Plantlet Recovery Research Articles

An Efficient Fluorescence-Activated Protoplast Sorting (FAPS) and Regeneration Protocol for Canola (Brassica napus).

Protoplast sorting and purification methods are powerful tools enabling the enrichment of cellular subpopulations for basic and applied studies in plant sciences. Fluorescence-activated protoplast sorting (FAPS) is an efficient method to isolate specific protoplast populations based on innate features (size and autofluorescence) or expression of fluorescent proteins. FAPS-based methods have recently been deployed in single-cell purification for single-cell RNA sequencing-based transcriptional profiling studies. Protoplast sorting methods integrated with the ability to culture and recover whole plants add value to functional genomics and gene editing applications. Enriching cells expressing nucleases linked to fluorescent proteins can maximize knockout or knockin editing efficiencies and minimize toxic and off-target effects. Here, we report the protocol for protoplast preparation, sterile cell sorting, culture, and downstream regeneration of plants from canola protoplasts. This protocol can be successfully applied to all totipotent protoplast methods that can regenerate into whole plants. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Preparation of transfected canola protoplasts for sorting Basic Protocol 2: Fluorescence-activated protoplast sorting Basic Protocol 3: Bead culture of sorted protoplasts and recovery of plantlets.

Effect of salt stress on plant regeneration efficiency in primed and non-primed seed's calli of rice (Oryza sativa L.) variety Swarna.

This study leads with the primed seeds of rice (var. Swarna) with distilled water (D.W.) and various concentrations of Mg(NO3)2 (0-8mM)/Kinetin (0-5ppm) alone or in combination with screen out the regeneration medium induced tolerance level of NaCl. To fulfill the objective, the primed and non-primed rice seeds were inoculated in MS medium supplemented with 30gL-1 maltose + 1gL-1 casein hydrolysate and 2mgL-1 of 2,4-D for callus induction and cultured up to 45days in two sets: one set for regeneration purpose in NaCl-induced regeneration medium and another set was used to study the physiological potentiality of the callus. The 45-day-old calli were transferred into regeneration medium MSR (MS medium for regeneration) (BAP: NAA: Kinetin = 4:1:1) containing NaCl with a concentration range of 0 to 300mM. The number of regenerating calli and shoot regeneration percentage, number of plantlets obtained from one callus, recovery of plantlets from each concentration of NaCl and proline estimation from the leaf of the regenerated plantlets were determined from one set obtained after 45days. The calli obtained from another set after 45days, the frequencies of total and embryogenic calli induction percentage, fresh and dry weights, proline content, nitrate reductase and superoxide dismutase activities were measured. The calli obtained from 2.5ppm kinetin + 4mMMg(NO3)2 primed seeds were showed best result as compared to the other treatments for the above-mentioned parameters in different concentrations of NaCl-induced medium and survive up to 200mM concentrations of NaCl.

A simple, cost effective and novel method of establishment of seeds of wild species and interspecific crosses of niger

In niger, seeds of wild species are smaller than cultivated niger but, flowers of Guizotia scabra are large with more number of ray and disc florets. Seeds of wild species planted directly in the field often fail to establish and plantlet recovery is very low. To overcome these limitations, seeds are planted in different containers with various kinds of potting mixes. A simple method has been developed for raising the wild species of niger using peat pellets. Using this procedure, there was 100% germination as well as 100% plant establishment was observed. This procedure can be successfully be used for all the crops with such difficulties in initial establishment of the plants.

Agrobacterium-mediated transformation of THC-containing Cannabis sativa L. yields a high frequency of transgenic calli expressing bialaphos resistance and non-expressor of PR1 (NPR1) genes

We established transformation technologies using Agrobacterium tumefaciens (Smith & Townsend) Conn to insert foreign genes into high THC-containing cannabis ( Cannabis sativa L.). The Arabidopsis non-expressor of pathogenesis-related protein 1 ( AtNPR1) gene was selected as a potentially useful agronomic gene, which was linked to the bar gene from Streptomyces that encodes herbicide resistance. We investigated how Agrobacterium strains (EHA105 and GV3101), glufosinate concentrations, explant source, and light intensity affected transformation frequency (TF). Transformation was confirmed by RT-PCR with primers for the NPR1 or bar genes. Glufosinate at 0.5–1 mg/L inhibited growth of non-transformed calli within 8 weeks following A. tumefaciens infection. Strain EHA105 yielded a higher TF compared to strain GV3101. Whole leaflets yielded a higher TF compared to sectioned leaf explants with strain GV3101. However, this effect was not seen with EHA105. Petiole segments showed a higher TF than leaf sections with strain EHA105. Placing explants under light or dark conditions did not affect TF, which ranged from 5% to 95% in different experiments. This is the first report of successful transformation of two high THC-containing C. sativa genotypes with two foreign genes simultaneously— AtNPR1 and bar. The recovery of plantlets from transgenic calli was not attempted and awaits further research.

