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In vitro organogenesis and histomorphological investigations in senna (Cassia angustifolia) - A medicinally valuable shrub
Abstract
A protocol for in vitro recurrent shoot production was established for Cassia angustifolia, a valuable medicinal plant which is used as laxative and in the treatment of a wide range of human ailments. Among the various seedling derived explants, cotyledonary node showed highest multiplication rate (2.41 shoots per explant) on Murashige and Skoog's (MS) medium supplemented with 1 μM N6-benzyladenine (BA). Nodal explants of the in vitro raised shoots too induced a maximum average number of shoots (2.5) with average shoot length (4.7 cm) on 1 μM BA. Of the cytokinins tried, N6-benzyladenine was found more effective than kinetin (Kn). The excised shoots were transferred to MS medium augmented with α-naphthalene acetic acid (NAA) for rooting. Nearly 80% shoots organized roots in half strength MS + 10 μM NAA within twenty days. The plantlets have been successfully transferred to soil. Histological examination of in vitro raised nodal explants revealed that multiple shoots formed both from preexisting and de novo buds on MS medium containing growth regulators. Morphological characteristics of in vitro and in vivo Cassia leaf have also been studied. Tissue-culture derived plants had functional stomata on both the surfaces of leaf and scattered wax particles on dorsal surface, whereas non-tissue-cultured plants had normal stomata with dense wax particles on the dorsal surface, the ventral surface showing only abnormal stomata which appeared non-functional.
... Multiple shoots were developed successfully from different explants (Cotyledonary node, nodal and shoot tips) excised from in vitro grown seedlings of C. angustifolia (Agrawal and Sardar 2003; Siddique and Anis 2007 a, b). Among the various explants, cotyledonary node gave the best response (Agrawal and Sardar, 2003), (Siddique and Anis, 2007a, b). ...
... Multiple shoots were developed successfully from different explants (Cotyledonary node, nodal and shoot tips) excised from in vitro grown seedlings of C. angustifolia (Agrawal and Sardar 2003; Siddique and Anis 2007 a, b). Among the various explants, cotyledonary node gave the best response (Agrawal and Sardar, 2003), (Siddique and Anis, 2007a, b). In addition, plant regeneration has been successfully developed by using root explants (Parveen and Shahzad 2011). ...
... µM was common in most of the in vitro micropropagated plants. According to Agrawal and Sardar (2003) 1.0 µM 6- benzyladenine (BA) was found best to induce multiple shoots in CN. However, at higher concentration of BA (10.0 µM), a decreasing trend in response in terms of percentage responding explants, average shoot number per explants as well as average shoot length was seen. ...
... BA is commonly preferred cytokinin for in vitro propagation, as it stimulates cell division as well as cell elongation, activates RNA synthesis, stimulates protein synthesis and enzyme activities (Al Malki and Elmeer 2010). The stimulating effect of BA on bud break and multiple shoot formation has been reported in many plants, e.g., Cassia angustifolia (Agrawal and Sardar 2003), Tylophora indica (Faisal and Anis 2007) and Bauhinia tomentosa (Naz et al. 2011). Explant browning is usually attributed to the production of phenolic compounds released from the cut surfaces of the explants. ...
... Our study indicates that the half MS medium supplemented with 1.5 μM NAA gave a lesser (50 %) rooting frequency, whereas quite a high percentage (80 %) of shoots rooted well when IBA was used, and it was found to be most effective at the 1.5 μM concentration. The promotive effect of IBA in rooting is well documented in Cassia angustifolia (Agrawal and Sardar 2006). ...
The objective of the current study was to develop an efficient and reproducible protocol for plant regeneration using nodal (1.0-1.5 cm) explants excised from a field grown mature plant of Cassia occidentalis L. The highest shoot regeneration frequency (80 %) with a maximum number of shoots (11.66) and shoot length (3.83 cm) after eight weeks of culture were observed on a Murashige and Skoog (MS) medium amended with 5.0 μM 6-benzyladenine, 100 μM citric acid, and 1.0 μM α-naphthalene acetic acid. A half-strength MS medium supplemented with 1.5 μM indole-3-butyric acid proved best for the induction of maximum roots (8.33) per shoot. Plantlets with well-developed shoots and roots were successfully acclimatized in plastic pots containing sterile Soilrite under two irradiances of 50 and 300 μmol m−2 s−1 (LI and HI, respectively) in a culture room, and after transfer to the field, the survival rate was 70 %. A significant increase in chlorophyll, carotenoid, and malondialdehyde content was found during acclimatization under both the irradiances but higher under HI. Similarly, the activities of superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase increased more under HI. Plantlets acclimatized under HI exhibited a better growth than those under LI.
... Due to its multifold uses, the plant is being highly exploited by the pharmaceutical companies and it has therefore, become imperative to develop a protocol for mass propagation through tissue culture for large scale production of sennosides and their further elevation using elicitors and through genetic transformation. Though, some work on in vitro regeneration of C. angustifolia has been published (Agrawal and Sardar 2003, 2006, 2007Siddique et al. 2010;Parveen and Shahzad 2011;Parveen et al. 2012) using different explants, none of them have established protocols pertaining to genetic and biochemical fidelity of the mature regenerants. Since the in vitro plant regeneration can cause genetic instability and somaclonal variation due to culture stress (Haisel et al. 2001), the direct development of a plant regeneration system and assessment of clonal fidelity of the in vitro raised plants of Cassia angustifolia will be of great significance in this crop. ...
... BA if added exogenously shortens the duration of S-phase of cell division through recruitment of latent origin of DNA replication both in vitro or in vivo (Francis and Sorell 2001). BA was also reported for induction of multiple shoots during in vitro culture of cotyledonary node, nodal explant (Agrawal and Sardar 2003), leaf explant (Agrawal and Sardar 2006), petiole explant (Siddique et al. 2010), root explant (Parveen and Shahzad 2011) and cotyledon explant (Agrawal and Sardar 2007;Parveen et al. 2012) of C. angustifolia. Significance of BA in inducing multiple shoots has been already reported in Simmondsia chinensis (Link) Schneider (Agrawal et al. 2002) and Spilanthes spp. ...
In vitro protocol has been established for clonal propagation of Cassia angustifolia Vahl which is an important source of anticancerous bioactive compounds, sennoside A and B. Nodal explants excised from field raised elite plant (showing optimum level of sennoside A and B) of C. angustifolia when reared on Murashige and Skoog's medium augmented with different cytokinins, viz. N(6)-benzyladenine (BA), N(6)-(2-isopentenyl) adenine (2iP) and 6-furfuryl aminopurine (Kn) differentiated multiple shoots in their axils. Of the three cytokinins, BA at 5 μM proved optimum for differentiating multiple shoots in 95 % cultures with an average of 9.14 shoots per explant within 8 weeks of culture. Nearly, 95 % of the excised in vitro shoots rooted on half strength MS medium supplemented with 10 μM indole-3-butyric acid (IBA). The phenotypically similar micropropagated plants were evaluated for their genetic fidelity employing random amplified polymorphic DNA (RAPD) markers. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 36 scorable bands, ranging in size from 100 to 1,000 bp were generated amongst them by the RAPD primers. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant proving their true to the type nature. Besides, high performance liquid chromatography evaluation of the sennoside A and B content amongst leaves of the mature regenerants and the elite mother plant too revealed consistency in their content.
