Micropropagated Banana Plants Research Articles

Tissue Culture in Banana Cultivation: A Review of its Impact on Disease Management, Yield Improvement, and Sustainable Production

In several tropical and subtropical areas, bananas (Musa spp.) are an important food source and commercial crop. Disease transmission and production uncertainty are common problems with traditional propagation techniques such as suckers. By supplying consistent, high-quality, and disease-free planting material, tissue culture, known as micro propagation, represents a potential alternative. The application of tissue culture has greatly enhanced disease management in banana cultivation. Reducing the incidence of ailments like Fusarium wilt, bacterial wilt, and Banana Bunchy Top Virus (BBTV), pathogen-free plantlets are grown in controlled conditions. This strategy lessens the demand for artificial fertilizers while also promoting healthier crops and lessening the impact on the environment. Micropropagated banana plants demonstrated consistent growth, early fruiting, and increased yield in contrast to conventionally propagated plants. Tissue-cultured plants exhibit strong development and genetic uniformity, which contribute to these advantages and more consistent and dependable harvests. By promoting efficient land use and reducing the need for chemical inputs, tissue culture enhances sustainability in the production of bananas. The growing demand for bananas may be met without requiring the increase of agricultural land thanks to tissue culture techniques, which enable the large-scale production of enhanced banana cultivars at a quick rate. Furthermore, using planting material free of disease lowers input costs and crop losses, supporting more environmentally friendly farming methods.

Yield and postharvest of ‘BRS Platina’ banana not irrigated under different types of soil mulches

Banana (Musa spp.) is a crop with outstanding economic and social expression throughout the world. The objective of this study was to investigate the effect of different mulches types in growth production and postharvest of banana ‘BRS Platina’ not irrigated. The experiment was conducted in the farm of the Federal University of Lavras (UFLA). Micropropagated banana plants of the cultivar BRS were planted in the field with a spacing of 2.5 × 3.0 m (1333 plants ·ha-1), without irrigation. About 20 days after planting, different mulches were installed: black polyethylene films, double-sided polyethylene films (white and black), organic mulch (from the own crop) and control (bare soil). The experimental was set up in a randomized block design with four treatments, six blocks and six plants per treatment, totaling 144 plants. Among the six plants used in each treatment, two constituted the border, therefore four plants were evaluated. The analysis performed in the first and second crop were: growth analysis, postharvest quality, production and productivity. Both organic and inorganic (plastic film) mulch provide greater growth and yield of banana ‘BRS Platina’ not irrigated, in two years of production and can be used in the cultivation of this crop.

Biopriming of micropropagated banana plants at pre- or post-BBTV inoculation stage with rhizosphere and endophytic bacteria determines their ability to induce systemic resistance against BBTV in cultivar Grand Naine

ABSTRACTRhizospheric and endophytic bacteria isolated from the roots and corms of banana were tested to find out their efficiency in controlling against banana bunchy top virus (BBTV). Bioformulations of mixtures of endophytic Bacillus pumilus and B. subtilis isolated from banana cv. Grand Naine and rhizobacterial isolate Pseudomonas fluorescens (Pf1) were found to be effective in increasing the growth and physiological parameters such as pseudostem girth and height, number of leaves, phyllochron, and leaf area in biohardened plants under greenhouse study. The consortia of bioformulation mixture of B. pumilus, B. subtilis, and P. fluorescens I showed 61.62% disease reduction over control. The defence enzymes such as peroxidase (POX), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), and total phenol were induced to an elevated level in biohardened plants. The applications of bioformulations to plants led to delay the symptom expression for 63.75 to 70.50 days compared to control after challenge inoculation with the virus in 34–67% of plants that exhibited the symptoms till 150 DAI. However, biohardening of plants with the same combinations of bacteria three days after BBTV inoculation led to express the symptoms 29.16 to 36.71 days and there was a significant decrease in plant growth parameters. Biopriming prior to BBTV infection has attributed to the enhanced plant growth and resistance against BBTV whereas, the same treatments after virus inoculation did not induce resistance. This study has proved that the time of application of consortia of bio-inoculants determines their effect of induced resistance to BBTV in micropropagated plants.

