Micropropagation Of Sugarcane Research Articles

Callus stage and morphology affect the DNA yield on the DNA extraction from the sugarcane in vitro callus

The main concern with the micropropagation of sugarcane (Saccharum officinarum) with a complex polyploid genome is the frequent occurrence of somaclonal variations. Therefore, genetic stability analysis is often required to ensure the uniformity of the micropropagated sugarcane, as early as in the callus stage. DNA extraction is the initial and important step of the genetic stability analysis. In this experiment, we compared the yield of DNA extraction from the sugarcane embryogenic and non-embryogenic calli. The sugarcane non-embryogenic callus was initiated from immature rolled leaf explants of the 6 months-old plant cane in the callus induction media, that were incubated in the dark room. The embryogenic calli were obtained by routine subcultures of the non-embryogenic calli every six weeks. The results showed that the extraction yield from the non-embryogenic calli was far lower than that from the embryogenic calli, most likely due to the high-water content of the non-embryogenic calli. Furthermore, histological analysis using Safranin and Fast Green staining revealed the low lignin content and nuclei in the non-embryogenic calli cell mass, causing the transparent and watery appearance of the calli and might influence the low DNA extraction yield.

Synergetic Effect of Different Plant Growth Regulators on Micropropagation of Sugarcane (Saccharum officinarum L.) by Callogenesis

The response of different plant growth regulators on callus induction and regeneration on three sugarcane genotypes (YT-53, CP-77-400, and NSG-59) was evaluated. Different concentrations of 2,4-D alone and in combination with other plant growth regulators (Kinetin and BAP) were used for callus induction. Kinetin along with IBA, BAP and NAA were analyzed with respect to shoot induction, while NAA and IBA were used for root induction. The best callus response in terms of number of days, callus fresh weight, and frequency in YT-53 was observed on MS media provided with 2,4-D (3 mg L−1) + Kinetin (0.5 mg L−1), while in NSG-59 the best response was on MS+2,4-D (4 mg L−1) + Kinetin (0.5 mg L−1), and in CP-77400, MS+2,4-D (5 mg L−1). For shoot induction, 2 mg L−1 Kinetin was found to be the best for YT-53 and NSG-59, while 1 mg L−1 BAP was found to be the best for CP-77-400 in terms of number of days, shoot numbers, and shoot length. The best media for root induction in terms of number of days, root numbers, and root length was 1 mg L−1 NAA + 1 mg L−1 IBA for YT-53, while this was 3 mg L−1 NAA for NSG-59. The highest root frequency and maximum root length in the minimum number of days was observed in CP-77-400 on MS media provided with 2 mg L−1 NAA.

Influence of Vitrofural® on sugarcane micropropagation using temporary immersion system

Sugarcane cultivation is of great economic and social importance worldwide. In vitro cloning using temporary immersion systems is an option to produce vitroplants of high genetic and phytosanitary quality to establish stock seedbeds. However, the sterilization of culture media through autoclaving increases production costs and limits the efficiency of temporary immersion systems. For that reason, the objective of this study was to evaluate the morphological and physiological changes caused by Vitrofural® in sugarcane plants under temporary immersion. We compared two types of culture media sterilization: chemical sterilization using Vitrofural® and autoclaving using Temporary Immersion Bioreactors. After four weeks of cultivation, we evaluated morphological (number of shoots, shoot length, and number of leaves per shoot) and physiological indicators (soluble and bound phenolics, and phenolics excreted to the culture medium). As well as chlorophyll-a, -b and total chlorophyll content. The results show that when Vitrofural® was added to culture medium, a higher number of shoots per explant, length of the shoots and number of leaves were observed in comparison with the shoots obtained in autoclaved sterilized medium. Furthermore, a reduced the amount of phenolics compounds were found in Vitrofural® treatent .In addition, the use of the Vitrofural® increased the synthesis of photosynthetic pigments respect to autoclaving. In conclusion, Vitrofural® has a positive effect in morphological and physiological of sugarcane cultured in vitro and is a low-cost alternative for the commercial micropropagation of sugarcane. Vitrofural® produced a positive effect on the growth, content of photosynthetic pigments and compounds phenols during in vitro culture of sugar cane.