Molecular and histological validation of modified in ovulo nucellus culture based high-competency direct somatic embryogenesis and amplitude true-to-the-type plantlet recovery in Kinnow mandarin.

Kinnow (Citrus nobilis Lour. × Citrus deliciosa Ten.) needs to be genetically improved for traits such as seedlessness using biotechnological tools. Indirect somatic embryogenesis (ISE) protocols have been reported for citrus improvement. However, its use is restricted due to frequent occurrences of somaclonal variation and low recovery of plantlets. Direct somatic embryogenesis (DSE) using nucellus culture has played a significant role in apomictic fruit crops. However, its application in citrus is limited due to the injury caused to tissues during isolation. Optimization of the explant developmental stage, explant preparation method, and modification in the in vitro culture techniques can play a vital role in overcoming the limitation. The present investigation deals with a modified in ovulo nucellus culture technique after the concurrent exclusion of preexisting embryos. The ovule developmental events were examined in immature fruits at different stages of fruit growth (stages I-VII). The ovules of stage III fruits (>21-25 mm in diameter) were found appropriate for in ovulo nucellus culture. Optimized ovule size induced somatic embryos at the micropylar cut end on induction medium containing Driver and Kuniyuki Walnut (DKW) basal medium with kinetin (KIN) 5.0 mg L-1 and malt extract (ME) 1,000 mg L-1. Simultaneously, the same medium supported the maturation of somatic embryos. The matured embryos from the above medium gave robust germination with bipolar conversion on Murashige and Tucker (MT) medium + gibberellic acid (GA3) 2.0 mg L-1 + ά-naphthaleneacetic acid (NAA) 0.5 mg L-1 + spermidine 100 mg L-1 + coconut water (CW) 10% (v/v). The bipolar germinated seedlings established well upon preconditioning in a plant bio regulator (PBR)-free liquid medium under the light. Consequently, a cent percent survival of emblings was achieved on a potting medium containing cocopeat:vermiculite:perlite (2:1:1). Histological studies confirmed the single nucellus cell origin of somatic embryos by undergoing normal developmental events. Eight polymorphic Inter Simple Sequence Repeats (ISSR) markers confirmed the genetic stability of acclimatized emblings. Since the protocol can induce rapid single-cell origin of genetically stable in vitro regenerants in high frequency, it has potential for the induction of solid mutants, besides crop improvement, mass multiplication, gene editing, and virus elimination in Kinnow mandarin.

Cryopreservation of coconut plumule using droplet vitrification

In the present investigation, four types of explants from mature zygotic embryos of coconut, viz., whole upper cotyledonary region without haustorium, half of the upper cotyledonary region without haustorium, plumule with a portion of radicle and exclusively plumular tissue, were cultured in 12 different media combinations to find a suitable explant which could be regenerated after cryopreservation. Explants were pre-cultured in medium with 0.4 and 0.5 M sucrose for three days followed by dehydration in PVS3 solution for different durations on a sterile aluminum strip after treating with loading solution. Strips were treated with liquid nitrogen inside a cryoflask until bubbling stopped and quickly transferred to a cryovial and stored for a minimum period of 24 hours in liquid nitrogen. It was observed that plumule alone or with a small portion of outer tissue was ideal for fast in vitro growth and recovery of whole plantlets of coconut in a medium supplemented with NAA alone. Addition of glutamine (5 mg L-1), TDZ (1 mg L-1) and NAA (18 mg L-1) aided the vigorous growth of plantlets. In control, the survival rate ranged from 60 to 90 per cent in plumule pre-grown in media containing 0.5 M sucrose after dehydration with PVS3 for various durations, whereas it was 14 to 75 per cent in cryopreserved ones. Considering the high survival (75%) and regrowth (35%) of cryopreserved plumule in the present study, there is much scope for further improvement of the procedure to find the right combination of factors so as to enhance complete recovery of plantlets without much injury to plumules during cooling and rewarming.