Application of electrotherapy for the elimination of cucumber mosaic virus (CMV) in micropropagated banana plants (Musa spp.)

Application of electrotherapy for the elimination of cucumber mosaic virus (CMV) in micropropagated banana plants (Musa spp.)

Isozyme analysis based genetic fidelity assessment of micropropagated banana plants

Isozyme studies of micropropagated and mother plants of banana cvs. Matti, Ney Poovan, Kechulepa, Dwarf Cavendish, Malbhog, Champa, B.B. Battisa and FHIA-1 were done to test their genetic fidelity. The banding patterns as revealed by electrophoretic variations were evaluated with respect to isozymes of acid phosphatase, catalase, esterase and peroxidase as markers. The genetic fidelity of micropropagated plants and the relationship of the different cultivars were determined by dendrogram using numerical taxonomy and multivariate analysis system(NTSYS). A clustered dendrogram was prepared by unweighted pair group method using averages (UPGAMA) method. At 87% similarity, the micropropagated and mother plants were clustered in four groups reflecting their genomic constitution. Cvs. Matti (AA) and Dwarf Cavendish (AAA) with similar ‘A’ genome were categorized in Cluster I. Cluster II comprised of cvs. Ney Poovan (AB), B.B. Battisa (ABB) and FHIA-1(AAAB) with genomic constitution of both ‘A’ and ‘B’ type. Cvs. Champa (AAB) and Malbhog (AAB) with similar genome were grouped inCluster III. Cluster IV contained the cv. Kechulepa (BB) having only ‘B’ genome. However, there was no somaclonal variation among the micropropagated plants and they showed 100% genetic similarity. Thus, the isozyme studies could be a reliable marker for testing the genetic fidelity of micropropagated plants and for evaluating the diversity among the banana germplasm.

Effect of microbial inoculants on the growth and yield of micropropagated banana (Musa indica) cv. Grand Naine

SummaryMicropropagated plants generally exist in a sterile medium or environment and can thus benefit from inoculation with beneficial microbes to enhance their rate of survival following transfer from the laboratory to field conditions. The present investigation studied the beneficial effects of microbial inoculants (i.e., Azotobacter chroococcum and/or Piriformospora indica) on the growth and nutrition of micropropagated banana plantlets under glasshouse, as well as under field conditions (with 75% and 100% of the recommended doses of N and P). Dual inoculation resulted in the maximum rate of survival (84.6%) of micropropagated banana plantlets 2 months after transfer to a glasshouse. The maximum rates of colonisation by P. indica (76.5%) or A. chroococcum (at 147 × 104 cfu g–1) were observed 2 months after inoculation. The maximum mean root length (15.5 cm), shoot length (33.3 cm), root dry weight (DW; 2.0 g), and shoot DW (3.6 g) were observed in double-inoculated, micropropagated plantlets 2 months after transfer to the glasshouse. Plantlets were then transferred to the field. Suckers from the year-1 experiment were also transferred to another field and re-inoculated with either one or both microbial cultures for the year-2 experiment. In both years, the maximum mean numbers of leaves, stem diameters, plant heights, numbers of suckers, leaf areas, phosphorus contents, nitrogen contents, total chlorophyll contents, numbers of fingers, and fruit yields were observed in plants which had been co-inoculated with P. indica plus A. chroococcum, compared to non-inoculated control plants. Microbial inoculation (single or dual) improved plantlet survival rates, as well as the growth and yields of micropropagated banana plants compared to non-inoculated control banana plants.