In-Vitro Management of Phytohormones for Micropropagation of Sugarcane

In-Vitro Management of Phytohormones for Micropropagation of Sugarcane

Sodium azide mutagenesis within temporary immersion bioreactors modifies sugarcane in vitro micropropagation rates and aldehyde, chlorophyll, carotenoid, and phenolic profiles

Chemical mutagens such as sodium azide (NaN3) have been widely used to increase genetic variability in crops, but the undirected mutations induced can have undesirable effects, which need to be characterized. This study investigated the effects of in vitro NaN3 (0–0.45 mM) exposure (30 days) on the micropropagation of sugarcane within temporary immersion bioreactors (TIB). Shoot multiplication rate and cluster fresh weight, and aldehyde, phenolic, carotenoid, and chlorophyll levels were measured on in vitro produced shoots. The soluble phenolic content of the culture medium was also assessed. NaN3 concentration was negatively correlated with sugarcane shoot multiplication rate and fresh weight; at 0.45 mM NaN3, these parameters were only 20% and 39% that of the untreated control, respectively. Shoot multiplication rate and fresh weight, and chlorophyll a and b levels were negatively correlated with NaN3 concentration. In contrast, malondialdehyde, other aldehyde, carotenoid, and exuded phenol levels were positively correlated with NaN3 concentration. Statistical comparisons suggest that shoot multiplication rate and the biochemical parameters that were positively correlated with NaN3 concentration may be the most suitable indicators of stress when optimizing the concentration of NaN3 for sugarcane explants. An interpolated 50% reduction of multiplication rates at 0.23 mM NaN3 suggests that this concentration to be suitable for TIB-based induction of mutagenesis in shoots and eventual production of agriculturally useful mutants.

A Protocol for <i>In-vitro</i> direct plant regeneration from leaf tissues for micropropagation of sugarcane

An efficient protocol to produce higher number of shoots was developed for Sugarcane (Saccharum Hybrid spp.) variety SL 96 328. In-vitro direct shoot regeneration from sugarcane leaf spindle tissues was achieved in modified Murashige and Skoog medium supplemented with 1.5 mg/L of Benzylaminopurine, 0.5 mg/L of Kinetin, 0.5 mg/L of 2,4-D, 1 mg/L of Indole-3-acetic acid and 400 mg/L of Cysteine Hydrochloride. The survival rate of explants and number, length and vigour of shoots generated from explants, and minimal number of chlorophyll-mutated shoots per explant were recorded. Explants incubated in the dark for two weeks enhanced direct shoot regeneration and produced the highest number of shoots (25) from a single explant. The best diameter and the thickness of the explant were 3 mm and 2 mm, respectively. The amplification of Simple Sequence Repeat primer using Polymerase Chain Reaction (PCR) revealed that the plants obtained through in-vitro directly-regenerated shoots were genetically more identical than those generated from stem cuttings. Nested PCR confirmed that the plantlets obtained from direct shoot regeneration were free from sugarcane white leaf disease (WLD) phytoplasma. Thus, the in-vitro protocol suggested for rapid micropropagation of sugarcane through this study can be adopted in producing genetically-homogenous and WLD-free planting material in establishing sugarcane nurseries.

Assessment of somaclonal variation during sugarcane micropropagation in temporary immersion bioreactors by intersimple sequence repeat (ISSR) markers

Somaclonal variation refers to the genetic and epigenetic changes in plants regenerated from plant tissue culture. In this study, using intersimple sequence repeat (ISSR) molecular markers, the somaclonal variation during micropropagation of sugarcane using temporary immersion bioreactors (TIBs) was evaluated. Apices of the cultivar Mex 69-290 were established and multiplied by ten subcultures in TIBs. After 30 d in each subculture, the number and length of shoots per explant were recorded. For the molecular analysis, ten plants were taken per subculture, and a total of 109 bands from ten ISSR primers were obtained. For each subculture, the polymorphism (%) was calculated. A dendrogram of genetic distances between subcultures and the donor plant was obtained using a matrix of Nei’s genetic distances and the unweighted pair group method with arithmetic mean (UPGMA). The results showed that the production of sugarcane shoots tends to increase until subculture 8, while shoot length decreases. ISSR markers showed the existence of somaclonal variation during micropropagation of sugarcane. The subcultures with the highest percentage of polymorphism (%) and genetic distances (GD) were the 1°, 9°, and 10° (with 10.1, 15.6, and 10.1% and 0.0222, 0.0181, and 0.0181 GD, respectively). The molecular and statistical analysis showed that in vitro establishment and the number of subcultures are both factors that affected the frequency of somaclonal variation during the micropropagation of sugarcane using TIBs. Thus, it is important to determine the optimal number of subcultures that can be made from an explant for each species to be micropropagated.

