An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.) (original) (raw)
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Efficient Plant Regeneration using Mature and Immature Embryos of Maize (Zea mays L.)
Abstract – Under this study an efficient protocol on callus induction (CI) and plant regeneration (PR) was developed using immature (IE) and mature embryos (ME) of four maize varieties viz. Barnali, Mohar, Khoi bhutta and Shuvra. For callus induction three basal media viz. MS, N6 and 6N1 were used where different concentration of 2,4-D (0.5, 1.0, 1.5, 2.0 mg/l) were used. Among the tested varieties, highest 75.28 and 77.16% callus were recorded for Mohar in MS supplemented with 1.5 mg/l BAP using immature and mature embryos respectively. The lowest value (25.48%) was recorded for Khoi bhutta on 6N1 + 0.5 2,4-D in ME. For PR, 3-4 weeks old calli were transferred to regeneration media (MS, N6 and 6N1) with BAP (0.5, 1.0, 1.5, 2.0 mg/l) and/or IAA (0.0, 0.5, 1.0, 1.5 mg/l). The maximum regeneration (77.44%) was found in MS that supplemented with 1.0 BAP and 0.5 IAA for Shuvra by IE and from ME, Mohar showed 76.09% regeneration. In the same medium combination, the highest elongated shoot (20.47cm) was recorded for Shuvra in IE; and from ME the value was 22.45 cm for Mohar. The variety Mohar also exhibited the maximum rooting (82.84%) in MS supplemented with 1.0 BAP and 0.5 IAA when it was regenerated from ME derived calli. Analysis of variance (ANOVA) showed significant differences among genotypes, media and hormonal combinations for callus induction and regeneration at P<0.01.
Indian Journal of Genetics and Plant Breeding (The), 2015
Thirteen subtropical maize genotypes were evaluated for in vitro callus induction and whole plant regeneration. Seeds were germinated on MS-based medium supplemented with 10 mg l-1 picloram and 3.0 mg l-1 BAP which induced the formation of nodes. The developed nodes were excised about 0.25 cm above and below and were split longitudinally into two halves. The split nodes were placed on the callusing medium supplemented with 2.2 mg l-1 picloram and 0.5 mg l-1 2, 4-D, the cut surface facing down and touching the media. Among the thirteen genotypes, embryogenic calli were induced in seven genotypes ranging between 10.78-65.65%. Out of seven genotypes, only one genotype i.e., VQL 2 regenerated in to a whole plant with a frequency of 34% on MS basal medium under 80 µM m-2 s-1 of light intensity. Regeneration medium and light intensity played key role for the initiation of somatic embryogenesis and regeneration of whole plants. Survival efficiency of in vitro regenerated plantlets was found to be 95% out of which 85% of the plants grew normally without any morphological abnormality.
Journal of Genetic Engineering and Biotechnology, 2016
Maize is one of the important cereal crops around the world. An efficient and reproducible regeneration protocol via direct organogenesis has been established using split nodes as ex-plants derived from 7 to 10 day old in vitro grown seedlings. Surface sterilized maize seeds were germinated on MS medium lacking plant growth regulators. Nodal sections of 7-10 day old seedlings were isolated, split longitudinally into two halves and cultured on regeneration medium containing different concentrations of 6-benzyladenine (2.20, 4.40, 6.60, 8.80, 11.0 and 13.2 lM) or kinetin (2.32, 4.65, 6.97, 9.29, 11.6 and 13.9 lM). Inclusion of 8.80 lM BA into MS supplemented medium triggered a high frequency of regeneration response from split node explants with a maximum number of shoots (12.0 ± 1.15) and the highest shoot length (3.0 ± 0.73) was obtained directly (without an intervening callus phase) within 4 weeks of culture. Further shoot elongation was achieved on medium containing 4.40 lM BA. The elongated micro shoots were rooted on MS medium fortified with 1.97 lM indole-3-butyric acid. The regenerated plantlets with roots were successfully hardened on earthen pots after proper acclimatization under greenhouse conditions. This new efficient regeneration method provides a solid foundation for genetic manipulation of maize for biotic and abiotic stresses and to enhance the nutritional values.