... Traditional morphological features, molecular bar-coding utilizing ITS sequences, RAPD analysis, and metabolic variables by GC-MS analysis validate the taxonomic diversity of Cassia and Senna (TuAbdel-Hameed et al., 2013;Eldemerdash et al., 2022). The shoot induction and multiplication are a precious method for the species of Senna alexandrina, in which micropropagation studies have been conducted using various explants (Table 1) (Agrawal and Sardar, 2003a, 2003bAnis 2007a, 2007b;Siddique et al. 2010;Parveen and Shahzad, 2011). George and Hussein (2014) ...
Senna is a medicinal herb widely utilized in Ayurveda, Unani, and allopathic medicine. The leaves and pods of Senna are the most valuable portions, as they contain the compounds sennoside-A and sennoside-B. The laxative properties of sennoside-A and sennoside-B are the fundamental reasons for their use. The demand for Senna on a global scale provides opportunities to cultivate this plant commercially. Farmers are more lucrative towards this crop owing to the low cost of cultivation, drought tolerance crop, being a good source of income, and requiring less maintenance. The availability of improved cultivars and agronomic practices provides an excellent opportunity for the farmers to take up the cultivation under residual moisture during the Rabi season will enhance the yield and socioeconomic status of the farmers.
... Numerous scientific reports provide clear shreds of evidence on the clinical use of senna for the treatment of chronic constipation in adults (Ulbricht et al., 2011). Furthermore, a summary of different herbal formulations Agrawal and Sardar (2003) 6-benzylaminopurine ( Murashige and Skoog's medium + (12.2) shoots per explant Parveen and Shahzad (2012) 6-benzylaminopurine (1.0 μM) Direct regeneration (Continues) and crude extracts of Cassia plants involved in the medication therapy of various diseases is presented in Table 2. ...
Nature gifts medicinal plants with the untapped and boundless treasure of active chemical constituents with significant therapeutic potential that makes these plants a beneficial source in the development of phytomedicines. Genus Cassia, with approximately 500 species, is a large group of flowering plants in the family Fabaceae. Cassia species are widely distributed throughout different regions mainly tropical Asia, North America, and East Africa. In the folk medicinal history, these plants are used as laxative and purgative agents. In the Ayurveda system of medicine, they are used to cure headache and fever. Cassia plants exhibit pharmacological activities at large scales such as antimicrobial, anticancer, antiinflammatory, antioxidant, hypoglycemic, hyperglycemic, antimutagenic, and antivirals. The phytochemical investigations of genus Cassia demonstrate the presence of more than 200 chemical compounds, including piperidine alkaloids, anthracene derivatives (anthraquinones), flavonoids, pentacyclic triterpenoids, sterols, phenylpropanoids, and γ‐naphthopyrones. The literature illustrated anthraquinones and flavonoids as major secondary metabolites from this genus. However, some Cassia plants, with rich contents of anthraquinones, still show toxicology properties. As Cassia plants are used extensively in the herbal system of medicine, but only senna dosage forms have achieved the status of the pharmaceutical market as standard laxative agents. In conclusion, further investigations on isolating newer biologically active constituents, unknown underlying mechanisms, toxicology profiles, and clinical studies of Cassia species are needed to be explored. This review article specifies the systematic breach existing between the current scientific knowledge and the fundamentals for the marketization of genus Cassia products.
... The technique has been widely applied in all the sectors of horticulture, plantation and forestry and has contributed significantly towards the enhanced production of high quality planting material to the market. Some in vitro regeneration protocol are available for Cassia angustifolia through different explants by different methods, such as direct regeneration through cotyledonary node and nodal segments (Agrawal and Sardar 2003;Siddique and Anis 2007 a and b), indirect plant regeneration via cotyledon and leaflet derived calli (Agrawal and Sardar 2006;Siddique et al. 2010) and somatic embryogenesis from cotyledon (Agrawal and Sardar 2007) but to the best of our knowledge, there is no report on direct organogenesis using shoot tip explants. Therefore, the objective of the present study was to compare the relative efficiency of shoot tip explants with previously available reports for in vitro micropropagation from different explants and to standardize the optimal concentration of cytokinin, or cytokinin and auxin combinations for maximum shoot production and establishment of complete plantlets. ...
An effective and improved plant regeneration system was successfully developed using shoot tip explants taken from a two years old mature plant of Cassia angustifolia. The effect of different cytokinins, [6-benzyladenine (BA), Kinetin (Kin) and thidiazuron (TDZ)] at different concentrations (0.5-10 µM) were evaluated as augmented with Murashige and Skoog (MS 1962) medium. Among all the cytokinins tested, TDZ (5.0 µM) was optimum in inducing multiple shoots as compared to BA and Kin. The rate of shoot multiplication was increased when optimal concentration (5.0 µM) of BA and Kin was tested with different concentration (0.1-1.0 µM) of Indole-3- acetic acid (IAA). Among all the combinations tested, the maximum rate of shoot multiplication was obtained on MS medium supplemented with 5.0 µM BA and 0.5 µM IAA. The number of the shoots and shoot length developed in TDZ was increased when transferred to MS medium devoid of TDZ. After every subculture, rate of the shoot multiplication and shoot length showed increment and continued even after fifth subculture without any decline rate. In vitro rooting in regenerated shoots were best obtained in half-strength MS medium supplemented with 2.0 µM indole-3- butyric acid (IBA). Plantlets with well-developed shoot and roots were successfully hardened off in earthen pots containing garden soil and grown in greenhouse with 80% survival rate.
... angustifolia have been developed using various explants derived from aseptic seedlings. Agrawal and Sardar ( 2003 ) used cotyledonary node (CN) explant for regeneration of senna on MS medium supplemented with 1.0 μM BA. Only 2.4 shoots per explant were induced however, the number of shoots increased to a maximum of 17.6 shoots per CN explants when cultured on MS basal medium after giving a treatment of 1.0 μM TDZ for 4 weeks (Siddique and Anis 2007 ). ...
