Improvement Of In Vitro Regeneration Of Brassica Oleracea Subsp. Italica Cv. Green Marvel Through Organogenesis

Broccoli (Brassica oleracea subsp. italica) cv. Green Marvel is an important vegetable crop grown in Cameron Highlands, Malaysia. Being an F1 hybrid crop, selfing will result in segregation in the F2 generation. In Malaysia, the production of hybrid seeds of cv. Green Marvel cannot be carried out due to the absence of parental stock material for hybridization. Therefore, the development of in vitro regeneration system is necessary to produce uniform and true-to-type plants of the cultivar. Currently, in vitro regeneration of other broccoli cultivars have been carried out but none has been reported on the cultivar Green Marvel. This study was carried out with the main objective of establishing a protocol for plant regeneration of broccoli cv. Green Marvel through multiple shoot formation from hypocotyl, shoot tip and cotyledon explants, followed by rooting and acclimatisation. All experiments were conducted and arranged in a Randomised Complete Block Design (RCBD). In the experiment on multiple shoot formation from hypocotyl explants, the concentrations of 6-benzylaminopurine (BAP) alone or in combination with α-naphthaleneacetic acid (NAA) or indol-3-butyric acid (IBA) were assessed. In treatments containing only BAP, 3 mg/l of the plant growth regulator produced the highest percentage of explant with shoots (96.7%) and the highest mean number of shoots per explant (6.03). In treatments containing BAP and NAA, the highest percentage of explant with shoots (93.3%) occurred on 1 and 3 mg/l BAP while the highest mean number of shoots (6.0) was obtained on 5 mg/l BAP. In treatments containing BAP and IBA, BAP alone enhanced shoot multiplication. The highest percentage of hypocotyl explant with shoots (100%) and highest mean number of shoots produced per explant (6.33) occurred on 3 mg/l BAP. Therefore, 3 mg/l BAP was considered the most suitable for adventitious shoot formation from hypocotyl explants of broccoli cv. Green Marvel. In the experiment on shoot multiplication from shoot tip explants, the concentrations of BAP either alone or in combination with NAA or IBA were assessed. The result showed that BAP at 5 mg/l produced the most number of shoots than any combinations of BAP with NAA or IBA. In treatments containing BAP and NAA, the highest percentage of explants with shoots (100%) and the highest mean number of shoots per explant (3.76) were on 5 mg/l BAP. In treatments containing BAP and IBA,, the highest percentage of shoot tip explants with shoots (96.7%) and the highest mean number of shoots produced per explant (2.83) also occurred on 5 mg/l BAP. BAP at 5 mg/l BAP was therefore the optimum hormonal treatment for shoot proliferation from shoot tip explants of broccoli cv. Green Marvel. In the experiment on multiple shoot formation from cotyledon explant using different combinations of BAP and NAA, the highest percentage of explant producing shoots (53.3%) and the highest mean number of shoots produced per explant (0.43) occurred on treatment combination consisting of 3 mg/l BAP and 1 mg/l NAA. The treatment combination consisting of 3 mg/l BAP and 1 mg/l NAA is therefore the most suitable for high frequency of shoot regeneration from cotyledon explant of broccoli cv. Green Marvel. In the experiment on rooting of shoots using different concentrations of auxins, the highest percentage of shoots with roots (100%) and the highest mean number of roots produced per explant (6.5) occurred on 0.2 mg/l IBA, but did not differ significantly compared to MSO medium. Meanwhile the highest mean length of root (2.46 cm) was attained on hormone-free MS medium (MSO). Thus, MSO medium was considered the most suitable medium for rooting of broccoli cv. Green Marvel shoots. In the acclimatisation study, various types of potting mixture were assessed. The highest percentage of survival (83.3%) occurred on medium containing sand+ soil (1:1) while the maximum root length (4.37 cm) and the highest plant height (7.87 cm) were attained in potting medium containing peatmoss+ perlite+ vermiculite (3:1:1). The mixture of peatmoss+ perlite+ vermiculite (3:1:1) was therefore considered the most suitable potting medium for broccoli cv. Green Marvel. Plantlets were well rooted and ready for field transfer after four weeks of acclimatisation. The study on multiple shoot formation concluded that BAP alone at 3 and 5 mg/l were more effective in inducing shoot proliferation on the hypocotyl and shoot tip explants of broccoli cv. Green Marvel, respectively, while BAP in combination with NAA was required for effective shoot formation from the cotyledon explants. The study on rooting showed that MSO was most suitable for high percentage of root formation, mean number of roots produced per explant and root length attained. In the acclimatisation study, the most suitable potting mixture consisted of peatmoss+ perlite+ vermiculite