Immature embryo-derived callus is more efficient for plant regeneration in maize but appears difficult to obtain in all seasons of the year compared to mature embryos from dry seeds which are readily available throughout the year. This study investigated the effect of seed size on in vitro seed germination, seedling growth, callus induction and plantlet regeneration, as well as the relationships between these parameters in five maize varieties. Seeds were designated either as large or small for each variety based on its 100-seed weights, while seed germination were obtained in petri-dishes placed between two sheets of pre-wetted filter paper. Seeds were disinfected, and mature embryos were excised from the maize endosperm and inoculated on the Murashige and Skoog salt (MS medium) supplemented with 30 g/l sucrose, 8 g/l agar, 0.1 g/l myo-inositol and 3 mg/l 2,4-D for callus induction, while embryogenic calli were transferred to medium containing 0.5 mg/l Benzylaminopurine (BAP) and 0.5 mg/l Kinetin for plant regeneration. The study showed that large seed size had significant effect on almost all the traits studied, while positive and significant correlations were observed between in vitro germination, seedling growth, callus induction and plantlet regen-eration. It can be concluded that callus fresh weight may be used as a marker for improving regeneration efficiency in maize. The results from this study suggest that genetic control of in vitro regeneration from maize mature embryo can be utilized to determine inherent genotypic potentials of maize varieties with tissue culture traits for maize improvement.
Direct in vitro multiple shoot regeneration in Maize (Zea mays) inbred lines
In vitro regeneration in maize, 2017
In vitro regeneration system of six maize inbred lines (EH1, EH2, EH3, EH6, EH8, EH9) for direct shoot multiplication was established using 1st collar node as explants of one-week-old in-vitro plantlets. The Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) in combination with Indole acetic acid (IAA) and GA3 were used. Results showed that the addition of BAP, IAA, and GA3 greatly enhanced the formation of multiple shoots in the tested lines. Significant differences were observed between the concentration of growth hormone and inbred lines. Among the six treated inbred lines, EH6 performed best for direct multiple shoot regeneration. Maximum number of shoots (11 shoots per explant) was observed on the medium containing BAP (0.7 mg/l) in combination with IAA (0.2mg/l) and GA3 (0.5 mg/l). Regenerated shoots were rooted in MS supplemented with 2 mg/l NAA. The rooted in vitro raised plantlets were acclimatized and successfully transferred to the greenhouse. The regeneration protocol developed in this study can play an important role in the mass propagation of elite genotypes of maize after successful transformation of the desired gene.
Regeneration of Plant through Embryo Culture from Promising Maize (Zea mays L.) Inbred Lines
Acta Scientific Agriculture, 2019
four different concentrations and combinations of hormones viz. 1.0 mgl-1 2,4-D (2,4-Dichlorophenoxyacetic Acid), 1.0 mgl-1 BAP (6-Benzyl Amino Purine), 1.0 mgl-1 Kinetin and BAP (1.0 mgl-1)+ Kinetin (1.0 mgl-1) supplemented with N6 (Chu) medium were used for embryogenic callus formation and plantlet regeneration. The inbred lines ML15, ML22 and ML9 revealed 100% direct somatic embryogenesis (germination) under treatments BAP (1.0 mgl-1) + Kinetin (1.0 mgl-1), Kinetin (1.0 mgl-1) and control treatments, respectively. The root length among the regenerated plantlet ranged from 0.66 to 4.83cm. Here, the longest root length was recorded in inbred line ML10 (4.83 cm) under 2,4-D (1.0 mgl-1) treatment. The shoot length was ranged from 1.40 to 5.43 cm. The inbred line ML-15 of BAP (1.0 mgl-1) treatment exhibited maximum shoot length (5.43cm) followed by ML15 of treatment BAP (1.0 mgl-1) + Kinetin (1.0 mgl-1).After hardening, the highest establishment rate was observed on ML10 as 100% under all treatment derived plants. And the plantlet derived from BAP and Kinetin combinations produced the maximum establishment rate (100%) for all the genotypes. The maximum root length was observed from 2,4-D regenerated inbred line ML15 (5.8cm) followed by the same genotype (5.5cm) from BAP and Kinetin combinations. The plantlet derived from BAP and Kinetin revealed maximum shoot length by genotype ML9 (9.2cm). Overall, in pot conditions, the superior shoot length performances