Direct Shoot Bud Regeneration Research Articles

Somatic Embryogenesis and Direct Shoot Bud Formation from in vitro Root Segments of Garlic (Allium sativum L.)

In vitro techniques are unconventionally used for garlic improvement. In garlic tissue culture, numerous roots are produced in vitro. Segments (1 cm) of those in vitro roots were cultivated on MS medium with 5.0 μM 2, 4-D for callus initiation followed by transfer to 0.5 μM 2, 4-D for somatic embryogenesis. Root segments had 51.67% callus and somatic embryo formation. Plantlet regeneration was obtained from 90% calli after being transferred to MS medium supplemented with 5.0 μM kinetin. Within 6 months, 54.8 plants per root segment and 42.2 plants per root tip explant were found. MS media supplemented with 1.0 or 5.0 μM NAA in combination with 10.0 or 50.0 μM BA were used for direct shoot bud induction from root segments. The highest percentage (23.33 %) of direct shoot bud regeneration was found in 5.0 μM NAA with 50.0 μM BA within 2 months. The number of shoots per explant was varied from 1-81 with the highest average of 15.75 shoots per explant. The shoots produced bulblets upon transfer to a medium containing higher amount of sucrose (12%). The largest bulblets weighed 356.57 g in the medium with 5.0 μM NAA and 50.0 μM BA. In both pathways, regeneration occurred from only the apical (distal) end of the root segments. The protocols are promising for continuous regeneration of propagules and genetic transformation study for improvement of garlic. Plant Tissue Cult. & Biotech. 33(2): 135-142, 2023 (December)

Detection of Variation in Long-Term Micropropagated Mature Pistachio via DNA-Based Molecular Markers.

Determination of genetic stability of in vitro-grown plantlets is needed for safe and large-scale production of mature trees. In this study, genetic variation of long-term micropropagated mature pistachio developed through direct shoot bud regeneration using apical buds (protocol A) and in vitro-derived leaves (protocol B) was assessed via DNA-based molecular markers. Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), andamplified fragment length polymorphism (AFLP) were employed, and the obtained PIC values from RAPD (0.226), ISSR (0.220), and AFLP (0.241) showed that micropropagation of pistachio for different periods of time resulted in "reasonable polymorphism" among donor plant and its 18 clones. Mantel's test showed a consistence polymorphism level between marker systems based on similarity matrices. In conclusion, this is the first study on occurrence of genetic variability in long-term micropropagated mature pistachio plantlets. The obtained results clearly indicated that different marker approaches used in this study are reliable for assessing tissue culture-induced variations in long-term cultured pistachio plantlets.

Direct shoot bud regeneration from shoot tip explants of Enicostema axillare: an important medicinal plant

An efficient protocol has been developed for in vitro propagation of Enicostema axillare using shoot tip explants. The shoot tip explants were cultured on MS medium supplemented with various combinations of (BAP, KIN) and (NAA/IAA & IBA) in different concentrations between 0.5 and 2.0 mg/l for multiple shoot bud induction. The highest percent of (98.51 %) was observed at 1.0 mg/l BAP in combination with 0.2 mg/l KIN while maximum number of shoot buds (8.41 shoots/explant) was noticed on MS medium containing 1.0 mg/l BAP and 0.2 mg/l KIN combination. The highest frequency (90.82 %) of multiple shoot bud regeneration was observed at 1.0 mg/l BAP and 0.5 mg/l IBA with 15.12 ± 2.12 shoots/explants. The regenerated multiple shoots were transferred to half-strength MS medium augmented with different concentration of 0.5–2.5 mg/l IBA for rooting. Among the different concentrations of IBA tested, maximum percentage of rooting (100 %) was observed in MS medium augmented with 1.5 mg/l IBA. The rooted plantlets were successfully transferred into plastic cups containing soil and sand in the ratio of 1:1. Subsequently established in the field conditions with 90 % of survival rate. The protocol developed can be utilized for both large scale plant production and conservation of germplasm of this species. The described method can be successfully employed for large-scale multiplication and in vitro conservation as well as production of secondary metabolites of E. axillare.

Evaluation of Genetic Homogeneity in Tissue Culture Regenerates of Jatropha curcas L. using Flow Cytometer and DNA-based Molecular Markers

The present investigation aimed to evaluate the reliability of in vitro propagation methods for elite genotypes of Jatropha curcas L., that maintain genetic integrity of tissue culture (TC) regenerates among two regeneration systems developed through direct shoot bud regeneration using nodal/apical shoot segments (protocol-A) and in vitro-derived leaves (protocol-B) as explants. Random amplified polymorphic DNA (RAPD), intersimple sequence repeat (ISSR), simple sequence repeat (SSR) molecular markers, and flow cytometery (FCM) were employed to evaluate genetic homogeneity in TC-regenerates at different passages of subcultures. RAPD markers showed genetic homogeneity in fifth-generation TC-regenerates of both protocols. ISSR markers showed genetic stability of leaf regenerates (protocol-B) at 10th generation. FCM analysis of TC-regenerates at 10th generation in protocol-B and at 20th generation in both protocols, showed stability of ploidy level. SSR assessment of TC-regenerates at 20th generation in both protocols confirmed genetic homogeneity. The results confirmed the genetic stability of the TC-regenerates and demonstrated the reliability of the regeneration systems developed so far using explants of two different origins, for large-scale multiplication of elite genotypes of Jatropha.