A competent protocol for large scale production of sugarcane (Saccharum officinarum L.) through meristem culture

A rapid micro propagation and acclimatization response of two different varieties of sugarcane Co86032 and CoN 04131(Saccharum officinarum L.) was obtained in this study. The shoot apical meristem of different sizes wascultured on Murashige and Skoog medium supplemented with different concentrations and combinations of ben-zylaminopurine and kinetin either alone or in combination with each other alongwith GA3. Best shoot formation response in Co 86032 was obtained on MS medium containing 1.5mg/l BAP while in CoN 04131 the combination of 0.5 mg/l BAP with 0.25 mg/l Kinetin showed best shoot formation response from apical meristem. Meristem of 3.0 mm size proved to be the best size for micropropagation of sugarcane. Excellent multiplication response of In vitro formed shoots was obtained when the concentration of BAP was decreased to 1.0 mg/l in Co 86032and 0.25 mg/l BAP and Kin in CoN 04131 (i.e. 0.25 mg/lBAP + 0.25 mg/l Kinetin. MS medium containing 1.0 mg/l NAA and 2.0 mg/l IBA showed 100% rooting response of In vitro regenerated shoots of both the varieties of sugarcane within eight days of inoculation. Best hardening response was obtained in sand+ soil + pressmud (1:1:1) media.

Use of antibiotics to control bacterial contamination during in vitro micropropagation of sugarcane

The role of antibiotics in controlling bacterial contamination and their effect on multiplication and growth of shoot cultures was investigated using sugarcane variety Co 05011. The results showed that Cefotaxime was the most effective antibiotic followed by Streptomycin and Tetracycline in controlling the bacterial contamination. Addition of Cefotaxime (250 mg/l) significantly reduced the frequency of bacterial contamination (38.3 percent) whereas all the cultures subcultured on the medium devoid of any antibiotic, showed the development of bacteria in the medium. The frequency of contaminated cultures was further reduced to 2.7 and 2.3 percent in presence of 500 and 750 mg/l of Cefotaxime, respectively. However, the antibiotic appeared to be detrimental to the shoot growth at the highest concentration (i.e. 750 mg/l). The rate of shoot multiplication in terms of number of shoots per culture was also highly influenced by the type and concentration of different antibiotics. The highest number of shoots (19.6 shoots per culture) could be recorded in presence of 500 mg/l Cefotaxime indicating the highest rate of shoot multiplication. The shoots developed on this medium were vigorous in growth and healthy in appearance. On the basis of above results it may be concluded that Cefotaxime at a concentration of 500 mg/l can be used for controlling bacterial contamination without any adverse effect on multiplication and growth of shoot cultures duringinvitro micropropagationof sugarcane.

Effects of Polyvinyl Pyrrolidone and Activated Charcoal to Control Effect of Phenolic Oxidation on In Vitro Culture Establishment Stage of Micropropagation of Sugarcane (Saccharum Officinarum L.)

Sugarcane isgrown in Ethiopia as both cash and industrial crop. Although it plays a great role in the economy of the country, there are no enough sugarcane varieties under production and it is not an ideal crop for conventional breeding. Hence, it takes around ten years for its breeding and extra years to scale up the planting material for commercial by vegetative propagation. To circumvent these limitations, biotechnological tool (Plant tissue culture) was born as best alternative. Though the tissue culture (micro propagation) plays a reliable role, culture establishment stage of sugarcane micro propagation has a serious phenolic oxidation problem which can kill the whole culture. Hence, these experiments were conducted to optimize the appropriate concentration of Polyvinylpyrrolidone (0.0, 0.1, 0.2, 0.3, 0.4 and 0.5 gL-1) and Activated charcoal (0.0, 0.1, 0.2, 0.3, 0.4 and 0.5 gL-1) on (C86-12, C86-56) genotypes in completely randomized design with 2 × 5 factorial treatment combinations arrangements to control the effect of phenolic oxidation. Analysis of variance showed that interaction effect of Polyvinylpyrrolidone and genotypes. Activated charcoal and genotypes on percentage of survived and dead explants due to phenolic oxidation were highly significant at (P=0.001). Murashige and Skoog medium supplemented with 0.2 gL-1 and 0.3 gL-1 of Polyvinylpyrrolidone has gave 100% and 80% survived explants of C86-56 and C86-12genotypes respectively while 0.4 gL-1 and 0.3 gLl-1of activated charcoal resulted in 46% and 40% of survived explants of C86-56 and C86-12 genotypes respectively, after 30 days of culturing. Thus, these optimized concentrations of Polyvinylpyrrolidone and activated charcoal are useful to control the effect of phenolic oxidation on culture establishment of micro propagation, which helps to have enough starter culture for further multiplication stage of micro propagation.

