In vitro Regeneration by Indirect Organogenesis of Selected Kenyan Maize Genotypes using Shoot Apices

Prunus spp. is one of the most recalcitrant fruit tree species in terms of in vitro regeneration and transformation, mostly when mature tissues are used as explants. The present study describes the in vitro regeneration via indirect organogenesis, and Agrobacterium tumefaciens-mediated transformation of the peach rootstock Hansen 536 (Prunus persica × Prunus amygdalus) through the use of meristematic bulks (MBs) as starting explants. Efficient adventitious shoot regeneration was obtained when Hansen 536 MBs were cultured on an optimized medium consisting of modified McCown Woody Plant medium (WPM) enriched with 4.4 M 6-Benzyladenine (BA), 0.1 M 1-Naphthaleneacetic acid (NAA) and 6.0 g L−1 plant agar S1000 (B&V). MB slices were used later as starting explants for Agrobacterium-mediated transformation to introduce an RNAi construct “ihp35S-PPV194” against PPV virus. Transgenic events were identified by both green fluorescent protein (GFP) screening and kanamycin selection at different concentrations (0, 17 or 42 M). GFP-fluorescent proliferating callus lines were selected and confirmed to stably express the ihp35S-PPV194::eGFP gene construct by molecular analysis. Although shoot regeneration from these transgenic calli has not been obtained yet, this represents one of the few examples of successful attempts in peach genetic transformation from somatic tissues, and also serves as a useful in vitro system for future gene functional analysis in peach.

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In-vitro Regeneration from Direct and Indirect Organogenesis of Crescentia alata Kunth an Important Multipurpose Medicinal Tree

European Journal of Medicinal Plants ◽

10.9734/ejmp/2021/v32i330381 ◽

2021 ◽

pp. 48-58

Author(s):

R. Abinaya

Keyword(s):

Shoot Organogenesis ◽

In Vitro Regeneration ◽

Basal Medium ◽

Direct Organogenesis ◽

Ms Medium ◽

Indirect Organogenesis ◽

Medicinal Tree ◽

Short Period ◽

In Vitro Clonal Propagation

In this present work, an in-vitro regeneration protocol for Crescentia alata (C. alata) was developed using various explants on Murashige and Skoog (MS) medium augmented with different concentrations and combinations of plant growth regulators (PGRs) for direct and indirect regeneration. The direct organogenesis was established from nodes and internodes on MS medium supplemented with cytokinins and auxins. The indirect organogenesis via callus phase was obtained from leaf, nodes and internodes on MS medium supplemented with different concentrations of PGRs. The high frequency shoot organogenesis were achieved directly from nodal explants were cultured on MS medium supplemented with 3.0 mg/L BAP+0.5 mg/L KIN +1.0 mg/L NAA. Indirect organogenesis callogenic frequency was optimized at the concentration of MS medium containing 1.0 mg/L BAP + 5.0 mg/L IAA. The callus was obtained from all the explants were used, among these explants internodal explants gave best result on MS medium supplemented with different concentrations of cytokinins and auxins for indirect organogenesis experiment. Indirect organogenesis the highest number of shoot regeneration was obtained in MS Basal Medium with 4.0 mg/L BAP + 0.5 mg/L KIN + 2.0 mg/L NAA from internodal explants. For root formation the regenerative shoots which were sub cultured on MS medium containing different ratios of auxins. The rooted plantlets were transferred successfully to the pots containing sterilized soil and were successfully hardened at greenhouse condition for 20 days then exposed to the natural environment. This is the first successful micropropagation report of an efficient and rapid in-vitro clonal propagation protocol for C. alata by direct and indirect shoot organogenesis through various explants, which can be employed for conservation of this important medicinal tree species as well as the utilization of an biologically important active biomolecules. This protocol can be very useful to obtain plants from various explants, without the requirement of meristematic regions, enabling the obtainment of a higher number of plants in short period.

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In Vitro Regeneration of Miscanthus x giganteus through Indirect Organogenesis: Effect of Explant Type and Growth Regulators

Plants ◽

10.3390/plants10122799 ◽

2021 ◽

Vol 10 (12) ◽

pp. 2799

Author(s):

Aušra Blinstrubienė ◽

Inga Jančauskienė ◽

Natalija Burbulis

Keyword(s):

Growth Regulators ◽

Root Formation ◽

In Vitro Regeneration ◽

Agricultural Crop ◽

Explant Type ◽

Breeding Methods ◽

Miscanthus X Giganteus ◽

Indirect Organogenesis ◽

Morphogenic Callus

Miscanthus x giganteus is a spontaneous sterile hybrid therefore the creation of useful genetic diversity by conventional breeding methods is restricted. Plant regeneration through indirect organogenesis may be a useful approach to create genetic variability of this important agricultural crop. The present study aimed to evaluate the effect of the explant type and growth regulators on indirect organogenesis of Miscanthus x giganteus and to determine the ploidy level of plant regenerants by flow cytometry. On average, the highest percentage of morphogenic callus tested explants formed in the medium supplemented with 2.5 mg L–1 IBA + 0.1 mg L–1 BAP + 4.0 mg L–1 l-proline. The most intensive secondary differentiation of callus cells was observed in the medium supplemented with 4.0 mg L–1 ZEA + 1.0 mg L–1 NAA. The highest root formation frequency with the highest number of roots was determined in the MS nutrient medium supplemented with 0.4 mg L–1 IBA, where more than 95% of plant regenerants survived and were growing normally.

