Abstract
The effect of three concentrations of MS-micro, macronutrients (MS-full, MS½, MS¼) and a range of cytokinins (0.00, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 mg dm-3 of KIN) and auxins (0.2 mg dm-3 IAA, IBA, NAA, and 2, 4-D) on shoot-root growth, initiated from stem nodal segments, were investigated. G. glabra shooting was significantly influenced by MS media strength. The MS¼ medium supplemented with 0.2 mg dm-3 KIN exhibited maximum shoot initiation and shoot length followed by MS½ at the same concentration of KIN. Best rooting and percentage were attained in MS¼ medium supplemented with 0.2 mg dm-3 IAA followed by NAA for KIN derived shoots.
Ex vitro acclimatization was provided special attention. Three different anchoring media were used singly and in combination. The results lead to conclusion that those plantlets whose roots were treated with IAA for15 min. before transferring to rooting media (at 2.0 mg dm-3 IBA), showed best to established themselves by transferring to anchoring medium containing sand+silt+peat (1:1
Problem Statement
Medicinal plants are important to health of many people in the developing countries. Approximately, 80% of the people in the developing countries still rely on the traditional medicine for their healthcare needs. Since the biggest problems with modern medicine is the overuse of antibiotics and contraindications of chemicals used as medicines. Many medicinal plants have long history of their use. For example, Licorice (Glycyrrhiza glabra) root is often used to prevent and treat stomach ulcers. Its active components are also used to prevent and treat hepatitis. Because of its extensive use the plant is now under the category of endangered species in Pakistan. Ginkgo biloba is an extremely popular medicinal plant that millions of people use for better health. It is called an antiaging because it has been proven to increase blood circulation to the entire body, especially the brain, increasing mood, mental alertness, memory and overall stamina. Indeed, it is the need of the time to cultivate these valuable medicinal plants due to their economic importance. In the recent years, micro-propagation is widely recommended as a biotechnological tool for the multiplication of valued plants. Many reports provide us tremendous information that this technique makes it possible to produce a large number of uniform plants. Such plants can be used for the extraction of medicinal compounds or for pharmacological studies
Objectives
- To develop an efficient in vitro protocol that will maintain and propagate potential medicinal plants, not endemic to Faisalabad like Licorice and Ginkgo
- To transfer acclimatized plants to field for growth under natural environmental conditions
Micropropagation of Licorice:
Shoots with some nodes were cut and placed in water containing in a beaker before brought in Lab. Nodal segments of 2-3 cm were cut in length, washed thoroughly with running tap water and then rinsed three times with distilled water. Disinfected the node segments first in 70% alcohol for 15 seconds, followed by 20% (v/v) sodium hypochlorite solution, and finally, with 0.01% mercuric chloride solution for 5 min.
The basic culture medium which was used in this study consisted of MS (Murashige and Skoog, 1962) salts (micro and macronutrients) and vitamins with 100 mg dm-3 inositol, 3% sucrose and 0.8% agar. The adjustment of pH of 5.8 and agar was added, before autoclaving. To study the effect of MS macronutrients and micronutrients, on micropropagation, three different concentrations of micro and macronutrients 1) MS-full (full strength), 2) MS½ (half strength) and 3) MS¼ (one-fourth strength) with or without cytokinins and or auxins, were used for shoot initiation and rooting, respectively. The cultures were placed in growth room, and maintained at 25 ± 2o C with 16-h photoperiod with irradiance of 60 µmol m-2 s-1.
Shooting
Shoot length, under control conditions showed lowest values at all the three levels of MS-nutrient media, except MS-full control, where no value was observed. All the three MS-nutrient media when supplemented with different concentrations KIN, that enhanced in shoot length compared to their respective controls. Increasing concentration of KIN in the MS-nutrient media consistently reduced the shoot length. Minimum reduction was observed at MS¼ nutrient medium supplemented with 0.2 mg dm-3 of KIN, followed by MS½ and MS-full. On the other hand, more the dilution in the concentrations of MS macro and micronutrients more was the length of shoot.
