Improving the quality of tissue-cultured plants by fixing the problems related to an inadequate water balance, hyperhydricity

In vitro tissue culture is a technique for accelerating plant propagation and supplying high- quality starting material which has a positive impact on product commercialization. Several obstacles may occur during the culture process, one of which is hyperhydricity. Hyperhydric shoots are characterized by extensive accumulation of water in the apoplast, the continuum of cell walls and intercellular air spaces which is almost completely flooded. The occurrence of hyperhydricity is a major problem in the micropropagation industry, since it reduces the quality and multiplication rate of microplants. Although numerous studies have been put forward to explain hyperhydricity, the underlying mechanism and causative factors of hyperhydricity are still debated. Understanding the underlying mechanisms and factors involved in the control of plant growth in vitro can greatly improve the quality of micropropagated plants. The research presented in this thesis succeeded in elucidating aspects of the mechanism, causality factors and methods to prevent hyperhydricity in in vitro grown Arabidopsis thaliana and Limonium sinuatum. Our study found that hypolignification of cell walls was an important causative factor in the development of hyperhydricity. The specific interaction of the plantlets, medium components and microenvironments were found to affect lignin biosynthesis, to lead to irregular stomatal features, abnormal anatomy of mesophyll cells and large intercellular spaces, to affect the water retention capacity and the transpiration rate. Exogenously applied calcium in combination with a specific lignin biosynthesis precursor, p-coumaric acid, and a stomatal opener (ALA) as supplements to the medium proved capable of reducing the occurrence or delaying the onset of hyperhydricity by stimulating cell wall lignin biosynthesis and modifying the pectin content of the leaves

Hypolignification: a decisive factor in the development of hyperhydricity

One of the characteristics of hyperhydric plants is the reduction of cell wall lignification (hypolignification), but how this is related to the observed abnormalities of hyperhydricity (HH), is still unclear. Lignin is hydrophobic, and we speculate that a reduction in lignin levels leads to more capillary action of the cell wall and consequently to more water in the apoplast. p-coumaric acid is the hydroxyl derivative of cinnamic acid and a precursor for lignin and flavonoids in higher plant. In the present study, we examined the role of lignin in the development of HH in Arabidopsis thaliana by checking the wild-types (Ler and Col-0) and mutants affected in phenylpropanoid biosynthesis, in the gene coding for cinnamate 4-hydroxylase, C4H (ref3-1 and ref3-3). Exogenously applied p-coumaric acid decreased the symptoms of HH in both wild-type and less-lignin mutants. Moreover, the results revealed that exogenously applied p-coumaric acid inhibited root growth and increased the total lignin content in both wild-type and less-lignin mutants. These effects appeared to diminish the symptoms of HH and suggest an important role for lignin in HH

Effect of cytokinin types, concentrations and their interactions on in vitro shoot regeneration of Chlorophytum borivilianum Sant. & Fernandez

Background: Chlorophytum borivilianum is a rare medicinal plant originally distributed throughout the forest of India. The tubers of C. borivilianum are used as an aphrodisiac and impotence supplement. The propagation of C. borivilianum is possible through seeds and tubers, but conventional methods may take several months. Hence in vitro technique of shoot regeneration could be an efficient alternative means of propagating the species. Latest study reported microtuberization of C. borivilianum but there is no sufficient study on a rapid method for shoot multiplication and elongation. Results: Young shoot buds of C. borivilianum were cultured on MS medium containing 6-benzylaminopurine (BAP) and Kinetin (Kn), both at 0, 8.88, 17.8 and 26.6 \u3bcM, either individually or in combinations. Proliferated shoots were subcultured on fresh medium of the same constituents on week 3 of culture for further shoot multiplication and elongation. BAP alone (8.88\u201326.6 \u3bcM) was significantly effective on shoot multiplication, while Kn alone (8.88\u201326.6 \u3bcM) was significantly effective on shoot elongation compared to the control containing MS basal medium without any plant growth regulator. However, combination of both cytokinins stimulated an interaction producing higher shoot number and shoot length compared to their individual application. Conclusions: The most suitable combination was 8.88 \u3bcM BAP + 8.88 \u3bcM Kn, reaching a mean shoot number of 10.83 and shoot length of 6.85 cm

In vitro plant regeneration and RAPD assessment of somaclonal variations in safed musli (Chlorophytum borivilianum Santapau & R.R.Fernandes)