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In vitro Regeneration of Japanese Burdock (Arctium lappa L.) from Cotyledon and Leaf Derived Explants

Plant Tissue Culture and Biotechnology ◽

10.3329/ptcb.v31i2.57340 ◽

2022 ◽

Vol 31 (2) ◽

pp. 123-134

Author(s):

Mustafa Abul Kalam Azad ◽

Md Arifuzzaman ◽

Md Mobarok Hossain ◽

Md Sohel Arman ◽

Muhammad Nurul Amin

Keyword(s):

In Vitro Regeneration ◽

Adventitious Shoots ◽

Leaf Explants ◽

Direct Shoot Regeneration ◽

Indirect Organogenesis ◽

Arctium Lappa ◽

Sand Mixture ◽

The Third ◽

Rooting Medium

Considering the vegetable and medicinal values, a micropropagation protocol has been established for Japanese Burdock (Arctium lappa L.) by culturing the explants of cotyledon and leaf obtained from in vitro grown seedlings. Direct shoot regeneration was achieved from cotyledon and leaf explants on MS fortified with 4.0 μM BAP and 2.0 μM IBA or NAA after 5 weeks of culture. In addition, both the explants also formed callus from their cut margins within 6 weeks of cultivation on medium complemented with 6.0 μM BAP and 4.0 μM IBA or NAA. Adventitious shoots were also redeveloped through indirect organogenesis from the cotyledon and leaf-derived callus within 10 weeks of culture on MS containing 4.0 μM BAP and 2.0 μM IBA or NAA. The highest rate of shoot reproduction was attained at the third subculture, and more than 12.6 shoots were formed per callus clump. Within 4 weeks of transfer to the rooting medium on MS containing 6.0 μM IBA, the cultured micro-shoots produced highest 5.3 roots per cultured shoot. Rooted plantlets were successfully established on a soil-composed-sand mixture under natural condition with 93.3% survival rate Plant Tissue Cult. & Biotech. 31(2): 123-134, 2021 (December)

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In vitro Regeneration of Vitex negundo L. - A Multipurpose Woody Aromatic Medicinal Shrub

Plant Tissue Culture and Biotechnology ◽

10.3329/ptcb.v18i1.3263 ◽

1970 ◽

Vol 18 (1) ◽

pp. 37-42 ◽

Cited By ~ 6

Author(s):

M. Jawahar ◽

S. Ravipaul ◽

M. Jeyaseelan

Keyword(s):

Callus Induction ◽

In Vitro Regeneration ◽

Leaf Explants ◽

Shoot Proliferation ◽

Optimal Level ◽

Vitex Negundo ◽

Indirect Organogenesis ◽

Shoot Bud ◽

Bud Initiation

A rapid and efficient protocol was developed for inducing indirect organogenesis using leaf explants of Vitex negundo L. Explants were cultured on MS with different concentrations of 2,4-D and IAA in combination with BAP for callus induction. The frequency of callus induction increased with increasing concentration of IAA (0.3 mg/l) and BAP (0.3 mg/l) at optimal level. The shoot buds appeared emerging as green coloured protuberances on the callus. The high frequency of shoot bud initiation and shoot proliferation was observed on MS containing 0.3 mg/l IAA and 0.3 mg/l BAP. The regenerated shoots were successfully rooted on MS supplemented with 0.5 mg/l IBA. Rooted plants were transferred to pots containing sand, soil and manure in the ratio of 1 : 1 : 1. Nearly 90% survival of in vitro plants were recorded. Key words : Vitex negundo, In vitro, Leaf, Callus, Regeneration D.O.I. 10.3329/ptcb.v18i1.3263 Plant Tissue Cult. & Biotech. 18(1): 37-42, 2008 (June)

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Improved procedures for in vitro regeneration and for phenotypic analysis in the model legume Lotus japonicus

Functional Plant Biology ◽

10.1071/fp05015 ◽

2005 ◽

Vol 32 (6) ◽

pp. 529 ◽

Cited By ~ 10

Author(s):

Ani Barbulova ◽

Enrica D'Apuzzo ◽

Alessandra Rogato ◽

Maurizio Chiurazzi

Keyword(s):

