The potential of double haploid cassava via irradiated pollen

Transcription

1 The potential of double haploid cassava via irradiated pollen Mary Buttibwa 1,2, Robert Kawuki 1, Arthur K. Tugume 2, Yona Baguma 1 1 NaCRRI, Namulonge, Uganda, 2 Makerere Unversity, Uganda World congress on Root and Tuber crops Nanning, Guangxi, China, January 18-23, 2016

2 Doubled haploid technology (DH) v Haploids????: v Haploids are defined as saprophytes with gametophytic chromosome number. v They have a single genome or chromosome set. v Mainly involves: v Gynogenesis (ovule culture ) v Androgenesis (Anther /pollen culture) v Chromosome elimination (Irradiated pollen and wild crosses (chromosome elimination following interspecific hybridization (bulbosum technique). v Reported as early as 1964, 1966 in Datura innoxia and Nicotiana tabacum

3 Overview of DHs production methods in Plants

4 Why inbreeding in cassava is important v Allows identifying useful recessive traits. v Germplasm exchange and storage greatly facilitated. v Elimination of undesirable alleles. v However inbreeding is time consuming. v DH shorten the time to homozygosity. v Genetic process is faster and more consistent.

5 Comparison of allele segregation in F 2 cross and in DH0 line Parents AAbb aabb GeneraHon F 1 AaBb Allele recombination F 1 gamets: AB, ab, Ab, ab Doubled haploid lines Self-pollination AB ab Ab ab AB ab Ab AABB ab aabb AAbb Generation DH0 efficient selection AABB : aabb : AAbb : aabb 1 : 1 : 1 : 1 Homozygotic in both loci 1 : 4 Not only we have higher chance to find what we need but also the line is stable! AB ab Ab aabb ab AB ab Ab ab AABB AaBB AABb AaBb aabb aabb aabb aabb AAbB AabB AAbb Aabb aabb aabb aabb aabb GeneraHon F 2 A-B- : aab- : A-bb : aabb 9 : 3 : 3 : 1 Homozygotic in both loci 1 : 16 Expressed phenotype

6 Pollen irradiation technique v UV, gamma rays and X-rays currently used to induce in situ haploid plants. v Pollen is irradiated with a high irradiation dose v Irradiated pollen is genetically inert, but physiologically active and germinates on the stigma v Reported in snake cucumber (Yanmaz et al., 1999) and squash (Kurtar et al., 2002).

7 Significance of the research v Since inbreds carry only one allele of each gene mutations and recessive characteristics are expressed in cassava. v Plants with lethal alleles are eliminated from the population. v Can produce homozygous diploid or polyploid plants - valuable in breeding. v Shorten the time for inbreeding for production of superior hybrids genotypes.

8 Objectives General objective v The overall objective of the study is to develop an optimized protocol for the production of double haploid cassava of selected cultivars in Uganda. Specific Objectives v To determine the correlation between the sizes(age) of cassava flower buds and the developmental stage of the pollen grain. v To stimulate cassava asexual embryo development and subsequent invitro embryo rescue.

9 Regular fertilization Embryo sac Developing seed Egg cell Sperm cells After double fertilization Egg cell (n) + sperm cell (n) = embryo (2n) Central cell (2n) + sperm cell (n) = endosperm (3n)

10 Methodology v Harvested mature male flowers were irradiated using a Co60 gamma ray source v Irradiation dosages: 0, 50, 100, 150, 200 and 250 Gy; 1 Gray (Gy) = 100 Rad). 300 Gy, 350 Gy, 400 Gy & 500 Gy v For each irradiation treatment represented by 5 petri dishes, each petri dish having ~ 40 male buds. v Bud detachment from mother plants(mm96/4271), irradiation and pollination were all done on the same day (within 14 hours).

