Improved Anther Culture Media for Enhanced Callus Formation and Plant Regeneration in Rice (Oryza sativa L.)

Anther culture technique is the most viable and efficient method of producing homozygous doubled haploid plants within a short period. However, the practical application of this technology in rice improvement is still limited by various factors that influence culture efficiency. The present study was conducted to determine the effects of two improved anther culture media, Ali-1 (A1) and Ali-2 (A2), a modified N6 medium, to enhance the callus formation and plant regeneration of japonica, indica, and hybrids of indica and japonica cross. The current study demonstrated that genotype and media had a significant impact (p < 0.001) on both callus induction frequency and green plantlet regeneration efficiency. The use of the A1 and A2 medium significantly enhanced callus induction frequency of japonica rice type, Nipponbare, and the hybrids of indica × japonica cross (CXY6, CXY24, and Y2) but not the indica rice type, NSIC Rc480. However, the A1 medium is found superior to the N6 medium as it significantly improved the green plantlet regeneration efficiency of CXY6, CXY24, and Y2 by almost 36%, 118%, and 277%, respectively. Furthermore, it substantially reduced the albino plantlet regeneration of the induced callus in two hybrids (CXY6 and Y2). Therefore, the improved anther culture medium A1 can produce doubled haploid rice plants for indica × japonica, which can be useful in different breeding programs that will enable the speedy development of rice varieties for resource-poor farmers.

Peach palm plantlet growth in different culture media in a temporary immersion system

ABSTRACT: Peach palm is a domesticated palm commercially important for the production of fruits and hearts of palm. Somatic embryogenesis, an effective technique for mass propagation, was successfully established for this species. Furthermore, a temporary immersion system improved plant regeneration. However, production can be further improved by understanding the peach palm’s growth dynamic and modifications of culture media. The aims of this study were to evaluate the growth of plantlets cultured in different culture media in a temporary immersion system and to correlate the results with nutrient uptake during the growth period. Somatic embryo-derived young plantlets approximately 1 cm in length were cultivated for 12 weeks in a twin flask system containing MS, Y3 or N6 salts, Morel and Wetmore vitamins and 3% sucrose, with a monthly medium refreshment. Growth was measured and mineral analysis of the plantlets was carried out after 12 weeks of culture. The Y3 and MS salts were the most appropriate for the plant growth. Number of roots was 52.52% higher and the root size was 40.42% between the N6 and MS medium and the root number in Y3 medium was 37.74% greater than in MS medium, which is important for post acclimatization survival. K and Na are important elements for peach palm. N is not required at such a high concentration as in Murashige and Skoog formulation. The Chu (N6) medium did not generate high quality plantlets, possibly due to the absence of some micronutrients, like Mo, Cu and Co.

Optimization of Conditions for Callus Induction from BC2F1 Population (Ranbir Basmati x PAU148) through Anther Culture

With different culture conditions and concentrations of growth regulators and supplements, Anther culture technique can be easily employed for the production of haploids under in vitro conditions. Aims: The present study was undertaken with the objective to optimize the development of doubled haploids using anthers for in vitro induction of callus on N6 medium. Place and Duration of Study: The samples (BC2F1 seeds) were raised previously in Skuast-J. From total degree program of 3 years, this work related to tissue culture technique was done in one year from January 2018 to January 2019. Methodology: The effect of levels of 2, 4-dichlorophenoxy acetic acid (2, 4-D) i.e. 0 to 3 mg/L in basal N6 media was observed on callus induction frequency (CIF). The effect of duration of cold pre-treatment was observed on callus induction frequency at 2.5 mg/L of 2, 4-D by giving the cold pre-treatment at 4ºC from 8 to 12 days. Also the effect of different amino acids was checked on callus induction frequency. Results: Highest callus induction frequency of 9.39 per cent was observed in N6 medium fortified with 2.5 mg/L 2, 4-D and lowest callus induction frequency of 2.52 per cent at the concentration of 1.0 mg/L. The cold pre-treatment for 10 days gave highest callus induction frequency of 1.44 per cent and lowest callus induction frequency of 0.44 per cent was obtained at cold pre treatment for 8 days. The highest callus induction frequency of 12.55 per cent was observed in case of media supplemented with 25 mg/L tryptophan and 40 mg/L cysteine and lowest callus induction frequency of 7.18 per cent was observed when media was supplemented with 560 mg/L proline. Conclusion: The cold pre-treatment of 10 days at 4ºC on media supplemented with 2.5 mg/L of 2, 4-D and combination of 25 mg/L tryptophan and 40 mg/L cysteine proves to provide best androgenesis conditions for anthers from BC2F1 population.

