Rownak Afza

Generation of transgenic banana (Musa acuminata) plants via Agrobacterium-mediated transformation

An Agrobacterium-mediated plant transformation system was developed for the generation of transgenic banana (Musa spp. van Grand Nain). This system allowed for the recovery of putative transformants within four weeks after co-cultivation of tissue samples with Agrobacterium. Two or more cycles of meristem rooting and micropropagation allowed for the selection of plants from this putative transformant population which demonstrated chromosomal integration of foreign DNA by Southern analysis with no indication of chimeric tissues. Since plant breeding strategies aimed at banana crop improvement are extremely complex and long-term, virtually all commercial production is from clonal derivatives of naturally occurring variants. The genetic transformation technology reported herein will provide an additional tool for crop breeders who wish to introduce value-added traits into the banana and plantain cultivars that serve as vital food sources and a means of generating export income for producing nations.

Physical and Chemical Mutagenesis

Important methods to artificially induce mutations are the use of chemical and physical agents. Most chemical mutagens are alkylating agents and azides. Physical mutagens include electromagnetic radiation, such as gamma rays, X rays, and UV light, and particle radiation, such as fast and thermal neutrons, beta and alpha particles. Mutagenic treatment of seeds is the most convenient and, therefore, the standard method in seed propagated crops. Seeds can be treated in large quantities and are easily handled, stored, and shipped. It is fairly easy to repeat the conditions of mutagenic treatment, pre- and post-treatment, and hence, to obtain reproducible results within practical limits. Besides seed treatment, whole plants, cuttings, tubers, pollen, bulbs, corms, or in vitro plants or tissues can be treated. This chapter is restricted to the commonly applied techniques of mutation induction in seeds by ethyl methanesulfonate (EMS) treatment and by gamma and fast neutron irradiation.

Salinity tolerant performance and genetic diversity of four rice varieties

The genetic diversity of three salinity tolerant rice varieties Pokkali, Nona-Bokra and Bicol was investigated using random amplified polymorphic DNAs(RAPDs). High yielding susceptible variety IR29 was used as check for comparison. The salinity performance of these varieties were tested by using rapid screening techniques at seedling stage. One hundred primers tested of which 42 revealed differences between Pokkali & Nonabokra, 43 between Pokkali & Bicol and 50 between Nonabokra and Bicol. Polymorphism differences between IR29 - Pokkali, IR29 - NonaBokra and IR29 - Bicol were 47%, 53% and 31%, respectively. Four primers amplified specific fragments that appeared in all the three salt tolerant varieties but not in the salt susceptible variety IR29. Primer UBC 9 (5′-CCTGCGCTTA-3′ produced a prominent diagnostic fragment of approximately 1600 bp; primer UBC 244 (5′-CAGCCAACCG-3′) generated a fragment of about 800 bp in the salt-tolerant varieties; primer UBC 251 (5′-CTTGACGGGG-3′) amplified one polymorphic band of 1100 bp and primer UBC 267(5′-CCATCTTGTG-3′) yielded a relatively weak polymorphic band of 1100 bp.

Anther culture in connection with induced mutations for rice improvement

Doubled haploids have long been recognized as a valuable tool in plant breeding since it not only offers the quickest method of advancing heterozygous breeding lines to homozygosity, but also increases the selection efficiency over conventional procedures due to better discrimination between genotypes within any one generation. Ten cultivars of japonica rice and nine cultivars of indica rice were evaluated for androgenic response. Various doses (10–50 Gy) of gamma rays were applied to investigate the effect of radiation on callus formation, green plant regeneration and the frequency of selected doubled haploid mutants. Similarly, the effects of colchicine concentration (10–200 mg/l) on callus induction, regeneration and fertility of green plants were observed. It was demonstrated that the dose of 20 Gy gamma rays and 30 mg/l concentration of colchicine have significant stimulation effect on regeneration of green plants from rice anther culture. The high frequency of observed doubled haploid mutants indicates that anther culture applied in connection with gamma rays is an effective way to improve rice cultivars.

Histology of somatic embryo initiation and organogenesis from rhizome explants of Musa spp.

The origin of somatic embryos derived from rhizome explants of triploid Musa cv. Grand Nain was the subject of histological studies during different phases of ontogenetic development. The investigation revealed that the majority of somatic embryos showed normal root formation and consisted of highly vacuolated cells in the poorly structured shoot apex. The embryogenic mass and somatic embryos were mostly derived from several morphologically competent cells. Single cell origin depended on the presence of organogenetically functional vascular cells of rhizome explants and occurred infrequently. The implications of these findings for genetic improvement of banana and plantain by in vitro mutation breeding and gene technology are discussed.

Effect of spikelet position on rice anther culture efficiency.

The potential of anthers from different parts of the panicle to induce callus was investigated with the japonica rice variety Taipei 309. The results showed that the callusing abilities of anthers from different spikelet positions were significantly different. After plating 4483, 4496, 4348 anthers from the basal, middle and top parts, the percentage of anthers forming calli was 20% in the basal part, 12% in the middle part and 8% in the top part. The anthers of basal parts containing pollen at all uninucleate stages, including early, middle and late, showed higher callus induction frequency than those from middle and top parts. The green plantlet regeneration frequencies of top, middle and basal spikelets were around 18% in all three cases. From the results it would appear that anthers from the basal part of the panicle should be used in anther culture of rice in order to obtain higher efficiencies, and thereby optimise the usefulness of this technique in rice breeding programmes.