Greenhouse evaluation on the performance of heat tolerant transgenic broccoli and genetic diversity analysis using inter simple sequence repeat (ISSR) markers

Background: Broccoli, Brassica oleracea subsp. italica is one of the many valuable Brassica species which is still less cultured under in vitro condition. Heat tolerant transgenic and non-transgenic broccoli cv. Green Marvel plantlets with well-developed root system obtained through in vitro culture were transferred into disposable plastic pots containing sterilized potting mixture consisting of (peatgroTM) + coconut dust (2:1) and maintained in a growth chamber. Results: After one month, the hardened plantlets were transferred and maintained in a transgenic greenhouse. After four months of acclimatization in the transgenic greenhouse, the efficacy of HSP101 gene in increasing the heat tolerance of the transgenic broccoli was evaluated. Results showed that the transgenic plants could survive and performed normally, producing flower heads even at the highest tested temperature of 34\ubaC. Seven transgenic broccoli lines with different gene copy number of the AtHSP101 gene as well as the control plant were assessed for genetic diversity using inter simple sequence repeat (ISSR) markers. Conclusions: ISSR results showed polymorphism and phylogenetic relationship between the transgenic and non-transgenic (control) Brassica oleracea cv. Green Marvel

Plant regeneration of Brassica oleracea sub sp. italica (Broccoli) CV Green Marvel as affected by plant growth regulators

Hypocotyls and shoot tips were used as explants in in vitro plant regeneration of broccoli (Brassica oleracea subsp. italica) cv. Green Marvel. The explants were excised from sterile germinated seedlings and placed on shoot induction medium containing basal salts of Murashige and Skoog (MS) and various concentrations of 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA). The highest percentage of hypocotyl explant producing shoot (96.67%) and the highest mean number of shoots produced per hypocotyl explant (6.03) were obtained on 3 mgL-1BAP. Meanwhile, the highest percentage of shoot tip explant producing shoot (100%) and highest number of shoot produced per shoot tip explant (3.76) were recorded on 5 mgL-1 BAP. For rooting of shoots, NAA, indoleacetic acid (IAA) and indolebutyric acid (IBA) at 0, 0.2 and 1 mgL-1 were applied. Highest percentage of shoots with roots (100%) and highest mean number of roots produced per shoot (6.5) occurred on medium with 0.2 mgL-1 IBA, while the maximum root length (2.46 cm) was attained on MS medium without plant growth regulator (MSO). Plantlets were successfully acclimatized in potting medium containing peatmoss, perlite, and vermiculite (3:1:1)

Greenhouse evaluation on the performance of heat tolerant transgenic broccoli and genetic diversity analysis using inter simple sequence repeat (ISSR) markers

Background: Broccoli, Brassica oleracea subsp. italica is one of the many valuable Brassica species which is still less cultured under in vitro condition. Heat tolerant transgenic and non-transgenic broccoli cv. Green Marvel plantlets with well-developed root system obtained through in vitro culture were transferred into disposable plastic pots containing sterilized potting mixture consisting of (peatgro™) + coconut dust (2:1) and maintained in a growth chamber. Results: After one month, the hardened plantlets were transferred and maintained in a transgenic greenhouse. After four months of acclimatization in the transgenic greenhouse, the efficacy of HSP101 gene in increasing the heat tolerance of the transgenic broccoli was evaluated. Results showed that the transgenic plants could survive and performed normally, producing flower heads even at the highest tested temperature of 34°C. Seven transgenic broccoli lines with different gene copy number of the AtHSP101 gene as well as the control plant were assessed for genetic diversity using inter simple sequence repeat (ISSR) markers. Conclusions: ISSR results showed polymorphism and phylogenetic relationship between the transgenic and non-transgenic (control) Brassica oleracea cv. Green Marvel