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM AN ELITE TROPICAL MAIZE INBRED LINE

ABSTRACT – The improvement of tropical maize inbred lines by genetic transformation techniques remains a difficulttask since not all genotypes are capable of regenerating efficiently in vitro. The objective of this study was to evaluatethree different callus induction media, based on N6 or MS salts containing either 2,4-D (0, 2.5, 5.0, 10.0, 15.0, 30.0mg .L-1) or Dicamba (0; 0.25; 0.5; 1.0; 2.0; 4.0 mg.l-1) in the production of embryogenic callus from immature zygoticembryos of the tropical maize inbred line L3. Callus maturation was tested in MS medium containing 60 g.L-1 sucroseand supplemented with different combinations of BAP (0; 0.1; 0.5; 1.0 mg.L-1), NAA (0; 1.0 mg.L-1) and CuSO4 (0;1.25 mg.L-1). The L3 inbred line presented higher capacity for Type II callus formation on N6 medium content 10mg.L-1 2,4-D. For the maturation of callus, absence of plant growth regulators and addition of CuSO4 allowed higherpercentage of regeneration. The protocol developed presented 85% production of Type II embryogenic callus and 45%plant regeneration.Keywords: 2,4-D, dicamba, embryogenic callus, Zea mays.EMBRIOGÊNESE SOMÁTICA E REGENERAÇÃO DE PLANTAS A PARTIR DE UMA LINHAGEM DE MILHO TROPICAL ELITERESUMO – O melhoramento de linhagens de milho tropical através de técnicas de transformação genética continua aser uma tarefa difícil uma vez que nem todos os genótipos são capazes de regenerar eficientemente in vitro. O objetivodeste estudo foi avaliar três meios diferentes para a indução de calos embriogênicos, baseados em N6 ou MS saiscontendo 2,4-D (0; 2,5; 5,0; 10,0; 15,0; 30,0 mg.L-1) ou Dicamba (0; 0,25 ; 0,5; 1,0; 2,0; 4,0 mg.l-1) na produção decalos embriogênicos a partir de embriões zigóticos imaturos da linhagem de milho tropical elite L3. A maturação doscalos foi testada em meio MS com 60 g.L-1 de sacarose suplementado com diferentes combinações de BAP (0; 0,1; 0,5;1,0 mg.L-1), ANA (0; 1,0 mg.L-1) e CuSO4 (0; 1,25 mg L-1). A linhagem L3 apresentou alta capacidade para produçãode calos do Tipo II em meio N6 contendo 10 mg.L-1 de 2,4-D. Para a maturação dos calos, ausência de reguladoresde crescimento vegetal e adição de CuSO4 possibilitou maior porcentagem de regeneração. O protocolo desenvolvidoapresenta produção de 85% de calos embriogênicos do Tipo II e 45% de regeneração de plantas.Palavras-chave: 2,4-D, dicamba, calos embriogênicos, Zea mays.