Safed musli (Chlorophytum borivilianum), is a traditional medicinal plant which belongs to liliaceae family and has an increasing demand due to its wide range of uses. It has an aphrodisiac property and form an important ingredient of herbal tonics prescribed in the Ayurvedic systems of medicine. Due to its medicinal properties and high demand in herbal industry, Safed musli has been introduced in Malaysia by Felda in 2008 under Felda Herbal Corporation Sdn. Bhd. The major constraint in the cultivation of Safed musli is long tuber dormancy at about 6 -7 months, so it’s only one crop per year can be grown. Besides, the other constraints include shortage of quality planting material, lower tuber multiplication rate and impossible to obtain true to type and disease free plants either from seed tuber or seed. A study was carried out using in vitro propagation and molecular characterization of Safed musli. In vitro culture has shown the advantage over the conventional methods of vegetative production as it ensures rapid rate of multiplication and disease free abundant plantlets. Young shoot buds of Safed musli were collected and sterilized. 100% of clean culture and plant survived in sterilization ent with 70% alcohol (6 minutes), 50% Sodium hypochlorite (20 minutes), 0.1% aqueous mercuric chloride (15 minutes). Mass production of shoots was achieved on MS medium supplemented with 3 mg/L BAP which produced the highest mean number of shoots (18.9) and shoot length (6.0) cm. MS medium supplemented with 1 mg/L IBA which produced the highest mean number of roots (52.4) and root length (6.6) cm. In acclimatization, plantlets on media containing vermiculite : organic matter (1:1) showed the highest percentage of survival plants (83%) and mean number of shoot length (18.69) cm. Callus of Safed musli were induced from young shoot bud on MS medium containing 5 mg/L 2,4-D with the highest percentage of callus formation (66.66%) and mean weight of callus (10.65) g. For shoot regeneration, the highest percentage (66.67%) and the highest mean number of shoots per vial (9.6) was obtained on MS medium with 0.5 mg/L BAP . The RAPD assessment of somaclonal variations among Safed musli plantlet was performed. Out of 90 bands, 55 were polymorphic bands and 35 were monomorphic bands ranging between 115.43bp – 3388.24bp. RAPD marker effectively recognized the genetic difference among the in vitro shoot

Rapid multiplication of Safed musli (Chlorophytum borivilianum) through shoot proliferation

Young shoot buds were used as explants for rapid multiplication of Safed musli (Chlorophytum borivilianum). The explants were cultured onto medium containing basal salts of Murashige and Skoog (MS) and various concentrations of 6-benzylaminopurine (BAP) and kinetin (KIN) for shoot induction. Treatment containing 3.0 mg/l BAP produced the highest mean number of shoots per explants (18.90) and a mean length of shoots (6.0 cm) after 28 days of culture. Regenerated shoots were successfully rooted on MS medium supplemented with 1.0 mg/l indole-3-butyric acid (IBA) and 30 g/l sucrose. For ex vitro establishment, well-rooted plantlets were transferred in potting medium containing vermiculite : organic matters (1:1)

Effect of cytokinin types, concentrations and their interactions on in vitro shoot regeneration of Chlorophytum borivilianum Sant. & Fernandez

Background: Chlorophytum borivilianum is a rare medicinal plant originally distributed throughout the forest of India. The tubers of C. borivilianum are used as an aphrodisiac and impotence supplement. The propagation of C. borivilianum is possible through seeds and tubers, but conventional methods may take several months. Hence in vitro technique of shoot regeneration could be an efficient alternative means of propagating the species. Latest study reported microtuberization of C. borivilianum but there is no sufficient study on a rapid method for shoot multiplication and elongation. Results: Young shoot buds of C. borivilianum were cultured on MS medium containing 6-benzylaminopurine (BAP) and Kinetin (Kn), both at 0, 8.88, 17.8 and 26.6 μM, either individually or in combinations. Proliferated shoots were subcultured on fresh medium of the same constituents on week 3 of culture for further shoot multiplication and elongation. BAP alone (8.88–26.6 μM) was significantly effective on shoot multiplication, while Kn alone (8.88–26.6 μM) was significantly effective on shoot elongation compared to the control containing MS basal medium without any plant growth regulator. However, combination of both cytokinins stimulated an interaction producing higher shoot number and shoot length compared to their individual application. Conclusions: The most suitable combination was 8.88 μM BAP + 8.88 μM Kn, reaching a mean shoot number of 10.83 and shoot length of 6.85 cm

Induction, subculture cycle and regeneration of callus in Safed musli (Chlorophytum borivilianum) using different types of phytohormones

Background: Chlorophytum borivilianum is an industrially valued medicinal crop. Propagation through seeds is not feasible because of low germination percentage and long dormancy period. Therefore, callus culture and plant regeneration can be an alternative to improve this crop production. Also, callus can serve as an alternative source of bioactive compounds. Objective: To evaluate the effect of different phytohormones on callus induction, subculture cycle, and regeneration studies of callus in C. borivilianum. Materials and Methods: Young shoot buds of C. borivilianum were inoculated on Murashige and Skoog medium fortified with 3% sucrose and different concentrations (0, 1, 5, 10, and 15 mg/L) of either naphthalene acetic acid or 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid and callus induction was evaluated up to four subcultures cycles. Shoot regeneration from callus was studied on Murashige and Skoog media fortified with 6-benzylaminopurine andkinetin or thidiazuron at varied levels (0, 0.5, 1, 2, and 3 mg/L). Microshoots were rooted on Murashige and Skoog media supplemented with 1.0 mg/L indole-3-butyric acid and plantlets were acclimatized before transferred to the natural conditions. Results: Callus induction was better evidenced on Murashige and Skoog media containing 5 mg/L 2,4-dichlorophenoxyacetic acid up to fourth subculture. Callus differentiated into shoots on Murashige and Skoog media fortified with 6-benzylaminopurine or kinetin, whereas thidiazuron completely failed to regenerate shoots. Furthermore, microshoots rooted on 1.0 mg/L indole-3-butyric acid containing Murashige and Skoog media. The rooted plantlets were successfully acclimatized and established in soil with 88.3% survivability. Conclusion: The type of auxins played an important role in inducing callus tissue from shoot bud explants of Safed musli. In future, this in vitro protocol could benefit in crop improvement programs and serve as a new source of bioactive compounds from Safed musli callus tissue for various therapeutic applications