In vitro adventitious shoot regeneration from cotyledon explant of Brassica oleracea subsp. italica and Brassica oleracea subsp. capitata using TDZ and NAA

Broccoli(Brassica oleracea subsp. italica) cv. Green Dragon King and cabbage (Brassica oleracea subsp. capitata) cv. Gianty are important vegetable crops grown in Cameron Highlands, Malaysia. The cotyledons of both cultivars were used as explant source for in vitro shoot regeneration. The objective of this research was to examine the influence of the growth regulators thidiazuron (TDZ) and -naphthaleneacetic acid (NAA) on adventitious shoot formation in these cultivars. This system of adventitious shoot regeneration from cotyledon explants could be useful as a tool for genetic transformation of the subspecies. otyledon explants of both cultivars excised from 5-day-old in vitro germinated seedlings were placed on shoot induction medium containing basal salts of Murashige and Skoog (MS) and various concentrations of TDZ and NAA. The highest percentage of cotyledon explant of broccoli cv. Green Dragon King producing shoot (76.66%) and the highest mean number of shoots produced per explant (0.9) were obtained on 0.1 mg/l TDZ with 0.1 mg/l NAA. Meanwhile, the highest percentage of cotyledon explant of cabbage cv. Gianty producing shoots (86.67%) and highest numberof shoots produced per explant (1.1) were recorded on 0.5 mg/l TDZ with 0.1 mg/l NAA. Therefore, 0.1 mg/l TDZ with 0.1 mg/l NAA and 0.5 mg/l TDZ with 0.1mg/l NAA are the recommended combinations for adventitious shoot regeneration from cotyledonary explants of broccoli cv. Dragon King and cabbage cv. Gianty respectively

Molecular progress on the mapping and cloning of functional genes for blast disease in rice (Oryza sativa L.): current status and future considerations

Rice blast disease, which is caused by the fungal pathogen Magnaporthe oryzae, is a recurring problem in all rice-growing regions of the world. The use of resistance (R) genes in rice improvement breeding programmes has been considered to be one of the best options for crop protection and blast management. Alternatively, quantitative resistance conferred by quantitative trait loci (QTLs) is also a valuable resource for the improvement of rice disease resistance. In the past, intensive efforts have been made to identify major R-genes as well as QTLs for blast disease using molecular techniques. A review of bibliographic references shows over 100 blast resistance genes and a larger number of QTLs (∼500) that were mapped to the rice genome. Of the blast resistance genes, identified in different genotypes of rice, ∼22 have been cloned and characterized at the molecular level. In this review, we have summarized the reported rice blast resistance genes and QTLs for utilization in future molecular breeding programmes to introgress high-degree resistance or to pyramid R-genes in commercial cultivars that are susceptible to M. oryzae. The goal of this review is to provide an overview of the significant studies in order to update our understanding of the molecular progress on rice and M. oryzae. This information will assist rice breeders to improve the resistance to rice blast using marker-assisted selection which continues to be a priority for rice-breeding programmes

In vitro propagation and detection of somaclonal variation in Phalaenopsis gigantea as affected by chitosan and thidiazuron combinations