Interest and support for the conservation and development of medicinal plants is increasing in all parts of the world. This is due, in part, to a growing recognition given to the role of medicinal plants in the provision of culturally relevant and affordable health care in creating sustainable livelihoods and in the vital conservation of biodiversity. This has also drawn the attention of the world community towards the need for creating mechanisms to ensure sustained development of the sector and to allow sharing of information between countries, organizations and agencies. The value of medicinal plants to human livelihoods is essentially infinite. The special significance of medicinal plants in conservation stems from the major cultural, livelihood or economic roles that they play in many people’s lives. Many of the threats to medicinal plant species are similar to those causing endangerment to plant diversity generally. The most serious proximate threats generally are habitat loss, habitat degradation and over-harvesting. In order to protect such endangered species from possible extinction, the exploitation of medicinal plants must be accompanied by conservation measures. Application of tissue culture of plant cells, tissues and organs is the most promising tool for medicinal plant conservation.
... Among various concentrations of BAP tested, the maximum number of shoots (13.5±0.07) per explants was observed on medium containing 1.0 mg l -l BAP (Table-1 , Fig.1A) as compared to other concentrations of BAP. Superiority of BAP in inducing shoots from cotyledonary nodes has been described previously in various woody species like Dalbergia sissoo (Pradhan et al., 1998), Simmondsia chinesis (Agrawal et al., 2002) Cassia angustifolia (Agrawal and Sardar, 2003), Albizia odoratissima (Rajeshwari and Paliwal, 2006) Melia azadirachta (Hussain and Anis, 2009) and Bauhinia cheilantha (Gutierrez et al., 2011). ...
Suman Kumari and Narender Singh (2012) In vitro plantlet regeneration from cotyledonary node explants of Salvadora persica L. a medicinally important desert tree. Journal of Agricultural Technology 8(5): 1839-1854. An efficient and reproducible in vitro propagation protocol has been developed for rapid micropropagation of Salvadora persica L. a medicinally and economically important desert tree. The present investigations were carried out with an aim for the development of a tissue culture method for the clonal propagation of this tree. The seeds were germinated on MS half strength medium devoid of growth regulators. The cotyledonary node explants were excised from twenty days old seedlings and inoculated on MS medium supplemented with different concentrations (0.25-2.0 mg l -1) of auxins (NAA, IAA and 2,4-D) and cytokinins (BAP & Kinetin) individually as well as in various combinations. The frequency of shoot regeneration from cotyledonary node was affected by various concentrations of auxins, cytokinins and successive transfer of mother explant. Maximum numbers of shoots (17.5±0.02) were obtained on MS medium containing BAP (2.0 mg l -1) in combination with IAA (0.5 mg l -l). The in vitro raised shoots (>3cm long) were excised aseptically and implanted on MS half and full strength medium fortified with auxins (IAA, NAA and IBA) at the concentrations of 0.5, 1.0 and 2.0 mg l -l for root formation. Maximum per cent roots induction and length was obtained on MS full strength medium supplemented with 1.0 mg l -l IAA. Seventy per cent plantlets were successfully established in earthen pots containing soil and sand mixture (3:1). The plantlets were then transferred to the field conditions. The regenerated plants were morphologically uniform and exhibited similar growth characteristics.
... Thus, it is necessary to develop an alternative source of propagation and efficient method for easy distribution of in vitro raised quality propagules in the form of small beads to fulfill the demands of pharmaceutical industry. During past years, several reports are available on its in vitro regeneration exploiting different strategies of micropropagation (Agrawal and Sardar 2003Siddique and Anis 2007;Siddique et al. 2010;Parveen and Shahzad 2011;Parveen.et al. 2012). ...
The present study described the encapsulation of nodal segments of Cassia angustifolia Vahl. excised from 1-month-old in vitro raised cultures for short-term conservation and propagation. Various concentrations and combinations of gelling matrix (sodium alginate) and complexing agents (calcium chloride) were tested to pre-pare uniform beads. The ideal beads were obtained through a combination of 3 % sodium alginate and 100 mM cal-cium chloride. The maximum conversion response (94 %) of encapsulated beads was obtained in Murashige and Skoog's medium (MS medium) supplemented with 2.5 lM benzyladenine (BA) and 0.4 lM a-naphthalene acetic acid (NAA) after 6 weeks of culture. The encapsulated and non-encapsulated nodal segments were also stored at 4 °C for different time periods (0, 1, 2, 4, 6 and 8 weeks). The regenerated microshoots were best rooted in optimized rooting medium that comprised half-strength MS ? 1.0 lM indole-3-butyric acid (IBA) ? 5.0 lM phloroglucinol (PG) for the production of complete plant-lets. The regenerated plantlets were successfully hardened and acclimatized in natural conditions with 70 % survival rate. Keywords Acclimatization Á Cassia angustifolia Á In vitro conservation Á Plantlet conversion Á Synthetic seeds Abbreviations CaCl 2 Á2H 2 O Calcium chloride BA Benzyladenine IBA Indole-3-butyric acid NAA a-naphthalene acetic acid MS Murashige and Skoog medium PG Phloroglucinol PGR Plant growth regulator Introduction
... Thus, it is necessary to develop an alternative source of propagation and efficient method for easy distribution of in vitro raised quality propagules in the form of small beads to fulfill the demands of pharmaceutical industry. During past years, several reports are available on its in vitro regeneration exploiting different strategies of micropropagation (Agrawal and Sardar 2003Siddique and Anis 2007;Siddique et al. 2010;Parveen and Shahzad 2011;Parveen.et al. 2012). ...
The present study described the encapsulation of nodal segments of Cassia angustifolia Vahl. excised from 1-month-old in vitro raised cultures for short-term conservation and propagation. Various concentrations and combinations of gelling matrix (sodium alginate) and complexing agents (calcium chloride) were tested to pre-pare uniform beads. The ideal beads were obtained through a combination of 3 % sodium alginate and 100 mM cal-cium chloride. The maximum conversion response (94 %) of encapsulated beads was obtained in Murashige and Skoog's medium (MS medium) supplemented with 2.5 lM benzyladenine (BA) and 0.4 lM a-naphthalene acetic acid (NAA) after 6 weeks of culture. The encapsulated and non-encapsulated nodal segments were also stored at 4 °C for different time periods (0, 1, 2, 4, 6 and 8 weeks). The regenerated microshoots were best rooted in optimized rooting medium that comprised half-strength MS ? 1.0 lM indole-3-butyric acid (IBA) ? 5.0 lM phloroglucinol (PG) for the production of complete plant-lets. The regenerated plantlets were successfully hardened and acclimatized in natural conditions with 70 % survival rate. Keywords Acclimatization Á Cassia angustifolia Á In vitro conservation Á Plantlet conversion Á Synthetic seeds Abbreviations CaCl 2 Á2H 2 O Calcium chloride BA Benzyladenine IBA Indole-3-butyric acid NAA a-naphthalene acetic acid MS Murashige and Skoog medium PG Phloroglucinol PGR Plant growth regulator Introduction
... Till to date, legumes appears to be the most recalcitrant to in vitro regeneration (Somera et al. 2003;Popelka et al. 2006). Although in vitro regeneration via direct and indirect organogenesis using various explants viz., cotyledonary node (Agrawal and Sardar 2003;Siddique and Anis 2007a), nodal (Siddique and Anis 2007b), petiole derived callus (Siddique et al. 2010), leaflet and cotyledon derived calli (Agrawal and Sardar 2006) has been reported. Somatic embryogenesis has also been reported from cotyledon derived callus (Agrawal and Sardar 2007). ...
