Shrish C. Gupta

In vitro development of plantlets from axillary buds of Acacia auriculiformis — a leguminous tree

Multiple shoots have developed from axillary buds excised from in vitro grown seedlings of Acacia auriculiformis on Gamborgs (B5) basal medium supplemented with coconut milk (5 or 10%) and benzylaminopurine (10-6M). These shoots, if transferred individually to indole-3-acetic acid (10-7M) or naphthaleneacetic acid (10-6 or 10-7M) augmented B5 medium, produced roots at their base.

Somatic embryogenesis and organogenesis in callus cultures of a tree legume — Albizia richardiana King

Hypocotyl explants of 1 and 10 mm lengths were excised from 12-day-old in vitro-grown seedlings of Albizia richardiana. The larger pieces, after 40 days of culture, developed shoots along with green calli on B5 + BAP (10−7–10−5M), while the smaller segments produced only green calli on B5+BAP (10−7–10−4M) medium. Some of the green calli turned morphogenic and started producing somatic embryos with the 2nd sub-culture and shoots from 7th sub-culture onwards. Calli retained the morphogenic potential even after repeated sub-culturing for over two years. The number of embryos in an embryogenic culture varied from 2 to 20 per callus mass of 5–6.5 cm3. Sucrose at the 2% level in MS medium was optimal for embryogenesis while 4% was optimal for shoot bud differentiation. Higher levels of sucrose (6–10%) caused browning of green calli and also inhibited differentiation into embryos and shoot buds. By selective sub-culturing of 0.1 cm3 pieces of embryogenic calli on MS+10−5M BAP, 46% of the cultures produced somatic embryos. The latter germinated into plantlets on Knops medium.

Effective Protocol for in Vitro Shoot Production Through Nodal Explants of Simmondsia Chinensis

Nodal explants excised from 18 to 20-year-old female plants of Simmondsia chinensis if cultured on Murashige and Skoogs medium supplemented with 20 μM N6-benzyladenine (BA) differentiated an average of 2.7 ± 0.4 shoots in 11.5% explants. The percentage of nodal explants inducing multiple shoots enhanced significantly if in vitro raised shoots were used as source of explants. Nearly 100% cultures differentiated an average of 4.7 ± 2.0 shoots per explant on the same medium. Nearly 85% of the shoots induced roots when a pulse treatment of 50 μM indole-3-butyric acid (IBA) was given prior to their transfer to semi-solid MS medium containing 10 μM IBA + 0.5% activated charcoal + 1 μM BA. Plantlets were gradually hardened in Soilrite and acclimatized to soil.

Development of shoots and roots in anther-derived callus of Azadirachta indica A. Juss. - a medicinal tree

Callus originated in microsporangial wall layers and connective tissues of anthers containing uninucleate microspores on Nitschs or Murashige and Skoogs medium supplemented with growth regulators. A higher percentage of cultures (43) produced callus on Nitschs medium containing 10 μM indole-3-acetic acid + 1 μM 6-benzyladenine. After 13–15 weeks, green nodular structures and prominent roots developed in 25% of the cultures on Murashige and Skoogs medium + 10 μM α-naphthaleneacetic acid + 1 μM kinetin. Multiple shoots were induced in this anther-derived callus when subcultured on Murashige and Skoogs medium augmented with 4.44 μM 6-benzyladenine + 0.53 μM α-naphthaleneacetic acid along with 18.75 μM polyvinylpyrrolidone. The excised shoots formed roots after subculturing on Murashige and Skoogs medium + 4.90 μM indole-3-butyric acid + 18.75 μM polyvinylpyrrolidone, thus developing complete plantlets. Examination of callusing anthers also revealed two- to multi-celled pollen masses with intact exine.

Commercialization of plant tissue culture in India

Commercial application of plant tissue culture started in USA with micropropagation of orchids in 1970s. It has seen tremendous expansion globally from 1985 to 1990 in the number of production units as well as the number of plants produced. With an estimated global market of 15 billion US dollars per annum for tissue cultured products, even with exponential expansion in the industry, the demand far exceeds production, leaving enough scope for expansion. This industry appears to be undergoing a pause in growth presently in developed countries as it is finding difficult to remain cost–effective. In US, only half the production capacity is being utilized currently due to high labour costs. In developing countries, with lower wage scales, plants are being produced at much cheaper rates. Indian micropropagation industry, though a late starter by almost a decade, compared to its western counterparts, has expanded exponentially from 5 million annual capacity in 1988 to 190 million in 1996. The facilities now created are at par with the best in leading countries like the Netherlands and USA. To remain in profitable business and to earn the much needed foreign exchange, Indian units need to judiciously mix steady revenue generating items with unique speciality items based on demand in domestic and international markets.