Induced Mutations For Enhancing Salinity Tolerance in Rice

Salt accumulation in soil surfaces, known as soil salinity, could lead to the impairment of plant growth and development and is manifested mostly under irrigated and dryland agriculture. Excess salts in the soil affects plants through osmotic stress; accumulation to toxic levels within the cells; and through the interference with the uptake of mineral nutrients. Rice productivity in several parts of the world is therefore severely limited by salinity on account of the prevalence of irrigation in rice farming. Tolerance to salt toxicity in plants is a genetic and physiologically complex trait. Halophytes (salt tolerant plants) are different from the salt-sensitive glycophytes in terms of peculiarities in their anatomy, ability to sequester otherwise toxic ions, and other physiologic processes. It is logical therefore to infer complexity also at the genetic level on account of the several pathways involved in these mechanisms. These complexities have confounded genetic improvement strategies for salinity tolerance in plants resulting in a paucity of saline tolerant plants, with only about 30 officially released saline tolerant crop varieties world-wide. Only one saline tolerant rice variety, Bicol, has been officially released to farmers. We review strategies being currently employed in the development of saline tolerant rice varieties. These include conventional plant breeding which is hampered by the lack of suitable genetic variation for this trait; the modest progress made through doubled haploidy; and the reliance on somaclonal variation, an unsustainably unpredictable strategy. This review also posits that while genetic transformation has led to the modification of certain physiological indices implicated in salinity tolerance in rice, in isolation, these modifications have not been translated to improved yield under salt stress. A more recently adopted strategy, induced mutagenesis, has led to some promising results. We argue that the production of induced rice mutants holds the greatest promise of these strategies for mitigating the scourge of soil salinity considering the relative ease with which other traits in this crop have been modified using this methodology. The underlying principles of induced mutagenesis; the modes of action of different mutagenic agents; and procedures for the rapid production and detection of mutants are also summarised. In order to enhance efficiency in the production, detection and incorporation of induced mutants into crop improvement programmes, we suggest the coupling of in vitro (such as doubled haploidy and cell suspension cultures) and molecular genetic techniques to this methodology. It is posited also that the efficiency of this process can be greatly enhanced by marker-aided selection while high throughput reverse genetics strategies could lead to the rapid detection of mutation events in target genes. It is concluded that with the plethora of genomics resources available for rice, the use of induced mutations for improving salinity tolerance (and other traits) would rely significantly on the concerted application of efficiency enhancing in vitro techniques and functional genomics strategies (including reverse genetics)

Detection of androclonal variation in anther-cultured rice lines using rapds

SummaryRandom amplified polymorphic DNA analysis was used to determine the occurrence and extent of variation in rice (Oryza sativa L.) plants regenerated from anther culture. Androclonal variation in morphologically uniform progenies was detected using 40 10-mer oligonueleotide arbitrary primers. Among 27 plants from nine anther culture-derived lines, variation was detected in three plants from two lines by two primers, namely UBC 160 and UBC 209. Primer UBC 160 amplified a polymorphic band on one of the three progenies from DH-34, while UBC 209 detected polymorphisms on two out of three progenies from line DH-58. Apart from these, the amplification produets were monomorphic across all the regenerants from anther culture-derived plants. Out of 40 tested primers, no difference in the banding pattern was observed in three seed-derived plants. The significance of possible androclonal variation at the DNA level in rice doubled haploid breeding and genetic mapping is discussed.

Genotypic variability for carbon isotope discrimination in the mutant and improved lines of barley

Drought tolerance is an important breeding objective in dry and semi-dry conditions. Carbon isotope discrimination (Δ) is a tool that may be used to improve water-use efficiency (WUE) as an indirect selection criterion. The study investigated the variability for Δ in improved F7 lines and their parents (three cultivars and two mutant lines), which were sampled randomly from an F6 nursery performing well under semi-dry conditions. In total, 40 entries were grown in sand culture, arranged in three-replicated randomized complete block designs in two sets of experiments in Antalya, Turkey. There were statistically significant differences (p<0.01) among genotypes in both sets of experiments for Δ, and Δ values range from 20.14 to 21.86. Low coefficient of variation (C.V.) values, i.e. 2 and 1.65 %, for both data sets revealed efficient control of experimental error for Δ and indicated little effect of environment. Consequently, broad-sense heritability estimates for Δ were 0.63 and 0.74. As Δ showed a considerably high heritability and consistency over the two sets of experiments and low C.V. values, it was concluded that this trait could be used in breeding programmes aimed at developing drought tolerance lines. The early heading mutant, M-K-88, and the cultivar selected from land race, Tokak 157-37, showed lowest Δ values, indicating that they had the best water-use efficiency. Low Δ values of these two genotypes were inheritable.