This report describes a protocol for the in vitro shoot induction and plant regeneration from epicotyl explants of Cassia angustifolia on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), Kinetin and 2-iP (0.5–10.0 μM). MS medium supplemented with BA (5.0 μM) was the most effective in inducing adventitious shoots and growth. The highest rate of shoot multiplication was achieved on MS medium supplemented with BA (5.0 μM) and Indole-3-acetic acid (IAA, 1.0 μM). The nodal segments excised from the shoots regenerated from BA (5.0 μM) and IAA (1.0 μM) were used as explants for next three round of micropropagation. The number of shoots significantly increased at successive round of micropropagation. For rooting, MS medium supplemented with 2.0 μM indole-3-butyric acid proved to be better than that supplemented with IAA or α-naphthalene acetic acid. The in vitro raised plantlets with well developed shoot and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse. About 52 plants (85 %) survived out of 60 plants transferred in garden soil.
... Establishment of genetic fidelity among the regenerants using various molecular markers is yet another significant parameter toward this direction. In the present investigation, it is clearly (Agrawal et al. 2002), Cassia angustifolia Vahl (Agrawal and Sardar 2003), Hollarhena antidysenterica (L.) Wall (Kumar et al. 2005) and Spilanthes acmella L. (Pandey and Agrawal 2009). ...
An efficient in vitro protocol has been established for clonal propagation of elite plant of Spilanthes calva DC., an important source of spilanthol, an antimalarial larvicidal compound. Nodal explants excised from field grown plant of S. calva DC. when reared on Murashige and Skoog's medium augmented with different cytokinins, viz. N6-Benzyladenine (BA), N6-(2-isopentenyl) adenine (2iP) and 6-furfuryl aminopurine (Kn), differentiated multiple shoots from the axils. BA at 10 μM proved optimum for elicitation of multiple shoots in 91.6 % cultures with an average of 7.12 shoots per explant within 6 weeks. The excised shoots rooted on half strength Murashige and Skoog's medium supplemented with 0.1 μM IBA. Micropropagated plants were hardened and transferred to field for acclimatization, where 95 % plants survived and were phenotypically similar to the donor plant. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to evaluate the genetic fidelity amongst the regenerants. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 71 scorable bands, ranging in size from 100 bp to 1,100 bp were generated by a combination of the two markers in the aforesaid plants. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant. The similarity values amongst the aforesaid plants varied from 0.967 to 1.000. The dendrogram generated through UPGMA (Unweighted Pair Group Method with arithmetic mean) analysis revealed 98 % similarity amongst them, thus confirming the genetic fidelity of the in vitro clones. © 2012 Prof. H.S. Srivastava Foundation for Science and Society.
... Large number of medicinal plants have been successfully regenerated through micropropagation either directly (Raghu et al. 2006; Bouhouche and Ksiksi 2007; Shahzad et al. 2011;) or indirectly via callus formation (Reddy et al. 2001; Faisal and Anis 2005; Rout 2005; Khanna et al. 2006; Shahzad et al. 2006). As far as literature is concerned, there are only few reports on in vitro regeneration of C. angustifolia using different explants, either through direct shoot regeneration or callus formation (Agrawal and Sardar 2003; Siddique and Anis 2007; Siddique et al. 2010; Parveen and Shahzad 2011). Although the number of shoots produced was very less in all these earlier reports. ...
In vitro regeneration was achieved through callus
culture derived from cotyledon explants of Cassia angustifolia
Vahl. on MS (Murashige and Skoog, 1962) medium.
Calli were induced from cotyledon explants excised from
aseptic 14 days old seedlings on MS medium containing
2,4-D (2,4-dichlorophenoxy acetic acid) and 2,4,5-T (2,4,5-
trichlorophenoxy acetic acid) at different concentrations
with 3% sucrose and 0.8% agar. Optimal growth of callus
was obtained at 5.0 μM 2,4-D, which was proved to be the
best for shoot regeneration when sub cultured onto MS
medium supplemented with cytokinins either alone or in
combination with an auxin. Maximum number of shoots
(23.2±1.4) were produced at 5.0 μM 6-benzylaminopurine
(BA) and 0.4 μM α-naphthalene acetic acid (NAA).
Regenerated shoots produced prominent roots when transferred
to half strength MS medium supplemented with
1.0 μM indole-3-butyric acid (IBA) and 5.0 μM phloroglucinol
(PG). Rooted plantlets thus developed were
hardened and successfully established in the soil. This
protocol yielded an average of 23 plants per cotyledon
explant over a period of 4 months.
... Till to date, legumes appears to be the most recalcitrant to in vitro regeneration (Somera et al. 2003;Popelka et al. 2006). Although in vitro regeneration via direct and indirect organogenesis using various explants viz., cotyledonary node (Agrawal and Sardar 2003;Siddique and Anis 2007a), nodal (Siddique and Anis 2007b), petiole derived callus (Siddique et al. 2010), leaflet and cotyledon derived calli (Agrawal and Sardar 2006) has been reported. Somatic embryogenesis has also been reported from cotyledon derived callus (Agrawal and Sardar 2007). ...
This report describes a protocol for the in vitro shoot induction and plant regeneration from epicotyl explants of Cassia angustifolia on Murashige and Skoog (MS) medium supplemented with 6-ben-zyladenine (BA), Kinetin and 2-iP (0.5–10.0 lM). MS medium supplemented with BA (5.0 lM) was the most effective in inducing adventitious shoots and growth. The highest rate of shoot multiplication was achieved on MS medium supplemented with BA (5.0 lM) and Indole-3-acetic acid (IAA, 1.0 lM). The nodal segments excised from the shoots regenerated from BA (5.0 lM) and IAA (1.0 lM) were used as explants for next three round of micropropagation. The number of shoots significantly increased at successive round of micropropagation. For rooting, MS medium supplemented with 2.0 lM indole-3-butyric acid proved to be better than that supplemented with IAA or a-naphthalene acetic acid. The in vitro raised plantlets with well developed shoot and roots were successfully established in earthen pots contain-ing garden soil and were grown in greenhouse. About 52 plants (85 %) survived out of 60 plants transferred in garden soil.