Kultur Antera untuk Mendapatkan Galur Padi Toleran Salinitas

<p>ABSTRACT<br /><br />Haploid breeding through anther culture allows shortening of the breeding cycle and production of homozygous lines from a segregating population in the immediate generation. This technique has been used for crop improvement especially in rice. The objective of this research was to determine regeneration ability of twelve F1s, derived from reciprocal crossing between high yielding rice variety and rice tolerance to salinity, through anther culture. Completely randomized design with 20 replications was used in this research. Medium for callus induction was based on N6 medium + 2.0 mg NAA L-1 + 0.5 mg kinetin L-1 + 1 mM putrescine, while regeneration medium was based on MS + 0.5 mg NAA L-1 + 2.0 mg kinetin L-1 + 1 mM putrescine. Rooting were done in MS medium + 0.5 mg IBA L-1 + 1 mM putrescine. The result indicated that F1 derived from IR77674/Inpari 29 (3.1% green plants/total anther) was the most responsive genotypes in rice anther culture (high anther culture ability). After greenhouse grow out 125 putative double haploid plants were obtained (41.5% from total acclimated green plantlets). <br /><br />Keywords: double haploid, green planlets, indica rice, salt tolerance <br /><br /></p>

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM AN ELITE TROPICAL MAIZE INBRED LINE

ABSTRACT – The improvement of tropical maize inbred lines by genetic transformation techniques remains a difficult task since not all genotypes are capable of regenerating efficiently in vitro. The objective of this study was to evaluate three different callus induction media, based on N6 or MS salts containing either 2,4-D (0, 2.5, 5.0, 10.0, 15.0, 30.0 mg .L-1) or Dicamba (0; 0.25; 0.5; 1.0; 2.0; 4.0 mg.l-1) in the production of embryogenic callus from immature zygotic embryos of the tropical maize inbred line L3. Callus maturation was tested in MS medium containing 60 g.L-1 sucrose and supplemented with different combinations of BAP (0; 0.1; 0.5; 1.0 mg.L-1), NAA (0; 1.0 mg.L-1) and CuSO4 (0; 1.25 mg.L-1). The L3 inbred line presented higher capacity for Type II callus formation on N6 medium content 10 mg.L-1 2,4-D. For the maturation of callus, absence of plant growth regulators and addition of CuSO4 allowed higher percentage of regeneration. The protocol developed presented 85% production of Type II embryogenic callus and 45% plant regeneration.Keywords: 2,4-D, dicamba, embryogenic callus, Zea mays.EMBRIOGÊNESE SOMÁTICA E REGENERAÇÃO DE PLANTAS A PARTIR DE UMA LINHAGEM DE MILHO TROPICAL ELITERESUMO – O melhoramento de linhagens de milho tropical através de técnicas de transformação genética continua a ser uma tarefa difícil uma vez que nem todos os genótipos são capazes de regenerar eficientemente in vitro. O objetivo deste estudo foi avaliar três meios diferentes para a indução de calos embriogênicos, baseados em N6 ou MS sais contendo 2,4-D (0; 2,5; 5,0; 10,0; 15,0; 30,0 mg.L-1) ou Dicamba (0; 0,25 ; 0,5; 1,0; 2,0; 4,0 mg.l-1) na produção de calos embriogênicos a partir de embriões zigóticos imaturos da linhagem de milho tropical elite L3. A maturação dos calos foi testada em meio MS com 60 g.L-1 de sacarose suplementado com diferentes combinações de BAP (0; 0,1; 0,5; 1,0 mg.L-1), ANA (0; 1,0 mg.L-1) e CuSO4 (0; 1,25 mg L-1). A linhagem L3 apresentou alta capacidade para produção de calos do Tipo II em meio N6 contendo 10 mg.L-1 de 2,4-D. Para a maturação dos calos, ausência de reguladores de crescimento vegetal e adição de CuSO4 possibilitou maior porcentagem de regeneração. O protocolo desenvolvido apresenta produção de 85% de calos embriogênicos do Tipo II e 45% de regeneração de plantas.Palavras-chave: 2,4-D, dicamba, calos embriogênicos, Zea mays