11 Pollination with irradiated pollen v Female flowers were bagged to avoid undesirable pollinations. v Control pollinations with non-irradiated pollen were performed at the same time v Field was laid-out in plots such that each dosage rate was assigned a separate set of plants/plot. v Records taken on numbers of surviving fruits per treatment up to 42 days, when embryo rescue was done. v Records were taken on seed-set (seeded or seedless embryos); embryo shape (irregular, globular, heart and cotyledonary) and number of embryos recovered. v 5 rounds of pollinations were undertaken

12 Methodology for SSR & ploidy analysis v DNA extractions v Irradiated plants v Leaf samples from mother plants for comparison v Extraction followed the Dellaporta et al. (1983). v Genotyping v 16 SSRY markers (EME425, NS158, NS169, SSRY100, SSRY103, SSRY106, SSRY135, SSRY148, SSRY179, SSRY181, SSRY19, SSRY20, SSRY215, SSRY240, SSRY28 and SSRY59). (Mba et al., 2001). v BecA Nairobi using DNA sequencer ABI 3730 v Ploidy analysis v Eight weeks old plantlets were subjected flow cytometry technique according to Otto (1990)

13 Results :Pollen germination Dosage Rep1/300 Rep2/300 Rep3/300 Fresh pollen v Fresh pollen had the highest germination frequency v 500GY had the lowest frequency

14 Pollen viability and germination tests Figure 1: Pollen size and pollen tube length following irradiation; higher irradiation doses (> 50 Gy) reduced pollen size and pollen tube length; A = 0 Gy; B = 50 Gy; C = 100 Gy; D = 150 Gy; E = 200 Gy; and F = 250 Gy. v There was germination at all the dose ranges. v Higher dosage reduced pollen viability.

15 Histology analysis A: Early uninucleate microspore B: Mid-uninucleate C: Late uninucleate D: Binucleate gametophyte E:Binucleate gametophyte F-I: Proress to mature pollen grains

16 Influence of irradiation dosage on cassava pollen germination s and mean number of fruit Dosage Total no. of pollen grains No. of pollen grains with pollen tube a Fresh pollen (80.8) Percent reduction in pollen germination Total pollinations Mean pollinations Percent abortion, 15 DAP Average no. of fruits at 42 DAP Fruit length (cm) Fruit width (cm) No. of seeds Average no. of seeds per fruit Dose 0 Gy (61.3) Dose 50 Gy (55.7) Dose 100 Gy (30.2) Dose 150 Gy (46.9) Dose 200 Gy (26.7) Dose 250 Gy (56.9) LSD CVs No. of embryos v By 15DAP Most of the fruits had aborted

17 Number of surviving cassava fruits following pollination with irradiated pollen v By 15DAP, over 80%of the fruit had aborted

18 Plant regeneration from irradiated pollen v v v v A and B : Opened and unopened cassava locules. C Freshly extracted embryo. D,Eand F Embryo germination after 3 days. G-J : Regenerated plantlets.

19 Zygosity and ploidy analysis of 62 cassava plantlets Dosage No. of plants No. of homozygous loci Mean (%) homozygous loci Channel mean Dose 0 Gy 5 7 to Dose 50 Gy 9 7 to Dose 100 Gy 6 2 to Dose 150 Gy 4 1 to Dose 200 Gy 2 2 to Dose 250 Gy 4 2 to Dose 300 Gy 25 2 to Dose 500 Gy 7 6 to Nase 14 a Mean ratio of sample to diploid Calcutta 4 v Nase14 had 100% homozygosity

20 Conclusions v Germination protocol for cassava pollen germination was established. v Follow-up studies plantlets. aimed at weaning and hardening of the v Exploring higher irradiation doses by use of high activity sources. v Rescuing embryos are earlier stages of development.

21 Acknowledgements v Personnel of Mulago Hospital, Kampala, Uganda, who assisted with the gamma irradiation v Mr. Moses Mugisha of the (IITA-Uganda) for technical assistance in ploidy analysis v ICESI University, Cali, Colombia

22 Thank you