... Among various concentrations of BAP tested, the maximum number of shoots per explant as well as highest shoot length was observed on medium containing 1.0 mg l -1 BAP (Fig. 1b;Table 1). The majority of literatures have also reported BAP as the most active cytokinin for shoot multiplication in various woody trees like Dalbergia sissoo (Gulati and Jaiwal 1996; Pradhan et al. 1998), Simmondsia chinensis (Agrawal et al. 2002; Prakash et al. 2003), Cassia angustifolia (Agrawal and Sardar 2003), Melia azadirachta (Hussain and Anis 2009), etc. Effect of successive transfer of mother explants on shoot multiplication A significant (P B 0.05) enhancement in the shoot multiplication was observed after each successive transfer up to1 a Seedling node showing shoot proliferation on MS medium containing 1.0 mg l -1 BAP. ...
An efficient in vitro propagation is described for Spondias mangifera Willd., a medicinally important tree, using nodal explants obtained from 4-week-old seedlings. The frequency of shoot regeneration
from seedling node was affected by various concentrations of BAP and successive transfer of mother explant. MS (Murashige
and Skoog, Physiol Plant 15:473–497, 1962) medium supplemented with 1.0mgl−1 of 6-benzylaminopurine (BAP) was optimal for shoot multiplication. Upon this medium, highest number of shoots (about 10.6)
per explants was obtained after fourth subculture of mother explants. Half-strength MS medium containing IAA (1.0mgl−1) was most effective for rooting of shoots. Regenerated plantlets were successfully acclimatized and transferred into soil
with 80–90% survival rate. The regenerated plants were morphologically uniform and exhibited similar growth characteristics
and vegetative morphology to the mother plants. This is the first report on micropropagation of S. mangifera, which can be applied for further genetic transformation assays and pharmaceutical purposes.
KeywordsAnacardiaceae-Organogenesis-Seedling node-Shoot multiplication-
Spondias mangifera
... Though the species which belong to the Leguminosae are known for their recalcitrant nature, yet some successful attempts have been made on in vitro organogenesis of Cassia fistula, C. siamea (Gharyal and Maheshwari 1990) and C. alata (Fett et al. 2000). Incidentally, there is no report on in vitro regeneration of C. angustifolia except our earlier paper (Agrawal and Sardar 2003) on seedling derived explants. Reports on its cultivation and genetic improvement programme are also Iimited (Jambhale et al. 1998, Lal et al. 1992. ...
High efficiency shoot regeneration was achieved through leaflet and cotyledon derived calli in Cassia angustifolia - an important medicinal plant. Dark brown compact callus was induced at the cut ends of the explants on Murashige and Skoog's
(MS) medium augmented with 1 µM N6-benzyladenine (BA) + 1 µM 2,4-dichlorophenoxyacetic acid (2,4-D). Such callus pieces on transfer to cytokinins (BA or kinetin)
supplemented medium differentiated shoots within 10 – 15 d. Of the two cytokinins, 5 µM BA was optimum for eliciting morphogenic
response in 83.33 and 70.83 % cultures with an average of 4.16 ± 0.47 and 3.70 ± 0.56 shoots in cotyledon and leaflet derived
calli, respectively. The addition of 0.5 µM α-naphthaleneacetic acid (NAA) to MS + 5 µM BA further elevated the maximum average
number of shoots to 12.08 ± 1.04 and 5.37 ± 0.52 for cotyledon and leaflet calli, respectively. The excised shoots were transferred
to a rooting medium containing either IAA (indole-3-acetic acid), IBA (indole-3-butyric acid) or NAA. Nearly 95 % shoots developed
an average of 5.4 ± 0.41 roots on half strength MS medium supplemented with 10 µM IBA.
... Thus, there is an urgent need to develop a suitable in vitro regeneration system for its propagation. There are only few reports available for in vitro plant regeneration of C. angustifolia using various explants like cotyledonary node (Agrawal and Sardar 2003), nodal segment (Siddique and Anis 2007), cotyledon and leaflet derived calli ( Sardar 2006, 2007). However, there is no report available on using root explant for regeneration of this plant species, therefore, to search a newer tissue for the development of high-efficiency regeneration system, we have attempted to utilize the root explant in the present study. ...
The aim of this study was to develop a new micropropagation system for Cassia angustifolia Vahl., an important medicinal legume using root explant as starting material. Root explants taken from 30-day-old aseptic
seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators: 6-benzyladenine
(BA), kinetin (Kn), and thidiazuron (TDZ). Organogenic nodular calli obtained on MS+TDZ (1.0μM) were transferred to shoot
regeneration medium supplemented with different cytokinins (BA, Kn or TDZ) either alone or in combination with auxin:indole-3-acetic
acid or α-naphthalene acetic acid. Maximum shoot regeneration frequency (90%) was obtained on MS+BA (2.5μM)+NAA (0.6μM)
wherein a maximum of 42.76±1.47 shoot buds per explant were induced with a maximum conversion rate of 35.63±0.75 shoots
per explant and average shoot length of 5.43±0.20cm. Elongated microshoots were successfully rooted under ex vitro conditions
by pulse treatment in 200μM of indole-3-butyric acid for half an hour. Microshoots were rooted, acclimatized and hardened
off simultaneously in sterilized soilrite inside the growth room and then established in pots containing sterilized soil and
manure (1:1) and grown under greenhouse condition with 90% survival rate. The histological sections at different developmental
stages of shoot buds revealed the organization of nodular meristematic zone leading to the orientation and differentiation
of shoot buds in large number and thereafter conversion into healthy shoots.
Keywords
Cassia angustifolia
–In vitro organogenesis–Root culture–Ex vitro rooting–Histology–Plant establishment
... Amongst the various cytokinins, BA was the most effective for shoot multiplication. Similar observations have been reported for various apocynaceous taxa and other woody taxa, such as jojoba (Agrawal et al., 2002;Prakash et al., 2003) and senna (Agrawal and Sardar, 2003). However, Kn promoted maximum shoot elongation in this species. ...
In vitro clonal propagation of 18–20-yr-old Holarrhena antidysenterica tress has been achieved by employing nodal explants. The tree explants showed marked seasonal variation in their morphogenic
response under in vitro conditions. Maximum response was obtained from the beginning of May to the end of July, followed by a gradual decline, finally
dropping to zero from October to February. The explants induced multiple shoots only on cytokinin-containing medium. Several
cytokinins [N6-benzyladenine (BA), N6-(2-isopentenyl) adenine (2ip), 6-furfuryl aminopurine (Kn), and adenine sulfate (Ads)] were assayed. The best response was
achieved with 15 μM BA in which 62.5% of cultures produced 2.75±0.2 shoots per explant with 3.56±0.2 cm average length. Amongsth the three heavy
metals assayed, silver nitrate (AgNO3) significantly improved the response. This compound enhanced both the percentage of responding cultures (86.6%) and the average
shoot number (4.73±0.2) at a concentration of 20mgl−1. Further improvement in the morphogenic response occurred when explants from in vitro shoots were employed instead of mature trees. In this case, the percentage of morphogenic cultures was increased to 100%
at the third subculture with an average of 11.45±0.3 shoots per explant. Regenerated shoots were rooted in half-strength Murashige
and Skoog medium with 10 μM indole-3-acetic acid. The plantlets were successfully acclimatized in soil.
