Alteration of media enables efficient in vitro cloning of mature Elaeocarpus serratus L. (Ceylon olive): a commercially important fruit tree.

Elaeocarpus serratus is a fruit tree able to propagate through conventional vegetative means to a limited extent restricts its wide cultivation by the farmers. In the present report, we have developed an efficient in vitro propagation protocol using mature nodal explants from a 17-year-old tree for the first time with 6.6 shoots/culture. Explants cultured on agar (0.8%) gelled standard Murashige and Skoog (MS) medium, ½ MS, ¾ MS, White's, Gamborg's B5 or woody plant medium (WPM) supplemented with 2.5 µM benzyl adenine (BA) and 0.1 µM α-naphthalene acetic acid (NAA) showed the superiority of ½ MS medium in terms of explant response and number shoots (6.6). Further optimization of ½ MS medium by altering nutrient elements (macros, micros, vitamins and Fe EDTA) were undertaken, and MS medium composed of half-strength major salts, original strength of minor salts and vitamins were supplemented with BA (2.5 µM) and NAA (0.1 µM), produced enhanced axillary bud proliferation (8.88/explant) and shoot elongation (3.83 cm). Reculturing of original explant on this medium after IV passages produced more than 16 healthy shoots per culture which attained a length of 4.13 cm. Microshoots raised through this way were rooted (86.11%) ex vitro by pulse treatment with 2 mM indole-3-butyric acid (IBA) for 5 min followed by planting in nursery pots containing a 1:1:1 (v/v/v) mix of sand, soil, and farmyard manure. The hardened plants were successfully planted in the fruit tree garden of the Department. Genetic fidelity of micropropagated and mother plants were tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers which showed a high degree of monomorphism thus supported morphological uniformity of micropropagated plants.

Effective reversal of hyperhydricity leading to efficient micropropagation of Dianthus chinensis L.

The effective reversion of hyperhydricity (HH) in Dianthus chinensis L. facilitated efficient in vitro production of hyperhydricity-free plantlets. Under routine sub-culture practice, the problem of HH arises after third sub-culture in agar (0.85%) gelled Murashige and Skoog (MS) medium containing 2.5 µM 6-benzyladenine (BA). To confirm the role of ethylene on hyperhydricity induction, an ethylene releasing compound ethephon (5 µM) was used in combination with 2.5 µM BA and demonstrated 100% HH with reduced stomatal aperture. Supplementation of 10 µM silver nitrate (AgNO3) to 2.5 µM BA containing medium resulted HH reversion with reduced shoot number (19.0); however, addition of 5 µM cobalt chloride (CoCl2) produced highest microshoots (202.0). The combination effect of AgNO3 (10 µM), CoCl2 (5 µM), and BA (2.5 µM) showed complete HH reversion and upheld normal microshoots (55.0) with reduced relative water content (78.3%). The Ag and Co salts regulate ethylene biosynthesis and thereby 50% reductions in H2O2 content characterized by formation of green healthy shoots with proper stomatal morphology. The gene expression profile of 1-Amminocyclopropane-1-carboxylase synthase (ACS1) and 1-Amminocyclopropane-1-carboxylic acid oxidase (ACO1) showed reduced expression after the retroversion of microshoots in anti-ethylene reversion medium compared to hyperhydric shoot. In vitro raised shoots were rooted (93.3%) ex vitro by 10 mM IBA treatment and 92.2% plants were survived. The genetic stability of micropropagated plants were analyzed and proved that addition of low levels of heavy metal salt in the medium does not cause any variation in banding pattern. The protocol forwards a novel method to revert HH of in vitro cultures by adopting intermittent exposure of anti-ethylene compounds added in the medium and the procedure can be applied to many other plants facing similar HH problems. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02645-7.

Influence of polyamines on hyperhydricity reversion and its associated mechanism during micropropagation of China pink (Dianthus chinensis L.).

Hyperhydricity (HH) is a physiological disorder that frequently occurs in plant tissue cultures, affecting healthy growth and development of clonal plants. The primary cultures raised in Murashige and Skoog (MS) medium supplemented with 2.5 µM N6-benzyladenine (BA) produced normal microshoot (6.3 shoots/ culture) with least HH. However, the third subculture onwards, HH becomes a major problem. The role of ethylene on HH induction through stomatal closure mechanism were proved by the supplementation of ethephon (5 µM) in the culture medium containing 2.5 µM BA. In the present study, the application of polyamines (putrescine, spermidine, or spermine) to minimize the HH was examined. Supplementation of 5 µM spermine in MS medium significantly reduced the percentage of HH to 0.33%, in contrast to control (100%), while a maximum number of healthy reverted shoots (11.0) were observed in 5 µM spermidine treatment. The addition of polyamines effectively reduced H2O2 content (50%) characterized by increased chlorophyll content with proper stomatal morphology. The relative gene expression profile of ethylene biosynthesis genes, 1-Aminocyclopropane-1-carboxylase synthase (ACS1) and 1-Aminocyclopropane-1-carboxylic acid oxidase (ACO1) at 5 µM spermine added medium was 1.09 and 1.3 over normal (1) or HH cultures (1.93 and 2.53) respectively, and thus directed restoration of normal morphology of shoots. The present finding in brief, forward a novel method to regulate HH in terms of endogenous ethylene by adopting polyamines exposure and the procedure can be applied to many other plants facing similar HH problems.