Shoot bud regeneration from different explants of Bacopa monniera (L.) Wettst. by trimethoprim and bavistin

A mass in vitro propagation system devoid of growth regulators for Bacopa monniera (L.) Wettst., a traditional Indian medicinal plant, has been developed. Direct shoot bud regeneration was induced by culturing internode and leaf explants on Murashige and Skoog's (MS) medium supplemented with an antibiotic (trimethoprim) or a fungicide (bavistin). Bavistin showed a marked cytokinin-like activity, as evident from high number of shoot buds induced in node, internode and leaf explants. Optimum adventitious shoot buds induction occurred at 300 mg/l bavistin from internode explants. In vitro regenerated shoots were elongated and rooted before transferred to field with 85% survival. The regeneration protocol developed in this study illustrates the usefulness of additives for mass propagation and germplasm conservation of B. monniera.

PkMADS1 is a novel MADS box gene regulating adventitious shoot induction and vegetative shoot development in Paulownia kawakamii

Direct regeneration of shoot buds in vitro is an important technique in plant genetic manipulation. We describe the isolation and functional characterization of a novel MADS box cDNA (PkMADS1) from Paulownia kawakamii leaf explants undergoing adventitious shoot regeneration. mRNA gel blot analysis confirmed the expression of PkMADS1 in the shoot-forming cultures, but no signal was observed in the callus-forming cultures. PkMADS1 transcripts were also detected in shoot apices, but not in root apices, initial leaf explants or the flower. In situ hybridization revealed that its expression was restricted to developing shoot primordia in the excised leaf cultures, suggesting a role for this gene in adventitious shoot formation. Transgenic Paulownia plants over-expressing the PkMADS1 gene showed some changes in phenotype, such as axillary shoot formation. In the antisense transformants, shoots were stunted and had altered phyllotaxy, and, in some lines, the shoot apical meristem appeared to have been used up early during shoot development. Leaf explants from the antisense transgenic plants showed a tenfold decrease in shoot regeneration compared with explants from sense transformants or wild-type. Our results show that PkMADS1 is a regulator of shoot morphogenesis.

Direct shoot regeneration from node, internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l−1 BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l−1 TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l−1 BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l−1 BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l−1 TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l−1 BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success.

Rapid and High-Frequency In Vitro Plant Regeneration from Leaflet and Petiole Explants of Groundnut ( Arachis hypogaea L.)

An efficient protocol for regeneration of groundnut plantlets from immature and mature leaflet and petiole explants excised from axenic seedlings has been developed. The highest frequency of callus induction obtained from leaflet explants was on Murashige and Skoog (MS) medium containing 2.0 mg/L of α-naphthalene acetic acid (NAA) and 0.5 mg/L of kinetin combination. MS medium containing different auxins in combination with 6-benzylaminopurine (BAP) induced shoot buds. A BAP (2.0 mg/L) and NAA (0.5mg/L) combination resulted in the highest frequency of shoot-bud regeneration. Subsequent shoot multiplication was obtained on MS medium supplemented with either BAP or kinetin (5.0 mg/L) in combination with NAA (1.0 mg/L). Immature leaflet explants were found to be more responsive to shoot induction than mature leaflet explants. Direct shoot-bud regeneration was also observed from petiole explants on the same regeneration medium used for leaflet callus. Regenerated shoots were rooted on MS medium containing indole-3-butyric acid (2.0 mg/L) and kinetin (0.5 mg/L). Plantlets obtained were successfully established in the field, where they grew to maturity and set viable seeds.

The role of ethylene on direct shoot bud regeneration from mangosteen ( Garcinia mangostana L.) leaves cultured in vitro

The role of ethylene in the regulation of shoot bud regeneration from leaf explants of mangosteen ( Garcinia mangostana L.) was studied by culturing leaf segments (2 mm) on woody plant medium supplemented with BA (20 μM), ethylene inhibitors AgNO 3, AVG or ethylene precursor ACC, under aerated or airtight condition. In aerated cultures, the first emergence of shoot buds occurred on midribs within 18 days of incubation and 92% of the explants regenerated buds by day 42. With airtight cultures sealed with rubber septa stoppers, a large accumulation of ethylene, callus production and a marked delay of at least 12 days in shoot bud regeneration were observed. The addition of ACC to the culture medium also dramatically delayed shoot regeneration and enhanced callus proliferation. Under airtight conditions, both AgNO 3 and AVG were effective in preventing the delay of shoot regeneration and callus formation. However, only AVG suppressed ethylene production. Ethylene or ethylene inhibitors did not affect the percentage of regenerating explants after 49 days of culture. As ethylene only delayed the emergence of shoot buds, it is presumed to regulate some of the early developmental stages associated with shoot bud regeneration in mangosteen leaf explants.