Variables Affecting Shoot Growth and Plantlet Recovery in Tissue Cultures of Drug-Type Cannabis sativa L.

Tissue culture approaches are widely used in crop plants for the purposes of micropropagation, regeneration of plants through organogenesis, obtaining pathogen-free plantlets from meristem culture, and developing genetically modified plants. In this research, we evaluated variables that can influence the success of shoot growth and plantlet production in tissue cultures of drug-type Cannabis sativa L. (marijuana). Various sterilization methods were tested to ensure shoot development from nodal explants by limiting the frequency of contaminating endophytes, which otherwise caused the death of explants. Seven commercially grown tetrahydrocannabinol (THC)-containing cannabis genotypes (strains) showed significant differences in response to shoot growth from meristems and nodal explants on Murashige and Skoog (MS) medium containing thidiazuron (1 μM) and naphthaleneacetic acid (0.5 μM) plus 1% activated charcoal. The effect of Driver and Kuniyuki Walnut (DKW) or MS basal salts in media on shoot length and leaf numbers from nodal explants was compared and showed genotype dependency with regard to the growth response. To obtain rooted plantlets, shoots from meristems and nodal explants of genotype Moby Dick were evaluated for rooting, following the addition of sodium metasilicate, silver nitrate, indole-3-butyric acid (IBA), kinetin, or 2,4-D. Sodium metasilicate improved the visual appearance of the foliage and improved the rate of rooting. Silver nitrate also promoted rooting. Following acclimatization, plantlet survival in hydroponic culture, peat plugs, and rockwool substrate was 57, 76, and 83%, respectively. The development of plantlets from meristems is described for the first time in C. sativa and has potential for obtaining pathogen-free plants. The callogenesis response of leaf explants of 11 genotypes on MS medium without activated charcoal was 35% to 100%, depending on the genotype; organogenesis was not observed. The success in recovery of plantlets from meristems and nodal explants is influenced by cannabis genotype, degree of endophytic contamination of the explants, and frequency of rooting. The procedures described here have potential applications for research and commercial utility to obtain plantlets in stage 1 tissue cultures of C. sativa.

Vegetative Propagation of Phytophthora cinnamomi-Tolerant Holm Oak Genotypes by Axillary Budding and Somatic Embryogenesis

Holm oak (Quercus ilex) is one of the most widely distributed tree species in the Mediterranean basin. High mortality rates have been observed in holm oak populations in the southwest of the Iberian Peninsula as a result of oak decline syndrome. Selection and propagation of genotypes tolerant to this syndrome could aid the restoration of affected areas. In this article, we report micropropagation and conservation procedures based on axillary budding and somatic embryogenesis (SE) of holm oak plants, selected for their tolerance to Phytophthora cinnamomi—the main biotic factor responsible for oak decline. Forced shoots were obtained from potted plants of eight different genotypes, and used as stock material to establish in vitro shoot proliferation cultures. Reliable shoot proliferation was obtained in seven out the eight genotypes established in vitro, whereas multiplication rates were genotype-dependent. The highest rooting rates were obtained by culturing shoots for 24 h or 48 h on rooting induction medium containing 25 mg L−1 indole-3-butyric acid, followed by transfer to medium supplemented with 20 µM silver thiosulphate. Axillary shoot cultures can be successful conserved by cold storage for 12 months at 4 °C under dim lighting. Shoot tips, excised from axillary shoot cultures established from tolerant plants, were used as initial explants to induce SE. Somatic embryos and/or nodular embryogenic structures were obtained on induction medium with or without indole-acetic acid 4 mg L−1, in two out the three genotypes evaluated, and induction rates ranged between 2 and 4%. Plantlet recovery was 45% after two months cold stratification of somatic embryos and eight weeks of culture on germination medium. Vegetative propagation of P. cinnamomi-tolerant Q. ilex trees is a valuable milestone towards the restoration of disease-affected areas.

Efficient Generation of CRISPR/Cas9-Mediated Homozygous/Biallelic Medicago truncatula Mutants Using a Hairy Root System.