were recorded in inbred regenerated plants from BAP + Kinetin and BAP treatments. Among the six inbred line of maize, ML-10 of treatment 2, 4-D and ML-22 of treatment BAP were successfully established in soil with the highest survival rate (100%). Again, the correlation studies revealed significant positive relationship between root length and shoot length. Considering the seedling vigour index, the genotype ML10 and ML 28 revealed superior index value in most of the treatments. Overall, the inbred lines ML10 and ML28 identified as superior genotypes for plant regeneration and the treatment combination BAP (1.0 mgl-1) +Kinetin (1.0 mgl-1) were identical for immature embryos generation. Furthermore, this novel protocol might play a significant role in overcoming further interspecific and intrageneric maize hybrids. Maize (Zea mays L.) is a member of the Gramineae or grass family. It is a monocotyledonous plant with a diploid chromosome number of 2n = 2x = 20 [1]. It is a particularly convenient plant for genetic studies because it can readily be either self or cross-pollinated with large numbers of seeds per plant. Globally it is top ranking cereal in worldwide significance as human food, animal feed and fodder as well as source of large number of industrial products like corn starch and starch-based products, and fermentation and distillation industries [2]. Conventional breeding method is insufficient to satisfy the goal as it demands more land, labor and capital. It also takes a long time, seed dormancy, early abortion of embryo and also infection of several diseases occur [3,4]. Usually it is a lengthy process but embryo culture can shorten the breeding cycle by overcoming dormancy in seeds. All maize tissue culture and transformation involves the use of immature zygotic embryos as an explants source for regeneration [5]. However, immature embryos are seasonally available and have strictly limited suitable duration of culture, 14-19 DAP [6]. During the past decades, many types of media have been developed for in vitro plant culture [7,8] with different formulations for the specific plants and tissues[9]. The most commonly used media for potato maize embryo culture is N6 basal media [10]. In culture media plant growth regulators generally play a vital role in embryo culture. Exogenous auxins seems to be required for plant embryo growth in vitro [11]. Cytokinins, when used as the sole hormone, are effective or promote young embryo growth. However, they promote growth and differentiation of embryos when they are combined with auxins [12]. Many researchers have investigated different plant regeneration through mature or immature embryos using
Tissue culture dependent regeneration of maize (Zea mays L.)
2018
Maize (Zea mays L.) is the third most important cereal crop in the world. It is a major cereal crop for livestock feed, human nutrition and important raw material for several agro-based industries, there is great demand for maize of both quality and quantity. An efficient plant tissue culture procedure with high regeneration frequency is prerequisite for this approach. In light of the situation an experiment was conducted with an objective to optimize regeneration of plants using different explants of maize. The experiment was conducted using two hybrids (GS-802, 27P17) and one composite (BVM-2) for protocol development for in vitro regeneration of maize (Zea mays L.) using three different explants viz., shoots, endosperm, ovules. Shoots of all the genotypes were inoculated in MS media supplemented with 1mg/l BAP, 2mg/l BAP, 3mg/l BAP, 4mg/l BAP and 5mg/l BAP along with a control. Endosperm of all the genotypes were cultured in callusing media containing MS basal salts supplemented with 1mg/l 2, 4-D+1mg/l NAA and 0.2mg/l 2, 4-D+ 2mg/l NAA. Ovules were inoculated in the N6 medium. Better response of kernels regeneration was observed when treated with HgCl2for 10 minutes. The response was observed for shoot elongation with 2mg/l BAP in GS-802. Highest shoot survival was observed in GS-802 with 2mg/l BAP and 4mg/l BAP. In endosperm culture, callusing observed on MS medium with different concentration of 2, 4-D+NAA. Better response of GS-802 was recorded in the hormonal combination of 2, 4-D-0.5mg/l + NAA-2mg/l and for BVM-2 in the hormonal combination of 2, 4-D-1mg/l + NAA-1mg/l. In case of ovule culture 85.71% of sprouting was observed in GS-802 genotype followed by BVM-2 and 27P17 with 76.91% and 47.61% respectively.