Soft rot inciting Pectobacterium carotovorum (syn. Erwinia carotovora) is unlikely to be transmitted as a latent pathogen in micropropagated banana

This study was undertaken to ascertain if the soft rot inciting Pectobacterium carotovorum/Erwinia carotovora would pass through the micropropagated bananas as a latent pathogen and cause disease during or post acclimatization. In vitro cultures of ‘Grand Naine’ were exposed to the pathogen by providing 100 μl of inoculum (0.001–1.0 at OD600 nm) at the lower leaf axil. These cultures showed a gradual development of soft rot symptoms coupled with obvious bacterial colony growth on banana proliferation medium and consequent plant mortality within a month irrespective of the inoculum level employed. Plants carried forward to acclimatization following inoculation in vitro failed to establish ex vitro. Monitoring the normal field-grown suckers at culture initiation through PCR screening employing soft rot Erwinia primers did not show the amplification of the 119-bp fragment as seen with the pure cultures of pathogen. Further testing of micropropagated banana plants through soil inoculation, in vitro culturing and PCR screening ruled out the possibility of the pathogen surviving in micropropagated stocks in latent form as the organism outgrew and killed the cultures. It emerged that the infection possibly takes place in the nursery. This information will be of particular value for the plant tissue culture industry, plant pathologists and quarantine agencies.

Development of an in vitro culture system adapted to banana mycorrhization

The beneficial impact of arbuscular mycorrhizal (AM) fungi on banana nutrition and resistance against abiotic and biotic stresses is well documented. However, most studies were conducted under greenhouse or field conditions and none reported the life cycle of the AM fungi on banana roots. It is obvious that any system associating both organisms under strict controlled in vitro culture conditions may help to comprehend the role of AM fungi in banana physiology. Here we developed an in vitro culture system associating autotrophic micropropagated banana plants with an AM fungus (Glomus intraradices). Intraradical root colonization, with production of arbuscules and vesicules, as well as extraradical development with production of new spores was observed. This study opens the door to investigate the role of AM fungi in banana physiology in particular for the control (through e. g. the elicitation of defence genes in bananas) of major banana root pathogens under strict in vitro culture conditions.

Anatomical and physiological modifications of micropropagated 'Caipira' banana plants under natural lighy

Research about the use of natural light associated to changes in sucrose levels demonstrated potential in promoting in vitro hardiness of tropical climate species, as well as reducing production costs. However, little is known about physiological and structural changes that happen in the process. This study evaluated the physiological and anatomic performance, and ex vitro survival of micropropagated banana plants in response to cultivation conditions, in the stage of in vitro rooting. Shoots of the 'Caipira' cultivar were cultivated in MS medium, supplemented with 1 mg L-1 NAA and 6 g L-1 agar, in which the following treatments were applied: two sucrose concentrations (15 g L-1 or 30 g L-1) and two cultivation conditions (Natural light - greenhouse and Artificial light - growth chamber). At the end of 45 days, the contents of chlorophyll a, b and total, the relative water content in the tissues, anatomic characteristics and the ex vitro survival were evaluated. Effects of growth environment and sucrose concentration were observed on micropropagated 'Caipira' banana anatomy, physiology and survival. In vitro rooting of the shoots under natural light in the medium containing 15 g L-1 or 30 g L-1 sucrose promoted major alteration in the increase of palisade and spongy parenchyma, as well as reducing leaf water loss and plant death. The results obtained in the present study confirm the potential of the use of natural light as a substitute for artificial light for micropropagation of tropical species.

Alterações anatômicas de bananeiras micropropagadas em resposta a aclimatização ex vitro