Commercial Scale Micropropagation of Sugarcane: Constraints and Remedies

Tissue culture techniques are now becoming important tools of crop improvement and rapid multiplication of selected clones. There has been considerable interest in applying these techniques in breeding, propagation, disease elimination, rejuvenation of older varieties, development of clones suitable for abiotic and biotic stresses, conservation of genetic resources etc. In vitro micropropagation is now drawing special attention for multiplication of new varieties on large scale in comparatively shorter period of time in several crops including sugarcane. Despite several advantages of applying micropropagation technique in sugarcane such as quick multiplication of newly released varieties; rejuvenation of old deteriorated varieties; production of disease free seed; easy transportation of seed material; elimination of viruses; high cane productivity and sugar yield etc., this technique is not gaining popularity up to the desired extent. There are several constraints like microbial contamination, phenolics secretion from cultured explants, shoot vitrification, thin canes in tissue culture raised crops, high cost of production and appearance of some variants in micropropagated population. The present article describes some of the problems encountered during in vitro micropropagation of sugarcane on commercial scale. Though some problems have now been resolved to considerable extents which have been described in this review. However, some constraints still require intensive research work to be resolved so that a safe and efficient exploitation of this technique can be ensured in sugarcane seed production programme.

High throughput in vitro micropropagation of sugarcane (Saccharum officinarum L.) from spindle leaf roll segments: Cost analysis for agri-business industry

High throughput micropropagation (HTM) protocol in sugarcane through direct shoot regeneration comprising five stages, was developed for agri-business industry. The distinction of the protocol lies in direct adventitious shoot regeneration without an intervening callus phase and liberty over the number of subculture passages leading to high rates of synchronous plant production. Stage 0 dealt with selection and maintenance of field-grown stock plants; Stage I marked the initiation of in vitro propagation on culture initiation medium using spindle leaf roll segments from apical tops; Stage II involved five passages (of 14 days each) on shoot multiplication medium followed by one additional passage (of 10 days) on shoot multiplication-cum-elongation medium. The shoot multiplication rates ranged from 4 to 25-fold in CoPb 91 and CoJ 83 cultivars, respectively; Stage III involved in vitro rooting on root induction medium and hardening for 14 days at each step; Stage IV comprised separation of clumps into single plantlets and transfer to soil. The complete plants were produced in 157 days with 97 percent survival rate. The fidelity of the protocol for agri-business industry was tested by producing approximately 0.1 million saleable HTM sugarcane plants in a small scale (150 m2) tissue culture unit. The recurring (energy, labour, media, culture container, transplanting material) and non-recurring (laboratory infrastructure, glasshouse, equipment) parameters accounted for 75.56 and 24.44 percent production costs, respectively. The low-cost options for media, culture containers and transplanting material were incorporated and cost per plant was calculated to be US$ 0.13. The HTM protocol was adopted by two sugar mills, and the HTM plants produced in our tissue culture unit were sold for seed multiplication to eight sugar mills and 430 farmers throughout North India.

Screening of Some Chemical Disinfectants for Media Sterilization During In Vitro Micropropagation of Sugarcane

Three antimicrobial compounds viz. mercuric chloride (HgCl2), sodium hypochlorite (NaClO) and bleaching powder, (CaOCl2) were tested at various concentrations to find out an effective chemical agent for sterilization of nutrient media for in vitro micropropagation of sugarcane. The best result was obtained in presence of NaClO at 0.1 % concentration where maximum 96 % shoot cultures were found contamination free, grew normally and were comparable to the cultures grown on autoclaved nutrient medium (control). Mercuric chloride (0.1 %), though prevented microbial contamination in about 85 % cultures, the shoots remained green for 4–7 days only and thereafter gradually turned brownish-yellow, showed rotting appearance and ultimately approximately half of the sterile shoots died within next one week. No microbial contamination was observed at higher concentrations (0.15 and 0.2 %) of HgCl2 and NaClO both, however the survival and growth of shoots were affected adversely. None of the chemicals was able to prevent microbial contamination up to the desired extent at concentrations below 0.1 %. Shoot cultures could be micropropagated successfully up to 10 subcultures (cycles) on medium supplemented with 0.1 % of NaClO, without any adverse effect on shoot growth. Addition of NaClO in rooting medium at a concentration of 0.1 % was also found equally effective. The results suggested that chemical sterilization of media with sodium hypochlorite, can be a cost effective option of autoclaving during micropropagation of sugarcane at commercial scale.