In the process of acquiring mutants mediated by CRISPR/Cas9, plantlets are often regenerated from both mutated and non-mutated cells in a random manner, which increase the odds of chimeric mutated plant. In general, it’s necessary to infect more explants or grow to next generation for the need of generating more biallelic or homozygous mutants. In present study, an efficient way of obtaining biallelic or homozygous mutated lines via fast-growing hairy root system without increasing numbers of infected explants or prolonging sexual propagation generation is reported. The fast growing lateral branches of hair roots are originated deep within the parental root from a small number of founder cells at the periphery, and therefore were employed as a library that classify different editing types in different lateral branches in which the homozygous or biallelic lines were screened. Here, MtPDS was employed in a proof-of-concept experiment to evaluate the efficiency of genome editing with our hairy root system. Homozygous/biallelic mutations were found only 1 of the 20 lines in the 1st generation hairy roots, and 8 lines randomly selected were cultured to obtain their branch roots, homozygous/biallelic mutations were found in 6 of the 8 lines in their branch roots. We also tested the method with MtCOMT gene and got the same result. All of the seedlings regenerated from the homozygous/biallelic hairy root mutation lines of MtPDS displayed albino phenotypes. The entire process from vector design to the recovery of plantlets with homozygous/biallelic mutations took approximately 4.5–6.5 months. The whole process could bring inspiration for efficiently generating homozygous/biallelic mutants through CRISPR/Cas9 system from the hairy root or root system of a chimeric mutated transformants, especially for the rare and endangered plants whose explants sources are very limited or the plants that lack of tissue culture and rapid propagation system.

Genetic Transformation of Common Wheat (Triticum aestivum L.) Using Biolistics.

The following protocol describes the genetic transformation of wheat using the BioRad PDS/1000-He particle delivery system. Immature embryos are isolated 12-16days post-anthesis, the embryonic axis is removed, and the immature scutella are precultured for 1-2days prior to particle bombardment. Gold particles are coated with plasmid DNA containing the gene(s) of interest plus a selectable marker gene, in this instance bar (bialaphos resistance), and are fired into the cells to deliver the DNA. Subsequent tissue culture and regeneration steps allow recovery of plantlets, assisted by the inclusion of PPT (phosphinothricin tripeptide), the active ingredient of glufosinate-ammonium containing herbicides, to help select transformants. This updated method introduces selection earlier in the regeneration process which provides a shortened protocol while maintaining high transformation efficiencies.

Somatic embryogenesis and regeneration of Kenyan wheat (Triticum aestivum L.) genotypes from mature embryo explants

Wheat (Triticum aestivum L.) improvement via genetic transformation depends on an efficient regeneration system for recovery of transgenic events. This study reports a somatic embryogenesis-based regeneration system for two Kenyan wheat genotypes (Eagle 10 and Njoro bread wheat II) from mature embryos. The study investigated the efficiency of mercuric chloride, commercial bleach, and chlorine gas in surface sterilizing explants prior to in vitro culture. Callus induction and somatic embryogenesis were done by culturing wheat mature embryos in Murashige and Skoog (MS) medium supplemented with 2, 4-dichlorophenoxyacetic acid (2,4-D) used either singly or in combination with 1-naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid (4-CPA) or 6-benzylaminopurine (BAP). Embryo germination and plantlet recovery were done by culturing embryogenic callus in MS medium without plant growth regulators (PGRs). Chlorine gas was significantly (p<0.001) the most effective in surface sterilization and maintenance of explant viability. All 2, 4-D concentrations tested (1, 2, 4 and 8 mg/l) induced embryogenic callus. A significantly higher callus induction rate and callus fresh weight were obtained when 2,4-D was used in combination with either NAA or 4-CPA than when it was used singly. Combining 2,4-D with BAP led to a significantly lower callus induction frequency. Somatic embryo germination was achieved in MS medium without plant growth regulators. These findings have the potential to inform future efforts in the application of modern biotechnology for accelerated wheat cultivar improvement. Key words: Wheat, somatic embryogenesis, mature embryos.