Pesquisas acerca das modificações estruturais e fisiológicas inerentes ao processo de micropropagação são fundamentais para compreender os efeitos desta técnica, desenvolver protocolos mais eficientes e, sobretudo, reduzir perdas ex vitro. O objetivo neste trabalho foi avaliar e quantificar as modificações na anatomia foliar de bananeiras provenientes de micropropagação, durante a fase de aclimatização em casa de vegetação. Para tanto, brotações axilares de bananeira cv. Japira, provenientes da multiplicação in vitro, foram enraizadas em meio MS, acrescido de ANA (1mg L-1) e ágar (6g L-1), e mantidas à temperatura de 25°C±2°C e 16 horas de irradiância a 35µmol m-2 s-1, por 35 dias. Posteriormente, as plantas foram submetidas a diferentes períodos de aclimatização (zero, 21, 42, 63, 84 e 120 dias) e avaliadas quanto à anatomia, por meio de seções transversais e paradérmicas foliares. O delineamento experimental foi inteiramente casualizado. Verificou-se que as maiores alterações anatômicas ocorrem após 42 dias do transplantio ex vitro, com acentuado espessamento dos parênquimas clorofilianos e limbo foliar, bem como diferenciação da maioria dos tecidos. Quanto aos estômatos, estes estão distribuídos em ambas às faces da epiderme, com maior número na face abaxial e em folhas oriundas de primórdios foliares formados in vitro.

Perda de água e modificações anatômicas em folhas de plantas de bananeiras micropropagadas durante a aclimatização

Estudos sobre os fatores envolvidos na adaptação das plantas micropropagadas ao ambiente ex vitro são imprescindíveis para definir quais os procedimentos devem ser utilizados durante a fase de aclimatização. O objetivo deste trabalho foi avaliar a contribuição da densidade estomática e da presença de cera epicuticular no controle da perda de água, em folhas de bananeiras micropropagadas. Para tanto, brotações axilares oriundas da etapa de multiplicação in vitro foram enraizadas por 24 dias, em meio MS, contendo 1mg L-1 de ácido naftalenoacético (ANA) e 6g L-1 de ágar e, posteriormente, foram aclimatizadas por 120 dias. Os tratamentos consistiram de folhas formadas in vitro e em diferentes estádios de aclimatização, tais como: T1 - folhas de plantas ao final da fase de enraizamento in vitro; T2 - folhas persistentes de plantas aos 30 dias de aclimatização; T3 - novas folhas de plantas aos 30 dias de aclimatização (folhas de transição); T4 - folhas de transição de plantas aclimatizadas por 60 dias; T5 e T6 - novas folhas de plantas aclimatizadas por 60 dias e 120 dias, respectivamente. Foram avaliados os seguintes parâmetros: a densidade estomática, o conteúdo relativo de água e a presença de cera epicuticular. Foi verificado que folhas de plantas provenientes da fase de enraizamento in vitro, em ambiente mixotrófico, apresentam reduzido controle sobre a perda de água e alta densidade estomática. A reduzida transpiração das folhas formadas na fase de aclimatização pode ser atribuída ao menor número de estômatos por unidade de área foliar, à maior capacidade destes em restringir a perda de água e à presença de cera epicuticular.

Substratos e fungo micorrízico arbuscular em mudas micropropagadas de bananeira na fase de aclimatação

Visando a avaliar diferentes substratos na formação de mudas de bananeira e seu efeito na resposta da planta à inoculação do fungo micorrízico Gigaspora margarita, foi conduzido um experimento em estufa de aclimatação da Biofábrica CAMPO - CPA/Embrapa Mandioca e Fruticultura, Cruz das Almas, Bahia. Foram testados 13 substratos, com e sem inoculação do fungo, em plântulas na fase de aclimatação. A inoculação foi realizada no momento do transplante e, após 55 dias de cultivo, obtiveram-se dados de crescimento, nutrição mineral e colonização micorrízica. O fungo micorrízico arbuscular (FMA) Gigaspora margarita colonizou intensamente e mostrou-se benéfico para o desenvolvimento das mudas de bananeira, sendo o seu efeito modulado pelo substrato de crescimento; o substrato turfa + vermiculita + 5% de esterco destacou-se entre os melhores para a formação de mudas normais e sadias, mas só quando associado à inoculação do FMA; o uso de substrato comercial Rendmax Citrus promoveu o melhor desenvolvimento das mudas, mas inibiu a colonização e o efeito da micorriza; a complementação mineral do Rendmax Citrus não se mostrou necessária para o cultivo de mudas de bananeira; o uso de vermicomposto mostrou-se promissor para a produção de mudas de bananeira, permitindo o efeito da inoculação com FMA.