... Superiority of BAP for shoot multiplication may be attributed to the ability of the plant tissues to metabolize BAP more readily than other synthetic growth regulators (Mallik et al. 2005). The majority of literature have also reported BAP as the most active cytokinin for shoot multiplication in various woody taxa, such as Simmondsia chinensis (Agrawal et al. 2002;Prakash et al. 2003), Cassia angustifolia (Agrawal and Sardar 2003) and Holarrhena antidysenterica . ...
An efficient in vitro propagation system has been developed for rapid micropropagation of Soapnut (Sapindus trifoliatus Linn.), a medicinally and economically important tree from nodal (axillary bud) segments of seedlings. The frequency of shoot
regeneration from seedling node explant was influenced by the age of the seedlings, growth regulators and successive transfer
of the mother explant. Explants from 4-week-old seedlings yielded the maximum shoot regeneration frequency (97.22%) on full-strength
MS medium supplemented with 1.0mgl−1 of 6-benzylaminopurine (BAP). After harvesting the newly formed shoots, the mother explants transferred to same medium subsequently
produced a maximum of 5.16 shoots per explant after third passage. Further improvement in the morphogenic response occurred
when the nodal explants excised from in vitro regenerated shoots were employed, and 6.89 shoots per explant were obtained
on the same medium after the third subculture. Optimal rooting (91.67%) was obtained by placing the micro-shoots in liquid
MS medium with 1.0mgl−1 IBA for 24h and then transferring to the agar solidified MS medium devoid of IBA. The micropropagated shoots with well-developed
roots were acclimatized and successfully transplanted to soil with 90% survival rate. Genetic stability of the regenerated
plants was assessed using random amplified polymorphic DNA (RAPD). The amplification products were monomorphic in micropropagated
plants and similar to those of mother plant. No polymorphism was detected revealing the genetic integrity of micropropagated
plants. This is the first report of an efficient protocol for regeneration of S. trifoliatus through organogenesis, which can be applied for further genetic transformation assays and pharmaceutical purposes.
KeywordsMicropropagation–Organogenesis–Sapindaceae–Saponin–Seedling node–Soapnut tree
An efficient in vitro method has been established for the production of whole plantlets of Bauhinia tomentosa using cotyledonary node (CN) explants excised from 15 d-old aseptic seedlings. Proliferating shoot cultures were obtained by placing CN explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), kinetin (Kn), and 2-isopentenyl adenine (2-iP), singly or in combination, with either of the auxins, indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). MS medium augmented with 5.0 μM BA was found to be most effective in providing the highest frequency (84%) of shoot regeneration, associated with the maximum number of shoots (9.0 ± 0.6) per explant.The highest efficiency of shoot proliferation was observed using a combination of 5.0 μM BA and 0.5 μM NAA, where the number of shoots increased to 16.2 ± 0.2 per explant after 8 weeks of culture. Improved shoot multiplication and elongation rates were achieved when 20.0 mg l-1 adenine sulphate (AdS) was added to the combined treatment of 5.0 μM BA and 0.5 μM NAA, where an average shoot number of 17.0 ± 0.6 and an average shoot length of 5.8 ± 0.3 cm were obtained.The maximum frequency of root formation (70%) and greatest root lengths (2.3 ± 0.4 cm) were obtained on filter-paper bridges in liquid MS medium augmented with 2.5 μM IBA and 5.0 μM chlorogenic acid (a phenolic compound) after 4 weeks of culture. Regenerated plantlets were successfully acclimatized in a growth room and subsequently transferred to a greenhouse.This is the first report on in vitro plantlet regeneration in B. tomentosa using CN explants.
Synthetic seed technology is an alternative to traditional micropropagation for production and delivery of cloned plantlets. Synthetic seeds were produced by encapsulating nodal segments of C. angustifolia in calcium alginate gel. 3% (w/v) sodium alginate and 100 mM CaCl2 · 2H2O were found most suitable for encapsulation of nodal segments. Synthetic seeds cultured on half strength Murashige and Skoog medium supplemented with thidiazuron (5.0 μM) + indole-3-acetic acid (1.0 μM) produced maximum number of shoots (10.9 ± 0.78) after 8 weeks of culture exhibiting (78%) in vitro conversion response. Encapsulated nodal segments demonstrated successful regeneration after different period (1-6 weeks) of cold storage at 4 °C. The synthetic seeds stored at 4 °C for a period of 4 weeks resulted in maximum conversion frequency (93%) after 8 weeks when placed back to regeneration medium. The isolated shoots when cultured on half strength Murashige and Skoog medium supplemented with 1.0 μM indole-3-butyric acid (IBA), produced healthy roots and plantlets with well-developed shoot and roots were successfully hardened off in plastic pots containing sterile soilrite inside the growth chamber and gradually transferred to greenhouse where they grew well with 85% survival rate. Growth performance of 2 months old in vitro-raised plant was compared with in vivo seedlings of the same age. Changes in the content of photosynthetic pigments, net photosynthetic rate (PN), superoxide dismutase and catalase activity in C. angustifolia indicated the adaptation of micropropagated plants to ex vitro conditions.
An in vitro propagation system for Cassia angustifolia Vahl. has been developed. Due to the presence of sennosides, the demand of this plant has increased manyfold in global market. Multiple shoots were induced by culturing nodal explants excised from mature plants on a liquid Murashige and Skoog [8] medium supplemented with 5-100 μM of thidiazuron (TDZ) for different treatment duration (4, 8, 12 and 16 d). The optimal level of TDZ supplemented to the culture medium was 75 μM for 12 d induction period followed by subculturing in MS medium devoid of TDZ as it produced maximum regeneration frequency (87%), mean number of shoots (9.6 ± 0.33) and shoot length (4.4 ± 0.46 cm) per explant. A culture period longer than 12 d with TDZ resulted in the formation of fasciated or distorted shoots. Ex vitro rooting was achieved when the basal cut end of regenerated shoots was dipped in 200 μM indole-3-butyric acid (IBA) for half an hour followed by their transplantation in plastic pots filled with sterile soilrite where 85% plantlets grew well and all exhibited normal development. The present findings describe an efficient and rapid plant regeneration protocol that can further be used for genetic transformation studies.