Regenerasi dan Aklimatisasi Kultur Antera Enam Persilangan F1 Padi Sawah

ABSTRACT<br /><br />The breeding of rainfed rice tolerant to drought can be accomplished using anther culture. The objectives of this research were to determine regeneration abilities of six F1 anther culture and its acclimatization ability. The experiment was arranged in completely randomized design with 14 replications. The treatments consisted of six F1 derived from crossing: INPARI 18 x IR83140-B-11-B (G1), INPARI 18 x B12825E-TB-1-25 (G2), INPARI 18 x IR87705-14-11-B-SKI-12 (G3), INPARI 22 x IR83140-B-11-B (G4), Bio-R81 x O18b-1 (G5), Bio-R82-2 x O18b-1 (G6). Media for callus induction was based on N6 medium + 2.0 mg L-1 NAA + 0.5 mg L-1 kinetin + 1.0 mM putresin + 60 g L-1 sucrosa, media for regeneration was based on MS + 0.5 mg L-1 NAA + 2.0 mg L-1 kinetin + 1.0 mM putresin, and media for rooting was based on MS + 0.5 mg L-1 IBA + 30 g L-1 sucrosa. The result indicated that all six F1 had different ability in anther culture. Bio-R82-2 x O18-b1 (G6) and Bio-R81 x O18-b1 (G5) F1 genotype had good response both of callus induction and plant regeneration. These two F1 genotypes also gave the highest ratio of green planlet production to number of anther inoculated (GP:AI) were 5.50% and 4.65%, respectively. In this research, there were identified doubled haploid plants were developed from 4 F1 derived cross namely G2 (2 plants), G3 (4 plants), G5 (21 plants), and G6 (26 plants).<br /><br />Keywords: Callus induction, doubled haploid, rice<br /><br />

Plant regeneration through callus initiation from mature and immature embryos of maize (Zea mays L.)

An efficient regeneration was developed using mature and immature embryos by using Maize (Zea mays L) variety MU 2092. Mature embryos are removed from surface sterilized seeds, slice them into halves and immature embryos are detached from seed endosperm. Both are used as explants to initiate callus on N6 medium supplemented with 2,4 D @ 4.0 mg.L-1. The induction frequency of primary calli i.e embryogenic callus was 90% in maize. The embryogenic calli on N6 medium supplemented with 6-benzylaminopurine (BAP) @ 0.5 mg.L-1 and kinetin @ 0.5 mg.L-1 was more effective in producing shoots. The culture expressed maximum plant regeneration potential with eight shoots per embryo on regeneration. Green shoots thus developed were successfully rooted within 20 days on MS media containing IBA (Indole-3-Butyric acid) 1mg L-1. Over 86 % of rooted plants grew well and produced seeds normally when transferred to green house. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for mature and immature embryos.

In vitro germination of zygotic embryos of hybrid BRS Manicoré (E. guineensis X E. oleifera)

ABSTRACT The interspecific oil palm hybrid BRS Manicoré (E. guineensis x E. oleifera) has superior agronomic characteristics. However, the germination rate is low (30%) and the process is slow when the seeds are sown in a conventional form. The purpose of this study was to optimize the in vitro germination of zygotic embryos of this hybrid comparing seed lots. The viability of zygotic embryos was evaluated by the tetrazolium test (0.075%) for 4 h. The embryos were cultured on MS and Y3 culture media, with and without the addition of NaH2PO4, as well as on MS, MS1/2 and N6 medium. In MS medium containing NaH2PO4, the germination rate was increased from 40 to 70% in comparison with the medium without sodium phosphate. The comparison between the culture media MS, MS 1/2, N6 and Y3 showed that 75% of zygotic embryos cultured in the Y3 medium formed whole plants (with roots and shoots defined), a higher percentage than embryos cultured on MS, MS 1/2 and N6 media (46, 35 and 17% respectively). In the same Y3 culture medium, the embryos were larger (36% ≥ 2 cm and 30% ≥ 5 cm) than in the other media. Results obtained by the tetrazolium test were similar to those of germination, showing the effect of the genotype of each seed lot. For the germination and development of plantlets it is essential to add NaH2PO4 to a culture medium containing no phosphate or with a low phosphate concentration.