Utilização de Gigaspora margarita em plantas micropropagadas de bananeira em diferentes estádios de enraizamento

Visando a avaliar o efeito de fungos micorrízicos arbusculares (FMA) na diminuição do tempo de formação de mudas de bananeira micropropagadas, foi conduzido um experimento em estufa de aclimatação da Biofábrica CAMPO - CPA/Embrapa Mandioca e Fruticultura, Cruz das Almas, Bahia. Foram testadas plântulas em três diferentes estádios de desenvolvimento do sistema radicular, inoculadas ou não com o fungo Gigaspora margarita e cultivadas em dois diferentes substratos à base de turfa, vermiculita e esterco. A inoculação foi realizada no momento do transplantio para a estufa e as plantas, cultivadas por 55 dias, quando foram coletadas para obtenção dos dados de avaliação. O fungo Gigaspora margarita colonizou intensamente e mostrou- se benéfico para o desenvolvimento das mudas de bananeira, sendo seu efeito modulado pelo substrato de crescimento; o substrato turfa + vermiculita + 5% de esterco, quando associado à inoculação do FMA, promoveu a formação de mudas normais e sadias; o início da fase de aclimatação de mudas micropropagadas de bananeira pode ser antecipado pelo uso da inoculação com fungos micorrízicos arbusculares, em substrato adequado.

Indole-3-acetic acid metabolism in normal and dwarf micropropagated banana plants (Musa spp. AAA)

Nanism is one of the most frequent type of mutant in micropropagated banana plants from the Cavendish subgroup. The present study aimed at studying some of the hormone factors involved in this type of mutation. Rhizomes from normal and dwarf plants from the cultivar Grand Naine were incubated for 5 d in the presence of [³H]-L-tryptophan, [³H]-indole-3-acetic acid and gibberellin, to quantify the endogenous levels of indole-3-acetic acid-ester, indole-3-acetic acid-amide, free indole-3-acetic acid, and cytokinins. The endogenous levels of indole-3-acetic acid and its ester- and amide-conjugated forms were measured in normal and dwarf plants incubated for 30 d with gibberellin, indole-3-acetic acid, and L- and D-tryptophan. In normal plants, the use of [³H]-L-tryptophan resulted in higher levels of radioactivity in the retention times corresponding to indole-3-acetic acid, indole-3-acetic acid-aspartate, indole-3-acetic acid-glycine and indole-3-acetic acid-alanine. These values were higher than those observed in dwarf plants. Higher quantities of radioactive indole-3-acetic acid and of amide-forms in dwarf plants occurred in rhizomes treated with [³H]-L-tryptophan and gibberellin simultaneously. The endogenous levels of total cytokinins were the same in both materials, while the levels of indole-3-acetic acid in normal plants were 1.5 times higher than in dwarf plants. Moreover, in these ones application of tryptophan and gibberellin coincided in an increase in the levels of free indole-3-acetic acid in dwarf plants and to a decrease in the levels of indole-3-acetic acid-ester and indole-3-acetic acid-amide.

Greenhouse response of micropropagated bananas inoculated with in vitro monoxenically produced arbuscular mycorrhizal fungi

The effect of an in vitro monoxenically produced arbuscular mycorrhizal fungus (AMF), i.e. Glomus intraradices on the growth and phosphorus content of micropropagated banana ( Musa spp. c.v. Grand Naine) plantlets was investigated in a pasteurized and non-pasteurized Vertisol. The experiment was conducted as a 2×2 randomly factorial design with the Vertisol pasteurized or non-pasteurized and the plants inoculated or non-inoculated with the AMF. The plants colonized by AMF had larger shoot and root dry weight and P content than the non-mycorrhizal plants. The largest growth increment was observed in the plants inoculated during the weaning phase with the monoxenic AMF inoculum and subsequently grown in the pasteurized soil. The native AMF population also induced a marked increase in plant growth supporting further the efficiency of this natural population, whereas both inoculum sources in combination did not yield the largest growth increment. The latter suggests a competitive phenomenon between the introduced AMF and the other soil micro-organisms including the native AMF population. Our results demonstrate, for the first time, the potential benefits of a monoxenically produced AMF in micropropagated banana plants.

Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism (MSAP)

The extent of DNA methylation polymorphisms was evaluated in micropropagated banana ( Musa AAA cv. ‘Grand Naine’) derived from either the vegetative apex of the sucker or the floral apex of the male inflorescence using the methylation-sensitive amplification polymorphism (MSAP) technique. In all, 465 fragments, each representing a recognition site cleaved by either or both of the isoschizomers were amplified using eight combinations of primers. A total of 107 sites (23%) were found to be methylated at cytosine in the genome of micropropagated banana plants. In plants micropropagated from the male inflorescence explant 14 (3%) DNA methylation events were polymorphic, while plants micropropagated from the sucker explant produced 8 (1.7%) polymorphisms. No DNA methylation polymorphisms were detected in conventionally propagated banana plants. These results demonstrated the usefulness of MSAP to detect DNA methylation events in micropropagated banana plants and indicate that DNA methylation polymorphisms are associated with micropropagation.

Endogenous Levels of Cytokinins, Indoleacetic Acid, Abscisic Acid, and Pigments in Variegated Somaclones of Micropropagated Banana Leaves

The banana (Musa spp. AAA) micropropagation shows a high incidence of off-types, among whose variegated plants are very common. Endogenous levels of growth regulators and pigment content were measured in normal and variegated leaves of the micropropagated banana plants growing in a greenhouse. Growth regulators were separated by high pressure liquid chromatography and submitted to enzyme-linked immunosorbent assay for quantification. Pigment content was measured using the colorimetric method. Green leaves contained 1.9 and 10 times more cytokinins compared with green and yellow sectors of variegated leaves, respectively. The levels of indoleacetic acid in normal leaves were significantly higher than those found in green and yellow sectors of variegated leaves; however, the levels of abscisic acid were lower in normal leaves. The lower content of chlorophylls in variegated leaves coincided with decreased endogenous levels of cytokinins, which indicated that variegation in banana leaves may be associated with alterations in the metabolism of this growth regulator.

Effects of light intensity and CO 2 concentration on growth of banana plants ( Musa AAA, cultivar ‘Petite Naine’) in vitro and subsequent growth following acclimatization

The development of micropropagated banana plants during the in vitro growth phase prior to acclimatization was studied both in tight vessels under two different photosynthetic photon flux densities (PPFD of 30 and 240 μmol m −2 s −1) and in continuously flushed vessels under three atmospheric CO 2 concentrations (0.034, 0.24 and 4.0%) at 240 μmol m −2 s −1 PPFD. In tight vessels at low PPFD, the CO 2 originating from dark respiration was partially fixed during the light period, indicating photosynthetic activity by the plants in vitro. At high PPFD, CO 2 originating from dark respiration was rapidly fixed in the early hours of the light period and CO 2 concentration became the limiting factor for photosynthetic activity. Plants in vitro grown under high PPFD accumulated 2.3 times the dry matter achieved by plants in low PPFD. However, this developmental advantage acquired in vitro was not maintained ex vitro at the end of the acclimatization phase (on a leaf area basis). In continuously flushed vessels, treatments with 0.24% and 4.0% CO 2-enriched atmospheres enhanced dry matter accumulation in vitro by 1.6 and 2.3 times, respectively, as compared to a 0.034% CO 2 treatment. Twenty days after transfer ex vitro, the development of plants (on a leaf area basis) from these CO 2 treatments was no longer significantly different. The relative growth rate ex vitro was lower for plants cultured in a CO 2-enriched atmosphere in vitro than for those cultured at 0.034% CO 2. Plants cultured in vitro in continuously flushed vessels, after 8 weeks ex vitro, produced as much as five times the foliar area of plants grown in vitro in tight vessels.