Highly efficient transformation and regeneration of aspen plants through shoot-bud formation in root culture

The natural capacity of aspen (Populus tremula L.) roots for direct shoot-bud regeneration was harnessed to establish a highly efficient transformation and regeneration procedure that does not require a pre-selection stage on antibiotics. Aspen stem segments were transformed using wildtype Agrobacterium rhizogenes (LBA9402) with the binary p35SGUSINT plasmid carrying the genes coding for β-glucuronidase (GUS) and neomycin phosphotransferase II. High levels of transient GUS expression were found in the basal cut surface of 87% of the segments, and 98% of these formed well-developed adventitious roots. Proliferating root cultures were established in liquid culture, and GUS expression was found in 75% of the roots. Shoot-bud regeneration in root cultures was very high: 99% of the roots yielded shoot-buds (4.3 buds per root), of which 91% expressed GUS. Southern blot analysis and polymerase chain reaction confirmed the transgenic nature of the plants expressing GUS. Kanamycin resistance of transformants was tested with respect to callus growth and bud regeneration. Callus from transgenic plants exhibited a high growth rate in the presence of up to 100 μg/μl kanamycin, and bud regeneration from transformed roots occurred in the presence of up to 30 μg/μl kanamycin. Callus and buds from control (non-transformed) plants failed to proliferate or regenerate, respectively, in the presence of kanamycin at concentrations above 10 μg/μl. Ninety-four independent clones from different transformation events were established, of which 52 were phenotypically true-to-type.

High frequency direct shoot bud regeneration from excised leaves of mangosteen ( Garcinia mangostana L.)

An improved procedure for direct shoot bud regeneration from young leaves of mangosteen ( Garcinia mangostana L.) has been developed. Regeneration was achieved by culturing 3-mm transverse sections of about 10-day-old leaves on woody plant medium enriched with 20 μM benzyladenine (BA), 20 g/l sucrose and 2.5 g/l Phytagel. A wound response in the presence of BA at the time of culture was found to be essential for shoot bud induction. Explant size, concentration of BA in the medium, timing of addition of BA to the medium as well as the time of wounding of explant significantly influenced shoot bud regeneration. Leaves from field-grown seedlings produced an average of 45 shoot buds per leaf after 50 days of culture, compared with 8 shoot buds per leaf from in vitro shoots. Regenerated shoots (5–6 mm tall) excised from the explants required BA (5 μM) for further growth. Rooting was induced with indolebutyric acid (IBA) when the shoots reached 10–15 mm and were established in vermiculite/sand mixture in pots.

Direct shoot bud formation from leaf explants of seedlings and mature mangosteen ( Garcinia mangostana L.) trees

This paper reports the direct regeneration of shoot buds from leaf segments of mangosteen ( Garcinia mangostana L.) cultured in vitro. Explants were taken from 1–3-year-old seedlings and 16 year-old mature fruiting trees. The optimal level of benzylaminopurine (BAP) for shoot bud development from seedling leaf explants was 5 mg l −1. Higher concentrations were also effective but shoot buds were clustered and stunted. Very young red leaves, 2–3 cm long, from mature trees also produced shoot buds on this medium. When leaves from mature trees that had turned from red to green were used, the frequency of shoot bud formation decreased. The number of vegetative buds was higher in red leaves from seedlings compared with young red leaves from mature trees. Secondary shoot buds also developed on leaves of cultured epiphyllous shoots from both seedlings and mature trees. Shoots from leaves of seedlings and mature trees, measuring more than 2 cm were rooted with an acute auxin treatment. Plantlets from seedling leaf explants were successfully acclimatised and established in soil.

An alternative approach to plant regeneration from protoplasts of sour cherry ( Prunus cerasus L.)

Mesophyll protoplasts, of sour cherry ( Prunus cerasus), clones CAB 4D, CAB 5H and CAB 11E, gave differential cultural responses. Protoplasts in media based on Murashige and Skoog (MS) salts, supplemented with 9% (w/v) mannitol plus growth regulators underwent cell wall regeneration, cell colony and callus formation. Zeatin (Z) was needed in order to induce cell division for all three sour cherry clones. The protoplast-derived calli, of clones CAB 4D and CAB 5H, underwent rhizogenesis as an intermediate step towards shoot bud differentiation. Clone CAB 5H also gave direct shoot bud regeneration from the protoplast callus.

Experimental Control of Growth and Differentiation in Organ Cultures of Physalis minima LINN.

Summary Morphogenesic responses of explanted stem and leaf tissues of Physalis minima to various phytohormones were investigated. The effect of various concentrations and combinations of auxin-cytokinin were tested on organ formation. Either the direct regeneration of shoot buds or the development of a callus which subsequently differentiated into shoot buds took place. Plantlets developed from the buds. The effect of gamma rays on the morphogenetic potentiality of stem segments was also investigated.

Bud formation and embryo differentiation in in vitro cultures of Petunia

Summary Excised stem and leaf explants of Petunia inflata and two varieties of Petunia hybrida, „rose du ciel“ and „cascade“ were grown on a defined nutrient medium. The morphogenetic responses of the expiants varied with the growth substances added to the medium. There was either the direct regeneration of shoot buds or the development of a friable callus which differentiated into embryos. Plantlets were produced from the embryos.