The efficiency of Cytochrome P450 gene-based markers in accessing genetic variability of drumstick (Moringa oleifera Lam.) accessions.

Drumstick (Moringa oleifera Lam.) is an important vegetable as well as forage crop of arid and semi-arid zones of the tropics. The leaves and pods of the plant are rich sources of minerals and vitamins. In the present work, genetic diversity study of 23 genotypes of M. oleifera collected from Kerala, Tamil Nadu and Karnataka states of India was carried out using seven cytochrome P450 (CytP450) markers. By using seven pairs of CytP450 gene-based markers, 88.25% of polymorphism was recorded among the 23 sampled genotypes. The Polymorphic Information Content (PI), Marker Index (MI) and Resolving Power obtained for seven primers were estimated 0.23, 2.96 and 9.83, respectively. The Unweighted Pair Group Method with Arithmetic mean (UPGMA) dendrogram based on this marker data indicate that genotypes from different geographical regions are placed in the same clusters. The dendrogram and Principal Coordinates Analysis (PCoA) plots derived from the binary data matrices were highly concordant. The investigation, in brief, proved that CytP450 based marker system is efficient in the elucidation of genetic diversity in M. oleifera accessions.

The effect of silver nitrate on micropropagation of Moringa oleifera Lam. an important vegetable crop of tropics with substantial nutritional value.

An improved micropropagation protocol facilitating continuous multiplication of elite germplasm of Moringa oleifera has been developed. Initial culture of nodal explant in MS medium supplemented with 2.5 µM BA resulted in the formation of 12.5 shoots per explant with high frequency of leaf fall (84.3%). To confirm whether the leaf fall is due to accumulation of ethylene in the culture vessel, effect of ethylene releasing agent CEPA in the medium was tested. In order to reduce leaf fall and improve multiplication, varying concentration of anti-ethylene agent, AgNO3 was incorporated in the medium. Addition of 2.5 µM AgNO3 in combination with 2.5 µM BA produced maximum number of shoots (17.6) including shoots originated from the base of the explant and shoots from the axillary buds of the primary shoots, where significant reduction in leaf fall (20.6%) was noticed. This enabled sustained multiplication of M. oleifera through continuous subculture without adversely affecting shoot number or shoot quality in terms of shoot length. Microshoots obtained from fourth subculture onwards were used for ex vitro rooting and found that by treating 50 µM NAA for 30 s, maximum numbers of microshoots (83.3%) were rooted. Rooted plants were acclimatized, survived and were successfully transferred to field. Genetic fidelity analysis using 10 ISSR primers revealed more than 95% monomorphic bands among plants raised in MS medium containing low concentration (2.5 µM) of AgNO3 and BA (2.5 µM). The addition of AgNO3 in the medium sustained in vitro growth and effectively prevented leaf fall compared to control, thus demonstrating efficient micropropagation of M. oleifera.

An improved micropropagation and ex vitro rooting of a commercially important crop Henna (Lawsonia inermis L.).

An improved micropropagation protocol has been developed for a cosmetically important, dye yielding crop, henna (Lawsonia inermis). Quality of henna product is governed by naphthoquinone based pigment lawsone, thus in vitro multiplication of superior healthy plant to achieve enhanced productivity in terms of dye content and biomass deserve due attention. In the present study, nodal explants collected from an elite plant screened on the basis of superiority in lawsone content was cultured on MS medium supplemented with 0.5 µM benzyl adenine (BA) gave significantly (p < 0.05) high number of shoots (24.33). The explants placed on MS medium augmented with 0.5 µM BA and 2-isopentenyladenine (2-iP) resulted in the formation of maximum number of shoots (43.67) and was elongated (12.57 cm) within 4 weeks of culture period. Enhanced axillary bud proliferation and production of mass number of micro shoots was achieved by the continuous subculture in MS medium containing 0.5 µM BA and 2-iP. In vitro raised micro shoots were dipped in 0.44 mM NAA for 5 min followed by planting in polyethylene pots containing a soil: vermiculite (1:1 v/v) mixture produced rooted plantlets (100%). Different auxin types and its concentrations had significant role rooting of L. inermis. Rooting response of various size shoots of L. inermis treated with 0.44 mM NAA showed 100% rooting in 4.1-5 cm size class shoots. After two months of potting, survived (95%) plants were successfully transferred to medicinal plant garden of the Department. The lawsone content of one-year-old micropropagated plants (23.04 mg/g dw) growing in normal environmental conditions and elite mother plant (22.84 mg/g dw) was almost similar. Through the present study, efficient cloning of superior germplasm of L. inermis was established.

Auxin and nutritional stress coupled somatic embryogenesis in Oldenlandia umbellata L.