In vitro regeneration through somatic embryogenesis as well as organogenesis using cotyledon of a woody medicinal legume, Cassia angustifolia is reported. The cotyledons dissected from semi-mature seeds, if inoculated on Murashige and Skoog’s medium (MS) supplemented
with auxin alone or in combination with cytokinin, produced direct and indirect somatic embryos. A maximum of 14.36 ± 2.26
somatic embryos per 20mg of explants including callus were produced in 70% cultures on MS medium with 2.5μM benzyladenine
(BA) + 10μM 2,4-dichlorophenoxyacetic acid (2,4-D). Although the percentage of embryogenic cultures was higher (83.33%) at
10μM 2,4-D + 1μM BA, the average number of somatic embryos was much less (7.6 ± 0.85) at this level, whereas at 2.5μM BA
and 5μM 2,4-D, there was a simultaneous formation of both somatic embryos and shoots. The somatic embryos, although started
germinating on the same medium, developed into full plantlets only if transferred to MS basal with 2% sucrose. Cytokinins
alone did not induce somatic embryogenesis, but formed multiple shoots. Five micromolar BA proved optimum for recurrently
inducing shoots in the competent callus with a maximum average of 12.04 ± 2.10 shoots and shoot length of 2.26 ± 0.03cm.
Nearly 91.6% shoots (2–2.5 cm in size) organized an average of 5.12 ± 0.58 roots on half strength MS + 10μM indole-3-butyric
acid. All the plantlets have been transferred successfully to soil. Types of auxin and its interaction with cytokinin significantly
influenced somatic embryogenesis.
An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants excised from 14-day-old aseptic seedlings. Of the two cytokinins, 6-benzyladenine (BA) and thidiazuron
(TDZ) evaluated as supplements to Murashige and Skoog (MS) medium, TDZ at an optimal concentration of 5.0μM was effective
in inducing multiple shoots. The highest rate of shoot multiplication was achieved on MS medium supplemented with 5.0μM TDZ
and 1.0μM indole-3-acetic acid (IAA) at pH 5.8. The regenerated shoots when subcultured on hormone free MS medium considerably
increased the rate of shoot multiplication and shoot length by end of fourth subculture passage. Rooting was achieved on the
isolated shoots using MS medium with 60μM indole -3- butyric acid (IBA) and 1% activated charcoal for 1week and subsequently
transferring the shootlets to half strength MS liquid media without IBA and activated charcoal. The in vitro raised plantlets
with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in greenhouse.
This study was carried out at Dept. of Ornamental Hort. Fac. Agric., Cairo Univ.,
and the applied part was carried out at the Tissue Culture Laboratory, Horticulture
Research Institute, Agriculture Research Center, during 2007-2009 seasons to develop an
efficient protocol of Polygala myrtifolia L. propagation by tissue culture technique.
Different disinfection agents (sodium hypochlorite and ethanol), explant types (apical,
middle and basal), cultural seasons (summer, autumn, spring and winter), plant growth
regulators (BA, kin, NAA, IAA and IBA), MS medium strengths (1/4, ½ and ¾),
commercial fertilizer (Nofatrein) concentrations as well as supporting agents (agar, gelrite
and filter paper bridge) and acclimatization medium substrates (peatmoss, vermiculite and
sand) were employed in these experiments. Important results could be summarized as
follows: To produce aseptic cultures, soaking apical explants in 20% Clorox (sodium
hypochlorite) solution either for 10 or 15 minutes without presoaking in 70% ethyl
alcohol was the best in producing the lowest contamination percentage, the highest
survival% and the lowest mortality%. During establishment stage, apical explants
cultured during spring season recorded the highest survival%, while middle explants were
more successful in other parameters, i.e. number of shoots/explant, shoot length and
number of leaves/shoot, when they were cultured during autumn season. Multiplication
experiments showed that the highest survival% was obtained either by BA at 1 mg/l or kin
at 2 mg/l without NAA addition or by BA at 1 mg/l with NAA at 0.5 mg/l. BA at 1 mg/l
without NAA addition presented the highest number of shoots/explant. In the same
regard, Nofatrein at 1 mg/l with ¾ MS medium strength gave the highest number of
shoots/explant and leaves/shoot. This was accompanied by an increase in survival%.
Results of rooting experiments demonstrated that IBA at 1 mg/l when applied as a
presoaking solution presented the highest rooting% and number of roots/plantlet, while
IBA at 1.5 mg/l added to the medium presented the longest roots. On the other hand, IBA
free medium solidified with agar+gelrite produced the highest survival% and rooting%.
Number of roots reached the maximum value by using IBA at 1 mg/l with agar. IBA at 1
mg/l with filter paper bridge presented the longest roots, the highest carotenoids, the
highest phenols and the highest total soluble sugars content. Peatmoss+vermiculite (1:1,
v/v) was superior in increasing most characteristics as survival%, plant height, number of
leaves/plantlet, leaf area, number of roots, plantlet fresh weight, total chlorophylls, total
indoles and total soluble sugars during acclimatization stage.
An effective protocol was developed for in vitro regeneration of the Cassia angustifolia via indirect organogenesis from petiole explants excised from 21-day-old axenic seedlings. Organogenic callus were induced on Murashige and Skoog (MS) medium supplemented with 5.0 µM 2,4-dichlorophenoxy acetic acid and 2.5 µM thidiazuron (TDZ). Adventitious shoot regeneration was achieved on MS medium supplemented with 5.0 µM TDZ as it induced 8.5 ± 0.98 shoots in 85% cultures. The number of shoots and shoot length was significantly enhanced when cultures were subcultured on auxin-cytokinin-containing medium. The highest number of shoots (12.5 ± 1.10) and shoot length (4.3 ± 0.20 cm) was recorded on MS medium supplemented with 5.0 µM TDZ and 1.5 µM indole-3-acetic acid. Regenerated shoots were rooted best on MS medium supplemented with 10.0 µM indole-3-butyric acid followed by their transfer to liquid MS filter paper bridge medium. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 70% survival rate. The plants showed normal morphological characteristics similar to the field grown plants.