In Vitro Regeneration ◽

Lotus Japonicus ◽

Shoot Formation ◽

Direct Somatic Embryogenesis ◽

Phenotypic Analysis ◽

Indirect Organogenesis ◽

Model Legume ◽

Mesorhizobium Loti ◽

Efficient Gene

As a prerequisite for the development of an efficient gene transfer methodology, the possibility of inducing direct somatic embryogenesis in Lotus japonicus (Regel) K. Larsen explants was investigated. Petiole bases, cotyledons, hypocotyls and stem segments were cultivated in the presence of different amounts of benzylaminopurine (BAP) and / or thidiazuron (TDZ). Regeneration was achieved differentially in the different explants and a higher efficiency of shoot formation was obtained with TDZ. By maintaining the same TDZ regime a second cycle of morphogenesis was achieved and the histological analysis of these structures indicated unambiguously their somatic embryogenic nature. Thidiazuron was also tested as an agent to improve the kinetics of shoot formation in a Lotus japonicus transformation–regeneration procedure based on indirect organogenesis. A very significant, highly reproducible, increase in the rate of the shoot formation was observed in independent transformation experiments. We also present an extensive analysis of the feasibility and reproducibility of an in vitro procedure, which can be very useful for the screening of symbiotic phenotypes in transgenic Lotus plants and for the analysis of the cascade of molecular and cytological events occurring soon after Mesorhizobium loti infection.

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In vitro Regeneration of Alstroemeria cv. ‘Balance’ by Indirect Organogenesis

Biosciences Biotechnology Research Asia ◽

10.13005/bbra/2484 ◽

2017 ◽

Vol 14 (2) ◽

pp. 607-614

Author(s):

Hossein Nazarian ◽

Maryam Beigi Harchegani ◽

Mahmoud Otroshy ◽

Ali Motamedi

Keyword(s):

Callus Induction ◽

Callus Formation ◽

In Vitro Regeneration ◽

Culture Technique ◽

Stem Explants ◽

Indirect Organogenesis ◽

High Concentrations ◽

Two Phases ◽

Positive Effect

ABSTRACT: This study was designed in order to optimize the indirect organogenesis (during callus induction and regeneration) of Alstroemeria cv. ‘Balance’ through tissue culture technique in two phases; the first stage: callus induction by rhizome segments, leaf and nodal stem which in the start, callus formation media were examined using two types of auxins; 2,4-D and NAA and a cytokinin; BAP in four different experimentations. In the second stage, calli derived from rhizome segments and nodal stem explants were transferred to regeneration media. The results revealed that 2,4-D in combination with BAP in the rhizome segments and nodal stem explants were efficient as compared to NAA. The highest yield of callus formation was also obtained in the rhizome segments explants. According to the results, it can be suggested that NAA as auxin, does not have direct positive effect on cell division in Alstroemeria. The 2,4-D is toxic at high concentrations and may bring about cell death. Eventually, the composition of 0.5 mg/l NAA with 3 mg/l BAP and callus derived from nodal stem explants may be introduced as the best combination for regeneration. These results indicate the necessity of the BAP cytokinin presence for regeneration. In addition, the maximum length of the shoot was obtained from combination of BAP with nodal stem explants, without the presence of NAA.

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Plant Regeneration from Leaf-derived Callus Cultures of Primrose (Primula vulgaris)

HortScience ◽

10.21273/hortsci.51.5.558 ◽

2016 ◽

Vol 51 (5) ◽

pp. 558-562 ◽

Cited By ~ 7

Author(s):

Sadiye Hayta ◽

Mark A. Smedley ◽

Jinhong Li ◽

Wendy A. Harwood ◽

Philip M. Gilmartin

Keyword(s):

Flower Development ◽

In Vitro Regeneration ◽

Leaf Explants ◽

Callus Cultures ◽

Indirect Organogenesis ◽

Horticultural Crops ◽

Donor Material ◽

Primula Vulgaris ◽

Inbred Parent

Efficient micropropagation of Primula species is important both for fundamental scientific studies and commercial applications. Primula vulgaris (Huds), along with other Primulaceae species, exhibits floral heteromorphy with two distinct forms of hermaphroditic flower. Studies to identify genes that control heteromorphic flower development require propagation of floral mutants, and efficient regeneration is a key requirement for plant transformation. Several species, including P. vulgaris cultivars and P. ×polyantha hybrids, are important horticultural crops in Europe, United States, and Japan and semidouble/double Primula varieties offer a high-end product. Vegetative propagation of sterile double forms, and as a means to increase numbers of inbred parent plants for F1 seed production is, however, slow. Micropropagation offers the most efficient way of increasing these varieties quickly and efficiently. To date, most Primula micropropagation protocols require explant material derived from in vitro grown seedlings or use floral parts as donor material with seasonal limitations. Therefore, an effective and efficient protocol was developed for in vitro regeneration of P. vulgaris via indirect organogenesis from adult leaf–derived explants. Exposure of leaf explants of P. vulgaris to media containing synthetic cytokinin, thidiazuron (TDZ), and auxin [1-naphthylacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D)] resulted in undifferentiated cell proliferation and followed by differentiated growth as shoot organogenesis. Silver nitrate improved in vitro callus growth and increased shoot regeneration further, with up to 72% of explants producing shoots. Regenerated plants developed normally and produced normal fertile flowers within 7 months. The system was also successfully applied for the micropropagation of sterile double-flowered P. vulgaris ‘Sue Jervis’. The protocol reported here enables propagation of P. vulgaris without seasonal limitation or destruction of valuable parent donor material. The protocol, with further development, has the potential to underpin development of a transformation system for Primula, which would be of value in studies on flower development and disease resistance in laboratory grown plants.

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