Micropropagation of sugarcane (Saccharum spp. hybrids) in vitro: A comparison between semi-solid and liquid RITA® temporary immersion culture systems with respect to plant production and genotypic and phenotypic fidelity

Micropropagation of sugarcane (Saccharum spp. hybrids) in vitro: A comparison between semi-solid and liquid RITA® temporary immersion culture systems with respect to plant production and genotypic and phenotypic fidelity

Development of a temporary immersion system (RITA®) for mass production of sugarcane (Saccharum spp. interspecific hybrids)

Commercial micropropagation of sugarcane is largely determined by the clonal fidelity and the cost of plants produced. Rapid production of plants in vitro reduces the frequency of offtypes in many species. By exploiting the concept of transverse thin cell layer culture, we have developed a rapid, high frequency direct plant regeneration system, called SmartSett®, for commercial sugarcane cultivars grown in Australia. Similar to conventional micropropagation, labour remains the major cost of this plant production system. Hence, to reduce the labour component, we have integrated the SmartSett® system with the RITA® temporary immersion bioreactor. Thin transverse leaf sections or fragmented leaves cultured on agar-based SmartSett® shoot induction medium were used as the starting material for RITA®. Shoot initiation on semi-solid medium prior to transferring to RITA®, culture immersion frequency, explant size and genotype determined the productivity (number of plants produced per unit culture) of the system. Results obtained with cultivar Q165 indicate that explants cultured for 45 d on SmartSett® shoot induction medium were the most prolific, producing on average 275 shoots per vessel after 45 d of culture in RITA with 1 min immersion every 12 or 24 h. Using the fragmented tissue, 14-d-old explants and 3-mm leaf tissue fragments were the most productive. Experiments with three cultivars (Q117, Q165 and Q205) showed that RITA® culture conditions need to be optimised for each cultivar for maximum plant production.

Use of the temporary immersion RITA® bioreactor system for micropropagation of sugarcane

Use of the temporary immersion RITA® bioreactor system for micropropagation of sugarcane

EFFECT OF ATRAZINE ON ALGAL CONTAMINATION AND SUGARCANE SHOOTS DURING PHOTOAUTOTROPHIC MICROPROPAGATION

A laboratory procedure was developed for obtaining and maintaining photoautotrophic cultures of sugarcaneshoots in vitro for three generations. Algae contamination in vessels was a problem for photoautotrophic growth under septicconditions. Atrazine, a herbicide, was effective in controlling algae at a concentration of 10 ppm, but the growth of shootsin herbicidetreated sugarless Murashige and Skoog (MS) medium decreased to about half of the growth in untreated medium.As a result, atrazine was not considered to be a practical solution for algae control. Additional work is needed to optimizephotoautotrophic micropropagation of sugarcane for both septic and aseptic conditions. Photoautotrophic micropropagationof sugarcane may be economical only if culture can be perfected under septic conditions.

An efficient protocol for micropropagation of sugarcane using shoot tip explants

An efficient protocol for micropropagation of sugarcane from shoot tip explants has been described. Shoot regeneration from inoculated shoot tip explants (40–60%) was obtained on MS medium containing BAP, Kinetin and GA3 (0.5 mg/l each). Numerous shoots were developed from the established explants on MS liquid medium + BAP + Kinetin (0.5 mg/l each). Profuge rooting of shoots was achieved on 1/2 strength MS salts + NAA (5.0 mg/l) + sucrose (60g/l) within 30–40 days. After hardening, the plantlets were field transplanted at 60 cm distances in rows 75 cm apart. The plants showed uniform growth and asynchronous tillering within 60 days of transplantation.

Effects of Genotype andin VitroMedia Composition on Micropropagation of Sugar Cane

Effects of Genotype and<i>in Vitro</i>Media Composition on Micropropagation of Sugar Cane

Commercial Plant Tissue Culture in India—Current Status

In recent years, there has been an explosion in the number of commercial plant tissue culture (TC) units in India. More than 25 such companies have production capacity of two to five million plants per annum. Almost all units are export oriented, but the target crops are the same. Indoor foliage plants dominate the export market. Micropropagation industry in India is providing major support to Indian agriculture in four crop groups: Fruits, ornamentals, spices, forestry/plantation crops. Banana is the largest selling TC fruit crop. TC papaya plants are now marketed for extraction and processing of papain. TC anthuriums, orchids, and gerberas have attained commercial importance. TC rose plants are used as pot plants. Nearly 500 ha are under TC cardamom cultivation in southern India recording 20% to 30% increase in yield. Vanilla cultivation is expected to increase from the existing 50 ha to more than 400 ha in the coming years using TC plants. Sugar companies have in-house units for micropropagation of sugarcane. There is demand for bamboo and eucalyptus for selective reforestation. The TC Industry is constrained by the non-availability of international varieties, high infrastructure and electricity costs, and lack of managers with commercial experience. A shake-up is imperative, during which many of the existing TC units may not survive the year 2000.