The leaf anatomy of an aseptically cultured red raspberry clone ( Rubus idaeus L.) was studied before and after transfer to soil under controlled environmental conditions. Leaves of plantlets formed in culture were smaller, thinner, had a less compact arrangement of palisade and mesophyll cells, and an altered palisade cell shape compared to leaves formed on plants in soil. The number of epidermal hairs, especialy the filiform type, was lower in vitro and the distribution of colleters was affected. Trichome number was greater in new leaves formed after transplantation and greatest in greenhouse- and field-grown control plant leaves. Calcium oxalate crystals were present in the leaves of in vitro plantlets and more numerous in the leaves formed on plants in soil. Stomata were fixed open, slightly raised, and occurred on the upper leaf surface of in vitro plantlets with many on the periphery of the leaf. Amphistomatous and the peripheral stomatal condition persisted in new leaves formed during the first month when cultured plantlets were transferred to soil at 3 or 6 klx. However, new leaves, like all greenhouse and field-control plant leaves, had few adaxial stomata at 9 klx and peripheral leaf stomata were rare. Anatomy of new leaves formed during the first month after transplanting in soil at 3, 6, or 9 klx was similar to those in culture. Parenchyma tissue was less compact than in control plant leaves and palisade cell shape remained abnormal. More than half the leaves from culture died within the first month of transferring plantlets to soil. Some survived for almost 3 months. Reduced trichome numbers, almost complete lack of filiform trichomes, and presence of peripheral and adaxial as well as unprotected, open, abaxial stomata would all contribute to transplant shock and water loss in cultured plantlets transferred to soil. New leaves of transplants, formed during the first month in soil, had transitional leaf anatomy and surface features. With time, in the soil environment, the appearance of subsequently formed leaves approached that of controls.
Leaf surfaces of non-tissue-cultured, vitrified and non-vitrified plantlets of Gypsophila paniculata (Babies Breath) were examined using an environmental scanning electron microscope. Non-tissue-cultured plants had a complete epidermal surface, recessed stomata and wax present on the leaf surface. The surface of tissue-cultured plantlets appeared similar to non-tissue-cultured plants excepting stomata were slightly protruding and less wax appeared to be present. In both non-tissue-cultured and tissue-cultured plants stomata were found both opened and closed and were observed closing. In contrast vitrified plantlets had abnormal, malformed stomata which appeared non-functional. The ventral surfaces of leaves seemed more normal than the dorsal, this may be due to the former receiving more light. Additionally, discontinuities were found in the epidermis. Often epidermal holes were found in association with stomatal apertures. It is suggested that the main cause of desiccation of vitrified G. paniculata plantlets ex vitro is due to loss of water from the discontinuity in epidermis and not because of non-functional stomata. Liquid water could be seen through the epidermal holes indicating that at least some of the extra water in vitrified plantlets is contained in the intercellular spaces.
Trichomes have been modified in a number of crops to develop insect-tolerant genotypes. Pigeonpea, Cajanus cajan (L.) Millsp., is often heavily damaged by insect pests, and trichomes provide a potential insect resistance mechanism. The following study was conducted to identify and characterize the distribution of trichomes on pigeonpea and two wild species, C. platycarpus (Bentham) van der Maesen and C. scarabaeoides (L.) Thours. Three glandular (Types A, B, and E) and two nonglandular (Types C and D) trichome types were identified with light and electron microscopy. Types A, B, C, and D were found on leaves, pods, and calyxes of all three Cajanus spp., except for Type A, which was not found on pods and calyxes of most C. scarabaeoides accessions examined. Because of their small size and rarity, Type E trichomes were not considered in this study. Pods of C. scarabaeoides were the most densely pubescent, followed by POdS of C. cajan and C. platycarpus. Trichome density on pods varied significantly among pigeonpea genotypes and different accessions of C. scarabaeoides. Differences across seasons and in greenhouse versus field-grown plants were also significant. Leaves of C. platycarpus possessed the fewest trichomes, while C. cajan and C. scarabaeoides had highly pubescent leaves. The resistance of C. scarabaeoides pods to Helicoverpa armigera (Hubner) larvae reported in an earlier study is due to the high density of nonglandular trichomes. This wild species may thus be an important source for developing insect resistant pigeonpea.
Shoot cultures of Quercus robur have been established and multiplied in vitro using original material from both juvenile seedlings and mature trees. The juvenile explants were embryos and shoot apices from 3–4-month-old seedlings. Shoot tips from the new growth of stump sprouts were used to establish initial cultures of mature trees. Of the various macronutrient formulae tried, the most satisfactory were Heller’s medium with 1 mM of (NH4)2SO4, and Gresshoff and Doy’s. A dose of 6-benzylaminopurine at 1 mg l−1 was used in initial cultures and at 0.1 mg l−1 in shoot multiplication cultures. A rooting rate of 83% was achieved with juvenile material and 63% with adult material by briefly dipping the basal ends of shoots regenerated in vitro in concentrated solutions of IBA.
Plants of apple (Malus domestica Borkh.) were regenerated from proliferating meristem-tips grown on nutrient medium. Only benzyladenine (BA), at an optimum of 5 × 10−6 M, was required for initial growth and development of meristem-tip explants which produced proliferating shoot cultures in high frequency. Naphthaleneacetic acid (NAA) at 10−5 M was used to initiate roots. Plantlets were then transferred to a growth regulator free medium where roots developed fully before potting. Temperatures below 28°C and high salt concentration decreased rooting efficiency.
The metabolic fate of [8-14C]benzyladenine applied to the excised organs of tomato (Lycopersicon esculentum Mill. cv. Heinz 1370) was investigated after 2 and 6 h of feeding. Although the roots were the most effective at uptake of the cytokinin the leaves metabolised it the most efficiently. The predominant metabolite in all of the tissues was an unknown compound which did not have a retention time corresponding with any of the standards used. The roots contained the most extensive range of metabolites which included the unknown metabolite and compounds co-eluting with adenine, and the riboside, nucleotide and 9-glucoside of benzyladenine. The 9-glucoside was detected only in the root material. The stem yielded the highest levels of radioactivity at the retention times of benzyladenosine-5-monophosphate and benzyladenosine. The radioactivity associated with these two cytokinins was transient in the leaf extract. This organ ultimately yielded radioactivity only at the retention times of the unknown metabolite and adenine. Since only the roots and leaves contained relatively large peaks of radioactivity at the elution volume of adenine it seems that degradative metabolism was more predominant in these organs than in the stem.
Procedures for micropropagation of Centaureaspachii (Compositae), an endangered rosulate plantendemic from the mediterranean Spain area, have beendeveloped using inflorescence nodal segments asexplants for in vitro establishment. Only 15%of explants remained contaminated using this materialto start the in vitro axenic cultures. Higherproliferation of shoots and multiplication coefficientwas obtained on Murashige and Skoog (MS) mineralmedium supplemented with 1.0 mg lminus 16-benzyladenine. However, shoot elongation decreasedwith the addition of this cytokinin.Rooting of shoots with only one auxin was very lowafter 6 weeks on the majority of rooting media tested.The best rooting result (60%) was obtained on MSmedium with a combination of 2 mg lminus 1indole-3-acetic acid plus 2 mglminus 1 indole-3-butyric acid. Moreover, in thisculture medium 50% of shoots rooted during the thirdweek of culture. High survival, over 80%, wasobtained when the plantlets were transferred togreenhouse conditions. The endangered Centaureaspachii can be successfully micropropagatedbeginning with a single inflorescence stem and withoutsignificant damage to the mother plant.