Rooting
All the shoots cultured on various concentrations of IAA, and IBA showed root initiation except NAA where two concentrations (0.2 and 0.4 mg dm-3) was showed root initiation, while 2,4-D totally failed to do so at all its tested concentrations.
Root length showed highly significant differences among the concentrations of IAA and IBA individually. Both auxins enhancement in shoot length after supplemented with MS- micro- and macro-nutrients (MS¼), compared to their respective controls. Increasing concentration of both the auxins increased consistently shoot length up to 2.0 mg dm-3 after that a decline was observed at 3.0 mg dm-3 . Maximum increase was observed at MS¼ nutrient medium supplemented with 2.0 mg dm-3 both for IBA and IAA. At this concentration (2.0 mg dm-3) IBA exhibited more shoot length than IAA
ACCLIMATIZATION
Acclimatization (hardening) was adapted for those plantlets which were well rooted (2 month old, developed on 2.0 mg dm-3 IBA). Plantlets were washed thoroughly by distilled water to removed completely remains of medium and immersed up to root in a solution of 0.2 mg cm-3 IAA (Table 3), for 15 minutes and transferred to Polyvinyl pots containing six different autoclaved anchoring media (soil texture) 1) sand; 2) silt; 3) peat; 4) Sand + silt (3:1); 5) Sand + peat (1:1) and 6) sand + silt + peat (1:1:1). After transplantation, pots were covered with polythene bags for first 15 days and watering at alternate day and finally, shafted under direct sun light (natural photoperiod) (Plate 4). The plantlets considered to acclimatize had at least one leaf in good condition.
Micropropagation of Ginkgo biloba
Seed germination
The results revealed that all the media under all temperatures showed zero germination except only one seed (2.00%) germinated that was sown in sand incubated at 4oC.These results indicated that quality of supplied was not good at all purchase times
Shoot initiation from nodal segment
Shoots initiation were observed on both the concentrations of BA. It was also observed that growth rate was slow at both the concentration. The 30 d period of growth revealed that 2.0 mg dm-3 BA showed a little faster growth rate than 1.0 mg dm-3. During the second period (60 days) growth rate was noted also was in first period. Furthermore shoots were become brown or burned due to unavoidable environment of growth room.
Conclusions
The effect of three concentrations of MS-micro, macronutrients (MS-full, MS½, MS¼) and a range of cytokinins (0.00, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 mg dm-3 of KIN) and auxins (0.2 mg dm-3 IAA, IBA, NAA, and 2, 4-D) on shoot-root growth, initiated from stem nodal segments, were investigated. G. glabra shooting was significantly influenced by MS media strength. The MS¼ medium supplemented with 0.2 mg dm-3 KIN exhibited maximum shoot initiation and shoot length followed by MS½ at the same concentration of KIN. Best rooting and percentage were attained in MS¼ medium supplemented with 0.2 mg dm-3 IAA followed by NAA for KIN derived shoots.
Ex vitro acclimatization was provided special attention, since during this process, micropropagated plantlets under go structural as well as physiological changes. Three different anchoring media were used singly and in combination. The results lead to conclusion that those plantlets whose roots were treated with IAA for15 min. before transferring to rooting media (at 2.0 mg dm-3 IBA), showed best to established themselves by transferring to anchoring medium containing sand+silt+peat (1:1:1).
Recommendations
The overall best and efficient resulted protocol for mass propagation of Glycyrrhiza glabra L. through nodal segments as selected during course of studies is as follow
Shooting
-Nodal segment (explants)
-MS-¼ strength of basic salts+30 g sugar+vitamins+0.8% agar
-0.2 mg dm-3 KIN
Rooting
-MS-¼ strength of basic salts+30 g sugar+vitamins+0.8% agar
-2.0 mg dm-3 IBA
Acclimatization
-Plantlet’s roots treated with IAA
-Pots containing anchoring medium well water and left for two days before plantlets transformation
-Transfer to anchor media containing sand+silt+peat (1:1:1)
-Initially cover with polyethylene bag for 15 days
-Irrigate with alternate day
-Uncover the pots
-Shift to natural environment
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