Micropropagation of Sesbania rostrata from the Cotyledonary Node

Multiple shoots were induced from the cotyledonary nodes derived from seedling of Sesbania rostrata on Nitsch (1969; N) medium supplemented with various concentrations of benzyladenine (BA). 1 mg dm−3 BA proved to be the best, eliciting 5.8 ± 1.0 shoots per explant in 100 % cultures. The elongation of shoots was best at 2.0 mg dm−3 BA. The shoot proliferation capacity increased to 7.5 shoots per explant following transfer of explants to the fresh shoot multiplication medium (MS + 1.0 mg dm−3 BA), after an initial incubation of 30 d. To further enhance number of shoots per explant an alternative strategy of cultivation of mother explant on fresh shoot multiplication medium after excision of shoots was adopted. Following the repeated harvesting of shoots an average of 33 shoots per explant could be obtained. The in vitro regenerated shoots produced roots when transferred to half-strength MS medium supplemented with 3 % sucrose and 1 mg dm−3 IBA. The developed plantlets were planted in the soil and transferred to the field after an acclimatization period of 3 – 4 months. These plants produced flowers and fruits in the field and exhibited the development of prominent and more organized stem nodules as compared to the in vivo raised plants of the same age.

In vitro plant regeneration of leguminous trees (Albizia spp).

Hypocotyl explants of three leguminous forest tree species, Albizia amara, A. lucida and A. richardiana, have differentiated shoot buds on B5 basal medium. Maximum number of shoots per explant developed on basal medium augmented with 2,4-D (0.1 μM) in A. amara (2) and BA (10 μM) for both A. lucida (2) and A. richardiana (1.6). Higher concentrations of auxins in the medium, in general, enhanced rooting and callusing but cytokinins promoted the growth of green calli. BA enchanced the differentiation of shoots in the three species. The in vitro grown shoots of A. amara and A. richardiana, after subculturing on B5+1 μM IAA developed roots (up to 30–40%). These plants have been successfully transferred to the field.

Influence of some adjuvants on in vitro clonal propagation of male and female jojoba plants

SummaryThe influence of different adjuvants, activated charcoal (AC), casein hydrolyzate (CH), coconut water (CW), polyvinylpyrrolidone (PVP), and triiodobenzoic acid (TIBA), has been assessed on the shoot production potential of the nodal explants derived from in vitro-raised male and female jojoba (Simmondsia chinensis) shoots. Nodal explants of each sex were cultured separately on Murashige and Skoog medium supplemented with different levels of AC, CW, CH, PVP, and TIBA either alone or along with optimum levels of N6-benzyladenine (BA; 10 μM for male, 20 μM for female). Some differences in response of the explants of both the sexes have been observed in terms of (1) percentage of explants developing shoots, (2) average shoot number, and (3) average shoot length. AC alone proved beneficial for elevating morphogenic response in male as well as female explants in comparison to basal medium or media containing AC and the optimum level of BA. When used alone, CH proved inhibitory for shoot differentiation in both sexes, especially in male explants. Addition of PVP to MS enhanced shoot proliferation in female explants only, but along with BA it increased the response of male explants. BA in combination with different levels of TIBA promoted shoot multiplication in female explants. Thus, explants of both male and female shoots exhibited differential morphogenic behavior under the influence of various adjuvants. However, BA alone proved to be the best for differentiation of shoots in both male (10 μM) as well as female (20 μM) explants.

Effect of Carbon Source on Datura metel Microspore Embryogenesis and the Growth of Callus Raised from Microspore-derived Embryos

Summary The effects of various carbohydrates (fructose, glucose, sorbose, lactose, maltose, mannose, sucrose, mannitol, sorbitol and glycerol) on microspore embryogenesis and the growth of embryoderived callus of Datura metel were studied. Among the carbohydrates tested, sucrose, mannitol and sorbitol promoted the androgenic response, whereas, glucose, fructose, maltose and mannose inhibited it. On lactose containing medium, the anther response was not affected significantly, but the majority of embryos, developed on this medium, failed to grow beyond globular or heart-shaped stage. Sorbose was the only carbohydrate which completely inhibited the androgenic response. The effects of most of these carbohydrates on callus growth were not similar to what is being recorded for plantlet yield of cultured anthers. Among the sugars supporting appreciable growth of callus, sucrose was the best, followed by glucose, mannose, fructose and maltose. In addition, slight growth, in terms of increase in either fresh or dry weight, was also observed on media containing glycerol, mannitol, sorbose or sorbitol. However, lactose proved to be inhibitory.

Occurrence and Histochemistry of the Anther Tapetal Membrane

Abstract Several angiosperm and one gymnosperm taxa were investigated. The development and localization of the membrane was studied and differences were found between genera with an amoeboid or with a secretory tapetum. Several histochemical tests and fluorescence microscopy were used to investigate the chemical composition of the membrane. These studies indicate that the membrane consists of (a) insoluble polysaccharides, mainly callose, pectin and a cellulose-like substance, in addition to (b) an acetolysis-resistant cemponent (probably sporopollenin).