a 2.5-fold increase in somatic embryogenesis (up to 80%) when 50 mg L−1 daminozide was included in the callus induction medium together with 3 mg L−1 2,4-D. It was found that the dark cultivation for 20–30 days was superior in terms of achieving maximum culture efficiency; moreover, switching to light in under 2 weeks from culture initiation significantly increased the number of albino plants, suppressed somatic embryogenesis, and decreased the regeneration of green plants. Media containing higher levels of copper ions did not have a positive effect on the regeneration of green plants; contrarily, the elevated concentrations caused albinism in plantlets. The results and relevant conclusions of the present study might be valuable for establishing an improved protocol for the regeneration of green plants in tissue cultures of T. timopheevii.

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In vitro bulblet regeneration from immature embryos of endangered and endemic Muscari azureum

Archives of Biological Sciences ◽

10.2298/abs1101209u ◽

2011 ◽

Vol 63 (1) ◽

pp. 209-215 ◽

Cited By ~ 1

Author(s):

S. Uranbey

Keyword(s):

Callus Induction ◽

High Frequency ◽

Ornamental Plant ◽

Immature Embryos ◽

Induction Medium ◽

Ms Medium ◽

Culture Initiation ◽

Mineral Salts ◽

Font Color

A high frequency of bulblet regeneration was achieved for the endemic and endangered ornamental plant Muscari azureum using immature embryos. Immature embryos of M. azureum were cultured on a callus induction medium consisting of N6 mineral salts and vitamins, 400 gL-1 casein + 40 gL-1 sucrose + 2 mgL-1 L-proline, 2 mgL-1 2,4-D and 2 gL-1 Gelrite. Then the embryogenic callus clusters were transferred to a bulblet induction medium consisting of MS mineral salts and vitamins containing different concentrations and combinations of BAP, KIN, TDZ, Zeatin, IAA, NAA, 30 gL-1 sucrose and 7 gL-1 agar. Prolific bulblet multiplication (over 13 bulblets/embryo) was achieved from immature embryos after 5-6 months of culture initiation. Well-developed bulblets were excised and individually rooted on ? strength MS medium supplemented with 1 mgL-1 IBA, 0.5 gL-1activated charcoal, 20 gL-1sucrose and 6 gL-1agar and acclimatized. This article has been retracted. Link to the retraction <a href="http://dx.doi.org/10.2298/ABS150608072E">10.2298/ABS150608072E</a>

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Establishment of Morphogenic and Regeneration Ability in two Indica Rice Cell Suspension Cultures after Exposure to NaCl

Plant Tissue Culture and Biotechnology ◽

10.3329/ptcb.v19i2.5436 ◽

1970 ◽

Vol 19 (2) ◽

pp. 185-197

Author(s):

T.L. Aditya

Keyword(s):

Cell Suspension ◽

Callus Induction ◽

Somatic Embryos ◽

Indica Rice ◽

Cell Suspension Cultures ◽

Nacl Stress ◽

Callus Cultures ◽

Regeneration Ability ◽

Rice Varieties

An efficient protocol was developed for in vitro morphogenic ability along with plantlet regeneration of two Bangladeshi indica rice varieties (BR24 and BR26) via somatic embryogenesis by applying 50 mM NaCl stress in callus induction and suspension initiation media. Osmotic stress was induced by NaCl (50, 100, 150, 200 and 250 mM) on the cell growth in suspension maintenance media. In viability test stress adapted cells showed 85 - 95% viability up to 200 mM NaCl compared with stress shocked (MS1-50) and control (MS1-0) treatments. Higher stress adapted cells showed growth retardation and the induction of plasmolysis. For both genotypes somatic embryos were obtained in both MS based liquid and semisolid media with or without 50 and 100 mM NaCl. Cell suspension-derived micro-calli were partially desiccated (6 - 12 hr) and subsequently maintained in MS1 callus induction media supplemented with proline (12 mM), ABA (2 mg/l) and 0.6% phytagel in the presence or absence of 50 and 100 mM NaCl. Subsequently, desiccated somatic embryos were transferred in MS based regeneration media with or without 50 and 100 mM NaCl. Proline mediated callus was found to be more effective in embryo differentiation than ABA. Partial desiccation dramatically enhanced callus growth and partially increased regeneration percentage. BR24 showed a better regeneration response producing plantlets in presence of proline in control media while BR26 restored regeneration potential in the presence of ABA and 100 mM NaCl. Plantlets regenerated from salt stressed callus cultures were then grown in compost in a glasshouse and produced normal, fertile plants. Key words: Indica rice, Cell suspension, Morphogenic, Regeneration D.O.I. 10.3329/ptcb.v19i2.5436 Plant Tissue Cult. & Biotech. 19(2): 185-197, 2009 (December)

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