Development of an efficient protocol for ‘Brazos’ blackberry in vitro multiplication

In vitro culture is a very important technique in plant mass propagation, as well as an auxiliary tool to germplasm conservation and gene transfer. The present study developed an efficient multiplication protocol for 'Brazos' blackberry. Here, its multiplication ability was evaluated using in vitro shoots grown on medium supplemented with different growth regulators: thidiazuron (TDZ), 6-benzylaminopurine (BAP), kinetin (KIN), zeatin (ZEA) and isopentyladenine (2iP) in two concentrations (5 and 10 μM) plus growth regulator free medium and five different basal media: Murashige and Skoog (MS), Murashige and Skoog with half-strengthened salts (MS/2), Anderson (AND), Quoirin and Lepoivre (QL) and woody plant medium (WPM). Number of shoots formed, shoot length and number of leaves per shoot were evaluated. Shoots were evaluated and subcultured three times, at every four weeks into a fresh medium. Among the different cytokinins, BAP in both concentrations was the best for shoot multiplication with 4.4, 6.1 and 7.9 shoots per explant with 5 μM and 4.3, 4.7 and 4.8 shoots per explant with 10 μM, in the three subcultures. Thidiazuron was not viable because of shoot malformation and great callus formation. Among the medium salts tested, MS medium was slightly better, reaching 3.9, 4.3 and 2.5 shoots per explant in the first, second and third subculture. The results of this investigation indicate that blackberry multiplication is viable and represents a potential technique for rapid and efficient multiple shoot induction and plantlet recovery. In vitro multiplication and a culture medium MS supplemented with BAP 5 µM can be recommended for 'Brazos' blackberry.

Sugar Beet Artificial Seeds an Overview

Artificial seed propagation of crops broadens the horizon of plant biotechnology and farming. The technology offers techniques for micropropagulated seed analogs such as axillary leaves, embryogenic calli, somatic embryos, apical shoot tips, and protocorm-like organs. Micropropagules are embedded in gelling medium and carboxyl methyl cellulose active coatings. A variety of plant species, such as mulberry, sandalwood, cardamom, banana, sugar beet, maize, and relative, have recorded encapsulation of micro shoots and somatic embryos and subsequent recovery of full plantlets. This knowledge has shown that artificial seed manufacturing is possibly helpful for the propagation of economically significant species ' inferior hybrids on a big scale. Artificial seed development can only succeed with effective upstream manufacturing of micropropagules and downstream germination procedures for an elevated proportion of plant regeneration as one of the significant value-added plant tissue culture goods. Different micropropagules were regarded for the manufacturing of artificial seeds; however, mostly favored were somatic embryos and axillary stem buds. As micropropagules, somatic embryos were used to create artificial seeds in a wide range of fruit and plant organisms, which include Daucus carota, Picea abies, Arachis hypogaea, Medicago sativa, Psidium guajava, and Vitis vinifera. The review illustrated the concept of synthetic seeds and encapsulation procedure of sugar beet

High frequency conversion of non-embryogenic synseeds and assessment of genetic stability through ISSR markers in Gymnema sylvestre

The present study describes the first attempt of exploiting encapsulation technology for high plantlet recovery, short-term storage and conservation of Gymnema sylvestre—an antidiabetic liana. Indole-3-butyric acid (IBA) pretreated nodal segments (NS) were encapsulated in sodium alginate (Na2-alginate) matrix and the optimal culture conditions were evaluated in terms of maximum conversion capacity of synseeds into complete plantlets. Highest conversion frequency of synseeds was obtained on Murashige and Skoog’s (MS) medium supplemented with 5.0 µM 6-benzyladenine (BA). Augmentation of Na2-alginate matrix with plant growth regulators (PGRs) and additive further improved in vitro conversion rates and the synthetic endosperm composed of 3% Na2-alginate in MS + 2.5 µM BA + 2.5 µM gibberellic acid (GA3) + 50 µM adenine sulphate (AdS) stimulated maximum recovery (88.2 ± 0.48%) of complete plantlets from synseeds. Studies on short term cold storage of synseeds showed that nutrient encapsulation maintains the viability of NS for a storage period of 8 weeks. Ex-vitro conversion of synseeds was also carried out on soilrite and vermicompost (3:1) mixture under culture room conditions. Monomorphic DNA profiles produced through Inter-Simple Sequence Repeat (ISSR) markers confirmed the genetic uniformity between synseed derived and mother plantlets.

