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

In vitro adventitious shoot regeneration and acclimatisation of Brassica oleracea subsp. italica cv. Green Marvel

Cotyledonary explants of Brassica oleracea subsp. italica (broccoli) cv. Green Marvel were cultured on Murashige and Skoog (MS) medium containing different combinations of the growth regulators 6- benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) for shoot regeneration. The optimal medium for inducing shoots contained 3 mgl-1 BAP and 1 mgl-1 NAA, which produced a shoot induction percentage of 53.33% and a mean number of 0.43 shoot per explant. The shoots were subsequently rooted in MS medium that contained 0.2 mgl-1 of indol-3-butyric acid (IBA). Different potting media were assessed during plantlet acclimatization. The highest percentage of plant survival (83.33%) was on the medium that contained sand and soil (1:1), while maximum root length (4.37 cm) and plant height (7.87 cm) were attained in potting medium that consisted peat moss, perlite and vermiculite (3:1:1).Key words: Brassica oleracea, broccoli, 6-benzylaminopurine, α-naphthalene acetic acid, indole-3- butyric acid

Genetic stability of in vitro multiplied Phalaenopsis gigantea protocorm-like bodies as affected by chitosan

Chitosan is a carbohydrate polymer derivative of chitin which presents in shell of crustaceans. This biopolymer is a non toxic and environmentally friendly, considered as a plant growth stimulator in some plant species. The present study investigates the effects of chitosan and media types on multiplication and genetic stability of Phalaenopsis gigantea protocorm-like bodies (PLBs). PLBs were inoculated in liquid New Dogashima Medium (NDM) and Vacin and Went (VW) supplemented with various concentrations of chitosan (0, 5, 10, 15, 20 and 25 mg/L). The highest PLB multiplication was observed on VW and NDM supplemented with 10 mg/L chitosan with mean number of PLBs 177 and 147, respectively. Chitosan promoted the formation of juvenile leaves and the highest number was observed in NDM supplemented with 20 mg/L chitosan with mean number of 66 leaves after 8 weeks of culture. Genetic stability was assessed among mother plant and secondary PLBs after 2, 4, 6, and 8 weeks of culture in liquid media. 8 out of 10 ISSR markers produced a total of 275 clear and reproducible bands with mean of 6.9 bands per primer. The secondary PLBs produced during sub-culturing process of chitosan treated liquid culture were genetically uniform and similar to mother plant

A Novel Damage Index for Online Monitoring of RC Slabs under Monotonic Loading by Integration of Process Controlling into Acoustic Emission Technique

This study introduces a novel structural health monitoring scheme for cementitious composite slabs with the aid of acoustic emission (AE) technique coupled with statistical process controlling (SPC) method. The adopted framework is an integrated monitoring solution that effectively relates current state (damaged) to reference state of the structure. Evaluation of the latter was made possible using autoregressive model incorporating a set of damage-sensitive feature. In order to provide a benchmark damage indicator, the collected data were processed using control chart analysis. The damage indicators for the former was similarly obtained and then compared with the benchmark to gauge the structural damage. These control charts offer a robust framework meticulously identifying inconsistency in the damage-sensitive feature imposed over the monitoring period. Linear and quadratic projections were also incorporated into SPC model to enhance identification of system transition to other damage states

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 (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

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

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