Plant Science, 1986
Friable callus (type 2) was selected from three genotypes (A188, hybrid A188 x B73, and hybrid B73 x A188) of Zea mays L. The three genotypes of type 2 callus doubled in fresh weight after 1 week, and growth was better on N6 than on Murashige-Skoog (MS) medium. Type 2 callus of hybrid B73 x A188 was maintained in culture longer than A188 type 2 callus, and it regenerated higher numbers of plants than the other two genotypes. Type 2 callus of the hybrid B73 × A188 was used to establish cell suspensions. Suspension cells initially grew better on N6 than on MS medium, but after several months of subculture, cells in either N6 or MS medium grew at similar rates. Suspension cells were in mid-log phase by 5-7 days and in stationary phase by about 10 days depending on inoculum density. Growth rate was optimal when cells were transferred at mid-low phase and dry weight of the suspension cells increased at least 10-fold during a 10-day period. Suspension cells from 9-month-old cultures plated on solid medium regenerated plants at an efficiency similar to that of the friable type 2 callus but with more phenotypic abnormalities. Thus, cell suspensions derived from type 2 B73 x A188 callus, in culture for over 1 year, were capable of regenerating plants when 9-months old.
2009
An in vitro propagation protocol was established for the Dendrobium Serdang Beauty orchid. The propagation protocol utilized calli tissues that were successfully initiated from protocorm-like bodies (PLBs) explants, while the leaf and root tip explants died. The percentage of protocorm-like bodies explants responding to calli formation was 100% in all tested levels of IAA, IBA and NAA auxin treatments. The highest amount of calli (49.59 gram) proliferated on MS medium containing 1.5 mg/L IBA. These calli successfully regenerated on media supplemented with either KIN or BAP cytokinins and combined treatments of KIN and IAA (4 mg/L) or NAA (1.5 mg/L). However, media supplemented with only 1 mg/L KIN was sufficient to produce significantly high percentage of plantlet formation (80%), high number of planlets per explant (4-5 plantlets) and high mean fresh weight per plantlet (11.128 g). These plantlets were acclimatized on all tested media and obtained satisfactory rate of plantlet survival (80-100%), mean number of leaves per plant (4-6 leaves), and mean leaf length (4 -5 cm). Among these media, charcoal was considered the most economical and available material in the local market. During the development of this protocol, substantial necrosis of calli were observed when cultures were treated with 2,4-D and BAP. It was proposed that the presence of ethylene within the cultures, which is known to be emitted by plant growth regulators into the micro-climate of in vitro culture vessels, is the determining factor of a suitable plant growth regulator for the survival and growth of the Dendrobium Serdang Beauty calli cultures in our study.
Five elite tropical maize inbred lines; CML395, CML443, CML442, MAS [MSR/312]-117-2-2-1-B-5-B) and CML216 as a control, were evaluated for their regenerability making use of calli derived from immature zygotic embryos. Murashige and Skoog basal salts supplemented with 1.0, 1.5, 2.0 and 2.5 mg/L 2,4-dichlorophenoxyacetic acid were used to induce callus. Callus induction frequency and formation of embryogenic callus varied significantly (p<0.01) depending on genotype and level of 2,4-dichlorophenoxyacetic acid. Shoot regeneration efficiency also differed significantly (p<0.01) depending on genotype. Significantly (p<0.05) higher callus induction and frequency of embryogenic callus were obtained at 1 mg/L 2,4-dichlorophenoxyacetic acid, indicating this as the optimal level for regenerating these inbred lines. CML395 and CML442 revealed significantly (p<0.05) higher callus induction and embryogenic callus frequency compared to CML443 and MAS [MSR/312]-117-2-2-1-B-5-B), while they were at par with the control inbred line CML216. Plants were regenerated from all the inbred lines except CML443 and were successfully acclimatized and grown to maturity. CML395 was the best regenerable line with significantly (p<0.05) higher regeneration efficiency of 109.3%. It was concluded that CML395, CML216 and CML442 can be used in in vitro genetic transformation.