Formación in vitro de rizomas en caña flecha (Gynerium sagitatum Aubl.) y recuperación de plantas

La caña flecha Gynerium sagitatum Aubl. (Poaceae) es una especie de gran importancia ambiental, cultural y económica para ciertas comunidades indígenas del Norte de Colombia, empleando su fibra en la fabricación apreciables artesanías. La cosecha de la planta y la no reposición de esta, viene contribuyendo en la disminución de poblaciones naturales de la esta especie. La micropropagación ha emergido como una de las la única posibilidades de producir de forma eficiente material vegetal para el establecimiento de cultivos y restaurar zonas afectadas. Con el fin de mejorar la eficiencia económica del protocolo de micropropagación, estructuras del tipo rizomas fueron inducidas in vitro a partir de explantes consistentes de plantas stablecidas in vitro bajo tres niveles de sacarosa, cuatro de benzilaminopurina – BAP y cuatro de ácido abscísico – ABA en MS con (en mg L-1) myo-inositol (100), tiamine HCL (0,4) y solidificados con Phytagel® (3.000). Los tratamientos (48) fueron distribuidos con un diseño completamente al azar con seis repeticiones por tratamiento. Los cultivos fueron mantenidos durante ocho semanas a 25 °C con 12 h de fotoperíodo (40 μmol fotones m-2 s-1) suministrada con lámparas de luz fría fluorescente. Diferencias estadísticas se observaron con respecto al número de rizomas formados y longitud de los rizomas como efecto de la interacción de los tres factores. La recuperación de las plantas ex vitro ocurrió en mayor número (6,0) cuando los rizomas se desarrollaron en medios suplidos con 263000 µM de sacarosa combinado con 4,44 y 8,88 µM de Benzilaminopurina – BAP. Los resultados evidencian la posibilidad de inducir in vitro rizomes de caña flecha para ser utilizados en conservación y propagación de esta especie.

Induction of haploid plants in citrus through gamma-irradiated pollen and ascertainment of ovule age for maximum recovery of haploid plantlets

The present investigation was carried out for the induction of haploid plants in Citrus grandis through in situ parthenogenesis by pollination with gamma-irradiated pollen of C. limetta and C. sinensis, treated with 50, 100, 200, 300, and 400 Gy gamma ray doses followed by in vitro ovule culture. Ovule culture at 50 days after pollination (DAP) was found optimum for the maximum recovery of in vitro-raised plantlets as compared to 35 and 20 DAP. At 50 DAP, irrespective of the pollen parent, plantlet regeneration capacity decreased at 300 and 400 Gy irradiation treatment (0.62% and 0.60%, respectively) with maximum recovery in the nonirradiated control (3.07%). Chromosome counting in actively growing root tips of all the in vitro-raised ovule cultured plantlets revealed that two haploid plants with chromosome number of nine were induced in C. grandis from the ovule culture that was cultured at 50 DAP following pollination with irradiated pollen of C. sinensis at 400 Gy and C. limetta pollen at 300 Gy. However, irradiation doses below 300 Gy were incapable of inducing any haploids in either cross combination. Similarly, all the plantlets regenerated from in vitro embryo culture of mature seeds were found to be diploid in nature, irrespective of the pollen parent and irradiation dose. Molecular analysis of the in vitro-raised haploid and diploid plants using SSR markers confirmed the maternal origins of the haploid plants; however, the diploid plants were found zygotic in nature with one allele from the seed parent and the other one from the pollen parent.

Response interactions in grape somatic embryogenic cultures to cold and gibberellic acid treatments to overcome embryo dormancy

Somatic embryogenesis (SE) is a widely used method in grape (Vitis vinifera) biotechnology as it permits the rapid multiplication of clonal material from a single explant source. A commonly observed difficulty with this technique, however, is that many of the newly derived embryos either cease development entirely or they develop abnormally and fail to form a plantlet capable of survival beyond the in vitro environment. Previous studies have reported that somatic embryos of grape enter into a state of endodormancy similar to that seen in grape seeds. Grape seeds require a cold period of several weeks to induce germination and the release of the embryo from dormancy is associated with a rapid rise in endogenous gibberellins. We tested the influence of cold and/or a pulse treatment of gibberellic acid (GA3) on embryogenic callus cultures of the variety Pinot noir. In the most efficient treatment tested (8 weeks in cold), a 4.6-fold improvement in plantlet recovery was recorded compared with that in the untreated controls. The improvement in yield was the result of combined increases in embryo differentiation and embryo germination. Treatment with GA3 improved embryo germination compared to the untreated controls, but only when GA3 was used in combination with sub-optimal cold treatments. The GA3 pulse treatments tested had no measurable impact on embryo differentiation or on the differentiation of plantlets from young germinated embryos.