Somatic embryos were induced from internodal segment derived callus of Oldenlandia umbellata L., in MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). Initially calli were developed from internodes of microshoots inoculated in 2.5 µM NAA supplemented medium. Then calli were transferred to 2,4-D added medium for somatic embryogenesis. Nutritional stress coupled with higher concentration of 2,4-D triggered somatic embryogenesis. Nutritional stress was induced by culturing callus in a fixed amount of medium for a period up to 20 weeks without any external supply of nutrients. Addition of 2.5 µM 2,4-D gave 100% embryogenesis within 16 weeks of incubation. Callus mass bearing somatic embryos were transferred to germination medium facilitated production of in vitro plantlets. MS medium supplemented with 2.5 µM benzyl adenine and 0.5 µM α-naphthalene acetic acid produced 15.33 plants per culture within 4 weeks of culture. Somatic embryo germinated plants were then hardened and transferred to green house.

Micropropagation of annatto (Bixa orellana L.) from mature tree and assessment of genetic fidelity of micropropagated plants with RAPD markers.

An in vitro propagation technique based on axillary bud proliferation was developed for the first time to mature annatto (Bixa orellana L.) tree. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with 1.0 µM benzyl adenine (BA) and tender coconut water (10 %) showed significantly high (P < 0.05) explant response (67.0 %), development of elongated shoots (3.36), shoot buds (8.9) and shoot elongation (3.53 cm). Cytokinins like zeatin, isopentenyl adenine (2-iP), kinetin, or thidiazuron (TDZ) were inferior to BA to induce multiple shoots. Seasonal variations significantly affected the in vitro response of nodal explants. In vitro rooting experiments have showed 55.6 % rooting on MS medium containing 15 µM indole-3-butyric acid (IBA). Alternatively, in vitro raised shoots were rooted (61.1 %) ex vitro, by 10 mM indole-3-butyric acid (IBA) for 30 s. The results of the RAPD marker system revealed the genetic stability among the micropropagated plants. The present protocol in brief, can be used for the clonal propagation of the superior genotype and preservation of germplasm.

An efficient in vitro propagation methodology for Annatto (Bixa orellana L.).

Efficient methods were developed for both in vitro seed germination and micropropagation of an economically important dye yielding multipurpose tree, Bixa orellana L. Mature seeds were inoculated onto Murashige and Skoog (MS) medium supplemented with different concentrations of gibberellic acid (GA3). Highest frequency of germination (93.3 %) was recorded on medium supplemented with 3 µM GA3 against 13.33 % in control. Nodal explants cultured on MS medium fortified with 5 µM isopentanyl adenine (2-iP) produced maximum explants response (93.3 %) and highest number of shoots (35.71). Addition of relatively higher concentration (15 µM) of benzyl adenine (BA) resulted in the production of significantly (P < 0.05) reduced number of shoots (12.66). Sucrose at 87.6 mM was found to be the best carbohydrate source for multiple shoot induction compared to glucose and fructose. Regenerated shoots (3-4 cm) were rooted (95.5 %) on agar gelled MS medium supplemented with 10 µM indole-3-butyric acid (IBA). In vitro developed plantlets with well-developed roots were potted and acclimatized initially in the growth chamber and then moved to a green house with 83.3 % survival. The present protocol avoids the use of auxins in shoot multiplication medium, which will lower the cost, avoid callus formation and thus reduces the possibility of somaclonal variation in the regenerated plants. The method is efficient to produce over 32,000 hardened plants within a 10-month culture period starting from a single nodal explant.

Micropropagation of mature Chinese tallow tree (Sapium sebiferum Roxb.).

An in vitro propagation technique based on axillary bud proliferation has been developed for matureSapium sebiferum trees. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (1-10 µM and α-naphthaleneacetic acid (0-0.5 µM showed axillary bud proliferation. Shoots proliferated in vitro were multiplied on Murashige and Skoog medium containing 2.5 µM benzyl adenine and 0.25 µM α-naphthaleneacetic acid. Seasonal changes affected the shoot proliferation potential of the initial explant. Shoots were rooted on a half-strength, growth-regulator-free, agar-gelled, MS medium after a 48-h treatment on half-strength MS liquid medium with 10 µM indole-3-butyric acid. Rooted plantlets were potted and acclimatized in a growth chamber and then moved to the greenhouse. Four-month-old plants were transplanted to the field.

A highly efficientin vitro regeneration methodology for mature Chinese tallow tree (Sapium sebiferum Roxb.).

A highly efficientin vitro regeneration methodology for mature chutese tallow tree (Sapium sebiferum Roxb.) has been developed. Shoot segments cultured on MS medium supplemented with 7.5 µM NAA produced light green callus. Optimum shoot differentiation resulted when callus was transferred to MS medium with 1 µM BA and 0.25 µM NAA. Shoot forming ability of callus was higher on MS medium compared to B5, half-MS or WPM. A continuous shoot harvest system at four-week intervals was established. Shoot yield continued for six months without loss of vigour. Regenerated shoots were rooted by culturing on half strength agar-gelled MS medium containing 1 µM IBA. Rooted plantlets were transferred to 1:1 soil vermiculite mixture and acclimatized with 67 % survival rate. Fully acclimatized plants were planted in the field, and performance is being evaluated.