Protocorm-like bodies (PLBs) multiplication is one of the most preferable in vitro methods to increase the number of orchids that produce very few seeds or seeds that are not able to germinate. In the present study the effects of chitosan and thidiazuron (TDZ) combinations on multiplication, differentiation, and genetic stability of Phalaenopsis gigantea PLBs were investigated using different media. Initial PLBs were cultured in solid New Dogashima (ND) medium and Vacin and Went (VW) medium supplemented with different concentrations of chitosan (0, 5, 10, 15, 20, and 25 mg·L–1) and TDZ (0, 0.1, and 0.5 mg·L–1). The highest mean number of PLBs (353 PLBs) was observed in ND medium with 10 mg·L–1 chitosan and 0.1 mg·L–1 TDZ combination after 20 weeks of culture. Some PLBs differentiated into mature PLBs with a profusion of leaves on the apical region, and tiny plantlets started to develop after 10 weeks of culture. The highest mean number of shoots was observed in VW supplemented with 10 mg·L–1 chitosan and 0.5 mg·L–1 TDZ (16 shoots). Intersimple sequence repeat (ISSR) markers were used to determine the genetic fidelity among mother plant and PLBs obtained from each subculture stage of solid ND medium supplemented with 10 mg·L–1 chitosan and 0.1 mg·L–1 TDZ (the optimal treatment for PLB proliferation). Dissimilarity of 5% occurred between the mother plant and PLBs obtained after 16 weeks of culture. The range in the similarity coefficient varied from 0.80 to 1.0, and only 20% dissimilarity occurred between mother plant and PLBs after 20 weeks of culture

Development of plant regeneration system and agrobacterium-mediated transformation of Brassica oleracea L. subsp. italica cv. green marvel with HSP101 gene

Broccoli seeds are among the most commonly imported vegetable seeds in Malaysia. In Malaysia due to the humid climate, production of hybrid seeds is almost impossible. Consequently, improvement of in vitro culture method for clonal propagation of broccoli plants having the F1 hybrid characteristics is essential. Broccoli plants respond adversely to extreme temperatures and high humidity in the lowland thus, gene transformation for heat tolerance would be beneficial. Cameron Highlands is the main broccoli producing area in Malaysia because of the suitable cool climate and the cultivar that is commonly grown is Green Marvel. Therefore, the main objectives of this study were to improve the shoot regeneration system for Brassica oleracea subsp. italica cv. Green Marvel and to introduce Arabidopsis thaliana HSP101 (Athsp101) cDNA into broccoli through Agrobacterium tumefaciens-mediated transformation in order to increase its heat-tolerance characteristic. Multiple shoot formation from hypocotyl and shoot tip explants were assessed using different concentrations of TDZ (thidiazuron), zeatin and kinetin. In the experiment on multiple shoot formation on hypocotyl explants, TDZ at 0.1 mg/l induced the highest percentage of explant with shoot (96.67%) and the highest mean number of shoots per explant (6.17). In the experiment on shoot multiplication from shoot tip explants, the highest percentage of shoot tip explant producing shoots (100%) was on medium with 0.1 mg/l TDZ followed 1.5 mg/l zeatin (96.67%), while, the highest number of shoots per explant (4.27) was on 1.5 mg/l zeatin. Therefore, 0.1 mg/l TDZ was considered the most suitable for adventitious shoot formation from hypocotyl explants and 1.5 mg/l zeatin from shoot tip. In the determination of minimum inhibitory concentration (MIC) of kanamycin for effective screening of broccoli transformants, the lowest percentage (0.0%) and mean number of survived hypocotyl explants (0.00) was on shoot regeneration medium (SRM) containing 60 mg/l kanamaycin, while the lowest percentage (0.0%) and mean number of survived shoot tip explants (0.00) occurred on SRM containing 90 and 100 mg/l kanamycin. Therefore, 50 mg/l and 80 mg/l kanamaycin were the chosen MIC for hypocotyl and shoot tip explants, respectively. In the optimization of factors affecting Agrobacterium mediatedtransformation of broccoli with AtHSP101 gene and the regeneration of putative transformed plantlets, hypocotyls explants precultured on SRM with 200μM acetosyringone produced the highest percentage (13.33 %) and mean number of putative transformants (0.17), while shoot tip explants precultured on callus induction medium (CIM) with 200μM acetosyringone produced the highest percentage (23.33%) and mean number of putative transformants (0.27) after 8 weeks of culture. Optimization of bacterial dilution and inoculation time showed that the inoculation of hypocotyl segments in 1:5 bacterial dilution for 30 min produced the highest percentage (20 %) and mean number (0.27) of putative transformants. The same bacterial dilution and inoculation time also produced the highest percentage (30%) and mean number (0.33) of putative transformant from shoot tip explants. Thus, preculture with 200μM acetosyringone followed by inoculation in (1:5) bacterial dilution for 30 min was the most successful for transformation of broccoli with AtHSP101 gene. PCR analysis showed the expected fragment size of the AtHSP101 gene, while Southern blot analysis showed different hybridization bands in the hypocotyl (1 and 2 gene copy number) and shoot tip (3 gene copy number) derived transformants. The gene expression was confirmed through reverse transcriptase (RT-PCR) assay.Consequently, the transgenic broccoli plantlets were transferred to different temperature regimes (20ºC, 30ºC and 34ºC) in the transgenic greenhouse to evaluate the efficacy of HSP 101 gene in increasing their heat tolerance. Results showed that the transgenic plants could survive and performed normally, producing flower heads even at the highest tested temperature of 34ºC. In conclusion, an improved regeneration system has been established from hypocotyl and shoot tip explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature

Shoot tip regeneration and optimization of Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleraceae var. italica) cv. Green Marvel

A protocol of plant regeneration from shoot tips and optimization of Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica) cv. Green Marvel have been developed. Shoot tip response was assessed on Murashige and Skoog (MS) medium supplemented with different concentrations of zeatin. The highest regeneration with a maximum of 13 shoots per explant was obtained on MS medium containing 1.5 mg l-1 zeatin. Primary selection of putative transformed explants was performed on the optimized regeneration medium (MS medium containing 1.5 mg l-1 zeatin and 80 mg l-1 kanamycin) for 60 days. The effects of preculture, acetosyringone and growth of bacterial culture were studied. Explants precultured on callus induction medium for 4 days prior to inoculation with A. tumefaciens with 200 lM acetosyringone resulted in improved transformation frequency. The Agrobacterium culture dilution of 1:5 and inoculation time of 30 min increased the efficiency of transformation of shoot tip explants. The results also indicated that 150 mg l-1 ampicillin alone was adequate to eradicate Agrobacterium growth in the SRM incorporated with the respective minimum inhibitory concentration of 80 mg l-1 kanamycin. The polymerase chain reaction (PCR) and Southern blot assays confirmed the transgenic status of the broccoli cv. Green Marvel regenerants. A transformation efficiency of 5 % was achieved based on the positive PCR results using the optimized procedure. The expression of luciferase reporter gene in the transformed cells and the transcription of AtHSP101 using RT-PCR further confirmed the transgenic status of the regenerated plants

Influence of phenyl-urea and adenine-type cytokinins on direct adventitious shoot regeneration of cabbage (Brassica oleracea subsp. capitata) “KY Cross”

In this study, the effects of phenyl-urea (Thidiazuron) and adenine (6-benzylaminopurine) and 6- y,y,dimethylally-amino purine type cytokinins alone or in combination with indole-3-butyric acid on shoot regeneration from hypocotyl and cotyledonary explants of Brassica oleracea ssp. capitata “KY Cross” were investigated. For hypocotyl explants, medium containing 2.27 μM Thidiazuron showed the highest mean number (18.15) of separable shoots per explant with 80% shoot formation. In the case of cotyledonary explants, the highest mean number of shoots (3.03) was obtained on medium containing 12.30 μM 6-y,y,dimethylally-amino purine with a percentage of 56.67% shoot formation. Plantlets were successfully acclimatized with 70% survival in potting medium consisting of coconut husk+vermicompost (7 : 1 v/v). The regeneration system developed herewith will be a valuable tool for genetic improvement of cabbage “KY Cross”