Temperature induction response technique: a screening tool for evaluation of banana cultivars for thermotolerance

Banana is a succulent tropical fruit crop. Screening and identification of cultivars for high temperature stress conditions is essential under changing environment. Temperature induction response (TIR) technique, is based on the principle of subjecting plantlets to sublethal temperatures and subsequent exposure to challenging temperatures, and further assessing growth and recovery. In this study, 5–6 weeks old micro-propagated banana plantlets were subjected to various temperatures from 55 to 70 °C for a duration of 2 h. Based on least survival (11%) and highest growth reduction (92%) during recovery of plantlets, 55 °C for 2 h was identified as challenging temperature or lethal temperature. The induction temperature, 42 °C for 2 h 30 min, at which more than 50% of the plantlets survived and a lower reduction in growth (20%) during recovery after exposure to lethal temperatures (55 °C for 2 h) were identified as the optimum induction temperature. Twenty banana cultivars were screened using TIR. Using Z distribution analysis the cultivars were grouped into tolerant (cvs. Grand Naine and Rasbale), which clustered in quadrant I, and susceptible (cvs. Red Banana and Kunnan), which clustered in quadrant III. Injury index was lower in cv. Grand Naine (tolerant) compared to cv. Red Banana (susceptible). Hence, we propose this technique as a potential tool to screen and identify temperature-tolerant genotypes in banana.

In vitro shoot tip multiplication of bambara groundnut [Vigna subterranea (L.) Verdc.

A rapid, simple and efficient protocol for direct in vitro multiple shoot induction and plantlet recovery was achieved from shoot tip explants of Bambara groundnut [Vigna subterranea (L.) Verdc.]. Shoot tips, were isolated from in vitro-grown seedlings and cultured on Murashige and Skoog basal medium (MS) containing Gamborg’ vitamins (B5) and supplemented with different concentrations of the plant growth regulators Thidiazuron (TDZ), N6-benzylaminopurine (BAP), Kinetin (KIN) and Adenine sulfate (ADS). TDZ 0.45 μM was found to be best for shoot multiplication with a mean of 11shoots per explant. Among the carbon sources tested, the best response in terms of mean number of shoots per explant was obtained with sucrose (11). The mean number of shoots per explant induced among the studied landraces of Bambara groundnut varied from 9 to 13. Individual shoots, aseptically excised, produced normal roots within 2 weeks on the basal MS medium supplemented with Indole Butyric Acid (IBA) or Naphthalene acetic acid (NAA). The highest number of roots per shoot and the longest roots were obtained on MS medium with 2 mg/L IBA. Rooted plantlets were successfully hardened under greenhouse conditions and subsequently established in the field conditions, with a recorded survival rate of 70 and 80 %, respectively. The transferred plants in the field were morphologically normal and fertile. This protocol can be efficiently used for mass propagation, germplasm preservation and probably also for gene transfer of Vigna subterranea (L.).

Pengaruh Retardan Paklobutrazol terhadap Pertumbuhan dan Pemulihan Dua Aksesi Ubi Kayu

&lt;p&gt;Normal growth medium is not effective for in vitro conservation due to the risk of somaclonal variation that may increase due&lt;br /&gt;to short interval between subculture. Two experiments involving growth retardant paclobutrazol (PBZ) were conducted to&lt;br /&gt;reduce explants growth and extend subculture interval. In order to develop medium-term conservation of cassava, the&lt;br /&gt;recovery of plantlets after in vitro storage was also observed. Accession 433 and 450 were used in two independent&lt;br /&gt;experiments. Completely Randomized Design was used with three replications. PBZ at 0, 3.4, 6.8, and 10.2 μM were&lt;br /&gt;supplemented onto MS medium + arginin 100 ppm. Observation was done on shoot length, number of nodes and leaves, and&lt;br /&gt;number of white and senescence leaves. The results showed that after nine months without subculture, both cassava&lt;br /&gt;accessions showed different results in in vitro growth and their recovery. PBZ 3.4 μM performed as the best treatment in&lt;br /&gt;accession 433 and 450 to reduce in vitro growth and their recovery after storage.&lt;/p&gt;